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Influences of social housing conditions on chemotherapeutic efficacy : tumor, host and temporal factors Kerr, Leslie Roxanne 2000

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INFLUENCES OF SOCIAL HOUSING CONDITIONS ON CHEMOTHERAPEUTIC EFFICACY: TUMOR, HOST AND TEMPORAL FACTORS by Leslie R. Kerr B.Sc , Carleton University, 1990 M . S c , Carleton University, 1993 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in The Facu l ty of Graduate Studies Department of Anatomy, Neuroscience Program We accept this thesis as conforming to the required standard A p r i l 2000 THE UNIVERSITY OF BRITISH C O L U M B I A © Leslie Roxanne Kerr , 2000 UBC Special Collections - Thesis Authorisation Form http://www.library.ubc.ca/spcoll/thesautli.html In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I further agree that permission for extensive copying of t h i s t h e s i s for s c h o l a r l y purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or p u b l i c a t i o n of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. The U n i v e r s i t y of B r i t i s h Columbia Vancouver, Canada lof 1 3/17/00 1:04 PM ABSTRACT Stressful life events and the ability to cope with stress may play an important role in both cancer progression and treatment. A better understanding of the factors that influence tumor and host responses to cancer treatments, including chemotherapy, is essential for the development of improved treatment strategies. Utilizing the transplantable, androgen-responsive Shionogi carcinoma (SCI 15), this thesis tested the hypothesis that psychosocial stressors (social housing conditions), tumor growth rate and/or size, and the temporal relationship between formation of experimental housing conditions and chemotherapy initiation would differentially influence chemotherapeutic efficacy. The possible roles of corticosterone, testosterone and growth hormone in mediating the effects of social housing conditions on tumor and host responses to chemotherapy were also examined. The experimental housing conditions used in these studies included DD/S mice that were reared individually then group-housed (IG), mice that were reared in groups then individually housed (GI) and mice that did not experience a change from their original rearing conditions (II or GG). The studies in this thesis demonstrated that housing conditions differentially influence both SCI 15 tumor and host responses to chemotherapy. Importantly, the temporal relationship between formation of experimental housing condition and chemotherapy initiation significantly influenced the direction and magnitude of the effects of social housing conditions on chemotherapeutic efficacy. The differential effects of housing conditions on tumor and host responses to chemotherapy may be mediated by alterations in basal corticosterone and growth hormone but not testosterone levels within the first week following formation of experimental housing conditions and initiation of chemotherapy. However, the differential effects of housing conditions on tumor and host responses to chemotherapy were independent of tumor growth ii rate and, at least in terms of host response to chemotherapy, were also independent of tumor burden. The final study of this thesis examined whether social housing conditions and gender interact to affect differentially the growth of a variant of the SCI 15 tumor, designated SCI 15V, that grows equally well in male and female DD/S mice. For both males and females, a change in social housing condition and the direction of change were critical in determining whether SCI 15V tumor growth rates increased or decreased compared to those of mice that did not experience a change. Moreover, social housing condition and exposure to acute daily novelty stress appeared to interact to produce differential effects on SCI 15V tumor growth rates in males and females. The data presented within this thesis indicate that social housing conditions can influence significantly tumor and host responses to chemotherapy. Moreover, these data demonstrate that the direction and magnitude of the effects of social housing conditions on chemotherapeutic efficacy is dependent on the temporal relationship between the formation of experimental housing conditions and initiation of chemotherapy and that the differential effects observed may be mediated, in part, through alterations in basal hormone levels. in TABLE OF CONTENTS ABSTRACT ii LIST OF FIGURES vii LIST OF TABLES viii LIST OF ABBREVIATIONS ix ACKNOWLEDGMENTS xi FORWARD xii CHAPTER 1: GENERAL INTRODUCTION A. Psychosocial Stressors and Psychosocial Oncology 1 1. Stress, Stressors and the Stress Response 2 2. Stress and Cancer: Human Studies 4 3. Stress and Cancer: Animal Studies 9 B. Psychoneuroendocrinology and Cancer 17 1. Hormones and Stress 17 2. Hormones and Cancer 23 3. Hormones and Cancer: Human Studies 25 4. Hormones and the SCI 15 Tumor 27 C. Psychosocial Stressors and Chemotherapy 28 1. Chemotherapy 28 2. Hormones and Chemotherapy 32 3. Tumor Response to Chemotherapy 32 4. Host Response to Chemotherapy 33 5. Stressors and Chemotherapy: Human Studies 36 6. Stressors and Chemotherapy: Animal Studies 38 D. Mouse-tumor Model for Studying the Effects of Psychosocial Stressors 39 E. General Rational and Thesis Objectives 45 CHAPTER 2: GENERAL METHODS A. Dissociation 49 B. Freezing and Thawing of Tumor Cells 50 C. Transplantation of Tumor Cells and Housing Conditions 50 iv D. Chemotherapy Treatment, 51 E. Radioimmunoassays (RIAs) 52 F. Statistical Analyses 53 CHAPTER 3. EFFECTS OF SOCIAL HOUSING CONDITIONS ON THE RESPONSE OF THE SHIONOGI CARCINOMA (SCI 15) TO CHEMOTHERAPY A. Introduction 55 B. Methods 58 C. Results 59 D. Discussion 61 CHAPTER 4. CHEMOTHERAPEUTIC EFFICACY IN THE SCI 15 MOUSE-TUMOR MODEL: TEMPORAL FACTORS A. Introduction 73 B. Methods 75 C. Results 77 D. Discussion 79 CHAPTER 5. EFFECTS OF SOCIAL HOUSING CONDITIONS ON SCI 15 TUMOR AND HOST RESPONSES TO ADJUVANT CHEMOTHERAPY A. Introduction... 94 B. Methods 96 C. Results 97 D. Discussion 100 CHAPTER 6. EFFECTS OF SOCIAL HOUSING CONDITIONS AND CHEMOTHERAPY ON HORMONE LEVELS IN THE SHIONOGI CARCINOMA (SCI 15) MOUSE TUMOR MODEL: TEMPORAL FACTORS A. Introduction 113 v B. Methods 1. Experiment 1 117 2. Experiment 2 118 C. Results 1. Experiment 1 120 2. Experiment 2 122 D. Discussion 124 CHAPTER 7. INTERACTIVE EFFECTS OF SOCIAL HOUSING CONDITIONS AND GENDER ON MOUSE MAMMARY TUMOR GROWTH A. Introduction 146 B. Methods 149 1. Experiment 1 149 2. Experiment 2 150 C. Results 1. Experiment 1 151 2. Experiment 2 152 D. Discussion 153 CHAPTER 8. GENERAL DISCUSSION A. General Discussion of Studies 162 B. Future Directions 170 REFERENCES 176 LIST OF FIGURES Fig. 1 Experimental design Fig. 2 Tumor growth in drug-vehicle treated mice Fig. 3 Survival probability in tumor-bearing mice receiving chemotherapy (TC) or drug vehicle (TV) and in non-tumor-bearing mice receiving chemotherapy (NTC) Fig. 4 Experimental design Fig. 5 Tumor growth in drug vehicle-treated mice Fig. 6 Survival probability in A: tumor-bearing mice receiving either chemotherapy (TC) or drug vehicle (TV) and B: non-tumor-bearing mice receiving chemotherapy (NTC) Fig. 7 Experimental design Fig 8 Tumor growth in drug vehicle-treated mice Fig 9 Survival probability in A: tumor-bearing mice receiving either chemotherapy (TC) or drug vehicle (TV) and B: non-tumor-bearing mice receiving chemotherapy (NTC) Fig. 10 Experimental designs Fig. 11 Tumor growth in drug vehicle-treated mice Fig. 12 Basal plasma hormone levels in tumor-bearing, drug vehicle-treated mice Fig. 13 Basal plasma hormone levels in tumor-bearing, chemotherapy-treated mice Fig. 14 Tumor growth in drug vehicle-treated mice Fig. 15 Basal plasma hormone levels in tumor-bearing, drug vehicle-treated mice Fig. 16 Basal plasma hormone levels in tumor-bearing, chemotherapy-treated mice Fig. 17 SC 115V tumor weights over 4 measurement times post tumor cell injection, for male and female mice exposed to acute daily novelty stress (S) or left undisturbed (no novelty stress; NS) LIST OF TABLES Table 1 Tumor growth delay (TGD) in chemotherapy-treated mice Table 2 TGD in days (mean ± SEM) in chemotherapy-treated mice Table 3 Percent body weight loss (mean ± SEM) in mice over the course of chemotherapy or drug vehicle treatment Table 4 Percent body weight loss (mean ± SEM) in mice over the course of chemotherapy or drug vehicle treatment Table 5 Tumor response to chemotherapy (mean ± SEM) in mice 4 d post chemotherapy treatment Table 6 Percent body weight loss (mean ± SEM) in mice 4 d post chemotherapy or drug vehicle treatment L I S T O F A B B R E V I A T I O N S ACTH - adrenocorticotrophic hormone AD - Adriamycin AIGF - androgen-induced growth factor ANOVA - analysis of variance ANS - autonomic nervous system AR - androgen receptor b-FGF - basic fibroblast growth factor cm - centimeter CNS - central nervous system CORT - corticosterone CRH - corticotrophin-releasing hormone CS - calf serum CTL - cytotoxic T-lymphocyte CY - cyclophosphamide d-day DHT - dihydrotestosterone DMEM - Dulbecco's minimum Eagle's medium DMSO - dimethylsulfoxide DNA - deoxyribonucleic acid DSM-IV - diagnostic and statistical manual of mental disorders, revision 4 E - epinephrine E 2 - estrogen ER- estrogen receptor ERE - estrogen response element FSH - follicle-stimulating hormone GG - group to group housed GH - growth hormone GI - group to individually housed GnRH - gonadotrophin-releasing hormone GR - glucocorticoid receptor HPA - hypothalamic-pituitary-adrenal HPG - hypothalamic-pituitary-gonadal hr - hour HRE - hormone response element HSP - heat shock protein ICV - intracerebroventicular II - individually to individually housed IG - individually to group housed IGF - insulin-like growth factor i.p. - intraperitoneal p.o. - peroral LC - locus coeruleus LH- luteinizing hormone LN 2 - liquid nitrogen MEL - melphan min - minute MR - mineralocorticoid receptor mRNA - messenger ribonucleic acid NE - norepinephrine NK - natural killer NTC - non-tumor bearing, chemotherapy POMC - proopiomelanocortin PNS - parasympathetic nervous system PVN - paraventricular nucleus RIA - radioimmunoassay sec - second s.c. - subcutaneous SCI 15 - Shionogi carcinoma 115 SEM - standard error of the mean SNS - sympathetic nervous system STV - saline-trypsin-versine T - testosterone TC - tumor-bearing, chemotherapy TGD - tumor growth delay TV - tumor-bearing, drug vehicle p-END- p-endorphin ACKNOWLEDGEMENTS I would like to thank my supervisors Drs. Joanne Emerman and Joanne Weinberg for their advice, guidance and support over the years. I would also like to thank the members of my supervisory committee, Drs. Gail Bellward, Peter Leung, Peter Reiner and Doug Waterfield, for their patience, advice and guidance throughout the course of my thesis. I am indebted to Heather Andrews, Dr. Karen Strange and Candace Hofmann for their thoughtful discussions, advice and friendship. Thanks also to Glenn Edin and Darcy Wilkinson for their technical support and help. A special thanks is extended to Dr. Webber for his advice and support. A large thanks to all the graduate students and my other friends in the Department, many of whom have helped to make me smile. I would like to extend my special gratitude to Mr. Roman Babicki for his generous financial support over two years of my doctoral studies through the Roman M. Babicki Scholarship for Medical Research. I would like to thank my parents and my sister for their love, support and encouragement. Finally, I would like to thank my fiancee, Rick Tinker, for his unwavering support and belief in me. Thank you for your love, friendship and willingness to stand by me during the trials of graduate life. This research was supported by grants from MRCC and NIH/NCI. xi FORWARD Portions of this thesis have been published previously or submitted for publication: Kerr, L.R., Grimm, M.S., Silva, W.A., Weinberg, J., and Emerman, J.T. Effects of social housing condition on the response of the Shionogi mouse mammary carcinoma (SCI 15) to chemotherapy. Cancer Research 57: 1124-1128, 1997. Kerr, L.R., Wilkinson, D.A., Emerman, J.T., and Weinberg, J. Interactive effects of psychosocial stressors and gender on mouse mammary tumor growth. Physiology and Behavior 66: 277-284, 1999. Kerr, L.R., Emerman, J.T., and Weinberg, J. Chemotherapeutic efficacy in the Shionogi carcinoma (SCI 15) mouse tumor model: temporal relationship between formation of social housing conditions and chemotherapy initiation. Psychosomatic Medicine, submitted, 2000. Kerr, L.R., Hundal, R., Silva, W.A., Weinberg, J., and Emerman, J.T. Effects of social housing condition on tumor and host responses to adjuvant chemotherapy in a Shionogi carcinoma (SCI 15) mouse tumor model. Psychosomatic Medicine, submitted, 2000. CHAPTER 1. GENERAL INTRODUCTION PSYCHOSOCIAL STRESSORS AND PSYCHOSOCIAL ONCOLOGY In general, psychosocial stressors include stressful intensive as well as possibly more subtle but complex social interactions or the lack of social interactions (Koolhaas et al, 1997a; Koolhaas et al, 19976; Theorell, 1997). Psychosocial stressors (like other stressors) disrupt homeostasis that is maintained in a coordinated fashion by the central and autonomic nervous systems in conjunction with complex interactions with endocrine and immune systems. Data from both human and animal studies indicate that psychosocial stressors can cause significant alterations in hormone levels (Armario & Lopez-Calderon, 1986; Riley, 1981; Rowse et al, 1992; Sachser, 1987; Sklar & Anisman, 1981) and immune functioning with or without hormonal mediation (Bartrop et al, 1977; Bonneau et al, 1990; Bovbjerg, 1991; Guyre et al, 1984; Hardy et al, 1990; Irwin et al, 1987; Kiecolt-Glaser & Glaser, 1995). Both endocrine and immune systems play integral roles in cancer growth (Bovberg, 1991; Musselman, 1998) and treatment (Emerman & Siemiatkowski, 1984; Goldin & Houchens, 1978; Levy et al, 1987; Lippman et al, 1985; Maclean & Longnecker, 1994; Mitchell, 1992; Mokyr & Dray, 1987; North, 1984; Peacock et al, 1987). Psychosocial stressors may influence both cancer growth and treatment by altering the activities the endocrine and immune systems through direct effects on the central and autonomic nervous systems. Psychosocial oncology is the study of the relationship between psychosocial stressors and cancer growth. Recently, both human and animal studies have also examined the relationship between psychosocial stressors and efficacy of cancer treatments. Thus, psychosocial oncology can be extended to the study of the relationship among stressors, cancer growth and treatment efficacy. 1 Stress, Stressors and the Stress Response The survival of an organism, and therefore the entire species to which it belongs, depends on its ability to adapt to a changing environment. Thus, life exists through the maintenance of a complex dynamic equilibrium, or homeostasis, that is constantly challenged by many endogenous and exogenous forces. Stressors are defined as the forces that disturb homeostasis, and stress is defined as a state of threatened homeostasis, which is typically characterized by alterations in an organism's hormonal and neural secretions. Following the threat of a stressor, homeostasis is reestablished by a complex repertoire of physiological and behavioral adaptive responses (together these constitute the stress response) generated by the organism. These responses can be influenced differentially by the type of stressor [broadly classified as physical (e.g. electric foot shock) or psychological (e.g. social housing condition)] as well as stressor duration (e.g. acute versus chronic) and intensity (Anisman et al, 1993; Armario et al, 1988; Armario et al, 1990; Harbuz et al, 1995; Patterson & Neufeld, 1987; Sklar& Anisman, 1981). The stress response may also be influenced differentially by the organism's perception of control over the stressor (i.e. the cognitive processing of the stimulus). These cognitive processes can blunt the stress response and therefore, may reduce the impact of a stressor on the organism (Ely, 1995; Lutgendorf et al, 1994; Steptoe, 1998; Weinberg & Levine, 1980). Examples of such cognitive processes include habituation and coping responses (Levine et al, 1978). Habituation is defined as the process whereby a novel stimulus, that is not intrinsically aversive, initially causes a stress response but following association of the stimulus with a non-threatening outcome, that stimulus no longer elicits a stress response. In contrast, coping can be defined as a process that enables an organism to control, therefore decrease, the levels of 2 arousal caused by a noxious or aversive stimulus. Coping can involve behavioral (e.g. escaping or moving away from the stimulus) as well as psychological (e.g. predictability of stimulus severity) responses. Importantly, factors such as past experiences, emotional states, environmental factors surrounding an event as well as genetic differences can influence the organism's perception of control over the stressor (Benus et al, 1991; Bouchard, T. 1994; Koolhaas et al, 1997b; Schwarzer, 1998). Generally, stress responses are advantageous for the survival of the organism, helping it to withstand stressors with a variety of relatively consistent changes in behavioral and physiological responses. The former include increased arousal and alertness, heightened and focused attention, and suppression of sexual and feeding behaviors. The latter are responsible for changes in metabolism (e.g. suppression of anabolic processes such as growth, increased pulse rate, blood pressure, lipolysis and gluconeogenesis) causing the redirection of energy (oxygen and nutrients) to the central nervous system (CNS) and stressed body site(s), where they are needed most. Although the coordination of the stress response is subserved by the CNS, the stress response system is composed of a complex network that includes communication among the CNS, autonomic nervous system (ANS), endocrine, and immune systems (Anisman et al, 1993; Brown, 1991; Chrousos & Gold, 1992; Jansen et al, 1995; Johnson et al, 1992). Interaction among these systems helps the organism to efficiently and flexibly coordinate the proper activation of physiological and behavioral stress responses that will return the organism to a state of homeostasis. Proper activation of these responses comprises both positive and negative feedback systems which are essential in preventing over response from both the central and peripheral components of the stress response system. If the interaction among these modulatory systems fails to contain the various elements of the stress 3 response, the adaptive physiological and behavioral responses may become inadequate, excessive or prolonged and may thus contribute to a variety of pathological conditions such as gastrointestinal ulcerations, myopathy, weight loss, fatigue, immunosuppression, steroid diabetes and reproductive dysfunction (McEwen et al, 1997; Stratakis & Chrousos, 1995; Szabo, 1998; Weissman, 1990). In addition, a variety of psychopathological conditions may occur including anxiety, depression, panic disorders, obsessive compulsive disorders and anorexia nervosa (Chrousos & Gold, 1992; Johnson et al, 1992). Stress and Cancer: Human Studies In humans, many psychosocial stressors/demands have been reported in the context of their relationship to the development and progression of cancer (Cooper & Faragher, 1993; Fox, 1998; Helgeson et al, 1998; Kornblith, 1998; Ramirez et al, 1989; Silberfarb & Greer, 1982). In recent years particular psychosocial stressors or factors directly related to these stressors have received special attention because they have been most often associated with increased risk of cancer development and increased probability of metastasis. These include: bereavement, depressive states, a sense of helplessness and hopelessness over life events, suppressed emotions (especially anger), and lack of social support networks (Fox, 1998; Silberfarb & Greer, 1982). However, possibly due to the complex nature of human studies, some reports have provided little or no support for the association between stressful life events and/or personalities and the risk of breast cancer (Cassileth, 1996; McKenna et al, 1999; Protheroe et al, 1999; Roberts et al, 1996; Tross et al, 1996). For example, retrospective studies reporting the relationship between stressful life events and cancer have been criticized because patients, knowing that they have cancer, may exhibit bias to negative interpretations of 4 life events (Fox, 1998; Levenson & Bemis, 1991). However, it has been suggested that recall bias may not be a significant factor in these studies (Geyer, 1992). Life stress, social support and cancer. Stressful life events, as well as the frequency of such events, have been associated with increased cancer risk and/or probability of metastasis and decreased survival probability. For example, Neale (1986) reported that being married as opposed to being widowed reduces cancer risk. Funch and Marshall (1983) demonstrated that death, illness or unemployment in the breast cancer patient's family predicts poorer survival and that increased social contacts increase survival probability. Retrospective studies have demonstrated that a stressful life event such as the death of a close friend or family member, are more common in a 1 to 6 year period preceding the diagnosis of cancer than in control populations (Cooper et al, 1989; Forsen, 1991; Geyer, 1992; Taylor et al., 1988). Also, Ramirez and colleagues (1989) demonstrated a positive correlation between the frequency of traumatic life events following the diagnosis of breast cancer and reoccurrence. Affective States, Bereavement, Personality Traits and Cancer. The diagnosis of cancer is in itself a stressful life event that is associated with increased distress among cancer patients as well as their friends and family. Psychological responses to cancer and its treatment have been categorized into (1) sadness and hopelessness, (2) anxiety, (3) anger and/or guilt and (4) avoidance or denial of the disease (Silberfarb & Greer, 1982; Spiegel, 1997; Temoshok, 1987). An important aspect of the psychological responses of cancer patients is the issue of autonomy and control (dependence/independence) over the disease and its treatment (Silberfarb & Greer, 1982). Overall, these psychological responses have been attributed to patients suffering from 5 severe distress/grief or depression. Depression has both psychological (mental) and somatic (physical) symptoms. The somatic indicators of depression in cancer patients are of little value as diagnostic criteria for depression are common to both cancer and depression. Because of this, the diagnosis of depression in the cancer patient depends primarily on the psychological, rather than somatic, symptoms (Massie & Popkin, 1998; Newport & Nemeroff, 1998). The measurement of depression in the cancer patient is challenging because the responses of the patient to the diagnosis of cancer are similar to major depression (Massie & Popkin, 1998; Newport & Nemeroff, 1998). To fulfill the DSM-IV (Diagnostic and Statistical Manual of Metal Disorders, revision IV) criteria for major depression, the patient must exhibit pervasively depressed mood or anhendonia (diminished interest or pleasure in nearly all activities) for at least 2 weeks. In addition to one of these two primary symptoms, the patient must also have at least four of the following symptoms (or three if the patient has both depressed mood and anhendonia): appetite or weight change (gain or loss); sleep disturbance (insomnia or hypersomnia); observable psychomotor agitation or retardation; fatigue; inappropriate guilt; poor concentration, and recurrent thoughts of death or suicide. These symptoms must represent a change from previous functioning and must cause clinically significant impairment in social or occupational functioning. In cancer patients, the evaluation of depression must determine whether the physiological effect of the cancer is underlying the depressive syndrome. Depressive syndromes which are caused by the direct physiological effects of cancer in DSM-IV, are called Mood Disorders with Depressive Features Due to Cancer. A prominent and persistent depressed mood that resembles major depression is a key feature of this disorder (Baraclough, 1999; Massie & Holland, 1990). 6 Interestingly, the DSM-IV recognizes an overlap in psychological and somatic symptoms between bereavement and a major depressive episode. Although several qualifying symptoms are listed, the DSM-IV states that there is no definitive description to distinguish between the diagnosis of bereavement and major depression (Newport & Nemeroff, 1998). Many studies have demonstrated that the psychological symptoms resulting from the diagnosis of cancer or bereavement can be resolved, in the majority of cases, over several weeks with support from family, friends and the health care providers including the physician who outlines a treatment plan that can offer hope (Chochinov et al, 1998; Massie & Popkin, 1998). However, some patients will develop persistent anxiety or depression. Reported rates of depressive states among random samples of hospitalized cancer patients vary from approximately 10-50% (Derogatis et al, 1983; Spiegel, 1997). Although depression has been estimated to be four times more frequent in cancer patients compared to the general population, this estimate becomes lower as the term "depression" is defined more rigorously (Barraclough, 1999; Spiegel, 1997). Clinically depressed patients, including cancer patients, are usually treated with a combination of supportive psychotherapy and antidepressants (Massie & Holland, 1988; Massie & Popkin, 1998). The goals of psychotherapy are to reduce emotional distress and to improve coping ability, self-esteem and the sense of control (Brown & Pedder, 1991; LeShan, 1977; Worden & Weisman, 1984). Importantly, factors that modify the impact of psychotherapy include the nature and availability of support from family, friends, and health care professionals as well as the personality traits or mental adjustment of the patient (Funch & Marshall, 1983; Silberfarb & Greer, 1982). For example, a three month post-operative assessment of mental adjustment to cancer demonstrated that cancer patients who were rated as 7 "stoic" or "helpless" tended to have worse outcomes in terms of disease free interval or cancer progression at five year (Greer et al, 1979), ten year (Pettingale et al, 1985) and fifteen year follow-up (Greer et al, 1991) than women who were rated as exhibiting "fighting spirit". Similarly, Temoshok (1987) suggested that a certain personality type, type C, may be associated with an increased risk of cancer (cutaneous malignant melanoma). The type C individual was hypothesized to be a cooperative and appeasing, unassertive patient, unable or unwilling to express negative emotions (particularly anger) and compliant with external authorities (Temoshok & Fox, 1984; Temoshok et al, 1985). In general, patients with a higher risk for poor coping include those who are socially isolated, have a history of recent losses, have multiple obligations, or use inflexible or fewer coping strategies (Cooper & Faragher, 1993; Spiegel, 1997). Cancer patients who learn to use more direct and confrontational coping strategies, through psychotherapy or behavioral therapy sessions, are less distressed and may have longer disease-free intervals and better quality of life than those who use avoidance and denial (Barraclough, 1999; Jacobsen & Holland, 1991; Spiegel, 1997). As outlined above, several studies have suggested an association among stressful life events, depression, bereavement and certain personality traits and the development or incidence of cancer. However, other studies have found no evidence or weak relationships among these factors and the development or incidence of cancer (e.g. Fox, 1989; Cassileth, 1996). Several reasons for inconsistent results in this area have been suggested. First, the range of assessment methods available with which to evaluate a patient's psychological state and/or mental adjustment can lead to considerable variability among studies (Goldberg & Cullen, 1985; Heim et al, 1997; Leigh et al, 1987; Spiegel, 1997). Similarly, the timing of these assessments relative to when the cancer was first detected, positively diagnosed, treated or reoccurred may 8 also result in inconsistent relationships between life stressors and cancer (Dean & Surtees, 1989; Jacobsen & Holland, 1991; Massie & Popkin, 1998; Spiegel, 1997). Also, independent biological variables (e.g. tumor type or site, gender, stage of disease and age) have been shown by some to impact significantly on cancer development, progression or survival probability (Redd et al, 1991; Silberfarb & Greer, 1982; Temoshok, 1987). Study design may also differentially affect results. For example, in retrospective studies the diagnosis of cancer may distort the patient's perception of stressful events due to the knowledge that he/she has cancer. Also, the clinical manifestation of cancer may occur years following neoplastic change and therefore, the onset of the disease may have occurred prior to the stressful event. Finally, premorbid psychological development (encompassing past experiences of the individual) and the cultural context in which that patient lives may also cause inconsistencies in the literature regarding the effects of psychosocial stressors and cancer development and/or progression. Stress and Cancer: Animal Studies In human studies, whether psychosocial stressors/demands or a patient's ability to cope with the demands, influence cancer risk and/or survival remains unresolved. Animal models allow investigation of the relationship among stressors, coping mechanisms and tumor growth or progression under more controlled conditions. However, even in animal models, the data are complex. Factors such as the type of tumor, stressor timing, duration and severity, the species or strain of the animal used and the ability to cope with the stressor can influence the effects of stress on tumor growth or progression. Animal-tumor models have demonstrated that stressors inhibit the growth of carcinogen-induced tumors whereas the growth of virally induced tumors is promoted by 9 stressors (Newberry, et al, 1991; Riley et al., 1981; Romero et al, 1992). It has been suggested that the differential effects of stressors observed among different tumor types may be due to the ability of the immune system to detect the tumor (Anisman et al, 1989; Justice, 1985; Riley, 1981; Steplewski et al, 1985). Thus, stressor-induced suppression of the immune system would permit rapid growth of immunogenic tumors (e.g. viral tumors) whereas growth of non-immunogenic tumors (e.g. carcinogen-induced) may be unaffected by changes in the immune system. Growth of non-immunogenic tumors has been suggested to be governed by other factors such as hormones or opioids (Justice, 1985; Peters & Kelly, 1977; Romero et al, 1992). Therefore, stressor-induced changes in hormone or opioid levels may be mediating growth of these tumors. Others have suggested that the differential effects of a stressor on carcinogen-induced compared to viral tumors may be due to the duration of stressor exposure; studies that used carcinogen-induced tumors typically involved repeated stressor sessions due to the length of time required for carcinogen-induced tumors to appear (Newberry et al, 1991; Sklar & Anisman, 1981). The importance of the duration of a stressor has also been demonstrated within the same tumor system. For example, Sklar and Anisman (1979) observed that a single session of inescapable footshock applied 24 hr following transplantation of the syngeneic P815 mastocytoma cells in mice enhanced tumor development whereas 5 or 10 successive days of footshock sessions failed to enhance tumor development (i.e. repeated or chronic stressor, antagonized the tumor enhancing effects of a single acute stressor). Importantly, the enhancing effects of acute stress on tumor development was also observed when the stressor was applied 3 or 5 days following tumor transplantation. Therefore, it was concluded that the antagonism of tumor development observed with the chronic stressors was not a result of the timing of stressor 10 application relative to tumor development, as has been observed in other studies (e.g. Amkraut & Solomon, 1972; Newberry, 1978; Riley, 1981). In addition to the type of tumor and stressor timing or duration relative to tumor induction, the type of stressor that an animal is exposed to can also differentially influence tumor growth. For example, restraint stress was found to decrease the growth of a transplantable mammary tumor (Walker carcinoma; Newton, 1964) as well as the number of tumors induced by the chemical carcinogen 7,12-dimethylbenz(a)anthracene (DMBA; Newberry et al, 1991). In contrast, rotational stress was found to increase the growth of both the transplantable allogenic lymphosarcoma (6C3HED; Kandil & Borysenko, 1988) and P815 mastocytoma (Sklar & Anisman, 1980). It is impossible to determine without exhaustive and systematic studies whether the type of tumor, the type and/or duration of the stressor, the species or strain of animal used, or an interaction among these (and possibly other) factors play key roles in modulating tumor growth rate. For this reason, the interpretation of animal-model data and the comparison of data among studies should be done with caution and consideration of the variables within each experimental paradigm. Housing conditions and tumor growth. In addition to foot shock and restraint stress, social housing conditions have also been shown to have significant influence on the development of tumors. In rodents, psychosocial stressors that have been studied in context of tumor growth and metastasis include individual or group housing conditions as well as changes in housing conditions. Studies have shown that individually housed animals typically have faster tumor growth rates than group housed animals (Riley, 1981; Riley et al, 1981). Additionally, a 11 change in housing condition may alter tumor growth rate. In one study, mice that were rehoused from group to individual housing had higher tumor growth rates than mice that remained group housed (Sklar & Anisman, 1980; Weinberg & Emerman, 1989). Interestingly, the adverse consequences of housing change on tumor growth were shown to be reduced by fighting, which has been suggested to act as a coping mechanism (Grimm et al, 1996; Sklar & Anisman, 1980; Sklar & Anisman, 1981); recall that coping can act to decrease the level and nature of behavioral and physiological responses to a stressor (Ely, 1995; Weinberg & Levine, 1980). Thus, fighting among group housed animals was shown to reduce tumor growth rate compared to groups where fighting did not occur (Sklar & Anisman, 1980). Similarly, other studies in which the organism's ability to cope is more clearly defined (e.g. ability to escape the stressor) also have demonstrated that the ability to cope with a stressor through behavioral means may be fundamental in determining the stressor-elicited variations in tumor growth. For example, whereas escapable footshock did not affect the growth of a P815 mastocytoma, an identical amount of inescapable footshock enhanced tumor growth rate (Sklar & Anisman, 1979). In addition, recovery from a stressor may reduce the growth rate of mammary tumors. Steplewski et al (1987) demonstrated that, following 11 days of restraint, tumor weights in female Lewis rats were similar to those of non-restrained control rats, but, after 12 days of recovery from the chronic restraint stress, tumor weights were lower than those of the control rats. Different housing conditions also have been shown to evoke distinctly different changes in both hormone levels and immune functioning which may mediate the differential in tumor growth rates observed (Rowse et al, 1990a; Rowse et al, 1995). 12 Individual housing. Studies have suggested that mice housed in individual housing conditions exhibit marked behavioral and physiological changes compared to group housed counterparts. In general, individual housing increases plasma testosterone (T) levels and immune responses and decreases plasma corticosterone (CORT) and norepinephrine (NE) levels as well as epinephrine (E) turn over in the adrenal glands (Brain, 1975). Individual housing in mice has been shown to increase both aggression in males (Brain, 1975) and response to stressors (Brain, 1975; Frances et al, 1989). The behavioral changes exhibited by individually housed animals have been suggested by some to be due to the inability of the animal to cope with the isolation due to the lack of social interactions (Ely, 1995; Henry et al, 1975). As mentioned earlier, individually housed animals typically have faster tumor growth rates compared to group housed animals. Although the hormone levels in individually housed animals are indicative of decreased stress levels, the hyperresponsiveness to stressors and increased tumor growth rates have been used as examples of increased stress levels in these animals (Brain, 1975; Dechambre & Gosse, 1973; Giralt & Armario, 1987). Thus, it is controversial whether individual housing constitutes a stressor in mice. Group housing. Group housed mice tend to form non-linear hierarchies with one dominant male and a number of equally-ranked submissive/defensive (subdominant) males in a cage (Haug et al, 1986). Among the submissive mice, two characteristic behaviors have been suggested: 'subdominant passive' and 'subdominant active'. The former constitutes the majority of mice housed in a group condition, who never initiate attacks against other group members whereas the latter, without being the dominant mouse, will attack other group members (Haemisch & Gartner, 1997). Generally, in stable dominance hierarchies, the 13 dominant male initiates and wins the majority of encounters (Brain, 1975; Sapolsky, 1995). Overall, it is suggested that in a stable dominance hierarchy, mice use specific and ritualized behaviors such as different postures, tail positions and vocalizations therefore minimizing the occurrence of overt fighting within the group. Thus, threats (usually without physical contact) replace actual attack. However, if an established dominance hierarchy is destroyed by the removal of an individual or the introduction of strangers, severe fighting will occur and will persist until a hierarchy is established (Grimm et al, 1996; Haug et al, 1986; Ortiz et al, 1984; Sapolsky, 1995). The degree and duration of overt fighting necessary to establish stable dominance hierarchies has been shown to be dependent upon several factors including the number of animals in the group in a defined area, genetic background and the time spent individually housed prior to being group-housed (Brain & Nowell, 1970; Bronson, 1973). In general, with increased population density, adrenal cortical activity, assessed by alterations in CORT levels, increases and luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels along with gonadal function (T levels) decrease. This general effect may be attributed to the increase in the proportion of subdominant male mice to the dominant male (Brain & Nowell, 1970). Compared to the subordinate mice, the dominant mouse in a stable hierarchy typically exhibits increased plasma T and NE levels and decreased CORT and dopamine levels (Ely, 1995; Haemisch & Gartner, 1997; Koolhaas et al, 1997a). The status-dependent differences in the activities of the hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-gonadal (HPG) axes have been suggested to be due to the higher frequency of fighting that occurs during the establishment of social hierarchies and following this, some aspects of subordination (Brain, 1975; Bronson, 1973; Ely & Henry, 1978). However, once the social hierarchies among groups is established, the number of aggressive encounters initiated by the 14 dominant male mouse is reduced (Brain, 1975) and the status-dependent difference in HPA and HPG activities may also be reduced (Bronson, 1973; Ely & Henry, 1978; Ortiz et al, 1984; Peng etal, 1989; Weinberg & Emerman, 1989). Temporal aspects of stress responses in group-housed mice. Increased fighting in unstable groups of male mice increases plasma CORT levels and decreases plasma LH and T levels, and to a lesser extent, FSH levels (Brain, 1975; Bronson, 1973). Interestingly, in dominant mice plasma CORT levels return to baseline in 1-3 days following group formation, whereas in subordinate mice this occurs 3-6 days following group formation (Bronson, 1973). FSH levels also return to baseline approximately 6 days following group formation in dominant but not subordinate mice. The return to baseline levels of hormones in these mice was related to a significant reduction in the frequency and intensity of fights that were initiated (Bronson, 1973). The differences in hormone levels between dominant and subordinate mice may be due to the fact that subordinate mice experience several episodes of defeat and therefore may experience higher levels of stress compared to the dominant mouse (Brain, 1975; Brain & Nowell, 1970; Haemisch & Gartner, 1997; Rodgers & Cole 1993). However, over the course of several days, adaptation to the stress of social defeat may develop (Bronson, 1973; Koolhaas et al, 1997a & b), resulting in a return to baseline levels of both physiological and behavioral responses. This adaptation to defeat among the subordinate mice may represent one factor in an established social hierarchy. Also, since overt fighting is replaced with aggressive postures and more structured and predictable behavior once a hierarchy is established, defeat responses in subordinate mice may consequently be replaced by defensive responses, with HPA and HPG activities similar to those observed in dominant mice. 15 Defeat and defense reactions are two important factors among group-housed mice that may contribute to status-dependent differences in HPA activity. The defense reaction is characterized by a stress response pattern mediated primarily by the ANS which includes increases in blood pressure and adrenal catecholamine biosynthetic enzymes (Ely, 1995; Ely & Henry, 1978). Conversely, the defeat reaction has been shown to be mediated primarily by the HPA stress response pattern, as indicated by increases in CORT levels (Ely & Henry, 1978). Importantly, the extent and duration of changes induced by both the defense and defeat responses have been shown to be related, in part, to genetic (strain or species) differences (Henry et al, 1993). The defense response in rodents is considered by some to be an active response whereas the defeat response is considered a passive response (Koolhaas & Bohus, 1989), and may be characterized as the loss of control over the environment (Koolhaas et al, 1997a). Interestingly, following 15 minutes of social defeat, E and CORT levels are similar to or higher than those observed following 15 minute exposure to restraint, footshock (0.5-2 mA) or forced swim (Koolhaas et al, 19976). Change in housing. We (Weinberg & Emerman, 1989) and others (Dechambre & Gosse, 1973; Sklar & Anisman, 1980) have shown that a change in housing condition can differentially affect tumor growth rates. Moreover, we have demonstrated that the direction of change [e.g. from individual to group (IG) or from group to individual (Gl)] may also play an important role in altering tumor growth rate as well as hormone and immune activities. Specifically, basal T levels and NK cell activity are higher and basal CORT levels are lower in Gl (fastest growing tumors) compared to IG (slowest growing tumors) mice (Rowse et al, 1990a; Rowse et al, 1995). Importantly, if an animal is allowed to adapt to its new social housing condition, the 16 impact of the change in housing condition on endocrine and/or immune functions is reduced (Hoffman-Goetz et ai, 1992; Weinberg & Emerman, 1989). PSYCHONEUROENDOCRINOLOGY AND CANCER Research conducted over the past 20 years, has made it increasingly apparent that a complex inter-relationship exists among neural, endocrine and immune functioning. Psychoneuroendocrinology examines the mechanisms involved in mediating stressor-induced alterations in endocrine function. As outlined earlier, environmental and cognitive factors can differentially influence endocrine functioning as well as alter adaptive behavioral responses, both of which may ultimately influence health and disease processes. Hormones, including steroid hormones and growth factors, play an important role in the development, growth and progression of many cancers. Importantly, circulating hormone levels may be altered due to exposure to a stressor, especially in the absence of effective physiological and/or behavioral coping responses. However, there remains limited data examining the interaction among stressor exposure (with or without coping mechanisms), alterations in hormone levels and cancer growth or progression. Hormones and Stress The two classical pathways of communication that are used by the CNS to modulate other bodily systems involved in the stress response include the endocrine system (humoral connections) and the ANS (direct neural connection). Two major axes control the activity of these systems: the HPA and the sympathetic nervous system/locus coeruleus (SNS/LC) axes. In addition, increased activity of these axes inhibits the activity of the HPG and growth axes as 17 well as that of the immune system. Hypothalamic-pituitary-adrenal axis. The level of activity of the HPA axis has been accepted as a central feature of the stress response (Chrousos & Gold, 1992; Johnson et al, 1992). In response to a stressor, activation of the HPA axis results in release of corticotrophic releasing hormone (CRH) from the paraventricular nucleus (PVN) of the hypothalamus which in turn promotes the synthesis of proopiomelanocortinin (POMC) and stimulates the release of POMC-derived peptides, adrenocorticotrophic hormone (ACTH) and beta-endorphin (p-END) from the anterior pituitary (Johnson et al, 1992; Stratakis & Chrousos, 1995). ACTH stimulates corticosterone (CORT, the main glucocorticoid in rodents; Cortisol in humans) secretion from the adrenal cortex whereas P-END has inhibitory effects on CRH secretion (Johnson et al, 1992; Stratakis & Chrousos, 1995). Glucocorticoids are the final effectors of the HPA axis and participate in the organism's stress response by providing energy substrates required by the organism, and function to help return the organism to a homeostatic state. For example, the highly catabolic glucocorticoids are responsible for: (1) producing lipolysis which increases the level of fatty acids; (2) glycogenolysis which increases blood glucose levels, and (3) protein catabolism which increases amino acid availability as substrates for gluconeogenesis, further increasing blood glucose levels (Johnson et al, 1992; Munck et al, 1984). In addition, hormones of the HPA axis can affect behavioral responses to stress. For example, central administration of CRH (intracerebroventricular; ICV) initiates a coordinated array of adaptive behavioral responses including facilitation of pathways that subserve vigilance, cautious avoidance, and anxiety (Chrousos, 1997; Johnson et al, 1992; Michelson et al, 1994). In contrast to CRH, ICV administration of ACTH does not appear to increase activity or 18 exploration but can affect social behavior in the rat by reducing social interaction and decreasing aggression (cf. Johnson et al, 1992). Following the threat of a stressor, it is important that the elevated activity of the HPA axis is returned to basal (baseline, non-stressed) activity. Suppression of the stressor-activated HPA axis occurs primarily through glucocorticoid negative feedback. Two receptor subtypes are involved in feedback: the type II, or 'classical' glucocorticoid receptor (GR) is located in the pituitary as well as specific brain regions including the hypothalamus, amygdala, and frontal cortex (Beaumont & Fanestil, 1983; Reul & deKloet, 1985); the type I, or mineralocorticoid receptor (MR) is located mainly in the hippocampus, and plays primarily a tonic role in HPA regulation but may also play some role in mediating stressor-induced activity of the HPA axis (Reul & deKloet, 1985; Spencer, et al, 1998). Interaction of the glucocorticoids with their cytosolic receptors initiates a cascade of effects including release of heat shock proteins from the previously inactive receptor and translocation of the steroid and receptor complex to the nucleus where it ultimately inhibits the release of ACTH or CRH from the anterior pituitary or PVN, respectively (Beato et al, 1989; Beato et al, 19966; Johnson et al, 1992). Thus, glucocorticoid-mediated inhibition of ACTH release occurs either directly at the level of the pituitary or indirectly via inhibition of CRH secretion. In addition to playing a key role in the termination of the stress response, glucocorticoids also play a vital role in the regulation of the diurnal rhythm of HPA axis activity, characterized by basal ACTH and CORT secretion (deKloet et al., 1990; Young et al, 1998). Receptor affinity studies, have shown that the MR binds corticosterone with 5-10 times greater affinity than that of the GR (deKloet et al, 1990; Reul & deKloet, 1985). Due to this differential affinity, the of MR is 50-90% occupied under basal levels of corticosterone, 19 rendering this receptor less sensitive to the dramatic stressor-induced increases of CORT levels (deKloet et al, 1990). It has been suggested that GRs may also play a role in regulating basal HPA activity. Sympathetic nervous system/ locus coeruleus axis. In addition to the HPA axis, the SNS/LC axis along with the related parasympathetic system is another important component of the stress response. In response to a stressor, the SNS/LC system activates both the cardiovascular and adrenal catecholamine systems to help with homeostatic adjustments. Generally, the magnitude of the SNS/LC response is modulated by the parasympathetic system which either enhances the response by withdrawing or antagonizes it by increasing its own activity. Activation of the SNS/LC system leads to increased release of NE from sympathetic nerves and E and some NE from the adrenal medulla (Axelrod & Resine, 1984; Chrousos, 1997; Yamaguchi et al, 1992), resulting in enhanced arousal and vigilance as well as increased anxiety. Like CORT, NE and E inhibit glucose uptake, fatty acid storage and protein synthesis and stimulate the release of energy substrates (e.g. glucose, amino acids and free fatty acids) from muscles, fat tissue, and liver (Johnson et al, 1992). The HPA and SNS/LC axes interact in a complex manner to mediate the stress response and can act at different levels during the stress response (Axelrod & Resine, 1984; Munck & Naray-Fejes-Toth, 1992). For example, the SNS/LC axis stimulates mesocortical and mesolimbic dopamine systems, influencing cognitive functions (e.g. habituation and coping responses) and aspects of motivation and reinforcement, respectively (Chrousos & Gold, 1992). The particular combination of neurons of the autonomic nervous system that are activated during the stress response is strongly affected by interaction with the CNS and components of 20 the HPA axis (Stratakis & Chrousos, 1995). Hypothalamic-pituitary-gonadal (HPG) axis. In general, the activity of the HPG axis involves secretion of gonadotrophin-releasing hormone (GnRH) by the arcuate neurons of the hypothalamus and stimulates the release of FSH and LH from the pituitary. FSH and LH stimulate the secretion of the sex hormones, estrogen (E2) and testosterone (T) from the gonads. The interaction between the HPA and HPG axes appears to be bi-directional and gender-specific. The activity of the HPG axis is inhibited at all levels by stressor-induced increases in HPA axis activity. For example, at the level of the hypothalamus, CRH suppresses the secretion of GnRH, whereas at the pituitary, CORT inhibits GnRH-induced FSH and LH release and at the gonads, CORT decreases gonadal sensitivity to gonadotrophins or influences sex steroid secretion (Chrousos, 1997). Conversely, T normally acts to inhibit HPA axis responses to a stressor, whereas E 2 increases the HPA response to a stressor (Handa et al, 1994). Influence of T on HPA function occurs via the androgen receptor (AR) which has been found in many brain regions known to mediate HPA function. For example, AR expression has been demonstrated in hippocampal CA1 pyramidal cells. Although the role of ARs in this region is not known, the overlap among AR, GR, and MR suggests that these receptors may actively regulate HPA functions (Handa et al, 1994). Moreover, cfos mRNA at the level of the PVN is increased following castration and reduced by administration of the AR agonist dihydrotestosterone (DHT) propionate (Handa et al, 1994). Therefore androgens may directly modulate the activity of neurons in the PVN thereby influencing HPA activity. The influence of the activity of the estrogen receptor (ER) on HPA function has been 21 defined more clearly than that of the AR. Estrogen receptor mRNA expression has been localized within the PVN suggesting that E 2 may stimulate CRH neuronal activity. Furthermore, an estrogen response element (ERE) has been identified in the promoter area of the CRH gene which directly stimulates CRH gene expression (Stratakis & Chrousos, 1995). Also, E 2 has been suggested to influence feedback mechanisms at the level of the hippocampus by altering GR and MR mRNA expression (Handa et al, 1994). Thus, the different locations of the ARs and ERs within the CNS may differentially affect HPA activity and may therefore also play an important role in influencing gender-specific patterns in both physiological and behavioral responses to stressors (Gallucci et al, 1993; Patchev era/., 1995). Growth Axis. During stress, the growth axis is also inhibited at many levels by HPA activity. However, this typically occurs only following prolonged activation of the HPA axis such as that seen with chronic exposure to a stressor. Similar to T and E 2, an acute elevation of plasma growth hormone (GH) levels is usually observed during the onset of the stress response, whereas prolonged activation leads to CORT-mediated suppression of GH levels and concomitant, but delayed, decreases in insulin-like growth factors (IGFs), such as IGF-1 (also called, somatomedin C; Ref. Chrousos & Gold, 1992; Weissman, 1990). In addition, CORT inhibits the effects of GH, IGF-1 and other growth factors on their target tissues (Chrousos, 1997). In several stress system-related disorders which involve HPA hyperactivity, such as chronic anxiety or depression, GH and/or IGF-1 levels are significantly decreased (Gallucci et al, 1993). Thus, upon exposure to a stressor, a complex response pattern is activated that is 22 composed of interconnected and bi-directional components that enable an organism to respond to the stressor and serve to reduce the physiological and/or psychological impact of the stressor on the organism. The ensuing stress response, although relatively consistent, can be differentially altered at many levels. Differential stressor characteristics, and genetic and experiential influences among other factors can cause broad variability in the level of activity of the HPA and SNS/LC axes. Nonetheless, it is clear that in order for an organism to return to a homeostatic state, it must be able to physiologically and behaviorally cope with the stressor. Hormones and cancer Control of cell growth is a subtly regulated process that responds to specific needs of the organism and is achieved through complex interactions of many factors such as steroid hormones, peptide hormones (e.g. growth factors), cytokines, their receptors and molecules involved in the transduction of their signals. Dysregulation of these factors may result in oncogenesis, exacerbation of cancer growth, or metastasis (Cross & Dexter, 1991; Mertani et al, 1998; Plotnikoff & Faith, 1994; Waterfield, 1991). Normal development and functioning of many adult tissues is regulated by hormones and growth factors. This is exemplified by the role of steroids, especially androgens and estrogens, in regulating the growth, differentiation and function of a variety of normal tissues including the gonads, pituitary and secondary sex organs (Miller & O'Neill, 1990). Steroids readily pass through cell membranes due to their lipophylic properties. In the cell, the steroids bind to specific intracellular receptors located in either the cytoplasm (e.g. AR and GR) or in the nucleus (e.g. ER). These receptors are normally bound in a complex with heat-shock proteins (HSPs) including HSP90, HSP70 and several minor species (Baniahmad & 23 Tsai, 1993). Steroid receptors are similar in structure: they contain a steroid binding domain, a hinge region, and a DNA binding domain (Jensen, 1992; Liao, 1992; Ribeiro et al, 1995). Hormone binding causes the HSPs to dissociate from the receptor, which in turn promotes conformational changes in the receptor exposing: (1) nuclear localization signal binding proteins, which aid in the translocation of the cytosolic receptor/ligand complex to the nucleus (Beato et al, 1996a) and/or (2) the DNA binding region of the receptor complex, allowing it to bind to hormone response elements (HRE) usually located in the promoter region of target genes (Jensen 1992; Liao, 1992; Ribeiro et al, 1995). Steroids bind to DNA as dimers. The HRE sites to which they bind have specific short consensus sequences which are imperfectly repeated in a palendromic (complemented and inverted) fashion. Each subunit of the dimer binds to one of the repeated elements (Tsai & O'Malley, 1994). Although there are specific HREs for each steroid receptor type, there is also overlap in the ability of different steroid receptors to bind to different HREs (Beato et al., 19966; Jensen, 1992; Liao, 1992). Binding of the receptor to an HRE may either stimulate or repress DNA transcription depending on the cell type and other genes involved (Beato & Sanchez-Pacheco, 1996; Liao, 1992). Growth factors are pleiotrophic peptide hormones that can act by autocrine and/or paracrine mechanisms. Their receptors are typically transmembrane tyrosine kinase receptors or are ligand-specific transmembrane receptors (e.g. GH and prolactin receptors) that associate with tyrosine kinases (Moutoussamy et al, 1998; Ullrich & Schlessing, 1990). Tyrosine kinase receptors have a single membrane spanning the a-helix, which divides the receptor into an extracellular ligand binding amino-terminal domain and a cytoplasmic carboxy-terminal domain that contains a tyrosine kinase (Ullrich & Schleesinger, 1990). Binding of a ligand to its 24 receptor usually results in dimerization between two similar receptors and results in tyrosine phosphorylation of the receptor itself and/or specific target proteins. The signaling pathways activated by tyrosine kinase and associated receptors are complex. The biological effects resulting from activation of these receptors, however, have a general effect of stimulating anabolic processes of growth and differentiation. Because of the important role these receptors play in regulating growth and differentiation, mutation of these receptors may result in tumor formation (Cantely et al, 1991). Hormones and Cancer: Human Studies Hormones regulate the growth of a number of cancers. For example, glucocorticoids play a role in mediating the growth of mammary carcinomas, leukemias and lymphomas (Lippman, 1985). Androgens influence the growth of prostatic, testicular, mammary and esophageal carcinomas (Daneshgari & Crawford, 1993; Lippman, 1985; Nakamura et al, 1989; Pasqualini, 1993; Rajpert-De Meyts & Skakkebaek, 1993; Ueo et al, 1990). Opioids, such as P-END retard the growth of mammary carcinomas, neuroblastoma and melanomas, whereas GH has been shown to accelerate the growth of osteogenic sarcomas, lymphomas as well as mammary and prostatic carcinomas (Goodman et al, 1978; Mertani et al, 1998; Nandi et al, 1995; Rogers et al, 1997). The effects of these hormones on cancer growth have been suggested to be due to their potent mitogenic influences on these tissues either independently (Aaronson, 1991; Mertani et al, 1998; Torosian & Donoway, 1991) or through synergistic activity with other hormones (Aaronson, 1991; Cross & Dexter, 1991; Goodman et al, 1978; Guzman et al, 1999; Russo et al, 1999). Although, the precise role of hormones in the promotion, growth or progression of cancer is not known, it is clear that hormones play a role 25 in both carcinogenesis and tumor growth (Bernstein & Ross, 1993; Cantley et al, 1991; Davies et al, 1994; Lippman, 1985; Nandi et al, 1995). For example, extensive epidemiological evidence has established that early menarche, late menopause and having the first pregnancy late in life are major risk factors for breast cancer. The risks of early menarche and late menopause are usually combined to emphasize the role of over-exposure to estrogens in increasing breast cancer risk, i.e. repeated surges of estrogen due to higher number of menstrual cycles or longer exposure to estrogens due to late menopause may provide the stimulus for carcinogenesis (Bernstein & Ross, 1993; Pike & Forman, 1991). Underexposure to progesterone has also been related to increased risk for breast cancer (Bernstein & Ross, 1993). Thus, the beneficial effects of having a child early in life could be due to the high concentrations of progesterone-like hormones involved in pregnancy which protect the breast cells from estrogen effects (Bernstein & Ross, 1993). Recent studies, however, have suggested that high concentrations of progesterone together with increased estrogen levels may reduce breast cancer risk. For example, administration of progesterone alone, at the levels seen during pregnancy, has been shown to increase breast cancer risk, but when administered together with estradiol, a significant reduction in risk is observed. Furthermore, estradiol alone was not as effective as estradiol plus progesterone in reducing breast cancer risk (Guzman et al, 1999; Russo et al, 1999; Sivaraman et al, 1998 ). Many hormone-responsive tumors (e.g. breast and prostate cancer) exhibit considerable variability in the level and combination of hormones required for proliferation (Bruchovsky & Rennie, 1978; Casagnetta et al, 1995; Darbe & King, 1987; Harper et al, 1974; Heppner, 1989; Heppner & Miller, 1981). In addition, most of these tumors will ultimately loose their requirement for hormones as they progressively become hormone non-responsive (Bruchovsky 26 & Rennie, 1978; Darbe & King, 1987; Lancaster et al, 1988). This progression may be due to the heterogeneous nature of the cancer cell population that is composed of a range of phenotypes including hormone-responsive and hormone non-responsive cells (Heppner, 1993; Ichikawa et al, 1989; Sutherland et al, 1993). The transition of a tumor cell population from hormone-responsive to hormone non-responsive may be due to the clonal selection of a hormone non-responsive phenotype (Kim & Depowski, 1975; Osborne & Arteaga, 1990; Rennie et al, 1990). However, even in hormone non-responsive tumors, altered hormone levels can indirectly influence tumor growth, by affecting host processes such as angeogenesis which can play a critical role in tumor growth (Folkman & Haudenschild, 1980). Hormones and the SCI 15 Tumor Like human cancers, the SCI 15 tumor is composed of a heterogeneous population of hormone-responsive and hormone non-responsive cells which are initially hormone-dependent but gradually lose hormone dependency and show autonomous growth (Ichikawa et al, 1989; Rennie et al, 1990). The SCI 15 tumor has been used by many investigators to study the mechanisms of hormonal control on cell proliferation and death in hormone-responsive tumors (Bruchovsky et al, 1990; Darbe & King, 1987; Ichikawa et al, 1989; Rennie et al, 1990; Sato et al, 1993; Yamaguchi et al, 1992; Yamanishi et al, 1995). Growth of the SCI 15 tumor is stimulated by physiological concentrations of androgens (Noguchi et al, 1987; Omukai et al, 1987; Yates & King, 1981) and growth hormone (Noguchi et al, 1993) and pharmacological concentrations of glucocorticoids (Omukai et al, 1987; Yates & King, 1981), all of which can be altered by stressors. Androgen-stimulated growth of the SCI 15 is mediated by androgen-induced growth factor (AIGF) that has marked homology to the basic fibroblast growth factor 27 (b-FGF) family of proteins (Hiraoka et al, 1987; Ruohola et al, 1995; Sato et al, 1993). This is similar to hormone-responsive human cancers. For example, growth stimulation of several human breast cancer cell lines has been shown to be mediated by an estrogen-induced growth factor, epidermal growth factor (EGF; Ref. Aaronson, 1991; Cross & Dexter, 1991; Sprone & Roberts, 1985). Following androgen withdrawal, regression of the SCI 15 tumor occurs due to the increased expression of the apoptotic gene, TRPM-2 (Rennie et al, 1988; Rennie et al, 1994). However, over time, tumors invariably reoccur in forms that are refractory to further hormonal manipulations, i.e. the tumor has progressed to an androgen-independent state (Bruchovsky et al, 1990). Interestingly, the androgen-independent SCI 15 tumor cells do not require androgen for growth and are also resistant to the apoptotic effects of the TRPM-2 gene (Rennie^ al, 1990). PSYCHOSOCIAL STRESSORS AND CHEMOTHERAPY Chemotherapy The goal in chemotherapy is to kill cancer cells while sparing normal tissue. Indeed, the factor limiting the effectiveness of chemotherapy is its toxicity to normal tissues. Presently in some cancers such as breast or ovarian cancer, small cell lung carcinoma and myeloma, it is realized that a cure cannot be realistically contemplated, but effective drugs or drug combinations are used to extend life considerably (Conley & Van Echo, 1996). Recent advances in combination therapies have increased the quality of life of cancer patients. Combination therapies include the use of different cytotoxic agents with synergistic or additive effects on tumor cell kill or alternatively the combination of different treatments such as surgery, radiation, immunotherapy or hormone therapy with chemotherapy. For example, one 28 form of combination chemotherapy, adjuvant chemotherapy, involves surgery to remove the primary tumor, followed by administration of cytotoxic drugs directed at the remaining malignant cells. Adjuvant treatment was founded on experimental data from the 1950's and 1960's that observed an inverse relationship between chemotherapeutic response and the number of tumor cells (Shapiro & Fugmann, 1957; Yarbro, 1996). Adjuvant chemotherapy has been shown in some cancers (e.g. Wilm's tumor, sarcomas, colorectal and breast cancers) to increase disease-free interval and survival probability (Yarbro, 1996). The cell cycle is an important concept in the understanding of the classification and effects of chemotherapeutic drugs. The body regulates the replication of all dividing cells by maintaining a balance between cell birth and death. The body's maintenance of this homeostasis depends on the synthesis of specific proteins, or signals that stimulate the entry and movement of dividing cells through the process of cell division as well as into programmed cell death (apoptosis). The following observations have been made with respect to tumor, growth and chemotherapy: (1) a single malignant cell can be lethal; (2) a relatively constant percentage of cells irrespective of tumor size, is destroyed with a constant dose of an effective drug; (3) a positive dose-response relationship exists between an effective drug and cancer cell population; and (4) treatment outcome is influenced by the number of malignant cells (Braverman, 1993; Goldin et al, 1956; Skipper, 1964; Wilcox, 1966). To achieve a cure, all tumors cells that are actively dividing or are capable of proliferating must be killed. Importantly, however, the number of cells present, the cell kinetics, the chemotherapeutic agent used and the tumor growth rate can all play key roles in the effectiveness of chemotherapy (Armitage, 1992; Skipper, 1977; Yarbro, 1996). There are two main categories of chemotherapeutic agents: cell cycle-specific drugs 29 which exert their effects at a certain stage(s) of the cell cycle and cell cycle-nonspecific drugs which are equally effective at all stages of the cell cycle. The two chemotherapeutic agents (Adriamycin and cyclophosphamide) used in the studies of this thesis are considered cell cycle-nonspecific. Adriamycin is an antitumor glycosidic antibiotic (anthracycline), has been in clinical use for over 25 years and is known to have cytotoxic activity on a wide spectrum of malignancies (Balis et al, 1983; Doroshow, 1996; Hoaland & Gastineau, 1996; Malpas, 1991). Adriamycin prevents cell division by interfering with DNA, RNA and/or protein synthesis (Balis et al, 1983; Doroshow, 1996; Malpas, 1991). Adriamycin itself is not associated with curative therapy, but in combination with cyclophosphamide, these two chemotherapeutic agents effectively contribute to high tumor response, due primarily to the low level of cross-resistance between these two drugs (Balis et al, 1983). Cyclophosphamide is an alkylating agent, has been used clinically for the treatment of malignant disease for over 40 years and remains one of the most widely used chemotherapeutic agents in combination chemotherapy (Balis et al, 1983; Malpas, 1991; Moore, 1991). Cyclophosphamide requires metabolic activation via hepatic mixed function oxidases (Moore, 1991). Its active metabolites (primarily phosphoramide mustard and to a lesser extent acrolein) alter DNA structure via their alkylating properties (H+ ion alkyl substitution), which results in crosslinking and strand breaking of DNA and destruction of its template (Balis et al, 1983; Moore, 1991). Thus, DNA replication does not occur and cell death via apoptosis is initiated. Phosphoramide mustard is efficiently catabolized by aldehyde dehydrogenase to non-active products. Aldehyde dehydrogenase is present in high concentrations in hepatocytes, intestinal mucosa, hematopoetic stem cells, and megakaryocytes and is believed to protect normal tissues from cyclophosphamide toxicity (Russoefa/., 1989). 30 Although Adriamycin and cyclophosphamide are cell cycle-nonspecific, the tumor cell cycle is still an important factor influencing the effectiveness of these drugs. In general, cells that are not dividing (i.e. resting, G 0 phase) as well as rapidly proliferating cancer cells are often resistant or insensitive to chemotherapy (Armitage, 1992; Skipper, 1977). The greater the fraction of cells in a growth phase, or the longer the susceptible growth phase of the cells, the higher the probability of tumor cell kill (Skipper 1977; Yarbro, 1996). In order to increase the effectiveness of a given drug dose, a variety of noncytotoxic drugs are used concurrently with other chemotherapeutic agents to potentiate their cytotoxicity. A potentiator may act by moving cells into a kinetic phase of greater susceptibility to cytotoxic agents (Emerman, 1988; Emerman & Siemiatkowski, 1984; Lippman et al, 1985). For example, androgens may be given to prostate cancer patients to increase the mitotic rate of the cancer cells so that the cells do not remain in G 2 phase a sufficient time to repair damaged DNA (Stewart & Evans, 1989). Many studies have demonstrated an inverse relationship between tumor response to chemotherapy and the toxic effects of chemotherapy on the host (e.g. weight loss, hair loss, myelo- and immuno-suppression; Ref. Blinson et al, 1984; DeWys et al, 1980; DeWys, 1982; Looney et al, 1980). Moreover, an increase in chemotherapeutic efficacy has been related to both positive tumor and host responses to chemotherapy (e.g. increased tumor regression and decreased toxic side effects). Thus, factors related to both the tumor and host can interact to influence chemotherapeutic efficacy (Chabot, 1994; Kopreski, 1996; Simpson-Herren et al, 1987). 31 Hormones and chemotherapy In addition to influencing cancer growth and progression, hormones with or without involvement of the immune system also play an integral role in cancer treatment (Abdul Hamied & Turk, 1987; Aylesworth et al, 1979; Black, 1994; Brenner & Margolese, 1991; English et al, 1987; Swain, 1996). Any tumor or host factor that alters, for example, drug metabolism and excretion (affecting active drug concentrations and the duration of drug exposure) or the sensitivity of tumor cell populations to drugs (e.g. alterations in cell cycle kinetics that alter the time in which cells are sensitive to the cytotoxic effects of the drugs) may contribute to differential tumor and host responses to chemotherapy. Hormones play an important role in maintaining homeostasis from the level of the whole organism to that of the single cell and may therefore affect chemotherapeutic efficacy. Indeed, changes in hormone levels have been shown to affect host metabolism (Johnson et al, 1992; Weissman, 1990) and/or tumor cell cycle kinetics (Emerman 1988; Lippman et al, 1985; Stewart & Evans, 1989; Swain, 1996) as well as the effectiveness of chemotherapy (Coleman, 1992; Emerman & Siemiatkowski, 1984; English et al, 1991; Kodama & Kodama, 1982; Lippman et al, 1985). Tumor Response to Chemotherapy For over 25 years, many experimental and clinical studies have examined the possibility of enhancing chemotherapeutic efficacy by expanding the fraction of cycling cancer cells (Conte et al, 1990; Kiang et al, 1981; Kiang & Kennedy, 1981; Lloyd et al, 1979; Sluyser et al, 1981). Theoretically, steroid hormones and/or growth factors should recruit cancer cells into an active cycling phase and this should result in a higher killing efficiency of anticancer drugs (Skipper, 1977; Yarboro, 1996). A slow-growing tumor may be more responsive to drugs 32 if it remains in the susceptible growth phase (Armitage, 1992; Emerman & Siemiatkowski, 1984). Thus, hormonal manipulations directed toward regulating cell growth, rather than producing cell death, combined with chemotherapy should be more effective in increasing tumor response to chemotherapy than either modality alone. Indeed, we (Emerman, 1988; Emerman & Siemiatkowski, 1984) and others (Conte et al, 1985; English et al, 1991; Hug et al, 1986; Lippman et al, 1985; Markaverich et al, 1983) have demonstrated that hormonal manipulation of cell cycle kinetics both in vitro and in vivo can alter the tumor response to chemotherapy. Unfortunately, conflicting results and only limited advantages to combined hormone administration and chemotherapy have been observed (Cocconi et al, 1983; Conte et al, 1990; Klijn et al, 1992). This may be due to the fact that after long-term use in several animal species, hormones can become co-carcinogenic or tumor promoters. Alternatively, tumors may progressively become less hormone-responsive due to the clonal selection of hormone non-responsive cells (Darbe & King, 1987). Finally, decreased cell proliferation (Benz et al, 1983; Hug et al, 1986) and defective transport mechanisms (Clarke et al, 1985; Goldenberg & Froese, 1985; Hug et al, 1985) induced by hormones and growth factors may increase resistance of tumors to chemotherapy. Host Response to Chemotherapy A complex interrelationship exists among tumor growth, the host environment and chemotherapeutic efficacy. Tumor growth as well as chemotherapy commonly cause the syndrome of cancer cachexia which consists of a series of symptoms including malnutrition/anorexia, nausea/vomiting, diarrhea, weight loss, weakness or loss of strength, severe tissue wasting, bone marrow depression, hair loss, and impaired organ function 33 (Bernstein, 1982; Costa, 1977; DeWys, 1982; Tisdale, 1999). The toxic side effects of chemotherapy are often disabling and may hamper severely the effectiveness of treatment by limiting both the dosages of therapeutic agents as well as the duration of therapy. Small increases (between 5-10%) in dose intensity (amount of drug delivered per unit time: Ref. Gilewski & Bitran, 1996) have been shown to result in disproportionately larger increases in response rate, cure rate or degree of control over cancer growth for Hodgkin's disease, ovarian, breast and colon cancers (Hryniuk et al, 1987; Longo et al, 1986; Smaaland et al, 1995). Although host tolerance to chemotherapy remains a major cause of treatment failure, several studies have demonstrated that a decrease in the toxic side effects of chemotherapy is related to an increase in tumor response to chemotherapy (Belinson et al, 1984; Corbett et al, 1978; Looney et al, 1980; Sullivan et al, 1995). Several factors have been shown to influence the severity of chemotherapeutic toxicity. These include: gender, tumor size and histological grade, timing of chemotherapy relative to surgery or stage of menstrual cycle and nutritional or metabolic states (Belinson et al, 1984; Bell et al, 1990; Boddy & Idle, 1993; Corbett et al, 1978; Goldin & Schabel, 1981; Henderson et al, 1988; Ueda et al, 1988; Wood & Hrushesky, 1996). Other studies have demonstrated that the toxic effects of chemotherapy can be reduced and its antitumor effects increased depending on the circadian stage in which the drug was administered (Hrushesky, 1985; Kerr et al, 1990; von Roemeling & Hrushesky, 1989). Changes in the therapeutic and toxic effects of chemotherapeutic agents during different stages of the circadian rhythm may be attributed to temporal variations in rapidly proliferating tissues. Importantly, the amplitude and the phasing of cell cycle kinetics has been shown to vary among distinct tissues; thus circadian rhythms in different tissues are asynchronous (Smaaland et al, 1995; Wood & Hrushesky, 1996). Recent human data indicate that asynchrony may also exist 34 between normal tissues and tumor proliferation. Thus, circadian phase differences between normal and malignant cells with respect to cell proliferation and possibly other susceptible rhythms could be used to maximize tumor response to chemotherapy and minimize host toxicity. Although it is known that exogenous application of hormones and growth factors as well as the removal of endogenous hormones can influence tumor response to chemotherapy, less is known of how perhaps more subtle alterations in endogenous hormone levels, such as those observed during the circadian rhythm or possibly following a stressor, may affect metabolism, distribution and excretion of cytotoxic drugs. It is obvious from chronotherapy studies that the homeostatic state of the organism may differentially affect chemotherapeutic efficacy. Although the factors of the circadian rhythm that are involved in altering the effectiveness of drugs remain to be determined, studies have suggested that circadian changes in endocrine levels may play an important role in mediating the differential effects of circadian rhythm periods on chemotherapeutic efficacy (Levi et al, 1980; Levi et al, 1981). For example, glucocorticoids have diverse physiological, metabolic and biochemical effects including inhibition of DNA, RNA, and protein synthesis. These processes are fundamental to the cell cycle and are also important in cellular repair processes and metabolic enzyme production (Harvey et al, 1994; Swain, 1996). Importantly, the HPA, HPG and growth axes are not only under circadian control but their activities may also be affected significantly by stressors. Thus, it is also possible that stressor-induced alterations in the activity of these axes and therefore the level of circulating hormone(s) may play an important role in influencing chemotherapeutic efficacy. 35 Stressors and Chemotherapy Stressors can disrupt physiological homeostasis that is maintained in a coordinated fashion by the central nervous system, endocrine and immune systems. There now is evidence that stressors may not only influence cancer growth but may also affect treatment possibly by altering the activities of the endocrine and/or immune systems. Few studies, however, have examined directly the effects of stressor-induced alterations in hormone levels or immune activity and chemotherapeutic efficacy. Human studies. Stressful life events such as divorce, bereavement or loss of a job may play a role not only in cancer growth and progression but also in the effectiveness of treatment (Cooper & Faragher, 1993; Levy et al., 1988; Spiegel, 1997). Reducing the impact of psychosocial stressors through active intervention (e.g. psychotherapy, behavioral therapy), social or family support and/or coping styles (e.g. disclosure, information seeking) has been shown to increase survival time and decrease the side effects of chemotherapy (Fieler et al, 1995; Heim et al, 1997; Heinrich & Schag, 1985; Leigh et al, 1987; Temoshok, 1987). However, similar to the studies examining the effects of life stressors on cancer growth and progression there are a number of studies that have reported no associations among psychosocial stressors and/or coping resources and survival time (Cassileth, 1985; Fox, 1998; Tross et al, 1996). The mechanisms underlying stressor-induced or coping-mediated effects on survival probability and toxic side effects of chemotherapy are unknown, but may include positive influences in behavioral responses such as health habits (e.g. diet, physical activity, sleep, adherence to the cancer treatment; Ref. Classen et al, 1998; Fox, 1998) as well as stressor-induced alterations in endocrine and/or immune functions (Fawzy et al, 1990; 36 Grossarth-Maticek & Eysenck, 1989; Levy et al, 1987; Spiegel et al, 1998). Importantly, active coping strategies and psychosocial intervention may reduce or possibly alleviate stressor-induced alterations in hormone levels and immune activity. Indeed, several studies have suggested that psychotherapeutic stress management techniques may have positive effects on endocrine and immune systems and may be related to enhanced survival probability (Bovbjerg, 1991; Fawzy etai, 1990; Grossarth-Maticek & Eysenck, 1989; Spiegel, 1997). However, only a limited number of studies have attempted to correlate stressor-induced or coping-mediated alterations in hormone levels or immune activity and the effectiveness of chemotherapy in terms of tumor or host responses. For example, Grossarth-Maticek and Eysenck (1989) demonstrated that women with breast cancer who had psychotherapy together with chemotherapy lived longer and had significantly higher lymphocyte counts than did patients receiving only chemotherapy. Also, the amount of social support that breast cancer patients experience or actively seek has been shown to be positively correlated with natural killer (NK) cell activity whereas lower NK cell cytotoxicity has been shown to predict cancer recurrence (Bovbjerg, 1991; Fawzy et al, 1990; Irwin et al, 1987; Levy et al, 1987). Importantly, a stress-induced decrease of NK cell activity is related to increases in CRH, ACTH and CORT levels (Berkenbosch et al, 1991; Bovbjerg, 1991; Hoffman-Goetz et al, 1992; Levy et al, 1987). In addition, recovery of the HPA axis following surgical stress has been related to cancer survival. Among patients operated on for breast or stomach cancer, the failure of morning Cortisol levels to decrease within two weeks after admission was associated with shorter survival probabilities (Audier, 1988) Also, it has recently been suggested that increased levels of CRH may alter the expression of breast cancer oncogenes influencing tumor growth (Licino et al, 1995) and possibly tumor response to chemotherapy. Finally, other studies have 37 demonstrated a relationship between psychosocial factors and estrogen and progesterone receptor status (Razavi et al, 1990); two important markers used to establish tumor response to either chemotherapy or endocrine therapy (Bryant et al, 1998; Howell et al, 1998; Miller & Sledge, 1999). Thus, a relationship among stressful life events and chemotherapeutic efficacy may involve alterations in endocrine levels and/or immune activity that may affect either tumor cells (e.g. proliferation, oncogene expression) or the host (e.g. metabolism, depression). However, the interrelationship is complex and requires systematic investigation. Animal studies. Exposure to rotation (spinning cages at 45 rpm for 10 minutes every hour from time of tumor inoculation until sacrifice) or restraint stress has been shown to decrease antitumor effects of chemotherapeutic agents (cyclophosphamide and razoxane; p.o.) in mice bearing Lewis lung carcinoma or TLX5 lymphoma in terms of tumor burden, extent of metastasis and survival probability (Perissin et al, 1991; Perissin et al, 1997). Importantly, the effects of these stressors on chemotherapeutic efficacy were attenuated in mice housed in protected housing (minimal acoustic, olfactory and visual communication among cages and entry into animal room restricted to once every five days) compared to those in conventional housing conditions (Perssin et al, 1997). The authors suggested that both the increased cytotoxic effects of cyclophosphamide and antimetastatic actions of razoxane in protected compared to conventional housing were due to neuroendocrine mediated increases in antitumor immune activity which can be modulated significantly by environmental and rotational stress (Giraldi et al, 1989; Giraldi et al, 1992a). Indeed, studies have demonstrated that the effectiveness of some chemotherapeutic agents (e.g. cyclophosphamide, Adriamycin) can be influenced significantly by the immunocompetence of the host (Giraldi, et al, 19926; North, 38 1984; Omukai, et al, 1983; Papa et al, 1988) or by modulation of hormone levels known to influence immune activity (Brenner & Margolese, 1991; English, 1987) or affect tumor cell cycle kinetics (Emerman & Siemiatkowski, 1984; English et al, 1991; Markaverich et al, 1983). MOUSE-TUMOR MODEL FOR STUDYING THE EFFECTS OF PSYCHOSOCIAL STRESSORS An animal-tumor model that uses the transplantable, androgen-responsive Shionogi carcinoma (SCI 15) has been developed in our laboratory to examine the effects of psychosocial stressors on tumor growth and chemotherapeutic efficacy (Kerr et al, 1997; Weinberg & Emerman, 1989). The SCI 15 tumor arose spontaneously in a female mouse of the DD/S strain. After 19 passages in male mice, an androgen-responsive variant arose that grows more rapidly in male than in female DD/S mice (Bruchovsky & Rennie, 1978; King & Yates, 1980). When 2 x 106 SCI 15 cells are injected subcutaneously in the interscapular region of a male mouse, a palpable tumor arises in approximately 8-10 days and grows to a mass of 2-3 grams in approximately 21 days. When a similar tumor inoculum is injected into a female DD/S mouse, tumor growth rate is considerably slower. In females, approximately 40 days is required for a tumor to reach approximately 1 gram in weight. Further, it has been demonstrated that morphological differences exist between tumors grown in male and female DD/S mice. Tumors grown in male mice exhibit a typical epithelial morphology, with cells appearing round to cuboidal and arranged in clumps or sheets with very little connective tissue separating the cells (Rowse et al, 19906). In contrast, tumors grown in female mice have a fibroblast-like morphology with spindle-shaped cells arranged in loose sheets or irregular cords, separated by 39 large amount of connective tissue. All studies to date, except one in this thesis, have used male mice to examine molecular, cellular, hormonal, or immune effects/interactions on growth of SCI 15 cells or subpopulations of these cells (e.g. Andrews et al, 1999; Kerr et al, 1997; Rowse et al, 1992; Rowse et al, 1995; Weinberg & Emerman, 1989). The SCI 15 tumor is similar to many hormone responsive human tumors such as breast and prostate cancer in its sensitivity to different classes of steroid hormones including androgens (King & Yates, 1980), estrogens (Noguchi et al, 1987) and glucocorticoids (Watanabe et al, 1982). Physiological concentrations of androgens both in vivo and in vitro stimulate the growth of SCI 15 cells by interaction with functional androgen receptors (Bruchovsky & Rennie, 1978; Emerman & Siemiatkowski, 1984; Jiang et al, 1993). Androgen-induced stimulation of SCI 15 cell growth is mediated by AIGF, a growth factor that has marked homology to the b-FGF family of proteins (Hiraoka et al, 1987; Ruohola et al, 1995; Sato et al, 1993). Castration of tumor-bearing male mice to remove endogenous androgens results in tumor regression followed by the outgrowth of a slower growing androgen-independent tumor (Bruchovsky & Rennie, 1978; Bruchovsky et al, 1990; Emerman & Siemiatkowski, 1984). Through the interaction with glucocorticoid receptors found within SCI 15 cells, pharmacological concentrations of glucocorticoids also stimulate growth SCI 15 cell growth both in vitro (Nakamura et al, 1989; Watanabe et al, 1982) and in vivo (Darbe & King, 1987; Jiang et al, 1993; Omukai et al, 1987). Similarly, SCI 15 cells also possess estrogen receptors and are stimulated to grow by pharmacological doses of estrogen in vivo (Noguchi et al, 1987; Nohno et al, 1982). However, the stimulatory effects of pharmacological estrogen levels are only apparent at suboptimal androgens levels and therefore have been suggested to be mediated by cross-reactivity with the androgen receptor (Noguchi et 40 al, 1987; Nohno et al, 1982). Finally, growth of SCI 15 cells has been shown to be stimulated by GH. Inhibition of GH secretion in male DD/S mice via somatostatin injections, decreases tumor growth rate and replacement of GH in a pulsatile fashion similar to that observed in male mice, restores SCI 15 tumor growth rates (Noguchi et al, 1993). Although a number of steroid hormones and growth factors affect SCI 15 cell growth, androgens are the primary stimulator of SCI 15 cell growth. We have demonstrated dramatic effects of social housing condition on the growth rate of this tumor. This model is unique in that both increased and decreased rates of tumor growth can be demonstrated in the same experiment where all variables, except the psychosocial stressor are constant. In our studies to date, male mice of the DD/S strain are housed either individually (I) or in sibling groups of 3 (G) at the time of weaning (3 weeks of age). When mice are 2-4 months of age, they are injected with tumor cells and experimental housing conditions are formed. Our studies demonstrate that being reared in a group then individually housed (GI) immediately following tumor cell injection increases tumor growth rate, whereas being individually then group-housed (IG) reduces tumor growth rate, compared to mice remaining in their original rearing conditions (II or GG). Furthermore, subjecting mice to acute daily novelty stress increases the differences in tumor growth rates among groups (Weinberg & Emerman, 1989). Importantly however, neither the size of the group-housed conditions (3 or 5) nor the sibling relationships within group-housed condition (housed with siblings or non-siblings) influenced tumor growth rates (Grimm et al, 1996). That is, regardless of group size (3 or 5) or sibling relationship (sibling or non-sibling), GI mice had the fastest and IG mice the slowest tumor growth rates, while II and GG mice had intermediate tumor growth rates (Grimm et al, 1996). The data of this study supported those of our previous studies demonstrating that 41 in our mouse-tumor model, both a change in housing condition and the direction of change plays an important role in mediating the differential tumor growth rates observed. These data also extended our previous findings by demonstrating that for mice housed in groups (IG or GG), dominance status had an effect on tumor growth. In the IG housing condition, we found that dominant mice had significantly faster tumor growth rates than subordinate mice, whereas in the GG housing condition, subordinate mice had significantly faster tumor growth rates than dominant mice. We suggested that these differential results may be related to the stability of the social groups at the time of tumor cell injection. Studies suggest that in stable social groups, dominance confers marked psychological advantages in that there is a high degree of control, predictability and social support for the dominant animals. Conversely, in an unstable or newly formed group the are high rates of aggression and dominance reversals, and dominance confers few psychological advantages (Sapolsky, 1992). Consistent with these data, mice in the IG housing condition experience acute social disruption when new groups are formed, resulting in the establishment of new dominance hierarchies, and therefore dominant animals may experience greater stress than the subordinate animals. In contrast, for mice in the GG housing condition, dominance has been established long before tumor cell injection, as mice in this housing condition have been together since weaning. Under these conditions, the dominant animal has an advantage over the subordinate animals and therefore may experience less stress. Thus, depending on the experimental housing condition, dominance status may be correlated with differential physiological changes which in turn may mediate the altered tumor growth rates observed. Examination of basal plasma levels of T and CORT at 1, 3, 7 and 21 days post tumor cell injection and experimental housing condition formation demonstrated that at 21 days, no 42 significant differences in plasma levels of T or CORT among mice in the different experimental housing conditions (Weinberg & Emerman, 1989). However, at 3 and 7 days, basal T levels were significantly higher in Gl mice (fastest tumor growth rate) compared to those in mice in all other housing conditions, whereas at 1, 3, and 7 days, basal CORT levels were significantly higher in IG mice (slowest tumor growth rate) compared to those in mice in all other housing conditions (Rowse et al, 1992). Thus, steroid hormone-induced alterations in SCI 15 tumor growth rates in mice among the different housing conditions are likely occurring within the first week post tumor cell injection and experimental housing condition formation. Furthermore, we have demonstrated that in mice housed under our standard laboratory conditions (group housed and not exposed to daily novelty stressors), T administration to castrated mice influences the growth of the SCI 15 tumor in a dose-dependent manner (Emerman & Siemiatkowski, 1984). Thus, these data suggest that T is a direct modulator of growth of the androgen-responsive SCI 15 tumor, whereas CORT likely plays an indirect modulatory role. That is, increased basal CORT levels may alter the activity of the HPG axis, resulting in inhibition of luteinizing hormone secretion and consequently, of T secretion. Recently, in a preliminary study, we have also demonstrated that at 3 days post tumor cell injection and experimental housing condition formation, GH levels are significantly higher in Gl mice (fastest tumor growth rate) compared to those in GG or IG mice. Thus, it is possible that increases in basal CORT levels may also alter the activity of the growth axis and therefore secretion of GH. Interestingly, SCI 15 tumor cells derived from mice in the different housing conditions are not significantly different from each other in terms of androgen and glucocorticoid receptor binding capacity and affinity (Rowse et al, 1992). Furthermore, in vitro growth of SCI 15 cells derived from IG mice is significantly greater in response to dihydrotestosterone and 43 hydrocortisone compared to that of cells derived from Gl mice. Thus, it appears that the hormonal milieu in vivo rather than alterations in the ability of the tumor cells themselves to respond to hormones is critical in the endocrine modulation of tumor growth rate. We have also investigated the possible involvement of alterations in immune activity in differentially affecting tumor growth rates among mice in the experimental housing conditions. We demonstrated that the presence of a tumor results in a general suppression of both B and T lymphocyte function in tumor-bearing mice relative to their non-tumor-bearing counterparts at 21 days post tumor cell injection and experimental housing condition formation (Weinberg & Emerman, 1989). In addition, we showed that the presence of a tumor significantly stimulates NK cell activity both at the spleen and at the tumor site at 3-7 days post injection (Rowse et al, 1990a; Rowse et al., 1995). Surprisingly, we found that NK cell activity was greater in Gl mice (fastest tumor growth rate) than in IG mice (slowest tumor growth rate). Also, in vivo stimulation of NK cell activity by polyinosinic:polyidylic acid (poly I:C) correlated with a corresponding increase in tumor growth rate in Gl mice (Rowse et al, 1995). However, poly I:C did not significantly affect tumor growth rate in IG mice compared to that of control mice, nor did antiasialo GM, (ASGM,; an NK suppressive agent) retard tumor growth rate in either Gl or IG mice (Rowse et al, 1995). These data suggest that NK cell activity may indirectly facilitate the growth of the SCI 15 tumor possibly through other immune mediators (e.g. CTL, cytokines), hormones or an interaction among these mechanisms. Finally, we have demonstrated that modulating SCI 15 growth both in vitro and in vivo alters the sensitivity of these tumor cells to chemotherapeutic agents. Investigating the effects of modulating androgen concentration on the cytotoxicity of Adriamycin (AD) and melphan (MEL) on cultured SC 115 cells, we demonstrated that cultured cells exposed to physiological 44 concentrations of DHT (10 ng/ml DHT), proliferated rapidly and were the most susceptible to the cytotoxic effects of A D and M E L prior to reaching confluence, at which time these cultures lost their sensitivity to the cytotoxic effects of A D and M E L (Emerman, 1988). In contrast, cultures incubated with concentrations of DHT (1 ng/ml) suboptimal for growth had a longer exponential growth phase resulting in an extended period of time in which they were sensitive to the cytotoxic effects of the drugs. In the absence of DHT, cultures had the slowest growth rate and were the least susceptible to the drugs. Our in vivo studies support and extend these data demonstrating that androgen withdrawal (castration) plus chemotherapy (AD and cyclophosphamide; CY) is superior to either modality alone. However, combining these cytotoxic agents with submaximal doses of T required to stimulate SCI 15 tumor growth in vivo, rather than with total T withdrawal, produced maximal tumor responses to chemotherapy (Emerman & Siemiatkowski, 1984). These results suggest that hormonal manipulations that maintain the slow growth of tumors may enhance the effectiveness of chemotherapeutic agents. GENERAL RATIONALE AND THESIS OBJECTIVES There is increasing evidence that stressful or adverse events and the ability to cope with these events may significantly influence the effectiveness of chemotherapy (Cooper et al, 1989; Fieler et al, 1995; Heim et al, 1997; Hislop et al, 1987; Levy et al, 1987; Ramirez et al, 1989; Scherge & Blomke, 1988; Spiegel et al, 1989). Unfortunately, there are limited data examining the mechanisms governing stressor-induced changes in chemotherapeutic efficacy. Several factors may play a role in mediating stressor-induced alterations in chemotherapeutic efficacy, including tumor size and growth rates, hormone levels and/or immune function or an interaction among these variables (Bassukas & Maurer-Schiltze, 1993; Boddy & Idle, 1993; 45 Chabot, 1994; North, 1984). In general, the variability in effectiveness of chemotherapy treatments is attributed to both tumor and host heterogeneity (Simpson-Herren et al, 1987; Simpson-Herren et al, 1988). Any factors in the tumor or the host that alter drug metabolism, distribution, and excretion (affecting concentrations of active drug and the duration of drug exposure) or alter the sensitivity of tumor cell populations to drugs may differentially influence chemotherapeutic efficacy (Goldin & Schabel, 1981; Lippman et al, 1985; Scheving et al, 1993; Wood & Hrushesky, 1996; Yarbro, 1996). The working hypothesis of this thesis is that social housing conditions significantly influence chemotherapeutic efficacy and that alterations in CORT, T and GH levels play a role in differentially altering tumor and/or host responses to chemotherapy. The major objectives of this thesis were to determine: (1) If tumor size and/or tumor growth rate at the time of chemotherapy initiation differentially affected the direction and magnitude of the effects of social housing condition on chemotherapeutic efficacy. Several factors including tumor size and tumor growth rate have been shown to influence the effectiveness of chemotherapeutic agents (Bassukas & Maurer-Schultze, 1993; Goldie & Coldman, 1979; Simpson-Herren et al, 1988; Skipper, 1977). In order to determine whether variations in tumor responses to chemotherapy were influenced by different tumor growth rates and/or tumor sizes at the time of chemotherapy initiation, chemotherapy was initiated: (a) when tumors were relatively large (lg) and growing at different rates; (b) when tumors were relatively large (lg) and growing at similar rates; and (c) when tumor burden was relatively undetectable, similar to the adjuvant situation in humans. Thus, the purpose of these studies (Chapters 3-5) was to test the hypothesis that tumor size and/or tumor growth rate would influence tumor response to chemotherapy (measured by tumor growth 46 delay; TGD) and/or the interaction between host and tumor responses to chemotherapy measured by overall survival probability. (2a) If altering the temporal relationship between formation of social housing condition and tumor cell injection differentially affected tumor growth rates among the housing conditions and; (2b) If altering the temporal relationship between formation of social housing condition and chemotherapy initiation differentially affected chemotherapeutic efficacy. The timing of operative stress relative to either tumor cell injection or chemotherapy has been shown to affect tumor growth rate as well as survival probability following chemotherapy (Audier, 1988; Corbett et al, 1978; Justice, 1985; Sklar & Anisman, 1981; Wood & Hrushesky, 1996). In order to examine whether tumor growth rate influenced chemotherapeutic efficacy (Chapters 3 and 4) it was necessary to alter the temporal relationship between formation of social housing conditions and (a) tumor cell injection and (b) chemotherapy initiation. Thus these studies also examined whether altering the temporal relationship between these variables differentially influenced tumor growth rate or chemotherapeutic efficacy, respectively, in mice among the different housing conditions. (3) If specific endocrine factors (CORT, T or GH) play a role in mediating the differential effects of social housing conditions on chemotherapeutic efficacy. Psychosocial stressors have been shown to alter hormone levels (Hoffman-Goetz et al, 1992; Levy et al, 1987; Rowse et al, 1992). Importantly, hormone levels have been related to variations in the effectiveness of chemotherapy (Emerman & Siemiatkowski, 1984; English et al, 1991; Kodama & Kodama, 1982; Levy et al, 1987; Markaverich et al, 1983; Rowse et al, 1992). However, the interaction between stressor exposure, altered hormone activity and the effectiveness of chemotherapy remains to be elucidated. Previously, we have demonstrated that 47 experimental housing conditions differentially influence CORT, T and, in a preliminary study, GH levels (Rowse et al, 1992). We have also shown that for male mice in our standard laboratory housing conditions (group housed and not subjected to daily novelty stressors), SCI 15 tumor response to AD and CY can be modulated by influencing tumor growth rate by altering the dose of testosterone administered following castration (Emerman & Siemiatkowski, 1984). This series of studies examined if CORT, T, and/or GH levels in mice in the experimental housing conditions are differentially altered following chemotherapy treatment. In addition, this study will help to determine whether altered hormone profiles among mice is related to differential tumor and/or host responses to chemotherapy. (4) In addition to these major objectives, an additional study in this thesis examined the interactive effects of social housing conditions and gender on the growth rate of a variant of the SCI 15 tumor designated SCI 15V, that grows equally as well in males as in females. Although gender differences have been reported to influence tumor progression and metastasis (Leigh et al, 1987; Maguire et al, 1996; Ueda et al, 1988), the interactions among psychosocial stressors, gender and cancer progression remain to be elucidated. This study was designed to compare directly the effects of social housing condition on tumor growth rates in male and female DD/S mice. Specifically, SCI 15V tumor growth rates in males and females housed in experimental housing conditions both with and without daily exposure to novel environments were examined. 48 C H A P T E R 2. G E N E R A L M E T H O D S A. Dissociation. Tumors weighing between 2-3 g were dissected free of subcutaneous tissue and finely minced. The pieces were transferred to a dissociation flask containing approximately 15 ml saline-trypsin-versine (STV; 0.05% trypsin (1:250) and 0.025% EDTA (Sigma Chemical Co., St. Louis MO) in Ca2+- Mg2+- free saline, pH 7.3). The flask was gently swirled for 2 min and the contents transferred to a 50 ml conical centrifuge tube (tube-#l) and spun at 80 x g for 1 min in a bench top clinical centrifuge. The supernatant was transferred to a second 50 ml centrifuge tube with an equal volume of Dulbecco's modified Eagle's medium (DMEM; Terry Fox Laboratory, Vancouver, BC.) and 5% calf serum (CS) to inactivate the trypsin. The tube was spun at 200 x g for 4 min to enrich for epithelial cells. The pellet (containing the epithelial cells) was resuspended in 5 ml DMEM and placed in a 37° C water bath. The tissue in the original centrifuge tube (tube-#l) was transferred to the dissociation flask and 15 ml STV was added. The flask was left shaking at 100 rpm on a gyrator shaker (Junior Orbit Shaker, Lab-line Instruments, Inc., 111.) in a 37° C incubator for 7 min. The contents of the flask were then transferred to the original 50 ml centrifuge tube (tube-#l) and centrifuged at 80 x g for 1 min. The supernatant was collected and combined with an equal volume of DMEM and 5% CS, spun for 5 min at 400 x g, the pellet was resuspended in 5 ml DMEM and placed in a 37° C water bath. The tissue remaining in the original centrifuge tube (tube-#l) was transferred to the dissociation flask for a third and final dissociation with STV for 7 min. The supernatant was collected as described above, combined with an equal volume of DMEM and 5% CS, and centrifuged. The resulting pellet was resuspended in 5 ml DMEM. All 3 cell suspensions were pooled and passed through a 50 um Nitrex filter (Tetko, Inc., Elmsford, NY) to remove cell 49 aggregates and debris. The resulting single cell suspension was centrifuged at 340 x g for 5 min and the pellet resuspended in 20 ml DMEM. An aliquot of this cell suspension was diluted 1:10 with DMEM and counted on a haemocytometer using trypan blue exclusion to determine the number of viable cells. Finally, the suspension was adjusted to the concentration desired for injecting cells into mice (see below). i B. Freezing and Thawing of Tumor Cells. In addition to maintaining the SCI 15 in vivo, tumor cells were also stored in a liquid nitrogen (LN2) tank (Locator Junior, Thermolyne, VWR/Canlab, Vancouver, BC, Canada). For freezing, SCI 15 cells were adjusted to a concentration of approximately lxlO7 cells/ml of freezing medium (50% DMEM: 44% calf serum: 6% dimethylsulfoxide (DMSO), Sigma-Aldrich Canada Ltd., Oakville ON, Canada) and slowly frozen according to the freezing tank (Union Carbide Canada Ltd., Richmond, BC, Canada). Once frozen, vials were transferred to the LN 2 storage tank. When required, selected vials of tumor cells were removed from the LN 2 storage tank and thawed quickly in a 37° C water bath. The tumor cells suspension was added to 10 ml of warmed DMEM to dilute the freezing medium. This dilute suspension was spun at 400 x g for 4 minutes to wash the cells. The supernatant was then discarded, the pellet was resusspended in 10 ml DMEM and spun at 400 x g for 5 min (wash step). This wash step was repeated, the pellet was resuspended in 10 ml DMEM and an aliquot was counted to establish viable cell numbers as described previously. Cells were resuspended at 2 x 107 cells/ml for tumor injection. C. Transplantation of Tumor Cells and Housing Conditions. Male mice of the DD/S strain were weaned at 3 weeks of age and housed either individually (I) or in groups (G). The androgen-responsive mammary carcinoma subline designated SCI 15 50 Class A (Bruchovsky & Rennie, 1978) is maintained in syngeneic male mice of the DD/S strain. At 2-4 months of age, mice in each rearing condition were injected sc in the interscapular region with either 2x10^  freshly dissociated SCI 15 tumor cells in 0.1 ml DMEM or 0.1 ml tumor cell vehicle (DMEM) alone. Except where indicated, experimental groups were formed immediately following sc injection: mice that were reared individually (I) or group (G) housed at weaning (21 d of age) were rehoused either from individual to group (IG) or from group to individual (GI) or remain in their original rearing conditions (II or GG). Except on the termination day, all mice were exposed for 15 min/day, 5 d/wk, to one of several novel environments, a treatment that enhances the differences in tumor growth rates among mice in the experimental groups (Weinberg & Emerman, 1989). The novel environments were: 1) a clear plastic container, 12 x 10 x 4 cm; 2) an opaque polypropylene container, 12 x 10x4 cm; 3) an opaque polyethylene container, 6 cm in diameter x 10 cm in height; 4) a cardboard box divided into compartments, 7 x 7 x 14 cm; and 5) a clean standard rodent cage, 18 x 29 x 13 cm, empty of bedding, food and water. Mice were placed individually into 1 of the 5 different novel environments. Times of stressor exposure were randomly varied each day between 0800 and 1200 h to increase stressor unpredictability. Body weights were measured and tumors were palpated every 2-3 days. When tumors were measurable (typically within 8-10 d following tumor cell injection), caliper measurements were taken, and tumor weights calculated according to the formula (Simpson-Herren & Lloyd, 1970): Tumor weight (g) = length (cm) x f width (cm)]2 2 D. C h e m o t h e r a p y T r e a t m e n t . Cyclophosphamide (Horner, Montreal Canada) and Adriamycin (Adria Laboratories Ltd. Ontario Canada) were dissolved in sterile 0.9% NaCl to make stock solutions of CY at 20 mg/ml and AD at 1 mg/ml. The stock solutions were stored at -70° C. Adriamycin, due to its 51 sensitivity to light, was stored in vials wrapped in tin foil. At the time of drug injections the vials were thawed and injection volumes were determined according to individual mouse weight. Chemotherapeutic agents (CY and AD) or drug vehicle (0.9% saline) were administered i.p. once a week for 3 weeks; except in the studies of Chapter 6, where mice received only one injection each of AD and CY. The timing of the drug injections in relation to both tumor cell injection and group formation varied among experiments (see specific experiments). The toxic effects of the chemotherapy treatment were monitored by morbidity (body weight loss) and mortality. The doses of drugs used have been shown to be optimal for SCI 15 tumor regression with minimal toxic side effects (Emerman & Siemiatkowski, 1984). Animals were terminated either when tumor weight reached 3.5 g or 30 or 70 days after initiation of an experiment (duration of separate experiments will vary). E. Radioimmunoassays (RIAs). Corticosterone levels were measured by radioimmunoassay (RIA) as described previously (Weinberg & Bezio, 1987; Weinberg & Emerman, 1989). Briefly, 33.3 ul of plasma was extracted in 300 ul of absolute ethanol. After incubation in dextran-coated charcoal (Fisher Scientific, Vancouver, BC, Canada) to absorb and precipitate free CORT, 100 ul each of antiserum (ICN Biomedicals Inc., Costa Mesa, CA) and tritiated tracer (New England Nuclear, Guelph, Ont., Canada) were added to all samples. Following an overnight incubation, all samples were counted with liquid scintillation counting (Scintisafe Econo2 Sx21 -5; Fisher Scientific). Testosterone levels were measured by RIA (ImmuChem; ICN Biomedicals). Samples were counted in Scintisafe Econo2 Sx21-5; Fisher Scientific). 52 Growth hormone levels were measured by RIA using purified mouse serum GH RIA immunoreagents distributed by the U.S. National Institute of Diabetes and Digestive and Kidney Diseases' National Hormone and Pituitary Program (Harbor-UCLA Medical Center Research and Education Institute, Torrance, CA). F. Statistical Analyses. Tumor response to chemotherapy was measured by tumor growth delay (TGD); defined as the mean time for tumors in chemotherapy-treated mice to reach a specific weight minus the mean time for tumors in drug vehicle-treated mice reach the same weight. Host response to chemotherapy was measured by: (a) the percentage of body weight lost over the course of chemotherapy. Percent body weight loss = [body weight at the second or third chemotherapy injection round minus body weight on the day of chemotherapy initiation] / [body weight on the day of chemotherapy initiation] x 100, where body weight = [gross body weight minus tumor weight]; negative values indicate weight loss between chemotherapy rounds; and (b) survival probability of tumor cell-vehicle injected (non-tumor-bearing), chemotherapy-treated mice. The interaction between tumor and host responses to chemotherapy was analyzed by overall survival probability of tumor cell injected (tumor-bearing), chemotherapy-treated mice. Survival probability was analyzed by Cox proportional hazards analysis and Kaplan Meier plots (Cox, 1972; Kaplan & Meier, 1958). For this analysis death, regardless of cause, was considered an event (i.e. mice were sacrificed when tumor weight exceeded 3.5 g or mice were found dead due presumably to the toxic side effects of chemotherapy). Mice that were still alive 30 or 70 days (depending on the experiment) were considered censored. In all experiments, at least 3 groups of animals from each housing condition (Gl, IG, and where 53 appropriate, II and GG) were tested. Tumor growth rate in drug vehicle-treated mice, tumor response to chemotherapy, body weight loss and hormone levels were analyzed using appropriate ANOVAs; significant main effects and interactions were analyzed further by Tukey's post-hoc tests. 54 CHAPTER 3. EFFECTS OF SOCIAL HOUSING CONDITIONS ON THE RESPONSE OF THE SHIONOGI CARCINOMA (SCI 15) TO CHEMOTHERAPY INTRODUCTION A number of human studies have demonstrated that stressful life events and the ability to cope with stress may play a role in cancer growth and metastasis. Psychosocial stressors such as divorce or bereavement have been associated with increased cancer risk (Bloom et al, 1978; Cooper et al, 1989; Jensen, 1991; Levy et al, 1987) and increased probability of metastasis (Hislop et al, 1987; Levy et al, 1988; Ramirez et al, 1989). Studies have also shown that social involvement with support groups or family members may be related to an increase in survival time (Greer et al, 1979; Greer et al, 1991; Levy, 1985; Northhouse, 1988) and a decrease in the rate of metastasis (Gruber et al, 1988; Spiegel et al, 1989), possibly by providing the patient with a form of coping strategy (Pettingale et al, 1985; Rogentine et al, 1979; Temoshok, 1987). There are, however, several studies that have reported no association among psychosocial stressors, coping strategies and survival time (Cassileth et al, 1985; Ewertz, 1986; Jamison et al, 1987; Muslin et al, 1966; Priestman et al, 1985) or rate of metastasis (Barraclough et al, 1992; Linn et al, 1982). A number of issues may affect the interpretation of these data. For example, in retrospective studies, the diagnosis of cancer may distort the patient's perception of past stressful events due to the knowledge that she/he has cancer. Also, the clinical manifestation of cancer may occur years following neoplastic change and therefore the onset of the disease may have occurred prior to the stressful event. Animal models allow investigation of the relationship between psychosocial stressors and tumor growth under more controlled conditions. However, even in animal studies the data are complex. Factors such as chronicity and timing of the stressor and the type of tumor being 55 examined can influence stressor effects on tumor growth and metastasis (Amkraut & Solomon, 1972; Baker & Jahn, 1976; Riley, 1981; Riley et al, 1981; Sklar & Anisman, 1979; Solomon et al, 1979; Steplewski et al, 1985). Psychosocial stressors such as housing condition or psychological stressors such as forced restraint have been shown to affect tumor growth and/or metastasis of either transplantable (Sklar & Anisman, 1980; Steplewski et al, 1987; Weinberg & Emerman, 1989) or chemically-induced tumors (Neiburgs et al, 1979; Newberry et al, 1976; Steplewski et al, 1987). Of relevance to this report, group housed animals typically have smaller tumors and increased rates of tumor regression or rejection than individually housed animals (Riley, 1981; Riley et al, 1981). In addition, a change in housing condition may increase tumor growth compared to that found in individually or group housed animals that do not experience change (Sklar & Anisman, 1980; Weinberg & Emerman, 1989). Interestingly, it has been suggested that the adverse consequences of housing change on tumor growth may be reduced by fighting (Amkraut & Solomon, 1972; Grimm et al, 1996; Sklar & Anisman, 1980), which may act as a coping mechanism and therefore reduce the impact of housing change on the animals. We have developed an animal-tumor model that demonstrates that a change in social housing condition can dramatically affect the growth of the transplantable androgen-responsive Shionogi mouse mammary tumor (SCI 15; Ref. Weinberg & Emerman, 1989). Moreover, we have shown that the direction of change in housing condition significantly influences tumor growth rate (Grimm et al, 1996; Weinberg & Emerman, 1989). Being reared in a group and then individually housed (GI) following tumor cell injection increases tumor growth rate, whereas being reared individually and then group housed (IG) reduces tumor growth rate, compared to that in mice remaining in their group rearing condition (GG). 56 Recently, epidemiological evidence suggests that psychosocial stressors may affect not only tumor growth and metastasis, but also tumor response to chemotherapy. Psychosocial stressors such as divorce or bereavement have been associated with decreased efficacy of cancer therapies (Lichtman et al, 1987; Waxier-Morrison et al, 1991). Reducing the impact of psychosocial stressors through social support or psychosocial intervention can extend survival time and decrease the toxic side-effects of chemotherapy in cancer patients (Burish et al, 1987; Grossarth-Maticek & Eysenck, 1989; Spiegel & Bloom, 1983; Spiegel et al, 1989). For example, in patients with metastatic non-small cell lung cancer, the combination of supportive care and chemotherapy offers a survival advantage over either modality alone (Cartei et al, 1993). In breast cancer patients, the combination of chemotherapy and psychotherapy (Spiegel & Bloom, 1983) or psychosocial support (Spiegel et al, 1989) increases survival time over chemotherapy alone. Recent animal studies (Giraldi et al, 1992; Giraldi et al, 1994; Perissin et al, 1991; Perissin et al, 1997) support these findings. It has been shown that exposure to rotational stress decreases the antitumor effects of orally administered cyclophosphamide or razoxane in mice bearing Lewis lung carcinoma in terms of tumor burden, extent of metastasis, and survival time (Perissin et al, 1991; Perissin et al,1997). The present study, using our animal-tumor model, extends these previous data in three important areas. First, we employ a psychosocial stressor, change in social housing condition from group to individual (Gl) or individual to group (IG). Second, we utilize a mouse mammary carcinoma that has similarities to hormone-responsive human cancers. Third, we use a chemotherapeutic regimen consisting of Adriamycin (AD) and cyclophosphamide (CY) administered intraperitoneally (i.p.). 57 METHODS Tumor propagation. Tumor cells were prepared and injected as described previously in "General Methods". Experimental animals. Male DD/S mice (n=112), between 2-4 mo of age were the experimental subjects in this study. Mice were reared either individually housed (I) or group housed (G). Immediately following tumor cell injection (s.c. injection of 2 x 106 cells suspended in 0.1 ml DMEM) or tumor cell-vehicle injection (s.c. injection of 0.1 ml DMEM), those mice reared in groups were rehoused individually (Gl) and those reared individually were rehoused in groups (IG), according to our published protocol (Weinberg & Emerman, 1989). Animals within each housing condition were randomly assigned into tumor cell injection groups receiving either chemotherapy (n=21 Gl, n=30 IG) or drug vehicle alone (n=20 Gl, n=25 IG), or into tumor cell-vehicle injection groups receiving chemotherapy (n=6 Gl, n=10 IG; Fig. 1). Beginning the day following tumor cell or tumor cell-vehicle injection, all animals were exposed to an acute daily stressor consisting of exposure for 15 min/d, 5 d/week to 1 of 5 different novel environments, a treatment that we have shown enhances tumor growth rate differences between experimental groups (Weinberg & Emerman, 1989). Body weights were measured and mice were palpated every second day; once tumors were measurable (approximately 8-10 d), caliper measurements were taken as described in "General Methods". Chemotherapy. Chemotherapy or drug vehicle administration (as described in "General Methods") was initiated when the mean tumor weight of mice within each housing condition (i.e. mean tumor weight among mice of a previously grouped condition [Gl] or within each newly formed group [IG]) reached 1.0 ± 0.2 g (Fig. 1). Mice in the no tumor control condition received the chemotherapeutic regimen starting at 16 d post tumor cell-vehicle injection for 58 mice in the GI condition (mean time for GI tumor-bearing mice to reach 1 g) or 20 d post tumor cell-vehicle injection for mice in the IG condition (mean time for IG tumor-bearing mice to reach lg). Statistical Analyses. Tumor response to chemotherapy in GI and IG housed mice was analyzed using survival probability and TGD, as described in "General Methods". Mice that were still alive at 30 d following the first round of chemotherapy were considered censored for survival probability analyses. Differences in TGD of mice in the experimental housing conditions was analyzed by analysis of variance (ANOVA) for the factors of Group and Weight. Tumor growth in drug vehicle-treated animals was also analyzed by ANOVA for the factors of Group and Days with repeated measures on Days; interactions were further analyzed by Tukey's post-hoc tests. Differences in the median survival times of mice in the two experimental housing conditions following chemotherapy initiation was analyzed by Student's t-test. RESULTS Consistent with our previous data (Weinberg & Emerman, 1989), tumor growth rate in drug vehicle-treated mice was significantly faster in mice in the GI housing condition than in mice in the IG housing condition (F[l,27]=26.096, pO.OOl; Fig. 2). Consequently, survival probability in these drug vehicle-treated mice was greater in the IG compared to the GI housing condition (£2=6.482, p=0.01; Fig. 3). As expected, tumor-bearing chemotherapy-treated mice survived longer than tumor-bearing drug vehicle-treated mice, regardless of experimental housing condition (5C2=32.816, p<0.001). As well, for all mice that received chemotherapy, survival probability was greater 59 for non-tumor-bearing than for tumor-bearing mice (X2=3.719, p=0.05; Fig. 3). Of importance is the observation that, similar to the drug vehicle condition, tumor-bearing chemotherapy-treated mice in the IG housing condition survived significantly longer than their counterparts in the Gl housing condition (X2=6.233, p=0.01; Fig. 3). Further inspection of the data suggests that the increased survival probability in tumor-bearing mice in the IG compared to the Gl housing condition was due to differential tumor responses to chemotherapy rather than to differences in the toxic side-effects of chemotherapy. First, the number of mice that were terminated due to tumor weights exceeding 3 g was greater for mice in the Gl (n=8; 38%) than in the IG (n=4; 13%) housing condition, resulting in a greater number of IG (n=14; 47%) than Gl (n=4; 19%) housed mice surviving to 30 d post chemotherapy or drug vehicle initiation (Fig. 3). Second, the median survival time from chemotherapy initiation was greater for mice in the IG than in the Gl condition (24.5 d versus 15.0 d respectively) (p<0.05; t-test). Finally, the number of deaths in tumor-bearing chemotherapy-treated mice (excluding mice whose tumors exceeded 3g) was similar in the IG (n=12; 40%) and Gl (n=9; 43%) housing conditions. Similarly, the number of deaths in non-tumor-bearing chemotherapy-treated mice was the same in the IG (n=3; 50%) and Gl (n=5; 50%) housing conditions (Fig. 3). Together, the data suggest that the differences in survival probability for tumor-bearing chemotherapy-treated mice in the two housing conditions was due to differential tumor responses to chemotherapy and that the toxic side-effects of chemotherapy affected mice in the Gl and IG housing conditions similarly, irrespective of tumor burden. This suggestion was supported by general gross autopsies performed on tumor-bearing, chemotherapy-treated mice found dead in their home cages between 1-30 days following chemotherapy initiation. Specifically, autopsies were similar among mice, regardless 60 of housing condition; the tissues and organs in both the peritoneal (i.e. liver, spleen, intestines, kidneys) and thoracic (lungs and heart) cavities were pink/red in color, the consistency of the tissues was similar to that of mice not treated with chemotherapy and no acrid odor was present upon opening the cavities. Finally, the differences in the TGD between mice in the Gl and IG housing conditions increased with increasing tumor weights (Table 1). Overall, TGD of mice in the IG condition is longer than the TGD of mice in the Gl condition (F[l,145]=3.727, p=0.055). These data support the finding that tumors in mice in the IG condition respond better to chemotherapy than do tumors in mice in the Gl condition. DISCUSSION The present data demonstrate that a psychosocial stressor, change in social housing condition, not only alters the SCI 15 tumor growth rate but also significantly affects the response of the SCI 15 tumor to chemotherapy. Data on tumor growth rate in drug vehicle-treated mice support and extend our previous findings (Weinberg & Emerman, 1989); mice that experience a change from group to individual (Gl) housing have a significantly faster tumor growth rate and a significantly reduced survival probability compared to mice that experience a change from individual to group (IG) housing. Importantly, the present data demonstrate that the direction of change in social housing condition also significantly influences survival probability following the initiation of chemotherapy. That is, chemotherapy-treated mice in the IG housing condition have a significantly greater survival probability than chemotherapy-treated mice in the Gl housing condition. The data suggest that this difference in survival probability is due to differential tumor responses to chemotherapy, as the number of mice in the 61 GI and IG conditions that died due to the toxic side-effects of chemotherapy was similar in both the tumor-bearing and the non-tumor-bearing conditions. Although it is difficult to extrapolate data from animal studies to humans, our animal-tumor model has relevance to the human situation. First, the androgen-responsive variant of the SCI 15 tumor, used in the present study, was derived from a mammary tumor that spontaneously arose in a female mouse (Bruchovsky & Rennie, 1978; Weinberg & Emerman, 1989). This heterogeneous solid tumor is similar to some hormone-responsive cancers in humans in its sensitivity to different classes of steroid hormones, including androgens (King & Yates, 1980), estrogens (Noguchi et al, 1987) and glucocorticoids (Watanabe et al, 1982). Second, this mouse mammary tumor is immunogenic (Rowse et al, 1990; Rowse et al, 1995), also characteristic of many human cancers. Finally, the SCI 15 mouse mammary tumor responds well to AD and CY, two chemotherapeutic agents used in the treatment of human cancers (Emerman & Siemiatkowski, 1984; Ihde et al, 1980; Lloyd et al, 1979). Thus, our data suggesting a relationship between psychosocial stressors and tumor response to chemotherapy may have potential clinical relevance. The mechanisms underlying the differential responses to chemotherapy observed in this study are unknown at present. One possibility is that stressor-induced and/or chemotherapy-induced changes in endocrine function are involved. We have previously shown that for male mice in our standard laboratory housing conditions (group housed and not subjected to daily novelty stressors), tumor response to AD and CY can be regulated by modulating the level of exogenous testosterone administered following castration (Emerman & Siemiatkowski, 1984). We have also demonstrated that the different housing conditions of our model are correlated with significant differences in basal testosterone and corticosterone levels (Rowse et al, 1992). 62 Basal levels of plasma testosterone are significantly higher and basal levels of plasma corticosterone are significantly lower in GI than in IG housed mice over the first seven days following tumor cell injection and rehousing. In the present study, it is not known if testosterone and/or corticosterone levels differ between mice in the two experimental housing conditions at the time when chemotherapy was initiated (between 14-20 d post tumor cell injection/rehousing) or during the course of chemotherapy. Studies investigating hormone levels at these times are in progress. In addition to the possible effects of social housing condition on circulating hormone levels, there is also evidence that alkylating chemotherapeutic agents such as CY may reduce plasma levels of both testosterone and corticosterone (Chang & Waxman, 1993; Johnston et al, 1995; LeBlanc & Waxman, 1990; McClure & Stupans, 1995). Although such effects have been demonstrated only following high, single dose chemotherapy treatments (Chang & Waxman, 1993; LeBlanc & Waxman, 1990; McClure & Stupans, 1995), it is possible that the dose of CY used in the present study may differentially alter hormone levels in mice in the GI and IG conditions, which may in turn alter tumor response to chemotherapy. Differential responses to chemotherapy in this study may also be mediated through changes in immune function. Such changes may occur either directly through psychosocial stressor- or chemotherapy-induced changes in immune function or indirectly through changes in hormonal activity that alter immunocompetence. We have shown that the SCI 15 tumor differentially stimulates natural killer (NK) cell activity in mice in the GI and IG conditions (Rowse et al, 1990; Rowse et al, 1995). In addition to alterations in NK cell activity, preliminary evidence from our laboratory suggests that the SCI 15 tumor stimulates a tumor-specific cytolytic immune response. Immune rejection of tumors in human and murine studies 63 has been shown to be mediated primarily by cytotoxic T lymphocytes (CTLs) (Knuth et al, 1992; Paramiani, 1990). Several studies examining the effectiveness of tumor-specific CTLs introduced into a tumor-bearing host demonstrate that optimal treatment includes the addition of chemotherapeutic agents (Berd et al, 1982; Cameron et al, 1990; Evans, 1983; Lafreniere et al, 1989; North, 1982; Sewell et al, 1993). On the other hand, chemotherapy itself may differentially affect the immune functions of mice in the two housing conditions. In animal studies, CY has been shown to alter cytokine levels (Abdul Hamied & Turk, 1987; Hasan et al, 1992; Kiberd & Young, 1994), T cell and natural killer (NK) cell activities (Brenner & Margolese, 1991; Stewart et al, 1990), and the accumulation of macrophages within the tumor (Dye & North, 1980; Nugent & Onofrio, 1987). Thus it is possible that the greater chemotherapeutic efficacy observed in mice in the IG housing condition may be due to an increase in immunoreactivity towards the tumor compared to that in mice in the Gl housing condition. The interactions among host environment, chemotherapy treatment and tumor growth are still to be elucidated. In general, chemotherapy has been shown to be more effective against fast growing than slow growing tumors (Skipper, 1977). Interestingly, our data indicate that chemotherapy is more effective against the slower growing (mice in the IG condition) than the faster growing (mice in the Gl condition) tumors. It is possible that tumor growth rate is so rapid in mice in the Gl housing condition that the tumor burden becomes too large for the chemotherapy to be effective. Since chemotherapy was initiated at a time when tumors were growing at different rates, albeit were at similar weights, it is difficult to determine if the differential responses to the chemotherapeutic drugs were due to different tumor growth rates, to psychosocial stressor-induced alterations in hormone levels and/or immune function, or to an interaction among these 64 factors. Studies to resolve these questions are presently being conducted. In summary, these data demonstrate that the psychosocial stressor of a change in social housing condition affects not only tumor growth rate but also tumor response to chemotherapy. These data highlight the possible impact of psychosocial stressors on the complex interrelationship among the host environment, tumor growth and progression, and the efficacy of chemotherapy. 65 Fig. 1. Experimental design M a l e D D / S m i c e 2 -4 m o o f age reared either i n d i v i d u a l l y (I) o r in g roups ( G ) S C I 15 t u m o r ce l l s in jected s.c. and m i c e rehoused into exper imenta l h o u s i n g cond i t i ons ( G I o r I G ) T u m o r c e l l - v e h i c l e in jected s.c. and m i c e rehoused into e x p e r i m e n t a l h o u s i n g c o n d i t i o n s ( G I o r I G ) E x p o s u r e to acute d a i l y nove l t y stressor 15 m i n / d a y , 5 d a y / w e e k , start ing the day f o l l o w i n g t u m o r c e l l or t u m o r c e l l - v e h i c l e i n jec t ion V C h e m o t h e r a p y o r d r u g - v e h i c l e r e g i m e n started w h e n tumors w e i g h e d a p p r o x i m a t e l y 1 g C h e m o t h e r a p y r e g i m e n started 16 or 20 d f o l l o w i n g t u m o r c e l l -v e h i c l e i n jec t ion M i c e te rminated 30 d after c h e m o t h e r a p y / d r u g v e h i c l e i n i t i a t i on o r w h e n tumor w e i g h t exceeded 3.5 g 67 Fig. 2. Tumor growth in drug vehicle-treated mice. Tumor weights (mean ± SEM) over 5 measurement days for drug vehicle-treated (9 g NaCl per 100 ml in distilled water) mice in the Gl (n=21) and IG (n=25) housing conditions. Tumor growth rate was significantly greater for mice in the Gl compared to mice in the IG housing condition (F[l,27]=26.096, p<0.001). 68 1 2 3 4 5 Days of tumor measurement 3. Survival probability in tumor-bearing mice receiving chemotherapy (TC) or drug vehicle (TV) and in non-tumor-bearing mice receiving chemotherapy (NTC). Day 0 is the day of initiation of chemotherapy (AD at 4.0 mg/kg and CY at 61.5 mg/kg) or drug vehicle administration. The first symbol represents the time at which the first death(s) occurred in each condition. For tumor-bearing mice receiving either chemotherapy (X2=6.233, p=0.01) or drug vehicle (X2=6.482, p=0.01), survival probability was significantly greater for mice in the IG than for mice in the GI housing condition. As expected, tumor-bearing chemotherapy-treated mice (n=21 GI, n=30 IG) had a significantly greater survival probability than tumor-bearing drug vehicle-treated mice (n=20 GI, n=25 IG) (X2=32.816, pO.OOl). Additionally, for all mice receiving chemotherapy, non-tumor-bearing mice had a significantly higher survival probability than tumor-bearing mice (X2=3.719, p=0.05). 70 0 5 10 15 20 25 Days post chemotherapy or drug vehicle initiation 30 71 Table 1. Tumor growth delay (TGD)a in chemotherapy-treated mice. Tumor Growth Delay in Days (±SEM) Housing Conditions Tumor Weights (g) 1.5 2.0 2.5 3.0 Gl 4.62 + 2.58 (21) 7.39 + 2.79 (18) 8.69 ± 2.96 (16) 13.93 + 3.05 (15) IG 4.17 + 2.47 (23) 10.14 + 2.52 (22) 15.35+2.65 (20) 19.89 + 2.79 (18) a TGD is defined as the mean time for tumors to reach a specific weight in chemotherapy-treated mice minus the mean time for tumors in drug vehicle-treated mice to reach the same weight. ( ) = n per group. b TGD for IG>GI, p=0.055 72 CHAPTER 4. CHEMOTHERAPEUTIC EFFICACY IN THE SC115 MOUSE-TUMOR MODEL: TEMPORAL FACTORS INTRODUCTION In humans, stressful life events and the ability to cope with stress may play a role not only in cancer growth and metastasis but also in response to chemotherapy. Psychosocial stressors such as divorce or bereavement have been associated with both increased cancer risk (Cooper & Faragher, 1993; Graves et al, 1986; Ramierez et al, 1989) and increased probability of metastasis (Hislop et al, 1987; Levy et al, 1988), as well as decreased efficacy of cancer therapies (Ramirez et al, 1989; Spiegel, 1997). Reducing the impact of psychosocial stressors through active intervention (e.g. psychotherapy), social or family support, and/or coping styles (e.g., disclosure, information seeking) has been shown to decrease the rate of metastasis (Gruber et al, 1988; Spiegel et al, 1989), increase survival time (Spiegel et al, 1989; Temoshok, 1987; Waxier-Morrison et al, 1991), and decrease the side effects of chemotherapy in cancer patients (Fieler et al, 1995; Redd et al, 1991; Shapiro et al, 1997; Spiegel & Bloom, 1983). There are, however, a number of studies that have reported no associations among psychosocial stressors and/or coping resources and rate of metastasis or survival time (Cassileth et al, 1985; Chabot, 1994; Fox, 1983; Heinrich & Schag, 1985; Jacobs et al, 1983; Tross et al, 1996). A number of issues may affect the interpretation of the data. For example, in retrospective studies, the diagnosis of cancer may distort the patient's perception of stressful events due to the knowledge that he/she has cancer. Also, the estimates of the psychosocial stressors or demands experienced by the cancer patients can vary widely depending on the nature of the assessment (e.g. retrospective versus prospective studies, psychological testing or psychiatric interviews; Ref. Goldberg & Cullen, 1985; Leigh et al, 73 1987). Moreover, psychosocial demands may change over the stages of cancer treatment (e.g. hospitalization, treatment, reoccurrence, metastasis), possibly differentially taxing the coping resources/styles of the patient (Heim et al, 1997; Hurny et al, 1996; Liang et al, 1990). Thus, the stage of treatment in which the patients are examined as well as the form of psychosocial assessment(s) employed may alter the association among psychosocial stressors, coping resources/strategies and cancer treatment. In addition, the timing of a stressful event relative to the initiation of treatment as well as tumor and host factors may affect interpretation of the data (Boddy & Idle, 1993; Corbett et al, 1978; Ell et al, 1992; Simpson-Herren et al, 1987; Simpson-Herren et al., 1988; Waxier-Morrison et al, 1991). Animal studies have also shown that stressors can influence chemotherapeutic efficacy. Exposure to rotational and/or restraint stress (psychological stressors) was shown to decrease antitumor effects of chemotherapeutic agents in mice bearing Lewis lung carcinoma or TLX5 lymphoma in terms of tumor burden, metastasis, and/or survival probability (Perissin et al, 1991; Perissin et al, 1997). Our data demonstrate that a change in social housing condition can influence both tumor response to chemotherapy, measured by tumor growth delay (TGD), and the interaction between tumor and host responses to chemotherapy, measured by overall survival probability (Kerr et al, 1997). In that study, we used our original animal-tumor model, as described above; mice were reared either individually (I) or in groups (G) until 2-4 months of age, at which time tumor cells were injected and experimental housing conditions (IG or GI) were formed. Chemotherapy was initiated when the mean tumor weight of mice in each housing condition reached 1 g (approximately 14 or 18 d post tumor cell injection and social housing condition formation for GI and IG mice, respectively). Under these conditions, TGD and overall survival probability were greater in IG than in GI mice. Since chemotherapy was 74 initiated when tumors, albeit at similar weights (1 g), were growing at different rates (i.e. faster in Gl than in IG mice), the differential responses to chemotherapy may have been due to differences in social housing conditions, differences in tumor growth rates, or to an interaction between these factors. The present study was undertaken to begin to address this issue. Experimental variables were manipulated such that chemotherapy was initiated when SCI 15 tumors weighed approximately lg (as in our previous study; Ref. Kerr et al, 1997) but were growing at similar rates. To accomplish this, tumor cells were injected but mice remained in their original rearing conditions (I or G) until the mean tumor weight of mice within that rearing condition reached 1 g; this occurred approximately 14 d post tumor cell injection for both I and G housed mice. At that time, mice were rehoused either from individual to group (IG) or from group to individual (Gl) or remained in their rearing conditions (II, GG). Chemotherapy was initiated 1 d following formation of experimental housing conditions. Thus, unlike our first study in which experimental housing conditions were formed immediately following tumor cell injection and 14-18 d prior to chemotherapy initiation (Kerr et al, 1997), in the present study, experimental housing conditions were formed approximately 14 d following tumor cell injection and 1 d prior to chemotherapy initiation. METHODS Tumor Propagation. Tumor cells were prepared and injected as described in "General Methods". Experimental Animals. Male DD/S mice (n=176) were the experimental subjects in this study. Following weaning, mice were reared either individually housed (I) or group housed (G; 75 Fig. 4). At 2-4 mo of age, mice in each housing condition were injected with either tumor cells (2 x 1()6 cells suspended in 0.1 ml DMEM s.c.) or tumor cell-vehicle injection (0.1 ml DMEM s.c), and remained in their I or G housing condition. When mean tumor weights of mice within each housing condition reached 0.8 ± 0.2 g (approximately 14 d post tumor cell injection) mice were either rehoused from individual to group (IG) or from group to individual (GI), or remained in their original housing conditions (II, GG), according to our published protocol (Weinberg & Emerman, 1989). Tumor cell-injected mice in each of the experimental housing conditions (II, IG, GG, and GI) were randomly assigned to receive either chemotherapy (TC; n= 9 II, n= 23 IG, n=13 GG, n=16 GI) or drug vehicle alone (TV; n=12 II, n=19 IG, n-12 GG, n=14 GI); tumor cell-vehicle-injected mice within each housing condition received chemotherapy (NTC; n=12 II, n=20 IG, n= 15 GG, n=ll GI). In addition, all animals were exposed to an acute daily stressor consisting of exposure for 15 min/d, 5 d/week to 1 of 5 different novel environments, as described in "General Methods". Chemotherapy/drug vehicle administration as well as acute daily novelty stress were initiated 1 d following formation of experimental housing conditions (approximately 14 d following tumor cell/tumor cell-vehicle injection; Fig. 4). Body and tumor weights were measured every second day as described in the "General Methods". Chemotherapy. Chemotherapy was administered i.p. every 7 d for a total of 3 injection rounds as described I the "General Methods". Statistical Analyses. Tumor and host responses to chemotherapy were measured as described in the "General Methods". Mice that were still alive 70 d following the first round of chemotherapy were considered censored for survival probability analyses as described in the "General Methods". Tumor growth rates for tumor cell-injected, drug vehicle-treated mice, 76 body weight loss and TGD were analyzed by ANOVAs for the factors of Group and Days, with Days treated as a repeated measures factor; significant effects were further analyzed by Tukey's post-hoc tests. RESULTS Tumor growth rates for tumor-bearing, drug vehicle-treated mice were similar for I and G housed mice from 0 d until approximately 14 d following tumor cell injection when mean tumor weights of I and G mice reached approximately 1 g; at which time experimental housing conditions (II, IG, GG and Gl) were formed (Fig. 4). Analysis of tumor growth rates in II, IG, GG, and Gl mice revealed significant main effects of housing condition [Group; F(3,46)=2.781, p=0.05] and Days [F(2,92)=241.660, p<0.001] as well as a significant Group x Days interaction [F(6,92)=2.395, p<0.05; Fig. 5]. Post-hoc analysis indicated that 5 d following formation of experimental housing conditions (approximately 19 d post tumor cell injection) tumor growth rates were significantly faster in II mice compared to either IG or GG mice (II>IG, p<0.001; II>GG, p<0.01; Fig. 5). Gl mice had intermediate tumor growth rates that did not differ significantly from those of mice in the other experimental housing conditions (Fig. 5). Although there were no significant differences in survival probabilities in tumor-bearing, drug vehicle-treated mice, mice with the fastest (II) and the slowest (IG) tumor growth rates showed a difference in survival probability that approached significance (II < IG; x^=3.243, p=0.072; Fig. 6A). For body weight loss, analysis of the main effect of Group [F=(3,48)=4.207, p=0.01] revealed that both II and IG mice lost significantly more weight than GG mice (II=IG>GG; p'sO.05), suggesting that body weight loss did not relate to tumor growth rate. Weight loss for Gl mice did not differ significantly from that of mice in any other housing condition (Table 2). 77 As expected, overall survival probability was greater for tumor-bearing, chemotherapy-treated mice than for tumor-bearing, drug vehicle-treated mice (TOTV; x2=57.778, pO.OOl; Fig. 6A), as well as for non-tumor-bearing than for tumor-bearing, chemotherapy-treated mice (NTOTC; x2=32.561, pO.OOl; Fig. 6B), regardless of experimental housing condition. Contrary to our previous data (Kerr et al., 1997), social housing condition did not influence the overall survival probabilities among tumor-bearing, chemotherapy-treated mice. However, the data revealed that social housing condition significantly affected both tumor and host responses. Analysis of tumor responses to chemotherapy, measured by TGD, revealed a significant effect of Group [F(3,159)=3.624, p=0.01]; Gl mice had a significantly longer TGD than II mice (GI>II, p<0.01) and a marginally longer TGD than IG mice (p=0.098; Table 3). Similarly, analysis of host response to chemotherapy, measured by percent body weight loss over the duration of chemotherapy treatment, revealed a significant main effect of Group [F(3,35)=4.170, p=0.01]; Gl mice lost significantly less weight compared to IG (GKIG; p<0.05) and marginally less weight compared to II mice (p=0.07; Table 2). For GG mice, both TGD and percent body weight change did not differ significantly from those of mice within the other social housing conditions. Gross autopsy examinations performed on mice found dead in their home cages 1-70 days following the initiation of chemotherapy (presumably due to the toxic effects of chemotherapy) revealed that compared to drug vehicle-treated mice, both tumor-bearing and non-tumor-bearing, chemotherapy-treated mice had dark red to almost black livers, dark and distended intestinal tracts, red lungs, and dark red to black colored hearts. The extent of discoloration/damage to these organs varied among animals and appeared to be similar among tumor and non-tumor-bearing mice. Due to the fact that autopsies were performed only on a 78 subset of animals, it was not possible to determine whether organ/tissue damage following chemotherapy treatment was worse for mice in one housing condition than in another. For non-tumor-bearing, chemotherapy-treated mice, overall survival probability for IG mice was significantly less than for either II or GG mice {%}=!.626, p<0.01 and %2=5.752, p<0.05, respectively) and marginally less than for GI mice (x^ =2.812, p=0.094; Fig. 6B). Similarly, analysis of body weight loss over the course of chemotherapy revealed a significant Group x Days interaction [F(3,52)=3,918, p=0.0T]; IG mice lost significantly more weight than mice in all other housing conditions (IG>II=GG=GI; p'sO.Ol; Table 2). It is possible that the decreased survival probability in non-tumor-bearing, chemotherapy-treated IG mice was due to a poor host response to chemotherapy (possibly reflecting greater toxic side-effects of chemotherapy), at least as assessed by body weight loss. D I S C U S S I O N The present study demonstrates that social housing condition significantly affects both SCI 15 tumor and host responses to chemotherapy. For tumor-bearing, chemotherapy-treated mice, both tumor response (TGD) and host response (weight loss) were better in GI than in II and IG mice. Specifically, GI mice showed significantly greater TGD than II and marginally greater TGD than IG mice, and GI mice lost significantly less weight than IG and marginally less weight than II mice. The interaction between tumor and host responses (overall survival probability) was not significantly different among mice from the different housing conditions. However, for non-tumor-bearing mice receiving chemotherapy, overall survival probability was significantly greater for II and GG and marginally greater for GI than for IG mice. Also, IG mice lost significantly more weight than II, GG, and GI mice. 79 The present data are in contrast to those of our previous study (Kerr et al, 1997) in which experimental housing conditions (IG, Gl) were formed immediately following tumor cell/tumor cell-vehicle injection (rather than 14 d later as in the present study), and chemotherapy was initiated 14-18 d following the formation of experimental housing conditions (rather than 1 d later as in the present study). Under those conditions, IG mice had a better tumor response to chemotherapy (longer TGD), a better host response to chemotherapy (less weight loss, unpublished results), and a greater overall survival probability compared to Gl mice. In addition, for non-tumor-bearing, chemotherapy-treated mice, body weight loss (unpublished results) as well as overall survival probability were similar (Kerr et al., 1997), suggesting that the toxic side effects of chemotherapy did not differ between the two housing conditions. Together, these data demonstrate that the temporal relationship between formation of experimental housing conditions and initiation of chemotherapy appears to be a critical factor in determining the effects of social housing condition on chemotherapeutic efficacy. The temporal relationship between tumor cell injection and formation of experimental housing conditions also differentially affects SCI 15 tumor growth rate (assessed in drug vehicle-treated mice). In our previous study in which social housing conditions were formed immediately following SCI 15 tumor cell injection, we found that Gl mice had significantly faster tumor growth rates than IG mice, and that II and GG mice had intermediate tumor growth rates (Grimm et al, 1996; Weinberg & Emerman, 1989). In contrast, in the present study, in which mice remained in their original I or G housing conditions following tumor cell injection, and experimental housing conditions were formed approximately 14 d later, II mice had significantly faster tumor growth rates than both IG and GG mice, whereas Gl mice had intermediate tumor growth rates. 80 The differential effects of social housing condition on both tumor growth rates and chemotherapeutic efficacy in our two experimental paradigms may also be due, in part, to the differential timing of acute daily novelty stress relative to formation of experimental housing conditions, initiation of chemotherapy and/or tumor cell injection. Although in both studies novelty stress was initiated 1 d following formation of experimental housing conditions, in the previous study this occurred 1 d after tumor cell/tumor cell-vehicle injection and approximately 13-17 d before initiation of chemotherapy, whereas in the present study this occurred approximately 15 d after tumor cell/tumor cell-vehicle injection and concurrently with the initiation of chemotherapy. Previous data have demonstrated that the timing of a stressor relative to tumor cell injection or tumor induction (e.g. DMBA- or NMU-induced tumors) can significantly affect tumor growth rates or tumor counts (Justice, 1985; Sklar & Anisman, 1981) as well as endocrine levels and/or immune activity (Anisman et al, 1989; Hoffman-Goetz et al, 1992; Riley, 1981). Modifying influences of hormone levels and/or immune activity at the level of both the tumor and the host have been shown to alter tumor growth rates as well as the cytotoxic effects and/or toxic side effects of chemotherapeutic agents (Emerman & Siemiatkowski, 1984; English et al, 1982; English et al, 1991; Grossarth-Matiecek & Eysenck, 1989; Hengst et al, 1981; Kandil & Borysenko, 1988; Omukai et al, 1983; Riley, 1981; Spiegel et al, 1998; Vogel & Bower, 1991). Therefore, it is possible that the timing of experimental variables in relation to each other (formation of experimental housing conditions, daily novelty stress, tumor cell injection and/or initiation of chemotherapy) differentially altered physiological profiles (e.g. endocrine levels or immune activity) and thus played a role in mediating the differential tumor growth rates as well as the differential tumor responses to 81 chemotherapy between our present and previous (Kerr et al, 1997) studies. This possibility requires further investigation. Our data further suggest that tumor growth rate at the time of chemotherapy initiation, at least when the tumors are of similar weight, does not play an important role in determining tumor response to chemotherapy. In our previous study, although tumors were of similar weights (1 g) at the time of chemotherapy initiation, tumor growth rate was faster in GI than in IG mice. Under those conditions, IG mice had a better tumor response to chemotherapy (longer TGD) compared to GI mice (Kerr et al, 1997). In the present study, in which mice remained in their I or G housing conditions following tumor cell injection, chemotherapy was also initiated when tumors were 1 g in weight but were growing at similar rates. In contrast to our previous data, GI mice had a longer TGD compared to II mice and a marginally longer TGD compared to IG mice. Moreover, it appears that the differential tumor growth rates observed in drug vehicle-treated mice following the formation of experimental housing conditions cannot be used to predict tumor response to chemotherapy. In our previous study, mice that had the slowest tumor growth rates had the best tumor response to chemotherapy (TGD) whereas in the present study, mice that had intermediate tumor growth rates (GI) had the best tumor response to chemotherapy (longer TGD). The mechanisms underlying the differential SCI 15 tumor responses to chemotherapy are unknown at present. Psychosocial stressor-induced and/or chemotherapy-induced changes in endocrine function may be involved. We have shown previously that for male mice in our standard laboratory housing condition (group housed and not subjected to daily novelty stressors), SCI 15 tumor response to AD and CY can be modulated by altering the level of exogenous testosterone administered following castration (Emerman & Siemiatkowski, 1984). 82 It has also been shown that the anti-tumor effects of CY on ascitic Ehrlich tumors in mice can be suppressed by increased activity of endogenous or exogenous corticosterone through acceleration of drug metabolism (Kodama & Kodama, 1982). Furthermore, we have demonstrated that basal plasma testosterone and corticosterone levels are significantly altered among mice in the different experimental housing over the first 7 d following tumor cell injection and formation of experimental housing conditions; basal testosterone levels were higher in GI than in IG mice whereas basal corticosterone levels were higher in IG mice than in mice in all other housing conditions (Rowse et al, 1992). Therefore in the present study, altered hormone profiles among mice in the different housing conditions at the time of chemotherapy initiation may have differentially affected tumor responses to chemotherapy. We are currently investigating this possibility. Differential tumor response to chemotherapy may also be mediated through changes in immune function. Such changes may occur either directly through chemotherapy-induced changes in immune function or indirectly through psychosocial stressor-induced changes in hormonal activity that in turn alter immune function. We have shown that the SCI 15 tumor differentially stimulates NK cell activity in mice the different housing conditions at 7 d following tumor cell injection and formation of experimental housing conditions (Rowse et al, 1995). In addition, preliminary evidence from our laboratory suggests that the SCI 15 tumor stimulates a tumor-specific cytolytic immune response (unpublished data). Several studies have shown that chemotherapy treatment is optimized when combined with an increase in immune activity (Giraldi et al, 1992; Grossarth-Maticek & Eysenck, 1989; Hengst et al, 1981; Levy et al, 1987; North, 1984; Rosenberg et al, 1986). Thus, differential immune activity in mice in the different experimental housing conditions could alter tumor response to chemotherapy. 83 Alternatively, chemotherapy in itself may differentially affect immune activity of mice in the different housing conditions thereby altering tumor response to chemotherapy. In animal studies, both AD and CY have been shown to affect the immune response (Abdul Hamied & Turk, 1987; Kopreski, 1996; Martin*?/al, 1975). In the present study, percent body weight change over the duration of chemotherapy in both tumor-bearing and non-tumor-bearing, chemotherapy-treated mice was used to analyze the general host response to chemotherapy. Body weight change has been shown to influence the effectiveness of chemotherapy (DeWys et al, 1980; DeWys, 1982; Fichtner & Tanneberger, 1987; Sullivan et al, 1995), possibly through alterations in hormone (e.g. glucocorticoids, insulin) levels that may modulate, directly or indirectly, the activity of drug metabolizing enzymes (Boddy & Idle, 1993; Chabot, 1994; Desoize & Robert, 1994; Heber et al, 1992; Kopreski, 1996; Shepherd & Harrap, 1983) or change the growth kinetics of tumor cells (English et al, 1991; Hug et al, 1986). Similar to other studies (Bellinson et al, 1984; Looney et al, 1980; Sullivan et al, 1995), the present study demonstrated that body weight loss over the duration of chemotherapy was inversely proportional to the tumor response to chemotherapy; Gl mice lost less weight and had a better tumor response to chemotherapy than mice in other housing conditions. Similarly, for non-tumor-bearing, chemotherapy-treated mice, body weight loss was related to a reduction in survival probability; IG mice lost the most weight and had the lowest survival probability compared to mice in other housing conditions. Interestingly, although significant differences in body weights over the course of chemotherapy were observed among tumor-bearing, chemotherapy-treated mice in the different experimental housing conditions, no significant differences in survival probabilities were observed. These data demonstrate that although social housing condition can affect both tumor and host 84 responses to chemotherapy as well as survival probability, the interaction among tumor, host, and chemotherapy is complex. In summary, our data highlight the possible impact of social housing condition on the complex interrelationship among the host environment, tumor growth, and chemotherapeutic efficacy. Furthermore, our data suggesting that the temporal relationship between psychosocial events and chemotherapy initiation is critical in determining the effectiveness of chemotherapy may have potential clinical relevance. 85 Fig. 4. Experimental Design Male DD/S mice 2-4 mo of age reared either individually (I) or in groups (G) 2 x 106 SCI 15 tumor cells injected s.c. and mice remain in their rearing conditions (I or G) Experimental housing conditions (II, IG, GG, GI) formed when tumor weights of mice within a rearing condition (I or G) reached a mean of 0.8 ± 0.2 g; approximately 14 d following tumor cell injection Tumor cell-vehicle injected s.c. and mice remain in their rearing conditions (I or G) Experimental housing conditions (II, IG, GG, GI) formed approximately 14 d following tumor cell-vehicle injection Chemotherapy or drug vehicle regimen and exposure to acute daily novelty stress (15 min/d, 5 d/wk) initiated 1 d following formation of experimental housing conditions (II, IG, GG, GI); approximately 15 d after tumor cell injection Mice sacrificed 70 d following chemotherapy/drug vehicle initiation or when tumor weight exceeded 3.5 g 87 Fig. 5. Tumor growth in drug vehicle-treated mice. Arrow represents formation of experimental housing conditions. Tumor weights (mean ± SEM) over 5 measurement times for drug vehicle-treated mice in the 4 experimental housing conditions (n=12 II, n=19 IG, n=12 GG, n=14 GI). Tumor growth rate by 19 d post tumor cell injection was significantly greater in II compared to GG (pO.OT) and IG (pO.001) mice. 88 6. Survival probability in A: tumor-bearing mice receiving either chemotherapy (TC) or drug vehicle (TV) and B: non-tumor-bearing mice receiving chemotherapy (NTC). Day 0 is the day of initiation of chemotherapy (AD at 4.0 mg/kg and CY at 61.5 mg/kg) or drug vehicle administration. The first symbol represents the time at which the first death occurred in each housing condition. A: Tumor bearing mice. For mice receiving drug vehicle, survival probability was marginally greater for IG compared to II mice (X2=3.243, p=0.072). For mice receiving chemotherapy, no significant differences were observed among the different housing conditions. As expected, chemotherapy-treated mice (n=9 II, n=23 IG, n=13 GG, n=16 GI) had significantly greater overall survival probability than drug vehicle-treated mice (n=12 II, n=19 IG, n=12 GG, n=14 GI; x2=57.778, pO.OOl). B: Non-tumor-bearing mice. For mice receiving chemotherapy (NTC; n=12 II, n=20 IG, n-15 GG, n=ll GI), survival probability was significantly lower in IG mice than in II (x2=7.626, p<0.01) and GG (X2=5.752, p<0.05) mice, and marginally less than in GI mice (x2=2.812, p=0.094). As expected, for all chemotherapy-treated mice, non-tumor-bearing mice had significantly higher survival probability than tumor-bearing mice (X2=32.561,p<0.001). 90 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Days post chemotherapy or drug vehicle initiation 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Days post chemotherapy initiation 91 Table 2 . Percent body weight lossa (mean ± SEM) in mice over the course of chemotherapy or drug-vehicle treatment TCb NTC TV Housing Condition C1-C2 C1-C3 C1-C2 C1-C3 C1-C2 II -13.38 ± 1.66 (9) -19.72 ± 3.18 c -8.22 ±0.71 (12) -14.28 ±0.84 -3.70 ± 1.02 e (12) IG -12.00 ±0.95 (23) -16.78 ± 1.55 c -12.96 ±0.83 (20) -17.50 ±0 .80^ -3.64 ± 1.22 e (19) GG -10.00 ± 1.70 (13) -13.68 ±2.83 4.12 ±0.87 (15) -11.84 ± 1.37 2.05 ± 0.60 (12). Gl -5.22 ± 1.20 (16) -12.29 ± 1.96 -4.93 ± 1.00 01) -12.01 ±2.43 -0.41 ± 1.78 (14) a Percent body weight loss from first (Cl) to second (C2) or third (C3) chemotherapy injection rounds = ([body weight at C2 or C3 minus body weight at Cl] / [body weight at Cl]) x 100, where body weight = gross body weight minus tumor weight; negative values indicate weight loss between chemotherapy rounds. Numbers in parentheses, n per group; b TC: tumor-bearing, chemotherapy-treated mice, NTC: non-tumor-bearing, chemotherapy-treated mice, TV: tumor-bearing, drug vehicle-treated mice; c IG>GI; p<0.05, II>GI; p=0.07; d IG>II=GG=GI; p'sO.Ol; e II=IG>GG, p'sO.05 92 Table 3. TGD a in days (mean ± SEM) in chemotherapy-treated mice Tumor weights (g) Housing Condition 1.5 2.0 2.5 3.0 II 0.75 ± 0.39 (8) 1.33 ±0.56 (8) 2.43 ±0.91 (7) 5.67 ±2.11 (9) IG 4.00 ± 1.13 (16) 4.15 ± 1.59 (13) 5.18 ±0.77 (11) 5.00 ±2.03 (6) GG 0.50 ±0.57 (10) 8.67 ±5.27 (9) 8.30 ±4.15 (10) 10.38 ±4.81 (8) GlA c 6.19 ±3.22 (16) 7.53 ±2.98 (16) 9.38 ±3.08 (16) 12.40 ±4.24 (11) a TGD is defined as the mean time, in days, for tumors to reach a specific weight in chemotherapy-treated mice minus the mean time for tumors in drug vehicle-treated mice to reach the same weight. Numbers in parentheses, n per group; b GI>II; p=0.01; c GI>IG; p=0.098 CHAPTER 5. EFFECTS OF SOCIAL HOUSING CONDITIONS ON SCI 15 TUMOR AND HOST RESPONSES TO ADJUVANT CHEMOTHERAPY INTRODUCTION In humans, stressful life events and the ability to cope with stress may play a role not only in cancer progression or metastasis but also in the effectiveness of treatments for this disease (Fieler et al, 1995; Heim & Schaffner, 1997; Hislop et al, 1987; Leigh et al, 1987; Redd et al, 1991; Spiegel, 1997; Spiegel et al, 1989). Relevant to this study, adjuvant chemotherapy has been shown to extend significantly both disease-free and overall survival time of cancer patients (Bonadonna, 1989). However, unexplainable differences in the toxic side effects experienced by patients receiving the same chemotherapeutic protocols have been observed (Boddy & Idle, 1993; Chabot, 1994). Due to increased toxic side effects of chemotherapy in some patients, drug treatment is halted, drug dose is reduced, or patient non-compliance to the treatment regimen occurs, all of which may decrease tumor responses to chemotherapy and possibly increase the probability of cancer progression and decrease survival probability (Bonadonna, 1989; Henderson et al, 198 ; Richardson et al, 1990; Spiegel, 1997; Wood et al, 1994). Psychosocial stressors or demands, as well as the coping resources used by the patient, may play a role in the unpredictable and highly variable incidences of the toxic effects of chemotherapy (Fieler et al, 1995; Heim et al, 1997; Hurny et al, 1996; Ramirez et al, 1989; Shapiro etal, 1997; Spiegel, 1997). The complex relationships between psychosocial stressors and the progression of cancer and/or chemotherapeutic efficacy are difficult to investigate in humans. Although the data in animals also are complex, animal models provide a way to investigate the relationship among stressors, tumor growth and chemotherapeutic efficacy under more controlled conditions. In 94 animals, psychosocial stressors such as change in social housing condition and psychological stressors such as forced restraint and rotation have been shown to affect both tumor growth rates or metastasis and both tumor and host responses to chemotherapy (Giraldi et al, 1992; Kerr et al ,1997; Perissin et al, 1991; Perissin et al., 1997). We have developed an animal-tumor model utilizing the transplantable, androgen-responsive Shionogi mouse mammary carcinoma (SCI 15; Ref. Weinberg & Emerman, 1989). We have shown that a change in social housing condition can significantly influence tumor growth rate as well as both tumor and host responses to chemotherapy and overall survival probability (Grimm et al, 1996; Kerr et al, 1997; Weinberg & Emerman, 1989). Moreover, data from our two previous studies (Chapters 3 and 4 of this thesis) demonstrate that the temporal relationship between formation of experimental housing condition and initiation of chemotherapy can differentially alter the effects of social housing condition on both the tumor and host responses to chemotherapy (Kerr et al, 1997; Kerr et al, 1999a). The present study is the last in a series of three studies examining the effects of varying temporal relationships among tumor cell injection, formation of experimental housing conditions and initiation of chemotherapy on tumor and host responses to chemotherapy. The results from our first study demonstrated that if experimental housing conditions are formed immediately following tumor cell injection, and chemotherapy is initiated approximately 14 d later, when tumors weigh approximately lg and are growing at different rates, IG mice (slower tumor growth rate) have a better tumor response to chemotherapy (longer TGD), better host response to chemotherapy (lost less weight) and a greater overall survival probability compared to Gl mice (faster tumor growth rate; Ref. Kerr et al, 1997). In addition, for non-tumor-95 bearing, chemotherapy-treated mice, host response in terms of body weight loss and survival probability are similar for IG and GI mice (Kerr et al., 1997). In contrast, the study in Chapter 4 (Kerr et al., 1999a) showed that if experimental housing conditions are formed approximately 14 d following tumor cell injection and chemotherapy is initiated 1 d later, when tumors weigh approximately lg and are growing at similar rates, GI mice have better tumor (longer TGD) and host (less weight loss) responses to chemotherapy compared to both II and IG mice, yet no differences in survival probabilities are observed among experimental housing conditions. However, for non-tumor-bearing, chemotherapy-treated mice, IG mice have the poorest host response (greater weight loss) and the lowest survival probability compared to mice in all other housing conditions. Together, these data indicate that chemotherapeutic efficacy is influenced by the temporal relationship between formation of experimental housing condition and initiation of chemotherapy but not by tumor growth rate at the time of chemotherapy initiation. The present study was designed to examine whether initiating chemotherapy 1 d following tumor cell injection and formation of experimental housing conditions, a time when tumor burden is undetectable (similar to an adjuvant situation in humans), differentially influences the effects of social housing condition on tumor and host responses to chemotherapy. METHODS Tumor Propagation. Tumor cells were prepared and injected as described in "General Methods". Experimental Animals. Male DD/S mice (n=113) between the ages of 2-4 months of age were the experimental subjects in this study. Mice were reared and housed as described in the "General Methods". The II housing condition was not used in the present study since, under 96 experimental conditions where tumor cells are injected and social housing conditions are formed concurrently, we have shown tumor growth rate and hormone levels in II mice are similar to those in GG mice (Grimm et al, 1996; Rowse et al, 1992; Weinberg & Emerman, 1989). In the interest of the number of mice used, the II condition was eliminated from this study. Mice within each housing condition were randomly assigned into tumor cell injection groups receiving either chemotherapy (TC; n= 9 GG, n= 14 Gl, n=20 IG) or drug vehicle (TV; n=6 GG, n=8 Gl, n-10 IG), or into tumor cell-vehicle injection groups receiving chemotherapy (NTC; n=l 1 GG, n=15 Gl, n= 20 IG; Fig. 7). Beginning the day following tumor or tumor cell-vehicle injection, all animals were exposed to an acute daily stressor. Body and tumor weights were measured every second day as described in the "General Methods". Chemotherapy. Chemotherapy (see "General Methods") was administered i.p. every 7 d for a total of 3 injection rounds. Statistical Analyses. No tumor growth was observed in tumor cell-injected, chemotherapy-treated mice. Therefore, tumor response to chemotherapy was not analyzed. Host response to chemotherapy was analyzed as described in the "General Methods". Mice that were still alive 70 d following the first round of chemotherapy were considered censored for survival probability analyses. Tumor growth rate in drug vehicle-treated mice and body weight loss were analyzed by ANOVAs for the factors of Group and Days with Days treated as a repeated measures factor; significant effects were further analyzed by Tukey's post-hoc tests. RESULTS Consistent with the data from the study in Chapter 3 in which social housing conditions were formed immediately following SCI 15 tumor cell injection, analysis of tumor growth rate 97 in drug vehicle-treated mice in the present study revealed a significant Group x Days interaction (F[8,72]=24.209, pO.OOl; Fig. 8). Tukey's post hoc analysis indicated that on days 15 and 17 following tumor cell injection and formation of experimental housing conditions, tumor growth rates were significantly faster in both GI and GG mice compared to IG mice (GI=GG>GI; p'sO.001). Consequently, survival probability was also greater in IG mice compared to both GI and GG mice (IG>GI=GG; %2=\2A2 and 20.18, respectively, p'sO.001; Fig. 9). Analysis of the percent body weight loss among mice in the different housing conditions similarly revealed a significant Group x Days interaction [F(2,14)=3.629, p=0.05], probably reflecting the somewhat greater initial weight loss in IG and GG mice compared to GI mice. However, post hoc analyses failed to reach significance. For tumor cell-injected, chemotherapy-treated mice, tumor response to chemotherapy was similar among mice in all experimental housing conditions; no tumors were palpable for up to 70 d after chemotherapy initiation. Similarly, no significant differences in overall survival probability were observed among mice in the different experimental housing conditions. However, analysis of the effects of social housing condition on weight loss over the course of chemotherapy revealed significant effects of both Group (F[2,32]=10.760, pO.OOl) and Days (F[2,32]=312.936, pO.OOl). IG mice lost significantly more weight than both GI and GG mice (IG>GI=GG, p'sO.01). Overall, body weight loss in all housing conditions was significantly greater at the third compared to the second chemotherapy round (pO.OOl; Table 4). Autopsies performed on a random subset of mice found dead in their home cages between 1 -70 days following the initiation of chemotherapy revealed that similar to the study in Chapter 4, chemotherapy treatment caused the intestinal tract, liver and heart to look discernibly darker compared to those in drug vehicle-treated mice. The degree of discoloration 98 as well as the number of discolored organs appeared to be greater following subsequent chemotherapy rounds. Overall, however, the extent of discoloration/damage to organs varied among animals and appeared to be similar among tumor and non-tumor-bearing mice. Also, due to the random nature in which the autopsies were performed, it was not possible to determine whether organ/tissue damage following chemotherapy treatment was worse for mice in one housing condition than in another. For non-tumor cell-injected, chemotherapy-treated mice, survival probability was significantly less for IG mice than for GI mice (IG<GI; x2=4.588, p<0.05; Fig. 9). Survival probability for GG mice did not differ significantly from that of IG and GI mice. Analysis of body weight loss over the course of chemotherapy revealed significant main effects of Group [F(2,43)=18.055, p<0.001] and Days [F(l,43)=97.661, pO.OOl] as well as a Group x Days interaction [F(2,43)=4.332, pO.05]. Similar to the results in their tumor cell-injected counterparts, non-tumor cell-injected, chemotherapy-treated IG mice lost significantly more weight at each chemotherapy treatment interval compared to both GI and GG mice (IG>GI, GG; p's=0.001), and the percentage of body weight lost in all housing conditions was significantly greater at the third compared to the second chemotherapy round (pO.OOl; Table 4). In addition, GI mice lost significantly more weight than GG mice (pO.Ol) from the initiation of chemotherapy to the third (final) chemotherapy round. As expected, survival probability for chemotherapy-treated mice was significantly greater than for drug vehicle-treated mice (x2=38.371, pO.OOl; Fig. 9) and tumor cell-injected mice treated with chemotherapy lost significantly more weight than those receiving drug vehicle, regardless of treatment interval examined (p'sO.001; Table 4). In addition, for mice treated with chemotherapy, tumor cell-injected mice lost significantly more weight than non-99 tumor cell-injected mice, regardless of experimental housing condition or treatment interval examined (p's<0.05). However, no significant differences in survival probabilities were observed between tumor cell-injected and non-tumor cell-injected mice receiving chemotherapy. D I S C U S S I O N The present study demonstrates that when chemotherapy is initiated 1 d after SCI 15 tumor cell injection and formation of social housing conditions, a time when tumor burden is undetectable (similar to the adjuvant situation in humans), tumor response to chemotherapy is similar among mice in all housing conditions; no tumor masses are palpable over the duration of the experiment (up to 70 d post chemotherapy initiation). In contrast, host responses to chemotherapy including both morbidity (body weight loss) and mortality (survival probability) are differentially affected by housing condition. Our previous two studies demonstrated that if chemotherapy is initiated when tumor burden is relatively large (approximately 1 g), social housing condition differentially influences tumor response to chemotherapy (Kerr et al.,. 1997; Kerr et al., 1999a), and importantly, that the effects of social housing condition are dependent on the temporal relationship between formation of social housing condition and the initiation of chemotherapy. In contrast, the present study demonstrates that if chemotherapy is initiated when tumor burden is undetectable, social housing condition does not influence tumor response to chemotherapy. This is not unexpected as it is recognized that when tumor burden is small, there is a greater chance for drugs to eradicate all tumor cells, possibly due to a closer proximity of the blood supply to the tumor cells and thus greater exposure of the cells to cytotoxic drugs, increased growth fraction 100 of tumor cells, and/or to a reduced probability that alterations in host physiological profiles (e.g. endocrine and/or immune activity) will influence the development of tumor cell populations resistant to chemotherapy (Heppner, 1989; Heppner, 1993; Miller et al, 1993). In terms of host response to chemotherapy, the present study demonstrates that rehousing mice from individual to group conditions (IG) 1 d prior to chemotherapy initiation can significantly increase body weight loss (morbidity) and/or decrease survival probability (mortality) compared to that of mice in both GG or GI housing conditions. Interestingly, these data are consistent with those of the study in Chapter 4 in which experimental housing conditions also were formed 1 d before initiation of chemotherapy, but when tumors were approximately 1 g (Kerr et al, 1999a). Importantly, these data are in contrast to those of our first study in Chapter 3, in which the formation of experimental housing conditions occurred 14-18 d before the initiation of chemotherapy and at a time when tumors were approximately 1 g. Under those conditions, morbidity and mortality were significantly lower in IG compared to GI mice (Kerr et al, 1997). Together, these studies demonstrate that the temporal relationship between formation of social housing conditions and initiation of chemotherapy is critical in determining the influence of social housing condition on the host response to chemotherapy and that the direction of these effects is predictable, regardless of tumor burden. Human and animal studies have shown that severe body weight loss over the course of chemotherapy can potentiate treatment-related toxicity and consequently, may decrease survival probability (Bassukas & Maurer-Schultze, 1993; DeWys, 1982; Ottery, 1994; Roth et al, 1988). For example, mice that lost more than 20% of their pre-treatment (300 mg/kg cyclophosphamide i.p.) body weight were shown to have an increased risk of dying of the toxic effects of chemotherapy (Bassukas & Maurer-Schultze, 1993). Both this and the previous study 101 in Chapter 4, in which chemotherapy was initiated 1 d following the formation of experimental housing conditions demonstrate that, regardless of tumor burden, social housing condition differentially affects body weight loss over the course of chemotherapy, suggesting possible differences in the degree of toxic effects of chemotherapy among mice in the different experimental housing conditions. Moreover, in both of these studies, for all chemotherapy-treated mice (tumor- and non-tumor cell-injected), mice in the IG condition showed the greatest weight loss (an average loss of 18.0 ± 1.3%) over the course of chemotherapy compared to mice in all other housing conditions. These data are similar to other studies demonstrating that being group-housed or rehoused into a larger group may increase drug toxicity. For example, the lethal dose of methamphetamine was over 7 fold lower in group-housed compared to individually housed rats and rehousing rats from 5 to 10 per cage significantly increased drug-induced mortality rate by over 4 fold (cf. Vogel, 1993). The increased weight loss, reduced survival probability and possibly increased toxic effects of chemotherapy experienced by IG mice in the present and preceding studies may be influenced by the increased fighting that occurs when the IG condition is formed. Previously, we have shown that 1 d following formation of experimental housing conditions, fighting and defensive behaviors are significantly increased in IG mice compared to mice in all other housing conditions (Grimm et al, 1996). In mice that are not treated with chemotherapy, we (Grimm et al, 1996; Weinberg & Emerman, 1989) and others (Sklar & Anisman, 1980) have suggested that fighting may represent a form of coping response and this may play a role in reducing tumor growth rate and possibly increasing chemotherapeutic efficacy in established social groups (Kerr et al, 1997). However, the initial physiological effects of fighting on the day of chemotherapy initiation may play a role in increasing the probability of weight loss and 102 toxic effects of chemotherapy and decreasing survival probability. A single experience with a stressor in the form of social defeat has been shown to have marked physiological consequences lasting from hours up to weeks (Koolhaas et al, 1997). The submissive, defeat reaction in mice and rats is characterized by an increase in corticosterone secretion, together with impaired production of sex steroids (e.g. testosterone) and suppressed immune function (Koolhaas et al, 1997; Michelson et al, 1994). Consistent with these findings, we have demonstrated that up to 7 d following formation of experimental housing conditions, IG mice have higher basal levels of corticosterone, lower basal levels of testosterone, and reduced natural killer cell activity compared to mice in the other social housing conditions (Rowse et al, 1992; Rowse et al, 1995). In addition, we and others have demonstrated that for mice allowed to adapt to new social housing conditions, the impact of the change in social housing condition on endocrine and/or immune functions is reduced (Grimm et al, 1996; Hoffman-Goetz et al, 1992; Koolhass et al, 1997). Importantly, stressor-induced changes in hormones and cytokines may influence the toxic side effects of drugs, possibly including chemotherapeutic agents (Bassukas & Maurer-Schultze, 1993; Guideri et al, 1974; Harvey et al, 1994; Kodama & Kodama, 1982; Kopreski, 1996; Vogel, 1993; Wood & Hrushesky, 1996). Interestingly, the present and previous studies demonstrate that if chemotherapy is initiated 1 d following the formation of experimental housing conditions (at a time when hormone and immune activity is differentially altered among social housing conditions; Ref. Rowse et al, 1992; Rowse et al, 1995), IG mice lose more weight and/or have a lower survival probability compared to Gl or GG mice. Conversely, if chemotherapy is initiated 14-18 d following formation of social housing condition (at a time when hormone and immune activity may be similar among social housing conditions), mice in the IG housing 103 condition lose less weight and have a higher survival probability compared to G l mice (Kerr et al, 1997). By 14 d following the initiation of chemotherapy, social hierarchies among mice in the IG housing condition have become established and mice have adapted to the new housing condition, as evidenced by the reduction in fighting among mice within the IG housing condition (Grimm et al, 1996). Thus, different physiological profiles may exist between IG mice rehoused 1 d, compared to those rehoused 14 d, prior to initiation of chemotherapy, and as a consequence, differential tumor and/or host responses to chemotherapy were observed. Although the mechanisms remain to be elucidated, housing-induced changes in endocrine and/or immune activity may play a role in influencing the differential effects of social housing condition on chemotherapeutic efficacy. In summary, the present study, together with our previous studies (Chapters 3 and 4 of this thesis), demonstrate that social housing conditions can significantly influence chemotherapeutic efficacy, and highlight the importance of the temporal relationship between formation of social housing conditions and initiation of chemotherapy on chemotherapeutic efficacy. Specifically, these studies show that i f chemotherapy is initiated at least 14 d after formation of social housing conditions and tumor cell injection, a time when tumors are relatively large (1 g) and growing at different rates, chemotherapy is more effective in mice that were reared individually then group-housed (IG) than in mice that were reared in groups and then individually housed (Gl; Ref. Kerr et al, 1997). In contrast, our second study demonstrated that i f chemotherapy is initiated when 1 d after formation of social housing conditions and at least 14 d following tumor cell injection, a time when tumors also weigh 1 g but are growing at similar rates, tumor and host responses to chemotherapy are the poorest for IG mice compared to mice in all other housing conditions (Kerr et al, 1999a). Finally, we 104 found that if chemotherapy is initiated 1 d after formation of social housing conditions and tumor cell injection, a time when tumor burden is undetectable, social housing condition does not affect tumor response to chemotherapy, but IG mice lose the most weight and have a lower survival probability compared to mice in all other social housing conditions. Importantly, these studies demonstrate that the effects of social housing conditions on chemotherapeutic efficacy appear to be independent of tumor growth rate at the time of chemotherapy initiation and, at least in terms of host response to chemotherapy, are also independent of tumor burden. These studies demonstrate the complex effects of social housing condition on both the tumor and host responses to chemotherapy. Although it is difficult to extrapolate from the animal to the human situation, the present data may help to emphasize the role that psychosocial stressors may play in the unpredictable and highly variable differences in the toxic effects of chemotherapy in humans. 105 Fig. 7. Experimental design. Male DD/S mice 2-4 mo. of age reared either individually (I) or in groups (G) 2 x 106 SCI 15 tumor cells injected s.c. and experimental housing conditions (IG, GG, GI) formed Chemotherapy or drug vehicle regimen and exposure to acute daily novelty stress (15 min/d, 5 d/wk) started 1 d following SCI 15 tumor cell injection and formation experimental housing conditions • Tumor cell-vehicle injected s.c. and experimental housing conditions (IG, GG, GI) formed Chemotherapy regimen and exposure to acute daily novelty stress (15 min/d, 5 d/wk) started 1 d following tumor cell-vehicle injection formation of experimental housing conditions • Mice sacrificed 70 d following chemotherapy drug vehicle initiation or when tumors exceeded 3.5 g 107 Fig. 8. Tumor growth in drug vehicle-treated mice. Tumor weights (means ± SEM) over 5 measurement times for drug vehicle-treated mice in the experimental housing conditions (n=10 IG, n=6 GG, n=8 Gl). Tumor growth rates for both days 15 and 17 were significantly faster in both Gl and GG compared to IG mice (p'sO.001). 108 Fig. 9. Survival probability in A: tumor cell-injected mice receiving chemotherapy (TC) or drug vehicle (TV) and B: in non-tumor cell-injected mice receiving chemotherapy (NTC). Day 0 is the day of initiation of chemotherapy (AD at 4.0 mg/kg and CY at 61.5 mg/kg) or drug vehicle administration. The first symbol represents the time at which the first death(s) occurred in each condition. A: For tumor cell-injected mice receiving drug vehicle, survival probability was greater in IG mice compared to either Gl or GG mice (%2 =12.42 and 20.18 respectively; p's<0.001). For tumor cell-injected mice receiving chemotherapy, no significant differences in survival probability was observed among the different housing conditions. As expected, chemotherapy-treated mice (TC; n= 9 GG, n= 14 Gl, n=20 IG) survived significantly longer compared to drug vehicle-treated mice (TV; n=6 GG, n=8 Gl, n=10 IG; x2=38.371, pO.OOl). B: For non-tumor cell-injected mice receiving chemotherapy (NTC; n=l 1 GG, n=15 Gl, n= 20 IG), survival probability was significantly lower in IG compared to Gl mice (%2 =4.588, p<0.05). No significant differences in survival probabilities were observed between tumor cell-injected and non-tumor cell-injected, chemotherapy-treated mice. 110 10 15 20 25 30 35 40 45 50 55 60 65 70 Days post chemotherapy or drug vehicle initiation 10 15 20 25 30 35 40 45 50 55 60 65 70 Days post chemotherapy initiation 111 Table 4. P e r c e n t b o d y w e i g h t l o s s 0 ( m e a n ± S E M ) i n m i c e o v e r the c o u r s e o f c h e m o t h e r a p y o r d r u g v e h i c l e t rea tment H o u s i n g T C * N T C T V C o n d i t i o n C 1 - C 2 C 1 - C 3 C 1 - C 2 C 1 - C 3 C 1 - C 2 C 1 - C 3 I G -12.58±1.27 -19 .33 ± 1 . 3 6 c - 12 .08 ± 1.20 -19 .00 ± 1 .40^ - 3 . 7 8 + 1 . 3 2 -2 .84 ±3.04 (15) (20) (5) G G -5 .99 ±1.23 -11 .96 ± 1.63 - 3 .32 ± 1.19 -6 .39 + 2.02 -4 .13 ±0.56 0.98 ±2.37 (9) (12) (6) G I - 6 .06 ± 0.90 - 12 .96 ± 1.22 -5 .89 ± 1.05 - 1 0 . 9 9 ± 1 .58 e 0.68 ± 1.30 -0 .17 ± 1.87 (12) (15) (6) a Percent b o d y w e i g h t change f r o m f i rst ( C I ) to second ( C 2 ) or to th i rd ( C 3 ) c h e m o t h e r a p y rounds = ( [body w e i g h t at C 2 or C 3 m i n u s b o d y w e i g h t at C I ] / [body w e i g h t at C I ] ) x 100, w h e r e b o d y we igh t = gross b o d y w e i g h t m i n u s t u m o r w e i g h t ; negat ive va lues ind icate w e i g h t loss between chemotherapy rounds. N u m b e r s in parentheses, n per g roup ; b T C : t u m o r - b e a r i n g , chemotherapy - t reated m i c e , N T C : n o n - t u m o r - b e a r i n g , chemotherapy - t rea ted m i c e , T V : t u m o r - b e a r i n g , d rug veh ic le - t reated m i c e ; c fo r C 1 - C 2 and C 1 - C 3 : I G > G I = G G ; p*s<0.01; d f o r C 1 - C 2 and C 1 - C 3 : I G > G I , G G ; p ' s O . O l ; e f o r C l - C 3 measurement interva l on l y : G I > G G ; p<0.01 112 Chapter 6. EFFECTS OF SOCIAL HOUSING CONDITIONS AND CHEMOTHERAPY ON HORMONE LEVELS IN THE SHIONOGI CARCINOMA (SCI 15) MOUSE TUMOR MODEL: TEMPORAL FACTORS INTRODUCTION In both humans and animals, exposure to a stressful event typically causes physiological changes including increased synthesis of corticosterone (CORT; Cortisol in humans), decreased synthesis of gonadal and growth hormones, and suppression of the immune response (Chrousos & Gold, 1992; Dantzer, 1991; Koolhaas et al, 1997a; Michelson et al, 1994; Rivier & Rivest, 1991). Reducing the impact of a stressful event through cognitive (e.g. perceived control over the stressful situation) or behavioral (e.g. disclosure, information seeking, expression of anger or aggression) means may reduce the stressor-induced physiological changes (Cobb, 1976; Henry, 1992; Koolhaas et al, 1997b; Steptoe, 1998). Relevant to this study, stressful events and the ability to cope with these events may influence cancer growth, metastasis and the effectiveness of cancer treatments (Baltrusch et al, 1991; Cooper & Faragher, 1993; Grossarth-Maticek & Eysenck, 1989; Heim et al, 1997; Sklar & Anisman, 1979; Spiegel et al, 1989; Temoshok, 1987; Watson & Greer, 1998). In addition, altered hormone levels have been related to increased cancer growth rates and metastasis as well as alterations in response to cancer treatments (Bernstein & Ross, 1992; Coleman, 1992; Emerman & Siemiatkowski, 1984; English et al, 1991; Hug et al, 1986; Lippman et al, 1985; Maraverich et al, 1983; Miller, 1990; Peacock et al, 1987; Rosenberg et al, 1985; Shapiro et al, 1995; Torosian & Donoway, 1991; Uchida et al, 1981). Thus, a possible mediator of the effects of a stressful life event on cancer growth, metastasis and/or treatment may be stressor-induced alterations in endocrine functioning. Few studies examining the effects of stressful events on cancer growth, metastasis or chemotherapeutic efficacy have concurrently examined stressor-induced changes in 113 endocrine levels. We have developed an animal tumor model which utilizes the transplantable, androgen-responsive Shionogi mouse carcinoma (SCI 15) to investigate the effects of social housing conditions on both tumor growth rate and chemotherapeutic efficacy. Mice are reared individually then group housed (IG), reared in groups then individually housed (Gl) or experience no change from their original rearing conditions (II or GG). Our studies have demonstrated that tumor growth rate as well as both tumor and host responses to chemotherapy (Adriamycin; AD, and cyclophosphamide; CY) are significantly influenced not only by housing conditions but also by the temporal relationship between the formation of experimental housing conditions and (a) tumor cell injection or (b) Chemotherapy initiation (Kerr et al, 1997; Kerr et al, 1999a; Kerr et al, 19996). Specifically, when housing conditions are formed immediately following tumor cell injection, Gl mice have significantly faster tumor growth rates compared to IG mice, and II and GG mice have intermediate tumor growth rates. In addition, if chemotherapy is initiated approximately 14-20 d following tumor cell injection and housing condition formation, IG mice have a better tumor response to chemotherapy (longer tumor growth delay; TGD), a better host response to chemotherapy (lost less weight) and a greater overall survival probability compared to Gl mice (Kerr et al, 1997). In contrast, if mice remain in their original rearing conditions following tumor cell injection, and experimental housing conditions are formed approximately 14 d later, II mice have significantly faster tumor growth rates compared to IG and GG mice whereas Gl mice have intermediate tumor growth rates (Kerr et al, 1999a). Under these conditions, when chemotherapy is initiated 1 d after the formation of experimental housing conditions, Gl mice have better tumor (longer TGD) and host (lose less weight) responses to chemotherapy compared to both II and IG mice, yet no 114 differences in survival probabilities are observed among mice in the different housing conditions (Kerr et al, 1999a). For GG mice, both TGD and weight loss did not differ significantly from those of mice in the other housing conditions. We have shown also that social housing conditions may differentially influence basal hormone levels in mice in the different experimental housing conditions. When experimental housing conditions are formed immediately following tumor cell injection, IG mice have elevated basal levels of CORT whereas GI mice have elevated basal levels of testosterone (T), compared to mice in the other housing conditions (Rowse et al, 1992). These changes are seen at 1-7 days but not 21 days after tumor cell injection and formation of experimental housing conditions (Rowse et al, 1992; Weinberg & Emerman, 1989). Also, in a preliminary study we have shown that growth hormone (GH) levels at 3 d post tumor cell injection and formation of social housing conditions are significantly higher in GI mice compared to GG and IG mice. Together these data suggest that alterations in basal hormone levels may play a role in mediating the differential tumor growth rates observed in our model. Data from our laboratory have also suggested that T-induced alterations in SCI 15 growth rate may differentially affect the cytotoxic effects of chemotherapeutic drugs. Following castration of mice housed in our standard laboratory housing conditions, SCI 15 tumor response to AD and CY was increased significantly by administering exogenous T at levels submaximal for tumor growth (Emerman & Siemiatkowski, 1984). Importantly, the timing of T administration relative to chemotherapy initiation may differentially influence tumor response to chemotherapy. For example, T administered before but not after CY treatment has been shown to increase the cytotoxic effects of CY (English et al, 1991). The antitumor effects of CY may also be markedly decreased by an increase in CORT levels prior to CY administration 115 (Kodama & Kodama, 1982; Shepherd & Harrap, 1982). However, an increase in CORT levels may also have beneficial effects in terms protecting the animal from the toxic effects of cytotoxic drugs (English et al, 1987; Levi et al, 1980). Finally, increased GH levels may increase tumor cell proliferation, including SCI 15 tumor proliferation (Nandi et al, 1995; Noguchi et al, 1993) as well as reduce chemotherapy-induced decreases in immune activity and body weight (Harrison et al, 1995; Ng et al, 1993; Wolf et al, 1994) and therefore may play a role in augmenting chemotherapeutic efficacy. Since CORT, T and GH levels influence SCI 15 growth rate and are altered among mice in the different housing conditions in our animal tumor model (Rowse et al, 1992; Weinberg & Emerman, 1989), these hormones may play a role in influencing tumor response to chemotherapy. In addition, both CORT and GH significantly modulate metabolic pathways of the host (e.g. white blood cell or bone marrow production; Ref. Fauci et al, 1976; Guyre et al, 1984; Merchav, 1998; Munke et al, 1984; Weissman, 1990; Woody et al, 1999) as well as enzymes responsible for drug metabolism (Lund et al, 1991; Zaphiropoulos et al, 1989) and therefore may also play a role in influencing host responses to chemotherapy. The present study examined whether basal plasma levels of CORT, T and GH, measured on the day of (day 0) or 1 or 5 d following chemotherapy or drug vehicle administration, were differentially altered in mice among the experimental housing conditions. In addition, we examined whether the temporal relationship between the formation of experimental housing conditions and chemotherapy or drug vehicle administration differentially affected CORT, T and GH levels. 116 METHODS Tumor Propagation. Tumor cells were prepared and injected as described in "General Methods" Experimental Animals. Experiment 1. Male DD/S mice (n=136) were used as experimental subjects in this study. Following weaning, mice were reared either individually housed (I) or group-housed (G; Fig 10A). At 2-4 mo of age, mice in each housing condition were injected s.c. with tumor cells (2xl06 cells suspended in 0.1 ml DMEM). Immediately following tumor cell injection, mice were either rehoused from individual to group (IG) or from group to individual (GI), or remained in their original housing conditions (II, GG) according to our published protocol (Weinberg & Emerman, 1989). Mice within each housing condition were randomly assigned into tumor cell injection groups receiving either chemotherapy (TC; n= 10 II, n= 18 IG, n= 13 GG, n= 12 GI) or drug vehicle alone (TV; n=15 II, n=27 IG, n=23 GG, n=18 GI; Fig 10A). Beginning the day following tumor cell injection and formation of experimental housing conditions, all mice were exposed to an acute daily stressor consisting of exposure for 15 min/d, 5d/week to 1 of 5 different novel environments, as described in "General Methods". Beginning 11 d following tumor cell injection, body weights were measured and tumors were palpated or caliper measurements were taken every day. Once tumor weights in mice in each experimental housing condition reached a mean of 0.8 ± 0.2 g, body and tumor weights were measured every second day as described in "General Methods". Body and tumor weights were not measured the day of trunk blood collection for hormone analyses. Tumors reached 0.8 ± 0.2 g at approximately the following times post tumor cell injection: 17d - II; 22d - IG; 16d - GG; and 15d - GI. 117 Experiment 2. Following weaning, male DD/S mice (n=130) were reared either individually (I) or group-housed (G; Fig 10B). At 2-4 mo of age, mice in each housing condition were injected s.c. with tumor cells. In contrast to Experiment 1, mice remained in their original housing conditions (I or G) following tumor cell injection. Beginning 11 d following tumor cell injection, body weights were measured and tumors in mice were palpated or caliper measurements were taken every day until the mean tumor weight of mice within each housing condition (I or G) reached 0.8 ± 0.2 g (approximately 14 d post tumor cell injection). At this time, all mice were rehoused either from individual to group (IG) or from group to individual (Gl), or remained in their original housing conditions (II, GG) and tumor and/or body weights were measured every second day (as described in Experiment 1). Mice in each experimental housing condition were randomly assigned into tumor cell injection groups receiving either chemotherapy (TC; n=10 II, n=20 IG, n=l 1 GG, n=l 1 Gl) or drug vehicle alone (TV; n=15 II, n=29 IG, n=17 GG, n= 17 Gl; Fig. 10B). Beginning the day following the formation of experimental housing conditions, all mice were exposed to acute daily novelty stress as described in "General Methods". Chemotherapy. For both experiments, chemotherapy or drug vehicle (9 g NaCl in 100 ml distilled water) was injected i.p. the day after the mean tumor weight of mice within a housing condition reached 0.8 ± 0.2 g (Fig 10A & 10B). Chemotherapy as described in "General Methods" was administered once i.p. Plasma hormone levels. In both experiments, animals were terminated between 0800-1130 h on the day they were to receive chemotherapy/drug vehicle (day 0) and 1 or 5 d post chemotherapy/drug vehicle administration. Mice were not exposed to novelty stress on the termination day so that basal hormone levels could be measured. Trunk blood was collected in 118 heparinized tubes, centrifuged at 2200 g for 10 min at 4°C and plasma was stored at -70°C until assayed. On day 0 in both experiments, blood was collected before chemotherapy or drug vehicle was injected and therefore tumor-bearing mice in both the chemotherapy and drug vehicle treatment conditions would have received identical treatment. Thus, for both experiments, in order to reduce the number of experimental animals used in this study, the plasma hormone levels for day 0 are from the same tumor-bearing animals. Corticosterone (CORT), testosterone (T) and growth hormone (GH) levels were measured by radioimmunoassay (RIA) as described in "General Methods" . Statistical analyses. Tumor response to chemotherapy was analyzed using the difference in tumor weights between drug vehicle-treated mice and chemotherapy-treated mice within corresponding housing conditions; tumor weights for mice in all housing conditions in drug vehicle- and chemotherapy-treated mice were rank-ordered and paired accordingly. Thus, tumor response to chemotherapy (g) = [tumor weight 4 d post drug vehicle administration minus tumor weight 4 d post chemotherapy administration]. Positive values indicate tumor response to chemotherapy, the greater the difference, the better the tumor response to chemotherapy. Host response to chemotherapy was measured by the percentage of body weight lost 4 d following chemotherapy/drug vehicle administration. Percent body weight loss = [body weight 4 d post chemotherapy/drug vehicle administration minus body weight on the day of chemotherapy/drug vehicle administration] / [body weight on the day of chemotherapy/drug vehicle administration] x 100, where body weight = [gross body weight minus tumor weight]. Negative values indicate weight loss. Hormone levels, tumor growth rates, tumor response to chemotherapy and body weight loss were analyzed by ANOVAs for the factors of Group, Days, and Treatment, where applicable. For tumor growth rate analysis, Days was treated as a 119 repeated measures variable. Significant effects were analyzed by Tukey's post-hoc tests. Analyses of tumor response to chemotherapy and body weight loss were performed on the subset of mice that were alive up to 5 d post chemotherapy/drug vehicle administration. RESULTS Experiment 1. Tumor growth rate in mice in the drug vehicle treatment condition was consistent with our previous findings using the same experimental paradigm (Kerr et al., 1997). Tukey's post hoc analysis of the significant Group x Day interaction [F (21,147)=5.92; pO.OOl; Fig. 11] revealed that tumor growth rates 16-18 d post tumor cell injection and housing condition formation were significantly faster in GI, II, and GG mice compared to IG mice (p'sO.001, 0.01 and 0.05, respectively), and significantly faster in GI mice compared to II and GG mice (p's < 0.05; Fig. 11). Tumor response to chemotherapy (tumor weight 4 d post drug vehicle administration minus tumor weight 4 d post chemotherapy administration) was consistent with and extended our previous data (Kerr et al., 1997). Analysis of the significant main effects of Group [F (3,22) = 3.589; pO.05] revealed that tumor response to chemotherapy was significantly greater in IG and GG mice than in GI mice (p'sO.05; Table 5). Tumor response to chemotherapy for II mice was not significantly different from those of mice in the other housing conditions. General host response to chemotherapy, analyzed by body weight loss from day 0 to 4 d post chemotherapy administration revealed a significant effect of Group [F (3,22) = 4.35; p = 0.01]. Similar to our previous data (Kerr et al., 1997), GI mice lost significantly more weight than GG mice (p=0.01) and marginally more weight than IG mice (p=0.061; Table 6). 120 Similarly, for tumor-bearing, drug vehicle-treated mice analysis of the significant effect of Group [F(3,20)=6.15; p<0.01] revealed that Gl mice lost significantly more weight than IG mice (p<0.01; Table 6). Comparisons of the effects of treatment conditions revealed that weight loss was similar among drug vehicle- and chemotherapy-treated mice (Table 6). Analyses of basal plasma hormone levels for tumor-bearing, drug vehicle-treated mice, revealed significant Group x Days interactions for CORT [F (6,63) = 2.57; p<0.05], T [F (6,64) = 4.74; p<0.001] and GH [F(6,51)=4.107; pO.OOl]. Post hoc analyses revealed that at day 0 (16-23 d post housing condition formation and tumor cell injection), there were no differences in hormone levels in mice among the different experimental housing conditions (Fig. 12 A-C). Following drug vehicle administration, compared to other housing conditions, transient but significant increases in T levels at 5 d in GG mice and GH levels at 1 d in Gl mice were observed (p'sO.Ol; Fig. 12 B and C, respectively). No significant differences in CORT levels in mice among the different housing conditions were observed at either 1 or 5 d post drug vehicle administration. Chemotherapy in itself altered hormone levels compared to those in drug vehicle-treated mice. Significant Treatment x Group x Day interactions for CORT [F(6,128)=2.710; pO.05] and GH [F(6,109)=3.205; pO.Ol] were observed. Post hoc analyses revealed that CORT levels were significantly higher in chemotherapy- compared to drug vehicle-treated II mice at 1 d and IG mice at 5 d post chemotherapy/drug vehicle administration (pO.Ol and pO.05, respectively). Also at 1 d post chemotherapy/drug vehicle administration, GH levels were significantly higher in drug vehicle-treated compared to chemotherapy-treated Gl mice (pO.Ol). T levels did not differ significantly between drug vehicle- and chemotherapy-treated mice. 121 Analysis of hormone levels in tumor-bearing, chemotherapy-treated mice, revealed significant effects for CORT and GH. Analysis of CORT levels revealed a significant effect of Day [F(2,65) = 15.72; p<0.001] and a significant Group x Day interaction [F (6,65) = 2.30; p<0.05]. Overall, CORT levels peaked at 1 d post chemotherapy administration (dl>d0; pO.OOl and dl>d5; p=0.01; Fig. 13A) and this effect was the greatest in II and GG mice (IIdl > IId0; pO.01 and GG d l > GGd 0; pO.05; Fig.l3A). Post hoc comparisons of the interaction revealed no significant differences in CORT levels in mice among the different housing conditions. Analysis of GH levels revealed a significant effect of Day [F(2,63)=4.974; pO.01]. GH levels were significantly higher at 1 d post chemotherapy administration than at day 0 (pO.01; Fig. 13C). Analysis of T levels revealed no significant differences over days or among experimental housing conditions. Experiment 2. For mice in the drug vehicle treatment condition, tumor growth rates were similar in I and G-housed mice (p=0.2; Fig. 14) prior to the formation of experimental housing conditions. Following formation of experimental housing conditions, a significant Group x Day interaction [F(6,18)=4.989; pO.01] was observed. Tumor growth rates at 18 d post tumor cell injection (4 d post formation of experimental housing conditions) were significantly faster in GI than in II and IG mice (p'sO.01) and were faster in GG than in IG mice (pO.05; Fig. 14). Tumor response to chemotherapy, measured at 4 d post chemotherapy administration (18 d post tumor cell injection and 5 d post formation of experimental housing conditions) were similar among mice in the different experimental housing conditions (Table 5). General host response to chemotherapy, analyzed by body weight loss prior to (day 0) 122 to 4 d post chemotherapy administration, revealed a marginal effect of Group [F(3,21)=2.73; p=0.070]. Post hoc analysis indicated that consistent with our previous study (Kerr et al, 1999a), IG mice lost marginally more weight than Gl mice (p=0.072; Table 6). There were no significant differences in weight loss for II and GG mice. Similarly, for tumor-bearing, drug vehicle-treated mice, no significant differences in body weight loss among mice in the different housing conditions were observed (Table 6). Overall, body weight loss was marginally greater for chemotherapy- than for drug vehicle-treated mice [TRT; F(l,40)=3.843; p=0.057; Table 6]. Analysis of basal plasma hormone levels in tumor-bearing, drug vehicle-treated mice revealed significant Group x Day interactions for CORT [F(6,60)=6.765; pO.OOl] and GH [F(6,51)=9.440; pO.OOl] and a significant effect of Group for T [F(3,52)=3.954; p=0.0.1]. Post hoc analyses revealed that at day 0 (approximately 14 d post tumor cell injection and 1 d post experimental housing condition formation), CORT levels were significantly higher in IG than those in all other housing conditions (p'sO.Ol; Fig. 15A). Also, for IG mice, CORT levels at day 0, were significantly higher than those at either at 1 or 5 d post drug vehicle administration (p'sO.Ol; Fig. 15A). GH levels at day 0 were significantly higher in Gl mice compared to those in all other housing conditions (p'sO.Ol; Fig. 15C) and overall, GH levels in Gl mice were significantly higher at day 0 compared to those at 1 or 5 d (p'sO.001). In addition, T levels, overall, were significantly higher in Gl than in II mice (p=0.01) and marginally higher than in IG and GG mice (p=0.060 and p=0.086, respectively; Fig. 15B). Analysis of the effects of chemotherapy treatment on hormone levels revealed a significant Treatment x Group x Day interaction for GH [F(6, 105)=2.195; p<0.05]. Significant differences in GH levels were observed only in Gl mice. GH levels at 5 d post drug vehicle/chemotherapy administration were significantly higher in chemotherapy-treated than in 123 drug vehicle-treated GI mice (p<0.05). Neither CORT nor T levels differed significantly among treatment conditions. Analysis of hormone levels in tumor-bearing, chemotherapy-treated mice revealed significant effects of CORT and GH. Analysis of CORT levels revealed significant main effects of Group [F(3,62)=7.380; pO.OOl] and Day [F(2,62)=3.517; p<0.05] as well as a Group x Day interaction [F(6,62)=3.713; pO.01]. CORT levels were significantly higher at day 0 in IG than in GG (pO.05), II and GI (p'sO.Ol) mice (Fig. 16A). By 5 d post chemotherapy, CORT levels in IG mice had dropped significantly compared to those at day 0 (pO.05; Fig. 16A). Regardless of housing condition, CORT levels were significantly higher at 1 d post chemotherapy than at day 0 (p<0.05). Examination of GH levels also revealed significant main effects of Group [F(3,54)=20.691; pO.OOl] and Day [F(2,54)=l 1.126; pO.OOl] as well as a significant Group x Day interaction [F(6,54)=7.240; pO.OOl]. GH levels at day 0 were significantly higher in GI than in II, IG and GG mice (p'sO.Ol) and at 5 d GH levels were significantly higher in GI than in IG mice (pO.05; Fig. 16C). For II mice, GH levels, overall, were significantly higher compared to those in IG mice (pO.01; Fig. 16C). Regardless of housing condition, GH levels were significantly reduced at 1 d compared to those at day 0 and 5 d post chemotherapy administration (p'sO.001). T levels did not differ significantly in mice among the experimental housing conditions (Fig. 16B). DISCUSSION Experiment 1 demonstrated that, consistent with our previous data, when experimental housing conditions were formed immediately following tumor cell injection (our original experimental paradigm; Ref. Kerr et al, 1997), tumor growth rates in drug vehicle-treated mice 124 were significantly faster in Gl than in IG mice, whereas II and GG mice had intermediate tumor growth rates. Similarly, tumor and host responses to chemotherapy were consistent with and extended our previous findings (Kerr et al, 1997); IG and GG mice had better tumor response to chemotherapy and lost less weight compared to Gl mice. In experiment 2, the temporal relationships between housing condition formation and (a) tumor cell injection or (b) chemotherapy administration differed from those of experiment 1. Specifically, housing conditions were formed approximately 14 d (not immediately, as in experiment 1) after tumor cell injection and 1 d (not 16-22 d, as in experiment 1) before chemotherapy/drug vehicle administration. Under these temporal conditions, tumor growth rates were fastest in Gl and slowest in IG mice. Tumor responses to chemotherapy at 4 d post chemotherapy administration, were similar among in mice the different housing conditions. However, examination of the general host response to chemotherapy revealed that Gl lost moderately less weight than IG mice. In our previous study (Kerr et al, 1999a), when tumor and host responses to chemotherapy were measured up to 70 d following the first injection of chemotherapy, we demonstrated that tumor response to chemotherapy was significantly greater for Gl than II mice and moderately greater for Gl than IG mice. In addition, Gl mice significantly less weight than IG mice and moderately less weight than II mice. In the present study, it may be too early following chemotherapy administration to observe significant differences in tumor and host responses to chemotherapy. It is of relevance, however, that a similar, albeit a moderate, difference was observed in body weight loss and the differences in tumor responses to chemotherapy were in a similar direction as those in our previous study. The temporal conditions of experiment 2 appear to increase the variability in tumor growth rates since the tumor growth rates observed in experiment 2 do not fully replicate those 125 of our previous study (Kerr et al, 1999a). In that study, tumor growth rates were fastest in II mice and slowest in IG mice whereas in the present study, tumor growth rates were fastest in GI mice and slowest in IG mice. Importantly, regardless of the variability in tumor growth rates, both tumor and host responses to chemotherapy are comparable to our previous data (Kerr et al, 1999a). In addition, although in the present study, tumor growth rates among mice in experiment 2 were similar to those observed in experiment 1, tumor and host responses to chemotherapy differed between these experiments. These studies support our previous data in demonstrating that tumor growth rate does not appear to play a direct role in mediating the differential effects of social housing conditions on chemotherapeutic efficacy. Furthermore, like our previous studies (Kerr et al, 1997; Kerr et al, 1999a), the present data also demonstrate that the temporal relationship between formation of social housing conditions and chemotherapy administration is critical in determining the direction and magnitude of the effects of housing condition on chemotherapeutic efficacy. Several factors, acting either independently or in an interactive fashion, may play a role in mediating stressor-induced alterations in chemotherapeutic efficacy (Calssen et al, 1998; Giraldi et al, 1992; Riley et al, 1981; Vogel et al, 1987). Previous studies suggest that alterations in tumor growth rate may influence tumor response to chemotherapy (Emerman & Siemiatkowski, 1984; English et al, 1991; Hug et al, 1986; Miller, 1990; Skipper, 1964). In addition, both tumor and host responses to chemotherapy may be influenced by alterations in circulating CORT, T and/or GH levels (Emerman & Siemiatkowski, 1984; English et al, 1987; Harrison et al, 1995; Kodama & Kodama, 1982; Ng et al, 1993; Shepherd & Harrap, 1982). We have shown that tumor growth rates as well as basal plasma levels of CORT, T and GH are differentially altered in mice among experimental housing conditions at 1 -7 days but not at 21 126 days following tumor cell injection and housing condition formation. (Rowse et al, 1992; Weinberg & Emerman, 1989). In addition, we have demonstrated that the temporal relationship between the formation of experimental housing conditions and tumor cell injection and chemotherapy initiation significantly influences tumor growth rates or chemotherapeutic efficacy, respectively (Kerr et al, 1997; Kerr et al, 1999a). Experiment 1 demonstrated that, similar to our previous study (Kerr et al, 1997), 16-22 d after formation of social housing conditions, basal plasma CORT, T and GH levels (measured prior to chemotherapy or drug vehicle administration; day 0) were similar in mice among the different housing conditions. However, at 1 or 5 d post drug vehicle administration (approximately 2-4 weeks post tumor cell injection and experimental housing condition formation), significant although transient increases in T levels in GG and GH levels in Gl mice and were observed. Our previous data (Rowse et al, 1992) suggested that alterations in hormone levels at much earlier time points, between 1-7 days following tumor cell injection and formation of experimental housing conditions, may meditate the differential tumor growth rates in mice among the different housing conditions. Alterations in hormone levels observed at later time points, as in the present study, do not appear to be related to the differential tumor growth rates. The occasional and transient alterations in basal hormone levels observed in mice among the different housing conditions in the present study, however, may play a role in maintaining tumor growth rates within these housing conditions. Under the temporal conditions of experiment 2, we were able to examined whether alterations in basal plasma levels of CORT, T and GH in tumor-bearing, drug vehicle-treated mice at 1, 2 and 6 d post housing condition formation (i.e. 0, 1 and 5 d post drug vehicle administration) were related to the different tumor growth rates observed in mice among the 127 different housing conditions: The data of the present experiment are comparable to but more transient than those observed in our previous study where hormone levels were measured 1 -7 d following formation of housing conditions and tumor cell injection (Rowse et al, 1992). Overall, T levels in Gl mice (fastest growing tumors) were higher than those in mice among the other housing conditions. In addition, CORT levels in IG mice (slowest growing tumors) and GH levels in Gl mice were higher at 1 d post housing condition formation compared to those in all other experimental housing conditions. These data support our suggestion that T, CORT and GH may play a role in modulating the differential tumor growth rates in mice among the experimental housing conditions. However, since in this study, tumor size at the time of housing condition formation was relatively large (lg), tumor-related factors also may be influencing the differential tumor growth rates observed (Heppner, 1989; Heppner et al, 1993). This possibility remains to be elucidated. Studies have shown that increased levels of CORT may suppress the anti-tumor activity of CY through acceleration of drug metabolism and if prolonged, elevated CORT levels may increase toxic side effects (Faber et al, 1974; Kodama & Kodama, 1982; Shepherd & Harrap, 1982), whereas increased GH levels may reduce toxic side effects such as immunosuppression and weight loss (Harrison et al, 1995; Ng et al, 1993; Wolf et al, 1994). For T, we have demonstrated that maintaining submaximal levels of T required for tumor growth, rather than withdrawing T completely, can enhance the antitumor activity of AD and CY (Emerman & Siemiatkowski, 1984). In experiment 1, chemotherapy itself increased CORT levels in II and IG mice and decreased GH levels in Gl mice compared to those of their drug vehicle-treated counterparts. However, no significant differences in hormone levels were observed in chemotherapy-treated mice among the different housing conditions. Although significant 128 effects of housing conditions on both tumor and host responses to chemotherapy were observed, the data suggest that these responses not appear to be directly influenced by alterations in CORT, T or GH levels, at least at the time points examined. In experiment 2, examination of basal plasma hormone levels revealed that prior to chemotherapy administration (day 0) CORT levels in IG mice and GH levels in GI mice were significantly higher compared to those in all other housing conditions. At 5 d post chemotherapy administration, GH levels were significantly higher in GI than in IG mice, whereas overall, GH levels were significantly higher in II than in IG mice. Interestingly, no significant differences in T levels in mice of the different housing conditions were observed. Together with our previous data demonstrating that both tumor and host responses to chemotherapy were best in GI mice and poorer in IG and II mice (Kerr et al, 1999a), the present data suggest that these effects do not appear to be directly related to differential hormone levels among the different housing conditions. However, alterations in CORT and GH levels within the first 7 d following the formation of housing conditions and administration of chemotherapy may play a role in mediating the differential effects of experimental housing condition on chemotherapeutic efficacy. It is unlikely that hormonal alterations in mice in the different housing conditions are the only factors mediating the differential effects of housing condition on chemotherapeutic efficacy. Future studies examining immune and tumor-specific factors as well as active drug to metabolite ratios and/or the interactions among these factors may elucidate further the mechanisms that are differentially influencing the effects of social housing conditions on tumor and host responses to chemotherapy. 129 Fig. 10. Experimental Designs A. Experiment 1 B. Experiment 2 Mice weaned and housed either individually (I) or in groups (G) I SCI 15 cells injected s.c. and experimental housing conditions (II, IG, GG, Gl) formed 16-22 d • Mean weight of tumors within an experimental housing condition reaches approximately 1 g I" Trunk blood collected (day 0) Chemotherapy or saline injected i.p I Trunk blood collected 1 d post chemotherapy or saline administration Trunk blood collected 5 d post chemotherapy or saline administration Mice weaned and housed either individually (I) or in groups (G) I SCI 15 cells injected s.c. ~ 14 d T Mean weight of tumors within a rearing housing condition reaches approximately 1 g and experimental housing conditions (II, IG, GG, Gl) formed W Trunk blood is collected (day 0); Chemotherapy or saline injected i.p. I Trunk blood collected 1 d post chemotherapy or saline administration Trunk blood collected 5 d post chemotherapy or saline administration 131 Table 5. Tumor response to chemotherapy0 (mean ± SEM) in mice 4d post chemotherapy treatment Housing Experiment 1 Experiment 2 Condition II 0.74 + 0.34 0.60±0.26 (5) (5) IG 0.8210.13 0.78 ±0.27 (9) (10) GG 0.85 ±0.52 0.6610.17 (5) (6) GI -0.2810.27* 1.23 10.20 (5) (5) a Tumor response to chemotherapy (g) = [tumor weight 4 d post drug vehicle administration minus tumor weight 4 d post chemotherapy administration]; positive values indicate tumor response to chemotherapy, the greater the positive difference, the better the tumor response; b IG=GG>GI, p's<0.05. Table 6. Percent body weight lossa (mean ± SEM) in mice 4 d post chemotherapy or drug vehicle treatment Experiment 1 Experiment 2 Condition T C b TV TC TV II -1.98 ±2.43 (5) 1.21 ± 1.20 (5) -0.75 ± 1.47 (5) 0.45 ±2.05 (5) IG 0.68 ± 1.45 (9) 3.40 ±0.85 (9) -5.62 ± 1.36e (10) 0.28 ± 3.42 (8) GG 2.69 ± 0.70 (6) 0.72 ± 1.58 (5) -3.29 ±2.93 (5) 2.54 ± 1.30 (6) GI -4.73 ± 1.79c (6) -2.72 ± \A6d (5) 0.88 + 1.64 (5) 1.87 ±2.49 (6) a Percent body weight loss = [body weight 4 d post chemotherapy or drug vehicle administration minus body weight on the day of chemotherapy or drug vehicle administration] / [body weight on the day of chemotherapy or drug vehicle administration] x 100; body weight = [gross body weight minus tumor weight]. Negative values indicate weight loss; * TC: tumor-bearing, chemotherapy-treated; TV: tumor-bearing, drug vehicle-treated; c GI>GG, p=0.01; GI>IG, p=0.061; ^GI>IG,p<0.01; e IG>GI, p=0.072 133 Fig. 11. Tumor growth in mice in the drug vehicle treatment condition. Tumor weights (means ± SEM) from 11-18 d post tumor cell injection and formation of social housing conditions for drug vehicle-treated mice in the experimental housing conditions (n=5, II; n=10, IG; n=6 GG; n=5, Gl). Tumor growth rates at 16-18 d post tumor cell injection and formation of social housing conditions were significantly faster in II, GG and Gl mice compared to IG mice (p'sO.Ol, 0.05, and 0.001, respectively). Also, tumor growth rates were faster in Gl mice compared to II and GG mice (p'sO.05). 134 Fig. 12 (A-C). Basal plasma hormone levels in tumor-bearing, drug vehicle-treated mice. Hormone levels (means; bars SEM) measured at 0, 1 and 5 d post drug vehicle administration. A: No differences in corticosterone (CORT) levels were observed among the experimental housing conditions. B: Testosterone (T) levels at 5 d were significantly higher in GG mice compared to all other housing conditions (p'sO.Ol). Also for GG mice, T levels were significantly higher at 5 d than those at 0 and 1 d (p'sO.05). C: Growth hormone (GH) levels at 1 d were significantly higher in GI mice compared to II, GG and IG mice (pO.05 and p'sO.001, respectively). Also for GI mice, GH levels were significantly higher at 1 and 5 d than those at day 0 (pO.OOl and pO.05, respectively). 136 Figure 13 (A-C). Basal plasma hormone levels in tumor-bearing, chemotherapy-treated mice. Hormone levels (means; bars SEM) measured at 0, 1 and 5 d post chemotherapy administration. A: Overall, corticosterone (CORT) levels were significantly higher at 1 d than at 0 and 5 d (p<0.001 and p<0.01, respectively). This effect was greatest in II and GG mice (IIld > II0d; p<0.01 and GG l d > GG0 d; p<0.05). No significant differences in CORT levels were observed among the experimental housing conditions. B: No significant differences in testosterone (T) levels were observed among the experimental housing conditions. C: Regardless of housing condition, growth hormone (GH) levels were significantly higher at 1 d than at day 0 (p<0.01). No significant differences in GH levels were observed among the experimental housing conditions. 138 Fig. 14. Tumor growth in mice in the drug vehicle treatment condition. Arrow represents formation of experimental housing conditions. Tumor weights (means ± SEM) from 11-18 d post tumor cell injection for drug vehicle-treated mice in the experimental housing conditions (n=5, II; n=9, IG; n=6, GG; n=6, GI). Tumor growth rates by 18 d post tumor cell injection (4 d post formation of experimental housing conditions) were significantly faster in GI mice than in II and IG mice (p's<0.01) and were also faster in GG than in IG mice (p<0.05). 140 Fig. 15 (A-C). Basal plasma hormone levels in tumor-bearing, drug vehicle-treated mice. Hormone levels (means; bars SEM) measured at 0, 1 and 5 d post drug vehicle administration. A: Corticosterone (CORT) levels at day 0 were significantly higher in IG than in II, GG or GI mice (p'sO.Ol). Also for IG mice, CORT levels were significantly higher at day 0 than those at I and 5 d (p'sO.Ol). B: Overall, testosterone (T) levels were significantly higher in GI than in II mice (p=0.01) and marginally higher than those in IG and GG mice (p=0.06 and p=0.086, respectively). C: Growth hormone (GH) levels at day 0 were significantly higher in GI mice compared to those in II, IG and GG mice (pO.01 and pO.OOl, respectively). Also for GI mice, GH levels were significantly higher at day 0 than those at 1 or 5 d (p'sO.001). 142 143 Fig. 16 (A-C). Basal plasma hormone levels in tumor-bearing, chemotherapy-treated mice. Hormone levels (means; bars SEM) measured at 0, 1 and 5 d post chemotherapy administration. A: Corticosterone (CORT) levels at day 0 were significantly higher in IG than in GG, II and Gl mice (p<0.05 and p'sO.Ol, respectively). By 5 d post chemotherapy administration, CORT levels in IG mice dropped significantly compared to those on at day 0 (p<0.05). Overall, CORT levels were significantly higher at 1 d post chemotherapy administration than at day 0 (p<0.05). B: No significant differences in testosterone (T) levels were observed among the experimental housing conditions. C: Growth hormone (GH) levels at day 0 were significantly higher in Gl than in II, IG and GG mice (p'sO.Ol). GH levels in Gl mice remained significantly higher than those in IG mice up to 5 d (pO.05). GH levels in Gl mice were significantly higher at day 0 than at 1 and 5 d (pO.Ol and pO.05, respectively). Regardless of housing condition, GH levels were significantly reduced at 1 d compared to levels at 0 and 5 d (p'sO.001). 144 CHAPTER 7. INTERACTIVE EFFECTS OF SOCIAL HOUSING CONDITIONS AND GENDER ON MOUSE MAMMARY TUMOR GROWTH INTRODUCTION Increasing evidence suggests that stress and the ability to cope with stress may play a role in malignant transformation and tumor progression (Cooper & Faragher, 1993; Greer et al, 1992; Jensen, 1991; Temoshok, 1987). Although the evidence that stressful life events are related to the development of cancer is controversial (e.g. Barraclough et al, 1992; Cassileth et al, 1985; Lin et al, 1982; Priestman et al, 1985), a number of human studies have correlated stressful life events with increased cancer risk (Cooper et al, 1989; Scherg & Blohmke, 1988) and decreased survival probability (Ramierez et al, 1989). Furthermore, various coping strategies have been related to decreased cancer recurrence (Greer et al, 1991; Hilakivi-Clarke et al, 1993; Hislop et al, 1987; Levy, 1985; Lippman, 1985) and increased survival time (Greer et al, 1979; Temoshok, 1987). Interestingly, gender differences have been reported to influence both tumor progression and metastasis (Leigh et al, 1987; Maguire et al, 1996; Ueda et al, 1988). However, the interactions among psychosocial stressors, gender, and cancer progression remain to be elucidated. The relationship between stressors and tumor growth may be demonstrated under more controlled conditions in animal models. However, even in animal models, this relationship is complex. Factors such as the ability to control the stressor, the timing, duration and severity of the stressor, the type of tumor, the animal's housing condition, and a variety of host related factors, including genetics, gender, and behavior, may influence the effects of stressors on tumor growth (Burchfield et al, 1978; Haseman et al, 1994; Justice, 1985; Newberry et al, 1984; Peters & Kelly, 1977; Riley etal, 1981; Sklar & Anisman, 1981; Steplewski et al., 1985). 146 Relevant to the present study, it has been shown that housing condition can significantly affect tumor growth in male mice. Typically, group-housed males have smaller tumors and show increased rates of tumor rejection or regression compared to individually-housed animals (Peraino et al, 1973; Riley, 1981). Data have also shown that a change in housing condition may increase tumor growth rate compared to that in animals that do not experience a change. Our data have demonstrated that the direction of change (from individual to group or from group to individual) as well as the change in social housing condition itself can significantly influence tumor growth rates in male mice (Grimm et al., 1996; Weinberg & Emerman, 1989). Utilizing the androgen-responsive Shionogi mouse mammary carcinoma (AR SCI 15) we have shown that being reared in a group (G) and then individually (I) housed (Gl) following tumor cell injection increases tumor growth rate, whereas being reared individually and then group housed (IG) reduces tumor growth rate compared to that in mice remaining in their original rearing conditions (II or GG). Differences in tumor growth rate between Gl and IG male mice in this animal-tumor model were most apparent in the presence of acute daily novelty stress (Weinberg & Emerman, 1989). Our data further suggest that one possible mediator of these differential tumor growth rates may be an alteration in endocrine function; mice in the Gl housing condition (largest tumors) have increased basal testosterone (T) and decreased basal corticosterone (CORT) levels whereas mice in the IG housing condition (smallest tumors) exhibit decreased basal T and increased basal CORT levels for at least one week following tumor cell injection and formation of experimental housing conditions (Rowse et al, 1992). Since both androgens and glucocorticoids have been shown to have significant effects on the growth rate of AR SCI 15 tumor cells both in vivo and in vitro (Bruchovsky & Meakin, 1973; Hiraoka et al, 1987; Jiang et al, 1993; Rowse et al, 1992; Yamanishi et al, 1992), it is possible that these.alterations in basal hormone levels play a role in 147 mediating the different tumor growth rates observed. Studies have also examined the effects of housing condition on tumor growth rates in females. Unlike the studies in males, the data on females are much more inconsistent. Studies have reported both increased (LaBarba et al, 1972; Riley, 1975) and decreased (Peraino et al, 1973) tumor growth rates in group housed compared to individually housed females. Few studies have directly examined whether social housing condition differentially affects tumor growth rates in males and females. Of the studies that have been done, males generally have higher tumor incidences or greater tumor growth rates compared to females. For example, both group and individually housed male mice had an increased incidence of spontaneous hepatic tumors compared to females (Peraino et al, 1973) and male rats subjected to a change from group to individual housing had higher growth rates of an injected mammary adenocarcinoma compared to females who experienced the same housing condition change (Steplewski et al, 1987). The present study directly compared the effects of social housing condition on tumor growth rate in males and females. We utilized a variant of the AR SCI 15 tumor, designated SCI 15V, that grows equally well in males and females. This variant arose spontaneously in male mice after many passages of the AR SCI 15. We examined SCI 15V tumor growth rates in males and females from GI, IG, and GG housing condition both with and without acute daily exposure to novel environments. For females, whether the stage of the estrous cycle at the time of tumor cell injection influenced tumor growth rates as well as possible interactive effects of social housing condition, tumor cell injection and acute daily novelty stress on estrous cycle periodicity were analyzed. 148 METHODS Tumor propagation. The SCI 15V tumor was maintained by serial transplantation in female mice of the DD/S strain as described previously for the SCI 15, see "General Methods". Experimental animals. Male (n=51) and female (n=106) mice were used in Experiment 1, and only female (n=88) mice were used for Experiment 2. All mice in these studies were of the DD/S strain and were 2-4 mo of age. Immediately following tumor cell injection (s.c. injection of 2 x 1()6 cells suspended in 0.1 ml DMEM), male and female mice that were reared as individuals (I) or in groups (G) of the same sex were rehoused either from group to individual (Gl) or from individual to same-sex group (IG) or remained in their original group housing condition (GG), according to our published protocol (Weinberg & Emerman, 1989). Mice were palpated every other day; once tumors were measurable (10-12 d post tumor cell injection), caliper measurements were taken every other day, as described in "General Methods". Experiment 1: Effects of social housing condition and exposure to acute daily novelty stress on tumor growth rates in male and female mice Male and female mice in each housing condition were randomly assigned to conditions that either received acute daily novelty stress (novelty stress, S; for males, n = 8 Gl, 6 GG, 15 IG; for females, n = 21 Gl, 18 GG, 34 IG) or were left undisturbed (no novelty stress, NS; males, n = 7 Gl, 6 GG, 9 IG; for females, n = 9 Gl, 9 GG, 15 IG). Beginning the day following tumor cell injection and the formation of experimental housing conditions, mice assigned to the acute daily novelty stress condition were exposed to 1 of 5 different novel environments as described in "General Methods". 149 Experiment 2: Effects of tumor cell injection, social housing, and acute daily novelty stress on estrous cycle periodicity Female mice (n=88) were reared either individually or in same-sex groups. To obtain base-line estrous cycle data for each experimental mouse, vaginal smears were taken every 3 d (0800-1200 h) for 18 d prior to tumor cell injection and formation of housing conditions. Vaginal smears were not taken every day since other studies have demonstrated that the estrous cycle can be influenced by stressors, including the frequency of examinations (Axelson, 1987; Briski & Sylvester, 1988; Emery & Schwabe, 1936; Marchlewska-Koj et al., 1994). On d 18, immediately following injection of SCI 15V tumor cells (T; s.c. injection of 2 x 106 cells in 0.1 ml DMEM) or tumor cell-vehicle (no tumor, NT; s.c. injection of 0.1 ml DMEM) females were rehoused into GI, IG and GG conditions (see Experiment 1). Females within each housing condition were randomly assigned to either the S or NS condition (as described in Experiment 1 above). N's per housing condition were: T/S - 6 GI, 6 GG, 10 IG; T/NS - 7 GI, 6 GG, 10 IG; NT/S - 6 GI, 6 GG, 10 IG; and NT/NS - 5 GI, 6 GG, 10 IG. Vaginal smears continued to be taken every 3 d for 21 d, beginning 2 d following tumor cell or tumor cell-vehicle injection and the formation of housing conditions. In addition, vaginal smears were taken every day for 14 d on a subset of non-experimental females reared either as individuals or in same-sex groups to determine the average estrous cycle length of females in the same housing condition. STATISTICAL ANALYSES Tumor growth rate. Tumor weights for males and females were analyzed separately by Treatment x Housing Condition x Days ANOVAs to analyze the effects of stress across days in 150 mice in the Gl, IG, and GG conditions. In addition, tumor weights for all mice were analyzed by Sex x Treatment (S, NS) x Housing Condition (Gl, IG, GG) x Days ANOVAs. Estrous cycle. The effects of both pre- and post-experimental housing and treatment conditions on estrous cycle length were analyzed by Chi-square tests. The expected values used in these analyses were derived from the estrous cycle data obtained from non-experimental female mice reared either as individuals or in same-sex groups. In addition, the effect of the estrous cycle stage at the time of tumor cell injection on subsequent tumor growth rate was analyzed by an ANOVA for the factors of Estrous Stage, Treatment, and Days. In all analyses, Days was treated as a repeated measures variable. Significant main effects or interactions in all ANOVAs were further analyzed by Tukey's post hoc tests. RESULTS Experiment 1: Tumor growth rates in male mice. The ANOVA revealed a significant Housing Condition x Days interaction, F(6,135)=2.372, p<0.05. Post hoc analysis indicated that, similar to our previous studies using the AR SCI 15 tumor (Weinberg & Emerman, 1989; Rowse et al, 1992), following exposure to acute daily novelty stress, SCI 15V tumor growth rates were significantly faster in males in the Gl compared to the IG condition (GI>IG, pO.OOl; Fig. 17). Males in the GG condition had intermediate tumor growth rates (not significantly different from Gl or IG; Fig. 17). In contrast to our previous data with the AR SCI 15, however, no significant differences in SCI 15V tumor growth rates were observed among non-stressed, IG, Gl, or GG males. Tumor growth rates in female mice. The ANOVA revealed a significant Treatment x Housing Condition x Days interaction, F(6,291)=3.733, p=0.001. In contrast to males, the post 151 hoc analysis indicated that following exposure to acute daily novelty stress, tumor growth rates were significantly faster in females in the IG compared to the GI condition (IG>GI; p=0.01; Fig. 17). Females in the GG condition had intermediate tumor growth rates (not significantly different from GI or IG; Fig. 17). In the non-stressed condition however, similar to our findings in stressed males, SCI 15V tumor growth rates were significantly faster in GI compared to IG females (GI>IG;p<0.01;Fig. 17). Comparison of tumor growth rates between male and female mice. The Sex x Treatment x Housing Condition x Days ANOVA revealed a significant 4-way interaction, F(6,426)=4.606, pO.OOl, as well as significant three-way interactions of Sex x Treatment x Housing Condition, F(2,142)=4.269, pO.05, and Sex x Treatment x Days, F(3,426)=l 1.597, pO.OOl. Post hoc analyses of the three-way interactions revealed that in the non-stressed condition, there were no significant differences in tumor growth rates between males and females. Following exposure to acute daily novelty stress however, males in the GI condition had significantly increased tumor growth rates compared to females in the GI condition (pO.01; Fig. 17). Furthermore, differential effects of tumor growth rates in GI males and females were observed. Whereas tumor growth rates for GI males were significantly faster for stressed than for non-stressed mice (pO.OOl; Fig. 17), the opposite was observed in females; tumor growth rates were significantly faster for non-stressed than for stressed GI females (pO.01; Fig. 17). Experiment 2 : Effects of experimental housing conditions on the estrous cycle. The estrous cycle length for non-experimental females, housed either as individuals or in same-sex groups varied between 93 and 126 h (3.88-5.25 d) with a mean of 108 V 2 h (4.5 d). Whether a mouse was housed individually or in a group did not influence the length of the estrous cycle, nor were estrous cycles synchronized consistently for group-housed females. A comparison of the 152 estrous cycle periodicity prior to and following tumor cell injection and formation of experimental housing conditions indicated that neither housing condition nor the direction of change in housing condition significantly affected the periodicity of the estrous cycle. Furthermore, there were no significant effects of exposure to acute daily novelty stress, tumor cell injection, or the interaction of these conditions on estrous cycle periodicity. Finally, the stage of the estrous cycle at time of tumor cell injection did not differentially affect the tumor growth rates of mice in the different experimental conditions. DISCUSSION Data from this study demonstrate that social housing condition and exposure to acute daily novelty stress interact to affect the growth rate of the SCI 15V tumor differentially in male and female DD/S mice. Consistent with our previous data from the male AR SCI 15 tumor model (Rowse et al, 1992; Weinberg & Emerman, 1989), in the presence of acute daily novelty stress, the growth rate of the SCI 15V tumor was significantly increased in GI compared to IG males. Unlike our previous data, however, there were no significant differences in SCI 15V tumor growth rates among GI, IG, or GG males under non-stressed conditions. For females, in contrast to males, following acute daily novelty stress, tumor growth rate was significantly decreased in GI compared to IG mice, whereas under non-stressed conditions, tumor growth was increased in GI compared to IG females. Previous studies conducted in our laboratory have demonstrated that following acute daily novelty stress, GI males have increased AR SCI 15 tumor growth rates as well as higher basal T and lower basal CORT levels compared to IG males (Rowse et al, 1992). Previous in vitro data have demonstrated stimulatory effects of physiological levels of dihydrotestosterone (DHT) and 153 pharmacological levels of hydrocortisone (HC) on the growth of AR SCI 15 cells whereas physiological levels of HC have no effect (Jiang et al, 1993). Together, these data suggest that T directly modulates AR SCI 15 tumor growth rates and that the hypothalamic-pituitary-adrenal (HPA) axis may be indirectly involved in the regulation of AR SCI 15 tumor growth via CORT induced modulation of T levels. Similar to our previous studies, in the present study SCI 15V tumor growth rate in Gl males exposed to acute daily novelty stress was significantly faster than those of IG males. However, unlike our previous results using the AR SCI 15, no significant differences in SCI 15V tumor growth rates for non-stressed males were observed. Although it appears that T and CORT may still be playing a role in modulating SCI 15V tumor growth rates observed in stressed Gl and IG males, the factors mediating the differential SCI 15V tumor growth rates remain to be determined. Nonetheless, these results demonstrate that social housing condition and exposure to acute daily novelty stress can significantly alter the growth rate of SCI 15V and that the SCI 15V responds differently to psychosocial and acute daily stressors compared to the AR SC 115. For females, preliminary studies conducted in our laboratory demonstrate no significant differences in basal plasma CORT levels among mice in different housing conditions, with or without exposure to acute daily novelty stress. Preliminary data also suggest that physiological levels of HC do not significantly affect the growth rate in vitro of female-derived SCI 15V cells. These data suggest that CORT may not be playing an important role in modulating tumor growth rates in females. That CORT may play a role in modulating SCI 15V tumor cell growth rates in males but not in females may reflect a complex interaction between biological (e.g. hormone levels and immune activity) and environmental (e.g. social housing condition) variables. That is, although 154 the same tumor cell suspension was injected into males and females, putting a heterogeneous tumor cell population into the different host environments of the male and female mice combined with gender-specific effect(s) of social housing conditions on endocrine and immune factors possibly allowed for the selection of distinct subpopulations within the SCI 15V with altered sensitivities to growth factors such as CORT. This possibility remains to be examined. Although estrogen levels in females were not directly analyzed, the present study did examine whether the stage of the estrous cycle at the time of tumor cell injection played a role in altering tumor growth rates as well as whether tumor cell injection, exposure to acute daily novelty stress, and/or housing condition affected estrous cyclicity. The estrous cycle is associated with rhythmic changes in hormone concentrations (Buckingham, 1982). Importantly, the stage of the estrous cycle at the time of exposure to a stressor has been shown to affect stress hormone levels differentially. For example, in intact females stress-induced increases in plasma adrenocorticotropic hormone (ACTH) and CORT levels are higher during proestrus, when concentrations of plasma estrogen, progesterone, and luteinizing hormone are high (Buckingham, 1982; Butcher et al, 1974; Carey et al, 1995; Viau & Meaney, 1991). In addition, the importance of estrogen in the stress-induced increases in plasma levels of ACTH and CORT has been demonstrated by sex steroid replacement in ovariectomized females (Burgess & Handa, 1992; Carey et al, 1995; Lesniewska et al, 1990; Viau & Meaney, 1991). Not only can the estrous cycle influence physiological responses to stressors, but tumor growth rates may also be influenced by estrous cycle stage at the time of tumor induction (Braun et al, 1989; Chang et al, 1993; Lindsey et al, 1981; Rivera et al., 1994). Furthermore, several studies also have shown that group housed females have abnormally prolonged estrous cycles or exhibit anestrous (Champlin, 1971; Gangrade & Dominic, 1984; Lamond, 1959; Marchlewska-Koj et al, 1994; Whitten, 1959) 155 compared to individually housed females. Thus, differences in the estrous cycle between grouped and individually housed females may alter sex steroid hormone levels, including estrogen, which may in turn influence the physiological effects of a stressor (e.g. plasma CORT levels; Ref. Buckingham, 1978). In the present study, the stage of the estrous cycle at the time of tumor cell injection did not influence tumor growth rates. Additionally, neither tumor cell injection, housing condition, nor exposure to acute daily novelty stress appeared to alter estrous cyclicity. Thus, in the present study, the estrous cycle probably did not play a significant role in the differential tumor growth rates observed. Furthermore, these results suggest that differences in the levels of estrogen among mice in the different housing conditions may be minimal and therefore, estrogen may not be playing an important role in modulating tumor growth rates among female mice in the different housing conditions. Although the specific mechanisms governing differential SCI 15V tumor growth rates between males and females remain to be identified, the results of this study demonstrate that exposure to acute daily novelty stress appears to interact with a change in social housing condition to alter tumor growth rates differentially in male and female mice. That is, following acute daily novelty stress, tumor growth rate was increased in Gl males but was decreased in Gl females compared to their similarly stressed IG as well as to their non-stressed Gl counterparts. In addition, for Gl mice exposed to acute daily novelty stress, males had increased tumor growth rates compared to females. These data are consistent with and extend those of Steplewski et al. (1987), who demonstrated greater tumor growth rates in Gl male compared to Gl female Lewis rats injected s.c. with mammary adenocarcinoma cells. Recently, Hilakivi-Clarke et al. (1993, 1994) have suggested that gender-specific differences in behavioral responses to stressors may be associated with differences in tumorigenesis. Male CD-I MT42 mice over expressing TGFcc, 156 display longer immobility in a swim test and are more aggressive to an intruder compared to females. It was suggested that increased tumorigenesis observed in male compared to female mice is associated with impaired behavioral responses of males to both stressors. Relevant to the present study, females in general, exhibit a greater preference for novelty than males (Gray & Cooney, 1982; Hughes, 1968; Russell, 1975; Shors & Wood, 1995), and for a number of behavioral tasks [e.g. activity in an elevated plus-maze (Steenbergen et al, 1989), open-field tests (Masur et al, 1980; Valle, 1970), active avoidance tasks (Barrett & Ray, 1970; Davis et al, 1976; Steenbergen et al, 1989)], females have been shown to exhibit more adaptive behavioral responses to stressors compared to males. Therefore, it has been suggested that females are less susceptible than males to the consequences of an aversive or stressful experience. Thus it is possible that the differences observed in the tumor growth rates between male and female mice in the present study may be dependent on sexually dimorphic behavioral responses to housing condition and/or exposure to acute daily novelty stress. In addition to behavioral responses, physiological responses to a stressor have also been suggested to influence tumor growth (e.g. Anisman et al, 1989; Kandil & Borysenko, 1988; Peters & Kelly, 1977; Riley, 1981; Romero et al., 1992). Gender differences in the response of the HPA axis to stress are well established. For example, in response to a stressful or aversive situation, females have been shown to have higher levels corticotropin releasing hormone, ACTH and/or CORT compared to males (e.g. Davis et al, 1976; Gallucci et al, 1993; Handa et al, 1994; Lesniewska et al, 1990; Livezy et al, 1985). Importantly, these responses have been shown to be modified by gonadectomy and/or gonadal hormone replacement (Davis et al, 1976; Lesniewska et al, 1990). Therefore, a factor that may be mediating the differential tumor growth rates observed between male and female mice in the present study is the sexually dimorphic 157 responses of the HPA axis to housing condition and/or exposure to acute daily novelty stress. These responses may differentially alter steroid hormone levels which could either directly influence tumor cells or, alternatively, indirectly influence tumor growth rates via alterations in immune activity and/or the level of growth factors (e.g. basic fibroblast growth factor or epidermal growth factor; Ref. Kandil & Borysenko, 1988; Yamaguchi et al, 1992) present at the tumor site. Preliminary studies in our laboratory have demonstrated that the morphological differences between the SCI 15V tumor grown in male and female mice were similar to those observed in our previous work using the AR SCI 15 tumor (Rowse et al, 1990). That is, tumors grown in intact males had a cohesive epithelial-like growth pattern, whereas tumors from females contained cells dispersed into loose connective tissue. Although histological and phenotypic differences between tumors have been suggested by others to account for altered biological properties of the tumor, including responsiveness of the tumor cell populations to hormones and/or growth factors (Boyd & Kaufman, 1990; Inoue et al, 1990; Lancaster et al, 1988; Medina & Kittrell, 1987; Yates & King, 1981), we have shown that morphological differences of AR SCI 15 tumor cells derived from Gl or IG housed male mice do not correlate with differential AR SCI 15 tumor growth rates observed in vivo (Rowse et al, 1990). Thus, as shown for the AR SCI 15 tumor, SCI 15V tumor growth rates and morphology appear to be independently regulated by the host environment. In summary, the present data demonstrate that for both male and female mice, a change in social housing condition and the direction of change appear to be critical in determining whether SCI 15V tumor growth rates increase or decrease compared to those mice that do not experience change. Moreover, social housing condition and exposure to acute daily novelty stress appear to 158 interact to produce differential effects on tumor growth rates in males and females. Finally, like some human cancers, the SCI 15 and its variants are heterogeneous, consisting of a biological variety of cells that can respond differentially to the complex interaction between biological and environmental variables. Thus, although it is difficult to extrapolate from the mouse to the human situation, our data may provide some insight to at least one possible source of variability in the human data on psychosocial stressors and cancer. 159 Fig. 17. SCI 15V tumor weights (mean SEM) over 4 tumor measurement times: days 10-12, 13-14, 15-16, and 17-18 post tumor cell injection, for mice exposed to acute daily novelty stress (S) and left undisturbed (no novelty stress; NS) males (left panels, n's per housing condition: S: 8 GI, 6 GG, 15 IG; NS: 7 GI, 6 GG, 9 IG) and females (right panels, n's per housing condition: S: 21 GI, 18 GG, 34 IG; NS: 9 GI, 9 GG, 15 IG). Tumor growth rates for male mice exposed to acute daily novelty stress were significantly faster by days 17-18 for GI compared to IG housed mice (pO.OOl). Group housed mice who did not experience a change in housing condition (GG) had intermittent tumor growth rates (not significantly different from GI or IG). Growth rates of the SCI 15V tumor for male mice in the GI housing condition were also faster by days 17-18 for stressed compared to non-stressed mice. In contrast to the male data, tumor growth rates for female mice exposed to acute daily novelty stress were significantly slower by days 17-18 for GI compared to IG housed mice (p=0.01). In the non-stressed condition however, tumor growth rates were significantly faster by days 17-18 for GI compared to IG housed mice (pO.01). Growth rates of the SCI 15V tumor for female mice in the GI housing condition were also faster by days 15-16 for non-stressed compared to stressed mice (pO.01). Interestingly, only within the GI housing condition did males have significantly faster tumor growth rates compared to females (days 15-16; pO.05 and 17-18; pO.OOl). 160 LU < U l \— Q. r - < ^ hCBjqni 00 vi i vi I IT/ i vi © UJ < j L fO «N 00 vi I vi VO I i n vi I ro i-^  <N © m fN 161 CHAPTER 8. GENERAL DISCUSSION In general, the variability of chemotherapeutic efficacy is attributed to tumor and/or host heterogeneity (Boddy & Idle, 1993; Metz et al, 1988; Simpson-Herren, et al., 1987; Simpson-Herren et al., 1988). That is, any factor in the tumor or host that alters drug metabolism, distribution or excretion (affecting drug concentrations and the duration of drug exposure), or alters the sensitivity of tumor cell populations to drugs may contribute to differential tumor and host responses to chemotherapy. Several factors, including tumor growth rate, tumor size as well as endocrine and immune variables may act either independently or in an interactive fashion to influence chemotherapeutic efficacy (Armitage, 1992; Bassukas & Maurer-Schultze, 1993; Bell et al, 1990; Corbett et al, 1978; Goldin & Houchens, 1978; Goldin & Schabel, 1981; Henderson et al, 1988; Lippman et al, 1985; Mitchell, 1992; Mokyr & Dray, 1987; Rosso et al, 1971; Ueda et al, 1988). We have shown that psychosocial stressors, social housing conditions, differentially influence SCI 15 tumor growth rates, basal T, CORT and GH levels as well as tumor and host responses to chemotherapy (Kerr et al, 1997; Kerr et al, 1999c; Rowse et al, 1992; Weinberg & Emerman, 1989). The studies in this thesis utilized the SCI 15 mouse-tumor model to examine whether: (1) social housing conditions, tumor growth rate, tumor size and alterations in basal levels of CORT, T, and GH at the time of chemotherapy initiation differentially influenced tumor and/or host responses to chemotherapy; and (2) the timing of the formation of experimental housing conditions relative to tumor cell injection or chemotherapy initiation differentially influenced tumor growth rate or chemotherapeutic efficacy, respectively. Data from the studies reported in chapters 3-5 demonstrated that both tumor and host responses to chemotherapy are significantly influenced by experimental housing conditions and 162 that the temporal relationship between the formation of housing conditions and chemotherapy initiation plays a critical role in determining the direction and magnitude of the effects of social housing conditions on chemotherapeutic efficacy. Moreover, these studies suggested that the effects of social housing conditions were independent of tumor growth rate at the time of chemotherapy initiation and, at least in terms of host response to chemotherapy, were also independent of tumor burden. Specifically, in the first study of this thesis (Chapter 3) experimental housing conditions (IG and GI) were formed immediately following tumor cell injection and chemotherapy was initiated 14-20 d later, at a time when tumors weighed 1 g and were growing at different rates. Under these conditions, IG mice (slowest tumor growth rate) had better tumor responses to chemotherapy (longer TGD), better host response to chemotherapy (less weight loss) and a greater overall survival probability compared to GI mice (fastest tumor growth rate). Also, for non-tumor-bearing, chemotherapy-treated mice, host responses in terms of body weight loss and survival probability were similar for IG and GI mice suggesting that the differences in survival probability observed in the tumor-bearing, chemotherapy-treated mice were due to differences in tumor responses to chemotherapy and not to differential toxic side effects in the host (Kerr et al, 1997). However, questions remained regarding the factors underlying these differential responses. Since chemotherapy was initiated when tumors, albeit at similar weights (lg), were growing at different rates (i.e. faster in GI than in IG mice), the differential responses to chemotherapy may have been due to differences in social housing conditions, differences in tumor growth rates, or an interaction between these factors. The next two studies (Chapters 4 and 5) were undertaken to begin to address these issues. In the study in Chapter 4, experimental variables were manipulated such that 163 chemotherapy was initiated when tumors weighed 1 g (as in the study in Chapter 3) but were growing at similar rates. To accomplish this, tumor cells were injected but mice remained in their original rearing conditions (I or G) until the mean tumor weight of mice within that rearing condition reached 1 g; this occurred approximately 14 d post tumor cell injection for both I and G housed mice. At that time, mice were rehoused either from individual to group (IG) or from group to individual (Gl) or remained in their original rearing conditions (II or GG). Chemotherapy was initiated 1 d following formation of experimental housing conditions. Thus, unlike the previous study in which experimental housing conditions were formed immediately following tumor cell injection and 14-18 d prior to chemotherapy initiation, in the study in Chapter 4, experimental housing conditions were formed approximately 14 d following tumor cell injection and 1 d prior to chemotherapy initiation. The data of Chapter 4 demonstrated that altering the temporal relationship between formation of social housing conditions and tumor cell injection differentially influenced tumor growth rates in mice of the different social housing conditions. In contrast to the data of Chapter 3, if mice remained in their original I or G housing conditions following tumor cell injection and experimental housing conditions were formed approximately 14 d later, when tumors weighed 1 g but were growing at similar rates, II mice had significantly faster tumor growth rates than both IG and GG mice, whereas Gl mice had intermediate tumor growth rates. Importantly, these data also demonstrated that under the temporal conditions of this study, Gl mice had better tumor (longer TGD) and host (less weight loss) responses to chemotherapy compared to both II and IG mice, yet no differences in survival probabilities were observed among housing conditions. However, for non-tumor-bearing, chemotherapy-treated mice, IG mice had the poorest host response (greater weight loss) and the lowest survival probability compared to mice in all other housing 164 conditions (Kerr et al, 1999a). The study in Chapter 5 continued to examine possible variables that may influence the differential responses to chemotherapy. Specifically, this study examined whether initiating chemotherapy 1 d following tumor cell injection and formation of experimental housing conditions, but at a time when tumor burden was undetectable (similar to the adjuvant situation in humans), differentially influenced the effects of housing conditions on tumor and host responses to chemotherapy. The data in this study demonstrated that when chemotherapy is initiated 1 d after formation of social housing conditions and tumor cell injection, social housing condition does not affect tumor response to chemotherapy. That is, chemotherapy was equally effective in inhibiting tumor growth in all mice. However, differential host responses to chemotherapy were observed among the experimental housing conditions; IG mice lost the most weight and had a lower survival probability compared to mice in all other social housing conditions (Kerr et al, 19996). These data are consistent with those of Chapter 4 in which experimental housing conditions were similarly formed 1 d before chemotherapy initiation, but at a time when tumors weighed approximately 1 g. Importantly, these data are in contrast to those of Chapter 3 in which formation of experimental housing conditions occurred 14-18 d before the initiation of chemotherapy but when tumors also weighed approximately 1 g. Under those conditions, morbidity and mortality were significantly lower in IG compared to Gl mice (Kerr etai, 1997). Together, the data of Chapters 3-5 demonstrate that social housing conditions can significantly influence chemotherapeutic efficacy and that tumor and host responses to chemotherapy interact to influence differentially TGD, weight loss, and survival probability. Moreover, these data highlight the importance of the temporal relationship between formation 165 of social housing conditions and initiation of chemotherapy on chemotherapeutic efficacy. However, the effects of social housing conditions on chemotherapeutic efficacy appeared to be independent of tumor growth rate at the time of chemotherapy initiation and, at least in terms of host response to chemotherapy, were also independent of tumor burden. The data in Chapter 6 of this thesis examined possible endocrine mediators that may play a role in differentially influencing the effects of social housing conditions on chemotherapeutic efficacy. We had shown previously that for male mice in our standard laboratory housing conditions (group housed and not subjected to daily novelty stressors), SCI 15 tumor response to AD and CY can be modulated by altering tumor growth rate via the administration of different doses of exogenous T following castration (Emerman & Siemiatkowski, 1984). Others have demonstrated that increased levels of CORT can suppress the antitumor activity of CY but may increase the toxic side effects of chemotherapeutic drugs such as AD and methotrexate (Kodama & Kodama, 1982). It has also been shown that increased levels of GH may directly affect tumor cell proliferation, including SCI 15 tumor proliferation (Mertani et al, 1998;Noguchi et al, 1993) as well as attenuate chemotherapy-induced decreases in immune activity and body weight (Harrison et al, 1995; Ng et al, 1993; Wolfe/ al, 1994) and may therefore play a role in augmenting chemotherapeutic efficacy. We have shown previously that social housing condition differentially alters the levels of CORT and T in mice at 1-7 days but not 21 days after the formation of social housing conditions (Rowse et al, 1992; Weinberg & Emerman, 1989). That is, up to one week following tumor cell injection and formation of social housing conditions, basal T levels were significantly higher in GI mice and CORT levels were higher in IG mice compared to mice in all other housing conditions. Also, in a preliminary study we demonstrated that GH levels at 3 d post 166 tumor cell injection and formation of social housing conditions are significantly higher in Gl mice compared to GG and IG mice. Others have shown that chemotherapy can significantly alter plasma levels of CORT, T and GH (Castagnatta et al, 1985; McClure & Stupans, 1995). Chapter 6 examined whether psychosocial stressor (housing condition)- or chemotherapy-induced alterations in basal plasma levels of CORT, T and/or GH either at the time of chemotherapy/drug vehicle initiation (day 0) or 1 or 5 d following chemotherapy initiation played a role in the differential tumor and host responses to chemotherapy. The two experiments in this study demonstrated that alterations in the levels of CORT and GH but not T within the first week following formation of experimental housing conditions and chemotherapy initiation may play a role in mediating the differential effects of social housing conditions on tumor and host responses to chemotherapy. In the first experiment in Chapter 6, experimental housing conditions (II, IG, GG and Gl) were formed immediately following tumor cell injection and chemotherapy was initiated 16-22 d later, at a time when tumors weighed 1 g and were growing at different rates. Under the conditions of this experiment, basal plasma levels of CORT, T and GH did not differ among tumor-bearing, chemotherapy-treated mice in the different housing conditions. Importantly however, the effects of social housing conditions on tumor and host responses to chemotherapy observed in this study, supported and extended our previous findings (Kerr et al, 1997) demonstrating that IG and GG mice had better tumor (longer TGD) and host (less weight loss) responses compared to Gl mice. Overall, these data suggest that at 2-3 weeks following tumor cell injection and experimental housing condition formation, the differences in tumor and host responses to chemotherapy observed among the different housing conditions were not due to alterations in CORT, T or GH levels. In the second experiment in Chapter 6, experimental variables were manipulated such 167 that chemotherapy was initiated when tumors weighed 1 g but were growing at similar rates. Thus, unlike the first experiment, tumor cells were injected but mice remained in their original rearing conditions (I or G) until the mean tumor weight of mice within each rearing conditions reached lg; this occurred approximately 14 d post tumor cell injection for both I and G housed mice. At that time, mice were rehoused into the experimental housing conditions (II, IG, GG or GI). Chemotherapy was initiated 1 d following formation of experimental housing conditions. Thus, in the first experiment of Chapter 6, experimental housing conditions were formed immediately following tumor cell injection and 16-22 d prior to chemotherapy initiation whereas in the second study, experimental housing conditions were formed 14 d following tumor cell injection and 1 d prior to chemotherapy initiation. The data of the second experiment in Chapter 6 demonstrated that in contrast to the first experiment, tumor and host responses were similar among housing conditions. Although the differences among tumor and host responses to chemotherapy (measured at only 4 d post chemotherapy initiation) in this experiment failed to reach significance, it is of relevance that the differences observed are in a similar direction as those in the study in Chapter 4 (tumor and host responses measured 30-70 d post chemotherapy initiation). This previous study demonstrated that GI mice had better tumor (longer TGD) and host (less weight loss) responses to chemotherapy compared to both II and IG mice; Ref. Kerr et al, 1999a). Moreover, in this previous study, when tumor and host responses to chemotherapy were measured at 4 d post chemotherapy initiation, no significant differences were observed among mice in the different housing conditions. Examination of basal hormone levels under the temporal conditions of the second experiment in Chapter 6 revealed that at the time of chemotherapy initiation (day 0), CORT levels were significantly higher in IG mice compared to those in all other housing conditions 168 and GH levels were significantly higher in Gl mice compared to those in all other housing conditions. Also, at 5 d post chemotherapy injection, GH levels were significantly higher in Gl than in IG mice. Interestingly, no significant differences in T levels in mice in the different housing conditions were observed. Thus, alterations in CORT or GH levels among mice in the different housing conditions may play a role in influencing tumor and host responses to chemotherapy. It is unlikely, however, that hormonal alterations in mice in the different housing conditions are the only factors mediating the differential effects of housing condition on chemotherapeutic efficacy. Future studies could also examine immune and tumor-specific factors as well as active drug metabolite ratios and/or the interaction among these factors to elucidate further the mechanisms that may be playing a role in differentially influencing tumor and host responses to chemotherapy. Overall, the data of Chapter 6 support and extend our previous studies suggesting that the temporal relationship between formation of social housing condition and chemotherapy initiation plays an important role in determining the effects of social housing condition on chemotherapeutic efficacy. However, further studies examining possible alterations in hormone levels among mice in the different housing conditions following subsequent chemotherapy injection rounds are required to elucidate further the possible effects of alterations in the temporal relationship between formation of social housing condition and initiation of chemotherapy on hormone levels and chemotherapeutic efficacy. In addition, other factors (e.g. immune activity, tumor cell population characteristics) may also be involved in mediating the differential temporal effects of housing condition on hormones levels and chemotherapeutic efficacy. These possibilities remain to be examined. The final study of this thesis (Chapter 7) examined the interactive effects of social 169 housing condition and gender on the growth of the SCI 15V tumor, that grows equally well in males and females. The data in Chapter 7 demonstrated that in the presence of acute daily novelty stress, the growth rate of the SCI 15V tumor was significantly increased in Gl compared to IG males. However, no significant differences in tumor growth rates among nonstressed Gl, IG or GG males were observed. For females, in contrast to males, acute daily novelty stress significantly increased tumor growth in IG compared to Gl mice, whereas under nonstressed conditions, tumor growth rate was significantly increased in Gl compared to IG females. Neither housing condition nor novelty stress altered estrous cyclicity, nor did the stage of the estrous cycle at the time of tumor cell injection influence tumor growth rates (Kerr et al, 1999c). These findings suggest that social housing condition and novelty stress interact to produce differential effects on the growth rate of the SCI 15V tumor in male and female DD/S mice. Although the specific mechanisms governing differential SCI 15V tumor growth rates between males and females remain to be identified, it is possible that sexually dimorphic behavioral and/or physiological responses to housing condition and/or exposure to acute daily novelty stressor may differentially influence tumor growth rates in male and female mice. Future Studies. The data presented within this thesis indicate that social housing conditions can influence significantly tumor and host responses to chemotherapy. Moreover, these data demonstrate that the direction and magnitude of the effects of social housing conditions on chemotherapeutic efficacy is dependent on the temporal relationship between the formation of experimental housing conditions and initiation of chemotherapy and that the differential effects observed may be mediated, in part, through alterations in basal hormone levels. However, other studies are required to elucidate further the mechanisms by which social housing 170 conditions differentially influence chemotherapeutic efficacy. Possible future studies include: (1) A longitudinal study examining corticosterone, testosterone and growth hormone levels over the full course of the chemotherapy regimen may further elucidate the possible roles of these hormones in mediating the influence of social housing condition on chemotherapeutic efficacy. In addition, in order to establish the possible roles of these hormones on tumor and host responses to chemotherapy (as well as on tumor growth rate), exogenous hormones levels, simulating those in mice in the different housing conditions, could be administered to mice depleted of endogenous hormones (either via pharmaceutical or surgical methods). At various intervals following hormone administration, tumor growth rate as well as tumor and host responses to chemotherapy could be monitored to ascertain whether certain hormone levels or combination of hormone levels differentially affected tumor growth rate or tumor and host responses to chemotherapy. If specific hormones or combinations of hormones are identified in affecting tumor growth rate or tumor and host responses to chemotherapy then other studies may examine possible mechanisms of these effects. For example, the effects of hormones on tumor growth may be investigated by examining the expression or activity of receptors for the identified hormones. Also, the expression of proteins or various second messenger systems that are associated with the hormone-mediated tumor growth could be examined to identify cellular mechanisms that may be related to tumor growth. (2) It is possible that alterations in hormone levels are not the only mediators of the differential effects of social housing conditions on chemotherapeutic efficacy; housing condition-induced alterations in immune activity may also play a role either with or without hormonal mediation 171 (Berkenbosch et al, 1991; Bonneau et al, 1990; Bovbjerg, 1991; Grossman, 1985; Kandil & Borysenko, 1988; Riley, 1981). To address this possibility, immune activity at the level of the spleen and of the tumor could be examined concurrently with hormone level measurements over the course of the chemotherapy regimen. Data from our laboratory have suggested that the SCI 15 tumor stimulates natural killer (NK) cell and cytotoxic T-lymphocyte (CTL) activities (Rowse et al, 1990; Rowse et al, 1995). These immune effector cells in turn play a role in modulating SCI 15 tumor growth rate (Rowse et al, 1995), and may also influence the cytotoxic effects of chemotherapeutic agents at both the level of the tumor (Brunda et al, 1978; Fearon et al, 1990; Ionnides & Whiteside, 1993; Lafrenier et al, 1989) and that of the host (Gruber et al, 1988; Gruber et al, 1993; Lechin et al, 1990; Levy et al, 1987). Examination of NK cell and CTL activities at the spleen as well as at the tumor site concurrently with hormonal alterations in mice in the different housing conditions may reveal whether housing condition-induced alterations these immune effector cells, hormone levels or an interaction between these factors plays a role in mediating the effects of social housing conditions on chemotherapeutic efficacy. In addition, the concurrent examination of immune activity at the spleen as well as at the tumor site would help to elucidate possible effects that the different housing conditions may have in immune effector cell cycling; other stressors have been shown to affect differentially the levels of immune effector cells within the spleen and in various other tissues (Keller et al, 1991). Differences in the number of immune effector cells within a given tissue compartment may alter tumor and/or host responses to chemotherapy (Maclean & Longnecker, 1994; North, 1982; North, 1984). (3) Both endocrine and immune activities are mediated by the activity of the central nervous 172 system (CNS). Importantly, stressors have been shown to affect differentially CNS, endocrine and immune system activities (e.g. Black, 1994; Bovbjerg, 1991; Kusnecov & Rabin, 1994; Rabin et al, 1989; Reichlin, 1993; Rivier & Rivest, 1991). It would be of interest to examine whether the differential effects of social housing conditions on chemotherapeutic efficacy are related to alterations in neurotransmitter levels. Possible neurotransmitters examined could include norepinephrine (NE), epinephrine (E) and serotonin (5-HT), three neurotransmitters known to be differentially affected by social stressors (Christensen & Jensen, 1995; Delbende et al, 1992; Ely, 1995), influence coping responses to stressors (Anisman et al, 1993; Baum & Posluszny, 1999; Henry, 1982; Steptoe, 1991) and mediate endocrine and immune activity (Anisman et al, 1993; Felten & Felten, 1991; Henry, 1982; Madden & Livnat, 1991; Roszman & Carlson, 1991). Possible brain regions in which to examine the expression or levels of these neurotransmitters include those related to the HPA axis (e.g. hypothalamus, hippocampus) as well as those that mediate emotional/social activities in the rodent (e.g. amygdala, prefrontal cortex). This experiment, examining possible alterations in neurotransmitter levels in brain areas associated with stressors, coping mechanisms and alterations in endocrine and immune activities could help to elucidate whether different CNS factors are involved in mediating alterations in endocrine or immune activities in mice among the different housing conditions as well as those that may influence the general effects of social housing conditions on chemotherapeutic efficacy. (4) It is also possible that alterations in gene expression within the tumor cell population in mice in the different housing conditions may directly affect tumor response to chemotherapy. Indeed, studies have demonstrated a correlation between expression of estrogen and 173 progesterone receptors and psychosocial variables; ER- or PR-negative tumors benefit from chemotherapy, whereas ER- or PR-positive tumors benefit from hormone therapy, e.g. tamoxifen. Razavi et al. (1990) demonstrated that of 93 patients with primary breast cancer, 75 with receptor-positive tumors were better adjusted psychologically than the 18 patients with receptor-negative tumors. Thus, patients' coping responses appear to correlate with tumor cell population characteristics and may help to determine the type of therapy from which they may benefit. Interestingly, these investigators suggested that the relationship of psychosocial variables and ER/PR status may be influenced by stressor-induced neurohormonal stimulation and/or alterations in immune function. Data from our laboratory have shown that SCI 15 tumor cell expression of heat shock proteins (HSPs) 25, 70 and 90 are differentially affected by social housing conditions and that corticosterone and testosterone, which are known to regulate SCI 15 growth, also alter HSP 25, 70 and 90 expression following direct exposure in vitro (Andrews et al, 1999). Importantly, elevated levels of HSP expression have been associated with both tumor growth (possibly due to alterations in hormone activity; Ref. Cai et al, 1993; Ciocca et al, 1993; Ferrarini et al, 1992) and tumor resistance to chemotherapy (Ciocca et al, 1992; Dunn et al, 1993; Fuqua et al, 1994; Oesterreich et al, 1993; Richards et al, 1996). Preliminary experiments in our laboratory (using the same experimental paradigms as that used in Chapter 4; experimental housing conditions were formed when tumors reached approximately 1 g and chemotherapy was initiated the following day) suggested that alterations in HSP expression in tumors may be associated with the differential effects of social housing conditions on tumor response to chemotherapy (Kerr et al, 1998). Specifically, prior to chemotherapy, HSP 25 expression was reduced in II mice compared to mice in all other housing conditions, whereas following chemotherapy, HSP 25 expression was increased in 174 mice in only the II condition. Together with the data of Chapter 4 demonstrating that II mice had the poorest tumor response to chemotherapy (i.e. shortest tumor growth delay), the results suggest that a reduction in tumor expression of HSP 25 at the time of chemotherapy initiation may be associated with poor tumor response to chemotherapy. Future studies could examine concurrently, the relationship among social housing condition, HSP expression in tumors, hormone levels and tumor response to chemotherapy over the chemotherapy regimen. In summary, the studies in this thesis highlight the possible impact of psychosocial stressors on the complex interrelationship among the host environment, tumor growth and chemotherapeutic efficacy. 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