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The effects of cyclosporin A, tamoxifen and medroxyprogesterone acetate on the enhacement of adriamycin… Claudio, Jerome Anthony A. 1995

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The Effects of Cyclosporin A, Tamoxifen and Medroxyprogesterone Acetate on the Enhancement of Adriamycin Cytotoxicity in Primary Cultures of Human Breast Epithelial Cells Jerome Anthony A. Claudio B.Sc. (Pharmacology and Therapeutics), University of British Columbia, 1991 A THESIS SUBMITTED FN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (DEPARTMENT OF ANATOMY) We accept this thesis as conforming t^p the required standa/dy THE UNi¥TiRSITY OF BRITISH COLUMBIA SEPTEMBER, 1995 © Jerome Anthony A. Claudio In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of AK.^ -iorny The University of British Columbia Vancouver, Canada Date (QeJcS&r 13. /<*<kf DE-6 (2/88) THESIS ABSTRACT Adriamycin (Adr), the single most active agent used in the treatment of breast cancer, may become ineffective as treatment progresses due to the development of multidrug resistant (MDR) tumors. A major mechanism associated with MDR is increased P-glycoprotein (Pgp) expression. This thesis examined the abilities of the anti-estrogen tamoxifen (TAM) and the progestin medroxyprogesterone acetate (MPA) as well as cyclosporin A (CsA), a known resistance modifier, to enhance the cytotoxic effects of Adr on human breast epithelial cells (HBEC) in primary culture. Pgp and estrogen receptor (ER) expression were determined in each of the cultures by immunocytochemical assays using the monoclonal antibodies C219 and H222 Spy, respectively. The Adr-sensitive, Pgp-, ER+ MCF-7 cell line and the Adr-resistant, Pgp+, ER- MCF7-AdrR cell line were used as controls. Primary cultures were categorized as HBEC from tissues with or without previous chemotherapy. Pgp was detected in 1 of the 15 cell cultures from tissues without previous chemotherapy and in 5 of the 6 cell cultures from tissues previously exposed to chemotherapy. Incubation with either CsA or MPA plus Adr enhanced Adr toxicity in Pgp+ but not Pgp- cell cultures, whereas T A M had no effect on the sensitivity of any of the cultures. Of the 21 primary cultures of HBEC, 3 were ER+. There was no correlation between the enhancement of Adr cytotoxicity and ER status. The data suggest that MPA as well as CsA may be useful as modifying agents in overcoming Pgp-associated multidrug resistance. TABLE OF CONTENTS Page THESIS ABSTRACT ii LIST OF TABLES vii LIST OF FIGURES : viii LIST OF ABBREVIATIONS ix ACKNOWLEDGEMENTS x CHAPTER 1. INTRODUCTION 1 1. Epidemiology of Breast Cancer 1 2. Treatment of Breast Cancer 3 3. Drug Resistance 4 4. Multidrug Resistance 6 5. Typical Multidrug Resistance: P-glycoprotein (Pgp) 8 5a. Structure and function of Pgp 9 5b. The mdr gene 11 5c. mdrl gene and Pgp in normal tissue 12 5d. mdrl gene and Pgp in human breast cancer 12 5d (i) Studies in breast cancer cell lines 13 5d (ii) Studies in clinical breast cancer 15 6. Atypical Multidrug Resistance 19 6a. Glutathione-S-transferases 19 6b. Topoisomerase II 21 6c. Cytochrome P450 22 6d. Glutathione peroxidase 22 6e. Multidrug resistance-associated protein 23 i i i 7. Circumventing Multidrug Resistance 24 7a. Cyclosporin-A (CsA) 25 7b. Tamoxifen (TAM) 26 7c. Medroxyprogesterone acetate (MPA) 27 8. Thesis Objectives 28 CHAPTER 2. M A T E R I A L S A N D METHODS 30 1. Processing Human Breast Tissue for Primary Cultures 30 2. Dissociation of Processed Tissue 31 3. Primary Cell Culture of Human Breast Epithelial Cells (HBEC) 32 4. Cell Line Culture 34 5. Immunocytochemistry 35 5a. Cytospinning cell culture samples for immunocytochemistry 35 5b. Pgp immunocytochemical assay (ICA).: 36 5c. Estrogen receptor (ER) ICA 37 5d. Evaluation of Pgp and E R content 38 6. Drug Testing Protocol 38 7. The M T T Assay 39 8. Statistics 40 CHAPTER 3. RESULTS 41 1. Pgp-ICA 41 la. Pgp-ICA in cell lines 41 lb. Pgp-ICA in primary cultures of H B E C 45 2. ER-ICA 49 2a. ER-ICA in cell lines 49 2b. ER-ICA in primary cultures of H B E C 53 3. Effects of Resistance Modifiers (RM) on Adriamycin (Adr) Cytotoxicity in the Cell Lines 56 iv 3a. Cytotoxic effects of Adr alone in cell lines 56 3b. Effects of CsA on Adr cytotoxicity in cell lines 56 3 c. Effects of T A M on Adr cytotoxicity in cell lines 58 3d. Effects of MPA on Adr cytotoxicity in cell lines 59 4. Effects of RM on Adr Cytotoxicity in Primary Cultures of HBEC With or Without Pgp Expression 62 4a. Cytotoxic effects of Adr alone in primary cultures of HBEC with or without Pgp expression 62 4b. Effects of CsA on Adr cytotoxicity in primary cultures of HBEC with or without Pgp expression 62 4c. Effects of T A M on Adr cytotoxicity in primary cultures of HBEC with or without Pgp expression 63 4d. Effects of MPA on Adr cytotoxicity in primary cultures of HBEC with or without Pgp expression 63 5. Effects of RM on Adr Cytotoxicity in Primary Cultures of HBEC With or Without ER Expression 67 5a. Cytotoxic effects of Adr alone in primary cultures of HBEC with or without ER expression 67 5b. Effects of CsA on Adr cytotoxicity in primary cultures of HBEC with or without ER expression 67 5c. Effects of CsA on Adr cytotoxicity in primary cultures of HBEC with or without ER expression 67 5d. Effects of CsA on Adr cytotoxicity in primary cultures of HBEC with or without ER expression 68 6. Effects of R M on Adr Cytotoxicity in ER- Primary Cultures of HBEC With or Without Pgp Expression 71 6a. Cytotoxic effects of Adr alone in ER- primary cultures v of HBEC with or without Pgp expression 71 6b. Effects of CsA on Adr cytotoxicity in ER- primary cultures of HBEC with or without Pgp expression 71 6c. Effects of TAM on Adr cytotoxicity in ER- primary cultures of HBEC with or without Pgp expression 71 6d. Effects of MP A on Adr cytotoxicity in ER- primary cultures of HBEC with or without Pgp expression 72 CHAPTER 4. DISCUSSION 74 REFERENCES 80 APPENDIX 1: Transport Medium 91 APPENDIX 2: Freezing Medium 91 APPENDIX 3: Dissociation Medium 91 APPENDIX 4: Attachment Medium for Human Breast Epithelial Cells (HBEC) in Primary Culture 91 APPENDIX 5: Preparation of Rat Tail Collagen 91 APPENDIX 6: Phenol red-free, Serum-free Medium 92 APPENDIX 7: Growth Medium for the MCF-7 Cell Line 92 APPENDIX 8: Growth Medium for the MCF7-AdrR Cell Line 92 APPENDIX 9: Preparation of Saline-Trypsin-Versene 94 APPENDIX 10: Preparation of Specimen Storage Medium for Primary Cultures of HBEC on Coverslips 92 APPENDIX 11: Preparation of Phosphate Buffered Saline (PBS) 92 APPENDIX 12: Preparation of Pooled Normal Human Serum 92 APPENDLX 13: Preparation of Dextran Charcoal-treated Serum 93 vi L I S T O F T A B L E S Table 1: Mechanisms of drug resistance observed in cancer cells 5 Table 2: Isoforms of Pgp found in mammalian cells .11 Table 3: Primary cell cultures with cells expressing Pgp 45 Table 4: Effects of resistance modifiers (RM) on Adr cytotoxicity in MCF-7 and MCF7-AdrR cell lines 61 Table 5: Normalized data of the effects of R M on Adr cytotoxicity in MCF-7 and MCF7-AdrR cell lines 61 Table 6: Effects of R M on Adr cytotoxicity in Pgp+ and Pgp- primary cultures of ER- human breast epithelial cells 73 Table 7: Normalized data of the effects of R M on Adr cytotoxicity in Pgp+ and Pgp- primary cultures of ER- human breast epithelial cells 73 vii LIST OF FIGURES Figure 1: A model for the transmembrane topology of P-glycoprotein (Pgp) 10 Figure 2: A 96-well microtiter plate 33 Figure 3: Pgp expression in a cytospin preparation of the MCF7-AdrR cell line 42 Figure 4: Pgp expression in MCF7-AdrR cells cultured on a coverslip 43 Figure 5: Pgp expression in MCF-7 cells cultured on a coverslip 44 Figure 6: Background peroxidase staining in the MCF7-AdrR cell line and a 6 day primary culture of HBEC 46 Figure 7: Pgp expression in a 6 day primary culture of HBEC 47 Figure 8: Pgp-negative HBEC in 6 day primary culture 48 Figure 9: ER expression in a cytospin preparation of the MCF-7 cell line 50 Figure 10: ER expression in MCF-7 cells cultured on coverslips 51 Figure 11: ER expression in MCF7-AdrR cells cultured on coverslips 52 Figure 12: ER expression in HBEC in a 6 day primary culture 54 Figure 13: ER-negative HBEC in 6 day primary culture 55 Figure 14: The effect of different concentrations of CsA on Adr cytotoxicity in the MCF7-AdrR cell line 57 Figure 15: The effect of different concentrations of MP A on Adr cytotoxicity in the MCF7-AdrR cell line 60 Figure 16: Effects of resistance modifiers (RM) on Adr cytotoxicity in Pgp+ (n=6) and Pgp- (n=15) primary cultures of HBEC 65 Figure 17: Effects of R M on Adr cytotoxicity in ER+ (n=3) and ER- (n=18) primary cultures of HBEC 69 viii L I S T O F A B B R E V I A T I O N S Adr Adriamycin CsA Cyclosporin-A DAB Diaminobenzidine tetrahydrochloride ER Estrogen receptor GST Glutathione-S-transferase GST-p The 7t-class glutathione-S-transferase HBEC Human breast epithelial cell ICA Immunocytochemical assay MDR Multidrug resistance mdrl The class I isoform of the multidrug resistance gene mitox Mitoxantrone MPA Medroxyprogesterone acetate MRP Multidrug resistance-associated protein MTT 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide PBS Phosphate buffered saline Pgp P-glycoprotein PgR Progesterone receptor PKC Protein kinase C R M Resistance modifier T A M Tamoxifen TopoII Topoisomerase II VP-16 Etoposide ix A C K N O W L E D G E M E N T S There are many people I wish to thank without whom the completion of this thesis would not have been accomplished. First and foremost, it is with my deepest thanks that I acknowldge my supervisor Dr. Joanne Emerman. Her patience, support and guidance were invaluable during my studies. I would also like to thank the members of my Supervisory Committee: Dr. Nelly Auersperg, who also allowed me the use of her Macintosh computer and ELIZA reader, and Dr. Joanne Weinberg. I wish to thank, too, the members of my Thesis Defense Committee, Dr. John Church, Chair, Dr. Donna Hogge, Dr. Calvin Roskelley and Dr. Joanne Weinberg. I know that their constructive comments after careful reading of my Thesis will aid in the final writing of this work of which I am very proud. Darcy Wilkinson is also owed thanks for her help in teaching me techniques in tissue culture and her help in other laboratory procedures. I would also like to thank Shannon Wilson for her technical assistance. A special thanks is owed to the graduate students, faculty and staff of the Department of Anatomy. Interacting with such a diverse group of individuals has made my graduate experience an exciting period of my life. Finally, I would like to give a great thanks to my family. I would never have been able to complete this work without their support, guidance and tolerance. This research was supported by a grant to Dr. Joanne Emerman from the B.C. Health Research Foundation. C H A P T E R 1 INTRODUCTION 1. Epidemiology of Breast Cancer The breast is a tubuloalveolar gland made up of a stromal and epithelial component. At rest, the breast is mainly made up of 90% connective tissue and 10% epithelial tissue [Russo and Russo, 1987]. The epithelial component of the mammary gland functions in the production and secrection of milk. Epithelial cells form the duct system of the human mammary gland, which consists of 15-20 major ducts called lactiferous ducts each of which drains a system of smaller ducts. The lactiferous ducts leave the body through individual openings at the nipple. Lactiferous ducts drain smaller intralobar ducts that, in turn, drain yet smaller interlobular ducts which terminate in lobuloalveolar regions called lobules. These are the secretory units of the gland and are made up of small ductules and the alveoli that they drain. It is the epithelial cells that line the ducts and lobules that give rise to breast cancer. The most common cause of cancer death in women is cancer of the breast. For women in the U.S. between the ages of 40 and 55 years, breast cancer is the leading cause of death [Colditz, 1993]. The incidence of breast cancer has been steadily increasing ever since the beginning of its data collection in 1973 at the National Cancer Institute, although mortality due to the disease has remained relatively stable [Newcomb and Lantz, 1993]. One reason for the increase in incidence can be attributed to increased use of mammography and, therefore, earlier detection of small tumors. It is hypothesized, however, that the mortality rate will eventually start to decline due to the impact of mammography. In fact, data from the National Center for Health Statistics reported that mortality rate decreased 2.4 percent between 1990 and 1991 [Newcomb and Lantz, 1993]. Nonetheless, one hopes that the incidence of the disease can also be decreased. Factors proposed to increase the risk of breast cancer are hormone levels, obesity and diet [Tretli l et al, 1990; Colditz, 1993; Howe, 1994; Byers, 1994; and Inskip, 1994], Furthermore, a familial link has been reported in some cases of breast cancer [Organ Jr., 1988; King, 1991]. Although many of these factors cannot be controlled, with the exception of diet, women can understand their own relative risk which may lead to an increased rate of early detection of breast cancer. Estrogen and progesterone are important hormones for the growth and development of the mammary gland. Estrogen and progesterone bind to estrogen receptors (ER) and progesterone receptors (PgR), respectively. During puberty, estrogen stimulates rapid growth of mammary connective tissue cells and ductal epithelial cells. Estrogen plus progesterone, in addition to growth hormone or prolactin, dramatically stimulates alveolar epithelial cell growth in the mammary gland during pregnancy [Topper and Freeman, 1980]. The duration of exposure to estrogen from puberty to menopause may be important with respect to the risk of breast cancer. The Nurses' Health Study reported that women who have a late menarche and an early menopause have a lower risk of developing breast cancer [Colditz, 1993]. Women who have an early menarche and/or a late menopause have an increased risk of developing the disease. Since increased hormone exposure is of great concern, the Nurses' Health Study also included women taking hormones as a contraceptive or who were involved in hormone-replacement therapy. Women involved in either use of these hormones also have an increased risk of getting breast cancer. Oral contraceptive users have an increased risk of 53% and woman undergoing hormone-replacement therapy have an increased risk of 36%. Time of pregnancy also seems to contribute to the incidence of breast cancer. Women who give birth to their first child at an early age have a reduced risk. It has been suggested that the reduction of breast cancer incidence may be explained by high levels of progesterone produced during pregnancy [Davidson and Lippman, 1989]. Another factor that contributes to the increased incidence of breast cancer is obesity [Tretli et al, 1990; Byers, 1994]. The adipocytes are a site of conversion of 2 androstenedione to estrone, an inactive precursor of estrogen [Davidson and Lippman, 1989]. This may contribute to elevated levels of estrogen in the breast [Witliff, 1974]. Although dietary fat intake has been proposed to be a factor in increased incidence of breast cancer, epidemiologic studies have not shown the two to be strongly linked [Howe, 1994]. However, a weak association between the two may exist. 2. Treatment of Breast Cancer For those patients who develop breast cancer, common treatment is removal of the solid tumor through surgery often followed by radiation and/or a chemotherapeutic regimen in order to kill any remaining cancer cells. The anthracycline Adriamycin (Adr) is the most effective single chemotherapeutic agent as it produces an initial response rate of 48% in patients with metastatic breast cancer. Unfortunately, this rapidly declines to 28% in patients who have previously been treated with the drug [Tormey et al, 1977]. Nonetheless, combination regimens have included the use of Adr because of its initial effectiveness. For example, CAF (cyclophosphamide, Adr and 5-fluorouracil) treatment results in an 82% response rate compared with CMF (cyclophosphamide, methotrexate and 5-fluorouracil), which produces a 68% response rate [Tormey et al, 1977]. The size of the tumor mass is important with respect to the effectiveness of chemotherapy in curing breast cancer. Surgical removal of a small tumor mass, less than 2 cm in diameter, followed by adjuvant chemotherapy is able to cure some women with primary breast carcinoma [Dalton, 1990]. Those who also have metastasis to less than 4 lymph nodes benefit from adjuvant chemotherapy. However, patients who have larger tumor masses and with greater lymph node metastasis benefit less from treatment [Dalton, 1990]. In addition to chemotherapy, endocrine therapy is used in the treatment of some breast cancers. Tamoxifen (TAM) is the most effective antiestrogen used in the treatment of postmenopausal breast cancer patients with a 5 year survival rate of 73% compared with a 68% survival rate if no treatment is administered [Early Breast Cancer Trialists' 3 Collaborative Group, 1988]. In premenopausal breast cancer, the use of T A M is less beneficial with less than a 1% difference in survival rate. The effectiveness of T A M as well as chemotherapy in the treatment of breast cancer was observed to be age-dependent by the Early Breast Cancer Trialists' Collaborative Group (1988). They reported that in patients 50 or older, T A M reduces mortality by 20%. In patients less than 50 years of age, treatment with T A M does not produce a significant reduction in the mortality rate. Conversely, chemotherapeutic treatment of women 50 and over does not produce a clear improvement in survival. However, women under 50 who are treated with chemotherapy have a reduced mortality of 26%. Furthermore, the group also reported that both older and younger patients have significantly decreased rates of recurrence when treated with either T A M or chemotherapy. 3. Drug Resistance As stated above, patients often respond well to the first course of chemotherapeutic treatment. However, one of the most frustrating problems in the treatment of cancer is the development of chemoresistance. Specific mechanisms for drug resistance in human tumors have been documented [Young, 1989]. Drug resistance occurs either by de novo synthesis of proteins (i.e. acquired drug resistance) or clonal selection of drug-resistant cells present in a heterogenous tumor [Goldie, 1992]. Examples of drug resistance mechanisms are listed in Table 1. 4 Table 1. Mechanisms of drug resistance observed in cancer cells. 1. Altered cellular pharmacokinetics of drug uptake or efflux. 2. Increased rate of D N A repair. 3. Changes in the levels of target enzymes or macromolecules. 4. Increased conversion of drug to inactive metabolites. 5. Decreased conversion of inactive prodrug to an active metabolite. Some antimetabolite anticancer agents are affected by altered cellular pharmacokinetics. It has been reported that impaired membrane transport in tumor cells results in resistance to methotrexate [Sirotnak et al, 1981]. Changes to the rate of D N A repair affect chemotherapeutic agents such as melphalan and cisplatin. For example, melphalan resistant human ovarian cell lines display increased repair of melphalan-induced damage [Young, 1989]. Furthermore, resistance to cisplatin in a rat ovarian tumor cell line is accompanied by increased poly(ADP-)ribose formation. Poly(ADP-ribose) polymerase is involved in D N A repair, hence elevated poly(ADP-)ribosylation may be connected to increased repair of cisplatin-mediated damage [Chen and Zeller, 1994]. In addition to altered pharmacokinetics, resistance to methotrexate can also be mediated by changes in the levels of the target enzyme. Elevated levels of dihydrofolate reductase from amplification of the gene encoding the enzyme is demonstrated in methotrexate-resistant tumor cells [Melera et al, 1980]. In support of this, investigators reported that transfection of dihydrofolate reductase into wild-type Chinese hamster ovary cells results in methotrexate-resistance [Banerjee et al, 1994]. Changes in the rate of conversion of a drug to an inactive or active metabolite are other mechanisms which can contribute to drug resistance. For example, cytosine arabinoside is activated by the enzyme deoxycytidine to its active form, a triphosphate metabolite. In human leukemia cells, this enzyme has been reported to have decreased 5 activity [Tattersall et al, 1974]. Other resistant human leukemia cells have been identified which have enhanced levels of cytidine deaminase, a degradative enzyme [Stewart and Burke, 1971]. To make matters worse, drug resistant cells may have more than one mechanism of drug resistance in operation. Additionally, cancer cells may exhibit cross-resistance to structurally and functionally unrelated drugs. More of this will be discussed in the section Multidrug Resistance. 4. Multidrug Resistance Human tumors are shown to exhibit several mechanisms of multidrug resistance (MDR). The best characterized of these mechanisms is associated with mdrl gene amplification and/or overexpression [Juranka et al, 1989]. This gene codes for a 170 kDa protein called P-170 or P-glycoprotein (Pgp). The expression of the mdrl gene results in resistance to multiple, structurally unrelated chemotherapeutic agents possessing different mechanisms of action [Gerlach, 1989]. In vitro models for the study of M D R have been developed in which vincristine (a Vinca alkaloid) and the antitumor antibiotics, Adr and actinomycin D, as well as other drugs have been used in order to isolate resistant sub-populations of a variety of human and rodent cell lines [Fojo, 1989; Schneider et al, 1990]. The M D R phenotype was observed in all of these cell lines and was subsequently characterized as cells which exhibit the following features: a) a broad range of resistance to structurally unrelated natural products including the anthracyclines, actinomycin D, Vinca alkaloids, epipodophyllotoxins and taxol. b) decreased intracellular accumulation of drug due to drug efflux mediated by PgP-6 c) either partial or complete reversibility of cross-resistance by administration of various agents including calcium-channel blockers (verapamil), phenothiazines and antiariythmic agents (quinidine, amiodarone). d) cytogenetic evidence of gene amplification in highly resistant cell lines, i.e. minute or double minute chromosomes, homogenous staining regions or abnormal banding regions. e) increased expression of the mdrl gene with or without amplification. The above features describe what is known as typical MDR and is associated with Pgp. When MDR is observed but Pgp is not present in the cancer, atypical MDR is said to be the mechanism of drug resistance. For instance, some cells exhibit resistance to multiple drugs and display decreased drug accumulation although Pgp expression is not detected [Taylor et al, 1991]. Atypical MDR may be mediated by intracellular events such as altered levels of glutathione-S-transferase (GST) [Cazenave et al, 1989], topoisomerase II (TopoII) [Lefevre et al, 1991] and multidrug resistance-associated protein (MRP) [Cole et al, 1992]. GSTs are Phase II enzymes which inactivate drugs via conjugation with glutathione. Enhanced levels of GST are observed in MDR cells with or without mdrl gene overexpression. In addition to Pgp, GSTs are elevated in MCF7-AdrR, an Adr-resistant cell line. Although Pgp appears to be the main contributor to Adr-resistance, GST may be associated with some Adr-resistance. Some GSTs possess intrinsic glutathione peroxidase and Adr is intracellularly activated and generates hydrogen and lipid peroxides [Fairchild and Cowan, 1991]. TopoII is a DNA binding enzyme that plays a role in DNA replication and cell proliferation. It is implicated in a type of MDR because it is a target for drugs such as anthracyclines and epipodophyllotoxins. These drugs bind to TopoII thus forming a stable drug-TopoII-DNA complex. This complex results in increased DNA strand breakage thus 7 leading to cell death. However, in cells displaying chemoresistance, there is a decrease in TopoTJ levels as compared with drug sensitive cells [Giai et al, 1991; K i m et al, 1991]. Another protein that has been studied with respect to M D R is protein kinase C ( P K C ) . Activation of this kinase with phorbol esters results in an increased resistance to drugs along with decreased drug accumulation [Giai et al, 1991]. It was proposed that P K C may directly or indirectly, via second messenger systems, alter levels of phosphoproteins that interact with drugs as they enter the cell. More recently, P K C has been demonstrated to modulate Pgp activity through phosphorylation [Chambers and Kuo , 1993]. M R P is a novel protein discovered by Cole et al (1992). They reported that M R P confers the M D R phenotype similar to Pgp. Although M R P has been studied in cell lines, the clinical relevance of M R P in M D R in cancer has yet to be elucidated. 5 . Typical Multidrug Resistance: P-glycoprotein (Pgp) Almost 20 years ago, Ling et al incubated a hamster cell line with increasing concentrations of colchicine [Juliano and Ling, 1976]. They subsequently discovered that the cell line developed cross-resistance to a variety of antitumor antibiotics. The acquired M D R was later associated with Pgp which actively pumps the drugs out of the resistant cells [Juranka et al, 1989]. Since these initial observations, the M D R phenotype has been observed in a variety of human cell lines and in primary human tumors. 8 5a. Structure and function of Pgp Pgp is an integral membrane protein that is made up of two identical domains consisting of 1280 amino acids in total [Gerlach, 1989] (Figure 1). Each domain spans the plasma membrane 6 times and both combine to form a pore or channel. The extracellular region is associated with a carbohydrate chain, whereas the intracellular portion has two ATP binding sites. There is a significant amount of homology between Pgp and bacterial transport proteins [Gerlach, 1989]. This provides evidence that Pgp functions as a pump. Furthermore, the ATP binding sites on the two domains have been demonstrated to be essential for what may promote drug efflux. Direct binding of drugs to Pgp may be an essential step in the action of drug efflux. The fact that so many different drugs are transported by Pgp may be explained by multiple binding sites on the protein [Tamai and Safa, 1990; Tamai and Safa, 1991]. Although the mechanism of drug binding is yet unknown, direct binding of drug analogs has been shown; for example, Pgp labels with photoactive vinblastine [Tamai and Safa, 1991]. Labeling can subsequently be inhibited by other drugs involved in the M D R phenotype as well as chemosensitizers such as verapamil [Ryffel et al, 1991]. The inabihty to prevent binding of all MDR-associated drugs may be due to differential binding affinities. Alternatively, multiple binding sites on the protein may explain this occurrence. Pgp seems to be heterogeneous in that some M D R cell lines display differential sensitivity patterns to drugs. For example, human K B carcinoma cells were selected for resistance to colchicine [Choi et al, 1988]. A single point mutation in the mdrl coding region was shown to alter the pattern of cross-resistance of Pgp [Safa et al, 1990]. The cells with the wild-type gene with Gly-185 were compared to a mutant transfectants with a valine residue at position 185. The transfectants have increased resistance to colchicine and etoposide accompanied by decreased resistance to Vinca alkaloids and actinomycin D. Furthermore, the suggestion that phosphorylation of Pgp may modulate the drug transport 9 600 Figure 1. A model for the transmembrane topology of P-glycoprotein (Pgp). This model is based on the human mdrl sequence. This is a reproduction taken from Juranka et al (1989), FASEB J, 3: 2585. 10 mechanism [Giai et al, 1991; Juranka et al, 1989] was recently confirmed [Chambers and Kuo, 1993] by showing that activity of Pgp is regulated through phosphorylation by PKC. 5b. The mdr gene The gene encoding Pgp is a member of a small, highly conserved multi-gene family in which three genes are represented in rodents and two in humans. Comparison of the cDNA and genome sequence indicate a large degree of homology between human, hamster and mice Pgp isoforms since protein structure and genome sequence exhibit highly conserved regions [Juranka et al, 1989; Bradley and Ling, 1994]. Each isoform, however, may have different functions. The Pgp isoforms have been grouped into three classes (Table 2). Studies using gene transfection implicate only class I and class U Pgp as conferring the M D R phenotype. Expression of class LU occurs mainly in hepatocytes, cardiac muscle and striated muscle [Bradley and Ling, 1994]. The MDR phenotype observed in human cancers corresponds only to mdrl expression although mdr3 is also expressed. Therefore, mdrl gene expression is of great concern since it is quite possibly the gene responsible for the M D R phenotype in human cancers. Table 2. Isoforms of Pgp found in mammalian cells Class I Class II Class UI Rodent + + + Human + - + 11 5c. mdrl gene and Pgp in normal tissue The expression of the mdrl gene product has been detected in normal tissue. It has been suggested that tissues exposed to carcinogens result in mdrl overexpression [Fairchild and Cowan, 1991]. Most cancers curable through chemotherapy, such as Hodgkin's disease and Burkitt 's lymphoma, occur in young patients. In contrast, adult related diseases such as breast, colorectal and lung cancer are less responsive and, therefore, less curable. Since younger patients have been exposed to carcinogens for a shorter time, it is possible that the difference in success rates between treatment of cancers associated with younger patients and those associated with adults may be due to levels o f Pgp [Fairchild and Cowan, 1991]. Furthermore, it has been shown that the gastro-intestinal tract receives a high degree of exposure to carcinogens which might explain the high levels o f Pgp in the colon. Pgp may occur naturally in normal tissue and may have specific functions. Pgp has been postulated to excrete metabolites and toxins in liver, colon and kidney [Fojo, 1989]. In tissues such as the adrenal glands and the pancreas, Pgp is implicated in roles involving hormone transport [Fojo, 1989]. Steroid transport is thought to occur through Pgp in adrenal cells from the zona fasciculata and zona reticularis [Fine et al, 1989]. 5d. mdrl gene and Pgp in human breast cancer Pgp has been observed in many types of tumor tissue and often correlates with the failure of chemotherapeutic treatment. As stated above, Pgp can be expressed in normal tissue prior to any development of cancer. Subsequently, cancer cells that arise from these tissues are therefore hypothesized to be resistant to MDR-related chemotherapeutic agents. For example, the colon exhibits high levels of mdr l expression and tumors resulting from this tissue are highly resistant to MDR-associated drugs [Sanfilippo et al, 1991]. 12 It has been suggested that carcinogens induce Pgp expression in normal cells. Unlike other tissues such as the colon, the mammary gland does not receive as much exposure to genotoxic agents. It is more likely that breast tumor cells are induced by drugs to express the membrane glycoprotein. One method of breast cancer treatment includes the administration of Adr in combination with other chemotherapeutic agents. Adr is the most active single agent used in the treatment of breast cancer. However, this drug is highly recognized for its capability to induce the M D R phenotype [Fairchild et al, 1987; Schneider et al, 1990]. It is clinically important to determine i f the M D R phenotype develops in human tumors as a result of exposure to MDR-associated drugs since many tumors become resistant to chemotherapy. 5d (i) Studies in breast cancer cell lines Cell lines derived from breast cancer cells have been extensively utilized as tools to develop models of multidrug resistance in breast cancer. Two methods have been used to confer the M D R phenotype in cell lines. The most direct method is mdrl gene transfection. Using this method, it is readily observable that previously drug-sensitive cells can become resistant to Adr and other MDR-related agents through expression of the mdr l gene. The degree of gene amplification and expression corresponds to the level o f drug resistance observed in the cell lines [Fairchild et al, 1987; Schneider et al, 1990]. The other method for developing M D R models in breast cancer cell lines is exposure of a cell line to increasing concentrations of a MDR-related drug with each passage of cells in culture. The M C F - 7 subline resistant to Adr is known as M C F 7 - A d r R [Fairchild et al, 1987]. Another breast cancer cell line, M D A - 2 3 1 , has a corresponding Adr-resistant subline, M D A - A d r [Merkel et al, 1989]. Cross-resistance to other drugs also developed in the cell lines with greatest resistance to the selecting agent. Early passages in the M D A - A d r cell line exhibited overexpression of the mdrl gene without 13 amplification. With subsequent passages, a higher magnitude of resistance was observed along with a 30-fold gene amplification [Merkel et al, 1989]. The MCF-7 cell line is a very intensively studied line with respect to MDR. Adr-resistance in the MCF7-AdrR subline has increased to a magnitude of 200-fold [Fairchild et al, 1987]. Both mdrl gene amplification and overexpression was observed in the resistant subline. Further studies in MCF7-AdrR cells resulted in the opening of a window into the complexity of Adr resistance. Pgp is not the only mechanism that accounts for resistance to Adr. These other mechanisms will be discussed in the section Atypical Multidrug Resistance. Cell lines made resistant to Adr also become resistant to the epipodophyllotoxin, etoposide (VP-16) [Politi and Sinha, 1989]. The role of this agent was studied in MCF-7 and HL60 cells as well as their Adr-resistant variants, MCF7-AdrR and HL60/Adr, in order to determine the involvement of Pgp in drug resistance [Politi and Sinha, 1989]. The effects of energy depletion on VP-16 efflux and accumulation were evaluated because Pgp is an ATP-dependent transport protein. In addition, the potential of vincristine and daunorubicin in modulating cellular pharmacokinetics of VP-16 were studied since these drugs compete for binding with Pgp. The resistance of MCF-7 and MCF7-AdrR cells at equitoxic concentrations of VP-16 were also determined. Resistant cells accumulate 171-fold more VP-16 and retain 536-fold more drug in comparison with their respective drug-sensitive parent cell lines. This implies decreased drug accumulation is not the major mechanism involved in VP-16 resistance. Energy depletion does not significantly alter drug efflux rates although drug accumulation slightly increases in resistant cells. In contrast, the addition of vincristine with VP-16 alters drug efflux, which suggests competitive binding for Pgp is occurring. Therefore, the overall results show that Pgp may neither be the only nor the major mechanism of VP-16 resistance in MDR cell lines. Another cell line was developed from MCF-7 cells in which cells were made resistant to mitoxantrone (mitox), a synthetic chemotherapeutic agent belonging to the 14 anthracenedione class of antineoplastics that is structurally similar to Adr [Taylor et al, 1991]. Tests were done in which MCF7-AdrR cells were compared to MCF7-mitox, the mitox resistant subline. What followed was that a unique MDR phenotype unrelated to Pgp was observed. For example, MCF7-mitox cells do not show a significant increase in sensitivity to mitox and Adr when verapamil is present. In contrast, MCF7-AdrR cells display increased Adr sensitivity with verapamil in culture. It has been shown that changes in mitox accumulation differs between the two drug-resistant cell lines; MCF7-mitox drug accumulation does not change when verapamil was present whereas the Adr-resistant subline displays increased drug accumulation. A different pattern of cross-resistance is also evident between the two cell lines. Therefore, different mechanisms of drug resistance were being exhibited between the two cell lines, one associated with Pgp and another unassociated with Pgp. Various studies in breast cancer cell lines as well as in other human cell lines have displayed results in which expression of the mdrl gene is evidently enough to confer the MDR phenotype. However, it is quite obvious that Pgp is not the only mechanism contributing to multidrug resistance in these cell lines. These results in cell lines model what occurs in vivo. Research using primary culture of cells from breast cancer tissue, which more closely mimics the in vivo situation should give greater insight into the relationship between Pgp and MDR in clinical breast cancer. 5d (ii) Studies in cinical breast cancer Breast cancer is initially responsive to a broad spectrum of single agent and combination chemotherapeutic regimens [Dalton, 1991]. As stated earlier, Adr is the most effective single agent utilized in treatment regimens for breast cancer. Unfortunately, most patients with metastatic breast cancer have recurrences which are difficult to treat because many of the cells become drug-resistant and resistance to Adr is a limiting factor in its use. Clinical resistance to anticancer agents arises due to a number of factors. For 15 example, tumors with slow growth rates are generally drug-resistant [Goldie, 1989]. Other factors of drug resistance include the development of pharmacological sanctuaries as a result of loss of vascular supply, the development of hypoxia in large tumors and the development of structural and metabolic changes in tumor cells [Taylor et al, 1991]. Whether or not MDR in the clinical setting is mainly due to Pgp overexpression is as yet unclear. A variety of techniques have been used to examine the level of Pgp and how it correlates with drug resistance. Southern, northern and western blot analyses have identified mdrl gene amplification and/or overexpression [Merkel et al, 1988]. Other studies have employed immunocytochemical techniques in which monoclonal antibodies are used in the staining procedure [Schneider et al, 1989; Wishart et al, 1990; and Verelle et al, 1991]. Merkel et al (1989) utilized various molecular biological techniques to study a total of 275 breast cancer specimens obtained from the San Antonio Breast Tumor Bank. Out of 248 cases of clinical breast cancer examined for mdrl gene amplification, 219 were not previously treated with chemotherapeutic agents, whereas 29 were treated cases (with Adr). None of the specimens exhibit any form of gene amplification. Furthermore, out of 95 breast cancers examined using northern blotting techniques, no samples contain detectable levels of mdrl gene expression. The northern blot analysis included 62 clinically sensitive tumors and 4 in vitro sensitive tumors, whereas resistant samples included 13 clinically resistant tumors and 16 in vitro resistant tumors. Thus, the results from clinical cases of breast cancer in this set of experiments do not correlate with those from studies involving cell lines with respect to the detection of mdrl gene amplification and overexpression. However, this study, which examined the D N A or RNA from homogenized tumors, cannot exclude the fact that small subpopulations of cells in the tumors may exist in which mdrl is amplified and/or overexpressed. Homogenization of a heterogeneous tumor may "dilute" the expression of mdrl in some cells and Pgp may not be detected. Therefore, it is difficult to determine using these techniques if Pgp is either 16 spontaneously expressed in cells in these tumor samples or if its expression is induced or selected for by Adr administration. Methods such as immunocytochemistry and flow cytometry which use anti-Pgp monoclonal antibodies are able to detect small clusters of MDR tumor cells [Merkel et al, 1988; Schneider et al, 1990]. Sanfilippo et al (1991) utilized an immunoblotting technique with the monoclonal antibody C219 to detect Pgp expression in breast tissue. Their results demonstrate that healthy gland and benign lesions are negative for Pgp. In contrast to the study by Merkel and co-workers (1989), they report 30% of previously untreated and 60% of previously treated breast cancers test positive for Pgp, which is in accordance with other investigators [Salmon et al, 1989; Schneider et al, 1989]. Furthermore, results of immunoreaction with C219 in western blots of breast cancer tissue correlate with low to intermediate detection of Pgp through immunocytochemistry [Salmon et al, 1989; Schneider et al, 1989]. This series of studies by Sanfilippo and co-workers (1991) also examined in vitro resistance to Adr and vincristine in 35 breast cancer samples with a metabolic assay utilizing [3H]-thymidine. Drug activity, expressed as the ability to inhibit incorporation of labelled precursor, shows that approximately 30% of Pgp-positive (Pgp+) tumors are resistant to both Adr and vincristine. In addition, the MDR phenotype is also observed when breast cancer samples are treated with MDR-unrelated agents. However, Pgp-negative (Pgp-) tumors also exhibit a high degree of resistance to both drugs (14 of 25 samples) suggesting that other mechanisms of MDR may also be operative. In order to compare Pgp levels between samples of breast cancer from patients previously and not previously treated with chemotherapy, Schneider and co-workers (1989) utilized an immunocytochemical technique to detect Pgp in cryostat sections of tumor samples. The monoclonal antibody C219, which recognizes an internal site on Pgp, was used [Grogan et al, 1990]. Schneider et al (1989) observed membrane-associated staining. Cytoplasmic staining was also observed and caused interpretative problems since Pgp is an integral membrane protein. However, recent studies have confirmed that Pgp 17 staining does occur in the cytoplasm and is localized in the Golgi [Veneroni et al, 1994]. The results presented by Schneider and co-workers show that the majority of their untreated samples (10 out of 12) are Pgp-. Pgp+ staining occurrs in a majority of the treated specimens regardless of treatment with MDR-related or -unrelated drugs, although samples with the greatest staining are those treated with MDR-associated agents. The positive staining detected in specimens treated with drugs unrelated to M D R was also observed by Sanfilippo et al (1991). These results suggest a relationship between M D R -related chemotherapy and Pgp expression. Comparing immunocytochemistry and western blotting methods, Schneider et al (1989) concluded that the latter may be impractical for clinical studies. It is a complicated technique and it requires use of sophisticated equipment that may not be available in the hospital. Furthermore, it is incapable of distinguishing Pgp expression between cancerous and non-cancerous lesions. These problems are not encountered with immunocytochemistry utilizing monoclonal antibodies directed against Pgp. C219 and another monoclonal antibody, MRK16, which recognizes an external epitope of Pgp, were used in the immunocytochemical experiments carried out by Wishart and co-workers (1990). Results from these experiments demonstrate Pgp expression in breast cancer before therapy. This observation is in support of work by Schneider et al (1989) who detect Pgp in a small proportion of cells from untreated cases of breast cancer. Therefore, these patients with Pgp+ tumor cells are examples of patients who may not benefit from MDR-associated drugs and perhaps should be treated with non-MDR related drugs. Investigation of Pgp expression using immunocytochemistry with C494 was undertaken by Verelle et al (1991). C494 is highly specific for the class I isoform of Pgp, the isoform that corresponds with the M D R phenotype. Twenty biopsies of untreated breast cancer were tested with C494: 17 samples from locally advanced disease and 3 from metastatic cases. Although the sample size is small in this study, a large majority 18 (86%) of the samples of untreated breast cancer express Pgp with most of the staining occurring in neoplastic cells. The results show that strong Pgp detection in untreated samples correlates with a shorter period of progression-free survival relative to patients in whom no Pgp is detected. Furthermore, strong positive staining in a large number of tumor cells correlates with AVCF (adriamycin, vincristine, cyclophosphamide and 5-fluorouracil therapy) resistance. 6. Atypical Multidrug Resistance Although Pgp is implicated as a mechanism of multidrug resistance, many of the previous experiments show that there may be other cellular events which can be altered and thus confer multidrug resistance. For example, some cells exhibit cross-resistance to drugs although mdrl is neither amplified nor overexpressed. In addition, differential patterns of cross-resistance to drugs exist even when Pgp is expressed, which suggests that other mechanisms may also come into play. Investigations into other mechanisms of drug inactivation show that these mechanisms are associated with altered levels of GSTs, TopoII, cytochrome P450 enzymes, glutathione peroxidase and MRP (discussed below). 6a. Glutathione-S-transferases GSTs are multifunctional enzymes known to be protective against cytotoxic agents by acting through several mechanisms [Cazenave et al, 1989]: 1) conjugation of electrophilic toxins to glutathione. 2) covalent or non-covalent protein binding of drug. 3) intrinsic glutathione peroxidase activity. There are 3 broad classes of cytosolic GSTs: alpha, mu and pi [Cazenave et al, 1989]. An overall elevation of GST activity was observed in cell lines selected for resistance to a variety of anti-cancer agents. It was thought that the pi class of GST (GST-p) was the most relevant to chemoresistance since it was found overexpressed in 19 MCF7-AdrR cells along with Pgp and in tumors of both human and animal origin [Moscow et al, 1989]. Similar to mdrl studies, the GST-p gene was transfected into MCF-7 cells by Moscow et al (1989) even though, in contrast to Pgp, wild type MCF-7 cells express GST-p. The purpose of these experiments was to determine the role of elevated levels of GST-p in MDR since the MCF7-AdrR cell line contains elevated Pgp. Transfection clones allow for observation of GST-p function without Pgp involvement. It was shown that GST-p transfected clones have less if not similar Adr resistance when compared to wild type MCF-7. In contrast, transfection of the mdrl gene is enough to confer cross-resistance to natural product chemotherapeutic agents. It was therefore proposed that an increase in GST-p activity or overexpression does not produce resistance to Adr and that other proteins may be involved in the mechanism of MDR. In experiments by Lefevre and co-workers (1991), another breast cancer cell line, CALcl8/AMSA, was selected for resistance to m-AMSA (amsacrine, an aminoacridine). The CALcl8/AMSA cells are also cross-resistant to Adr, NMHE (an ellipticine) and teniposide (VM26; an epipodophyllotoxin). No mdrl expression is detected but GST-p overexpression is evident. They conclude that GST-p is involved in the resistance process in this cell line. However, the drugs to which CALcl8/AMSA shows resistance act on the enzyme TopoU as well. As stated above, TopoU may be another mechanism of MDR and will be discussed in the following section. Forrester et al (1990) studied the involvement of GST in clinical cases of breast cancer in a total of 41 matching samples of normal and malignant breast tissue. In a majority of the samples, the levels of GST-p are similar between matching normal and tumor specimens. In some cases, GST-p content is lower in the tumor sample compared to the corresponding normal tissue. Therefore, GST-p is not uniformly elevated in human breast tumors. Keith and coworkers (1990) and Singh et al (1990) also show that GST-p expression is not elevated in all their samples of normal and matching tumor tissue. 20 However, experiments demonstrate that elevated GST-p occurs in the tumor samples compared to matching normal specimens [Cazenave et al, 1989]. With respect to other GST isozymes, mu class GST is subject to large inter-individual variation in that it is detected in only 50% of the samples; when it is detected in normal tissue, it is also detected in the corresponding tumor sample [Forrester et al, 1990]. GST-mu had comparably high levels relative to GST-p when expressed. In contrast, low levels of the alpha class GST are detected but are expressed in most of the samples. Thus GST-p seems to be the major isozyme in tumor cells although the role of GST-p overexpression in MDR in clinical breast cancer is unclear. To summarize, GST-p may play a minor role in MDR with respect to cross-resistance in cell lines and may have a role in the chemoresponsiveness of a small proportion of tumors. 6b. Topoisomerase TJ TopoII is a protein associated with the mitotic chromosome scaffold as well as the nuclear matrix [Earnshaw and Heck, 1985; Heller et al, 1986]. It is implicated in the function of cell proliferation since growth arrested cells, i.e. those cells that are fully differentiated or in G 0 , express low levels of enzyme, whereas replicating cells have a high TopoII content [Epstein et al, 1989]. TopoII also causes transient enzyme-bridged double strand DNA breaks to allow interconversion of topological DNA isomers [SuUivan and Ross, 1991]. This enzyme, as mentioned earlier, is targeted by anthracyclines, epipodophyllotoxins and aminoacridines. However, in contrast to Pgp, Vinca alkaloids do not affect TopoII. Therefore, MDR cells that are not resistant to Vinca alkaloids are subsequently thought to have modified TopoII activity. The AMSA-resistant cell line discussed in the previous section displays cross-resistance to a variety of drugs. It was hypothesized that low levels of TopoII are present such that stable DNA-TopoII complex formation can not occur and will subsequently spare DNA from being cleaved [Lefevre et al, 1991]. This is demonstrated by suppressed 21 single-strand DNA breakage and DNA-protein crosslinking in the CALcl8/AMSA cell line. Although diminished in resistant cells, TopoU is identical to wild-type TopoU in drug-sensitive cells. Thus, decreased TopoJJ activity rather than expression of a mutant TopoU is suggested to explain the phenomenon of MDR in this cell line. 6c. Cytochrome P450 In contrast to the overexpression of Phase II metabolizing enzymes, such as GST-p, Phase I enzymes are down-regulated in MCF7-AdrR cells. Phase I enzymes are primarily cytochrome P450s, which convert drugs to a more polar metabolite [Benet et al, 1990]. They either activate or detoxify xenobiotics. MCF7-AdrR cells exhibit suppressed expression of P450IAI in comparison to drug-sensitive MCF-7 cells [Fairchild and Cowan, 1991]. In human breast tissue samples studied by Forrester and co-workers (1990), neither tumor nor normal specimens express this class of P450 enzymes, although P450UC enzymes are detected. However, since P450 enzymes are implicated in the metabolism of a variety of xenobiotics, they may have a role in MDR. 6d. Glutathione peroxidase Some antineoplastic agents act through the formation of free radicals as does radiotherapy. Adr may be cytotoxic in tumors due to the formation of a semiquinone-free radical upon drug metabolism. This active metabolite subsequently causes the generation of superoxide anion radicals, hydrogen peroxide and hydroxyl-free radicals which are extremely toxic [Mimnaugh et al, 1989; Sinha et al, 1989]. Radical formation can be inhibited by glutathione peroxidase in MCF7-AdrR [Sinha et al, 1989], which provides support for this mechanism of action of Adr and the subsequent emergence of MDR. There are 2 types of glutathione peroxidase: selenium-dependent glutathione peroxidase (reduces H2O2 and organic hydroperoxides) and selenium-independent glutathione peroxidase (reduces organic hydroperoxides only). In both normal and 22 malignant breast tissue, glutathione peroxidase activity is almost completely mediated by the selenium-dependent class [Forrester et al, 1990]. Furthermore, the selenoproteins are found elevated in MCF7-AdrR cells [Chu et al, 1990], which is consistent with increased glutathione metabolism [Batist et al, 1990]. Elevated levels of glutathione peroxidase are observed in breast tumor samples relative to normal tissue counterparts [Singh et al, 1990], Therefore, resistance to Adr may be mediated through elevated glutathione peroxidase. It is important to remember that M D R refers to cross-resistance to structurally unrelated drugs. Glutathione peroxidase does not strictly fit into this classification of drug resistance since it only acts on drugs which act through quinone moieties. However, it is mentioned here because it may explain resistance to Adr which cannot be explained by other mechanisms of drug resistance. 6e. Multidrug resistance-associated protein Recently, Cole et al (1992) reported that one mechanism of M D R may be due to expression of a multidrug resistance-associated protein (MRP). This 190 kDa protein is distinctly different from Pgp as it is encoded by the MRP gene. However, it does share some similarities with Pgp. For example, MRP also belongs to the A B C superfamily of transporters and therefore is reported to be a transporter itself [Cole et al, 1992; Krishnamachary and Center, 1993]. As with Pgp, MRP expression confers M D R resulting in resistance to natural product agents [Grant et al, 1994]. Currently, studies have investigated the role of MRP in cell lines to explain M D R in those that do not express Pgp. MRP was thought to be the mechanism of M D R observed in mitox-resistant cells as discussed earlier [Taylor et al, 1991]. However, Futscher et al (1994) reported that M R P is not elevated in drug-resistant cells compared to the sensitive parent line. It remains to be shown how MRP relates to M D R in clinical cases of cancer. 23 7. Circumventing Multidrug Resistance Multidrug resistance, whether mediated by Pgp or by other mechanisms, presents a significant problem in the treatment of cancer patients. Although a large majority of individuals with breast cancer are initially responsive to chemotherapy, most will eventually develop resistance and die from the disease. Thus, techniques which circumvent drug resistance must be developed and incorporated into chemotherapeutic regimens for the purpose of treating these cases of malignant breast disease. In order to prevent resistance to the natural product agents described above, noncross-resistant drug combinations can be administered. Recently, a semi-synthetic Vinca alkaloid, vindesine, has been suggested as a form of chemotherapy in advanced breast cancer if tumor cells are resistant to Adr. Adachi et al (1991) demonstrated that continuous administration of vindesine has a large anticancer effect. Of course, one may eventually come across drug-specific resistance. An alternate technique would be to administer chemosensitizers early in the treatment regimen such that MDR cells will not predominate in the growing tumor. When two drugs are administered at the same time, they may be additive or synergistic in their effects. If the effects are additive and the tissue becomes resistant to one of the drugs, there is a net decreased effect that equals the effect of the tumor-sensitive drug alone. However, if two drugs work synergistically and the tissue becomes resistant to one of the agents, the two drugs still work together such that one drug will enhance the effects of the other. For example, both Adr and MP A are used in the treatment of breast cancer. If the two work additively and the tumor becomes resistant to either agent, then treatment is only as effective as the tumor-sensitive drug. If the two work synergistically, the tumor may be resistant to either of the drugs alone but when given together, there will be a significant antitumor effect, greater than the tumor-sensitive drug alone. The use of Pgp binding agents such as verapamil has been employed to increase the effect of Adr and/or vincristine in vitro. In the clinical situation, cardiotoxic side effects arise, although they 24 are readily reversed if verapamil administration is discontinued or if dosage is decreased [Pennock et al, 1991; Ries and Dicato, 1991]. However, the degree to which M D R reversal occurrs is dependent on the dose of verapamil. Unfortunately, to reduce risk of cardiotoxicity, the in vivo dose has to be reduced to a concentration less than that required to modify M D R in vitro. Other chemosensitizers, therefore, have to be used. Evidence suggests that cyclosporin-A (CsA) binds to Pgp [Beck, 1990] and enhances the effect of MDR-related drugs in Pgp expressing cells [Twentyman and Wright, 1991]. Furthermore, cyclosporin-H, an inactive analog, also prevents drug resistance in M D R cell lines [Ryffel etal, 1991]. Inactive derivatives may therefore be useful as chemosensitizers. Agents that affect the endocrine system, such as progestins and T A M , also can modify MDR. These agents are currently used in the treatment of breast cancer with minimal side effects. Investigators have reported interaction of progesterone [Naito et al, 1989; Yang et al 1989] and T A M [Fine et al, 1993; Kirk et al, 1993; Leonessa et al, 1994] with Pgp . 7a. Cyciosporin-A (CsA) Isolated from the fungus Tolypocadium inflatum Gams, CsA is a cyclic peptide found to have immunosuppressive properties [Borel et al, 1976]. As a result, CsA is used extensively to prevent immune rejection of transplanted tissues in humans. As discussed above, the use of verapamil as a resistance modifier (RM) is limited by its cardiotoxicity. In search of novel RMs, CsA became recognized for its ability to modulate MDR. In fact, studies by Silbermann et al (1989) show CsA to be superior to verapamil, since CsA restores daunorubicin accumulation to higher levels and more effectively on a molar basis. In order to determine its mechanism of action, Tamai and Safa (1990) studied the reversal of Vinca alkaloid resistance by cyclosporins in M D R Chinese hamster cells. They report that [3H]-vincristine uptake in membrane vesicles displays Michaelis-Menten kinetics with high and low affinity components. The high affinity uptake of [ H]-vincristine is shown by these investigators to be inhibited by CsA 25 through competitive interaction. These investigators also demonstrate that CsA inhibits 125 [ IJ-NASV, a vinblastine analogue, from photolabelling Pgp in a dose-dependent manner. That CsA directly binds with Pgp is further supported by Ryffel et al (1991). They used a [ H]-cyclosporin diazirine analogue and show that verapamil inhibits photolabelling of Pgp by this agent. In addition, the [ H]-cyclosporin derivative competed for binding with CsA as well as with a nonimmunosuppressive analogue, cyclosporin-H. 7 b . Tamoxifen (TAM) The most effective anti-estrogen used in the endocrine treatment of breast cancer is TAM [Early Breast Cancer Trialists' Group, 1988]. Its mechanism of action is through binding with the cytoplasmic ER [Legha, 1988]. The TAM-ER complex then gets translocated to the nucleus of the cell and results in decreased cytoplasmic ER. Furthermore, at low levels, TAM has mild estrogenic effects and can increase PgR synthesis in ER positive cells [Namer et al, 1980]. In addition to its anti-estrogenic effects, TAM exhibits an ability to enhance the cytotoxicity of Adr [Wasserman et al, 1992]. Using a cell line developed from ascitic fluid of an untreated ovarian carcinoma patient, Wasserman et al (1992) demonstrates that verapamil enhances Adr cytotoxicity in these Pgp+ cells. When verapamil is replaced by TAM, similar results are observed. Fine et al (1993) performed similar studies on Pgp+ human renal carcinoma cell lines. They observed that increasing the concentration of TAM from 4 uM to 8 uM increases the accumulation of [3H]-vinblastine from 20-30% to 70%, whereas the same changes in concentration of verapamil do not have a similar effect. Hence, maximum inhibition of MDR requires higher concentrations of TAM than verapamil, which can be clinically accomplished due to TAM's relatively low toxicity. This group also repeated these experiments on sensitive and MDR MCF-7 human breast cancer cells with similar 26 results. Furthermore, to determine its mechanism of action in reversing MDR, these investigators found that TAM competitively inhibits photoaffinity labelling of Pgp with [ H]-azidopine. Drug accumulation and Pgp binding with respect to TAM were also studied by Leonessa et al (1994) on sensitive and MDR MCF-7 cells. In support of the findings of Fine et al (1993), they report increased accumulation of [ FTJ-vinblastine in MDR cells due to the presence of TAM. Furthermore, these investigators also observed that TAM inhibits the binding of [ H]-azidopine to Pgp. Through isobologram analyses, Leonessa et al (1994) determined that 1 uM of TAM interacts synergistically with vinblastine and Adr. In addition to binding to Pgp, O'Brian et al (1986) showed that triphenylethylenes, such as TAM, affect the phosphorylation of Pgp by PKC. Chambers and Kuo (1993) determined that PKC stimulates the phosphorylation of Pgp in the MDR cell line, KB-V1. Furthermore, TAM affects plasma membrane fluidity, which subsequently alters drug accumulation within a cell [Ramu et al, 1991]. Therefore, TAM may be effective in modifying MDR by inhibiting Pgp function both directly and indirectly. 7c. Medroxyprogesterone acetate (MPA) Medroxyprogesterone acetate (MPA) is a synthetic progestin that binds to the PgR and is used in the treatment of breast cancer. Twenty-30% of patients resistant to TAM have undergone remissions with MPA [Cavalli et al, 1984]. The effects of MPA on Adr cytotoxicity in human breast cancer cells have also been studied [Shaikh et al, 1989]. It was reported that pre-exposure of MCF-7 to MPA results in synergistic enhancement of Adr cytotoxicity in wild-type MCF-7 cells. Since wild-type MCF-7 cells lack Pgp, elevated Adr toxicity may be due to the ability of MPA to influence membrane fluidity [Bojar et al, 1984]. MPA may also enhance Adr cytotoxicity by binding to Pgp. Naito et al (1989) studied the ability of various steroid hormones to inhibit binding of vincristine to Pgp. 27 Using the vindesine analogue [ Ffj-NABV, which binds to Pgp, they observed that photolabelling is reduced in the presence of vincristine and progesterone. Yang et al (1989) studied the effects of progesterone interaction with Pgp in the endometrium of the mouse gravid uterus and in MDR cells developed from murine macrophage-like cells. They report that [ H]-azidopine labelling is reduced by 66% in the endometrial cells and by 82% in the M D R cells. In addition, progesterone inhibits [ H]-vinblastine binding in the MDR cells by 69%. Furthermore, progesterone increased [ H]-vinblastine accumulation by 5-fold and increases vinblastine sensitivity of M D R cells. Since the majority of the work on the interaction between Pgp and progestins has been accomplished with progesterone, Ishida et al (1994) studied the effects of MP A on MDR. The IC 5 0 (the concentration of a drug required to inhibit cell growth by 50%) of each show that MP A is more potent than progesterone in modifying Adr toxicity in Adr-resistant MCF-7 cells. Furthermore, increased cytotoxicity is due to increased Adr accumulation as determined by measuring the intracellular level of [ I 4C]-Adr. 8. Thesis Objectives CsA is known to have resistance modifying activity. However, its use as a R M may be limited since CsA is nephrotoxic in vivo [Handschumacher, 1990] and has been shown to be toxic to cell lines in vitro [Tamai and Safa, 1990; Twentyman and Wright, 1991]. T A M and MP A have been used in the treatment of breast cancer with limited toxicity [Pannuti et al, 1982; Legha, 1988]. Furthermore, evidence suggests that these agents also interact with Pgp and may have resistance modifying capability [Shaikh et al, 1989; Fine et al, 1993; Leonessa et al, 1994; Ishida et al, 1994]. Therefore, this thesis compared the effects of T A M and M P A as resistance modifiers with those of a known resistance modifier. As discussed above, many of the previous studies of resistance modifiers have been performed on cell lines in vitro and have been directed at understanding their mechanism of action. In this thesis, experiments were performed on 28 primary cultures of human breast epithelial cells ( H B E C ) which more closely resemble cells in vivo than do cell lines. Thus, the principle objective of this thesis was to determine the effectiveness of CsA, T A M and M P A in enhancing Adr cytotoxicity in primary cultures of human breast epithelial cells. There are many studies that support a positive correlation between drug resistance and Pgp expression in cell lines [Fairchild et al, 1987; Merkel et al, 1989; Politi and Sinha, 1989; Schneider et al, 1990; and Taylor et al, 1991]. However, such a relationship is somewhat less clear in clinical breast cancer [Merkel et al, 1989; Schneider et al, 1989; Wishart et al, 1990; and Verelle et al, 1991]. Therefore, a second objective of this thesis was to correlate Pgp expression with Adr resistance in primary cultures of H B E C . The effectiveness of T A M and M P A in the endocrine treatment o f breast cancer appears to be due to their interaction with hormone receptors. T A M binds directly with E R and progestins bind to PgR, an effect of which is to decrease E R levels. This thesis investigated the E R status o f H B E C in primary culture. The objective o f this portion o f the thesis was to determine i f E R status correlated with the effects of CsA, T A M or M P A on H B E C growth and/or sensitivity to Adr. 2 9 CHAPTER 2 MATERIALS AND METHODS 1. Processing Human Breast Tissue for Primary Culture Normal breast tissues were obtained from reduction mammoplasties and benign and malignant tissues from biopsies and mastectomies. Surgical procedures were performed by collaborating surgeons at local hospitals. Information regarding whether or not a patient had received previous chemotherapy was provided by the hospitals' pathology departments. Sterile centrifuge tubes containing transport medium (Appendix 1), used continually in our laboratory, were placed on ice in an insulated container and delivered to the operating room the morning of the surgeries. After surgery, tissue samples were transferred into the centrifuge tubes by operating room nurses and brought back to the tissue culture room as soon as possible. Within a safety hood and under sterile conditions, a tissue specimen was transferred into a sterile petri dish using forceps. Excess fat was trimmed off of the sample with sterile scalpels. The tissue was minced into approximately 1 mm-* pieces using 2 scalpels in a cross-cutting manner. With larger samples from reduction mammoplasties, dehydration of the tissue was prevented by mincing the tissue with the addition of transport medium. Furthermore, scalpel blades had to be changed often. Using the scalpel blades as spatulas, the minced tissue was transferred into a 2 ml cryotube until they were 1/2 full. The vial was then filled to a final volume of 1.8 ml with freezing medium (Appendix 2), which is used routinely in our laboratory. The tissue and the freezing medium were then gently mixed by inverting the freezing vial several times. The tissue-containing vials were then slowly frozen and stored in liquid nitrogen until dissociated for cell culture. 30 2. Dissociation of Processed Tissue Once the desired tissue sample was selected, it was removed from liquid nitrogen and quickly thawed by immersion and agitation in a 37°C waterbath. Prior to opening the cryotube, it was wiped with a Kim-wipe dampened with 70% ethanol. This same Kim-wipe was held over the lid in order to prevent aerosol release when the tube was opened. The contents of the tube were transferred to a 15 ml centrifuge tube. The cryotube was subsequently washed twice with pre-warmed Dulbecco's modified Eagle's medium/Ham's F12 medium (DME/F12; 1:1; Terry Fox Laboratory) containing 10 mM Hepes (H; Sigma) using a pasteur pipette. Washes were also transferred to the centrifuge tube for a final volume of approximately 5 ml. The tissue mixture was spun in a clinical centrifuge at 100 x g for 3 min and the supernatant was discarded. The tissue then was washed in 5 ml DME/F12/H and centrifuged again at 100 x g for 3 min. The supernatant was discarded and the tissue was resuspended in 5 ml warmed dissociation medium (Appendix 3), which is used in our laboratory on a regular basis. The mixture was transferred from the centrifuge tube to a 125 ml dissociation flask. The centrifuge tube was washed with 5 ml dissociation medium which was also transferred to the dissociation flask. An additional 10 ml of dissociation medium was added to bring the total volume of dissociation medium to 20 ml. The flask was first covered with sterile tin foil followed by parafilm and placed in a gyrating shaker inside a 37°C dry incubator for approximately 18 h. After dissociation, the suspension was transferred to a 50 ml centrifuge tube labelled "A". The flask was rinsed 3 times with DME/F12/H: twice with 5 ml and once with 2 ml. Each rinse was added to tube A. Subsequently, the tube was centrifuged at 40 x g for 30 sec and the supernatant was transferred to a 50 ml centrifuge tube labelled "B". The speed at which the suspension was centrifuged at was chosen to preferentially to form a rich epithelial cell suspension. To maximize the collection of epithelial cells, washing and centrifugation of pellet "A" was repeated twice: once with 5 ml DME/F12/H and again with 2 ml DME/F12/H. After each spin, the supernatant was added to tube B. Pellet "A" was 31 resuspended in 2 ml growth medium (Appendix 4). The contents of tube "B" were then centrifuged at 80 x g for 4 min to obtain a pellet of epithelial cells separate from the stromal components which remained in the supernatant and were discarded. To ensure the removal of all enzymes from the dissociation medium, the pellet was washed in 5 ml DME/F12/H and centrifuged at 80 x g for 4 min. After discarding the supernatant, the pellet was resuspended in 2 ml DME/F12/H and viable cells, determined by trypan blue exclusion, were counted on a hemacytometer. The trypan blue dye is unable to penetrate the cell membrane of viable cells whereas dead cells stain blue due to decreased membrane integrity allowing the uptake of the dye. 3. Primary Cell Culture of Human Breast Epithelial Cells (HBEC) Once the viable cell number was determined, the cell suspension in tube "B" was centrifuged at 80 x g for 4 min and the supernatant was discarded. The pellet was resuspended in DME/F12/H and divided into 2 components: one for seeding onto collagen-coated 96-well microtiter plates and another for seeding onto collagen-coated tissue culture coverslips. Each fraction was subsequently resuspended and seeded onto collagen-coated surfaces at 2.5 x 103 cells/cm2 in attachment medium (Appendix 4). If there were not enough cells to do both, HBEC were seeded only onto collagen-coated 96-well microtiter plates. Cells were seeded onto the inner 10x6 matrix of the collagen-coated microtiter plate (Figure 2). Wells which were devoid of cells were filled with DME/F12/H to protect wells containing cells from evaporation of medium. One row was also left in order to have an "MTT control" on each plate to account for the absorbance of M T T (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide). Collagen coating of the microtiter plate is regularly done in our laboratory by adding a drop of rat tail collagen (Appendix 5) to each well and gently swirling the plate to make certain that the well is entirely covered. The coverslips were collagen-coated by placing the coverslips 32 Figure 2. A 96-well microtiter plate. The outer wells (green) contained DME/F12/H in order to prevent evaporation of medium in the inner wells (blue), which may contain cells. Wells which were devoid of cells were also filled with DME/F12/H. One row (orange) was left as an "MTT control" to account for the absorbance of MTT. 33 into a 4 well plate and adding one drop of rat tail collagen onto each coverslip and gently swirling the plate. Plates and coverslips were allowed to dry for at least 1 h in a laminar flow hood under U V light to ensure sterility. Pellet "A" was seeded onto collagen-coated tissue culture coverslips in a volume of 0.5 ml growth medium/well. Cultures were placed in a humidified incubator with 5% C02 and 95% air at 37°C. On day 1, the medium was changed to phenol red-free, serum-free medium (Appendix 6) and changed, thereafter, on alternate days. Due to the observation that fibroblasts grow rapidly in serum-containing media, a serum-free medium was chosen [Emerman and Wilkinson, 1990]. 4. Cell Line Culture The MCF-7 cell line was obtained from the American Type Culture Collection and an Adriamycin-resistant subline, MCF7-AdrR, was a gift courtesy of Dr. K. Cowan at the U.S. National Institutes of Health. The cell lines were maintained in 75 mm tissue culture flasks in 15 ml of phenol red-free DME/F12/H supplemented with 5% dextran charcoal-treated fetal bovine serum (Gibco Laboratories) and 0.014 M NaHC03 (Appendix 7). Medium for MCF7-AdrR was further supplemented with 5.0 pg/ml Adr (Adria Laboratories) (Appendix 8). Cultures were maintained in an incubator with a humidified atmosphere containing 5% C O 2 and 95% air at 37 C. Media was changed every other day. Cells were subcultured when flasks were 80% confluent by washing the cells with warmed DME/F12/H (to remove any residual serum) and treated with 10 ml of warmed saline-A, 0.05% trypsin and 0.025% E D T A (Appendix 9). The culture flasks were gently agitated for approximately 5 min to obtain a single-cell suspension. To terminate trysinization, 15 ml of serum-containing medium was added to the cell suspension and placed in a centrifuge at 100 x g for 3 min. After the supernatant was removed, cells were resuspended in 5 ml of the appropriate growth medium for each cell line (Appendices 7 and 8). A small aliquot of the cell suspension was removed, transferred to a glass test 34 tube and viable cells counted on a hemacytometer using the trypan blue dye as explained above. Some of the cells were frozen and stored in liquid nitrogen for future use or were discarded while another fraction was used to seed a new tissue culture flask. For experiments, a seeding density of 2.5 x 103 cells/cm2 was obtained. Cells were seeded onto collagen-free 96 well microtiter plates and collagen-free tissue culture coverslips. 5. Immunocytochemistry 5a. Cvtospinning cell culture samples for immunocytochemistry Primary HBEC and the MCF-7 and MCF7-AdrR cell lines were harvested from 25 cm2 flasks using 5 ml of a mixture containing saline-A, trypsin and E D T A (Appendix 9). Semm-containing medium (Appendix 4, 7 or 8 respectively) was added to terminate trypsin activity and the single-cell suspension was transferred to a 15 ml centrifuge tube and centrifuged at 100 x g for 3 min. The supernatant was removed and the cell pellet was resuspended in 5 ml of DME/F12/H. A small aliquot was removed and viable cells counted using the trypan blue exclusion method. The cell suspension was then divided into two fractions: one for storage in liquid nitrogen or for seeding into another tissue culture flask and another for making cytospin preparations. The fraction for cytospinning was centrifuged at 100 x g for 3 min. The supernatant was removed and the pellet was resuspended in DME/F12/H to obtain a final cell concentration of 100 cells/ul. The cell suspension was then cytospun at 1200 x g for 1 min onto glass slides and air dryed overnight. Cells then were fixed and permeabilized with 10% formalin, methanol and acetone. From this point, slides could undergo immunocytochemical staining or they could be stored in specimen storage medium (Appendix 11) in -20°C for up to 4 months. Specimen storage medium is used to store samples for ER-ICA as suggested by Abbot Laboratories. It was used for Pgp-ICA since storage in this medium resulted in coverslips that were free of debris compared to air drying and storage of the coverslips at -70°C as described by Chan et al (1988). Although this method was successful in assaying for Pgp 35 in M C F 7 - A d r R , the positive Pgp control, we were not able to identify E R in M C F - 7 , the positive E R control. Before we were able to modify the cytospinning procedure for E R , we found that we were able to immunocytochemically stain for E R in cells cultured on coverlsips. Therefore, we carried out all Pgp and E R immunocytochemical assays on cells cultured on coverslips (described below). 5b. P-gp immunocytochemical assay (ICA) Freshly obtained H B E C for primary culture and the cell lines were seeded in their respective growth media (Appendices 4, 7 and 8) at 2.5 x 10 3 cells/cm 2 onto collagen-coated or collagen-free plastic tissue culture coverslips, respectively, and maintained for 6 days with media changes on alternate days. The medium of primary cultures of H B E C was changed to phenol red-free, serum-free medium (Appendix 6) after 1 day in vitro. Immunocytochemical assays for Pgp were performed using a modification of the method of Chan et al (1988). The M C F - 7 cell line was used as a negative control for Pgp and the M C F 7 - A d r R cell line was used as a positive control. Cells on coverslips were fixed and permeabilized in 10% formalin for 10 min and washed in phosphate-buffered saline (PBS; Appendix 10) for 5 min. The coverslips then were placed in methanol for 4 min and then acetone for 1 min. Afterwards, coverslips were washed in P B S for 5 min and were either stored in specimen storage medium (Appendix 11) at -20°C or underwent the immunocytochemical staining protocol. Prior to addition of antibody, endogenous peroxidase activity was quenched using a series of solutions: (a) 20% methanol, 80% ethanol and 2% H2O2 for 10 min; (b) 0.02% periodic acid, 0.35% sodium azide and 3% H 2 0 2 for 20 min; (c) 0.05 M Tr i s -HCl in 0.1 M N a C l at p H 7.5 for 5 min. Cells, subsequently, were layered with the primary antibody for Pgp (C219; 1:71.4; Signet Laboratories, Inc.) and incubated overnight (a minimum of 18 h). Following 2 washes with phosphate buffered saline (PBS) and 0.05% Tween 20, the cells were incubated with a secondary antibody mixture o f rabbit antimouse IgG (1:149; Cedarlane Laboratories 36 Ltd.) and peroxidase conjugated F(ab')2 fragments of rabbit antimouse IgG (1:149; Jackson Immunoresearch Laboratories, Inc.) for 45 min. The cells then were washed with PBS and 0.05% Tween 20 and incubated with mouse monoclonal peroxidase-antiperoxidase (1:26; Sternberger Monoclonals Inc.) for 45 min. After 2 washes with PBS and 0.05% Tween 20, cells were incubated with peroxidase conjugated F(ab')2 fragments of rabbit antimouse IgG (1:149) for 45 min. The cells were washed as before and then incubated with 0.05% 3,3' diaminobenzidine tetrahydrochloride (DAB) in 0.05 M Tris, pH 7.6, and 0.05% H2O2 for 30 min at room temperature. Following this, the cells were washed with PBS solution and counterstained with hematoxylin. All antibody incubations were performed in a humidified chamber at 4°C, whereas all PBS washes were performed at room temperature. 5c. Estrogen receptor (ER) ICA Cell cultures were assayed for ER using the ER-ICA kit (Abbott Laboratories). The MCF-7 cell line was used as a positive control for ER and the MCF7-AdrR cell line was used as a negative control. Cells on coverslips were fixed using 10% formalin, methanol and acetone, then washed with PBS. Afterwards cell cultures were washed in PBS for 5 min and were either stored in specimen storage medium (Appendix 11) at -20°C or underwent the immunocytochemical staining protocol. Blocking reagent was layered onto the cells for 15 min. After removing excess solution, the primary antibody to the estrogen receptor (H222 Spy) was layered onto the cells for 1 h. After 2 washes with PBS, cells were incubated with bridging antibody for 1 h. The cells were washed twice with PBS and incubated with the peroxidase-antiperoxidase complex for 1 h. After 2 washes with PBS, the cells were incubated with the kit's Chromogen Substrate Solution plus DAB for 30 min. Cells then were rinsed gently for 5 min using distilled H 2 O and counterstained with hematoxylin. All procedures were carried out at room temperature. 37 5d. Evaluation of Pgp and E R content In each immunocytochemical assay, 3 categories of staining were observed: no staining; few positive staining cells (<20%); or numerous (>20%) positive. The cultures were categorized as Pgp+ i f greater than 10% of the cells in culture expressed Pgp. H B E C containing Pgp and E R were counted without knowing whether or not the cells were stained with control or primary antibody. 6. Drug Testing Protocol H B E C in primary culture and the cell lines were seeded at 2.5 x 103 cells/cm 2 on collagen-coated 96-well microtiter plates in phenol red-free, serum-containing attachment medium (Appendices 4, 7 or 8). On day 1, the medium was changed to phenol red-free, serum-free medium (Appendix 6) for primary cultures or phenol red-free serum-containing medium (Apendices 7 and 8) for the cell lines with or without 4.3 u M C s A (a gift from Sandoz Canada Inc.), 1.0 u M T A M (a gift from ICI Pharmaceuticals Canada) or 1.3 u M M P A (Sigma). A l l these concentrations are achievable in vivo. Twentyman and Wright (1991) observed that 0.086 u M is the minimum concentration of C s A needed to enhance Adr cytotoxicity in a Pgp-expressing mouse mammary carcinosarcoma cell line. However we were unable to enhance Adr cytotoxicity in M C F 7 - A d r R with C s A less than 4.3 u M . T A M at 1.0 pJVI was chosen because it is achievable in patients with niinimal side effects [Legha, 1988]. M P A at 1.3 u M was the drug concentration chosen since we were unable to observe enhancement o f Adr at lower concentrations. Since phenol-red, a p H indicator used in tissue culture medium, has estrogenic activity [Devleeschouwer et al, 1992], it may interact with T A M and/or M P A to affect the growth of cells in culture. To avoid this, phenol red-free medium was used in the culture medium. After 24 h, the medium was changed to that with or without the test agent plus or minus Adr (0.5 or 5.0 ug/ml). These concentrations of Adr were chosen since 5.0 ug/ml is the peak plasma level of A d r and 1/10 of this level is approximately the clinically relevant concentration to use in vitro 38 pmerman et al, 1990]. After a 48 h incubation, the medium was changed to remove the Adr. Following a 48 h recovery period, the cultures were terminated and the final cell densities were determined using the tetrazolium dye (MTT) reduction assay (described below) [Emerman and Eaves, 1994]. Each condition was replicated at least four times. M C F - 7 and M C F 7 - A d r R cells were used as controls for Adr sensitivity. These cell lines were seeded at a concentration of 2.5 x 103 cells/cm 2 on collagen-free 96-well microtiter plates in the medium described above for cell lines. On day 1 in culture, the medium was changed to that with or without the resistance modifying agent to be tested: 4.3 u M CsA, 1.0 u M T A M or 1.3 u M M P A The same drug testing protocol was used for the cell lines. 7. The M T T Assay In initial studies, viable cells from the cultures, assessed by trypan blue exclusion, were counted on a hemacytometer and cell counts compared the absorbance values determined in the M T T (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoUum bromide) assay [Emerman and Eaves, 1994]. This comparison showed that there was a linear relationship between the number o f cells present and the absorbance readings. Therefore, the M T T assay was used in subsequent experiments. Culture medium was removed and medium (phenol red-free D M E / F 1 2 / H ) containing 1 mg/ml M T T (Sigma) was added to each well in a volume of 100 ul and incubated in 95% air and 5% CO2 at 37°C for 5 h. Afterwards, 100 ul of 20% formol in saline was added to each well and incubated at room temperature and in the dark for 30 min. The medium was removed and 100 ul of anhydrous isopropanol was added to each well and incubated at room temperature and in the dark for 1 h. The plate was gently agitated for 30 sec prior to measuring absorbance. A 96-well microtiter plate reader (model E L 311, Biotek Instruments) was used to determine absorbance values at 540 nm. Cell yield was represented as a percent of control. 39 8. Statistics The data were statistically analyzed using two-way analysis of variance ( A N O V A ) followed by Tukey post hoc tests; all comparisons were at p<0.05, 2-tailed, using the statistical program Systat 5.03 for Windows (Systat Inc., Evanston, EL). In order to determine i f their was a significant interaction between the two drugs, the effects of the drug combinations used were compared to that of a control (no Adr and no resistance modifier). I f an interaction was found to be statistically significant and to determine i f this interaction was either additive or synergistic, the data were normalized such that the effects of A d r with or without R M were compared to the effects of no Adr with or without R M , respectively. If the results still showed significant difference from the effect of Adr alone and R M alone, then the drug interaction was classified as synergistic. I f no statistical significance was observed, then the interaction was classified as additive. 40 C H A P T E R 3 R E S U L T S 1. Pgp-ICA l a . Pgp-ICA in cell lines Initially, cell lines were cultured and harvested using trypsin. They then were cytospun onto a glass slide, fixed and immunocytochemically assayed for Pgp. The result of Pgp-ICA in the MCF7-AdrR cell line is shown in Figure 3. The presence of a brown coloration indicates that MCF7-AdrR is Pgp+. The method was changed to culturing the cells on coverslips since cytospinning could not produce results in ER-ICA in MCF-7, the positive ER control as discussed in the Materials and Methods. Hereafter, cell lines were cultured and assayed for Pgp on coverslips since Pgp-ICA could also be accomplished this way. MCF-7 and MCF7-AdrR cells were seeded on coverslips at 2.5 x 103 cells/cm2 in their respective growth media (Appendices 7 and 8). After 6 days in vitro, the cell lines were fixed and stained for Pgp using an immunoperoxidase technique. Since the C219 monoclonal antibody stains Pgp in the plasma membrane and the Golgi region of the cells [Schneider et al, 1989; Veneroni et al, 1994], both membrane and cytoplasmic staining were observed in MCF7-AdrR cells (Figure 4) indicated by the presence of a brown coloration in the cells. Figure 5 illustrates that MCF-7 cells were Pgp- as indicated by the abscence of brown coloration in the cells. 41 Figure 3. Pgp expression in a cytospin preparation of the MCF7-AdrR cell line. Immunoperoxidase staining of MCF7-AdrR cells cytospun onto a a glass slide is described in the Materials and Methods. The cell line was stained using the primary antibody C219. Arrowheads point to a brown coloration of the cytoplasm and/or cell membrane indicating Pgp+ staining. 42 Figure 4. Pgp expression in MCF7-AdrR cells cultured on a coverslip. Immunoperoxidase staining using the primary antibody C219 is described in the Materials and Methods. Arrowheads point to a brown coloration of the cytoplasm and/or cell membrane indicating Pgp+ staining. 43 Figure 5. Pgp expression in MCF-7 cells cultured on a coverslip. Lmmunoperoxidase staining using the primary antibody C219 is described in the Materials and Methods. The lack of a brown coloration of the cytoplasm and/or cell membrane suggests that these cells do not express Pgp.. 44 lb. Pgp-ICA in primary cultures of H B E C H B E C for primary cell cultures were obtained from breast tissue specimens from 15 patients who had not received chemotherapy and 6 patients who had received chemotherapy. In all experiments, cells were seeded on coverslips at 2.5 x 103 cells/cm2 in serum-containing attachment medium (Appendix 4). After 24 h, the medium was changed to phenol red-free, serum-free medium (Appendix 6). Cells on coverslips were fixed and assayed for Pgp after 6 days in vitro. Pgp was detected in 6 of the 21 samples; of the Pgp+ cultures, 5 of the 6 were from tissues previously exposed to chemotherapy (Table 3). Pgp+ cultures were not difficult to identify despite background peroxidase activity in the primary cultures compared to cultures of MCF7-AdrR cells, the Pgp+ control (Figure 6). Note the brown coloration of the Pgp+ H B E C in primary culture in Figure 7 compared to the Pgp- HBEC in primary culture in Figure 8. Table 3. Primary cell cultures with cells expressing Pgp Culture Tissue Previous Percent of type Chemotherapy Pgp+ cells in culture HMC217 malignant no 37% HMC227 malignant yes 15% HMC228 malignant yes 18% HMC229 malignant yes 32% FC73 benign, previous yes 28% carcinoma BT35 normal, previous yes 23% carcinoma 45 Figure 6. Background peroxidase staining in the MCF7-AdrR cell line and a 6 day primary culture of HBEC. These HBEC were obtained from breast tissue from a patient previously treated with chemotherapy. Immunoperoxidase staining of (a) MCF7-AdrR and (b) primary cultured HBEC using the primary antibody C219 is described in the Materials and Methods. Arrowheads point to the brown coloration of the cytoplasm and/or cell membrane indicating Pgp+ staining. Arrows indicate the background peroxidase staining. 46 Figure 7. Pgp expression in a 6 day primary culture of HBEC. These HBEC were obtained from breast tissue from a patient treated with chemotherapy. Immunoperoxidase staining using the primary antibody C219 is described in the Materials and Methods. Arrowheads point to the brown coloration of the cytoplasm and/or cell membrane indicating Pgp+ staining. 47 Figure 8. Pgp-negative HBEC in a 6 day primary culture. These HBEC were obtained from breast tissue from a patient not treated with chemotherapy. Immunoperoxidase staining using the primary antibody C219 is described in the Materials and Methods. The lack of brown coloration of the cytoplasm and/or cell membrane suggests that these cells do not express HBEC. 48 2. ER-ICA 2a. ER-ICA in cell lines As stated above, cell lines were initially cultured and harvested using trypsin. They then were cytospun onto a glass slide, fixed and immunocytochemically assayed for E R . The result of E R - I C A in the M C F - 7 cell line is shown in Figure 9. Using cytospinning did not identify E R using the E R - I C A in MCF -7 , the positive E R control, as discussed in the Materials and Methods. Therefore, the method was changed to culturing the cells on coverslips After 6 days in vitro, M C F - 7 and MCF7-AdrR cells cultured on coverslips were fixed and assayed for E R using an immunoperoxidase technique. M C F - 7 cells were positive for E R as indicated by the brown nuclear staining in the cells (Figure 10). This staining was seen in cells throughout the culture. In contrast, MCF7 -AdrR cells, which have been shown to lack E R , did not show staining in the nucleus (Figure 11). 49 Figure 9. ER expression in a cytospin preparation of the MCF -7 cell line. Immunoperoxidase staining using the primary antibody H 2 2 2 SPy is described in the Materials and Methods. Although MCF -7 are found to contain ER, we were not able to detect them in cytospin preparations of this cell line since the nucleus of these cells lack brown coloration. 50 Figure 10. ER expression in the MCF-7 cell line cultured on coverslips. Immunoperoxidase staining using the primary antibody H222 SPy is described in the Materials and Methods. Arrowheads point to the brown stained nuclei indicating that these cells are ER+. 51 Figure 11. ER expression in the MCF7-AdrR cell line cultured on coverslips. Immunoperoxidase staining using the primary antibody H222 SPy is described in the Materials and Methods. The lack of brown stained nuclei suggests that these cells do not express ER. 52 2b. ER-ICA in primary cultures of H B E C In all experiments, cells were seeded on coverslips at 2.5 x 103 cells/cm2 in serum-containing attachment medium (Appendix 4). After 24 h, the medium was changed to phenol red-free, serum-free medium (Appendix 6). Cells on coverslips were fixed and assayed for ER after 6 days in vitro. Of the 15 primary cultures of HBEC obtained from patients who had not received chemotherapy, 2 were ER+. Of 6 cultures from cells taken from patients who received chemotherapy, 1 was ER+. In contrast to ER staining in the MCF-7 cell line, ER+ HBEC in primary cultures were located in or near dense areas of HBEC (Figure 12). These areas or epithelial islands are sites at which a clump of cells attached to the coverslip. ER- staining of primary cultured HBEC is illustrated in Figure 13 by the lack of brown stained nuclei. 53 Figure 12. ER expression in HBEC in a 6 day primary culture. Cells were stained using the primary antibody H222 SPy as described in the Materials and Methods. Arrowheads point to the ER+ HBEC with darkly stained nuclei. 54 Figure 13. ER-negative HBEC in a 6 day primary culture. Cells were stained using the primary antibody H 2 2 2 SPy as described in the Materials and Methods. The lack of brown stained nuclei suggests that these cells do not express ER. 55 3. Effects of R M on Adr Cytotoxicity in the Cell Lines 3a. Cytotoxic effects of Adr alone in the cell lines MCF-7 and MCF7-AdrR cells were seeded in 96-well microtiter plates at 2.5 x 103 cells/cm2 in their respective growth media (Appendices 7 and 8). After 24 h, media was changed to that with either no RM, 4.3 uM CsA 1 MM T A M or 1.3 uM MPA. After 24 h, the medium was again changed and contained no Adr, 0.5 or 5.0 ug/ml Adr plus either no RM, 4.3 uM CsA, 1 uM T A M or 1.3 uM MPA. After 48 h, the medium was changed to that without Adr. Cell line cultures were terminated after another 48 h and cell growth determined using the MTT assay. Using growth curves for these cell lines previously established in our laboratory (Stingl, 1992), the control wells (no R M and no Adr) contained 11 x 103 MCF-7 cells and 16 x 103 MCF7-AdrR cells after 6 d when cultures were terminated. The effects of the drug combinations in the inhibition of cell growth of MCF-7 and MCF7-AdrR cells are illustrated in Table 4. As expected, the Pgp- MCF-7 cell line was sensitive to the cytotoxic effects of Adr at both 0.5 and 5.0 ug/ml, whereas the Pgp+ MCF7-AdrR cell line was resistant to Adr at both concentrations. MCF-7 cell growth was significantly reduced to 22.4 ± 3.4 % of control (p<0.001) and 16.7 ± 4.3 % of control (p<0.001) by 0.5 and 5.0 ug/ml, respectively. 3b. Effects of CsA on Adr cytotoxicity in the cell lines In initial experiments, two concentrations of CsA were tested for their ability to modify resistance to Adr in the cell line MCF7-AdrR: 0.86 and 4.3 uM CsA. However, no effect on Adr cytotoxicity was observed when the lower concentration of CsA was used in combination with Adr (Figure 14). Therefore, subsequent experiments were performed with 4.3 uM CsA. 56 140 o o U c CD o s-CM 120 100 80 60 40 20 0 0 0.86 CsA (juM) 0.0 ug/ml Adr 0.5 ug/ml Adr 5.0 ug/ml Adr 4.30 Figure 14. The effect of different concentrations of CsA on Adr cytotoxicity in the MCF7-AdrR cell line. The cells were cultured in medium with or without CsA at two concentrations (0.83 and 4.8 uM) in the presence or absence of 0.5 ug/ml or 5.0 ug/ml Adr as described in the Materials and Methods. Each bar displays the mean of growth of MCF7-AdrR as a percent of control (no CsA and no Adr) +/- SEM. Experiments at each concentration of CsA were replicated 4 times. 57 CsA significantly inhibited the growth of both cell lines (Table 4). CsA alone significantly reduced growth of MCF-7 cells and MCF7-AdrR to 29.1 ± 3.8 % of control (p<0.001) and 31.5 ± 4.8 % of control (p<0.001), respectively. As shown in Table 4, when Adr was added to the medium in combination with CsA, MCF-7 cells responded with a 7-fold decrease in cell growth to 0.5 ug/ml Adr compared to Adr alone (pO.001). Furthermore, the combination of the two drugs significantly inhibited cell growth compared to CsA alone (p<0.001; Table 5); therefore, a synergistic effect between the two drugs occurred. CsA plus 5.0 ug/ml Adr also resulted in significantly decreased cell growth compared to CsA alone (p<0.005) and Adr alone (p<0.05) (Table 4). However, Table 5 shows that the effect was only additive. MCF7-AdrR cells were sensitized to both concentrations of Adr when CsA was added to the medium. The data show a synergistic effect between the two drugs (Table 5). CsA combined with 0.5 ug/ml Adr inhibited cell growth to 12.4 ± 6.5 % of control (p<0.005; Table 4), a 3-fold reduction in growth compared to CsA alone (p<0.05). MCF7-AdrR cell growth was further inhibited by CsA plus 5.0 ug/ml Adr to 5.3 ± 2.0 % of control (p<0.005), a 6-fold reduction compared to CsA alone (p<0.001). 3c. Effects of T A M on Adr cytotoxicity in the ceil lines Table 4 shows that T A M alone significantly inhibited growth in MCF-7 cells (p<0.005) and MCF7-AdrR cells (p<0.05). T A M plus 0.5 and 5.0 ug/ml Adr resulted in decreased MCF-7 cell growth when compared to T A M alone (p<0.005); however, T A M did not enhance Adr cytotoxicity. The combination of T A M plus Adr resulted in decreased growth of MCF7-AdrR cells compared to Adr alone (p<0.005; Table 4). MCF7-AdrR cell growth with T A M plus 0.5 ug/ml Adr was significantly different from 0.5 ug/ml Adr alone, however, it was not different from T A M alone. TAM plus 5.0 ug/ml Adr acted synergistically to inhibit the 58 growth of MCF7-AdrR cells to 49.5 ± 7.6 % of control. Table 5 shows this result was significantly different from T A M alone (p<0.005) and Adr alone (p<0.005). 3d. Effects of M P A on Adr cytotoxicity in cell lines As with CsA different concentrations of MPA (0.013, 0.13 and 1.3 uM) were tested on MCF7-AdrR cells in combination with different concentrations of Adr (Figure 15). MPA at 1.3 uM increased the cytotoxicity of 0.5 ug/ml Adr. Therefore, this concentration was used in subsequent experiments. MPA did not significantly inhibit MCF-7 cell growth by itself (Table 4). Similar to TAM, MPA plus either Adr concentration resulted in decreased MCF-7 cell growth compared to MPA alone (p<0.005) but was not significantly different from cell growth with Adr alone. In MCF7-AdrR cells, MPA alone significantly inhibited cell growth (p<0.05; Table 4). MPA plus Adr significantly inhibited MCF7-AdrR cell growth compared to Adr alone (p<0.005 for 0.5 ug/ml Adr and p<0.05 for 5.0 ug/ml Adr) but not compared to MPA alone. Therefore, MPA plus Adr did not result in significantly increased Adr cytotoxicity, although there was a trend showing enhanced Adr cytotoxicity. 59 Figure 15. The effect of different concentrations of M P A on Adr cytotoxicity in the MCF7-AdrR cell line. The cells were cultured in medium with or without M P A at various concentrations (0.013, 0.13 and 1.3 uM) in the presence or absence of 0.5 ug/ml or 5.0 ug/ml Adr as described in the Materials and Methods. Each bar displays the mean of growth of MCF7-AdrR as a percent of control (no M P A and no Adr)+/- SEM. Experiments at each concentration of M P A were replicated 4 times. 60 Table 4. Effects of resistance modifiers (RM) on Adr cytotoxicity in MCF-7 and MCF7-AdrR cell lines [Adr] noRM CsA TAM MPA (u.g/ml) (4.3 nM) (1.0 (JM) (1.3 nM) Percent of Control MCF-7 0 100 (±2.4)* 29.1 (+3.8)2 62.8 (+ 3.8)2 80.3 (± 9.5) 0.5 5.0 22.4 (± 3.4)3 16.7 (±4.3)3 3.4 (±0.4)' 4.9 (±1.2)' 30.7 (± 2.4)3 11.4 (± 1.4)3 11.2 (±3.8)3 12.1 (± l . l ) 3 MCF7-AdrR 0 100(+2.3)1 31.5 (±4.8)2 74.2 (±6.5)2 72.1( ±9.5) 2' P <° 0 5 0.5 102.7 (+ 2.2) 12.4 (±6.5) 67.7 (± 7.5)2 63.2 (±9.4)2 5.0 89.0 (+ 3.0) 5.3 (±2.3) 49.5 (±7.6)4 55.7 (± 7.6)2 Control (no RM and no Adr) + SEM Significant inhibition of growth by RM alone (p<0.001 unless otherwise indicated) Significant inhibition of growth by Adr alone (p<0.005) Significant interaction between RM and Adr resulting in enhanced inhibition of growth (p<0.005) Table 5. Normalized data of the effects of R M on Adr cytotoxicity in MCF-7 and MCF7-AdrR cell lines [Adr] noRM CsA TAM MPA (ixg/ml) (4.3 nM) (1.0 M M ) (1.3 nM) Percent of Control MCF-7 0 100 (± 2.4)1 I O O ^ . S ) 1 I O O ^ . S ) 1 100 (+ 9.5)1 0.5 22.4 (± 3.4)2 11.8 (±0.4)A 48.9 (± 2.4)2 13.9 (±3.8)2 5.0 16.7 (+ 4.3)2 16.7 (± 1.2)3 18.2 (± 1.4)2 15.1 (±1.1)2 MCF7-AdrR 0 100 (+2.3)1 100(±4.8)1 100(±6.5)1 100 (± 9.5)1 0.5 102.7 (+ 2.2) 39.3 (±6.5)A 87.2 (± 7.5) 87.6 (± 9.4) 5.0 89.0 (+ 3.0) 16.9 (±2.3)4 4.3 (± 7.6)4 77.2 (± 7.6) Control ± SEM 2Significant inhibition of growth by Adr alone (p<0.005) 3 Additive effect of RM and Adr on inhibition of growth Synergistic effect of RM and Adr on inhibition of growth (p<0.005) 61 4. Effects of RM on Adr Cytotoxicity in Primary Cultures of HBEC With or Without Pgp Expression 4a. Effects of Adr in primary cultures of HBEC with or without Pgp expression Primary cultures of HBEC obtained from patients with or without previous chemotherapy were seeded in 96-well microtiter plates at 2.5 x 103 cells/cm2 in attachment medium (Appendices 4). After 24 h, medium was changed to phenol red-free, serum-free medium (Appendix 6) with either no RM, 4.3 uM CsA, 1 uM T A M or 1.3 uM MPA. After 24 h, the medium was again changed and contained no Adr, 0.5 or 5.0 ug/ml Adr plus either no RM, 4.3 uM CsA, 1 uM T A M or 1.3 uM MPA. After 48 h, the medium was changed to that without Adr. Cell cultures were terminated after another 48 h and cell growth determined using the MTT assay. Controls (no R M and no Adr) of the primary cultures of HBEC grew to an average of 13 x 103 cells. In primary cultures of HBEC, 0.5 and 5.0 ug/ml of Adr were cytotoxic to both Pgp- (n=15; p<0.005) and Pgp+ cell cultures (n=6; p<0.005) (Figure 16). However, 0.5 ug/ml Adr was significantly more effective in primary cultures of Pgp- HBEC (36.2 ± 3.9% of control) than primary cultures of Pgp+ HBEC (68.7 + 3.1 % of control) (p<0.005). 4b. Effects of CsA on Adr cytotoxicity in primary cultures of HBEC with or without Pgp expression As illustrated in Figure 16, CsA alone significantly inhibited growth of Pgp-primary cultures of HBEC (p<0.001). CsA plus 0.5 ug/ml Adr did not significantly inhibit HBEC growth further (11.5 + 3.5 % of control compared to CsA alone, 24.0 ± 4.3 % of control). However, CsA plus 0.5 ug/ml Adr significantly inhibited growth of HBEC compared to Adr alone (36.2 ± 3.9 % of control; p<0.001). Therefore, growth inhibition was due to the effects of CsA alone. CsA plus 5.0 ug/ml Adr inhibited growth of HBEC 6 2 to 7.2 ± 1.2 % of control which was significantly different from the effect of CsA alone (p<0.005) but not 5.0 ug/ml Adr alone (16.0 ± 3.4 % of control). Pgp+ primary cultures of HBEC also were significantly growth inhibited by CsA alone (35.2 + 4.9 % of control, no CsA and no Adr; pO.OOl). In combination with Adr, only CsA plus 0.5 ug/ml Adr interacted synergistically to enhance Adr cytotoxicity (p<0.005). There was an additive effect of CsA plus 5.0 ug/ml Adr on the growth inhibition of Pgp+ primary cultures of HBEC (p<0.05). 4c. Effects of T A M on Adr cytotoxicity in primary cultures of HBEC with or without Pgp expression T A M inhibited growth of Pgp- primary cultures of HBEC to 70.0 ± 4.8 % of control (p<0.001; Figure 16). T A M plus Adr significantly reduced cell growth compared to T A M alone (p<0.005), however, this combination did not enhance Adr cytotoxicity. Although T A M did not increase the inhibitory effect ofAdr on cell growth, it also did not interfere with the action of Adr. Pgp+ primary cultures of HBEC were not significantly growth inhibited by T A M alone. Both Adr concentrations inhibited the growth of these cells in culture to the same degree in the presence or absence of TAM. 4d. Effects of MPA on Adr cytotoxicity in primary cultures of HBEC with or without Pgp expression Figure 16 illustrates that MPA alone inhibited growth of Pgp- primary HBEC cultures to 80.0 ± 6.9 % of control (p<0.05), similar to TAM. Growth of Pgp- primary cultures of HBEC was reduced to 29.2 ± 5.4 and 12.4 + 3.0 % of control when MPA was combined with 0.5 and 5.0 ug/ml Adr, respectively. Although these drug combinations inhibited cell growth compared to MPA alone (p<0.005 for both Adr concentrations), Adr cytotoxicity was not enhanced compared to the effects of either Adr concentration alone. 63 The growth of Pgp+ primary cultures of HBEC was also inhibited by MPA alone (p<0.05). As with Pgp- primary cultures of HBEC, MPA plus either concentration of Adr resulted in decreased cell growth compared to MPA alone (p<0.005). More importantly, in contrast to the results obtained in Pgp- cultures, Adr cytotoxicity was enhanced in Pgp+ cultures when MPA was combined with 0.5 ug/ml (40.3 ± 4.3 % of control; pO.OOl). 64 Figure 16. Effects of resistance modifiers (RM) on Adr cytotoxicity in Pgp+ (n=6) and Pgp- (n=15) primary cultures of HBEC. The cells were cultured in medium with or without 4.3 uM CsA (panel A), 1.0 uM T A M (panel B) or 1.3 uM MPA (panel C) in the presence or absence of 0.5 ug/ml or 5.0 ug/ml Adr as described in the Materials and Methods. In each panel, unshaded bars - no RM, shaded bars - RM. Each bar displays the mean of growth of HBEC in primary culture as a percent of control (no Adr and no RM) +/- SEM. To distinguish between statistically relevant data, bars are labelled as: a = significant inhibition of growth by RM alone; b = significant inhibition of growth by Adr alone; c = additive effect of RM and Adr; or d = synergistic effect of R M and Adr. Experiments were replicated 4 times. 65 U. CsA (4.3 jiM) Pgp" HBEC 100 80 60 40 20 fllOO o ( J 80 O 60 a 4 0 QJ W 20 i . <D PL, 100 80 60 40 20 0 p. T A M (1.0 jaM) a b a a b h ^ b P2 C. MPA (1.3 nM) 0 0.5 b b h b b 5.0 i t 0 Pgpr+" HBEC b •5E 0.5 c b b b b 5.0 Adr (|ig/inl) 66 5. Effects of R M on Adr Cytotoxicity in Primary Cultures of HBEC With or Without ER Expression 5a. Cytotoxic effects of Adr in primary cultures of HBEC with or without ER expression Adr at 0.5 and 5.0 ug/ml reduced growth of ER- primary cultures of HBEC (n=18) to 53.5 ± 3.9 % of control and 8.4 ± 1.1 % of control, respectively (psO.OOl; Figure 17). Both concentrations of Adr were also cytotoxic to ER+ primary cultures of HBEC (n=3) reducing cell growth to 55.9 ±6.5 % of control and 25.1 ± 4.8 % of control, respectively (ps<0.001). 5b. Effects of CsA on Adr cytotoxicity in primary cultures of HBEC with or without ER expression In ER- primary cultures of HBEC, cell growth was inhibited to 35.2 + 4.9 % of control by CsA alone (p<0.005; Figure 17). CsA combined with either Adr concentration decreased cell growth compared to CsA alone (p<0.005). Additionally, significant enhancement of Adr cytotoxicity was observed when CsA was combined with 0.5 ug/ml Adr (p<0.005). At the higher concentration of Adr, the effects of CsA and Adr were additive (p<0.05). CsA alone also inhibited ER+ primary cultures of HBEC (p<0.005). The effects of CsA and 5.0 ug/ml Adr on growth inhibition were additive in these cultures (13.0 ± 2.4 % of control, p<0.005). 5c. Effects of T A M on Adr cytotoxicity in primary cultures of HBEC with or without ER expression Figure 17 shows TAM, by itself, significantly inhibited cell growth of primary cultures that were ER- (p<0.001). Combinations of T A M plus Adr resulted in increased 67 growth inhibition compared with T A M alone (p<0.005), but were not significantly different from the cytotoxic effect of Adr alone. Therefore, T A M was not interfering with cytotoxic effects of Adr. Interestingly, ER+ primary cultures of HBEC did not exhibit significant inhibition of growth with T A M alone (82.8 ± 6.8 % of control). Furthermore, the growth inhibition observed with both concentrations of Adr were not significantly affected by the presence of TAM. 5d. Effects of MPA on Adr cytotoxicity in primary cultures of HBEC with or without ER expression Similar to the effect of T A M by itself, MPA alone inhibited growth of ER- HBEC cultures to 77.0 ± 4.8 % of control (p<0.001; Figure 17). MPA combined with either concentration of Adr produced greater growth inhibition compared with MPA alone (p<0.001). Of greater importance is the observation that MPA enhanced Adr cytotoxicity when 0.5 ug/ml Adr was combined with MPA (29.1 ± 3.9% of control; p<0.005). ER+ primary cultures of HBEC were not inhibited by MPA alone. Although combinations of MPA with Adr resulted in decreased cell growth with respect to MPA alone (p<0.005), Adr cytotoxicity was not enhanced. 68 Figure 17. Effects of R M on Adr cytotoxicity in ER+ (n=3) and ER- (n=18) primary cultures of HBEC. In each panel, unshaded bars - no RM, shaded bars - RM. Each bar displays the mean of growth of HBEC in primary culture as a percent of control (no Adr and no RM) +/- SEM. To distinguish between statistically relevant data, bars are labelled as: a = significant inhibition of growth by R M alone; b = significant inhibition of growth by Adr alone ; c = additive effect of R M and Adr; or d = synergistic effect of R M and Adr. Experiments were replicated 4 times. 69 o u a o © a QJ S-PH 100 80 60 40 20 100 80 60 40 20 100 80 60 40 20 0 |A. CsA (4.3 iM) ER" HBEC b -=E-d |B. TAM (1.0 nM) a b b |C. MPA (1.3 |iM) 3 E -b •3E-d b b Eld" HBEC b a 2 •EE 0 0.5 5.0 0 0.5 Adr (jag/ml) b b h b b h 5.0 70 6. Effects of R M on Adr Cytotoxicity in ER- Primary Cultures of H B E C With or Without Pgp Expression 6a. Cytotoxic effects of Adr in ER- primary cultures of H B E C with or without Pgp expression Three of the 21 primary cultures of HBEC were ER+, 2 of which were Pgp- and one was Pgp+. These ER+ sample numbers were too small for statistical analyses. Of the ER- cultures, 13 of the 18 were Pgp- and 5 of the 18 were Pgp+. Table 6 shows data on the effects of the RM on Adr cytotoxicity in relation to Pgp expression in the ER-cultures. Adr at both 0.5 and 5.0 ug/ml was cytotoxic in both Pgp- and Pgp+ cultures (p<0.005), but Pgp+ cultures were significantly more resistant to the lower concentration of the chemotherapeutic agent (p<0.005). 6b. Effects of CsA on Adr cytotoxicity in ER- primary cultures of H B E C with or without Pgp expression CsA inhibited the growth of ER-, Pgp- cultures (p<0.001; Table 6). However, in combination with either 0.5 or 5.0 ug/ml Adr, the data show that only CsA had an effect on growth inhibition. ER-, Pgp+ HBEC cultures were also growth inhibited by CsA alone (p<0.001). CsA plus 0.5 ug/ml Adr produced a synergistic effect on inhibition of cell growth in these cultures (p<0.005; Table 7). 6c. Effects of T A M on Adr cytotoxicity in ER- primary cultures of H B E C with or without Pgp expression Cell growth in ER-, Pgp- cultures was inhibited by T A M alone (p<0.001; Table 6). However, Adr cytotoxicity was not enhanced by the combination of T A M plus either low or high Adr concentration. 71 T A M did not inhibit growth in cultures of ER-, Pgp+ HBEC. Although combination of T A M plus either 0.5 or 5.0 ug/ml Adr inhibited cell growth, they did not increase Adr cytotoxicity. 6d. Effects of MPA on Adr cytotoxicity in ER- primary cultures of HBEC with or without Pgp expression Primary cultures of ER-, Pgp- HBEC were growth inhibited to 70.0 + 7.3 % of control when exposed to MPA alone (p<0.001; Table 6). When combined with 0.5 ug/ml Adr, MPA produced an additive effect on growth inhibition of HBEC (p<0.05; Table 7). However, MPA plus 5.0 ug/ml Adr did not increase Adr cytotoxicity compared to Adr alone. ER-, Pgp+ HBEC were not growth inhibited by MPA alone. Similar to TAM, MPA combined with either concentration of Adr inhibited HBEC growth. However, Adr cytotoxicity was not enhanced. 72 Table 6. Effects of RM on Adr cytotoxicity in Pgp+ and Pgp- primary cultures of ER-human breast epithelial cells [Adr] noRM CsA TAM MPA (Ug/ml) (4.3 nM) (1.0 nM) (1.3 M M ) Percent of Control Pgp- (11=13) 0 100 (± 4.0)1 15.9(± 2.8)2 65.8 (± 4.9)2 70.0 (± 7.3)2 0.5 32.8 (± 5.4)3 5.7 (+ 1.7)2 19.5 (+4.8)3 4, p<0.05 13.9 (±2.4) ' y 5.0 8.3 (± 2.0) 4.0 (+ 1.5)2 6.8 (± 1.8)3 7.3 (± 2.2)3 Pgp+ (n=5) 0 0.5 100 (± 5.0)1 66.5 (± 3.3)3 31.7 (± 3.0)2 4,p<0.005 9.5 (±4.4) * 85.2 (+6.3) 54.1 (+4.5)3 82.9 (+ 6.4) 41.7 (± 5.1)3 5.0 8.4 (± 1.2)3 1.7 (±0.4)3 4.3 (± 0.6)3 6.3 (± l . l ) 3 Control (no RM and no Adr) + SEM 2Significant inhibition of growth by RM alone (p<0.001) Significant inhibition of growth by Adr alone (p<0.005) Significant interaction between RM and Adr resulting in enhanced inhibition of growth Table 7. Normalized data of the effects of R M on Adr cytotoxicity in Pgp+ and Pgp-primary cultures of human breast epithelial cells without ER expression [Adr] noRM CsA TAM MPA (Ug/ml) (4.3 nM) (1.0 pM) (1.3 nM) Percent of Control Pgp- (n=13) 0 100 (± 4.0)1 100 (± 2.8)1 100 (± 4.9)1 100(+7.3)1 0.5 32.8 (± 5.4)2 35.8 (+ 1.7) 29.6 (+4.8)2 19.9 (+ 2.4)3 5.0 8.3 (± 2.0)2 25.2 (+ 1.5) 10.3 (+ 1.8)2 10.4 (± 2.2)2 Pgp+ (n=5) 0 100 (± 5.0)1 100 (+ 3.0)1 100(+6.3)1 100 (± 6.4)1 0.5 66.5 (+ 3.3)2 30.0 (±4.4)4 63.5 (+4.5)2 50.3 (+5.1)2 5.0 8.4 (± 1.2)2 5.4(±0.4)2 5.0 (± 0.6)2 7.6 (± l . l ) 2 1 Control ± SEM 2Significant inhibition of growth by Adr alone (p<0.005) 3 Additive effect of RM and Adr on inhibition of growth Synergistic effect of RM and Adr on inhibition of growth (p<0.005) 73 C H A P T E R 4 D I S C U S S I O N The major purpose of this thesis is 2-fold. The first objective was to determine if T A M and MPA as well as CsA may be used as resistance modifiers to enhance the cytotoxic effects of Adr in cells. The second objective was to determine if there was a positive correlation between RM activity of T A M and MPA and Pgp expression. TAM, an anti-estrogen, and MPA, a synthetic progestin, are used as single agents in the treatment of breast cancer. They are effective in increasing disease-free intervals in patients with ER+ tumors [Sedlacek and Horowitz, 1984; Gundersen et al, 1990]. In addition to binding to hormone receptors, T A M and MPA alter cell membrane fluidity [Bojar et al, 1984; Ramu et al, 1991] and bind to Pgp [Naito et al, 1989; Yang et al, 1989; Fine et al, 1993; Leonessa et al, 1994]. T A M also affects Pgp phosphorylation by PKC [O'Brian et al, 1986]. Therefore, these agents may be effective as resistance modifiers in MDR tumors that are Pgp+, which would be alternatives to the more toxic chemicals such as CsA that are currently being tested for this purpose. The concentrations of T A M and MPA used in this study are those used clinically in endocrine treatment of breast cancer. A recent Phase I clinical trial has utilized higher concentrations of T A M to sensitize resistant tumors to vinblastine. Although T A M was more effective at higher doses, a side effect is severe ataxia [Fine et al, 1993]. Therefore, the lower concentration of T A M was used. Adr was used in the present study as it is the most active single chemotherapeutic agent in the treatment of breast cancer but it is ineffective in MDR tumors, including those that demonstrate increased Pgp expression [Tormey, 1975; Salmon et al, 1989]. The significance of the experiments in this thesis is that they were done on HBEC in primary culture, which are more relevant to the clinical situation than are cell lines. This laboratory has previously shown that the serum-free culture conditions used select for and stimulate the growth of HBEC from both normal and malignant breast tissue samples, 74 whereas fibroblast growth is inhibited. The epithelial cells generated under these conditions have been identified by their cuboidal morphology, the presence of apical microvilli, tight junctions, desmosomes and the presence of keratin filaments [Emerman and Wilkinson, 1990]. Primary cultures of HBEC most likely consist of both normal and malignant cells. No attempt has been made to distinguish between them here. Few in vitro assays have been described that can easily and reproducibly distinguish between nonmalignant and malignant HBEC [Petersen et al, 1992; Bergstraesser et al, 1993]. However, this does not detract from the important finding that the cells in cultures which were positive for Pgp expression showed increased sensitivity to the cytotoxic effects of the lower concentration of Adr in the presence of CsA or MPA. Furthermore, it has been shown that responses of normal cells to Adr are similar to those of cells from tumor tissue from the same patient, suggesting that a patient's normal cells may be cultured to determine drug sensitivities of their malignant counterparts [33]. The MCF-7 cell line and its MDR subline MCF7-AdrR were used as controls due to their contrasting properties of sensitivity to Adr, Pgp expression and ER expression. Pgp expression was evident mainly in breast tumors that had previously been exposed to chemotherapy. Importantly, Pgp expression was positively correlated with resistance to Adr. Primary cultures of HBEC that were Pgp+ were more resistant to 0.5 ug/ml Adr than those that were Pgp-, although all primary cultures were sensitive to 5.0 ug/ml Adr. Immunocytochemistry demonstrated that there were fewer numbers of Pgp+ cells in primary cultures than in cultures of the Pgp+ MCF7-AdrR cell line. In some of the primary cultures, a light brown staining was observed and was classified as background staining in this thesis. However, the use of an irrelevant antibody could have better determined if the light brown coloration was actually an indication of cells expressing lower levels of Pgp+. Nonetheless, the fewer numbers of Pgp+ cells in primary culture may explain why the primary cultures were less resistant to Adr than the cell line. On the other hand, MCF7-AdrR cells also overexpress GST-p, TopoU and glutathione peroxidase 75 which would contribute to the overall resistance of the cell line to Adr along with Pgp [Sinha et al, 1989; Fairchild and Cowan, 1991]. The presence of these atypical MDR mechanisms which may be operative in the HBEC that we placed in primary culture is not known but may also contribute to their resistance to Adr. A variety of techniques have been used to examine the level of Pgp in breast cancer and how it correlates with drug resistance. Southern, northern and western blot analyses have identified mdrl gene amplification and/or overexpression [Merkel et al, 1988]. However, the proportion and types of cells involved cannot be determined using these electrophoretic methods. Immunocytochemistry and flow cytometry using anti-Pgp monoclonal antibodies are able to detect small clusters of MDR cells [Merkel et al, 1988; Schneider et al, 1989]. Furthermore, the use of immunocytochemistry allows for identification of cell types. CsA is known to enhance the cytotoxicity of MDR-related chemotherapeutic agents but it does have toxic side effects in vivo [Tamai and Safa, 1990; Twentyman and Wright, 1991; Erlichman et al, 1993]. The effects of CsA as a resistance modifier have been studied in cell lines. However, in addition to increasing chemotherapeutic drug accumulation, CsA toxicity has been demonstrated in a Pgp+ Chinese hamster lung cell line [Tamai and Safa, 1990; Twentyman and Wright, 1991]. The purpose of its use in this study was to determine its cytotoxic effects on cells with and without Adr in primary cultures of HBEC, which are more relevant than cell lines to the in vivo situation. In addition, it was of interest to compare the effects of TAM and MPA as resistance modifiers with those of a known resistance modifier. We have shown that CsA itself significantly inhibited cell growth in all cultures, and in some cases, was more cytotoxic than Adr. Despite its toxicity, however, CsA significantly enhanced the cytotoxicity of 0.5 ug/ml Adr in Pgp+ primary cultures of HBEC. It has been reported that a low dose of CsA (0.086 uM) is sufficient to sensitize a Pgp-expressing mouse mammary carcinosarcoma cell line to Adr [Twentyman and Wright, 1991]. However, in our 76 experiments, increased sensitivity of the MCF7-AdrR cell line to this anthracycline was not observed at concentrations less than 4.3 uM CsA. T A M is known to affect cell growth by several mechanisms, which may affect chemo-hormonal regimens [Legha, 1988; Thomas and Monet, 1992]. One mechanism of action of TAM, an anti-estrogen, is competing with estrogen for binding to ER [Legha, 1988; Thomas and Monet, 1992]. To minimize growth inhibition by T A M via ER-related mechanisms, cells were cultured in phenol red-free medium containing dextran charcoal-treated serum or serum-free medium to minimize any estrogen-related growth stimulation [Devleeschouwer et al, 1992]. The data demonstrate that this was accomplished: T A M alone inhibited the ER+, Pgp- MCF-7 cell line and the ER-, Pgp+ MCF7-AdrR cell line to the same degree. This has been reported previously for T A M as well as for other anti-estrogens, suggesting the inhibition of growth by ER-independent mechanisms [Coradini et al, 1994]. When Adr was added to medium containing TAM, the cytotoxic effect of the higher concentration of Adr was enhanced only in MCF7-AdrR cells. Increasing the efficacy of Adr was positively correlated with Pgp expression in the drug-resistant cell line. Interestingly, T A M alone significantly inhibited growth of ER- but not ER+ primary cultures of HBEC. The reason for this observation is not known, but the data suggest that T A M is inhibiting growth by ER-independent mechanisms in these cultures as well as in the cell lines. In any event, ER status did not affect the role of T A M as a resistance modifier. As indicated in Table 6, TAM was not able to enhance Adr cytotoxicity. Chemosensitization of MDR cells by TAM has been studied previously in cell lines. T A M not only binds to Pgp [Fine et al, 1993; Leonessa et al, 1994], but also affects phosphorylation of Pgp by PKC [O'Brian et al, 1986], which activates the drug efflux pump [Chambers et al, 1993]. In addition, T A M affects plasma membrane fluidity, which subsequently alters drug accumulation within a cell [Ramu et al, 1991]. Therefore, T A M may be effective in modifying MDR by inhibiting Pgp function both directly and indirectly. Kirk et al have studied the effects of TAM and its metabolites in the modification of Adr 77 toxicity [Kirk et al, 1993]. A range of 1-20 uM T A M progressively shifts the Adr dose-response curve for the MCF7-AdrR cell line to the left. Other studies showed that T A M at 1 uM interacts synergistically with Adr and vinblastine in MCF7-AdrR and CL 10.3 transduced cells expressing Pgp and that this effect is mediated by Pgp [Leonessa et al, 1994]. However, higher concentrations of T A M (2.5-5 uM) results in non-significant interactions with the two MDR-related agents. We tested the ability of 1 uM T A M to enhance drug toxicity since this concentration is achievable in vivo with minimal side effects [Legha, 1988]. Consistent with these previous studies, we observed increased Adr toxicity in the presence of T A M in MCF7-AdrR cells. However, this result was not observed in primary cultures containing Pgp+ HBEC. The ability of T A M to interfere with MDR has also been studied in vivo [Trump et al, 1992; Fine et al, 1993]. A clinical study involving 53 patients with advanced refractory cancer suggests that T A M at high doses (4 uM) can be safely administered in the clinical setting. Although this high dose of T A M resulted in ataxia, it is reversible by terminating treatment. Only 3 patients showed a partial response to treatment with high-dose T A M plus vinblastine, although they did not measure Pgp expression in the patients' tumors. Our data show that 1 uM T A M did not enhance Adr cytotoxicity in primary cultures of HBEC. MPA may affect cell growth by one of several mechanisms [Shaikh et al, 1989; Classen et al, 1993]. Presumeably, its major mechanism of action is via interaction with progesterone receptors, which subsequently reduces ER content [Classen et al, 1993]. In this study, MPA inhibited the growth of primary cultures that contained ER- cells, whereas cultures that contained both ER- and ER+ cells were not significantly affected. Similar results were observed with TAM. These observations plus the fact that there was neither estrogen nor progesterone in the medium suggest that MPA was acting through an ER-independent mechanism. 78 M P A was used in this study since, in addition to T A M , progestins have been shown to be substrates for Pgp [Naito et al, 1989; Yang et al, 1989]. Naito et al demonstrated that photoaffinity labelling of Pgp can be inhibited by progestins, androgens, estrogens and glucocorticoids with progesterone having the highest binding affinity [Naito et al, 1989]. Furthermore, progesterone has the highest binding affinity for Pgp out of all of the steroid hormones tested. Whether or not MPA also interacts with a hormone receptor is not likely relevant here since studies have not yet indicated that hormone receptor content is related to Pgp expression [Kacinski et al, 1989; McGuire, 1990]. In fact, Ishida and co-workers (1994) demonstrated that MPA enhances the cytotoxicity of Adr in a synergistic manner in MCF7-AdrR cells as well as in the human nasopharyngeal carcinoma, KB, and its Adr-resistant cell line, KB-A1, all of which are negative for steroid receptors. Shaikh et al (1989) observed significant synergism between MPA and Adr in a MCF-7 cell line resistant to the growth-inhibitory effect of the progestin. It may be of greater importance that MPA affects membrane fluidity, which has been shown to affect drug uptake by MCF-7 cells [Bojar et al, 1984]. In our study, M P A did not enhance Adr cytotoxicity in MCF7-Adr cells, however, it did so in primary cultures of HBEC. Furthermore, this increased sensitivity to Adr was correlated with positive Pgp expression. In conclusion, our data indicate that expression of Pgp correlated with resistance of primary cultures of HBEC to Adr. 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Kluwer Academic Publishers, Boston, pp. 1-12. 9 0 APPENDIX 1: Transport Medium DME:F12 -(1:1) hEPES BUFFER - 10 mM Calf serum - 5% Insulin - 5 ug/ml APPENDIX 2: Freezing Medium DME - 50% Calf serum -44% Dimethylsulfoxide - 6% APPENDIX 3: Dissociation Medium DME:F12 -(1:1) Hepes buffer -10 mM Fetal calf serum - 2% Insulin - 5 ug/ml Collagenase - 300 U/ml Hyaluronidase -100 U/ml Appendix 4: Attachment Medium for Human Breast Epithelial Cells (HBEC) in Primary Culture DME:F12 -(1:1) Hepes buffer - lOmM Pooled, dextran charcoal-treated normal human serum - 5% Insulin - 5 ug/ml Appendix 5: Preparation of Rat Tail Collagen To prepare the collagen solution, rat tails were placed in 95% ethanol for 15 min. The tendons were dissected out, weighed and immersed in sterile deionized water under an ultraviolet light in a laminar flow hood for 24 h. The fibers were then bathed in a solution of 0.01 N acetic acid and stirred for 48 h at 4°C. For another 24 h, they remained in the acetic acid solution without agitation. The solution was transferred into 50 ml centrifuge tumbes and spun in a Sorvall ultracentrifuge for 30 min at 10,000 x g. The supernatant consisted of the collagen solution and was bottled and stored at 4°C. 91 Appendix 6: Phenol red-free, Serum-free Medium Phenol red-free DME/F12 -(1:1) Hepes buffer - lOmM NaHC0 3 - 14 mM Bovine serum albumin - 5 mg/ml Cholera toxin - 10 ng/ml Hydrocortisone - 0.5 ug/ml Insulin - 10.0 ug/ml Appendix 7 : Growth Medium for the MCF-7 Cell Line DME/F12 -(1:1) Hepes buffer - 10 mM NaHC0 3 -14 mM Dextran charcoal-treated fetal calf serum - 5% Appendix 8: Growth Medium for the M C F 7 - A d r R Cell Line Phenol red-free DME7F12 -(1:1) Hepes buffer - 10 mM NaHC03 - 14 mM Dextran charcoal-treated fetal calf serum - 5% Adriamycin - 5 ug/ml Appendix 9 : Preparation of Saline-Trypsin-Versene NaHC03 - 0.35 g NaCl - 8.0 g KC1 - 0.4 g Glucose - 0.35 g EDTA - 0.25 g Trypsin (Gibco) - 0.5 g Distilled water - 1000 ml The first 5 ingredients were weighed out and placed in a large Erlenmeyer flask. The flask was then filled with 950 ml of distilled water and stirred to dissolve the ingredients. Trypsin was then added and stirred until dissolved. Using either NaOH or Hcl, the pH of the solution was adjusted to 7.4 and the remaining distilled water was added. The solution was filter sterilized under aeseptic conditions using a Millipore perstaltic pump. The 92 sterilized solution was then stored as 10 ml aliquots in 17 x 100 mm propylene plastic test tubes in a -20°C freezer. Appendix 10; Preparation of Phosphate-Buffered Saline NaCl K 2 H P 0 4 K H 2 P 0 4 Distilled water -8 .5g - 1-43 g - 0.25 g - 1000 ml The ingredients were added to 900 ml distilled water. The pH was adjusted to 7.4 with either NaOH or Hcl. Then the remaining distilled water was added to make a final volume of 1000 ml. The solution was then stored at 4°C. Appendix 11: Preparation of Specimen Storage Medium for Primary Cultures of H B E C on Coverslips Sucrose - 42.8 g MgCl (Hexahydrate) - 0.70 g Glycerol - 250 ml Phosphate-buffered saline (PBS) - 250 ml The first two ingredients were dissolved in 200 ml PBS. The final volume was adjusted to 250 ml with the remaining PBS. The glycerol was then added and stirred until well mixed. The medium was then stored between -10°C to -20°C. Appendix 12: Preparation of Pooled Normal Human Serum Serum samples were collected in the mornings from patients who had fasted over the previous 8-12 h. Blood was received in non-heparininzed tubes, incubated for 30 min at 37°C and then centrifuged at 100 x g and the serum collected with a sterile pasteur pipette. Pooled serum was stored at -20°C. 93 

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