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Opioid inhibition of the growth of the transplantable androgen-responsive (AR) Shionogi mouse mammary… Jian, Yi 1993

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OPIOID INHIBITION OF THE GROWTH OF THE TRANSPLANTABLEANDROGEN-RESPONSIVE (AR) SHIONOGI MOUSE MAMMARY CARCINOMA(SC115)byYI JIANGM.D. (Medicine) Shanghai First Medical UniversityA THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIESDEPARTMENT OF ANATOMYWe accept this thesis as conformingto the required standardUNIVERSITY OF BRITISH COLUMBIAAUGUST 1993© Yi Jiang,^1993In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of The University of British ColumbiVancouver, CanadaDateDE-6 (2/88)IIABSTRACTWe investigated the effects of steroid hormones, growthfactors, and anti-growth factor antibodies on the growth of cellsin primary culture from the transplantable androgen-responsive(AR) Shionogi mouse mammary carcinoma (SC115). In addition, theeffects of opioid agonists and antagonists on the growth of SC115tumor cells in primary culture were examined. The possible roleof opioids in mediating the differential tumor growth rates ofmice exposed to experimental housing groups was also examined.SC115 tumor cells were cultured at 5 x 104 cells/cm2 ontocollagen-coated 96-well microtiter tissue culture plates. Thecells were incubated in medium containing 5% fetal bovine serum(FBS), 2% dextran charcoal-treated FBS (DCTFBS), or seurm-freemedium, with or without different concentrations ofdihydrotestosterone (DHT), hydrocortisone (HC), 178-estradiol(E2), basic fibroblast growth factor (bFGF), epidermal growthfactor (EGF), anti-bFGF antibody with either DHT or bFGF, oranti-EGF antibody with either E2 or EGF. After 48 h, some of thecultures were exposed to 10-14-10 -7 M of either 8-endorphin (5-EP), which binds to the 8 opioid receptor, cyclazocine (CZ),which binds to the k receptor, or morphine sulfate (MS), whichbinds to the g receptor. These opioids were added daily for 3 d.In some experiments, 10-8 and 10 -6 M of naloxone, an opioidreceptor antagonist, were added with the opioids. On day 5, thecultures were terminated and cell numbers determined by thetetrazolium dye reduction assay.iiiThe results show that AR SC115 tumor cells in serum-containing medium were significantly stimulated to grow in adose-dependent manner with DHT ranging from 3.5 x 10-1° - 3•5 x10-6 M and bFGF ranging from 1-100 ng/ml and in serum-free mediumwere significantly stimulated to grow by bFGF at allconcentrations examined (1-500 ng/ml). HC ranging from 10-9-10-5M also significantly stimulated SC115 tumor cell growth. Incontrast, E2 (10-9-10-6 M) and EGF (1-100 ng/ml) at allconcentrations examined significantly inhibited SC115 tumor cellgrowth in serum-containing medium, whereas EGF had no effect onSC115 tumor cell growth in serum-free medium at any of theseconcentrations. Anti-bFGF antibody had a significant inhibitoryeffect on the DHT- or bFGF-stimulated SC115 tumor cell growth inserum-free medium. Anti-EGF antibody also significantly inhibitedSC115 tumor cell growth with or without E2 or EGF in serum-freemedium. All 3 opioids at concentrations higher than 10-12 Msignificantly inhibited S0115 tumor cell growth (up to 40%) inmedium that maximally stimulated these cells (DHT at 3.5 x 10-8M, HC at 10-6 M, or 5% FBS), although inhibition also occured,albeit to a lesser degree, in steroid hormone-free medium. 8-EPat concentrations of 10-10-10-7 M also inhibited SC115 tumor cellgrowth when added to medium containing DCTFBS and 10 ng/ml bFGF.When naloxone was added with 8-EP or MS, the inhibitory effectsof the opioids were partially or totally blocked. Both 8-EP andCZ also significantly inhibited growth of SC115 tumor cells frommice exposed to different social housing conditions. Overall, 5-EP inhibited growth of SC115 tumor cells from IG mice (smallerivtumors) to a greater degree than SC115 tumor cells from GI mice(larger tumors) in 2% DCTFBS-containing and HC-containing media,whereas the result was reversed in DHT-containing medium. CZinhibited growth of SC115 tumor cells from GI mice to a greaterdegree than cells from IG mice in 2% DCTFBS-containing and DHT-containing media, whereas the result was the same in HC-containing medium.The results suggest that the growth of AR SC115 tumor cellsis sensitive to different steroid hormones and growth factors inprimary culture. Opioid peptides may be involved in regulatingendocrine control of growth of the AR SC115 carcinoma and theinhibitory effects of opioids may be mediated by multiple opioidreceptors. In our animal-tumor model, opioids may also play arole in the differential tumor growth rates of mice exposed tothe experimental housing groups.TABLE OF CONTENTSPAGEABSTRACTTABLE OF CONTENTSLIST OF FIGURESLIST OF ABBREVIATIONS^ xiiiACKNOWLEDGEMENTS xviI. Introduction^1.1^General Introduction^1.2^Human Breast Cancer121.3 Effects of Androgens on the Growth of the SC115Tumor 71.4 Effects of Glucocorticoids on the Growth of theSC115 Tumor 121.5 Effects of Estrogens on the Growth of the SC115Tumor 151.6 The Proliferation of Androgen-induced SC115 TumorCells may be Mediated by a FGF-like Polypeptidethrough an Autocrine Mechanism 171.7 The Endogenous Opioid Peptide^(EOP)^System 191.8 EOP Interaction with Endocrine Functions 231.9 EOP Interaction with Immune Functions 241.10 Opioids and Tumors 301.11 Animal-tumor Model for Studing Effects ofStressors on Mammary Tumor Growth 321.12 The Objectives of the Thesis 37viII. Materials and Methods^ 39^2.1^General Methods 392.1.1 Materials^ 39(1) Steroid Hormones and Growth Factors^39(2) Opioids^ 402.1.2 Maintaining the SC115 Tumor^ 402.1.3 Harvesting Cells^ 41(1) Hemacytometer Counts 42(2) The MTT Assay^ 432.1.4 Animal-tumor Model for Studing Effectsof Stressors on Mammary Tumor Growth^432.2^Methods for Preliminary Experiments^ 452.2.1 Seeding Cells on Day 0 Versus Day 1^452.2.2 Seeding Density Experiment^ 462.2.3 Selecting an Appropriate Serum-freeMedium for the Growth of SC115 TumorCells in Primary Culture^ 462.2.4 Determining Time Points for Studing theEffects of Opioids on SC115 TumorCell Growth^ 472.3^Experiments^ 482.3.1 Effects of steroid Hormones and GrowthFactors on the Growth of SC115 TumorCells in Serum-containing Medium^482.3.2 Effects of Steroid Hormones, GrowthFactors, and Anti-growth Factors on theGrowth of SC115 Tumor Cells in Serum-freeviiMedium^ 492.3.3 Effects of Opioids on the Growth ofSC115 Tumor Cells^ 502.3.4 Effects of the Opioid Antagonist Naloxoneon the Growth of SC115 Tumor Cells^512.3.5 Effects of Opioids on the Growth ofSC115 Tumor Cells from the Animal-tumor Model^ 51^2.4^Statistical Analysis 52III. Results^ 533.1^Results of Preliminary Experiments to DevelopProcedures^ 533.1.1 Seeding Cells on Day 0 Versus Day 1^533.1.2 Seeding Density Experiment^ 533.1.3 Selecting an Appropriate Serum-freeMedium for the Growth of SC115 TumorCells in Primary Culture^ 583.1.4 Determining Time Points for Studing theEffects of Opioids on SC115 TumorCell Growth^ 613.2^Experimental Results 693.2.1 Effects of Steroid Hormones and GrowthFactors on the Growth of SC115 TumorCells in Serum-containing Medium^69(1) Steroid Hormones^ 69(2) Growth Factors 72viii3.2.2 Effects of Steroid Hormones, GrowthFactors, and Anti-growth Factors on theGrowth of SC115 Tumor Cells in Serum-free Medium^ 72(1) Effects of DHT and bFGF on the Growth ofSC115 Tumor Cells^ 72(2) Inhibitory Effects of Anti-bFGF Antibodyon DHT- or bFGF-induced Growth of SC115Tumor Cells in Serum-free Medium^77(3)^^Effects of E2, EGF, and Anti-EGF Antibodyon the Growth of SC115 Tumor Cells inSerum-free Medium^ 803.2.3 Effects of Opioids on the Growth of SC115Tumor Cells^ 803.2.4 Effects of the Opioid Antagonist Naloxoneon the Growth of SC115 Tumor Cells^883.2.5 Effects of Opioids on the Growth of SC115Tumor Cells from the Animal-tumor Model^93(1) Effects of Steroid Hormones on the Growthof SC115 Tumor Cells from Mice inExperimental Housing Groups^ 93(2) Effects of Opioids and Naloxone on theGrowth of SC115 Tumor Cells from Micein Experimental Housing Groups^93IV. Discussion and Conclusions^ 1154.1^Culturing AR SC115 Tumor Cells on a CollagenixSubstrate^ 115^4.2^Growth-stimulation of AR SC115 Tumor Cellsby Steroid Hormones and Growth Factors^1164.3^Growth-stimulation of AR 5C115 Tumor Cellsby Steroid Hormones is Inhibited by OpioidAgonists^ 1204.4^Opioid Agonists Inhibit the Growth of AR SC115Tumor Cells from Animals in the ExperimentalConditions of our Animal-tumor Model^1244.5^Concluding Remarks^ 126V. References^ 130VI. Appendices^ 143LIST OF FIGURESxFigure 1 Tumor growth in male mice in the four housinggroups 34Figure 2 Seeding cells on day 0 versus day 1 54Figure 3 Seeding density experiment 56Figure 4 Growth of SC115 tumor cells in serum-free^(SF)medium 59Figure 5 Effects of opioids on the growth of SC115 tumorcells cultured on collagen-coated dishes after3 d in primary culture 62Figure 6 Effects of opioids on the growth of SC115 tumorcells cultured on collagen-coated dishes after5 d in primary culture 64Figure 7 Effects of opioids on the growth of SC115 tumorcells cultured on collagen-coated dishes after7 d in primary culture 67Figure 8 Effects of DHT, HC, or E2 on the growth of primarycultures of SC115 tumor cells 70Figure 9 Effects of bFGF and EGF on the growth of primarycultures of S0115 tumor cells 73Figure 10 Effects of DHT, bFGF,^or DHT^(3.5 x 10-8 M)plus bFGF on the growth of primary cultures ofS0115 tumor cells in serum-free medium 75Figure 11 Effects of anti-bFGF antibody on DHT- or bFGF-stimulated SC115 tumor cell growth in serum-freemedium 78xiFigure 12 Effects of EGF or E2 on the growth of primarycultures of SC115 tumor cells in serum-freemedium 81Figure 13 Effects of anti-EGF antibody on SC115 tumorcell growth in serum-free medium with or withoutE2 or EGF 83Figure 14 Effects of 8-endorphin^(8-EP),^cyclazocine^(CZ),and morphine sulfate^(MS)^on the growth of S0115tumor cells cultured in medium with or withoutDHT,^HC,^or 5% fetal bovine serum^(FBS) 86Figure 15 Effects of 8-EP on the growth of SC115 tumorcells cultured in medium containing bFGF(10 ng/ml) 89Figure 16 Effects of opioids and naloxone on the growthof primary cultures of SC115 tumor cells 91Figure 17 Effects of naloxone on the growth of primarycultures of S0115 tumor cells 94Figure 18 Effects of DHT and HC on the growth of primarycultures of SC115 tumor cells from mice inexperimental housing groups 96Figure 19 Effects of 8-EP or CZ on the growth of primarycultures of SC115 tumor cells from mice inexperimental housing groups 99Figure 20 Effects of 5-EP or CZ on the growth of primarycultures of SC115 tumor cells from mice inexperimental housing groups 101Figure 21 Effects of 8-EP or CZ on the growth of primaryFigure 22cultures of SC115 tumor cells from mice inexperimental housing groupsEffects of 8-EP plus naloxone in basic mediumon the growth of primary cultures of SC115 tumorxi i104cells from mice in experimental housing groups 106Figure 23 Effects of 8-EP plus naloxone in DHT-containingmedium on the growth of primary cultures of S0115tumor cells from mice in experimental housinggroups 109Figure 24 Effects of 8-EP plus naloxone in HC-containingmedium on the growth of primary cultures of SC115tumor cells from mice in experimental housinggroups 111Figure 25 Effects of naloxone on the growth of primarycultures of SC115 tumor cells from mice inexperimental housing groups 113LIST OF ABBREVIATIONSa-,13-,y-MSH alpha, beta, gamma-melanocyte-stimulating hormoneACTH^adrenocorticotropic hormoneAl androgen-independentANOVA^analysis of varianceAR androgen-responsiveB-EP^beta-endorphinbFGF^basic fibroblast growth factorB-LPH^beta-lipotropinBSA bovine serum albuminCA^cyproterone acetateCM conditioned mediumConA^concanavalin ACTL cytotoxic T lymphocyteCZ^cyclazocineDCT dextran charcoal-treatedDCTFBS^dextran charcoal-treated fetal bovine serumDex dexamethasomeDHT^dihydrotestosteroneDMEM^Dulbecco's modified Eagles mediumDNA deoxyribonucleic acidE 2^178-estradiolEDTA ethylenediamine tetraacetic acidEGF^epidermal growth factorEOP endogenous opioid peptide(s)ER^estrogen receptorxivER+^estrogen receptor positiveER- estrogen receptor negativeETCH^ethyl alcoholFBS fetal bovine serumFSH^follicle-stimulating hormoneHC hydrocortisoneHC1^hydrochloric acidIFN interferonIGF^insulin-like growth factorIGF-I^insulin-like growth factor-IIL interleukiniv^intravenouskDa kilodaltonLH^luteinizing hormoneLPS bacterial lipopolysaccharideMS^morphine sulfateMTT [3-(4,5-Dimethylthiazol-2-y1)-2,5-diphenyltetrazolium] bromideNK cell^Natural Killer cellPDGF^platelet-derived growth factorPgR+^progesterone receptor positivePHA phytohemagglutininPOMC^proopiomelanocortinPWM pokeweed mitogensc^subcutaneous5C115^Shionogi mouse mammary carcinomaSC-3^AR cell line derived from the 5C115 tumorXVSRBC^sheep red blood cellSTV saline-trypsin-verseneT^testosteroneTGF transforming growth factorTP^testosterone propionateACKNOWLEDGEMENTSI would like to thank my research advisors, Dr. Joanne T.Emerman and Dr. Joanne Weinberg, for all the time, effort, andhelp they have given me throughout my thesis research project. Ithas been a true learning experience working in theirlaboratories. Without their guidance and encouragement, thisthesis would still be in progress. I am very grateful to themembers of my thesis committee, Dr. Bruce Crawford and Dr. PeterLeung, for their time and helpful comments. Thanks to theexternal examiner, Dr. Donald Brunett, for his time and comments.Thanks also to Darcy Wilkinson and Shannon Wilson for theirtechnical assistance and Gerry Rowse for helping out with theexperiments and for providing advice on many aspects of thisthesis.I am grateful to my husband, Anming Zhang, for all the moralsupport, constant encouragement, and understanding throughoutthese years and to my parents for their caring and support. Thiswork is dedicated to them.xvi1I. Introduction1.1 General Introduction Since the endorphin-like drugs have become increasinglyuseful for managing pain in patients with advanced cancer, it isimportant to determine if these same drugs affect tumor growth,thereby ultimately affecting patient survival. This researchproject was designed to investigate the effects of opioids on thegrowth of cells from the transplantable androgen-responsive (AR)Shionogi mouse mammary carcinoma (SC115) in primary culture.The AR S0115 tumor, established by Minesita and Yamaguchi in1964, has been considered an excellent model to study theproliferative effects of steroid hormones as it contains severalsteroid hormone receptors, including androgen receptors(Bruchovsky & Rennie 1978; Matsumoto et al. 1982), estrogenreceptors (Noguchi et al. 1984; 1985b), and glucocorticoidreceptors (Watanabe et al. 1982) and has been shown to besensitive to androgens (Darbre & King 1987; Jung-Testae et al.1988; Matsumoto et al. 1982; Yates & King 1978), estrogens(Noguchi et al. 1984; 1985a; 1985b; 1987), and glucocorticoids(Omukai et al. 1987; Watanabe et al. 1982). This tumor originatedspontaneously in a female mouse of the DD/S strain. After 19passages in male mice, an AR variant arose and grew more rapidlyin males than in females. This AR SC115 tumor has maintained itsresponsiveness to physiological concentrations of androgens invivo (Matsumoto et al. 1982) and in vitro (Desmond et a/. 1976;Yates et al. 1980; Yates & King 1978) for many years.Furthermore, the growth of the SC115 tumor is also stimulated by2pharmacological doses of glucocorticoids both in vivo (Omukai etal. 1987; Watanabe et al. 1982) and in vitro (Omukai et a/. 1987)and by pharmacological doses of estrogens in vivo (Noguchi et al.1987). The growth-stimulatory effects of androgens, estrogens,and glucocorticoids are mediated through their receptors. Using acompetitive [3H]-binding assay, androgen receptor complexes canbe isolated from both nuclear and cytosol fractions of SC115tumor cells, which are taken from tumors or culture (Matsumoto etal. 1982). These androgen receptor complexes of SC115 tumor cellshave similar binding properties to receptors isolated from normalandrogen target tissues. The antiandrogen, cyproterone acetate(CA), which by itself is inactive, completely suppresses theandrogen-induced effects on growth in vitro (Jung-Testae et al.1985; 1988). Recent evidence suggests that the stimulatoryeffects of androgens and glucocorticoids on SC-3 cellproliferation (an AR cell line derived from the SC115 tumor) maybe mediated by the synthesis of a fibroblast growth factor-likepolypeptide that acts on the cells in an autocrine or paracrinemanner (Lu et a/. 1989; Nakamura et al. 1989; Nonomura et a/.1988; 1989). This mouse mammary tumor was selected for studing asit is similar to some human breast cancers in its sensitivity tosteroid hormones.1.2 Human Breast Cancer It is well known that 1 in 9 women in America will developbreast cancer sometime during their lifetime. This fact hasstimulated a major research effort to develop curative procedures3for this disease. Historically, treatments applied specificallyto breast cancer have been based on altering the endocrineenvironment of the tumors (Wakeling 1990). Estrogen receptors(ER) can be detected in 60-80% of human breast cancers. Thepresence of ER in breast cancer is used as a marker forpredicting likely responses to endocrine (i.e., antiestrogen)therapy. Of patients with ER positive (ER+) tumors, 50% will showa beneficial response; of those with ER+ and progesteronereceptor positive (PgR+) tumors, 75% will respond to therapy(Horwitz 1987). The presence of ER has also been known to reflectthe grade of this tumor; less differentiated, more aggressivetumors tend to be ER negative (ER-; Horwitz 1987). Endocrinetreatment of breast cancer classically involves both removal ofthe ovaries (i.e., reducing estrogens) in premenopausal women andadministration of pharmacological doses of estrogens inpostmenopausal women (Sutherland 1987). More recently, theantiestrogen tamoxifen has become the initial hormone treatmentof choice for both premenopausal and postmenopausal women(Patterson et al. 1981). The major antiestrogen effects oftamoxifen are due to competition with estrogen for the high-affinity ER. Binding of tamoxifen to this receptor leaves thecells refractory to further estrogen stimulaton (Dickson &Lippman 1989). Hormonal treatment on relapse involves either theadministration of progestins, androgens, or glucocorticoids, orthe surgical ablation of the adrenal or pituitary glands. Thesemethods act to decrease estrogen stimulation either directly(oophorectomy), through removal of the precursor necessary for4estrogen formation (adrenalectomy), or through removal of thetrophic hormones, adrenocorticotropic hormone (ACTH), follicle-stimulating hormone (FSH), or luteinizing hormone (LH), necessaryfor estrogen formation (hypophysectomy).Estrogens are mitogens for both normal and malignant breastepithelium (Lippman & Dickson 1989). In vitro, it has beenobserved that many ER+ human breast cancer cell lines, includingMCF-7, ZR-75-1, and T-47-D, are growth stimulated byphysiological concentrations of 178-estradiol (E2) and inhibitedby pharmacological concentrations of E2, whereas ER- lines do notshow these effects (Darbre et al. 1983). In the well-characterized MCF-7 human breast cancer cell line, physiologicalconcentrations of E2 induce cell growth, mitogenic enzymeactivity, and metabolic events (Dickson & Lippman 1987).Pharmacological concentrations of E2 inhibit these events to alevel similar to one that is seen with the antiestrogen tamoxifen(Lippman et al. 1976). However, tamoxifen can block cell growthin the absence of E2, suggesting either that tamoxifen might haveantimitogenic effects independent of ER (Wakeling 1988), or thatthe phenol-red containing medium used to culture these cellsmight have a weak estrogenic effect (Bindal et al. 1988). Inanother ER+ and PgR+ line, T-47-D, E2 has a biphasic effect oncell growth (Chalbos et al. 1982). E2 at concentrations of lessthan 10-8 M stimulates cell proliferation and at higherconcentrations progressively inhibits cell proliferation. Theantiestrogen tamoxifen inhibits the E2-induced stimulation anddecreases the growth of control cells. E2 increases ZR-75-1 cell5growth with half maximal stimulation of cell proliferation at 5 x10-12 M (Labrie et al. 1990).In addition to ER, glucocorticoid receptors have also beenfound in human mammary epithelial cells and in some human breastcancer cell lines, such as MCF-7 and ZR-75-1 (Horwitz et a/.1975). Glucocorticoids have stimulatory effects on normal humanmammary epithelial cell growth in primary culture. Yang et a/.(1980, 1982) demonstrate that hydrocortisone (HC) stimulatesmammary epithelial cell growth in a dose-responsive manner.Glucocorticoids have both stimulatory and inhibitory effects onbreast cancer cell line growth. A synthetic glucocorticoid,dexamethasome (Dex), inhibits the E2-stimulated cellproliferation of the MCF-7 cell line in a dose-dependent fashion,and this is antagonized by an antiglucocorticoid (Zhou et al.1989). Although glucocorticoids have inhibitory effects on theproliferation of MCF-7 cells in the presence of E2 (Lippman eta/. 1976), it has been reported that HC augments basal andinsulin-enhanced DNA synthesis (Linebaugh & Rallerma 1977).Darbre and Daly (1989) report that in the absence of phenol-red,glucocorticoids (eg. Dex) have stimulatory effects on the growthof several cell lines, including MCF-7, ZR-75-1, and T-47-D, butthis effect is less than the effect of E2. Glucocorticoids haveinhibitory effects on the growth of these cell lines in thepresence of phenol-red (Darbre & King 1987). Glucocorticoids andandrogens have cumulative inhibitory effect on ZR-75-1 cellproliferation (Labrie et al. 1990).Androgen receptors can be detected in 30-40% of human breast6tumors (Poortman et a/. 1975). The specific effects of androgenson breast cancer cell proliferation have also been investigated(Labrie et al. 1990; Poulin et al. 1988). In the ZR-75-1 cellline, physiological concentrations of androgens markedly decreasebasal and E2-induced cell proliferation as well as cellsaturation density through their androgen receptors (Poulin etal. 1988). Furthermore, the potent antiproliferative effect ofandrogens is additive to that achieved by antiestrogens.Dihydrotestosterone (DHT) has a biphasic effect on theproliferation of these cells incubated in the absence of E2.Concentrations of DHT ranging from 10-11-10-8 M inhibit basalcell growth to a maximam of 50% in a dose-dependent manner. Whenconcentrations of androgens are increased to 10-8-2 x 10-6 M, theinhibitory effects gradually decrease, although cell numbersremain below control levels. The inhibitory effect of DHT isneutralized by coincubating ZR-75-1 cells with an antiandrogen.Testosterone (T) has the same inhibitory effect on the growth ofthese cells. The mechanism of androgen's inhibitory effect may bedue to the fact that androgens exert a potent negative effect onER expression in this breast cancer cell line. The direct actionof androgens on breast cancer cells is also illustrated by thedemonstration of an inhibitory effect of androgens on E2-inducedPgR levels in MCF-7 cells (MacIndoe & Etre 1981). It is alsoillustrated by their stimulation of the secretion of Zna2-glycoprotein (Chalbos et al. 1982) and their stimulation of theexpression of the 15 kilodalton (kDa) gross cystic disease fluidprotein (GCDFP-15) in T-47-D cells (Chalbos et al. 1982). In the7absence of phenol-red, androgens (eq. T) have stimulatory effectson growth of several cell lines, including MCF-7, ZR-75-1, and T-47-D, but the effects are less than that of E2 (Darbre & Daly1989).The effects of E2 on the growth of normal and malignanthuman breast epithelial cells in primary culture in the presenceor absence of serum have also been studied. E2 at physiologicalconcentrations can stimulate the growth and at pharmacologicalconcentrations inhibit the growth of human breast cells in thepresence of serum (Emerman et al. 1987). In contrast, E2 has nogrowth-stimulatory effects in serum-free medium (Gabelman &Emerman, 1992). This may be due to the absence of serum-bornfactor(s) required for estrogenic stimulation of proliferation.There is considerable evidence that E2-stimulated growth ismediated by the production of growth factors such as epidermalgrowth factor (EGF) and insulin-like growth factor (IGF), and/ortheir receptors (Barker & Vinson 1990; Dickson et al. 1986; Huffet a/. 1988). An increase in EGF and/or IGF receptor levels by E2could enhance the effect on growth of EGF and/or IGF present inserum.1.3 Effects of Androgens on the Growth of the S0115 Tumor Androgen is the major steroid hormone that affects SC115tumor growth. Originally, the AR subline of the transplantableSC115 tumor grew only in intact male mice. It failed to grow ineither female or castrated male mice unless these mice were givenexogenous androgens (Bruchovsky & Rennie 1978; Emerman & Worth81985). However, due to the process of progression, the SC115tumor now also consists of a heterogeneous population of AR andandrogen-independent (Al) cells (Buchovsky & Rennie 1978; Emerman& Seimiatkowski 1984; Emerman & Worth 1985). The predominantsubpopulation of the SC115 tumor in intact male mice is AR;whereas the predominant subpopulation of the SC115 tumor grown infemale or castrated male mice is Al. These two subpopulations ofcells have different growth rates and morphologies (Emerman &Siemiatkowski 1984; Emerman & Worth 1985). The AR tumors aregenerally palpable in intact males 1 wk after tumor-cellinjection and grow to a weight of approximately 3 gm by 3 wk. Infemale or castrated males, the Al tumors are palpable after about1 mo and grow to almost 1 gm by 2 mo following tumor-cellinjection. The AR SC115 tumor grown in intact males is a soft,undifferentiated medullary carcinoma showing a compact polyhedralcell pattern characteristics of epithelial cells. In contrast,the Al tumors lose their cohesive growth pattern, and cells aredispersed into loose sheets and irregular strands which grow inloose stroma. In addition, the Al tumors are relatively hard.Rowse et a/. (1990b) show that tumors maintained in female micecontain osteoid-like regions which stain positive for sialic acidand sulphate moieties. It has also been shown by others that 83%of the tumors grown in females are slow growing and designated as"hard" tumor due to the remarkable bone formation among thesespindle-shaped cells (Kitamura et a/. 1979). The hard tumors growequally well in both male and female mice. The cells containcytosolic androgen receptors but show a postreceptor defect for9androgen actions (Bruchovsky & Rennie 1978).There are two possibilities that may explain the developmentof the Al spindle-shaped tumors (Emerman & Worth 1985): (a) TheS0115 tumor is composed of two different types of cells, AR andAT cells. The latter becomes predominant with androgen depletion.(b) AR cells change into AT cells in the absence of androgens. Asmall population of the latter derived from the AR cells cansurvive and proliferate with androgen depletion. In addition, alarge tumor mass leads to accelerated proliferation and autonomyfrom androgens, perhaps by leading to constitutive production oftumor-specific growth factors, the production of which isinitially triggered by androgens (Kitamura et a/. 1979; Luthy etal. 1988).AR growth of SC115 tumor cells is also maintained in cellculture. Since serum is an undefined mixture of hormones andgrowth factors, Stanley et a/. (1977) developed a dextran-charcoal method to remove all the endogenous steroid hormones,including androgens, estrogens, and glucocorticoids, from theserum. A cell line derived from the SC115 tumor cells cultured inmedium containing dextran charcoal-treated (DCT) serum show adose-dependent proliferative response to androgens (Yates & King1978; 1981). However, after 1 wk of androgen deprivation, theresponse of S0115 cells to androgen is greatly reduced, and after2 wk, there is no growth stimulation of androgen-deprived cellsby T. Restoration of T to the medium of short-term androgen-deprived cells results in an increase in the growth response toT, eventually reaching the level observed for cells maintained in1 0androgen. Long-term androgen deprivation causes an irreversibleloss of the androgen response.Androgen withrawal leading to a loss of androgenresponsiveness in these cells is accompanied by morphologicalchanges, increased sensitivity to serum, increased densitydependency (lower saturation density), and increased anchoragedependency (Yates et a/. 1980). AR SC115 tumor cells areelongated and rounded, fibroblastic in appearance, grow in anirregular manner and overlap to form multilayered foci (Yates &King 1981). In the absence of androgen, however, the cells areflatter and more epithelial-like with little overlapping.Although the cells are stimulated to grow by androgens, thisobviously does not reflect the SC115 tumor grown in vivo. Emermanand Worth (1985) demonstrate that the growth rates, morphologies,and tumorigenic potentials characterizing the AR SC115 tumor andits Al variant in vivo persist when cells are cultured oncollagen gels rather than on plastic. It has been shown thatnormal and malignant mammary epithelial cells from severalspecies retain their morphological and functional characteristicswhen grown on a collagen substrate (Emerman et al. 1990; Emerman& Pitelka 1977; Emerman & Worth 1985). Cells from the AR SC115tumor cultured in DHT-containing medium grow faster than cells inDHT-deprived medium and cells from Al tumors. AR tumor cellsmaintained in medium containing DHT form a confluent pavement,similar to the tumors in vivo. The culture morphologies of ARtumor cells grown in the absence of DHT and of Al tumor cellscultured in the presence or absence of DHT resemble the11histologic pattern of Al tumors in vivo. Cells form small clumpsor cords of cells or remain isolated. Tumors arising frominjection into male mice of cells from freshly dissociated ARtumors or cells of AR tumors cultured in the presence of DHTappear more rapidly and grow faster in intact males than infemales or castrated males. Tumors arising from cells cultured inthe absence of DHT or from freshly dissociated or cultured cellsof Al tumors have identical rates of appearance and growth in allhosts.Cells of the AR and Al SC115 tumors have been characterizedby flow cytometric analysis of their DNA content and bykaryotypic analysis of metaphase spreads (Emerman & Kalousek1987). Both tumors have diploid and near tetraploid populationsof cells. However, data suggest that AR and Al malignant cellsboth appear to be polyploid. A decrease in the polyploidpopulation of the AR tumor accompanies tumor regression followingcastration, but this population is restored when tumor growthresumes. Karyotypic analysis of metaphase spreads of cells fromboth AR and Al tumors shows a range of 55-88 chromosomes and theycontain the same chromosome anomalies. Differencies between thecell populations of the AR and Al tumors remain to be elucidated.Cells from the AR tumor cultured in androgen have the sameproportion of diploid and polyploid cells as the AR tumor in vivo(Emerman et al. unpublished results). If androgen is removed fromthe culture, growth slows and the percentage of polyploid cellsdecreases. When growth resumes following replacement of androgen,the polyploid population increases comparable to that in vivo.12The ratio of diploid to polyploid cells in cultures of cells fromthe Al tumor is similar to that of the Al tumor in vivo in mediumwith or without androgen. The karyotype is also maintained in thepolyploid populations in culture.1.4 Effects of Glucocorticoids on the Growth of the SC115 Tumor Watanabe et a/. (1982) studied the effect of glucocorticoidson the growth of the SC115 tumor in vivo. When castrated malemice are treated with Dex for 2 wk at daily doses of 1-5 mg/kgstarting 3 d before tumor inoculation, the tumor weight as wellas transplantability are significantly increased. Dailyinjections of high doses of Dex (4 mg/kg), but not physiologicaldoses (160 4g/kg) markedly stimulate tumor growth, approachingthe growth rate found in a normal male mouse (Omukai et a/.1987). The SC115 tumor grown in Dex-treated castrated male micehas morphplogical, biochemical, and biological characteristicssimilar to those of the S0115 tumor grown in intact male mice.Dex does not interact with androgen receptors. It may actdirectly through glucocorticoid receptors or its action may bemediated in part by the suppression of the immune system of thehost. Glucocorticoids have been shown to have effects onvirtually every component of the immune system in both animalsand humans (Munck et al. 1984). Generally, they haveimmunosuppressive and anti-inflammatory effects atpharmacological doses. There are two indirect pieces of evidencethat the immunological status of the host may affect the growthof the SC115 tumor. Kitamura et al. (1979; 1980) demonstrate that13the S0115 tumor grows in both castrated male and female nudeathymic mice. In addition, Nohno et al. (1986) reports that asingle administration of S. aureus increases host immunity anddecreases the transplantability of the SC115 tumor. It hasrecently been shown that the presence of the SC115 tumor has aneffect on the immune system (Rowse et al. 1990a). A study wasundertaken to determine if natural killer (NK) cell activity isinvolved in mediating the effects of differential housingconditions on SC115 tumor growth rate (see below). Splenic NKcell activity was assayed at 24 h, 3 d, and 1 wk post-injectionin both tumor- and vehicle-injected animals. Significantstimulation of splenic NK cell activity occurs 3 d post-injectionof SC115 cells. This study demonstrates that the SC115 tumor iscapable of stimulating NK cell activity. However, it is not yetclear if modulation of the NK cell activity has an effect onmediating the growth rate of this tumor from mice in differenthousing conditions.In addition to androgen receptors, glucocorticoid receptorshave also been found in the cytosol of SC115 cells (Waranabe etal. 1982). In serum-free cell culture, the proliferation of SC-3cells is markedly (up to 25 fold) stimulated by 10-11-10-8 M T,whereas proliferation is only slightly (up to 3.3 fold), butsignificantly stimulated by 10-8-10-5 M Dex (Omukai et al. 1987).Both androgens and glucocorticoids bind to their own receptors,but do not bind with each other's receptors. Dex shows bothinhibitory and stimulatory effects on androgen-inducedproliferation of SC115 cells in culture (Hiraoka et a/. 1987).14Stimulation of SC-3 cells induced by greater than 10-8 M T issignificantly inhibited by the addition of 10-8-10-5 M Dex in adose-dependent manner, whereas a lesser degree of stimulationinduced by less than 10-10 M T is significantly enhanced by theaddition of Dex. The mechanism of the biphasic effect of Dex isnot clear at this point, but it might occur at the post-receptorlevel. It has been hypothesized that Dex-glucocorticoid receptorcomplexes inhibit the binding of the more active T-androgenreceptor complexes to the acceptor sites on the chromatin. Thishypothesis is further supported by the finding that 10-8 M Dexstimulates the synthesis of five secretory proteins in SC-3 cellsthat are identical to those induced by 10-8 M T (Nakamura et al.1987a).The interaction between physiological doses of androgens andpharmacological doses of glucocorticoids in stimulating theproliferation of the SC115 tumor is very interesting. AR cellsare sensitive to both androgens and glucocorticoids, whereas AIcells are unresponsive to both steroids. Darbre and King (1987)find that the two steroids can interact to prevent theprogression of steroid insensitivity that occurs when one hormoneis removed. SC115 cells can be protected against a loss ofresponse to either androgens or glucocorticoids with eithersteroid alone. That is, androgen protects against loss ofglucocorticoid sensitivity and vice versa. Yates and King (1978)demonstrated that Dex has a biphasic effect on a cell line fromthe AR SC115 cell growth in the absence of T in vitro; at 10-8 M,15it inhibits proliferation by 30% but at 10 6 M, it stimulatesgrowth by 235%.1.5 Effects of Estrogens on the Growth of the SC115 Tumor Noguchi et al. (1984) and Nohno et a/. (1982) found ER inthe cytosol of subcutaneous (sc)-grafted SC115 tumors. When E2 ata daily dose of 2 mg/kg is given to intact male mice bearing theSC115 tumor, tumor growth as well as the weight gains of seminalvesicles and prostate glands are inhibited. Estrogen binds to ERbut also binds to androgen receptors which is likely responsiblefor its inhibitory effects on growth. In contrast, Noguchi et a/.(1984) found a stimulatory effect of E2 on SC115 tumor growth.Although a physiological concentration of E2 (40 gg/kg) does notsignificantly enhance tumor growth, daily injections of 160-400rig/kg of E2 significantly stimulate growth in a concentration-dependent manner (Noguchi et al. 1984). The growth ratestimulated by 4 mg/kg of E2 approaches the level induced by 4mg/kg testosterone propionate (TP) (physiological dose). Lowdoses of androgens alone (400 gig/kg of TP) fail to maintain theSC115 tumor (Noguchi et a/. 1985a). Tumor growth in castratedmale mice treated with a low dose of TP (400 gg/kg) is similar tothat of the Al tumor developed from the original SC115 tumorafter androgen removal. A dose of 160 gg/kg E2 can actsynergistically with low doses of TP (400 gg/kg) to stimulate thegrowth of the SC115 tumor. Addition of 400 gg/kg E2 to 400-1000gg/kg TP significantly augments growth.16The morphology of tumors was also examined (Noguchi et a/.1985b). Tumors grown in castrated males are composed of medullarycells if stimulated by daily injections of 4 mg/kg of TP whereastumors consist of spindle-shaped cells if the host is injectedwith vehicle only. If the SC115 tumors are grown in mice injectedwith 400 or 1000 gg/kg of TP or 400 jig/kg of E2, about one halfof the tumors are of the spindle-cell type and the other half, ofmixed type. In contrast, most tumors are composed of themedullary type when E2 is added to 400 or 1000 jig/kg of TP.The stimulatory effect of estrogen-induced proliferation ofSC115 cells is shown to be mediated by the ER but not theandrogen receptor (Noguchi et al. 1985b). Daily injection of highdoses of diethylstilbestrol, which does not bind to the androgenreceptor even at high concentrations, significantly stimulatestumor growth in a dose-dependent manner. Moreover, the anti-androgen, CA, has no inhibitory effect on E2-induced growth ofthe SC115 tumor but significantly inhibits in a dose-dependentmanner the TP-induced growth of the SC115 tumor and of seminalvesicles.In 1976, Jung-Testae et al. and King et a/. found ER inSC115 cells in culture, but did not correlate E2 with aproliferative response of the cells. In cell culture (Nakamura etal. 1987b; Noguchi et a/. 1987), E2 (1012_10-6 M) has no effect-9on SC-3 cells in serum- supplemented medium, whereas E2 (10 _10-6 M) as well as CA (10 -8 -10-6 M) inhibit the growth-stimulatory effect of T (10-10 M) on SC-3 cells in a dose-dependent manner in medium with DCT serum. This is probably due17to the competitive inhibition of T binding to its receptor by E2and CA.1.6 The Proliferation of Androgen-induced SC115 Tumor Cells maybe Mediated by a FGF-like Polypeptide through an Autocrine Mechanism It is suggested that androgen-induced growth of SC115 cellsmay be mediated by the production of a growth factor(s). Using[ 35S]-methionine labeling of SC115 cells in primary culture,Jung-Testae et al. (1988) have shown several intracellular andsecreted proteins with molecular weights of 55, 40, and 15 kDaspecifically induced by T. Furthermore, the culture mediumconditioned by T-treated SC115 cells has significant mitogenicactivity on L929 mouse fibroblasts. However, the growth factor(s)is not characterized (Jung-Testae & Baulieu 1985). Using serum-free (Lu et a/. 1989; Nakamura et a/. 1987b; 1989; Nonomura etal. 1988) and protein-free (Tanaka et a/. 1990) media,investigators have been able to demonstrate that androgen- andglucocorticoid-induced proliferation of SC115 tumor cells iscaused by a fibroblast growth factor (FGF)-like polypeptide in anautocrine fashion. Nakamura et a/. (1987b) found that both cellnumbers and DNA synthesis of SC-3 cells are significantlystimulated by conditioned medium (CM) from SC-3 cells exposed toT, whereas the CM from cells not exposed to T has no growthstimulatory effects. The CM was fractionated using gelfiltration. Specific fractions markedly stimulate theproliferation of SC-3 cells without T and the morphology changes18from epithelial to spindle-shaped (Sato et al. 1988). Theactivity present in CM is not antagonized with the anti-androgenCA at 10-6 M, eliminating the possibility that a residual amountof T remaining in the CM induces these events (Nakamura et a/.1987b; Sato et al. 1988). The purified androgen-induced growthfactor from the CM can be associated with FGF receptor on SC-3cells, strongly suggesting that this growth factor is an FGF-likepolypeptide (Nonomura et a/. 1990). The androgen-induced growthfactor eluted from the heparin-Sepharose column reacts with anti-FGF antibody when examined by radioimmunoassay and specificallyinhibited the binding of [1251,j bFGF to FGF receptor and theseactivities are exactly in parallel to the growth-stimulatoryactivity (Nonomura et al. 1989).Nakamura et al. (1989) further examined the effects ofvarious growth factors, including EGF, transforming growthfactor-alpha (TGF -a), platelet-derived growth factor (PDGF),insulin-like growth factor-I (IGF-I), insulin, TGF-B, bFGF, andaFGF on the growth of the SC-3 cell line in serum-free medium.The proliferation of SC-3 cells reaches a plateau with 10-8 M T(up to 200-fold), 10-7 M Dex (up to 30-fold), and 1 ng/ml of FGF(up to 50-fold). In contrast, all concentrations tested ofprogesterone, E2, EGF, PDGF, IGF-I, insulin, and TGF-a have noeffects. However, the addition of EGF (1 ng/ml) significantlyenhances the T-induced growth of SC-3 cells (Nakamura et al.1987a; Noguchi et al. 1987). TGF-8 (1 ng/ml) slightly stimulatesthe growth (up to 5-fold) of these cells but markedly inhibitsthe growth stimulation induced by T (Yamanishi et al. 1990).19Although the T-induced growth is almost completely inhibited byTGF-B, b- or a-FGF-like peptide-induced growth is only partiallyinhibited (45%) by TGF-B. This difference can be explained by thefact that TGF-B decreases the amount of T-induced FGF-likepeptide secreted from SC-3 cells to 18% of control. Thisinhibitory effect is found to be reversible.Androgen-induced proliferation of SC-3 cells (eitherdirectly or by CM) is neutralized by anti-bFGF antibody (Luthy etal. 1988; Nonomura et a/. 1989; Tanaka et al. 1990). Anti-bFGFantibody can significantly inhibit 10-8 M T-, 10-7 M Dex-, and0.1 ng/ml bFGF-induced proliferation of SC-3 cells by 70%. Thisis dose-responsive within the concentrations of 100-600 ng/ml ofanti-bFGF antibody. These results provide strong evidence thatthe physiological doses of androgens and pharmacological doses ofglucocorticoids produce a FGF-like polypeptide that stimulatesSC-3 cell growth through an autocrine mechanism.1.7 The Endogenous Opioid Peptide (EOP) System Since 1975, when EOP were first isolated from the brain(Hughes et a/. 1975), their anatomical distribution andphysiological functions have been extensively studied. Inmammals, these peptides can be derived from at least 3 distinctgenes: the ACTH/13-endorphin (5-EP) precursor (proopiomelanocortinor POMC), the enkephalin precursor (proenkephalin A), and thedynorphin/neo-EP precursor (proenkephalin B) (Akil et al. 1984;1988). Using molecular biology techniques, the POMC gene, firstdiscribed by Nakanishi et a/. (1979), has been found to contain20the 31 amino acid peptide 13-EP at its carboxy terminus and the 91amino acid precursor B-lipotropin (8-LPH, which contains 8-melanocyte stimulating hormone (MSH) in some species); themidregion of this precursor contains ACTH1_39 (which can becleaved into a-mSH and CLIP or ACTH18_39). At the amino terminusof this precursor is the active ACTH/MSH core, known as y-MSH. Inthe proenkephalin A precursor, all of the known active peptidesare oploid in nature. It contains 7 peptides with the Met- orLeu-enkephalin active core. Four of the 7 peptides produced aresimple Met-enkephalin, two are carboxyl extended Met-enkephalin-Arg-Phe-Arg-Ghy-Leu, and the final one is Leu-enkephalin (Comb eta/. 1982; Noda et al. 1982). The proenkephalin B precursorproduces 3 main Leu-enkephalin-containing peptides: a/1-neo--EP,dynorphin A, and dynorphin B (Kakidani et al. 1982). All of thesepeptides have one structural feature in common, namely, a N-terminal sequence beginning with the same four amino acids (Tyr-Gly-Gly-Phe).The EOP are widely distributed in both central andperipheral nervous systems. The major location of POMC is thepituitary (Bloom et al. 1977). In man, it is found mainly in theanterior lobe, but in most other species, it is found primarilyin the intermediate lobe. There are two cell groups in the brainthat produce B-EP/ACTH peptides: one is in the region of thearcuate nucleus of the medial basal hypothalamus (Bloch et al.1978) and the other is in the nucleus of the solitary tract andthe nucleus commissuralis (Schwartzberg & Nakane 1981).Proenkephalin A is also widespread in the hypothalamus,21pituitary, and central nervous system. In the brain, it is foundat every level from the cortex to the spinal cord (Elde et al.1976). Unlike POMC peptides, proenkephalin-like peptides are alsofound in the adrenal medulla, the gastrointestinal tract, theautonomic nervous system (with the catecholamines), and severalother structures. Recently, the pro-neo-EP/dynorphin precursorhas been found in the gut, posterior pituitary, and brain. It isfound in scattered cell groups throughout the brain stem and inseveral hypothalamic nuclei including vasopressin-producing cellsof the magnocellular portion of the paraventricular nuclei(Khachaturian et al. 1986; Watson et al. 1982).The final products produced by and stored within a givenneuron depend not only on the genetic code for the precursor, butalso on the program that directs enzymes to process the precursorin certain ways---the post-translational processing events. Theprecursor is translated from its mRNA using ribosomal machineryand emerges in the form of a protein. The opioid precursors havea molecular weight of about 25-30 kDa. The precursor is thenprocessed by proteolytic enzymes, which produce individualpeptide domains. POMC cells of the anterior pituitary and thoseof the intermediate lobe of the pituitary process the precursorquite differently (Akil et al. 1981; Zakarian & Smyth 1982). Inthe anterior lobe, ACTH1_39 is the major product; whereas in theintermediate lobe, this peptide is further processed to producethe highly modified a-MSH [N-acetyl ACTH1_13-NH2] and CLIP orACTH18_39. All of the 8-LPH in the anterior lobe is converted to13-EP in the intermediate lobe. The potent opioid peptide 8-EP1_3122undergoes a-N-acetylation at the tyrosine residue, or it iscleaved at the carboxyl-terminus to remove the last four to fiveresidues, or both. Thus the main product in the intermediate lobeis not 8-EP1_31, but rather N-acetyl-13-EP1_27, a peptide withgreatly decreased receptor affinity, which has been proposed as apossible endogenous antagonist.The processing of POMC in the brain is less understood andcontroversial. The 8-EP1_27 form exists in substantialquantities. While N-acetylation occurs in some brain regions, itis less common than in the intermediate lobe of the pituitary(Evans et al. 1982; Zakarian & Smith 1982).The post-translational processing of proenkephalin in thebrain often yields the enkephalin pentapeptides. In the adrenalgland, post-translational processing may yield large peptides,such as peptide E and peptide F which contain the enkephalinsequences at each terminus (Kimura et a/. 1980).Processing of prodynorphin yields two possible neo-EPs, aand 8, which differ by a lysine residue (Kanagawa et al. 1981),as well as dynorphin A1_17, which further cleaves to dynorphinA1_8. The latter is the major product in the brain, but not inthe pituitary (Weber et a/. 1982). Prodynorphin processing in thepituitary is less well known.The multiple opioid receptor system was first postulated byMartin et a/. (1976). The basic opioid receptors including mu(morphine), kappa (ketocyclazocine), delta (enkephalin), and aspecific 8-EP receptor (epsilon), show different degrees ofselectivity for the different peptides (Akil et al. 1988).23Pharmacological studies show that multiple opioid receptorsinteract with the multiple opioid peptides. 1-EP, except for itsunique epsilon receptor, recognizies both mu and delta sites,with a slight preference for delta. All of the proenkephalin-related peptides show delta receptor activity ranging from Leu-enkephalin, which is primarily delta, to the enkephalinoctapeptides, which appear to be equally mu and delta. Alldynorphins and neo-EPs show a preference for the kappa receptor.Dynorphin1_8, however, retains delta capability, whereasdynorphin1_23 acts potently at both mu and kappa receptors. Mostof the opioid peptides and drugs perform their functions byinteracting with at least one of the four receptor types.1.8 EOP Interaction with Endocrine Functions EOP are important in the control of many body functions,including analgesia (Jaffe & Martin 1980)), behavioral (Jaffe &Martin 1980), endocrine (Van Augt & Meites 1980), and immunefunctions (Fischer 1988; Fischer & Falke 1984; Weber & Pert1984), as well as in tumor biology (Lewis et al. 1983; Zogan &McLaughlin 1981a). The effect of morphine, an opioid agonist, inanalgesia was found a century ago. The dense concentrations ofopioid receptors in the hypothalamus and pituitary suggest thatthey have endocrine functions, that is, they may play aphysiological role in regulating pituitary hormone release (VanAugt & Meites 1980).In general, the EOP, morphine, and related drugs exertsimilar effects on acute release of pituitary hormones (Van Augt24& Meites 1980). A high dose of opioids produces a rapid increasein the release of prolactin, growth hormone, ACTH, antidiuretichormone, and possibly MSH, and a decrease in gonadotropins,thyrotropin, and oxytocin. Continous administration of opioidsmay lead to attenuation or even loss of their influence onsecretion of some pituitary hormones (Van Augt & Meites 1980).Rivier et al. (1977) confirm that 13-EP (120 gg/kg) and morphine(4 mg/kg) injected intravenously (iv) into male rats stimulateprolactin and growth hormone release; this effect can be reversedby the opioid antagonist, naloxone (1 mg/kg). Naloxone (250 mg/h,iv, for 8 h) alone has been shown to reduce basal serum levels ofprolactin and growth hormone and to elevate serum levels of LHand FSH, as well as to decrease a stress-induced rise ofprolactin in rats (Armstrong et al. 1988; Rivier et al. 1977).Further, Seigel et al. (1982) demonstrate that acute naloxone (5mg/kg) administration produces a basal and stress-inducedhypersecretion of ACTH and corticosterone in adult male rats. Itis generally believed that opioid peptide regulation of pituitaryhormone release occurs at the level of the hypothalamus, mainlyvia hypothalamic neurotrasmitters (dopamine, norepinepherine,serotonin, acetylcholine, and others) which regulate release ofthe hypothalamic-releasing hormones into the pituitary portalvessels (Van Augt & Meites 1980).1.9 EOP Interaction with Immune Functions Evidence accumulated in the past few years suggests thatopioids may influence the immune system. The EOP have been found25to influence the functions of most of the major cell types in theimmune system. The different EOP have different immunomodulatoryfunctions even though they have identical NH2-terminals. Opioidpeptides affect various immunocyte-mediated events such as NKcell activity (Faith et al. 1984; Mattews et al. 1983), antibodyproduction (Johnson et al. 1982), mononuclear cell chemotaxis(Van Epps et a/. 1983), and the generation of cytotoxic T cells(Carr & Klimpel 1986). Some effects can be blocked by an opioidreceptor antagonist, and some cannot, suggesting that both °plaidspecific and non-specific ligands exist on the immune cells.Data indicate that lymphocytes, phagocytic leukocytes, andterminal comlexes of complement possess opioid receptors (Carr etal. 1988; Hazum et a/. 1979; Schweigerer et a/. 1983). Early workdemonstrates the presence of high affinity opioid receptors onmurine splenocytes and human leukocytes (Johnson et al. 1982;Mehrishi & Mills 1983)). Using SUPERFIT, Carr et al. (1988)demonstrate that both human and murine splenic and peripheralleukocytes possess specific binding sites for the G-classligand, a specific receptor ligand for 0-receptor.(1) Antibody ProductionJohnson et a/. (1982) report that a--endorphin (>=0.05 11M)as well as Met- and Leu-enkephalin (>=0.02 nM) are potentsuppressors of antibody production. In contrast, 13- and 7-EPshave no effect on antibody production. Suppressive effects of a-EP, Met-, and Leu-enkephalins can be partially inhibited bynaloxone (3 I.LM) or 3-EP (12 gm). Others (Heijnen & Ballieux 1986)26have found that if added to the medium at the appropriate time,8-EP enhances the number of antibody-forming cells and that thiseffect is initiated by the COOH-terminal end of the peptide.(2) SRBC-RosettingMet- and Leu-enkephalins increase the percentage of T cellsthat form active rosettes with sheep red blood cells (SRBC), butdepress total T cell rosette formation (Miller et al. 1983;Wybran et a/. 1979). Morphine (0.1-100 nM) reduces the frequencyof active and total SRBC rosette formation. This effect ofmorphine is reversed by naloxone (Bayer et al. 1990).(3) Lymphocyte ProliferationGilman et a/. (1982) investigated the effects of a- and 1-EP and [D-Ala-Met]-enkephalin in modulating the proliferativeresponse of splenic T lymphocytes to mitogenic stimulation. 8-EPat doses of 1-100 ng/ml (rat physiological concentrations)enhances the proliferative response of rat splenic cells to the Tcell mitogens Concanavalin A (ConA) and phytohemagglutinin (PHA)but not to the B cell mitogens bacterial lipopolysaccharide (LPS)and dextran sulfate. This is not reversed by 10 11M naloxone.Neither a-EP nor [D-Ala-Met]-enkephalin has this effect. Thesedata suggest that the enhanced T cell proliferative response tomitogens may be mediated by a non-opioid but 8-endorphin-specificligand. However, others have reported (Kusnecov et a/. 1987) that8-EP (10-12-10-9 M) induces a dose-responsive enhancement of theproliferative response of rat splenic T cells to ConA, which is27inhibited by naloxone (10-6 M). The question of whether or notthe effects of opioid peptides on the immune system are mediatedby opioid or non-opioid receptors needs further investigation. Incontrast, in humans it has been shown that 8-EP has a suppressiveeffect on PHA-induced lymphocyte proliferation by a stimulatingsuppressor T cells and that this effect is not naloxone-reversible (McCain et a/. 1982). Conflicting results may be dueto the differences in species, lymphocyte source, andconcentrations used by particular researchers.Generally, in vitro, data suggest that high pharmacologicalconcentrations of morphine and methadone (0.1 mM or more) depressthe PHA-, ConA-, or pokeweed mitogen (PWM)-induced blastogenesisof both T and B lymphocytes (Bocchini et a/. 1983), whereas lowerconcentrations of morphine (0.1 gM) appear to have inconsistenteffects (Maravelias & Contselinis 1984).(4) Cytotoxic ActivityOpioid peptides enhance the generation of cytotoxic Tlymphocytes (CTL, Carr & Klimpel 1986) as well as the expressionof NK cell-mediated cytotoxicity (Faith et al. 1984; Mandler eta/. 1986). 2-EP as well as Met- and Leu-enkephalins have beendemonstrated to enhance NK cell activity (Faith et al. 1984;Mattews et a/. 1983). These effects can be inhibited by theopioid antagonist, naloxone, suggesting that enhanced activity ismediated via an opioid receptor. In normal volunteers and cancerpatients, Plotnikoff et al. (1986) found that Met-enkephalin (1-250 rig/kg) significantly increases total lymphocytes, T cell28subset, and NK cell activity and increases mitogen-stimulatedlymphocyte blastogenesis with PHA, ConA, and PWM. Interestingly,Williamson et al. (1987) found that 8-EP1_31 has a biphasiceffect on human NK cells. Preincubation of effector humanlymphocytes with 10-11-10-7 M 8-EP containing an unmodified N-terminal sequence increases NK cell activity. In contrast,preincubation with lower concentrations (10-18_10-13 ms) of 13-EPreduces NK cell activity, which bind only to non-opioidreceptors. Shavit et a/. (1987) report that a single exposure tothe °plaid form of footshock stress or a single high dose (35-501.1g/kg) of morphine induces suppression of rat splenic NK cellcytotoxicity. This effect appears 3 h after treatment, returningto normal by 24 h. The effect of morphine is blocked bynaltrexone, another opioid antagonist. Naltrexone-reversiblesuppression of NK cell activity has been seen in cells derivedfrom the spleen, bone marrow, and peripheral blood, suggestingthat this suppression does not result from a selective egress ofNK cells from the spleen. In contrast, Mandler et al. (1986)demonstrated that 5-EP (10-10-10-7 M), but not a-EP or y-EP,significantly augments human NK cytolytic activity by 63% in analoxone-reversible manner.Overall, the opioid peptides can both increase and reducelymphocyte cytotoxicity depending on the opioids, theconcentrations used, and whether binding occurs at the N- or C-terminal sequence.29(5) Chemotactic Response of Monocytes and NeutrophilsOpioid peptides are able to stimulate chemotaxis ofmonocytes and neutraphils both in vivo and in vitro. Van Epps etal (1983) show that 13-EP and Met-enkephalin (10-8 M) can interactwith and stimulate leukocyte migration, particularly monocytes,lymphocytes, and possibly neutrophils. Furthermore, in the caseof monocytes, this stimulation of migration is directional,indicating that 13-EP has chemotactic activity. Migration can beblocked by naloxone, suggesting opioid receptor involvement.(6) Factors Released by LymphocytesAnother effect of opioid peptides on the immune system isthat they can modulate production of lymphokines such asinterferon-7 (IFN), interleukin-2 (IL-2), and lymphocytechemotactic factors. Brown and Van Epps (1986) demonstrate that13-EP and Met-enkephalin (10- 14_10-10 M) significantly enhance IFNproduction by human peripheral lymphocytes incubated with ConA.This effect is not inhibited by naloxone (10 and varies fromdonor to donor. There is also no absolute correlation between anenhanced response to 13-EP and an enhanced response to Met-enkephalin. Gilmore and Weiner (1988) investigated the effect of13-EP on IL-2 production. 13-EP (10-6 M) enhances the production ofIL-2 from ConA-stimulated, unfractionated murine splenocytes aswell as from a mouse T cell lymphoma. Met- and Leu-enkephalins(10-12-10-6 M) do not have this effect. 5-EP-stimulated IL-2production is not reversed by naloxone and dependent on theintegrity of the C-terminal amino acids. The in vitro30proliferative expression of both CTL and NK cells is regulated byIL-2. IL-2 production may be a mechanism by which the cytotoxicactivity of CTL and NK cells is increased by B-EP.1.10 Opioids and Tumors Evidence suggests that opioid peptides are also involved intumor biology (Lewis et al. 1983; Zagon & McLaughlin 1981a).Opioid peptides have the ability to retard normal cell growth(Slotkin et a/. 1980; Willson et a/. 1976), particularly indeveloping neural tissues. Mechanisms involved includeantimitotic and cytotoxic activity, opioid-induced damage tochromosomes or suppression of cell division, and alterations inpolyamine, nucleic acid or protein systhesis. Several tumor celllines, including neuroblastomas (Kazmi & Mishra 1986), MCF-7, abreast cancer cell line, (Maneckjee et a/. 1990), and small cellcarcinomas (Roth & Barchas 1986) have been found to containmultiple opioid receptor subtypes.Opioid agonist-modulation of the growth of neuroblastoma hasbeen well studied. Chronic administration of heroin (3-15 mg/kgdaily), a narcotic opioid compound, effectively retards tumorgrowth and prolongs survival time in mice with transplantedneuroblastoma (Zagon & McLaughlin 1981a). The antitumor effect isblocked by simultaneous administration of the opioid antagonist,naloxone. Heroin's antitumor effect has been confirmed by invitro investigations (Zagon & McLaughlin 1981c). The addition ofheroin in concentrations ranging from 10-8-10-2 M to S20Y (aneuroblastoma cell line) 24 h after cell seeding, produces a31dose-dependent inhibition of growth. This growth-retarding effectis reversible, since removal of heroin allows cells to resumenormal growth. Analysis of mitotic figures reveals that heroinaffects cell division. The action of heroin in perturbing cellgrowth is also blocked by concomitant administration of naloxone(10 Moreover, °plaid antagonists alone may also havestimulatory or inhibitory effects on neuroblastoma growthdepending on the dosage used (Zagon & McLaughlin 1981a, 1981c,1985). For example, naloxone (5-20 mg/kg daily), a non-addictiveopioid antagonist, exerts a similar effect on neuroblastomagrowth in mice as heroin itself (Zagon & McLaughlin 1981b).Naltrexone, another opioid antagonist that is eight times asactive and three times as long-acting as naloxone (Blumberg &Dayton 1973), administered once daily at 10 mg/kg (a dose whichblockes the opioid receptor for 24 h), stimulates tumor growthand shortens survival time in mice innoculated with neuroblastomacells. In contrast, a dosage of 0.1 mg/kg (which blocks theopioid receptor for 4-6 h) has remarkable antitumor effects(Zagon & McLaughlin 1983).Besides neuroblastoma, opioid agonists and antagonists arealso involved in other carcinogenic processes, including mammarycarcinoma, lymphoma, melanoma, and tumor matastasis (Aylsworth eta/. 1979; Murgo 1985). Pretreatment with naloxone and naltrexonesignificantly inhibits growth of carcinogen-induced mammarycancer in rats (Aylsworth et a/. 1979) and causes a completeregression in mice with spontaneous and transplanted mammarytumors (Tsunshima et al. 1982). Similarly, a well characterized32human breast cancer cell line, MCF-7, has been found to possessmultiple opioid receptor subtypes (Maneckjee et a/. 1990). Opioidligands specific for the different receptor subtypessignificantly inhibit the growth of MCF-7 cells in a dose-relatedmanner. This inhibition is reversed by concomitant addition ofnaloxone to cell culture. Met- and Leu-enkephalins significantlyinhibit the growth rate of the B16-BL6 tumor in mice and decreasethe numbers of metastases in lung (Murgo 1985; Scholar et al.1987). Furthermore, clinical studies show that a high percentageof primary ductal carcinoma of the breast contains scatteredtumor cells with opioid peptide immunoreactivity (Scopsi et a/.1989) and two small cell carcinoma cell lines contain opioidpeptide receptors (Roth & Barchas 1986).1.11 Animal-tumor Model for Studing Effects of Stressors on Mammary Tumor Growth An animal-tumor model has been developed in our laboratory toexamine the effects of social housing condition on growth of theSC115 tumor (Weinberg & Emerman 1989). Following weaning at 3 wkof age, male mice of the DD/S strain are housed individually (I)or in groups of 3 (G). At 2-4 mo of age, animals are injectedwith tumor cells or vehicle and experimental groups formed asfollows. Mice reared individually remain individually housed (II)or are rehoused in groups of 5 (IG). Those reared in groupsremain in their groups (GG) or are rehoused individually (GI).Interactive effects of changes in housing conditions and an acutepsychological stressor (daily exposure to a novel environment)33are also examined. Thus half of the animals in each housingcondition are exposed for 15 min/day to 1 of 5 different novelenvironment (see 2.1.4 below). Data demonstrate that animalsreared individually and remaining individually housed (II) orreared in a social group and then singly housed (GI) followingtumor cell injection show markedly increased tumor growth ratecompared to that in mice remaining in the rearing group (GG), ifanimals are also exposed to acute novelty stress. In contrast,mice reared individually and then moved to a large social group(IG) following tumor cell injection show markedly reduced tumorgrowth, both in the presence or absence of acute daily noveltystress (Fig.1, Weinberg & Emerman 1989).As noted above, androgens and glucocorticoids, both stress-related hormones, have significant effects on the growth rate ofSC115 tumor cells. Therefore, it is hypothesized that analteration in the endocrine function of the mice may be involvedin the differential tumor growth rates observed in this model. Astudy was undertaken (Rowse et a/. 1992) to investigate thepossibility that an effect of housing condition on plasma levelsof androgens and glucocorticoids may, in part, mediate thedifferential tumor growth rates observed in this model. Plasma Tand corticosterone levels were assayed 24 h, 3 d, and 1 wk posttumor cell/vehicle injection and group formation. Basal levels ofplasma T are elevated in mice of the GG, GI, and II groups, butnot in mice of the IG group, at 1 d post-injection and groupformation. At 3 d and 7 d, T levels of mice in the GG and IIcondition decline, whereas basal T levels of mice in the GI34Fig.1 Tumor growth in male mice in the four housing groups:GG, raised and maintained in silbing groups of two to three; GI,raised in sibling groups of two to three, then seperated andhoused singly; IG, raised singly housed, then rehoused innonsibling groups of four to five; II, raised and maintainedsingly housed. 0 - n per group. On Day 18 following tumor cellinjection, GI = II > GG > IG, p < .05 (Weinberg & Emerman 1989).36condition remain elevated. In contrast, basal levels of plasmacorticosterone are significantly elevated in IG animals comparedwith animals in all other groups on all 3 test days. Overall,plasma corticosterone levels show significantly greaterelevations at 1 d post injection than at 1 wk post injectionalthough levels are within the physiological basal range at alltimes. This experiment demonstrates that changes in plasmahormone levels may be important mediators of the differentialtumor growth rates observed in mice housed under the differenthousing conditions in our model.Another possible mediator of the differential tumor growthrates observed in this model is a shift in the responsiveness ofthe tumor cells to hormones. As noted previously, the SC115 tumoris heterogeneous, containing AR and Al cells (Emerman 1988;Emerman & Worth 1985). It has been demonstrated that growingS0115 tumor cells in an androgen deprived environment (in afemale mouse or in vitro) results in the selection of Al cells(Darbre & King 1987; Emerman 1988; Emerman & Worth 1985). Thus itis possible that selection for cells with greater (AR) or lesser(Al) hormone sensitivity may occur in animals in the 4 housingconditions, resulting in the differential tumor growth.In a recent study, this possibility was investigated. Invitro proliferation of tumor cells from IG and GI animals inresponse to DHT and HC was examined (Rowse et a/. 1992). Datashow that cells from both groups are significantly stimulated byboth DHT and HC. In fact, tumor cells from IG animals have asignificantly greater response to DHT and HC stimulation than do37tumor cells from GI animals. This result suggests that the slowergrowth rates of tumors in mice of the IG conditions result fromalterations in the internal environment of the mice rather thanfrom a decrease in the tumor cells' ability to respond tohormones.1.12 The Objectives of the Thesis There are two main objectives of this thesis:(1) The first objective was to extend the study of hormonemodulation of SC115 tumor growth. Most of the previous in vitrowork characterizing the SC115 tumor has been done using an ARcell line derived from the SC115 tumor. The present study isunique in that SC115 primary cultures were examined. The effectsof steroid hormones, growth factors, and anti-growth factorantibodies on the growth of SC115 tumor cells in serum-containingand serum-free medium in primary culture were investigated. Inaddition, this is the first study to examine the effects ofopioid agonists and antagonists on the regulation of AR SC115tumor growth.(2) The second objective of this thesis was to examine thepossible role of opioid peptides in modulating the differentialtumor growth rates of mice from our experimental housing groups.For this study, tumors were taken from mice in GI (largesttumors) and IG (smallest tumors) conditions, dissociated tosingle cells and cultured in serum-containing medium. In vitroresponse to steroid hormones and opioids of AR S0115 tumor cellsfrom mice in GI and IG conditions was studied.38Two major sets of experiments have been completed.I. Preliminary experiments to develop precedures(1) to choose the better procedure for measuring cell growth: theMTT assay versus hemacytometer counts.(2) to choose the optimal seeding density for the cell culturestudies.(3) to find the appropriate serum-free medium for growing SC115tumor cells.II. Primary Experiments(1) to study the effects of steroid hormones and growth factorson the growth of SC115 tumor cells in serum-containing andserum-free medium.(2) to study the effects of anti-bFGF and anti-EGF antibodies onthe growth of SC115 tumor cells in serum-free medium.(3) to study the effects of opioid agonists and antagonists onthe growth of SC115 tumor cells.(4) to study the effects of opioids on the growth of SC115 tumorcells from mice in GI (largest tumors) and IG (smallesttumors) conditions.39II Materials and Methods2.1 General Methods 2.1.1 Materials(1) Steroid Hormones and Growth FactorsDihydrotestosterone^(DHT),^hydrocortisone (HC),^178-estradiol (E2), epidermal growth factor (EGF), mouse anti-EGFantibody, and insulin were purchased from Sigma Chemical Co. (St.Louis, MO). Basic fibroblast growth factor (bFGF) (humanrecombinant) was purchased from Upstate Biotechnology, Inc. (UBI,Lake Placid, NY). IgG fraction of bFGF antibody was purchasedfrom R & D Systems, Inc. (Minneapolis, MN). The anti-bFGFantibody was prepared in rabbits by injection of highly-purifiednative bovine brain bFGF. Rabbit IgG used as the control was akind gift from Dr. J. H. Ledsome, Dept. of Physiology, Universityof British Columbia (Vancouver, B. C. Canada).DHT, HC, and E2 were prepared by dissolving in 95% ethylalcohol (ETOH, Sigma) in 10, 5, and 2 mg/ml stock solution,respectively, diluted to working solutions of 100, 500, and 20gg/mi, aliquoted at 1 ml/tube into polypropylene tubes (1.8 mlsize, Falcon, Lincoln Park, NJ), and then stored at -20°C. Thefinal concentration of ETOH in the culture medium was less than0.05%. Insulin dissolved in 0.005 M HC1 was stored in a workingsolution of 0.5 mg/ml at 4°C for 2 wk. Basic FGF was diluted to125 ng/gl in serum-free medium containing 0.5% bovine serumalbumin (BSA, pH 7.0-7.5, Sigma), aliquoted to 10-20 .ti/tube(Falcon, 1 ml polypropylene tubes), and stored at 4°C for 1 mo.EGF at a concentration of 10 ng/gl in distilled H2O was aliquoted40to 100 gl/tube and stored at -20°C. Lyophilized bFGF antibody (1mg/vial) was reconstituted in 48.5 gl dH20 and used immediately.Mouse anti-EGF antibody (0.1 ml/vial) was diluted to a workingsolution of 1:10 in serum-free medium, aliquoted to 0.5 ml/tube(1.8 ml size polypropylene, Falcon), and stored at 20°C for 1 wk.(2) Opioids13-Endorphin (13-EP), cyclazocine (CZ), and morphine sulfate(MS) were supplied by Peninsula laboratories (San Carlos, CA),Sterling Drug Co., and BDH Inc. (Toronto, Ontario), respectively.13-EP was dissolved in 0.005 M acetic acid, aliquoted at 2-4gg/glass tube (12 x 17 cm), lyophilized, and stored at 4°C. 13-EPsolution was made fresh with each medium change. CZ was dissolvedin 0.005 M HC1 at a concentration of 1 mg/ml. MS was dissolved indH20 at a concentration of 10 mg/ml. Both CZ and MS stocksolutions were aliquoted at 1 ml/tube (1.8 ml) and kept at 4°C.Naloxone (Sigma) was dissolved in 0.005 M HC1 at a concentrationof 4 mg/ml, aliquoted at 1 ml/tube (1.8 ml), and stored at 4°C.2.1.2 Maintaining the SC115 TumorThe androgen-responsive^(AR)^Shionogi mouse mammarycarcinoma (SC115) was maintained in our laboratory by serialtransplantation in intact male mice (mice that are not castrated)of the DD/S strain. The dissociation of tumors was as describedin our standard laboratory protocol (Emerman 1988; Emerman &Worth 1985). Tumors weighing approximately 2 g were dissectedfree of subcutaneous (sc) tissue and finely minced. The pieces41were transferred to a flask containing 0.05% trypsin (1:250;Grand Island Biological Co. [GIBCO], Burlinton, Ontario) and0.25% ethylenediamine tetraacetic acid (EDTA) (Sigma) in Ca2+-and Mg2+-free saline A, pH 7.3. The flask was shaken at 37°C fortwo 7 min periods. At the end of each peroid, the supernatant wasdecanted and cells in suspension were collected by centrifugationat 80 x g for 4 min. The pellets were resuspended in Dulbeco'smodified Eagle's medium (DMEM, Terry Fox Laboratory, Vancouver,B.C.), combined, and then passed through a 150 gm Nitex (TetkoInc., Elmsford, N.Y.) to collect single cells or small cellaggragates. Viable cells, determined by trypan blue exclusion,were counted on a hemacytometer (described below). Suspensions of3 x 106 cells in 0.1 ml DMEM were injected sc into theinterscapular region of intact male mice 2-4 mo old. Theremaining cells were resuspended in freezing medium (Appendix 1),frozen in 1.8 ml tubes at concentrations of 1-1.5 x 107cells/ml/tube, and stored in liquid nitrogen for use in cellculture studies.Cell counting was conducted using a hemacytometer. One dropof trypan blue was added to a cell suspension (10 ml) and mixed.A drop of cell suspension was placed onto both side wells of thehemacytometer. Viable cells were counted from the 4 cornersquares of both wells.2.1.3 Harvesting CellsIn the initial experiments, cell viability was determined bytrypan blue exclusion and viable cells were counted using a42hemacytometer. In order to do this, cells had to be cultured onto35 mm or 16 mm tissue culture dishes (Falcon). Not only did thisprocedure require a large number of cells, which was sometimesdifficult to obtain from dissociations of fresh tumors, but itwas also time consuming.The tetrazolium dye reduction (MTT) assay (Carmichael et a/.1987) modified for our laboratory procedures (described below;Stingl et a/. 1992) was then used to measure viable cell number.This dye is a soluble yellow compound that is cleaved by thedehydrogenase enzymes of mitochondria in living cells to form aninsoluble purple formazan product. Therefore, the amount offormazan product is proportional to the number of viable cells inthe population. The cells were cultured in 96-well microtiterplates and the plates were counted using a 96-well microtiterreader (model EL 311, Biotek Instruments Inc., Winooski,Vermont). This assay was used in order to process large numbersof samples rapidly. There was a linear relationship between cellcounts and the MTT assay, so that the MTT assay was usedroutingly in subsequent experiments. Results were expressed as apercentage of the controls (control conditions specified in eachexperiment).(1) Hemacytometer CountsSC115 tumor cells were cultured onto collagen-coated 35 mmdishes or 16 mm wells. At the end of the experiments, media wereremoved and 1-2 ml of collagenase (Appendix 2) was added to eachdish for 5 h in order to detach the cells from the plates. This43was followed by adding 1 ml of saline-trypsin versene (STV,Appendix 3) for another 30 min. Cell viability was determined bytrypan blue exclusion and viable cells were counted on ahemacytometer.(2) The MTT Assay[3-(4,5-Dimethylthiazol-2-y1)-2,5-diphenyltetrazolium]bromide (MTT, Sigma) was dissolved in phenol red-free F12/DMEM/H(Sigma) and filter sterilized using a 0.22 gm filter (Falcon).Media were removed and 1 mg/ml of MTT solution was added to eachwell at 100 41/well for 5 h at 37°C. At the end of thisincubation, 100 41 of 20% formal in saline was gently added toeach well for 30 min at room temperature to fix the cells to theplates. After removing the MTT-formal-saline solution, 100 gl ofisopropanol (Sigma) was added to each well for 1 h to dissolvethe formazan crystals. The plates were read under a 96-wellmicroplate reader. The amount of formazan crystals formed wasdetermined by absorbance at 540 nm. Results were expressed as apercentage of the controls.2.1.4 Animal-tumor Model for Studying Effects of Stressors onMammary Tumor GrowthThe colony of DD/S mice was maintained in our laboratoryaccording to our standard protocol (Weinberg & Emerman 1989).Mice were reared in sibling groups of 3 from weaning (3 wk ofage) until adulthood. Animals were housed under conditions ofcontrolled temperature (22°C) and lighting (12 h light; 12 h44dark) in a colony room that was protected from extraneouslaboratory noise.Experimental conditions designed to study the effects ofstressors on SC115 tumor growth were described as in Weinberg &Emerman (1989). Mice were reared either individually housed or insibling groups of 3 from weaning until adulthood (2-4 mo of age).At the start of the experiment, animals were injected sc in theinterscapular region with a single cell suspension of 3 x 106SC115 cells in 0.1 ml of DMEM. Two groups, balanced for age, wereformed immediately following tumor-cell injection:(1) IG---male mice raised singly housed were rehoused into groupsof 5 and injected with tumor cells.(2) GI---male mice raised in sibling groups of 3 were separated,singly housed, and injected with tumor cells.These different housing conditions reliably result indifferential tumor growth rates (Weinberg & Emerman, 1989). Inaddition, animals in all groups were subjected to acute dailynovelty stress (being placed into 1 of the 5 novel environments).This treatment, which reliably raises corticosterone levels(Friedman & Ader 1967) and the corticosterone response is slow tohabituate (Hennessy & Lesine, 1977; Pfister & King 1976),maximizes the differential tumor growth rates observed (Weinberg& Emerman 1989). Five different novel environments were used: (1)a clear plastic container, 9 cm in diameter x 7 cm in height, (2)a polypropylene container, 12 x 10 x 4 cm, (3) a polyethylenecontainer, 6 cm in diameter x 10 cm in height, (4) a cardboardbox divided into compartments, 7 x 7 x 14 cm and, (5) a standard45rodent cage, 18 x 29 x 13 cm, empty of bedding, food, and water.Animals were exposed to one of the five environments (as orderedabove) each day, 15 min/day, 5 d/week, between 0800 and 1200 h.Animals were not subjected to stress on the day of theexperiments.2.2 Methods for Preliminary Experiments For the experiments described below, frozen SC115 tumorcells were used unless otherwise indicated.2.2.1 Seeding Cells on Day 0 Versus Day 1This experiment was designed to determine if tumor cellswould grow better if given a one day recovery period afterthawing and before seeding or if seeded immediately afterthawing. We thought that a one day recovery period might allowthe dead or dying cells to be easily separated from viable cells.SC115 tumor cells were quickly thawed and washed twice withDMEM. Half of the cells were seeded immediately at 5 x 104cells/cm2 onto 35 mm collagen-coated tissue culture dishes inDMEM containing 2% dextran charcoal-treated fetal bovine serum(DCTFBS, also called basic medium, Appendix 4) with or withoutDHT (3.5 x 10-8 M) or HC (10-6 M). The remaining cells were firstplated overnight onto 10 cm non-tissue culture Petri dishes(Falcon) in 15 ml basic medium at 37° C, 95% air: 5% CO2. Thenext day, the cell suspension was centrifuged and the supernatantdecanted to eleminate non-viable cells. The cells were thenseeded at 5 x 104 cells/cm2 onto collagen-coated dishes. Media46were changed 48 h after cell seeding and thereafter onalternative days. On day 7 (after cell seeding), the cultureswere terminated and viable cells, determined by trypan blueexclusion, were counted on a hemacytometer.2.2.2 Seeding Density ExperimentThis experiment was designed to choose the optimal seedingdensity for all cell culture studies in this thesis. SC115 tumorcells were quickly thawed and seeded at 2.5 x 104, 5 x 104, and 1x 105 cells/cm2 onto collagen-coated 16 mm wells in basic mediumwith or without DHT (3.5 x 10-8 M) or HC (10-6 M). Media werechanged every other day. On day 5, cultures were terminated.Viable cells, determined by trypan blue exclusion, were countedon a hemacytometer.2.2.3 Selecting an Appropriate Serum-free Medium for the Growthof SC115 Tumor Cells in Primary CultureThis experiment was designed to find the appropriate serum-free medium for growing 5C115 tumor cells. Three serum-free mediawere tested: serum-free-I (F12/DME/H-containing 0.1 % BSA,Appendix 5), which is the serum-free medium described in theliterature for growing SC-3 cells (Nonomura et al. 1988; Omukaiet a/. 1987), serum-free-II (F12/DME/H-containing 0.1 % BSA, 0.1lig/m1 insulin, and 1.0 ng/ml cholera toxin, Appendix 5), andserum-free-III (F12/DME/H-containing 0.1 % BSA, 1.0 p.g/m1insulin, and 10 ng/ml cholera toxin, Appendix 5) which are twoserum-free media used for growing human mammary epithelial cells47in our laboratory (Emerman et al., manuscipt in preparation).SC115 tumor cells were quickly thawed, washed twice with DMEM,and incubated in basic medium in non-tissue culture Petri dishesovernight. The cell suspension was centrifuged to separate viablecells from non-viable cells. Viable cells were seeded at 5 x 104cells/cm2 onto collagen-coated 96-well microtiter plates andincubated in basic medium. On day 1, the serum-containing mediumwas removed and serum-free-I, serum-free-II, and serum-free-IIImedia were added to the plates. The media were changed on day 3,4, and 5. The cultures were terminated and cell numbersdetermined by the MTT assay. Cell growth was expressed as apercentage of the controls.SC115 tumor cells grew optimally in serum-free-II medium(see Results Section). Therefore, the serum-free-II medium wasused for all experiments (in this thesis serum-free medium alwaysrefers to serum-free-II medium).2.2.4 Determining Time Points for Studing the Effects of Opioidson SC115 Tumor Cell GrowthIn a series of preliminary experiments, cultures wereterminated at different time points to identify the mostappropriate time to measure the effects of opioids on growth.SC115 tumor cells were grown for 3 d, 5 d, and 7 d.In the first set of experiments, SC115 tumor cells werecultured at 5 x 104 cells/cm2 in basic medium with or without DHT(3.5 x 10-8 M) or HC (10-6 M) on collagen-coated 96-well plates.Three opioids, 5-EP, CZ, and MS, at concentrations of 10-11-10-748M were added to cultures on days 1 and 2; cultures wereterminated on day 3 and growth determined by the MTT assay.In the second set of experiments, S0115 tumor cells werecultured at 5 x 104 cells/cm2 in basic medium with or without DHT(3.5 x 10-8 M) or HC (10-6 M) on collagen-coated 96-well plates.13-EP, CZ, and MS at concentrations of 10 -11 -10 -7 M were added tocultures on days 2 and 4; cultures were terminated on day 5 andgrowth determined by the MTT assay.In the third set of experiments, S0115 tumor cells werecultured at 5 x 104 cells/cm2 in basic medium with or without DHT(3.5 x 10-8 M) or HC (10-6 M) on collagen-coated 35 mm plates. 1-EP, CZ, and MS at concentrations of 10 -9 -10 -7 M were added ondays 2, 4, and 6. Cultures were terminated on day 7 and viablecell numbers determined by trypan blue exclusion andhemacytometer counts. Growth was expressed as a percentage of thecontrols.2.3 Experiments 2.3.1 Effects of Steroid Hormones and Growth Factors on theGrowth of SC115 Tumor Cells in Serum-containing MediumSC115 tumor cells were quickly thawed, washed twice withDMEM, and incubated in basic medium in non-tissue culture Petridishes overnight. The cell suspension was centrifuged to separateviable from non-viable cells. Viable cells were then seeded at 5x 104 cells/cm2 onto collagen-coated 96-well microtiter platesand incubated in DMEM under one of the following 3 conditions: 1)DMEM plus 2% DCTFBS (basic medium); 2) basic medium plus one of49the following steroid hormones: DHT ranging from 1 ng/m1-1 gg/m1(3.5 x 10-10-3.5 x 10-6 M), HC ranging from 3.6 ng/m1-3.6 gg/m1(10-10 or E2 ranging from 0.27 ng/m1-0.27 gg/ml (10-9 -10-6 M); or 3) basic medium plus one of the following growthfactors: bFGF or EGF at concentrations of 1, 10, and 100 ng/ml(5.7 x 10-11-5.7 x 10 and 1.65 x 10-10-1.65 x 10-8 M,respectively). The media were changed on days 2, 3, and 4. On day5, cultures were terminated and cell numbers determined by theMTT assay. Cell growth was expressed as a percentage of thecontrols.2.3.2 Effects of Steroid Hormones, Growth factors, and Anti-growth Factors on the Growth of SC115 Tumor Cells in Serum-freeMediumSC115 tumor cells were quickly thawed, washed twice withDMEM, and incubated in basic medium in non-tissue culture Petridishes overnight. The cell suspension was centrifuged to separateviable from non-viable cells. Viable cells were seeded at 5 x 104cells/cm2 onto collagen-coated 96-well microtiter plates andincubated in basic medium. On the next day (day 1), the serum-containing medium was removed and the serum-free medium alone orserum-free medium containing one of the following was added tothe plates: 1) steroid hormones: 0.01 gg/ml DHT (3.5 x 10-8 M) or2.7 ng/ml E2 (10-8 M); 2) growth factors: bFGF at concentrationsof 1, 10, 100, 250, and 500 ng/ml or EGF at concentrations of 1,10, 50, and 100 ng/ml; 3) bFGF at concentrations of 1, 10, 100,250, and 500 ng/ml with 3.5 x 10-8 M DHT; 4) 400 lig/m1 anti-bFGF50alone or with 3.5 x 10-8 M DHT or with 10 ng/ml bFGF; 5) controlantibody alone or with 3.5 x 10-8 M DHT or with 10 ng/ml bFGF; 6)1:100 dilution of anti-EGF alone or with 10-8 M E2 or with 10ng/ml EGF; and 7) control antibody alone or with 10-8 M E2 orwith 10 ng/ml EGF. The media were changed on day 4 and on day 6,cultures were terminated and cell numbers determined by the MTTassay.2.3.3 Effects of Opioids on the Growth of SC115 Tumor CellsSC115 tumor cells were quickly thawed, washed twice withDMEM, and incubated in basic medium in non-tissue culture Petridishes overnight. Viable cells, collected by centrifugation, wereseeded at 5 x 104 cells/cm2 onto collagen-coated 96-wellmicrotiter plates and incubated in 5% FBS (containing endogenousphysiological concentrations of steroid hormones) or basic mediumwith or without 0.01 gg/m1 (3.5 x 10-8 M) DHT, 0.36 gg/m1 (10-6M) HC, or 10 ng/ml bFGF. Concentrations of steroid hormones andgrowth factor were those shown to be optimal for growthstimulation in the experiments above. Beginning on day 2 inculture, 13-EP, CZ, or MS were added with each medium change forthe next 3 consecutive days at concentrations ranging from 10-11-10 -7 M. On day 5, cultures were terminated and cell numbersdetermined by the MTT assay. Cell growth was expressed as apercentage of the controls.512.3.4 Effects of the Opioid Antagonist Naloxone on the Growth ofSC115 Tumor CellsSC115 tumor cells were quickly thawed, washed twice with 10ml DMEM, and incubated in basic medium in non-tissue culturePetri dishes overnight. Viable cells, collected bycentrifugation, were seeded at 5 x 10 4 cells/cm2 onto collagen-coated 96-well microtiter plates and incubated in basic mediumwith or without 0.01 gg/m1 (3.5 x 10-8 M) DHT or 0.36 gg/ml (10-6M) HC. Beginning on day 2 in culture, naloxone, an opioidantagonist,^at concentrations of 10-8 and 10 -6 M (3.6 ng/ml and360 ng/ml)^was added with or without either 8-EP (10 or MS(l0 ^with each medium change^for the next^3 d. On day 5,cultures were terminated and cell numbers determined by the MTTassay. Cell growth was expressed as a percentage of the controls.2.3.5 Effects of Opioids on the Growth of SC115 Tumor Cells fromthe Animal-tumor ModelThis study examined the effects of opioids on hormoneresponsiveness of SC115 tumor cells taken from mice in ourexperimental housing conditions. Animals were terminated at 3 wkpost tumor-cell injection, when the faster growing tumors hadreached a mass of 2-3 g. Tumors were removed from GI (largesttumors) and IG (smallest tumors) animals, and dissociated tosingle cell suspensions (as described in 2.1.2). Cells wereseeded at 5 x 104 cells/cm2 onto collagen-coated 96-wellmicrotiter plates either with basic medium alone or with basicmedium plus either 3.5 x 10-8 M DHT or 10-6 M HC. Cells were52incubated for 5 d at 37°C 5% 002. 5-EP and CZ ranging from10-7 M were added to half of the wells in each condition on days2, 3, and 4 with medium changes. In another experiment, naloxone(10-1°, 10-8, and 10-6 M) was also added alone or combined with10 8-EP. On day 5, numbers of viable cells were determined bythe MTT assay.2.4 Statistical Analysis Statistical analysis of the data was carried out usinganalyses of variance (ANOVAs) to examine dose-responserelationships for steroid hormones, growth factors, and opioids.1 way ANOVA was used for the factors of group or dose asappropriate for each experiment. 2 way ANOVA was used for thefactors of group and dose for each experiment. Significant maineffects or interactions in ANOVAs were further analyzed usingTukey's post-hoc tests.53III. Results3.1 Results of Preliminary Experiments to Develop Procedures 3.1.1 Seeding Cells on Day 0 Versus Day 1In this experiment, Shionogi mouse mammary carcinoma (SC115)cells were thawed and seeded at 5 x 104 cells/cm2 onto collagen-coated tissue culture dishes with or without dihydrotestosterone(DHT, 3.5 x 10-8 M) or hydrocortisone (HC, 10-6 M). Seeding wasdone either immediately (day 0) or following pre-incubation withDMEM containing 2% dextran charcoal-treated fetal bovine serum(DCTFBS), hereafter called basic medium, in non-tissue culturePetri dishes for 24 h. Fig.2 shows that cells pre-incubated for 1d had a higher growth rate in all media than cells seededimmediately after thawing. Therefore, pre-incubation of cells for24 h prior to seeding was chosen as the standard method for allexperiments using frozen SC115 tumor cells.3.1.2 Seeding Density Experiment50115 tumor cells were seeded onto collagen-coated plateswith basic medium, or medium containing DHT (3.5 x 10-8 M), or HC(10-6 M) to determine the optimal initial seeding density. Fig.3shows the growth rates after 5 d in culture of cells seeded at2.5 x 104, 5 x 104, and 1 x 105 cells/cm2 in basic medium with orwithout DHT or HC. At seeding densities of 5 x 104 and 1 x 105cells/cm2, cell growth was optimally stimulated by DHT and HC.However, at a seeding density of 5 x 104 cells/cm2, culturesbecame 75-80% confluent whereas at a seeding density of 1 x 105cells/cm2, cultures were 100% confluent in these media.54Fig.2 Seeding cells on day 0 versus day 1. Shionogi mousemammary carcinoma (SC115) cells were grown for a total of 7 d.The growth of S0115 tumor cells seeded on day 1 was greater thanthat of cells seeded on day 0 in DMEM containing 2% dextrancharcoal-treated fetal bovine serum (DCTFBS, also called basicmedium) and in dihydrotestosterone (DHT; 3.5 x 10-8 M)-, andhydrocortisone (HC; 10-6 M)-containing media. Data represent theresults of 2 replications. The control group consisted of cellsgrown in basic medium alone. Data are expressed as a percentageof the controls.50004004--'C0(30 300G)0)co 200-1-,C(1.)043 100a55Basic medium^DHT^HC56Fig.3 Seeding density experiment. SC115 tumor cells, seededwith 2.5 x 104, 5 x 104, and 1 x 105 cells/cm2, were grown for atotal of 5 d. The growth of SC115 tumor cells was increased withincreasing seeding densities whether in basic medium, or in DHT(3.5 x 10-8 M)-, or HC (10-6 M)-containing medium. Data representthe results of 2 replications. Error bars are too small to show.The control group consisted of cells grown in basic medium. Dataare expressed as a percentage of the controls.Basic mediumDHTHC2.5^5^ 104^2Cell number x 10 /cm57800-"0+-,C 6000(304000)0)(t)I.C(I)20000_58Therefore, a seeding density of 5 x 104 cells/cm2 was chosen asthe standard density for all experiments, since culturesterminated after 5 d were still in the exponential growth phase.3.1.3 Selecting an Appropriate Serum-free Medium for the Growthof SC115 Tumor Cells in Primary CultureThis experiment was designed to determine the appropriateserum-free medium for growing SC115 tumor cells in primaryculture. Growth of SC115 tumor cells was examined in serum-free-Imedium, serum-free-II medium, and serum-free-III medium with andwithout DHT (3.5 x 10-8 M) or HC (10-6 M). The ANOVA showed amain effect of treatment (p<0.01). Post-hoc tests showed thatcell numbers were lower in serum-free-I medium than in basicmedium, p<0.01, basic medium with DHT, p<0.001, and basic mediumwith HC, p<0.001 (Fig.4). In contrast, cell growth wassignificantly stimulated in serum-free-II and serum-free-IIImedia compared to that in basic medium, p's<0.01, basic mediumwith DHT, p's<0.01, and basic medium with HC, p's<0.01.Furthermore, cell growth in serum-free-III medium was alsosignificantly stimulated compared to serum-free-II medium in all3 conditions, p's<0.01. Since the growth pattern of SC115 tumorcells in serum-free-II medium was closer to basic medium thanthat in either serum-free-I or serum-free-III medium, serum-free-II medium was chosen as the optimal serum-free medium for growingSC115 tumor cells (from now on, serum-free medium refers toserum-free-II medium).59Fig.4 Growth of S0115 tumor cells in serum-free (SF) medium.SC115 tumor cells were seeded onto collagen-coated 96-well plates(5 x 104 cells/cm2) containing 100 ul basic medium. On the nextday, medium was changed to basic medium with or without DHT (3.5x 10 -8 M) or HC (10 -6 M), or SF-I, SF-II, and SF-III media withor without DHT (3.5 x 10-8 M) or HC (10-6 M). Cell growth wasexpressed as a percentage of the controls (basic medium with orwithout DHT or HC). Values represent means (+SEM) of 6_determinations. 50115 tumor cell growth was significantlyinhibited in SF-I medium alone (p<0.01), or SF-I medium with DHT(p<0.01), or HC (p<0.01) compared to that in basic medium, butcell growth was significantly increased in both SF-II and SF-IIImedia with either DHT (p<0.001) or HC (p<0.001) compared to thatin basic medium. **, p<0.01 compared to controls.6003002500U 200150C 10050Basic mediumSF-ISF-IIMedium without^Medium with^Medium withDHT or HC DHT HC613.1.4 Determining Time Points for Effects of Opioids on SC115Tumor Cell GrowthIn a series of preliminary experiments, cultures wereterminated at different time points. In the first set ofexperiments, 5-endorphin (8-EP), cyclazocine (CZ), and morphinesulfate (MS) at concentrations of 10-11-10-7 M were added tocultures containing basic medium plus DHT (3.5 x 10-8 M) or HC(10-6 M) on days 1 and 2, and cultures were terminated on day 3by the tetrazolium dye reduction (MTT) assay. As shown in Fig.5a,B-EP, CZ, and MS at all concentrations examined had nosignificant effects on SC115 cell growth in DHT-containing medium(p's>0.10). Similarly, 8-EP and CZ had no effect on SC115 tumorcell growth in medium containing HC. However, the ANOVA indicateda main effect for treatment of MS, p<0.01. Post-hoc tests showedthat MS at concentrations of 10-10-10-8 M had a significantstimulatory effect on SC115 tumor cell growth in HC-containingmedium compared to the control, p's<0.05, (Fig.5b).In the second set of experiments, 5-EP, CZ, and MS wereadded to cultures containing basic medium plus DHT (3.5 x 10-8 M)or HC (10-6 M) on days 2 and 4, and cultures were terminated onday 5 and assayed by the MTT method. In medium containing DHT,SC115 tumor cell growth was stimulated by l0 8-EP, 10-10 MCZ, and 10-11-10-1° M MS compared to the controls, p's<0.01,(Fig.6a). None of the remaining concentrations of the 3 opioidshad any effect on cell growth (Fig.6a). In medium containing HC,post-hoc test indicated that 10-9-10-8 M 8-EP and 10-10-10-9 M CZsignificantly inhibited cell growth compared to that in the62Fig.5 Effects of opioids on the growth of SC115 tumor cellscultured on collagen-coated dishes after 3 d in primary culture.5C115 tumor cells were cultured in basic medium with DHT (3.5 x10-8 M) or HC (10-6 M). Three opioids, 8-endorphin (8-EP),cyclazocine (CZ), and morphine sulfate (MS) at concentrations of10 -11 -10 -7 M, were added with the medium changes. Cell growth wasexpressed as a percentage of the controls (medium withoutopioids). Values represent means (+SEM) of 11-19 determinationsin 2-4 experiments. (a) 8-EP, CZ, and MS at all concentrationsexamined had no effects on SC115 cell growth in DHT-containingmedium. (b) 8-EP and CZ at all concentrations examined had noeffects on SC115 cell growth in HC-containing medium. MS atconcentrations of 10-1°-10-8 M had a significant stimulatoryeffect on SC115 cell growth in HC-containing medium, p's<0.05. * rp<0.05, **, p<0.01 compared to controls. Vb-EP^CZ^MS2002c 15000100(1)0 500200c 1500100cu(T)C.)^50?1-)0Control- 1110- 1010^M- 910^M- 810^M- 710^M63Control- 1110- 1010-910-a10 M- 710^Mb-EP^CZ^MS64Fig.6 Effects of opioids on the growth of SC115 tumor cellscultured on collagen-coated dishes after 5 d in primary culture.SC115 tumor cells were cultured in basic medium with DHT (3.5 x10-8 M) or HC (10-6 M). Three opioids, 3-EP, CZ, and MS atconcentrations of 10-11-10-7 M, were added with the mediumchanges. Cell growth was expressed as a percentage of thecontrols (medium wthout opioids). Values represent means (+SEM)of 6-26 determinations in 2-5 experiments. (a) 13-EP at aconcentration of 10-7 M, CZ at a concentration of 10-10 M, and MSat concentrations of 10-11-10-10 M significantly stimulated SC115cell growth compared to the controls, p's<0.01, in DHT-containingmedium. (b) 5-EP at concentrations of 10 -9-10-8 M and CZ atconcentrations of 10-10-10-9 M significantly inhibited SC115 cellgrowth compared to the controls, p's<0.05, in HC-containingmedium. MS had no effect on SC115 cell growth in HC-containingmedium. *, p<0.05, ** p<0.01 compared to controls. Control- 1 110-1010^M-910^M-810^M- 710^MP" "1b—EP^CZ^MS65200c 1500100j .(1)(LI(I)C.) 500200c 1500(.)100.CD(v50 50Q-)0Control- 1110- 1010-910--a10-710 Mb—EP^CZ^MS66controls, p's<0.05, (Fig.6b). MS at all concentrations examinedhad no significant inhibitory effect on cell growth.In the third set of experiments, 50115 tumor cells weregrown for 7 d and 13-EP, CZ, and MS were added to culturescontaining basic medium plus DHT (3.5 x 10-8 M) or HC (10-6 M).Media were changed on days 2, 4, and 6. As shown in Fig.7a, 13-EPat all concentrations examined (l0-10 significantlyinhibited 5C115 tumor cell growth in DHT-containing mediumcompared to that in the controls, p's<0.01. CZ and MS had noeffects on cell growth in the DHT-containing medium (Fig.7a). Onthe other hand, statistical analyses revealed that 13-EP and CZ atall concentrations examined (10-10 had no effects on SC115tumor cell growth in medium containing HC. Post-hoc showed thatMS (10 -9 -10 -7 M)) significantly inhibited S0115 tumor cell growthcompared to that in control conditions (p's<0.01) in HC-containing medium (Fig.7b).In these preliminary experiments designed to determine thetime for the optimal effect of opioids on SC115 tumor cellgrowth, opioids were added every other day. The results of thepreliminary experiments were variable. 13-EP had the mostconsistent inhibitory effect, whereas CZ and MS had variableeffects, resulting in inhibition, no effect or slightstimulation. The reason for this is not clear. One possibleexplanation might be that the opioids are not stable in thesolution. Thus, adding the opioids every other day in medium maynot be sufficient. Therefore, in the experiments undertaken forthis thesis, opioids were added to cultures daily with each67Fig.7 Effects of opioids on the growth of SC115 tumor cellscultured on collagen-coated dishes after 7 d in primary culture.SC115 tumor cells were cultured in basic medium with DHT (3.5 x10 -8 M) or HC (10 -6 M). Three opioids, 8-EP, CZ, and MS atconcentrations of 10-9-10-7 M, were added with the mediumchanges. Cell growth was expressed as a percentage of thecontrols (medium wthout opioids). Values represent means (+SEM)of 6-14 determinations in 2-3 experiments. (a) 8-EP at allconcentrations examined significantly inhibited SC115 cell growthin DHT-containing medium compared to that in the control,p's<0.01. CZ and MS had no effects on cell growth. (b) 8-EP andCZ had no effects on SC115 cell growth in HC-containing medium.MS at all concentrations examined significantly inhibited SC115cell growth in HC-containing medium compared to that in thecontrol, p<0.01. **, p<0.01 compared to controls.68b—EP^CZ^MSb—EP^CZ^MSControl- 910- a10- 710 MControl-910- a10- 710 M150125100755025rA(a) DHT* -X- ** -X- * - (b) HCr "15012510075502569medium change. As described in the results below, more consistentinhibitory effects of the opioids were observed.3.2 Experimental Results 3.2.1 Effects of Steroid Hormones and Growth Factors on theGrowth of SC115 Tumor Cells in Serum-containing Medium(1) Steroid HormonesEffects of DHT (3.5 x 10- 10-3.5 x 10-6 M) and HC (10-9-10-5M) on the growth of SC115 tumor cells were examined. After 5 d inculture, the ANOVA revealed a significant dose-response curve forDHT, p<0.001 (Fig.8a). Post-hoc tests indicated that doses of 3.5x 10 -9-3.5 x 10 -7 M were significantly different from control(basic medium without hormones). Growth stimulation was maximalat the physiological concentration of DHT (3.5 x 10-8 M) andgrowth in response to this dose was significantly different fromgrowth in response to doses of 3.5 x 10-10, 3.5 x 10-9, and 3.5 x10-6 M, p's<0.05. There was also a significant main effect ofdose for HC, p<0.01 (Fig.8b). Growth of SC115 cells at a dose ofl0 HC was significantly stimulated over the control level,p<0.01.In contrast, statistical analyses showed that 1713-estradiol(E2), from physiological to pharmacological concentrations (10-9-10 -6 M), significantly inhibited SC115 tumor cell growth after 5d in culture compared to that in basic medium without E2,pfs<0.001 (Fig.8c).70Fig.8 Effects of DHT, HC, or E2 on the growth of primarycultures of SC115 tumor cells. Cells were cultured in basicmedium plus or minus DHT, HC, or E2 on collagen-coated 96-welltissue culture plates and cultures were terminated after 5 d.Cell growth, as determined by the MTT assay, was expressed as apercentage of the control cultures (basic medium without steroidhormones). Values (means + SEM) with DHT or HC represent means of_29-30 determinations in 5 experiments; values with E2 represent6-12 determinations in 2 experiments. (a) SC115 cell growth wassignificantly stimulated by 3.5 x 10-9-3.5 x 10-7 M DHT(p's<0.001) compared to that in the control. Growth stimulationwas maximal at the physiological concentration of DHT (3.5 x 10-8M) and growth in response to this dose was significantlydifferent from growth in response to doses of 3.5 x 10-10, 3.5 x10-9, and 3.5 x 10-6 M, p's<0.05. (b) SC115 cell growth was alsosignificantly stimulated by l0 ^HC compared to that in thecontrol (p<0.01).^(c) SC115 cell growth was significantlyinhibited by 10-9-10-6 M E2 compared to that in the control(p's<0.001). *, p<0.05, **, p<0.01 compared to controls.400-153000200It2a)u 100---10^-a^-7^-60^10^10^10^10^10Concentrations (x 35 M)300-3 -8^-7^---E100^10^10^10^loConcentrations (M)103000o 20048a)100a)-9^-a^-7^-60^10 10 10 10Concentrations (M)71-510-510(a) DHT\NNT7-,/(b) HC0o 200(11.)100(c) E 2—^ x72(2) Growth FactorsLiterature has shown that growth stimulation of SC115 tumorcells by physiological doses of androgens may be mediated by afibroblast growth factor (FGF)-like polypeptide via an autocrineor paracrine mechanism. Similary, growth stimulation of mammaryepithelial cells by estrogens in the female may be mediated byepidermal growth factor (EGF) via an autocrine mechanism.Therefore, effects of bFGF and EGF, at concentrations of 1, 10,and 100 ng/ml, on SC115 tumor cell growth were investigated. TheANOVA revealed that bFGF significantly enhanced SC115 tumor cellgrowth in a dose-responsive manner in serum-containing medium,p<0.001 (Fig.9a). Significant stimulation over the control leveloccurred at the physiological concentration of 10 ng/ml, p<0.01,but maximal stimulation occurred at the pharmacologicalconcentration (100 ng/ml), p<0.05. In contrast, EGF markedlyinhibited SC115 tumor cell growth compared to the control at allconcentrations tested, p's<0.01, (Fig.9b).3.2.2 Effects of Steroid Hormones, Growth Factors, and Anti-growth Factors on the Growth of SC115 Tumor Cells in Serum-freeMedium(1) Effects of DHT and bFGF on the Growth of SC115 Tumor CellsThe effects of DHT and bFGF on the growth of 5C115 tumorcells in serum-free medium were studied. Fig.10a shows that DHT(3.5 x 10-8 M) alone significantly increased SC115 tumor cellgrowth (p<0.01) over the control levels. In addition, bFGF at allconcentrations examined (1, 10, 100, 250, and 500 ng/ml)73Fig.9 Effects of bFGF and EGF on the growth of primarycultures of SC115 tumor cells. Cells were cultured as describedin Fig.8. Cell growth was expressed as a percentage of thecontrol cultures (basic medium without bFGF or EGF). Valuesrepresent the means (+SEM) of 12-18 determinations in 2-3experiments. (a) SC115 cell growth was significantly stimulatedby 1-100 ng/ml bFGF in a dose-responsive manner (p<0.01).Significant stimulation over the control level occurred at thephysiological concentration of 10 ng/ml, p<0.01, but maximalstimulation occurred at the pharmacological concentration (100ng/ml), p<0.05. (b) SC115 cell growth was significantly inhibitedby 1-100 ng/ml EGF at all concentrations examined (p's<0.01). **,p<0.01 compared to controls.50040030020010074Basic medium1 ng/ml10 ng/ml100 ng/ml**=i^1Basic medium1 ng/m I1 0 ng/mI100 ng/ml 4;-_-y ,2_,coc..)4-6a)(5)cc5-1---,Ca)c)4-)a2002E 1500U0100(1)0)cc5-i--,Ca)50(1)0_(a) FGF Concentrations(b) EGF Concentrations75Fig.10 Effects of DHT, bFGF, or DHT (3.5 x 10-8 M) plus bFGFon the growth of primary cultures of SC115 tumor cells in serum-free medium. Cells were cultured on collagen-coated 96-welltissue culture plates in serum-free medium plus or minus DHT orbFGF. After 5 d, cultures were terminated and cell growthdetermined by the MTT assay. Cell growth was expressed as apercentage of the control cultures [serum-free medium withoutbFGF or DHT (100% growth) which is not shown in this graph].Values represent the means (+SEM) of 4-8 determinations. (a) bFGFat concentrations of 1, 10, 100, 250, and 500 ng/ml and DHT (3.5x 10-8 M) significantly stimulated SC115 cell growth compared tothe control, p's<0.01. (b) bFGF at all concentrations examinedwhen added to cultures containing DHT significantly stimulatedSC115 cell growth compared to that in the control, p's<0.01. * rp<0.05, **, p<0.01 compared to controls.4000-1-,E 3000U4-6O 200C))a)+-,c00,._^100(1)a04000+-,E 3000U02(1)^000)0-4-,c(1)100CDU-076 DHT i -1 10 100 250 500 1oFGF (ng/m1)DHT) -1 10 100 250500}DHTH-bFGF (ng/m1)77significantly increased cell growth in serum-free medium,p's<0.05, to a level similar to that of the pharmacologicalconcentration of bFGF in basic medium. Furthermore, thepossibility that DHT and bFGF may have any synergestic effects onSC115 cell growth was also examined. Data indicated that bFGF atall concentrations, when added to the medium together with 3.5 x10-8 M DHT, significantly increased SC115 cell growth compared tothe control, p's<0.001, (Fig.10b). Fig. 10a and 10b indicatesthat the growth stimulation produced by the addition of both DHTand bFGF was not significantly greater than that produced by bFGFalone. Therefore, it appears that DHT and bFGF do not havesynergestic effects on SC115 cell growth. All concentrations ofbFGF alone or bFGF added with DHT had greater stimulatory effectson SC115 tumor cell growth than DHT alone, p's<0.01.(2) Inhibitory Effects of Anti-bFGF Antibody on DI-IT- or bFGF-induced Growth of SC115 Tumor Cells in Serum-free MediumThe effects of anti-bFGF antibody on DHT- or bFGF-stimulatedgrowth of SC115 tumor cells were examined in serum-free mediumusing the MTT assay. As shown in Fig.11, SC115 tumor cell growthwas markedly and significantly inhibited by the addition of 400ug/ml anti-bFGF antibody to medium containing DHT or bFGF(p's<0.01), whereas the same amount of control IgG had nosignificant inhibitory effects (data not shown). When anti-bFGFantibody was added to the medium containing DHT, it significantlysuppressed SC115 tumor cell growth compared to that in DHT alone78Fig.11 Effects of anti-bFGF antibody on DHT- or bFGF-stimulated SC115 tumor cell growth in serum-free medium. Cellswere cultured as described in Fig.10. Cell growth was expressedas a percentage of the control cultures (c; serum-free mediumalone). Values represent the means (+SEM) of 4 determinations._Anti-bFGF antibody at a dose of 400 ug/ml significantly inhibitedDHT (3.5 x 10-8 M)- or bFGF (10 ng/m1)-stimulated SC115 cellgrowth (p's<0.01) and also inhibited cell growth in controlmedium alone (sero; anti-bFGF in control medium alone), (p<0.05).*, p<0.05, **, p<0.01 compared to controls.U 200c 100a)CI5^C DHT bFGF^0 DHT bFGF^No anti—bFGF^anti—bFGF7980(by 40%), p<0,01, and growth was suppressed to the control level.However, when anti-bFGF antibody was added together with bFGF, itsignificantly inhibited SC115 tumor cell growth compared to thatin bFGF alone (by 70%), p<0.01, and growth was suppressed tobelow the control level. It should be noted, however, that anti-bEGF antibody also significantly inhibited growth in the absenceof DHT and bFGF, albeit to a lesser degree (by 33%).(3) Effects of E2, EGF, and Anti-EGF Antibody on the Growth ofSC115 Tumor Cells in Serum-free MediumThe ANOVA indicated a main effect of treatment for E2. Post-hoc tests showed that after 5 d in culture, E2 (10-8 M)significantly decreased SC115 tumor cell growth compared to theserum-free medium without E2, p<0.01, (Fig.12). EGF at allconcentrations examined (1, 10, 50, and 100 ng/ml) had no effecton SC115 tumor cell growth (Fig.12). In addition, after 5 d inculture, anti-EGF antibody at a concentration of 1:100significantly inhibited SC115 tumor cell growth in serum-freemedium alone and in serum-free medium containing E2, or EGF (10ng/ml), p's<0.01 (Fig.13).3.2.3 Effects of Opioids on the Growth of SC115 Tumor CellsEffects of the opioid agonists, 13-EP, CZ, and MS, on thegrowth of SC115 tumor cells were studied. SC115 tumor cells werecultured in basic medium with or without steroid hormones (DHT at3.5 x 10-8 M or HC at 10-6 M), growth factor (bFGF at 10 ng/ml),or 5% FBS. Beginning on day 2 in culture, 8-EP, CZ, and MS were81Fig.12 Effects of EGF or E2 on the growth of primarycultures of SC115 tumor cells in serum-free medium. Cells werecultured as described in Fig.10. Cell growth was expressed as apercentage of the control cultures (c; serum-free medium withoutEGF or E2). Values represent the means (+SEM) of 4_determinations. E2 (10-8 M) significantly decreased SC115 tumorcell growth compared to the serum-free medium without E2, p<0.01.EGF at all concentrations examined (1, 10, 50, and 100 ng/ml) hadno effect on SC115 tumor cell growth compared to the control.**, p<0.01 compsred to controls.C 2 I1 10 50 100 1EGF (ng/ml)1501005008283Fig.13 Effects of anti-EGF antibody on SC115 tumor cellgrowth in serum-free medium with or without E2 or EGF. Cells werecultured as described in Fig.10. Cell growth was expressed as apercentage of the control cultures (c; serum-free medium alone).Values represent the means (+SEM) of 4 determinations. Anti-EGFantibody at a concentration of 1:100 significantly inhibitedSC115 tumor cell growth in serum-free medium alone (sero; anti-EGF antibody in serum-free medium alone), serum-free mediumcontaining E2, or serum-free medium containing EGF compared tothat in the control, p's<0.01. **, p<0.01 compared to controls.Percentage of Controlo 01o 8o85added with each medium change at concentrations ranging from10 -11 -10 -7 M for 3 consecutive days. As shown in Fig.14a, growthof S0115 tumor cells cultured in the basic medium (withoutsteroid hormones) was significantly decreased (approximately 20%)with the addition of 13-EP, p<0.001 and CZ, p<0.001 at allconcentrations examined. MS had no effect on cell growth in thisbasic medium except at a concentration of 1O where it had aninhibitory effect (p<0.01).Growth of SC115 tumor cells in medium containing DHT (3.5 x10-8 M) or HC (10 -6 M) was significantly retarded (from 25%-40%and 10-35%, respectively) compared to the growth in the controls(no opioids) condition, p's<0.001 (Fig.14b and 14c,respectively). This was true for all 3 opioids at allconcentrations examined. The ANOVAs revealed no dose-responserelationships for 13-EP, CZ, or MS in medium containing DHT or HC.13-EP, CZ, and MS (10-11-10-7 M) were also added to S0115tumor cells cultured in medium containing 5% FBS (which containsphysiological concentrations of all the endogeneous steroidhormones). Statistical analyses indicated that all 3 opioids atall concentrations examined significantly inhibited SC115 tumorcell growth (by about 30-35%) after 5 d in culture, p's<0.001(Fig.14d).Finally, the effect of 13-EP on the growth of SC115 tumorcells in medium containing 10 ng/ml bFGF was also investigated.The ANOVA revealed a main effect of treatment. Post-hoc testsshowed that 13-EP at concentrations of 10-1°-10-7 M, but not inconcentrations lower than 10-10 M (10-14, 10-13, and 10-11 M),86Fig.14 Effects of 8-endorphin (8-EP), cyclazocine (CZ), andmorphine sulfate (MS) on the growth of SC115 tumor cells culturedin medium with or without DHT, HC, or 5% fetal bovine serum(FBS). Cells were cultured in basic medium without or with DHT(3.5 x 10-8 M), HC (10-6 M), or 5% FBS, and 8-EP, CZ, or MS, asdescribed in Fig.8, and terminated after 5 d. Cell growth wasexpressed as a percentage of the control cultures (medium withoutopioids). Open bar represents control which is medium withoutopioids, while patterned bars represent medium containing opioidswith doses of 10-11-10-7 M going from left to right. Valuesrepresent the means (+SEM) of 18-30 determinations in 3-5experiments. (a) SC115 tumor cell growth was significantlyinhibited by 10-11-10-7 M 8-EP (p's<0.001), and CZ (p's<0.001),and by 10-11 M MS (p<0,01) in basic medium. (b) SC115 tumor cellgrowth was significantly inhibited by all 3 opioids at all dosesexamined in medium containing DHT (p's<0.001). (c) SC115 tumorcell growth was significantly inhibited by all 3 opioids at alldoses examined in medium containing HC (p's<0.001). (d) SC115tumor cell growth was significantly inhibited by all 3 opioids atall doses examined in medium containing 5% FBS (p's<0.001). * Ip<0.01 compared to controls.0•0 D . O N 6th0Percentage of Control0cn 0rip • Ir I^V.VAo. Al• Ak A. A. AA.knt-oKCCe:CnokiC•NC.:;•;•:,'4•Ve•Xq. • • • • • • : •;"::11•V ****• •APercentage of controlth87Percentage of Control^Percentage of control88significantly inhibited cell growth (by about 20%) compared tothat in medium without 8-EP, p's<0.05 (Fig.15).3.2.4 Effect of the Opioid Antagonist Naloxone on the Growth ofSC115 Tumor CellsIn order to study the mechanisms of these opioid effects onSC115 tumor cell growth, a g opioid receptor antagonist, naloxone(10-8 and 10-6 M), was added to SC115 tumor cells culturedtogether with 8-EP (l0 or MS (l0 The ANOVA indicated asignificant effect of treatment on 50115 tumor cell growth inDHT-containing medium, p<0.001. Fig.16a illustrates that 5-EPsignificantly inhibited 50115 cell growth, p<0.001, and naloxoneat concentrations of 10-8 and 10-6 M totally blocked the effectof 5-EP. MS also significantly inhibited SC115 tumor cell growth,p<0.001. Naloxone at a concentration of 10-8 M completely blockedthe inhibitory effect of MS, and at a concentration of 10-6 Mpartially, p<0.05, blocked the inhibitory effect of MS in DHT-containing medium.The ANOVA also indicated a significant effect of treatmenton 50115 tumor cell growth in HC-containing medium, p<0.01.Fig.16b shows that both 5-EP and MS significantly inhibited SC115tumor cell growth, p's<0.01, and naloxone at doses of 10-8 and10-6 M totally blocked the inhibitory effect of 8-EP and MS inHC-containing medium.A main effect of treatment indicated that naloxone alonealso significantly inhibited the growth of SC115 tumor cells inbasic medium, p<0.01, as well as in medium containing DHT,89Fig.15 Effects of 1-EP on the growth of SC115 tumor cellscultured for 5 d in medium containing bFGF (10 ng/ml). Cells werecultured as described in Fig.8. Cell growth was expressed as apercentage of the control cultures (medium without opioids).Values are the means (+SEM) of 12 determinations in 2experiments. S0115 tumor cell growth was significantly inhibitedby 10-10-10-7 M B-EP compared to that in the control (p's<0.01).**, p<().01 compared to controls.97_Fig.16 Effects of opioids and naloxone on the growth ofprimary cultures of SC115 tumor cells. Cells were cultured for 5d in basic medium plus either DHT (3.5 x 10-8 M) or HC (10-6 M),and 8-EP (1O or MS (10 with or without naloxone (10-8and 10-6 M) as described in Fig.8. Cell growth was expressed as apercentage of the control cultures (medium without opioids ornaloxone). Values represent the means (+SEM) of 12-18determinations in 2-3 experiments. (a) Naloxone at concentrationsof 10-8 and 10-6 M totally blocked the inhibitory effect of 5-EPon the growth of SC115 tumor cells in DHT-containing medium.Naloxone at a concentration of 10-8 M totally blocked theinhibitory effect of MS on the growth of SC115 tumor cells inDHT-containing medium. Naloxone at a concentration of 10-6 Mpartially blocked the inhibitory effect of MS on the growth ofSC115 tumor cells in DHT-containing medium, p<0.05. (b) Naloxoneat concentrations of 10 -8 and 10-6 M totally blocked theinhibitory effects of 8-EP or MS on the growth of SC115 cells inHC-containing medium. *, p<0.05, **, p<0.01 compared to controls.(a) b—EP (10- M) MS (10- M)T^*0^4rO 4O 4O 4O 4O 4O 40^4O 4O 4O 444_-_921201008060402012010080604020C EP EP EP-80 10 10-6 1Nal (M)C MS MS MS-8 -60 10 10 1Na I (M)oC EP EP EP-80 110 10-6 INal (M)C MS MS MS-80 11 0 1 0-6 INa! (M)93p<0.001, but had no effect in medium containing HC, p>0.10,(Fig.17). Post-hoc tests showed that 10-6 M naloxonesignificantly inhibited SC115 tumor cell growth in basic mediumby approximately 20%, p<0.01 (Fig.17a) and that 10 1°, 10-8, and10-6 M naloxone significantly inhibited cell growth in DHT-containing medium by approximately 10-20%, p's<0.05 (Fig.17b).3.2.5 Effects of Opioids on the Growth of SC115 Tumor Cells fromthe Animal-tumor Model(1) Effects of Steroid Hormones on the Growth of SC115 TumorCells from Mice in Experimental Housing GroupsThis study examined hormone responsiveness of 5C115 tumorcells taken from mice in our experimental housing conditions.Cells from mice in both IG (small tumors) and GI (large tumors)groups were significantly stimulated by both 3.5 x 10-8 M DHT(p<0.01) and 10-6 M HC (p<0.01) compared to that seen in basicmedium as shown in Fig.18. There were no significant differencesbetween the 2 groups in response to either DHT or HC, p>0.10.However, in both GI and IG groups, DHT had a significantlygreater stimulatory effect on SC115 tumor cell growth than HC,p's<0.01.(2) Effects of Opioids and Naloxone on the Growth of SC115 TumorCells from Mice in Experimental Housing GroupsThis study examined the role of opioids in the differentialtumor growth rates of SC115 cells from IG (small tumor) and GI(large tumor) animals. Addition of 8-EP to cells cultured in94Fig.17 Effect of naloxone alone on the growth of primarycultures of SC115 tumor cells. Cells were cultured for 5 d inbasic medium with or without DHT (3.5 x 10-8 M) or HC (10-6 M) asdescribed in Fig.8. Cell growth was expressed as a percentage ofthe control cultures (medium without naloxone). Values representthe means (+SEM) of 16-42 determinations in 3-7 experiments. (a)10-6 M naloxone significantly inhibited SC115 cell growth in10,basic medium by approximately 20%, p<0.01. (b) 1°- 10-8, and10-6 M naloxone significantly inhibited cell growth in DHT-containing medium by approximately 10-20%, p's<0.05. (c) 10-12-10-6 M naloxone had no effect on SC115 cell growth in HC-contining medium. *, p<0.05, **, p<0.01 compared to controls.95Basic medium^DHT^HCNaloxone Concentrations1 1 Control-1210^M-1010^M--E3M 1 0 M1 0-6 M:::•:•:•:•:•:•:•::.•:•:!:•:::•::::96Fig.18 Effects of DHT and HC on the growth of primarycultures of S0115 tumor cells from mice in the experimentalhousing groups. Values represent the means (+SEM) of 6determinations. SC115 tumor cells from both GI and IG mice weresignificantly stimulated to the same degree by DHT (3.5 x 10-8 M,p's<0.01) and HC (10-6 M, p's<0.01). *, p<0.05 compared tocontrols.Percentage of Control_,^rv^w0^o o o^o0^0 o 098basic medium resulted in a significant main effect of group,p<0.01, as well as a group x dose interaction, p<0.05, (Fig.19a).Overall, tumor cells from IG mice showed a greater inhibition ofgrowth with 8-EP than tumor cells from GI mice. Allconcentrations of 8-EP significantly inhibited SC115 tumor cellgrowth compared to the controls in both GI and IG groups,p's<0.01. In addition, 10-8 M 8-EP had a greater inhibitoryeffect than 10-10 M 8-EP on cells from IG mice, p<0.05.With the addition of CZ, there was also a significant maineffect of group, p<0.05, and a group x dose interaction, p<0.001,(Fig.19b). In contrast to the results for 8-EP, with CZ, tumorcells from GI mice showed a greater suppression than tumor cellsfrom IC mice. All concentrations of CZ significantly inhibitedSC115 tumor cell growth compared to the controls in both GI andIG groups, p's<0.001. In addition, 10 CZ had a greaterinhibitory effect than 10 CZ on cells from IG mice, p<0.05.If 8-EP was added to medium containing DHT, there was asignificant main effect of group, p<0.001, (Fig.20a). Tumor cellsfrom GI mice showed a greater inhibition of growth with 8-EP thantumor cells from IG mice, p<0.01. All concentrations of 8-EPsignificantly inhibited SC115 tumor cell growth compared to thecontrols in both GI and IG groups, p'2s<0.01. No dose-responserelationships were observed for either animal group.If CZ was added to medium containing DHT, there was also asignificant main effect of group, p<0.001, (Fig.20b). Tumor cellsfrom GI mice showed a greater inhibition of growth with CZ thantumor cells from IG mice, p<0.001. All concentrations of CZ99Fig.19 Effects of 8-EP and CZ on the growth of primarycultures of SC115 tumor cells from mice in the experimentalhousing groups. Cells were cultured in basic medium. Cell growthwas expressed as a percentage of the control cultures (mediumwithout opioids). Values represent the means (+SEM) of 6determinations. (a) SC115 tumor cell growth from both GI and IGmice was significantly inhibited by 10-10-10-7 M 8-EP (p's<0.01).(b) SC115 tumor cell growth from both GI and IG mice wassignificantly inhibited by 10-10-10-7 M CZ (p's<0.01). **, p<0.01compared to controls.-650o-NJI O^0I 0^0^I ^Dil Lo^8^,--I-a_Percentage of Controlco___t.^__I.^_._■.^_t.0 0 0 0ICOI^I^1-..1  oK K K KPercentage of Control1\)^0")^CO^00 0 0^0 0 00101Fig.20 Effects of 13-EP and CZ on the growth of primarycultures of SC115 tumor cells from mice in the experimentalhousing groups. Cells were cultured in basic medium containingDHT (3.5 x 10-8 M). Cell growth was expressed as a percentage ofthe control cultures (medium without opioids). Values representthe means (+SEM) of 6 determinations. (a) Growth of SC115 cellsfrom both GI and IG mice was significantly inhibited by 10-10 -10 -7 M 13-EP (p's<0.01). (b) Growth of SC115 cells from GI micewas significantly inhibited by 10-1°-10-7 M CZ (p's<0.01),whereas growth of SC115 cells from IG mice was significantlyinhibited by 10-9-10-7 M CZ (p's<0.01). *, p<0.05, **, p<0.01compared to controls.Percentage of ControlcoPercentage of ControlcoK K K■•:;"*.XV._,.^"^ L^o0 o 0 0I^,^,^D_ CO LO o^INU00 0 0 0 0I■**103significantly inhibited SC115 tumor cell growth compared to thecontrols in GI mice, p's<0.001. CZ at concentrations of 10-9-10-7M significantly inhibited SC115 tumor cell growth compared to thecontrols in IG mice, p's<0.001. No dose-response relationshipswere observed for either animal group.If B-EP was added to medium containing HC, there was asignificant main effect of group, p<0.001, as well as a group Xdose interaction, p<0.001 (Fig.21a). Tumor cells from IG miceshowed a greater inhibition of growth with 8-EP than tumor cellsfrom GI mice. All concentrations of 8-EP significantly inhibitedSC115 tumor cell growth compared to the controls in both GI andIG groups, p's<0.01. In addition, growth inhibition was greater-10with concentrations of 10 -10-8 M 8-EP than with aconcentration of 10 -7 M 8-EP on cells from IG mice, p's<0.001.If CZ was added to medium containing HC, there was asignificant group X dose interaction, p<0.05 (Fig.21b). In GImice, CZ at concentrations of 10-9-10-7 M significantly inhibitedS0115 tumor cell growth, p's<0.01, whereas in IG mice, CZ atconcentrations of 10 -10 M significantly inhibited SC115tumor cell growth, p's<0.05, compared to that in controlcultures. No dose-response relationships were observed for eitheranimal group.Additionally, naloxone at concentrations of 10- 12_10-6 mtotally blocked the effects of 10 3-EP at all concentrationsexamined in the GI group both in basic medium and in HC-containing medium (Fig.22a and Fig.24a). However, naloxone at aconcentration of 10-6 M had a slight but significant stimulatory104Fig.21 Effects of 13-EP and CZ on the growth of primarycultures of SC115 tumor cells from mice in the experimentalhousing groups. Cells were cultured in basic medium containing HC(10-6 M). Cell growth was expressed as a percentage of thecontrol cultures (medium without opioids). Values represent themeans (+SEM) of 6 determinations. (a) Growth of SC115 cells fromboth GI and IG mice was significantly inhibited by 10-10-10-7 M1-EP (p's<0.01). (b) Growth of SC115 cells from GI mice wassignificantly inhibited by 10-9-10-7 M CZ (p's<0.01), whereasgrowth of SC115 cells from IG mice was significantly inhibited by10 -10 M CZ (p's<0.01). *, p<0.05, * *, p<0.01 compared tocontrols.co01r 1X•:•. _L ^_L^.-.^no o o o 0^1^clipklo^D^■J^8 ^-1- 0K <K K•r.r•. ^.4.^. . .^.' 11" "IPW11'.■ '4"_L77 -_L0 P-^+0koPercentage of Control^Percentage of Controlcn106Fig.22 Effects of 8-EP plus naloxone in basic medium on thegrowth of primary cultures of SC115 tumor cells from mice in theexperimental housing groups. Cells were cultured in basic mediumwith 8-EP (10-9 M) plus naloxone (10- 12_10-6 NI,.) Cell growth wasexpressed as a percentage of the control cultures (medium withoutopioid or naloxone). Values represent the means (+SEM) of 6determinations. (a) In GI mice, naloxone at concentrations of10 -12 -10-6 M totally blocked the inhibitory effect of 13-EP onSC115 cell growth compared to that in the control. (b) In IGmice, naloxone at a concentration of 10-12 M partially blockedthe inhibitory effect of 8-EP on SC115 cell growth compared tothat in the control (p<0.01), but naloxone at concentrations of- -1010 , 108 , and 10 -6 M totally blocked the inhibitory effect ofB-EP on cell growth. **, p<0.01 compared to controls.107Naloxone Concentrations- 1010Control- 1210X•:•:•:•:•:•:•;- 810 M- 610 M108effect on the growth of SC115 tumor cells from GI mice in mediumcontaining DHT (3.5 x 10-8 M), p's<0.01 (Fig. 23a). In the IGgroup, naloxone, at a concentration of 10-12 M partially blockedthe inhibitory effect of 13-EP (by about 92%) in basic medium,p<0.05 (Fig.22b) and at concentrations of 10- 10-10-6 M totallyblocked the inhibitory effect of 8-EP, p's>0.10. Naloxone at allconcentrations examined totally blocked the inhibitory effect of13-EP in HC-containing medium in both GI and IG animals, p's>0.10(Fig.24a and 24b) and at a concentration of 10-8 M partiallyblocked the inhibitory effect of 13-EP in DHT-containing medium(by almost 95%), p<0.01 (Fig.23b).Naloxone alone had no effect on the growth of tumor cellfrom either GI or IG groups in basic medium or in mediumcontaining DHT or HC, p>0.10 (Fig.25a and 25b, respectively).109Fig.23 Effects of 8-EP plus naloxone in DHT-containingmedium on the growth of primary cultures of SC115 tumor cellsfrom mice in the experimental housing groups. Cells were culturedin basic medium with 8-EP (l0 plus naloxone (10 12_10-6 m).Cell growth was expressed as a percentage of the control cultures(medium without opioid or naloxone). Values represent the means(+SEM) of 6 determinations. (a) In GI mice, naloxone atconcentrations of 10-12-10-8 M totally blocked the inhibitoryeffect of 8-EP on SC115 cell growth compared to that in thecontrol whereas naloxone at a concentration of 10-6 Msignificantly increased cell growth (p<0.01). (b) In IG mice,naloxone at a concentration of 10-8 M partially blocked theinhibitory effect of 8-EP on cell growth compared to that in thecontrol (p<0.05), but naloxone at concentrations of 10- 12, 10-10,and 10 -6 M totally blocked the inhibitory effect of 8-EP on cellgrowth. *, p<0.05, **, p<0.01 compared to controls.150A- 1010- 8M 10 M- 610 MControl- 1210":•?.•1.44WIV1 10Naloxone Concentrations111Fig.24 Effects of 8-EP plus naloxone in HC-containing mediumon the growth of primary cultures of S0115 tumor cells from micein the experimental housing groups. Cells were cultured in basicmedium with 8-EP (10-9 M) plus naloxone (10-12-10-6 M). Cellgrowth was expressed as a percentage of the control cultures(medium without opioid or naloxone). Values represent the means(+SEm) of 6 determinations. (a) In GI mice, naloxone atconcentrations of 10-12-10-6 M totally blocked the inhibitoryeffect of 8-EP on S0115 cell growth compared to that in thecontrol. (b) In IG mice, naloxone at concentrations of 10- 12_10-6M totally blocked the inhibitory effect of 8-EP on cell growthcompared to that in the control.1121202 •000U 8060Naloxone ConcentrationsControl- 1210- 1010- 810^M- 610^M113Fig.25 Effects of naloxone on the growth of primary culturesof SC115 tumor cells from mice in the experimental housinggroups. Cells were cultured in basic medium or medium containingDHT (3.5 x 10-8 M) or HC (10-6 M). Cell growth was expressed as apercentage of the control cultures (medium without naloxone).Values represent the means (+SEM) of 6 determinations. (a)Naloxone at concentrations of 10-10-10-6 M had no effect on thegrowth of SC115 tumor cells from GI mice. (b) Naloxone atconcentrations of 10-10-10-6 M had no effect on the growth ofSC115 tumor cells from IG mice.114Basic medium^DHT^HCBasic medium^DHT^HCNaloxone Concentrations12010080604020-4AControl-1010- 810^M-610 MControl- 1010-810- 6Ma 10120100806040200.11■"*"A115IV. Discussion and ConclusionsThe present study investigated the effects of steroidhormones, growth factors, and opioid peptides on the growth ofthe androgen-responsive (AR) Shionogi mouse mammary carcinoma(SC115) in primary culture.4.1 Culturing AR SC115 Tumor Cells on a Collagen Substrate Mammary tumor cells cultured on conventional tissue cultureplastic lose their tissue specific characteristics, making itdifficult to extrapolate data from experiments in vitro to the invivo situation. For example, Yates and King (1981) cultured acell line originating from the AR SC115 tumor on plastic, andobserved that changes in growth properties and morphologyaccompanied changes in hormone responsiveness which did notreflect tumor behavior in vivo. In contrast, it has been shownthat normal mammary epithelial cells from several species retaintheir normal morphological and functional characteristics whengrown on a collagen substrate (Emerman et al. 1990; Emerman &Pitelke 1977). Emerman and Worth (1985) demonstrated that thegrowth rates, morphological, and tumorigenic potentialcharacterizing the AR SC115 tumor and its androgen-independent(Al) variant persist when cells from these tumors are cultured oncollagen gels. Furthermore, a study of the interaction betweenhormones and chemotherapeutic agents on AR SC115 tumor cellsurvival demonstrated that results in the in vitro collagen gelsystem (Emerman 1988) parallel the results obtained in in vivo116studies (Emerman & Siemiatkowcki 1984). In the present study, ARSC115 tumor cells were cultured on collagen-coated plates in allexperiments. Therefore, we believe that our in vitro resultsaccurately reflect the in vivo situation.4.2 Growth-stimulation of AR SC115 Tumor Cells by SteroidHormones and Growth Factors Previous data have shown that physiological concentrationsof androgens and pharmacological concentrations ofglucocorticoids significantly stimulate the proliferation ofSC115 tumor cells in vivo (Bruchovsky & Meakin 1973; Hiraoka etal. 1987; Matsumoto et al. 1982; Watanabe et al. 1982) as well asthe growth of the AR cell line, SC-3 (derived from the SC115tumor) in vitro (Hiraoka et al. 1987; Omukai et a/. 1982; Yateset al. 1980; Yates & King 1978). Data from the presentexperiments with 5C115 tumor cells in primary culture confirmthat dihydrotestosterone (DHT) ranging from 3.5 x 10-1° - 3•5 x10-6 M and hydrocortisone (HC) ranging from 10-9-10-5 Msignificantly increase growth of SC115 tumor cells. With DHT, asignificant dose-response curve is observed. Maximal stimulationby DHT occurs with the physiological concentration (3.5 x 10-8 M)whereas maximal stimulation by HC occurs with a pharmacologicalconcentration (10Our data also show that 17B-estradiol (E2), ranging fromphysiological to pharmacological concentrations (10-9-10-6 M)inhibits the growth of SC115 tumor cells. Previous studiesindicate that E2 in concentrations even higher than 10-6 M has no117effect on SC-3 cell growth in vitro (Noguchi et al. 1987), butinhibits the growth-stimulating effect of testosterone on SC-3cells in a dose-dependent manner in serum-supplemented medium(Noguchi et a/. 1987). E2 also shows competitive effects on {3H]-testosterone binding to androgen receptors in SC-3 cells. Effectsof estrogens on growth of the AR SC115 tumor in vivo, however,show conflicting results. Nohno et al. (1982) showed that when E2at a daily dose of 2 mg/kg is given to intact male mice bearingthe original androgen-dependent SC115 tumor, tumor growth as wellas weight gains of seminal vesicles and prostate gland areinhibited. Estrogens bind to the estrogen receptor (ER) but alsobind to the androgen receptor which is likely responsible fortheir inhibitory effects on growth. In other studies,pharmacological concentrations of E2 (160-400 jig/kg) were shownto stimulate this androgen-dependent SC115 tumor cell growth incastrated male mice in a dose-dependent manner (Noguchi et al.1984; 1985a). E2 can act synergistically with low doses oftestosterone propionate (TP) to stimulate tumor growth (Noguchiet al. 1984). The stimulatory effect of estrogen on proliferationof SC115 tumor cells was shown to be mediated by ER, but notandrogen receptor in vivo. The inhibitory effect of E2 observedin our experiments may help to explain the fact that the originalAR SC115 tumor grows more rapidly in males than in females. It ispossible that E2 may bind to the ER as well as to the androgenreceptor (Nohno et a/. 1982).Recent studies have shown that growth stimulatory effects ofsex steroids on cell proliferation are mediated by specific118polypeptide growth factors (Lippman et al. 1986). Data suggestthat the effect of androgens on proliferation of SC115 tumorcells is mediated by a fibroblast growth factor (FGF)-likepolypeptide in an autocrine fashion (Nakamura et al. 1989). Ourresults show that bFGF also enhances S0115 tumor cell growth inprimary culture in a dose-dependent manner (by 3 fold) in serum-containing medium. Similarly, evidence suggests that E 2stimulation of mammary epithelial cell growth in the female(Davidson & Lippman 1989) may be mediated by epidermal growthfactor (EGF) through an autocrine mechanism (Bates et a/. 1986;Dickson et a/. 1986). In estrogen-responsive cancer cells,Lippman et a/. (1986) have suggested that all stimulatory effectsof estrogens on the growth of human breast cancer cells such asMCF-7 are mediated through transforming growth factor (TGF)-a orinsulin-like growth factor-I (IGF-I) through an autocrinemechanism. Our data demonstrate that EGF inhibits cell growth atall concentrations examined in serum-containing medium. Theseresults suggest that the inhibitory effect of estrogen on S0115tumor growth may also be mediated by EGF.In order to investigate the molecular mechanisms of tumorcell growth, however, a culture system using a serum-free mediumis desirable, since serum contains various growth factors andunknown other factors. Other investigators (Nakamura et al. 1987;Nonomura et a/. 1988) using a serum-free culture system (serum-free-I as defined in Methods), found that testosterone, highconcentrations of dexamethasone (Dex), and bFGF significantlystimulate the growth of SC-3 cells. For our studies, we developed119a serum-free medium which was optimal for growing SC115 tumorcells in primary culture (i.e., serum-free-II as defined inMethods). Consistent with previous studies, our data show thatDHT (3.5 x 10-8 M) and bFGF (1-500 ng/ml) at all concentrationsexamined significantly stimulate SC115 tumor cell growth inprimary culture in our serum-free culture medium. Furthermore,our data demonstrate a marked inhibitory effect of anti-bFGFantibody on bFGF- and androgen-induced growth of SC115 tumorcells. Therefore, the present study provides evidence suggestingthat SC115 tumor cells may produce a FGF-like peptide(s) whichmediates, at least partially, their androgen-induced growth.Anti-bFGF antibody also inhibited growth to a limited degree inthe absence of DHT or bFGF. It is possible that bFGF is anautocrine growth factor for these cells that is produced insmaller quantities in the absence of DHT. In contrast, EGF at allconcentrations examined (1-100 ng/ml) had no effect on the growthof SC115 tumor cells in serum-free medium whereas E2 had a markedgrowth inhibitory effect on SC115 tumor cells in serum-freemedium. So it is unlikely that the inhibitory effect of E2 on thegrowth of SC115 tumor cells is mediated by EGF. In addition,anti-EGF antibody also had a significant inhibitory effect on thegrwoth of SC115 tumor cells in serum-free medium, and this effectwas the same in medium containing EGF, E2, or in serum-freemedium alone. The reason for the non-specific effect of anti-EGFon SC115 tumor cell growth remains to be investigated. This non-specific effect may be due to the fact that the concentration ofanti-EGF antibody is too high, so it inhibits cell growth120nonspecifically. Alternatively, the antibody may bind to someunknown factors in the serum-free medium to inhibit the cellgrowth.4.3. Growth-stimulation of AR SC115 Tumor Cells by SteroidHormones is Inhibited by Opioid Agonists Opioids and opioid peptides can modulate both normal andmalignant cell growth (Lewis et a/. 1983; Slotkin et al. 1980;Willson et al. 1976; Zagon & McLaughlin 1981a). The present studyis the first to demonstrate that the opioid peptide system may beinvolved in modulating the growth of SC115 tumor cells in primaryculture. Three opioids, 13-endorphin (5-EP), cyclazocine (CZ), andmorphine sulfate (MS), at the minimal dose of 10-11 M were shownto retard SC115 tumor cell growth in basic medium, as well as inDHT-, HC-, 5% FBS-, and bFGF-containing media. The opioids had agreater inhibitory effect in the presence of DHT than in itsabsence. Furthermore, the inhibitory effect of 13-EP occurred atthe minimal dose of 10-10 M in bFGF-containing medium. No dose-response relationship was observed at doses of 10-11-10-7 M forall 3 opioids in all medium conditions.The various neuropharmacological effects of opioids andendogenous opioid peptides are known to occur through interactionwith at least one of the 4 opioid receptor subtypes (8, k, g, and0). Although multiple opioid receptor subtypes have not beencharacterized from 5C115 tumor cells at this time, it is possiblethat opioid modulation of tumor growth may be mediated by peptidebinding to opioid receptors. Multiple cellular receptors for121opioid peptides have been found in several other tumor lines,including neuroblastomas, the MCF-7 cell line, and small cellcarcinomas of the lung (Kazmi & Mishra 1986; Maneckjee et a/.1990; Roth & Bachas 1986). The effects of opioids on tumor growthhave been seen both in vivo (Lewis et a/. 1983; Zagon &McLaughlin 1981a) and in vitro (Maneckjee et a/. 1990; VonHoff &Forseth 1982; Zagon & McLaughlin 1981c). In vitro, opioidsprobably exert their inhibitory effects on tumor growth viadirect binding to tumor cell receptors. This has beendemonstrated in neuroblastoma (Zagon & McLaughlin 1981c) and inthe human breast cancer MCF-7 cell line (Maneckjee et a/. 1990).The effects of opioids on tumor growth in vivo may be stimulatoryor inhibitory. Chronic administration of heroin effectivelyretards tumor growth and prolongs survival time in mice withtransplanted neuroblastoma (Zagon & McLaughlin 1981a). Ratsinjected with a mammary ascites tumor and subjected to aninescapable footshock stress, which causes endogenous opioidrelease, manifest enhanced tumor growth indicated by decreasedsurvival time and decreased percent survival (Lewis et al. 1983).Three possible mechanisms may underlie opioid effects on tumorgrowth in vivo. First, they may alter tumor growth through directeffect on opioid receptors present on tumor cells. Multipleopioid receptors have been characterized from several tumor celllines, although further work needs to be done to characterize theopioid receptors in the SC115 tumor. Second, they may alter tumorgrowth through the release of hormones which can modulate tumorgrowth, such as prolactin and growth hormone. These two hormones122are known to have a significant impact on mammary tumor growth(Emerman et al. 1985; Shiu et a/. 1987). Third, they may altertumor growth indirectly through modulation of immune surveillancemechanisms. Opioids have been found to influence the functions ofmost of the major cell types within the immune system (Fischer1988; Weber & Pert 1984). Opioids can decrease antibodyproduction, depress mitogen-induced blastogenesis by both T and Blymphocytes, enhance or reduce CTL or NK cell activities, andmodulate the production of lymphokines (Bocchini et a/. 1983;Faith et al. 1984; Gilman et a/. 1982; Gilmore & Weiner 1988;Johnson et al. 1982, Shavit et al. 1987). The final effect ofopioids on tumor growth will be the net effect of these threeinfluences.Our data further demonstrate that when naloxone, a g opioidreceptor antagonist, is added to the culture medium, it totallyor partially blocks the inhibitory effects of 13-EP and MS ontumor cell growth. Such reversal of opioid effects has beendemonstrated in several other systems both in vivo (Zagon &McLaughlin 1981a) and in vitro (Maneckjee et al. 1990; Zagon &McLaughlin 1983), and may reflect both direct and indirectmechanisms of action of opioid peptides and their antagonists.The regression of growth of spontaneous and transplanted mammarytumors in 03H mice by intratumoral injections of naloxonesuggests that the effect of this drug may be through a directaction against the tumor itself (Tsunashima et al. 1982)involving the opioid receptor system. In addition, the opioidantagonist naltrexone has been shown to have both positive and123negative effects on the growth of neuroblastomas in mice (Zagon &McLaughlin 1983). These apparently conflicting effects resultfrom dose-dependent actions of the drug. Lower doses ofnaltrexone reduce tumor incidence and delay the appearance oftumors, whereas higher doses reduce the time before tumorappearance and decrease survival time compared to control, tumorbearing mice. In our system, naloxone alone also shows small butsignificant inhibitory effects on tumor cell growth in mediumcontaining physiological doses of DHT and in basic medium, butnot in medium containing HC. It is possible that if naloxone isadded to medium together with opioids, it acts as an antagonist,but if added alone, it may act as a partial agonist (throughopioid receptors). Alternatively, naloxone alone may act throughindependent channels (through different receptors) or may provokea compensatory release of endorphins, and these endogenouscompounds may in turn work in a fashion similar to that ofopioids. Evidence has shown that some tumor lines secreteendogenous opioids, including the MCF-7 cell line and small lungcarcinoma (Roth & Barchas 1986). Naloxone is also a potentinhibitor of prolactin secretion by the pituitary (Armstrong etal. 1988) and this hormone has been implicated in growth andmaintenance of mammary carcinoma.In these experiments, the inhibitory effect of opioids isgreater in the DHT-stimulated medium than in the basic steroidhormone-free medium. Since the majority of human breast cancersare ER positive and hence believed to be hormonally responsive,this observation may have clinical significance. In fact, in the124MCF-7 cell line, the inhibitory effect of opioids is restrictedto the hormonally responsive fraction (Maneckjee et a/. 1990).This observation may also have relevence for the choice ofendocrine therapy and/or chemotherapy. Evidence has already shownthat concentrations of androgen suboptimal for growth increasethe sensitivity of the AR SC115 tumor to chemotherapeutic agentswhereas the condition with no androgens present is leastsensitive to these agents (Emerman 1988). Whether or not theseeffects of opioids observed in culture translate to the in vivosystem of solid tumor growth remains to be determined. Howeverour data would suggest that a combination of endocrine ablationand administration of antineoplastic agents may not be beneficialto the clinical management of breast cancer.4.4 Opioid Agonists Inhibit the Growth of AR SC115 Tumor Cells from Animals in the Experimental Conditions of Our Animal-tumor Model The SC115 tumor is heterogenous, containing AR and Al cells(Emerman & Semiatkowski 1984; Emerman & Worth 1985). It has beendemonstrated that growing SC115 tumor cells in an androgendeprived environment (in a female mouse or in vitro) results inthe selection of Al cells (Darbre & King 1987; Emerman 1988).Therefore a study was undertaken to determine if selection forcells with greater (AR) or lesser (Al) hormone sensitivity mayoccur in animals in our different housing conditions resulting indifferential tumor growth rates (Rowse et al. 1992). That is, wewanted to determine if tumors from GI mice (large tumors) have a125greater response to DHT or HC than tumors from IG mice (smalltumors). The results of that study indicated that thedifferential tumor growth rates observed between animals from theGI and IG housing groups are not due to differential selection ofAl versus AR tumor cells. Whereas in vivo, tumors in IG mice havea slower growth rate than tumors in GI mice, in vitro, cells fromtumors of IG mice grow faster and are actually more responsive toDHT and HC than cells from tumors of GI mice. Data from ourexperiments replicate the findings of Rowse et al. (1992).Furthermore, the possible role of opioid peptides inmodulating the differential tumor growth rates of mice from theexperimental housing groups was also examined. Tumors were takenfrom mice in GI and IG groups. Our data show that SC115 tumorcells from animals in both IG and GI groups are significantlyinhibited by all 3 opioid peptides (8-EP, CZ, and MS). Overall,13-EP had a greater inhibitory effect on the growth of S0115 tumorcells from IG mice than on the growth of cells from GI mice inboth basic medium and HC-containing medium. However, in DHT-containing medium, 8-EP had a greater inhibitory effect on thegrowth of SC115 tumor cells from GI mice than on the growth ofcells from IG mice. CZ had a greater inhibitory effect on thegrowth of SC115 tumor cells from GI mice than on the growth ofcells from IG mice in basic medium and DHT-containing medium, butCZ had the same inhibitory effect on the growth of cells fromboth groups in HC-containing medium. The inhibitory effect of 13-EP on tumor cell growth was totally blocked by the presence ofnaloxone in DHT- or HC-containing medium. Naloxone alone had no126effect on the growth of SC115 tumor cell from experimental mice.As noted above, naloxone alone had a small but significantinhibitory effects on the growth of SC115 tumor cells from micereared under our standard laboratory housing conditions (i.e.housed in groups of 3, not subjected to stress) in some mediumconditions possibly due to naloxone's partial agonist effect.Naloxone had no effect on tumor cells from mice exposed topsychosocial stressors (different housing conditions). Stress maychange the response of tumor cells to naloxone.Evidence from previous work in our laboratory suggests thatthe differential tumor growth rates of mice from the differenthousing conditions in our animal-tumor model may be mediated byboth endocrine and immune changes in these mice. Plasma levels ofandrogens appear to be particulary important (Rowse et a/. 1993).Data from the present experiments now show that opioids areregulators of SC115 tumor cell growth in vitro. The relevance ofchanges in plasma and/or pituitary levels of 13-EP-likeimmunoreactivity in 5C115 tumor growth rates of mice housed inthe experimental housing conditions of our model remains to bedetermined.4.5 Concluding Remarks The experiments in this thesis were designed to investigatethe effects of steroid hormones and growth factors on the growthof the AR SC115 mammary carcinoma in primary culture. A secondobjective of this thesis was to study the effects of opioid127peptides on the growth of the AR SC115 carcinoma. The majorfinding from this thesis are as follows:1) DHT (3.5 x 10-10-3.5 x 10-6 M) and HC (10-9-10-5 M)significantly stimulate SC115 tumor cell growth in serum-containing medium. The stimulatory effect of DHT is dose-responsive. DHT (3.5 x 10-8 M) also stimulates SC115 tumorcell growth in serum-free medium. The maximal effect of DHT isat the physiological concentration (3.5 x 10-8 M) and that ofHC is at a pharmacological concentration (i02) bFGF (1-100 ng/ml) significantly stimulates the AR S0115 tumorcell growth in serum-containing in a dose-response manner andin serum-free medium at all concentrations examined (1-500ng/ml).3) Anti-bFGF antibody significantly inhibits the DHT- and bFGF-induced SC115 tumor cell growth. AR SC115 tumor cells mayproduce a FGF-like polypeptide which partially mediatesandrogen-induced growth.4) E2, from physiological to pharmacological concentrations,significantly inhibits the AR SC115 tumor cell growth inserum-containing medium. In addition, E2 (10-8 M) alsoinhibits SC115 tumor cell growth in serum-free medium.5) EGF (1-100 ng/ml) significantly inhibits the AR SC115 tumorcell growth in serum-containing medium, but not in serum-freemedium.6) 5-EP, CZ, and MS (10-11-10-7 M) significantly inhibit thegrowth of the AR SC115 tumor cells in medium containing DHT,HC, bFGF, or in medium containing 5% FBS at the minimal dose128of 10 -11 M. Inhibition by 13-EP occurs at a minimal dose of10-10 M in bFGF-containing medium. Inhibition of SC115 cellgrowth by 8-EP, CZ, and MS is maximal in DHT-containingmedium.7) Inhibitory effects of 8-EP (10 ^and MS (10 ^on the AR50115 tumor cell growth are totally or partially blocked by anopioid antagonist, naloxone (10-8 and 10-6 M). Thus, multipleopioid receptors may be involved in the inhibitory effects ofthe opioids on the growth of 5C115 tumor cells.8) Naloxone (10- 12_10-6 M) alone also significantly inhibitsS0115 tumor cell growth. Further experiments need to be doneto characterize the possible mechanisms involved in thisinhibitory effect of naloxone.9) The AR 50115 tumor cells from both GI and IG animals aresignificantly stimulated by DHT (3.5 x 10-8 M) and HC (10-6M). In vitro, cells from tumors of IC and GI mice areresponsive to DHT and HC to the same degree. These datasuggest that the slower growth rates of tumors in mice of theIG group result from alterations in the internal environmentof the mice rather than from a decrease or increase in thetumor cell's ability to respond to hormones.10) 8-EP and CZ (10-10-10-7 M) significantly inhibit the growthof AR SC115 tumor cells from both GI and IG animals. Theinhibitory effect of 8-EP (10 ^on the growth of S0115tumor cells from both GI and IG animals is reversed bynaloxone (10-12-10 -6 M).129In conclusion, we have demonstrated that the growth of ARS0115 tumor cells in primary culture is stimulated byphysiological concentration to pharmacological concentrations ofDHT, by pharmacological concentrations of HC, and by bFGF. Thesestimulatory effects are inhibited by opioid agonists. The opioidpeptide systems may be involved in regulating endocrine controlof the growth of the androgen-responsive Shionogi mouse mammarycarcinoma in vivo. Opioid peptide systems may also play a role inthe differential tumor growth rates of SC115 tumor cells frommice in the experimental housing groups of our animal-tumormodel.130V. 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Dextran Charcoal-treated Fetal Bovine SerumSerum:^100 mlCharcoal:^10 mg/ml serumDextran: 1 mg/ml serum143Shake water bath for 2 hSpin at 4000 RPM for 30 min at room temperaturePre-filter (0.45 g)Sterile filter (0.22 11)5. Serum-free (SF) MediumSF-I:^100 mlF12/DME/H^100 mlBSA^0.1%SF-II:^100 mlF12/DME/H^100 mlInsulin 0.1 lg/m1Cholera Toxin^1.0 ng/mlBSA^0.1%SF-III:^100 mlF12/DME/H^100 mlInsulin 1.0 gg/mlCholera Toxin^10 ng/mlBSA^0.1%144

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