ABSTRACT THE EFFECTS OF HORMONES ON THE GROWTH OF CARCINOGEN-INDUCED RAT MAMMARY CARCINOMA lN_VITRO By Gloria M. Iturri The effects of hormones on the growth of 7,12-dimethylbenz-(a)- anthracene (DMBA)-induced rat mammary carcinoma were studied in_yitrg, The primary hormones under investigation were ovine prolactin (5.0 pg per ml), estradiol-l7B (0.0l pg per ml), ovine and human growth hormone (5.0 ug per ml), while secondary interest was given to the effects of insulin (5.0 pg per ml) and corticosterone (1.0 pg per ml). Cell growth was evaluated by l) determining DNA synthesis (H3- thymidine incorporation in counts per minute per ug DNA) of explants (at l20 hours of culture) and 2) growing collagenase-dispersed cells and determining changes in cell number (at 24 and 48 hours of culture) and colony number and area (at 120 hours of culture). The medium used for organ and cell culture was Medium l99, containing either 50 I.U. per ml of penicillin G (organ culture) or l.0 ml of antibiotic-anti- mycotic mixture (cell culture). Cultures were continuously gassed (95% 0 :5% C02). 2 The major findings of the study were as follows. Gloria M. Iturri A. Prolactin l. Prolactin, in the presence of insulin and corticosterone, when compared to cultures containing only insulin and cortico- sterone, consistently enhanced DNA synthesis in explants of rat mammary carcinoma (120 hour culture) obtained from either intact or ovariectomized rats. Criteria for DNA synthesis were H3- thymidine incorporation into DNA as well as autoradiographic analysis. 2. Prolactin, in the presence of insulin and corticosterone, also increased the cell growth of dispersed cells at 24, 48, and l20 hours of culture. 3. Prolactin, in the presence of corticosterone alone initially (at 24 and 48 hours of culture) had a slight stimulatory effect on cellular growth. At l20 hours of culture, however, this hor- monal combination had a greater stimulatory effect on cell growth than did corticosterone alone. 4. When corticosterone was in the media, there was no significant difference between prolactin and insulin, upon cellular growth at 24 and 48 hours of culture. At 120 hours of culture, prolactin was slightly superior to insulin in stimulating cell growth. 5. Prolactin alone was slightly superior to insulin alone (media lacking corticosterone) in stimulating cell growth after l20 hours of culture. Gloria M. Iturri 6. Prolactin plus insulin, in the presence of corticosterone, significantly increased cell growth at 24, 48 and l20 hours of culture, in comparison to cultures containing only corticosterone. 7. Prolactin plus corticosterone, in the presence of insulin, had a greater stimulatory effect on cell growth than did insulin alone after five days of culture. 8. The triad of hormones, when compared with either prolactin plus corticosterone, or insulin plus corticosterone, was consider- ably more effective stimulating cell growth after 24, 48 and l20 hours of culture. 9. The triad of hormones was consistently superior to media lack- ing hormones at 24, 48 and 120 hours of culture. 8. Estrogen l. Estradiol-l78 failed to stimulate or reactivate DNA synthesis of explants obtained from ovariectomized rats after l20 hours of culture (media containing insulin and corticosterone). 2. Estrogen plus prolactin was as effective as prolactin alone in stimulating DNA synthesis of explants from ovariectomized rats after l20 hours of culture (media containing insulin and cortico- sterone). C. Growth Hormone l. Either ovine or human growth hormone alone slightly stimulated DNA synthesis after 120 hours of culture (media containing insulin and corticosterone). Gloria M. Iturri 2. Human growth hormone and prolactin combined, when compared with human growth hormone alone, markedly enhanced DNA synthesis after l20 hours of culture (media containing insulin and cortico- sterone). This hormonal combination was as effective as prolactin alone in stimulating DNA synthesis. D. Insulin l. Insulin plus corticosterone, when compared with corticoster- one alone, had either no effect (at 24 hours of culture), or a slight stimulatory effect (at 48 and 120 hours of culture) on cell growth. 2. Insulin, in the presence of prolactin and corticosterone when compared with prolactin plus corticosterone alone, was consistently superior in enhancing cellular growth (at 24, 48 and l20 hours of culture) and DNA synthesis (at l20 hours of culture). E. Corticosterone l. Corticosterone alone initially (at 24 and 48 hours of culture) stimulated cell growth and eventually (at l20 hours of culture) inhibited cell growth. 2. Either corticosterone plus prolactin or corticosterone plus insulin, when compared with cultures lacking hormones, had a greater stimulatory effect on cell growth after 24 and 48 hours of culture. At 120 hours of culture, these hormonal combinations had no appar- ent effect (perhaps some slight inhibition) on cell growth. Gloria M. Iturri 3. Corticosterone plus insulin, in the presence of prolactin, had a greater stimulatory effect on cell growth than did prolactin alone after l20 hours of culture. Rm 1. Fetal calf serum, either in cultures lacking or containing hormones (triad of hormones), consistently stimulated cell growth at 24, 48 and 120 hours of culture. 2. Fetal calf serum plus the triad of hormones markedly stimu- lated cell growth when compared with either serum alone or the triad of hormones alone at 24, 48 and 120 hours of culture. THE EFFECTS OF HORMONES ON THE GROWTH OF CARCINOGEN-INDUCEO RAT MAMMARY CARCINOMA IN VITRO By by Gloria M: Iturri A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Anatomy I975 Dedicated to my husband, Sergio, whose moral support, understanding, and his unfailing belief in his wife made this work possible. Dedicated also to my parents and my family whose encouragement made this work a much easier one. ii ACKNOWLEDGEMENTS I wish to express my heart—felt appreciation to my academic advisor, Dr. Clifford W. Welsch. His intellectual enthusiasm, demands for excellence, steadfast in my abilities have enriched my life greatly. His sensitive appreciation for life has made me appreciate him both as a professor and as a friend. His encouragement and his many kindnesses over the last past year will always be remembered. His personal drive and dedication have instilled in me a desire to pursue a career in cancer research. It is an honor to say with pride, "I was Dr. Welsch's student." I would also like to extend my sincere gratitude to the members of my committee, Dr. W. D. Collings, Dr. Robert Echt, Dr. Vance Sanger and Dr. Al W. Stinson for their constructive critical review of my manu- script, and their suggestions for its improvement. Their interest and encouragement during my doctoral program were very meaningful. My special thanks are given to Dr. W. D. Collings, my academic advisor, and Dr. Herbert Cox, my major professor during my master's program who provided me with the courage and determination to pursue my graduate studies. It is with sincere appreciation that I also wish to thank Dr. John Gill for his advise in the statistical analysis of the results. Acknowledgement is also due to the secretaries of the Department of Anatomy, Mrs. Diane Elo and Miss Diane Wardwell for their patience in the typing of the draft of my manuscript. I am also indebted to the National Institute of Health (Research Grant No. CA-l3777) and the American Cancer Society (Research Grant No. ET-59) for the support they provided me to do this work. The financial aid I received in the form of a research assistantship provided by the National Institute of Health (Research Grant No. CA-l3777) and a teaching assistantship provided by the Department of Anatomy were indispensable. Lastly, but not the least, I wish to express my gratitude to my husband, Sergio, for his enduring patience, encouragement and generous understanding throughout my graduate studies. TABLE OF CONTENTS INTRODUCTION .......................... REVIEW OF LITERATURE ...................... I. The Role of Hormones in Mammary Tumorigenesis in Mice: In_Vivo Studies. . . . ................ II. The Role of Hormones in Mammary Tumorigenesis in Rats: .In Vivo Studies .................... III. The Role of Hormones in Mammary Tumorigenesis in Humans: In Vivo Studies ............... IV. The Influences of Hormones on Mouse, Rat and Human Mammary Tumors in Culture: In_Vitro Studies . . . A. Mouse Mammary Tumors In_Vitro ........... B. Rat Mammary Tumors In_Vitro. . . ......... C. Human Mammary Tumors In_Vitro ........... MATERIALS AND METHODS ..................... 1. Organ Culture ..................... A. Preparation of Mammary Tumors for Organ Culture. . B. DNA Extraction and Analysis of Cultured Mammary Tumor Explants .................. C. Autoradiographic Analysis of Cultured Mammary Tumor Explants .................. D. Experimental Design ................ II. Cell Culture ..................... A. Preparation of Cell Suspensions from Mammary Tumors ...................... iv 12 l6 l6 I9 21 24 25 25 26 28 29 3T 3T TABLE OF CONTENTS-—continued B. Seeding of Cell Suspensions. Analysis of Growth by Measuring Number and Area of Colonies of Cells ...................... I. Experimental Design ............ Seeding of Cell-Suspensions. Analysis of Growth by Determining Cell Number ........ 1. Experimental Design ............. RESULTS ............................ I. II. Organ Culture . . . . . . . . ............ A. DNA Synthesis in DMBA-Induced Rat Mammary Carci— noma at 12. 24, 48. and 96 Hours of Organ Culture Effect of Prolactin and Insulin on DNA Synthesis in 5-Day Organ Cultures of DMBA—Induced Rat Mammary Carcinoma ................ Effect of Prolactin and Estrogen on DNA Synthesis in 5-Day Organ Cultures of DMBA-Induced Rat Mammary Carcinoma of Ovariectomized Rats ..... Effect of Prolactin and Ovine Growth Hormone on DNA Synthesis in 5-Day Organ Cultures of DMBA- Induced Rat Mammary Carcinoma .......... Effect of Prolactin and Human Growth Hormone, Alone and in Combination on DNA Synthesis in 5-Day Organ Cultures of DMBA-Induced Rat Mammary Carcinoma .................... Cell Culture ..................... A. Analysis of Growth by Measurement of Colony Number and Area ................. l. Effect of Combination of Insulin, Prolactin, and Corticosterone on Growth of Collagenase— Dispersed Cells of DMBA-Induced Rat Mammary Carcinoma after 2 Days of Culture ...... Page 32 34 36 38 4o 40 40 46 52 52 58 75 75 75 TABLE OF CONTENTS--continued 2. Effect of Combination of Insulin, Prolactin, and Corticosterone on Growth of Collagenase-Dispersed Cells of DMBA-Induced Rat Mammary Carcinoma after 5 Days of Culture ................ Effect of Combination of Insulin, Prolactin, and Corticosterone on Growth of Collagenase Dispersed Cells of DMBA-Induced Rat Mammary Carcinoma after 5 Days of Culture. Comparison with Cultures Containing Insulin Alone ............. Effect of Combination of Insulin, Prolactin, and Corticosterone on Growth of Collagenase Dispersed Cells of DMBA-Induced Rat Mammary Carcinoma after 5 Days of Culture. Comparison with Cultures Containing Insulin and Prolactin Alone. . . . Effect of Combination of Insulin, Prolactin, and Corticosterone on Growth of Collagenase Dispersed Cells of DMBA-Induced Rat Mammary Carcinoma after 5 Days of Culture ............... Effect of Fetal Calf Serum on Growth of Colla- genase Dispersed Cells of DMBA-Induced Rat Mammary Carcinoma after 5 Days of Culture . . . . 8. Analysis of Growth by Determining Number of Cells . . I. Effect of Insulin, Prolactin and Corticosterone on Growth of Collagenase Dispersed Cells of DMBA- Induced Rat Mammary Carcinoma ......... . a. After 24 Hours of Culture. Average Number of Cells per Well (Microplates) ......... b. After 24 Hours of Culture. Average Number of Cells per Petri Dish ............. c. After 48 Hours of Culture. Average Number of Cells per Petri Dish ............ d. After 24 and 48 Hours of Culture. Average Number of Unattached Cells per Petri Dish . . Effect of Fetal Calf Serum on Growth of Colla- genase-Dispersed Cells of DMBA-Induced Rat Mammary Carcinoma after 24 and 48 Hours of Culture ..................... vi Page 75 83 83 89 96 TDD 100 TDD 100 105 107 109 TABLE OF CONTENTS--continued Page DISCUSSION ......................... . . 112 SUMMARY ............................ 145 APPENDICES ........................... 151 I. DNA Synthesis in DMBA-Induced Rat Mammary Carcinoma at 12, 24, 48 and 96 Hours of Organ Culture ....... 151 II. Effect of Prolactin and Insulin on DNA Synthesis in 5—Day Organ Cultures of DMBA-Induced Rat Mammary Carcinoma ....................... l52 III. Effect of Prolactin and Estrogen on DNA Synthesis in 5-Day Organ Cultures on DMBA-Induced Mammary Carcinoma of Ovariectomized Rats ........... 153 IV. Effect of Prolactin and Ovine Growth Hormone on DNA Synthesis in 5-Day Organ Cultures of DMBA-Induced Rat Mammary Carcinoma ................... l54 V. Effect of Prolactin and Human Growth Hormone,Alone and in Combination,on DNA Synthesis in 5-Day Organ Cul- tures of DMBA-Induced Rat Mammary Carcinoma ...... l55 VI. Effect of Prolactin and Human Growth Hormone,Alone and in Combination,on DNA Synthesis in 5-Day Organ Cul- tures of DMBA-Induced Rat Mammary Carcinoma ...... l56 VII. Effect of Combination of Insulin, Prolactin and Corticosterone on Growth of Collagenase Dispersed Cells of DMBA-Induced Rat Mammary Carcinoma after Two Days of Culture .................... 158 VIII. Effect of Combination of Insulin, Prolactin and Corticosterone on Growth of Collagenase Dispersed Cells of DMBA-Induced Rat Mammary Carcinoma after Five Days of Culture .................... l59 IX. Effect of Combination of Insulin, Prolactin and Corticosterone on Growth of Collagenase Dispersed Cells of DMBA-Induced Rat Mammary Carcinoma after Five Days of Culture. Comparison with Cultures Containing Insulin Alone ..................... T60 X. Effect of Combination of Insulin, Prolactin and Corticosterone on Growth of Collagenase Dispersed Cells of DMBA-Induced Rat Mammary Carcinoma after Five Days of Culture. Comparison with Cultures Containing Insulin and Prolactin Alone .............. l6l vii TABLE OF CONTENTS--continued XI. XII. XIII. XIV. XV. XVI. XVII. Effect of Combination of Insulin, Prolactin and Corticosterone on Growth of Collagenase Dispersed Cells of DMBA-Induced Rat Mammary Carcinoma after Five Days of Culture ................. Effect of Fetal Calf Serum on Growth of Collagenase Dispersed Cells of DMBA-Induced Rat Mammary Carcinoma after Five Days of Culture .............. Effect of Insulin, Prolactin and Corticosterone on Growth of Collagenase Dispersed Cells of DMBA-Induced Rat Mammary Carcinoma after 24 Hours of Culture . . . Effect of Insulin, Prolactin and Corticosterone on Growth of Collagenase Dispersed Cells of DMBA-Induced Rat Mammary Carcinoma after 24 Hours of Culture . . . Effect of Insulin, Prolactin and Corticosterone on Growth of Collagenase Dispersed Cells of DMBA-Induced Rat Mammary Carcinoma after 48 Hours of Culture . . . Effect of Insulin, Prolactin and Corticosterone on Growth of Collagenase Dispersed Cells of DMBA-Induced Rat Mammary Carcinoma after 24 and 48 Hours of Culture ..................... Effect of Fetal Calf Serum on Growth of Collagenase Dispersed Cells of DMBA-Induced Rat Mammary Carcinoma after 24 and 48 Hours of Culture .......... BIBLIOGRAPHY ...................... viii Page 162 163 164 165 166 167 168 169 TABLE 10. 11. LIST OF TABLES DNA Synthesis in DMBA-Induced Rat Mammary Carcinoma at 12, 24, 48, and 96 Hours of Culture. Experiment #1. . DNA Synthesis in DMBA-Induced Rat Mammary Carcinoma at 12, 24, 48, and 96 Hours of Culture. Experiment #2. . DNA Synthesis in DMBA-Induced Rat Mammary Carcinoma at 12, 24, 48, and 96 Hours of Culture. Experiment #3. . Effect of Prolactin and Insulin on DNA Synthesis in 5-Day Organ Cultures of DMBA-Induced Rat Mammary Carcinoma. Experiment #1 ............... Effect of Prolactin and Insulin on DNA Synthesis in 5-Day Organ Cultures of DMBA-Induced Rat Mammary Carcinoma. Experiment #2 ............... Effect of Prolactin and Insulin on DNA Synthesis in 5-Day Organ Cultures of DMBA-Induced Rat Mammary Carcinoma. Experiment #3 ............... Effect of Prolactin and Estrogen on DNA Synthesis in 5-Day Organ Cultures of DMBA—Induced Mammary Carcinoma of Ovariectomized Rat. Experiment #1 ........ Effect of Prolactin and Estrogen on DNA Synthesis in 5-Day Organ Cultures of DMBA-Induced Mammary Carcinoma of Ovariectomized Rat. Experiment #3 ...... Effect of Prolactin and Estrogen on DNA Synthesis in 5-Day Organ Culture of DMBA-Induced Mammary Carcinoma of Ovariectomized Rat. Experiment #3 ......... Effect of Prolactin and Ovine Growth Hormone on DNA Synthesis in 5-Day Organ Cultures of DMBA-Induced Rat Mammary Carcinoma. Experiment #1 ........... Effect of Prolactin and Ovine Growth Hormone on DNA Synthesis in 5-Day Organ Cultures of DMBA-Induced Rat Mammary Carcinoma. Experiment #2 ........... ix Page 41 42 43 47 48 49 53 54 55 59 6O LIST OF TABLES—«continued 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. Effect of Prolactin and Ovine Growth Hormone on DNA Synthesis in 5-Day Organ Cultures of DMBA-Induced Rat Mammary Carcinoma. Experiment #3 ............ Effect of Prolactin and Ovine Growth Hormone on DNA Synthesis in 5—Day Organ Cultures of DMBA-Induced Rat Mammary Carcinoma. Experiment #4 ........... Effect of Prolactin and Human Growth Hormone Alone, and in Combination, on DNA Synthesis in 5-Day Organ Cultures of DMBA-Induced Rat Mammary Carcinoma. Experiment #1. . Effect of Prolactin and Human Growth Hormone Alone, and in Combination, on DNA Synthesis in 5-Day Organ Cultures of DMBA-Induced Rat Mammary Carcinoma. Experiment #2. . Effect of Prolactin and Human Growth Hormone Alone, and in Combination, on DNA Synthesis in 5-Day Organ Cultures of DMBA-Induced Rat Mammary Carcinoma. Experiment #3. . Effect of Combination of Insulin, Prolactin, and Corticosterone on Growth of Collagenase Dispersed Cells of DMBA—Induced Rat Mammary Carcinoma After 2 Days of Culture. Experiment #1 ................. Effect of Combination of Insulin, Prolactin, and Corticosterone on Growth of Collagenase DiSpersed Cells of DMBA-Induced Rat Mammary Carcinoma After 2 Days of Culture. Experiment #2 ................. Effect of Combination of Insulin, Prolactin, and Corticosterone on Growth of Collagenase Dispersed Cells of DMBA-Induced Rat Mammary Carcinoma After 5 Days of Culture. Experiment #1 ................. Effect of Combination of Insulin, Prolactin, and Corticosterone on Growth of Collagenase Dispersed Cells of DMBA-Induced Rat Mammary Carcinoma After 5 Days of Culture. Experiment #2 ................. Effect of Combination of Insulin, Prolactin, and Corticosterone on Growth of Collagenase Dispersed Cells of DMBA-Induced Rat Mammary Carcinoma After 5 Days of Culture. Experiment #3 ................. Effect of Combination of Insulin, Prolactin, and Corticosterone on Growth of Collagenase Dispersed Cells of DMBA-Induced Rat Mammary Carcinoma After 5 Days of Culture. Comparison with Cultures Containing Insulin Alone. Experiment #1 .................. X Page 61 62 65 66 67 78 79 8O 81 82 84 LIST OF TABLES-~continued 23. 24. 25. 26. 27. 28. 29. 30. 31. Effect of Combination of Insulin, Prolactin, and Corticosterone on Growth of Collagenase Dispersed Cells of DMBA-Induced Rat Mammary Carcinoma After 5 Days of Culture. Comparison with Cultures Containing Insulin Alone. Experiment #2 ................ Effect of Combination of Insulin, Prolactin, and Corticosterone on Growth of Collagenase Dispersed Cells of DMBA-Induced Rat Mammary Carcinoma After 5 Days of Culture. Comparison with Cultures Containing Insulin and Prolactin Alone. Experiment #1 ........... Effect of Combination of Insulin, Prolactin, and Corticosterone on Growth of Collagenase Dispersed Cells of DMBA-Induced Rat Mammary Carcinoma After 5 Days of Culture. Comparison with Cultures Containing Insulin and Prolactin Alone. Experiment #2 .......... Effect of Combination of Insulin, Prolactin, and Corticosterone on Growth of Collagenase Dispersed Cells of DMBA-Induced Rat Mammary Carcinoma After 5 Days of Culture. Comparison with Cultures Containing Insulin and Prolactin Alone. Experiment #3 ........... Effect of Combination of Insulin, Prolactin and Corticosterone on Growth of Collagenase Dispersed Cells of DMBA-Induced Rat Mammary Carcinoma After 5 Days of Culture. Experiment #1 ................. Effect of Combination of Insulin, Prolactin, and Corticosterone on Growth of Collagenase Dispersed Cells of DMBA-Induced Rat Mammary Carcinoma After 5 Days of Culture. Experiment #2 ................. Effect of Combination of Insulin, Prolactin, and Corticosterone on Growth of Collagenase Dispersed Cells of DMBA-Induced Rat Mammary Carcinoma After 5 Days of Culture. Experiment #3. ............... Effect of Fetal Calf Serum on Growth of Collagenase Dispersed Cells of DMBA-Induced Rat Mammary Carcinoma After 5 Days of Culture ............. Effect of Insulin, Prolactin and Corticosterone on Growth of Collagenase Dispersed Cells of DMBA-Induced Rat Mammary Carcinoma After 24 Hours of Culture. (Average Number of Cells Per Well) ........ xi Page 85 86 87 88 9O 91 92 99 101 LIST OF TABLES--continued Page 32. Effect of Insulin, Prolactin and Corticosterone on Growth of Collagenase Dispersed Cells of DMBA—Induced Rat Mammary Carcinoma After 24 Hours of Culture. (Average Number of Cells per Petri Dish) ........ 102 33. Effect of Insulin, Prolactin and Corticosterone on Growth of Collagenase Dispersed Cells of DMBA-Induced Rat Mammary Carcinoma After 48 Hours of Culture ..... 106 34. Effect of Insulin, Prolactin and Corticosterone on Growth of Collagenase Dispersed Cells of DMBA-Induced Rat Mammary Carcinoma After 24 and 48 Hours of Culture . 108 35. Effect of Fetal Calf Serum on Growth of Collagenase Dispersed Cells of DMBA—Induced Rat Mammary Carcinoma After 24 and 48 Hours of Culture ............ 110 xii FIGURE 1. 10(A-E). LIST OF FIGURES DNA synthesis in DMBA-induced rat mammary carcinoma at 12, 24, 48 and 96 hours of culture ........... Effect of prolactin (P) and insulin (1) on DNA synthe- sis in 5-day organ cultures of DMBA-induced rat mammary carcinoma ..................... . . Effect of prolactin (P) and estrogen (E) on DNA synthe- sis in 5-day organ cultures of DMBA-induced mammary carcinoma of ovariectomized rats ............ Effect of prolactin (P) and ovine growth hormone (GH) on DNA synthesis in S-day organ cultures of DMBA- induced rat mammary carcinoma ............. Effect of prolactin (P) and human growth hormone (HGH), alone and in combination, on DNA synthesis in 5-day organ cultures of DMBA-induced rat mammary carcinoma ..................... Effect of prolactin (P) and human growth hormone (HGH), alone and in combination, on DNA synthesis and labeling index in 5-day organ cultures of DMBA-induced rat mammary carcinoma . . .............. Representative explants of mammary tumors obtained from cultures containing insulin, corticosterone and prolactin ....................... Autoradiographs focused on (A) mammary tumor cells and (8) silver grains X 1080 ................ Effect of insulin (I), prolactin (P) and corticosterone (C) on growth of DMBA-induced rat mammary carcinoma . . Representative colonies of DMBA-induced rat mammary carcinoma cells obtained from cultures containing insulin, corticosterone and prolactin after 5 days of culture . . .................... . xiii Page 45 51 57 64 69 72 74 77 94 98 LIST OF FIGURES--continued Page 10(F). 11. Isolated cells (not colonies) of DMBA-induced rat mammary carcinoma obtained from cultures containing insulin, corticosterone and prolactin after 24 hours of culture (x 315) ................... 98 Effect of insulin (1), prolactin (P), and cortico- sterone (C) on growth of collagenase dispersed cells of DMBA-induced rat mammary carcinoma after 24 and 48 hours of culture .................... 104 xiv INTRODUCTION The 7, lZ-dimethylbenz-(a)-anthracene (DMBA)-induced rat mammary tumor system developed by Huggins (1965), like some human mammary cancers, is known to be hormone dependent. That is, the tumors respond to endocrine alterations in the host. It has been accepted for many years, that among the steroid hormones, the estrogens possess a special ability to influence growth and development of these neoplasms. However, relatively recent studies indicate that estrogen may act primarily on the pituitary stimulating prolactin secretion as well as influencing growth hormone secretion (Sterental gt_al,, 1963; Furth, 1961, 1968; and Welsch §t_al,, 1968). It has been suggested, therefore, that prolactin or growth hormone rather than estrogen may be the key hormones in mammary tumorigenesis (Kim and Furth, 1960a; Sterental t 1., 1963; Pearson gt_gl,, 1969; Welsch g_.al,, 1969, 1970; Nagasawa and Yanai, 1970; and Meites §t_al,, 1972). As a result of numerous investigations involving hypophysectomy and/or ovariectomy, and adrenalectomy of mammary tumor bearing rats and concurrent hormonal therapy, it is now fairly certain that the major influential mammary tumor growth-promoting hormones are prolactin, growth hormone and estrogen (Huggins gt_al,, 1959a; Kim and Furth, 1960b; Sterental _t_al., 1963; Dao, l964; Pearson gt_al,, 1969; Nagasawa and Yanai, 1970; and Welsch _e_t_g1_., 1973). The comparative mammary oncogenic activities of these hormones have not been resolved nor has it been determined whether they act primarily via a direct action on the tumor tissue or indirectly on the tissue, i.e., by influencing other endocrine parameters. With the relatively recent development of normal (Elias 1957, 1961; Prop, 1961; Barker et_a1:, 1964; Mishkinsky gt_al,, 1967; El-Darwish and Rivera, 1970) and neoplastic (Heuson §t_al,, 1967; Hollander, 1970; Welsch and Rivera, 1972) mammary organ culture tech- niques, the comparative and direct effects of these hormones on growth and development of mammary tissue can be evaluated. Thus, the purpose of this study was 1) to compare the direct effects of prolactin, growth hormone and estrogen on growth (DNA syn- thesis) of organ cultures of DMBA-induced rat.nmnnmry tumors and 2) to develop and determine the potential of using tumor cell disper- sions in lieu of organ culture as a technique for evaluating growth of DMBA-induced rat mammary tumors in_vitro. REVIEW OF LITERATURE I. The Role of Hormones in Mammary Tumorigenesis in Mice: In Vixo Studies Spontaneous mammary tumors in inbred mice may develop as a result of a number of different factors such as: genetic constitution, ade- quate hormonal stimulation and presence of mammary tumor virus (MTV) (Liebelt and Liebelt, 1967). Since 1913, several investigators have studied the hormonal factors related to spontaneous mammary tumorigenesis as reviewed by Shimkin (1945). In 1916, Lathrop and Loeb studied the effects of re- moving the ovaries. They found that ovariectomy at an early age decreased the incidence of mammary tumors in mice. However, ovariectomy had little or no effect when performed several months after birth (Lathrop and Loeb, 1916, Richardson, 1967). Mammary tumors could be in- duced in castrated males by ovarian grafts (Huseby and Bittner, 1951). When mice were ovariectomized and adrenalectomized, mammary tumors also rarely developed (Shimkin and Wyman, 1945). These studies suggest that ovarian hormonal stimulation is important in the induction of mammary tumors. Lacassagne (1932) was one of the first to indicate the prob- ability of correlation between estrogen and mammary tumors since injections of the steroid induced mammary tumors in R 111 strain of male mice. Subsequently, investigators found that estrogenic compounds induced mammary tumor formation in male mice (Burrows, 1935), and in- creased incidence of mammary tumors in male mice of high and low strains and in female mice (Shimkin and Andervont, 1942). The frequency of tumors appears to depend on the dose of hormones. Gardner (1941) found that increased doses of estrogen increased the incidence of tumors in mice until attainment of a level of toxicity which decreased both the incidence and growth of tumors. Other investi- gators have found that dosage levels of estrogen were not as important as duration of dosage (Burns and Schenken, 1940) and continuity of administration (MUhlbock and Boot, 1965). However, various strains responded differently to estrogen treatment indicating the possible presence of a hereditary factor in the induction of mouse mammary tumors (Suntzeff §£.Ql:: 1941; Bonser, 1944). Estrogen did not appear to in- duce mammary tumors in virus (MTV) free mice; however, in genetically susceptible animals, MTV accelerated and intensified tumor development after estrogen treatment (Mfihlbock, 1956). The role of progesterone is still questionable. Progesterone has been reported to have no effect (Burrow and Hoch-Ligeti, 1946), to decrease (Heiman, 1945), and to increase (Trentin, 1954) the incidence of mammary tumors in mice. In male mice of susceptible strains mammary tumors did not develop when progesterone was administered every 28 days for two years (Trentin, 1954). However, in another study progesterone- treated virgin females showed an increased incidence of tumors when compared to controls, suggesting a role for this hormone in mammary tumorigenesis (Trentin, 1959). Pseudopregnancy has been reported to in- crease the incidence of spontaneous mammary tumors (Law, 1941, Blair ._t'_l., 1960), suggesting that increased secretion of ovarian hormones particularly progesterone, can stimulate mammary tumorigenesis. Repeated pregnancies have also been reported to increase spontan- eous mammary tumor incidence in mice, although with considerable variance among strains (Lathrop and Loeb, 1913, Suntzeff gt_al,, 1941, Bruni and Montemurro, 1971). MUhlbock (1956) confirmed that mammary tumors were more prevalent in breeding females than in virgin animals. He further reported that incidence of mammary tumors increased with the number of pregnancies, and Blair gt al. (1960), found this increase proportionate to the number of litters born. Forced—bred females with more than three litters had also a higher incidence of mammary tumors (Mfihlbock, 1956). It is well-known that estrogen and progesterone secretion increase during pregnancy, which further emphasizes the role of these hormones in spontaneous mammary tumors. It has been generally accepted that lactation decreases the incidence and growth of mammary tumors. For instance, Fekete (1940) found that mammary tumor numbers were reduced by prolongation of nurs- ings. Bogen (1935) reported that mammary tumors developed in mice whose offspring were removed after birth, but did not develop in those nursing their offspring. Mfihlbock (1956) compared mammary tumor incidence in breeding females whose offspring were taken away after birth to prevent lactation and animals which were allowed to feed their young. In the group permitted lactation, the mammary tumor incidence was lower and the tumor age considerably higher than in the other group. Single or multiple pituitary grafts release large amounts of prolactin and very little adrenocorticotrophic hormone (ACTH), thyroid- stimulating hormone (TSH), growth hormone (GH), follicle-stimulating hormone (FSH) or luteinizing hormone (LH) (Meites and Nicoll, 1966). The early work of Loeb and Kirtz (1939) showed that subcutaneous graft- ing of several pituitaries increased the incidence of mammary tumors in mice, suggesting prolactin as the key hormone in mammary tumorigenesis. This work was subsequently confirmed when pituitary implants grafted subcutaneously, intraocularly as a single isograft (Liebelt and Liebelt, 1961) or, underneath the kidney capsule, as multiple isografts in mice (MUhlbock and Boot, 1959) increased the incidence of mammary tumors. Furthermore, several investigators showed that pituitary grafts stimulated mammary tumorigenesis in resistant strains as well as in MTV-free strains of mice (MUhlbock, 1956 and MDhlbock and Boot, 1959). Pituitary grafts also increased mammary tumorigenesis in male mice (Hagen and Rawlinson, 1964, Hagen, 1966) and in ovariectomized mice (Bittner and Cole, 1961), further suggesting a primary role for pro- lactin, and perhaps a secondary role for ovarian hormones, in mammary tumorigenesis. Increased mammary tumor incidence was also observed in female mice injected with prolactin for an extended period of time (Boot gt_al,, 1962). Hypothalamic lesions, regardless of location, also increased the incidence and decreased the latent period of mammary- tumor appearance in agent-bearing and agent-free mice (Bruni and Montemurro, 1971). These lesions disrupt the hypothalamic-hypophyseal portal system, thus preventing the neurohormones from reaching the anterior pituitary gland. This effect results in decreased secretion of all anterior pituitary hormones except prolactin which is increased (Meites and Nicoll, 1966). These studies further demonstrate that prolactin as well as ovarian hormones appear to be important in induc- tion of spontaneous mammary tumorigenesis. II. The Role of Hormones in Mammary Tumorigenesis in Rats: In Vivo Studies Mammary tumors can be induced in rats by polynuclear aromatic hydrocarbons, which possess little hormone activity, such as 3 methyl- cholanthrene (3MC) (Huggins gt__l,, 1959a) and DMBA (Huggins gt_al,, 1961). Huggins gt_al, (1959a), induced mammary tumors by administering 3MC to female rats for 20 days, every animal developed mammary tumors in 2 months. Shay et a1, (1949), reported similar findings. Huggins ‘_t‘al. (1961), also found that a single dose of DMBA always induced mammary tumors. The structural similarity of steroid hormones to the polycyclic aromatic hydrocarbons suggested that such hormones resemble these hydrocarbons irl their capacity to interact with the cellular constituents of the target cells. Therefore, some authors have indi- cated that the aromatic hydrocarbons may interact at the same cellular sites as do steroid hormones (Yang §t_gl,, 1961). The carcinogen- induced mammary tumors are hormone dependent since they respond readily to changes in hormone secretion. Induction of carcinogen-induced mammary tumors in rats appears to require the presence of ovarian and anterior pituitary hormones. A reduced incidence of tumors was observed in rats ovariectomized prior to carcinogen treatment (Huggins gt_al,, 1959a; Talwalker 25.91:, 1964; Welsch gt al,, 1968). When ovariectomy was performed 30 days before carcinogen administration in female rats, mammary tumors did not appear (Dao, 1962; Welsch gt_al,, 1968), suggesting that ovarian hormones were necessary for the initiation of the carcinogenic process by the polycyclic hydrocarbons. Ovariectomy of rats immediately after administration of carcinogens either inhibited or greatly reduced the incidence of mammary tumors (Huggins §t_a1,, 1959a; Dao, 1962; Welsch et 21,, 1971). Dao (1962), showed that ovariectomy preceding or immedi— ately following 3MC treatment inhibited mammary tumor incidence, whereas ovariectomy 7 days after carcinogen treatment was unable to prevent or reduce the incidence of these tumors. Thus, these experi— ments suggested that neoplastic transformation in mammary gland cells could not occur in the absence of ovarian hormones, since concurrent administration of these hormones from ovarian grafts will induce mammary tumors. Tumor incidence increased when ovaries were transplanted 10 or 15 days before carcinogen administration, but did not increase when ovaries were grafted as long after treatment as 25 days. Intact or castrated male rats were refractory to carcinogenic induction of mammary tumors, while ovarian grafts returned tumor incidence to normal. This was particularly evident when castration was performed prior to ovarian grafting and carcinogen treatment (Dao and Greiner, 1961), providing further evidence of the importance of ovarian hormones in induction of carcinogen-induced rat mammary tumors. Generally speaking, intense stimulation of the growth of rat mammary glands bgf9:e_carcinogen treatment results in a marked reduction of the incidence of tumors. Thus, treatment of rats prior to carcinogen administration with progesterone (Jabara, 1967); norethynodrel- mestranol, Enovid (Welsch and Meites, 1969); pregnancy and pseudopreg- nancy (Dao and Sunderland, 1959); median eminence lesions (Clemens gt_al,, 1968; Welsch gt _1., 1969); pituitary grafts (Welsch gt_al,, 1968); and reserpine (Welsch and Meites, 1970), inhibited incidence of mammary tumors. These studies suggest that intense stimulation of mammary growth by ovarian hormones and prolactin prior to carcinogen treatment protected the mammary tissue from the action of carcinogen. Established DMBA-induced mammary tumors grow at an accelerated rate after administration of progestational compounds (Huggins gt_al,, 1959b); pregnancy and pseudopregnancy (Dao and Sunderland, 1959); norethynodrel-mestranol combination (Welsch and Meites, 1969); median eminence lesions (Clemens gt_al,, 1968; Welsch gt_al,, 1969; Klaiber _t_al,, 1969); pituitary grafts (Welsch gt_al,, 1968); and reserpine (Welsch and Meites, 1970). Most of these treatments are associated with increased blood levels of ovarian hormones as well as prolactin, suggesting that these hormones are responsible for the effects of in- creased tumor-growth. Accelerated growth of mammary tumors in rats therefore, appears to require the presence of both ovarian and anterior pituitary hormones, m0re specifically, estrogen and prolactin. This is further illustrated by studies demonstrating that inhibition of carcinogen-induced mammary tumor growth results from hypophysectomy and/or ovariectomy and adrenalectomy (Huggins et_al,, 1959a; Sterental gt_al,, 1963; Dao, 1964; Pearson gt_al,, 1969; Nagasawa and Yanai, 1970; Welsch gt al,, 1973). These surgical procedures markedly reduce prolactin and ovarian secretions, and it is through this mechanism 10 that inhibition of the growth of mammary tumors occurs. That prolactin is the "key" pituitary hormone in the growth of rat mammary tumors is demonstrated by studying the effects of placing bilateral lesions in the median eminence in carcinogen-treated intact or ovariectomized tumor bearing rats. These lesions significantly promoted growth of these tumors (Clemens gt _l., 1968; Welsch gt_al,, 1969; Klaiber 23.21:, 1969) by increasing prolactin secretion from the anterior pituitary. Such studies demonstrate the importance of pro- lactin in this process; however, ovarian hormones also appear to be involved, since a diminished growth of tumors was observed in ovariec- tomized rats with median—eminence lesions when compared with intact rats, despite the presence of high levels of blood prolactin (Welsch _t_al,, 1969; Klaiber et al,, 1969). The study of Dao and Sinha (1972) also supports these findings, since ovarian grafts reactivated the growth of carcinogen-induced mammary tumors in these rats. Injections of prolactin alone reactivated mammary tumor growth (for approximately 15 days) in ovariectomized-adrenalectomized (Nagasawa and Yanai, 1970), ovariectomized-adrenalectomized-hypophysectomized (Pearson gt_al,, 1969) DMBA-treated, tumor-bearing rats. These results suggest that prolactin by itself can promote growth of carcinogen- , induced mammary tumors in rats. However, the effects observed observed may not have persisted over a long period of time. Injections of growth hormone to DMBA-treated rats did not reacti- vate growth of tumors regressing after adrenalectomy and ovariectomy (Nagasawa and Yanai, 1970) or adrenalectomy, ovariectomy and hypophysec- tomy (Pearson et_al,, 1969). However, Young (1961) reported an increase 11 of growth of 3MC-induced mammary tumors in hypophysectomized female rats treated with ovarian hormones and bovine growth hormone. The role of growth hormone in the growth of carcinogen-induced rat mammary tumors requires further investigation. Administration of drugs that increase prolactin secretion general- ly results in enhanced growth of carcinogen-induced mammary tumors in rats. Drugs that increase mammary tumor growth include reserpine (Welsch and Meites, 1970); perphenazine (Pearson gt_al., 1969); haloperidol (Quadri gt__l,, 1973a) and methyl-dopa (Quadri gt_al,, 1973b). A number of drugs that decrease prolactin secretion result in the inhibition of mammary tumor growth. For example, ergocornine or ergocryptine (Nagasawa and Meites, 1970; Heuson et_al,, 1970; Cassell __t 11., 1971; Stahélin §t_al,, 1971); iproniazid (Nagasawa and Meites, 1970); pargyline and lysergic acid (LSD) (Quadri gt_al,, 1973a); L-dopa (Quadri _t_al,, 1973b), all potent suppressors of prolactin secretion, have been reported to inhibit mammary tumor growth. Reserpine (Lacassagne and Duplan, 1959) and perphenazine (Ben-David, 1968) are tranquilizers that suppress hypothalamic activity, reducing the secretion of all anterior pituitary hormones except prolactin (Meites and Nicoll, 1966). Haloperidol and methyl-dopa increase pro- lactin release by reducing catecholamine activity in the hypothalamus (Lu gt__l,, 1971). Ergot drugs such as ergocornine and ergocryptine have been found to act on the hypothalamus (Wuttke gt_al,, 1971) and also to act directly on the pituitary (Lu gt_al,, 1971) to inhibit prolactin release. Iproniazid, a monoamine oxidase inhibitor, probably inhibits prolactin secretion by interfering with the catabolism of 12 catecholamines and thereby increasing catecholamine concentration in the hypothalamus (Lu and Meites, 1971). Pargyline, LSD and L-dopa probably act in a similar way by influencing catecholamines and conse- quently suppressing pituitary prolactin secretion (Lu and Meites, 1971; Quadri gt_al,, 1973a,b). Enhanced regression of mammary tumors was observed when reserpine was administered simultaneously with ergocornine to DMBA-treated rats (Welsch gt_al,, 1973), further suggesting that pituitary hormones other than prolactin participated in the growth of these tumors. III. The Role of Hormones in Mammary Tumorigenesis in Humans: In Viyg_§tudies Since 1833 when Astley Cooper reported that mammary cancers were stimulated during the premenstrual phase of each cycle, it has been known that hormones were involved in human mammary tumorigenesis. It was not until 1889 (Schinzinger) that the concept of hormones as chemical messengers was formulated, suggesting that there might be a relationship between the ovaries and human breast cancers. Beatson (1896) was first to perform an ovariectomy in human patients for the control of breast cancer. Other investigators con- firmed the beneficial effects of ovariectomy (Thompson, 1902; Lett, 1905; Treves and Finkbeiner, 1958; Fracchia gt_al,, 1969). About one third of the breast-cancer patients reSponded favorably with regression of mammary carcinoma after ovariectomy. There is evidence to suggest that premenopausal and postmenopausal breast cancers respond differently to endocrine manipulations as a result of having different hormonal 13 environment. Thus, moderate dosage levels of ovarian hormones appear to be stimulatory to mammary cancer growth and development whereas large doses inhibit tumor growth (Stoll, 1969). Ovariectomy is an effective measure in many premenopausal patients with advanced breast cancer (Taylor, 1962; Fracchia gt_al,, 1969). However, adrenal glands, as a source of estrogenic compounds have also been considered a pos- sible stimulant to breast cancer. Some investigators performed adrenalectomy in women suffering from advanced mammary carcinoma and reported some beneficial effects (Huggins and Bergenstal, 1952; Fracchia _th_l., 1967). Other investigators found that elimination of the two sources of ovarian hormones by ovariectomy and adrenalectomy resulted in a higher total rate of remission of tumor growth in patients with metastatic breast cancers (Huggins and Dao, 1953; Fracchia gt 21,, 1967). The reduction of ovarian hormone levels ob- tained by adrenalectomy combined with ovariectomy may be obtained in- directly by removal of the pituitary. Thus, Bulbrook gt_al, (1958) reported that estrogen excretion is reduced to a very low level in hypophysectomized patients, hypophysectomy first used by Luft and Olivecrona (1953). Other investigators have reported hypophysectomy as an effective therapeutic procedure in the management of advanced breast cancer (Pearson gt 11., 1956). Hypophysectomy was also effec- tive in patients suffering from recurrence of breast cancer growth after remission obtained from ovariectomy and adrenalectomy (Pearson and Ray, 1959; Kennedy and French, 1965), or in patients who did not respond to ovariectomy and adrenalectomy (Pearson, 1957), suggesting that pituitary hormones were involved in growth of breast tumors. 14 Treatment with high doses of estrogens was advised as the primary step in endocrine therapy in all patients showing no evidence of ovarian secretion. In postmenopausal women, estrogen therapy yields a higher rate of tumor remission than any other method of endocrine therapy (Stoll, 1969). Progesterone itself has been tried for the treatment of advanced breast cancer, but even when given in high doses, has failed to produce a remission (Co-operative Breast Cancer Group, 1964). New progestogens have been synthesized and used in clinical tests, but only a few pro- gestational compounds were effective in the treatment of human breast cancer (Co-operative Breast Cancer Group, 1964). A palliative effect has been observed after progestin therapy in 22.3% of cancer patients previously not responsive to estrogen therapy (Co-operative Breast Cancer Group, 1961). However, progestins in combination with estrogens were more effective in tumor regression than progesterone alone (Landau __ta_1_.,1962). It has been suggested that pregnancy may be protective against mammary cancer in humans. Women who had had a first pregnancy prior to age 20 had only about two-thirds of the breast cancer risk of those whose first pregnancy occurred after age 25. The association with age at first pregnancy appear to explain at least part of the association of decreasing breast cancer risk with increasing parity (Mirra _t_al,. 1971), suggesting that intense stimulation of ovarian hormones and pro- lactin can actually protect mammary tissue from breast cancer. Peller (1940) reported that breast cancer was more common in women bearing only one child than in multipare. There is general agreement that the 15 frequency of breast cancer decreases as the number of pregnancies in— creases, however, the frequency of the disease between nulliparous and parous women differs greatly according to the methods employed in presenting the statistical data (Peller, 1940; Bonser gt_al,, 1961). The reduced risk of breast cancer in women having their first child at an early age explains the previously observed inverse relationship between total parity and breast cancer risks, since women having their first birth early tend to become ultimately of high parity (MacMahon _t__l,, 1970). Reports have also been shown that pregnancy unfavorably influences existing tumors in patients with breast cancer by increasing growth of such tumors (Kilgore and Bloodgood, 1929). However, there is no evidence that pregnancy can stimulate development of new tumors (Fitzwilliams, 1925). It has been suggested that lactation may also protect humans against mammary cancer. Studies indicate age at first nursing and the total duration of nursing to be different among control groups than among various breast cancer groups. However, this effect cannot be separated from the factors of age at first pregnancy and the number of pregnancies (Wynder et_al,, 1960). Pharmacological agents acting by way of the hypothalamus can greatly influence production and discharge of pituitary hormones, thus influencing mammary tumor development. Therefore, certain drugs have been recently tested in humans for possible effects on suppression of prolactin secretion. Certain ergot alkaloids have been reported to suppress prolactin secretion in patients with galactorrhea (Lutterbeck gt_al,, 1971; Besser gt_al,, 1972). However, the Eur0pean Breast Cancer 16 Group (1972) failed to induce a significant number of objective remis- sions in patients with advanced breast cancer when treated with this drug. L-dopa, a potent prolactin suppressor, has also been tested in patients with advanced breast cancer. Stoll (1972) reported that seven postmenopausal patients with metastatic breast carcinoma failed to have a regression with either estrogen or L-dopa administration. However, when given combined therapy of estrogen and L-dopa, three of the seven patients showed significant tumor regression. Pearson _t_al. (1972), observed regression of mestastatic breast cancer in two out of seven patients after 2 months of treatment with L-dopa. These studies are among the first jn_vivg_studies which suggest that prolactin may have a stimulatory role in human breast cancers. IV. The Influences of Hormones on Mouse, Rat and Human Mammary Tumors in Culture: In Vitro Studies A. Mouse Mammary Tumors In_Vitno The nature of the hormonal influence on growth and differentiation of mammary glands in mice has been established by studies of animals deprived of various endocrine glands (Nandi, 1958). However, such studies do not permit delineation of the action of hormones on mammo- genesis, since the possible influence of the remaining endocrine gland can not be eliminated. Thus, methods in which organs are cultured jg_!jt§9_offer an advantage in analyzing the role of hormones in growth and differentiation in chemically defined media. l7 Organ cultures of mammary glands from embryonic (Lasfargues and Murray, 1959), young virgin (Prop, 1959, 1960) and adult (Elias, 1957, 1959; Elias and Rivera, 1959) mice have been cultured in partly or entirely synthetic media supplemented by hormones. The effectiveness of insulin on mammary cultures was first described for the lobular- alveolar stage of development of mammary glands (Elias, 1959) and for the viability and secretory activity of mammary duct end-buds (Elias, 1961, 1962) in culture. In organ cultures, insulin and cortisol permitted survival of late prelactating mouse mammary tissue (Elias, 1959), whereas prolactin and cortisol stimulated secretory activity (Elias 1957; Elias and Rivera, 1959). Rivera and Bern (1961) studied the effects of different hormones on mammary tissue of late-prelactating, early-prelactating and non- pregnant multiparous female mice jg_vitrg, They found that the 3 types of tissues were maintained with the addition of insulin and cortisol, to some extent with insulin alone, and not at all with cortisol alone. The more differentiated the tissue in the direction of secretory activ- ity, the greater the need for cortisol in addition to insulin for maintenance ig_vitro. The addition of prolactin and growth hormone to media containing insulin and cortisol maintained the secretory state of late prelactating tissue and stimulated early prelactating and non- pregnant tissues to secretory activity. It was further reported (Rivera, 1964) that regression of mammary glands occurred in the syn- thetic medium alone. However, when aldosterone, insulin, estradiol and progesterone were added, maintenance of these tissues was observed. When this combination was supplemented with prolactin and growth hormone, 18 ductal dilatation, lateral bud formation and irregular organization of duct endings were observed. Differentiation of mouse mammary glands appear to require insu- lin, hydrocortisone, and prolactin (Rivera and Bern, 1961; Juergens _t_al,, 1965, Stockdale et_al,, 1966; Lockwood et_al,, 1967a). But epithelial cells of mammary glands must divide first in order to syn— thesize casein in response to these hormones (Stockdale and Topper, 1966; Lockwood _t_al,, 1967a and b). Lockwood gt al. (1967a), reported that insulin was required for initiation of DNA synthesis and G1 phase (after mitosis); hydrocortisone activity preceded that of prolactin, and prolactin elicited differentiation of mammary glands ig_vitrg, Insulin appeared to be of primary importance in stimulating cell pro- liferation, as measured by incorporation of H3-thymidine into DNA, and by autoradiographic studies with H3-thymidine (Stockdale and Topper, 1966; Turkington, 1968). El-Darwish and Rivera (1970) studied the com- bined effect of insulin, corticosterone and prolactin on mammary explants from midpregnant mice. They confirmed that insulin was prim- arily responsible for the stimulation of DNA synthesis and found that its peak activity occurred on the first day of culture. After this time, prolactin and corticosterone interacted with the tissue to maintain the insulin stimulation. Mills and Topper (1970) studied the effects of these 3 hormones on the morphology of explants from midpregnant mouse mammary glands and reported that insulin promoted the formation of daughter cells within the alveolar epithelium. The addition of hydro— cortisone to the medium containing insulin brought the daughter cells to a new intermediate level of ultrastructural development; when prolactin 19 was added to the insulin, hydrocorticosterone medium, the daughter cells completed their ultrastructural differentiation. Elias and Rivera (1959) compared the role of hormones in the growth of normal, precancerous and neoplastic mammary tissue ifl_vitrg, before and after addition of various hormones alone, or in combination, in the chemical media. They reported that normal and precancerous mammary tissue degenerated in media-lacking hormones, but tissues were maintained when ovine prolactin and cortisol were added to the media. When high concentrations of prolactin and cortisol were added, secretory activity was induced in the normal and precancerous explants. However, estrone, progesterone or growth hormone alone or in combina- tion did not affect these structures. Also, higher hormone concentra- tion was more necessary for survival of normal tissue than precancerous explants. Rivera §t_al, (1963), later demonstrated that steroid hor- mones inhibited growth of mouse mammary gland tumors jn_vitrg, They found that estrone, estradiol-17B, progesterone, cortisol, cortico- sterone and deoxycorticosterone were toxic in high doses (especially progesterone). Turkington and Hilf (1968) found that DNA synthesis in mouse mammary carcinoma was stimulated by insulin and inhibited by estrogens as in normal mammary epithelial cells. B. Rat Mammary Tumors In Viggg In_vitro experiments have demonstrated the morphogenic activity of prolactin in rat mammary glands (Mishkinsky §t_al,, 1967). Dilley and Nandi (1968) reported that mammary glands from several strains of immature female rats formed lobules of alveoli when cultured in a 20 chemically-defined medium lacking steroid hormones but containing insulin and prolactin. The degree of lobulo-alveolar differentiation was increased if estradiol, progesterone and aldosterone were also included in the culture media. Insulin alone did not induce signifi- cant alveolar development, or maintain initial mitotic or DNA synthetic activity. However, insulin plus prolactin stimulated alveolar develop- ment and maintained initial mitotic activity (Dilley, 1971). The effect of insulin on cell proliferation in carcinogen-induced mammary carcinoma in vit§g_(Heuson gt__l,, 1967) was also studied. It was found that insulin stimulated cell proliferation as measured by thymidine-C14 incorporation into DNA. Heuson and Legros (1968) reported that this effect was not mediated through an increase of glucose uptake and utilization by the explants; therefore, it did not appear to be a simple permissive effect bearing on energy-yielding reactions. Instead, insulin appeared to act on processes more directly related to the control of DNA synthesis. It was further reported (1971) that, in addition to tumors with insulin—dependent cell pro- liferation, other tumors displayed an intense mitotic activity spon- taneously and independently of the presence of insulin jg_vitr9: It was also found that in most tumors DNA synthesis and DNA—polymerase activity ran a closely parallel time course. In tumors with insulin— independent DNA synthesis, DNA-polymerase was also insulin-independent. It was suggested therefore, that the stimulating property of insulin involved activation or induction of enzyme-systems responsible for DNA synthesis. 21 Hollander (1970) found that prolactin increased the incorporation of H3-thymidine into DNA in rat mammary tumors jg_vjtrg, but estradiol- 17B and growth hormone had no effect. Prolactin also increased incorporation of H3-thymidine into DNA in DMBA-induced rat mammary carcinoma in vitrg, but in the presence of insulin and corticosterone (Welsch and Rivera, 1972). Dao and Sinha (1972) studied the effects of insulin, estrogen and prolactin alone or in combination on DNA synthesis of DMBA-induced mammary tumor explants jg_vitrg, They found that these hormones when added to the culture media failed to stimulate DNA synthesis. However, when the tumors were cultured in the presence of insulin, estrogen, prolactin, together with progesterone, DNA synthesis was stimulated. C. Human Mammary Tumors In Vitgg Human mammary gland explants can be organ-cultured in a chemically defined medium and induction of mammogenesis and secretion can be in- duced by the addition of appropriate hormones or combinations of hor- mones to the medium. Ceriani §t_al, (1972), observed that most of the explants cul- tured in a hormone-free medium survived, while the addition of insulin gave full maintenance. Barker gt_al, (1964), reported that addition of insulin to the media resulted in marked morphological changes of ductal epithelium, including proliferation and hypertrOphy. Flaxman and Lasfargues (1973) reported that significant DNA synthesis and mitosis occurred in the absence of supplemental hormones, but addition of insulin and prolactin caused an increase in the number of cells 22 synthesizing DNA. Ceriani et_al, (1972) reported that prolactin and insulin induced epithelial cell growth, but addition of bovine growth hormone to insulin and prolactin magnified their effect on the epi- thelium of human mammary glands. Other studies have demonstrated morphological or biochemical evidence of inhibition or stimulation of mammary tumor growth by estrogenic substances. Thus, Kellner and Turcic (1962) reported that steroid hormones inhibited growth of breast tumors in_vjtrg, Chayen _t_al, (1970), found that five out of sixteen human breast cancer tis— sues survived well in the presence of estradiol. Hollander gt_al: (1958), found an estrogen-sensitive enzyme in normal and cancerous breast tissue, suggesting possible relationship between jn_vitrg_estro- gen sensitivity of carcinoma tissue and hormonal therapeutic response. Rienits (1959) studied the effect of estrone on the respiratory rate of 23 human mammary tumors both primary and metastatic. He found no effect on fourteen, inhibition in two, and stimulation to a significant degree in seven of such tumors. Barker and Richmond (1971) observed the effects of stilbestrol on tumors from cancer patients. The rate of growth of cells in vitro was assessed by glucose utilization and lactic acid production. It was unchanged in three cases, inhibited in two, and increased in one case. Rate of growth was also determined by rate of amino acid incorporation in vitrg_and the effect of estradiol-17B in this process was evaluated (Heuson and Legros, 1963). This steroid inhibited tumor growth in 21 out of 24 specimen studied. Burstein (1971) incubated tumor cells from metastatic breast 3 cancers for 48 hours jg_vitro with H -thymidine in the presence of 23 cortisol, estradiol-17B, progesterone or growth hormone. In 15 out of 40 primary or metastatic lesions there was a correlation between the clinical response to either ablative endocrine surgery or exogenous hormone administration and in_vitrg responses. Mioduszewska gt_al, (1967), studied the effects of cortisol, estradiol, progesterone, growth hormone and prolactin on cultures of mammary carcinoma in 26 patients. They also compared these results with the clinical course of the disease, and with the results of abla- tive hormonal therapy in persons with advanced breast cancers. Two types of reaction of mammary carcinoma to hormones in_vitrg_seemed to be related to prognosis. Thus, lack of response to prolactin and stimulation of growth by cortisol, both which were associated with a poor prognosis, and a positive response to prolactin and lack of response to cortisol, both which were associated with a good prognosis, provided an indication of the eventual course of the disease. Prolactin dependence has been demonstrated in 32% of fifty human breast—cancers in_vitro (Salih gt al,, 1972). For 20% of these malignant tumors, the only requirement found was prolactin; for 12% enhancement also occurred with estradiolv17B as well as prolactin, and in one case, androgen and prolactin could enhance the tumor growth, suggesting that a trial of management based on the in vitrg_findings may be possible. MATERIALS AND METHODS 1. Organ Culture A. Preparation of Mammary Tumors for Organ Culture 8. DNA Extraction and Analysis of Cultured Mammary Tumor Explants C. Autoradiographic Analysis of Cultured Mammary Tumor Explants 0. Experimental Design II. Cell Culture A. Preparation of Cell Suspensions from Mammary Tumors B. Seeding of Cell Suspensions. Analysis of Growth by Measuring Number and Area of Colonies of Cells 1. Experimental Design C. Seeding of Cell Suspensions. Analysis of Growth by Determin- ing Cell Number 1. Experimental Design 24 25 All animals used in these studies were mature virgin female Sprague-Dawley rats (Spartan Research Animals, Inc., Haslett, Michigan) housed in a temperature (75:2°F) and light (14 hrs/day) controlled room, and given a diet of Wayne Lab Blox (Allied Mills, Chicago, Ill.) and water ag_libitum. Virgin female rats, 55 days of age were given a single intra— venous injection of one m1 of a lipid emulsion containing 5 mg of 7, lZ-dimethylbenzathracene (DMBA). Palpable tumors began to appear 4 weeks after carcinogen treatment and by ten weeks, nearly 100% of the rats developed mammary tumors. Only in experiment ID-3, were animals ovariectomized 10 days before removal of the tumors. I. Organ Culture A. Preparation of Mammary Tumors for Organ Culture Tumors of approximately 1.5 cm in diameter were aseptically removed from anesthetized rats and cut into one mm3 pieces, yielding approxi- mately 120 explants. Each organ culture consisted of explants taken from 3 rat tumors. Each tumor was cut into pieces avoiding the con- nective tissue as much as possible. Explants were placed at random in each of small (10 X 30 mm) Falcon disposable petri dishes. Each small dish contained 10 pieces of tissue and 2 m1 of culture Medium 199, Earle Base (2170 mg NaHCO3 per liter) (Difco Labs; Detroit, Michigan). Three small uncovered petri dishes were placed inside a large Falcon disposable petri dish (100 X 15 mm) containing water-saturated filter paper. Covered large dishes containing the 3 petri dishes were placed 26 in a plexiglass gassing chamber which was continuously infused with gas (95% 02:5% C02). During the culture period, the gassing chamber was housed in an incubator maintained at 37°C. All culture-media contained 50 I.U. per m1 of penicillin G (Nutritional Biochemicals Corporation). Sterilization of the media was accomplished by passage through millipore filters (0.45 p) under vacuum. Five pg per m1 of insulin (bovine pancreas, 22.5 I.U. per mg; Nutritional Biochemicals Corporation); 1.0 pg per m1 of corticosterone (Nutritional Biochemicals Corporation); 5.0 pg per m1 of prolactin (NIH - S8 or $9 ovine, 28 I.U. and 33 I.U. per mg); 0.01 pg per m1 of estradiol-178 (Nutritional Biochemicals Corporation) and 5.0 pg per m1 of growth hormone (Upjohn human, Lot number 8717DADl48) were added to the experimental groups. Steroid hormones were added to the media in an ethanol base with a maximum final ethanol concentration of 0.5%. Protein hormones were added in a Medium 199 base. Explants were cultured for 5 days. Media were changed on days 2 and 4. On day 5, 4 hours prior to termination of culture, sterile H3- thymidine (New England Nuclear, 6.7 Ci/ml) was added to the explants in a final concentration of 0.5 p Ci/ml. Termination of the cultures con- sisted of a fast removal of the explants from the media and storage in 0.15 M KCl at -15°C for DNA extraction and analysis. DNA synthesis was estimated by measuring H3-thymidine incorporation into DNA. 8. DNA Extraction and Analysis of Cultured MammarygTumor Explants The explants were weighed and then homogenized in an all-glass homogenizer in cold 10% trichloroacetic acid (TCA). They were washed 27 3 times by centrifugation for 10 minutes in a Sorvall RC 2-8 Centrifuge (2500 X G) at -4°C with cold 10% TCA. Water and lipids were removed by washing the extracts twice with cold 1M Na acetate in methanol, twice with cold-methanol-chloroform (2:1), twice with cold absolute ethanol, and once with cold ethyl ether. The ether remaining in the extracts was evaporated in a hood, and the powder obtained was dried in a desiccator under vacuum. The dehydrated-defatted extracts (DDE) were hydrolysed in 0.3 N KOH for 3 hours at 37°C to remove RNA, tubes were then placed in cold, and all subsequent steps were carried out at 4°C. The alkaline digest was neutralized with 0.3 N hydrochloric acid, acidified with 10% perchloric acid (PCA) and kept in the cold for 1 hour. The supernatant was removed by centrifugation (2500 X G) and the pre- cipitate washed twice with 10% PCA. DNA was extracted from the pre- cipitate by treatment with 5% PCA at 70°C for 30 minutes. The precipitate was washed twice by centrifugation (2500 X G) for 10 minutes with 5% PCA, and the combined supernatants were adjusted to a final volume of 6 m1. Duplicate 2 m1 samples were analyzed for DNA by the diphenylamine reaction (Burton, 1956), using calf-thymus DNA standards. Four ml of diphenylamine reagent were added to 2 m1 samples of each group and then they were incubated at 30°C for 18 hours. Samples were analyzed at 600 mp in a Spectrophotometer (Beckman 08). The remaining 2 m1 of samples were neutralized with 3N NaOH to pH 7. From these samples, triplicates of 100 A were placed on filter-paper discs which were subsequently dried overnight. These discs were placed in scintil- lation vials containing 10 m1 of POPOP-PPO toluene. Each sample was counted for 10 minutes in a scintillation counter (Unilux Table Model, 28 Nuclear Chicago, 45% efficiency). H3 -thymidine incorporation (cpm) per pg DNA was statistically analyzed by Randomized Block Test. Parameters were further tested by Dunnet's Test (one-sided) and Orthogonal Contrast Test (Sokal and Rohlf, 1969). C. Autoradiographic Analysis of Cultured Mammary‘Tumor Explants Cultured mammary tumor explants were sectioned and prepared for autoradiographic analysis. Autoradiographs were made by the method of Messier and Leblond (1957) as modified by Walker (1959). Explants were fixed in Bouin's fluid. Paraffin sections were mounted on albuminized slides. Paraffin was removed in xylol and the slides were run through absolute ethanol and absolute ethanol—ethyl ether (1:1). The coating of slides with emulsion was performed in a darkroom at 60°F. Liquid NTB2 emulsion (Eastman Kodak, Rochester) was placed in a Naples stain- ing jar (Thomas, Philadelphia) and melted in a water bath at 42-44°C for one hour. The slides were placed on a warming plate at the same temperature for a few minutes, then dipped slowly in to the emulsion and held there about 4 seconds. After withdrawal of the slide, its base was held against absorbent paper briefly and then the slide was put on a plastic rack for about 4 hours in a dark room. The slides were then stored in the refrigerator in lightntight boxes for 3 weeks. Development of autoradiographs were carried out at 68°F for 3 minutes. The slides were rinsed briefly in water and cleared for 8 minutes in an acid fixer. Subsequent processing was carried out at room temperature in the light, and consisted of running water, 5 minutes; Harris 29 hematoxylin, 3 minutes; water rinse; acid bath (2 m1 concentrated hydrochloric acid in 300 ml water), 2 seconds; running water, 5 minutes; dehydration in graded alcohol series, including eosin Y counter— staining; 15 minutes in 2 changes of absolute ethanol; one hour in ethanol-cedarwood oil (1:1); one hour in dilute (2/3 toluene) Permount; one hour in stock Permount; slides were allowed to drain 5 seconds; cover glasses were dipped in dilute Permount and placed on the slides. Autoradiographic analysis were performed on explants of DMBA— induced rat mammary carcinoma obtained from the studies done on DNA synthesis (see section 0-4). Incorporation of H3-thymidine was analyzed by the presence of silver grains over the epithelial cells. A thousand cells were counted within 8 or 9 fields of each microsc0pic slide (15-20 slides per group), and cells showing silver grains were scored in each group. Autoradiographic studies were analyzed by 2 Way Analysis of Variance with Unequal Number and Sheffé Test (Sokal and Rohlf, 1969). 0. Experimental Design Five sets of experiments were designed to determine the effects of different hormones on DNA synthesis in organ cultures of DMBA- induced rat mammary carcinoma. 1) DNA Synthesis at 12, 24, 48 and 96 Hours of Culture Media contained corticosterone, insulin and prolactin. Each experiment consisted of 480 explants from 3 rat mammary tumors randomly distributed into 48 small petri dishes. This study was performed 3 times. 30 2) Effects of Insulin and Prolactin on DNA Synthesis of Rat Mammary Carcinoma Each experiment consisted of 360 explants from 3 mammary tumors randomly distributed into 36 small petri dishes. Group I and II received insulin or prolactin alone. Group III received a combination of both hormones. All media contained corticosterone. This study was performed 3 times. 3) Effects of Estrogen and Prolactin on DNA Synthesis of Rat Mammary Carcinoma Each experiment consisted of 480 explants from 3 rat mammary tumors distributed into 48 small petri dishes. Group I served as a control. Group II and III received estrogen or prolactin alone; group IV received a combination of both hormones. All media contained insulin and corticosterone. This study was performed 3 times. 4) Effects of Ovine Growth Hormone and Prolactin on DNA Synthesis of Rat Mammary Carcinoma Each experiment consisted of 360 explants from 3 rat mammary tumors randomly distributed into 360 small petri dishes. Group I and 11 received ovine growth hormone or prolactin alone; group III re- ceived a combination of both hormones. All media contained insulin and corticosterone. This study was performed 4 times. 5) Effects of Human Growth Hormone and Prolactin on DNA Synthe§is of Rat Mammary Carcinoma Each experiment consisted of 480 explants from 3 mammary tumors randomly distributed into 48 small petri dishes. Group I served as a control; group II and III received human growth hormone or prolactin alone; group IV received a combination of both hormones. All media con- tained insulin and corticosterone. This study was performed 3 times. 31 II. Cell Culture A. Preparation of Cell Suspensions from Mammary Tumors Each cell culture consisted of a suspension taken from a single rat tumor. Mammary tumors were aseptically removed and cut into pieces avoiding when possible the connective tissue. Each piece was minced with a scalpel blade in 1.0 m1 of Hanks balanced salt solution (Gibco, Grand Island Biological Company) containing sterile collagenase (Nutritional Biochemicals Corporation) at a concentration of 300 mg percent. One ml of Antibiotic-Antimycotic mixture (100 X) (Gibco, Grand Island Biological Company) was added to this media after being filtered through millipore filters (0.45 p). Minced tissue was then transferred into stoppered test tubes and additional Hanks balanced salt solution containing collagenase was added (1 part of tissue mince per 3.0 m1 of collagenase solution, final digestion concentration). Stoppered test tubes were then placed in a shaker for one hour (37°C) to digest the tissue (disperse the cell). At the end of one hour, the cell solution was centrifuged for 10 minutes in a Sorvall RC2-B centrifuge at 20°C (278 X G). The supernatant was discarded and the tissue resuspended in a fresh Hanks balanced salt solution containing collagenase equivalent in volume to the previous digestion procedure. Tissue was digested.under the same conditions for one more hour, then centrifuged and resuspended_in a fresh Hanks balanced salt solution containing collagenase solution. The tissue was further digested for 20 or 30 minutes and centrifuged again under the same conditions. Supernatant was discarded and cells were washed 2 times in Hanks 32 balanced salt solution (1:3) supplemented with 1.0 ml of the Antibiotic- Antimycotic mixture(100 X). Cells were thoroughly mixed with the solu- tion by means of a small orifice Pasteur pipette to break up any remaining chunks and strings of connective tissue. Tumor debris and cell aggregates were removed by filtering through 4 layers of sterilized cheese cloth and then through a Cytosieve Filter Capsule (Gelman Instru- ment Company, Ann Arbor, Michigan). The cytosieve, a disposable assembly, was attached to a syringe. Polypropylene filters, 25 mm in diameter, were placed in the cytosieve. On the top of each filter, rubber rings were used to tighten firmly the filter cap for an adequate seal. This assembly was autoclaved each time prior to use. Packed cells were resuspended in Medium 199 containing 50 I.U. per ml of penicillin G. B. Seeding_of Cell Suspensions. Analysis of Growth by Measuring Number and Area of Colonies of Cells Tumor cell counts on day 0 were made by using a hemocytometer counting chamber (Spencer Bright-Line Improved Newbauer, 1/10 mm depth). One tenth of the cell suspension was stained with 5.0 m1 crystal violet (0.2 gm crystal violet in 0.1 M citric acid) and incubated for 15 minutes at 37°C. Cell-suspension was shaken in a Vortex Genie Mixer. The stained cell-suspension was then placed into the hemocytometer by means of a 50 A micropipette. Counts of all calibrated volumes (9 large squares) were done in a Spencer American Optical Microscope (400 X magnification). The same volume of suspended cells was plated in small Falcon disposable petri dishes (10 X 30 mm) containing 2 ml of Medium 33 199. Three small uncovered petri dishes were placed in a covered, large Falcon disposable petri dish (100 X 15 mm) containing water~ saturated filter paper. The large dishes were placed in a plexiglass gassing chamber which was continuously infused with gas (95% 02: 5% C02). During the culture-period, the gassing chamber was housed in an incu— bator at a temperature of 37°C. Culture medium was changed on days 2 and 4 in the 5-day cultures. Medium was not changed in 2—day cultures. Termination of cell cultures consisted of discarding the media from the petri dishes and fixing the colonies of cells attached to the dishes with Spray-Cyte (Adams). This is a water—soluble fixative, con— taining polyethylene glycols, isopropyl alcohol and propellants. In order to identify the colonies growing in these petri dishes, they were stained with Harris Hematoxylin for 2 minutes followed by a) Mallory, or b) Papanicolaou stain (Humason, 1972). Mallory stain consisted of processing the dishes through distilled water, 3 dips; Mallory I (1% acid fuchsin) for 15 seconds; 0.1% phosphomolybdic acid for 3-4 minutes; distilled water, 1 dip; Mallory II (0.5 gm Aniline Blue plus 2 gm Orange G in 1000 m1 of distilled water) for 2 minutes; distilled water, one dip; 90% ethyl alcohol, 2 dips; distilled water, 2 dips; and then mounting the cover glass with glycerol jelly mounting brand (Allied Chemical). Papanicolaou stain consisted of processing the dishes through distilled water, 3 dips; 0.5% ammonium hydroxyde in 70% alcohol for 1 minute; graded alcohol series for dehydration; Orange G-6 stain for 3 minutes (consisted of Orange G and phosphotungstic acid); 95% ethanol, 2 dips; EA 50 (light Green SF Yellowish, Eosin Y, phosphotung- stic acid, acetic acid) for 4% minutes; graded alcohol series for 34 rehydration and mounting the cover glass slides (22 X 22 mm) with glycerol jelly. Counts of colony number were made microsc0pically, using a Spencer American Optical Microsc0pe (450 X magnification). Determina- tion of colony area was accomplished by using a Baush and Lomb, Model L, Optical microscope. A Keuffel and Esser compensating planimeter was used for determination of total area of colonies (p2). Photo- micrographs of these colonies were taken with a Standard Universal, Photomicroscope (Bright field), using Kodachrome II, type A (400 X magnification) Differences in total number of colonies were statis- tically analyzed by X2 test (Sokal and Rohlf, 1969). I. Experimental Design Six sets of experiments were designed to determine the effects of different hormones on growth of collagenase dispersed cells of DMBA-induced rat mammary carcinoma. 1. Effect of Combination of Insulin, Prolactin,yand Corticosterone on Growth of Collagenase Dispersed Cells: 2-Days of Culture This experiment was performed twice. Cultures were plated at a density of 1580 (study #1) and 3330 (study #2) tumor cells per petri dish respectively. There were 3 petri dishes in Group I (control) and 3 in Group II (insulin + prolactin + corticosterone). 2. Effect of Combination of Insulin, Prolactin, and Corticosterone on Growthiof Collagenase DiSpersed Cells: 5-Deys of Culture This experiment was performed 3 times. In study #1, there were 3 petri dishes in Group I (control) and 3 in Group II (insulin + 35 prolactin + corticosterone). Cultures were plated at a density of 1580 tumor cells per petri dish. In study #2, there were 14 petri dishes in Group I, and 14 in Group II. Cultures were plated at a density of 525 tumor cells per petri dish. In study #3, there were 12 petri dishes in Group I and 12 in Group II. Cultures were plated at a density of 3330 tumor cells per petri dish. 3. Effect of Combination of Insulin, Prolactin, and Corticosterone versus Insulin Alone on Growth of Collagenase Diepersed Cells: S-Days of Culture This experiment was performed twice. In study #1, there were 11 petri dishes in Group I (insulin), and 12 in Group II (insulin + prolactin + corticosterone). Cultures were plated at a density of 1600 tumor cells per petri dish. In study #2, there were 14 petri dishes in Group I, and 14 in Group II. Cultures were plated at a density of 3550 tumor cells per petri dish. 4. Effect of Combination of Insulin, Prolactin, and Corticosterone versus Insulin or Prolactin Alone on Growth of Collagenase Dispersed Cells: 5-Days of Culture This experiment was performed 3 times. In study #1, there were 12 petri dishes in Group I (insulin), 12 in Group II (prolactin) and 12 in Group III (insulin + prolactin + corticosterone). Cultures were plated at a density of 1850 tumor cells per petri dish. In study #2, there were 3 petri dishes in Group I, 3 in Group II, and 3 in Group III. Cultures were plated at a density of 190 tumor cells per petri dish. In study #3, there were 12 petri dishes in Group I, 12 in Group II and 12 in Group III. Cultures were plated at a density of 780 tumor cells per petri dish. 36 5. Effect of Fetal Calf Serum on Growth of Collagenase Diepersed Cells: 5-Days of Culture There were 15 petri dishes in Group I (control); 15 in Group II (10% fetal calf serum); 15 in Group III (insulin + prolactin + corticosterone); and 15 in Group IV (insulin + prolactin + cortico- sterone and 10% fetal calf serum). Cultures were plated at a density of 1820 tumor cells per petri dish. 6. Effect of Combination of Insulin, Prolactin, and Corticosterone on Growth of Collagenase Dispersed Cells: 5-Days of Cultures This experiment was performed 3 times. In study #1, there were 9 petri dishes in Group I (control);9 in Group II (corticosterone); 9 in Group III (corticosterone + insulin); 9 in Group IV (cortico- sterone + prolactin) and 8 in Group V (corticosterone + insulin + prolactin). Cultures were plated at a density of 2330 tumor cells per petri dish. In study #2, there were 8 petri dishes in Groups I and II, and 9 in Groups III, IV and V. Cultures were plated at a density of 1610 tumor cells per petri dish. In study #3, there were 6 petri dishes in Groups I, II, III, IV and V. Cultures were plated at a density of 2120 tumor cells per petri dish. C. Seedingyof Cell Suspensions. Analysis of Growth by Determining Cell Number Suspension of cells was obtained as explained in Section II A, with the following modifications: 1) Cells were dispersed in Hanks balanced salt solution containing sterile collagenase at a concentra- tion of 200 mg percent in lieu of 300 mg percent. 2) The medium used for these cultures was Medium 199, Earle's Base, Modified (1250 gm 37 NaHCO3 per liter) (Gibco, Grand Island Biological Company), in lieu of Earle's Base (2170 mg NaHCO3 per liter). 3) Medium was not changed during the culture period. 4) And discarded media from centrifugation at termination of cultures were further evaluated for unattached cell number as well as attached cell number. Tumor cell counts on day 0 were determined as explained in Section II A, with some modifications. One tenth of the cell suspension were stained with 1.0 m1 of crystal violet rather than 5.0 m1 of the stain. The same volume of suspended cells were plated in either small Falcon disposable petri dishes, as previously explained (Section II B), or 0.05 ml of suspended cells were plated in plastic microtest plates (BBL Microtest II Tissue Culture Plate) "microplates". Fixation and staining of the cells attached to the petri dishes and microplates were performed as indicated in Section II B. Staining and cell counts of unattached cells were made in the same way in which cells had been counted prior to dispersion (day 0) (Section II B). Microplates com- prised of 96 optically clear, flat-bottom wells, each 6 mm in diameter and approximately 0.3 m1 capacity. Each microplate consisted of 8 rows, 12 wells per row numbered 1-12 for accurate identification. Microplates protected by a lid were placed in a plexiglass gassing chamber as ex— plained previously (Section II B). Each well contained 0.05 ml of cell suspension and 0.05 ml of Medium 199. Cell counts were made by using an Inverted Microscope (Unitron Bright field, binocular model BR-BMIC) under 300 .X magnification. The average number of cells counted either per petri dish or per well was statistically analyzed by 38 X2 test. The average number of unattached cells per petri dish was also analyzed by X2 test. I. Experimental Design Four sets of experiments were designed to determine the effects of insulin, prolactin, and corticosterone on growth of collagenase dispersed cells of DMBA-induced rat mammary carcinoma. 1. Effect of Insulin, Prolactin, and Corticosterone on Growth of Collagenase Dispersed Cells: 24 Hours of Culture. Cells Grown in Petri Dishes This experiment was performed 3 times. In study #1, there were 6 petri dishes in Group I (control); 6 petri dishes in Group II (corticosterone); 9 petri dishes in Group III (corticosterone + insulin); 9 petri dishes in Group IV (corticosterone + prolactin); and 9 petri dishes in Group V (corticosterone + insulin + prolactin). Cultures were plated at a density of 174 cells per petri dish. In study #2, there were 6 petri dishes in Groups I and II, 8 petri dishes in Group III and IV; and 9 petri dishes in Group V. Cultures were plated at a density of 316 cells per petri dish. In study #3, there were 8 petri dishes in Groups I, II and III; and 6 petri dishes in Groups IV and V. Cultures were plated at a density of 1040 cells per petri dish. Only in study #3 was growth of unattached cells also analyzed in the culture media. 2. Effect of Insulin, Prolactin,yand Corticosterone on Growth of Collagenase Dispersed Cells: 48 Hours of Culture. Cells Grown in Petri Dishes This experiment was performed 3 times. In study #1, there were 9 petri dishes in Groups I, II, III, IV and V. Cultures were plated at 39 a density of 174 cells per petri dish. In study #2, there were 6 petri dishes in Groups I, II, III, IV and V. Cultures were plated at a density of 316 cells per petri dish. In study #3, there were 9 petri dishes in Groups I, II, III, IV and V. Cultures were plated at a density of 1040 cells per petri dish. Only in study #3 was growth of unattached cells also analyzed in the culture media the hormones added to Groups I-V were the same as in section CI-l. 3. Effects of Insulin, Prolactin, and Corticosterone on Growth of Collagenase Dispersed Cells: 24 Hours of Culture. Cells Grown in Microplates This experiment was performed 3 times. In study #1, there were 12 wells in Groups I and II; 24 wells in Groups III, IV and V. Cultures were plated at a density of 15 cells per well. In study #2, there were 8 wells in Group I and 10 wells in Groups II, III, IV and V. Cultures were plated at a density of 80 cells per well. In study #3, there were 8 wells in Group I and 10 wells in Groups II, III, IV and V. Cultures were plated at a density of 110 cells per well. The hormones added to groups I-V were the same as in section CI-l. 4. Effect of Fetal Calf Serum on Growth of Collagenase Dispersed Cells: 24 and 48 Hours of Culture Cells were grown in petri dishes, and were examined for growth in media only (unattached cells). This experiment was performed once. There were 3 petri dishes in Group I (control); 3 petri dishes in Group II (10% fetal calf serum); 3 petri dishes in Group III (insulin + prolactin + corticosterone); and 3 petri dishes in Group IV (insulin + prolactin + corticosterone and 10% fetal calf serum). Cultures were plated at a density of 610 cells per petri dish. RESULTS I. Organ Culture A. DNA Synthesis in DMBA—Induced Rat Mammary Carcinoma at 12, 24, 48, and 96 Hours of Organ Culture Preliminary studies were done to determine the effect of a combination of corticosterone, insulin, and prolactin on H3-thymidine incorporation during a 4-hour period of labelling, expressed as counts per minute (cpm) at 12, 24, 48, and 96 hours of culture. The results indicated that at 96 hours of culture these hormones significantly increased H3-thymidine incorporation per pg DNA in com- parison to 12, 24 and 48 hours of culture (P< 0.01) (Tables 1, 2 and 3). However, there was no significant difference between cultures at 12, 24 and 48 hours of culture on H3-thymidine incorporation. Figure l graphically represents the results of these experiments. Results of these experiments are in accord with the findings of Turkington and Hilf (1967), in which active DNA synthesis began in mouse mammary carcinoma at 12 hours of culture (Medium 199 + insulin) and continued to rise for at least 96 hours. It is interesting that in non-tumorous mammary tissue, i.e., mid—gestation mouse mammary gland, synthesis of DNA appears to peak at 48 hours of culture rather than at 96 hours (Lockwood et_el,, 1967a; El-Darwish and Rivera, 1970). Since 96 hours was found to be significantly superior to shorter time periods 40 41 weeps men on venue we: mcwewesgp-mz .ucmcmmwwu xfiycmowwwcmwm no: u U\n .U\m .n\m .e\a,.e\n .e\e " Fo.ova .Amzmmwu Loss“ mo uomcaxm umppmwmu.lvmpmcu>;mcv moo« ¥ .mmcsupau we co_um:wscmu op cowca mczoz e .xrco >H azocm :_ a new N xmn :o nmmcmco mcmz mwumz .azocm comm op umuum mew: A_E\mn o.mv :wuomFocm use .A_E\mn o.mv CWFzmcH .A_E\ma o.Pv mcogmumoowucou k. vo.¢nF N.Pm— o.o o.oF m.m_ mczoz om >H um.mm o._¢F m.m o.mm N.m~ meson we HHH nm.wm 0.0mm m.m o.o¢ q.¢~ mcaog am HH m¢.om m.mm m.~ o.om m.mp meson NF H Agauv AEQUV Amnv Amnv Amsv encasemmcp azoco <20 cowpmcoacoacH “no _co >H mzomm cw q ucm N awn :o ummcmno mcmz ovum: .pcmmmmmwu Xchmwamcmwm uoc u U\n .U\m .n\m .u\o .u\n .U\m .hmsmmwp Logan mo pummuxm umpumwmc..umumcc>;muV maoxk dem .mmm:p_=m mo cowumcwscmm op Loren mmso; e .maomw comm om vmcum mmmz Aps\m: o.mv :wuumpomm ccm .Aps\ma o.mv chzmcm .APE\m: o.Fv mcommpmoowumoum um.¢om m.mom N.N o.mF n.o mmzo; om >H om.om_ m.omm m.~ o.~m N.“ mcao; mm HHH no.mep m.mom _.m o.qm m.m mmzo; cm HH mm.omp m.m¢o~ m.m o.nm m.m memo; Np H Asmuv Asmmv Amnv Amnv Amev *pcmspmmmh mzocw (zo compmmoamoucH moo 5Humx PmpOH 0 00020 0w 2 020 m 200 so ummcmgo mmmz mwnmz .02020 260m om umvnm mmmz A_E\0n 0.0V cwuumFomm ucm .A_E\0n 0.0V :WFzmcH .AFE\02 0.Fv mcocmpmouwpmoom um.00< 0.00 um.00 0.0m< N.N 0.20 m.m~ mmzog 0< HHH 0~.Nm m.0m~ “.0 0.NF ~.0F mmso; 2N H0 m0.00 F.Nmm 0.0 0.0F 0.0 memo; NF H A5060 Asamv “may Amnv Amsv mpcmspmmmp 02020 <20 cowpmmogcoucH 000 <20 #4000 0: cm0 mcwuwsxzh-m: 05 cm0 Fmpop page» mcwuwsxgeum: Pmmop <20 m2 acmevcmaxm .mmappau cmmco mo mmzoz 00 use .0< .20 .m_ pm msocwummu 2wage: 002 nmuaucH-<0z0 cw mmmmgpcxm <20 .m mpnmh Figure 1. 44 DNA synthesis in DMBA-induced rat mammary carcinoma at 12, 24, 48, and 96 hours of culture. Corticosterone, insulin and ovine prolactin were added to each group. Media were changed on day 2 ~in the group cultured for 96 hours. H3-thymidine was added to the media 4 hoursprior to termination of culture. Figure 1 graphically represents the results shown in Tables 1, 2 and 3. 45 Exp. #I ZCHD - |6{)- |2()'- 8C):- 40- Exp. #2 Exp. #3 . . nu nu nu nu .4 420 - - Au 8 4 ZCHD - 2803 <20 at .30 05263.7»: 3€HD - SCH) - 24o - 18() b 12() - Hours in culture Figure 1 46 for DNA synthesis, 120 hours was chosen for termination of subsequent studies because this timing permits changing the hormonal media at 2 and 4 days. Total DDE; total DNA; DNA per mg DOE; and total H3- thymidine incorporation were determined but not analyzed statistically because these parameters are of lesser importance for the evaluation of DNA synthesis. 8. Effect of Prolactin and Insulin on DNA Synthesis in 5-Day Organ Cultures of DMBA-Induced Rat Mammary Carcinoma The results illustrated in Tables 4, 5 and 6 show the changes in H3-thymidine per pg DNA (cpm) which were derived from a series of experiments to determine whether insulin was prerequisite for the stimu- latory effect of prolactin on DNA synthesis in 5-day organ cultures of DMBA-induced rat mammary carcinoma. It is apparent that the combination of insulin and prolactin markedly increased DNA synthesis of rat mam- mary carcinoma compared with cultures containing insulin (P< 0.05) or prolactin (P<:0.01) (Tables 4, 5, and 6) as was indicated by the statistical analysis. Although H3-thymidine incorporation per pg DNA appeared to be consistently higher in the insulin-treated cultures than the prolactin-treated cultures, these differences were not found to be significant. Nevertheless, a significant synergism between insulin and prolactin on enhanced DNA synthesis has been demonstrated in this study (Figures 2 and 9). 47 .pcmcmuywv zpuc000002000 00: n 0\0 ._0.0V0 u 03 .000 v0 u 0\0 .Am0mmmp 20200 yo uo0cpxm umuu0mm0.lump0cvzsmuv 00044 .mmzarau we 00000005200 00 20000 00002 < 000mg mgp op um000 m0; m00002020102 .< 000 N 000 cc 0m00020 mmmz 00002 .00020 200m 0» 0m000 m0; APE\00 0.—v mcommpmouwpmoox 00.020 0.000 0.2 0.0_ 0.0 220\00 0.00 000002020 + 2_e\02 0.00 0__0000 000 00.00 0.00 0.0 0.0 0._2 220\00 0.00 000002020 00 00.202 0.000 0.2 0.02 0.22 220\02 0.00 0020000 0 A5000 Asaov Amnv A000 Amev 400mEp0mLH 0:020 <20 2000020020000 000 <20 44000 0: mma m000wszsplmz 02 mm0 F0000 F0000 00_020220-02 20002 <20 F0 acmswmmgxm .050000000 0000502 002 umozvcHl<020 0o mmngpzu :0020 200-0 :0 000020000 <20 no 0000020 000 200000000 00 uum000 .< 00000 .pcm200400 020:0uwmwcmwm no: u 0\0 .—0.0v0 u 03 .00.0V0 u 0\0 .2000000 20200 00 uu02uxm 0m000»m0.u0mu0200200v 00044 .0200200 00 00000000200 00 20020 0200; 0 000mg mg“ 00 00000 002 000005220102 .0 0:0 N 000 cc 0m02020 m2mz 000m: .00020 000m 00 0m000 003 AFE\02 0._v mco2mamoowp2o04 48 00.200 0.200 0.0 0.02 0.0_ 220\00 0.00 0_000_020 + 2_E\00 0.00 0020000 000 00.002 0.00 0.0 0.0 0.22 2_e\0< 0.00 200002020 00 0_.00_ 0.000 0.0 0.0 0.02 2_2\02 0.0V 2__0000 0 As0uv Agaov 200v Amnv A050 4ucmsp0m2h 00020 <20 0000020020000 000 <20 44000 0: 2m0 mcw0wsxghlmz 05 2m0 F0000 F0000 000000220-00 _0002 <20 N0 pcmsw2m0x0 .050000200 020500: 002 0m000001<020 mo 0m200200 :0020 000-0 :0 0_0m;pcxm <20 :0 =PF0000 0:0 00000Po20 mo pommmm .m m_000 .0 000 N 000 cc 0m00020 m2m3 000m: .pcm2mmww0 0puc000400000 00: u 0\0 .Ho.0v0 u 03 .0o.0v0 u 0\0 .Hm00000 20500 mo 0002uxm 000000m0.-0m002000m0v 00044 .m2spH00 mo cowp0cws2mp op 20020 0200; 0 000mg mg» 00 00000 003 m00002000-m: .00020 200m 0» 0m000 003 HHE\0: 0._V mco2m00oowp2ou4 0N. ””””””””” 240 - 200 - l60 .— IZO - 80 - _ _ — _ O O O 0 O m w. e 4 2:03 <20 oi .3 0525203»: Exp. #3 I40 l- IZO *- IOO - 80 '- 60 '- 40 - 20 - 52 C. Effect of Prolactin and Estrogen on DNA Synthesis in 5-Day Organ Cultures of DMBA-Induced Mammary,Carcinoma of Ovariectomized Rats Tables 7, 8 and 9 illustrate the different effects of estrogen and prolactin on DNA synthesis. The tumors at the time of culture were in a state of regression indicating a reduction of approximately 35% in mean tumor diameter during the l0 day period following ovariec- tomy. When prolactin was added to synthetic media containing explants of mammary carcinoma, a significant degree (P<:0.05) of increase in H3-thymidine incorporation into DNA was observed in comparison to con- trols. HOwever, estrogen under identical experimental conditions was without significant effect in comparison to controls. There was a significant effect on DNA synthesis when the prolactin-treated group was compared to the estrogen-treated group (P<:0.0l). Combined 3-thymidine incorpora- estrogen and prolactin increased significantly H tion (P< 0.05) in comparison to estrogen-treated cultures, the increase being an almost 95% significant effect on DNA synthesis when the combination-treated cultures were compared to controls. However, there was not a significant difference between estrogen + prolactin- treated cultures and cultures treated with prolactin alone (Figure 3). D. Effect of Prolactin and Ovine Growth Hormone on DNA Synthesis in 5-Day Organ Cultures of DMBA-Induced Rat Mammary Carcinoma Other experiments were carried out to determine whether ovine prolactin was significantly different from ovine growth hormone in its effect upon DNA synthesis in 5-day organ cultures of DMBA-induced :CJJUC—I<§z: +0 IULZJPZU :EC1C )C:Im Cm mwmfliuC)W <22 CC CZCCS&UL 1:! iv+§£n:$: KC «iiikl N =~Q=k 53 .000000000 0000000000000 000 u 000 .000 .000 .mo.ovn_ u U\0 ..mo.ov0_ u 03 .8.ov0 n 03 .0000000 00000 00 uu00px0 00pu0000..000000x000v 00040 .00000000 00 00000000000 op 00000 00:00 0 00000 000 on 00000 003 00000000pum: .0 000 N 000 00 0000000 0003 0000: .00000 0000 0» 00000 0003 000\0n 0.0v 0000000 000 00000: 0.00 000000000000004 00.00 0.00 0.0 0.00 0.00 000000 0.00 000000000 + 0_E\0: 00.00 00000000 >0 U0.00 0.00 0.0 0.00 0.00 00000: 0.00 000000000 000 00.0 0.0 0.0 0.00 0.00 00000 00.00 00000000 00 00.00 0.00 0.0 0.00 0.00 -- 0 00000 00000 0000 0000 0000 4000000000 00000 000 0000000000000 000 020 44000 0: 000 000000000- 2 00 000 00000 00000 000000000-00 00000 0 020 00 00000000x0 .000 000000000000>o 00 000000000 000000: 0000000-<020 mo 00000000 00000 000-0 00 000000000 <20 00 00000000 000 000000000 00 000000 .0 00000 54 .000000000 0000000000000 000 u 000 .000 .000 .mo.ov0 u 0\0 .mo.ov0 u 03 .—o.ovn_ n 03 .0000000 00000 00 00000x0 00000000.:00000000000 00004 .00000000 00 00000000000 00 00000 00:00 0 00000 000 00 00000 003 000000000- 0 .0 000 N 000 00 0000000 0003 00002 .00000 0000 00 00000 0003 APE\00 0.00 0000000 000 00000: 0.0 000000000000004 00.00 0.00 0.0 0.00 0.00 000000 0.00 000000000 + 000000 00.00 00000000 >0 00.00 0.00 0.0 0.00 0.00 000000 0.00 000000000 000 00.00 0.00 0.0 0.00 0.00 000000 00.00 00000000 00 00.0 0.00 0.0 0.00 0.00 -- 0 00000 00000 0000 0000 A005 400000000» 00000 000 0000000000000 000 020 00000 00 000 000000000- I 00 000 00000 0000» 000000000-00 00000 0 020 00 00000000x0 .000 000000000000>0 00 000000000 000000: 0000000-<020 00 00000000 00000 000:0 00 000000000 <20 00 00000000 000 000000000 00 000000 .0 00000 55 .pcmgmw$wv »_p=muw¥wcmwm go: " v\u .v\m .n\m .mo.ovm u u? .mo.ovm u n3 .Fodvm u 03 .Amsmmmp gosap mo “umgpxm umupmwmv.uumpmgvxsmnv mao«* .mmgzp_:o mo cowpwcwELmu op Lewga mgzo; c m_vme as» o» cmuvm mm: mcwcwsxcp- : .v new N mac co vmmcmgu mgmz mwvmz .asogm Loam 0p umuvm mgmz AFE\m: o.mv :WFamcH new AFE\m1 o.Pm mcogmumouwugou« n_.o¢ n.m¢ o._ o.m_ _.m AFs\m1 o.mv =_pum_ogm + A_E\m1 _o.ov cmmogpmm >H u¢.Nm m.0m_ m.~ m.o_ m.m Ays\m; o.mv cwuum_oga HHH nm.m F.5m m.F o.m_ N._F A_E\mn _o.ov :mmogpmm HH m¢._F m._¢ m.o N.¢F m.op -- H Asaov Asqov Anny Amav Amsv *pcmsummg» aaogo <20 cowgmgoqgoucH was <20 *;moo an Lag mcwnwsxgbumz ms Lma FmpOF page» mewuwsxgp-mx Pouch <20 M% ucwswgmaxm .pmm umNPEouumwgm>o we msocwugmu xngEc: nmoaucHu*- C) Figure 3 58 rat mammary carcinoma. The results illustrated in Tables l0, ll, 12 and 13 indicate that prolactin-treated cultures increased H3-thymidine incorporation significantly (P< 0.05) in comparison to controls. However, there was neither a significant effect between the growth hormone-treated group and the control group; nor between the prolactin- treated group and the growth hormone-treated group in increasing H3- thymidine incorporation per ug DNA (Figure 4). Ovine growthhormone appeared to increase DNA synthesis when compared to controls because 3/4 of the cultures treated with growth hormone, when analyzed 3 separately, showed an increase in uptake of H -thymidine incorporation. However, the 4 studies combined did not show significant differences. E. Effect of Prolactin and Human Growth Hormone, Alone and in Combination on DNA Synthesis in 5-Day,0rgan Cultures of DMBA-Induced Rat Mammary Carcinoma The results illustrated in Tables l4, l5 and 16 show the effects of prolactin and human growth hormone, alone and in combination, on H3 -thymidine incorporation into DNA (cpm). The prolactin-treated group showed significantly (P‘=0.05) increased H3-thymidine incorporation in comparison to controls. However, human growth hormone-treated groups did not respond significantly when compared with controls or the pro- lactin-treated groups. The combination of human growth hormone and prolactin significantly (P< 0.01) increase H3-thymidine incorporation when compared to controls; or human growth hormone-treated groups (P< 0.05). However, there was no significant difference between the combined hormone-treated groups and the prolactin-treated groups (Figure 5). 59 .ocmcmcc_u x_u=auwcwcmwm “a: u U\n .n\a .85 va u a}. .33qu LOEB we uomgaxm umpumwmv lumpmgucEwE moot}, .mczuFau mo co_pchEcmH op Lo_ca mesa; w ownme mg» op nmnum mm: mcwvwex5u- I .¢ new N Xmu :o uwmcmco wgmz mwumz .qzocm comm op cmvvm mcmz Ape\mn o.mv :w_:m:H can Afie\mn o._ mcogmumoowpgoo« um.mmF m._mP N.0 o.m_ P.NN A_E\ma o.mv accumFoca HHH no.mm_ N.0“ m.o m.© ¢.mp APE\m; o.mv acoeLo: guzocw m=_>o HH ao.mp m.m F.F o.“ 0.9 -- H Asaov Agaov Anny Anny Amev aucmEpmmgp azocm <20 cowpacoatoocH was <20 ..mao a: con mcwuwsxch-mz as con Pouch Pouch mequexgp-mI Pouch 42o _% acme_cmaxm .meocwocmo xngEm: pom umusucH-o new :Ppuupocq mo pumemm .o_ m_nah 60 .pcmcacc_u s.u=auwc_em_m be: u U\n .n\a .86 X. n u} .Amsmmwu Logsu mo pumguxm vmupmmmu.uumpmgua;muv moo¥« .mcappsu we cowpmcwscmu op Lowgg mgzo: v ovums asp op umuum mm: mcmuwexgp- I .e use N xmu co ummcogu mgmz mwvmz .aaocm :umm op umuum mcmz A_E\mn o.mv :WFzmcH ucm Aps\mn o._ mcocmpmoumagou« un.m- m.mm ~.N m.o N.0 A_E\mn o.mv cwuom_oga HHH no.mm N.o¢ m.F o.m_ N.m A_E\m: o.mv acoscox cpzoco acc>o HH mm.ON m.NF N.N m.FF m.m .. 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H Asauv Asaov “may Amnv Amev «pcmsummgp azogo o new cwpumpoga yo pumeem .Np m_nmp 62 .oc3HHoo Ho coHHocwscop op LoHLo mcoos o owoos on“ op ooooo mo: ocwowsxgp- I .ooocm zoom op omooo moo: AHE\m: o.mv cHHomcH oco AHE\o: o.H :o oomcogo moo: ovum: .ocmcaccHo HHpcaochcmHm o0: u U\n .n\a .mo.o vm u U\o .HoommHH Loss» mo poocuxo ooppommo..oouogo>zoov moo*« .o oco N xmo ocogogmooHHHouc m.on un.omm m.o o.m N.m HHE\ma o.mv CHHUMHOLQ HHH om.omN N.NNH v.0 o.m m.m AHE\ma o.mv ocoscoI cuzocu ocw>o HH mn.mn m.mm m.o m.m m.NH .. H HEoov “Eoov Anny Hazy Huey «pcmsuooch ooogw o oco cwuoopogo mo HomHmm .mH oHnoh Figure 4. 63 Effect of Prolactin (P) and Ovine Growth Hormone (GH) on DNA synthesis in 5-day organ cultures of DMBA- induced rat mammary carcinoma. ' Corticosterone and insulin were added to each group. Media were changed on day 2 and 4. H3-thymidine was added to that media 4 hours prior to termination of culture. Figure 4 graphically represents the results shown in Tables 10, ll, l2 and 13. 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Results also indicate that the number of cells was sig- nificantly increased in group II (corticosterone) (P<:0.05); group III (corticosterone + insulin) (P<:0.005); and group IV (corticosterone + prolactin) (P< 0.005) when compared to controls (Group 1). However, there were not significant differences between groups II and III; groups 11 and IV; or groups III and IV. Plating density did not sig- nificantly influence the average number of cells per well. b) After 24 Hours of Culture Average Number of Cells per Petri Dish The effects of combined insulin, prolactin and corticosterone on the growth of collagenase dispersed cells of DMBA-induced rat mammary carcinoma after 24 hours of culture in petri dishes are shown in Table 32 and illustrated in Figures 9 and ll. lOl .HzmzomHHo HHHcooHHH00H0 .00 u o\0 .o\0 .0H0 M0Ho n 00.0 V. 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HHH 000.0 0> HH 000.0 Hzm.mHHH0 xHuzooHHHzmHm Ho: .HHH 000.0 0> HH 000.0 H000.0”.0. 0 000 0H 0000.0 00 HHH 000 HH 0000.0 H000.0“v0. 0 000 0H 0000.0 00 HHH 000 HH 0000.0 H000.0_v0. 0 000 0H .HHH .HH 0000.0 00 H 000.0 H000.0 V0. 0 000 0H .HHH .HH 0000.0 00 H 000.0 “0.30: wv "wucmoHHHcmHm ..Ho Hw>mm "0.50: 0N "mucooCHcmHm ..Ho w~m>mm ..o..m 0.00:0H0.H zomII0 .mm mHaoh .00 .0E0H so.H 0m>H.m0 m.mz 0HHmo mmmzh .zmHo H.Hm0 .m0 mHHmo .0E0H 000H .0 HHHmzmo o Ho 0mHoH0 m.m3 0m.0HH00 .000.m zoom zH 0mz0H0 H.0m0 m m.m3 m.mzH* HHEH00 0.0. 0H000H0.0 I HHEH00 0.0. 0HH000H + 0.0.0 0.00H 0.00 + 0.0H0 HHEH00 0.H. 000.00000H0.00 , II I. HHE\00 0.0. :HHooHo.0 + 0.0 + 0.00 0.0H + 0.0HH HHEH00 0.H. 000.00000H0.00 0H I. I. HHE\00 0.0. 0H.000H + 0.H + 0.00 0.0 + 0.00 HHEH00 0.H. 000.00000H0.00 HHH 0.H.“ 0.00 0.H .H 0.00 HHEH00 0.H. 000.00000H0.00 HH 0.0.“ 0.00 0.HH 0.0.00 I- H 0.00: 00 0.00I 0N 00.09500... 000.0 ««z0Ho H.Hm0 .m0.0HHm0 0mzoouuoz: H0 .sz0z mmo.m>< m.0HH00 Ho 0.00I 00 00o 0N .mHHo osozHo.o0 H.oEEoI pom 0mo0ozHI<020 .0 0HHmo 000.m00H0 mmozmmoHHoo .0 zH20.0 :0 mzo.mpmooHH.00 00o cHHooHo.0 .zHH00zH Ho HomHHm .00 mHon l09 with groups 11 (corticosterone) and III (corticosterone + insulin) at 24 and 48 hours of culture (P<:0.005). The effect of insulin, in the absence of prolactin, was analyzed by comparing groups II and III at 24 and 48 hours. These results indicate that there was no signifi- cant effect of insulin at 24 hours of culture; but the presence of insulin at 48 hours of culture resulted in an increased number of cells (P< 0.00l). The effect of insulin, in the presence of prolactin, was analyzed by comparing groups IV and V at 24 and 48 hours of culture. Results indicate that the presence of insulin (Group V) significantly increased the number of cells in comparison to group IV at 24 and 48 hours of culture (P< 0.005). A sufficient number of free cells (unattached) were not found in any of the other colony-forming culture studies (Section II-A). 2. Effect of Fetal Calf Serum on Growth of Collagenase- Dispersed Cells of DMBA-Induced Rat Mammary Carcinoma after 24 and 48 Hours of Culture The effects of fetal calf serum on the growth of collagenase dispersed cells of DMBA-induced rat mammary carcinoma after 24 and 48 hours of culture are shown in Table 35. Only the average number of unattached cells per petri dish was analyzed in this experiment, because of insufficient number of attached cells. Results of these studies indicate that the combination of insulin, prolactin, corticosterone and fetal calf serum (Group IV) significantly increased the number of cells in comparison to all other groups (I, II, and III) (P< 0.005). Analysis of the data also indicates that group II (fetal calf serum), group III (insulin + prolactin + corticosterone) and group IV showed 110 .00 0H000 .0500 0000.0.v00 >H 000 HH 0000.0 0> HHH 000 H 0000.0 0000.0.v0v >H 000 HHH 0000.0 00 HH 000 H 0000.0 000 .0 0000000 0 00 000000 0.03 00.00000 0000.00v00 0H 000.0 00 HHH 000.0 Amoo.o_v0v >H .0 HHH 0000.0 0> HH 000.0 0000.00.00 >H .0 HHH .HH 0000.0 0> H 000.0 000000000000 00 000>00 ..0..0 0.000000.H 0002.3. .0000 0.000 .000.0 0000 :0 000000 0.000 m 0.03 0.0000 A0000 50.00 0000 ~0000 + AF5\00 o._v 000.0000000.00 + 0H0\00 0.00 0.000H0.0 + 0.H.“ 0.00H 0.0.0 0.000 000\00 0.00 00H000H 0H A—5\00 0.Hv 0:0.0000000.00 + I. I. 0H0\00 0.00 00000H0.0 + 0.0 + 0.00 0.0 + 0.00 A00\00 0.00 00H000H HHH 0.0.0 0.0. 0.0.0 0.00H .00H0 00.00 0.00 H0000 HH 0.H.“ 0.00 0.0.0 0.00 -- H 0.00: 00 0.00: cm 00005000: 000.0 «00000 0.000 .00.0H000 0000000000 00 .0050: 000.0>< yam UmuzbcHll.5 times the incorporation of H3-thymidine into DNA in comparison to cultures lacking prolactin. This stimulatory effect was consistent in all three replications (Tables 4—6; Figures 2 and 9). In another set of experiments (Tables lO-l3, Figure 4), prolactin in the presence of insulin and corticosterone consistently stimulated (> 2.3) H3-thymidine incorporation into DNA in 5—day organ cultures of DMBA-induced rat mammary carcinoma of intact rats, in comparison to cultures lacking prolactin; and in a third set of experiments, prolactin again consistently stimulated (> 3.8 times) H3-thymidine into DNA in comparison to cultures lacking prolactin (Tables l4-l6; Figures 5 and 6). Prolactin, in the presence of insulin and corticosterone, was also 3-thymidine into DNA able to stimulate 2.7 times the incorporation of H in 5-day organ cultures of DMBA-induced mammary carcinoma of ovariectomized rats, in comparison to cultures lacking prolactin (Tables 7-9; Figure 3). Autoradiographic studies of explants of mammary carcinoma ob- tained from intact rats confirmed the stimulatory effect of prolactin, in the presence of insulin and corticosterone, upon cell growth. ll4 The results of such experiments parallel the isotope studies, i.e., prolactin significantly increases the number of cells having silver grains (engaged in DNA synthesis) when compared to controls. The labeling index was 5.6 times greater in cultures containing the triad of hormones than in those cultures lacking prolactin (Figure 6). These studies also showed that epithelial cells and not connective tissue elements incorporated H3-thymidine into DNA, as was indicated by the presence of silver grains only over epithelial cells (Figure 8). These results therefore indicate that epithelial cells are the ele- ments responsive to hormonal stimulation in rat mammary carcinoma. These findings have been previously reported by Juergens gt_al, (1965), who found that the primary site of DNA synthesis was the epithelial cells when pregnant mouse mammary tissue was cultured in the presence of prolactin, insulin and corticosterone. Although it was clear that prolactin consistently stimulated H3 -thymidine incorporation into DNA of organ cultures of rat mammary carcinoma, it was not known whether this enhanced DNA synthesis truly reflected an increased cell number. When dispersed cells of DMBA-induced rat mammary carcinoma were cultured for five days in the presence of prolactin, insulin and corti- costerone, the number and area of colonies were 3.9 times and 7.3 times, respectively, greater than in cultures lacking prolactin (Tables 27-29; Figure 9). When dispersed cells were plated in micro- plates in the presence of prolactin, insulin and corticosterone for 24 hours, the number of cells was 2.4 times greater than cells cultured in media lacking prolactin (Table 3l, Figure 9). When dispersed cells ll5 were plated in petri dishes in the presence of all three hormones for 24 hours, the number of cells was also consistently greater (2.3 times) than in cells whose culture media lacked prolactin (Table 32; Figures 9 and ll). Furthermore, under similar conditions (cells cultured for 48 hours), the three hormones combined increased the number of cells to 2.5 times that of cells cultured in media lacking prolactin (Table 33; Figures 9 and ll). Studies of unattached dispersed cells of rat mammary carcinoma indicated that cells grown in the presence of prolac« tin, insulin and corticosterone increased the number of cells to 2.7 times and l.6 times that of cells cultured in media lacking prolactin, at 24 and 48 hours, respectively (Table 34). Thus, these studies pro— vide convincing evidence that the addition of prolactin to the culture media results in a profound increase in mammary tumor cell number, corroborating the results obtained in the H3-thymidine—incorporation culture studies. These results are in accord with other jn_vjtrg_experiments pro— viding evidence that prolactin actively stimulates DNA synthesis in organ cultures of DMBA-induced rat mammary carcinoma in the presence of corticosterone and insulin (Welsch and Rivera, l972). Hollander (l970) also showed that prolactin increased DNA synthesis, indicated by H3-thymidine incorporation into DNA of rat mammary carcinoma (MT/W9). Prolactin has also been shown to be mitogenic jn_vjtrg_when using normal rat mammary tissue.) Dilley (l97l) observed that insulin plus prolactin maintained initial mitotic activity in 5—day cultures of immature rat mammary glands; corticosterone was not used in those cultures. According to Topper and his group, prolactin has no mitogenic l16 effect in mouse mammary tissue in_vitrg_(0ka and Topper, l972). This is further documented by the work of Turkington (l968) showing that prolactin with or without insulin, induced no change in labeling and mitotic indexes of mouse mammary gland explants in_vitrg, The differ- ence between these findings and other reports could be attributable to species differences or perhaps to the age of the animals used. Dilley (l97l) for example, used rats one month of age, an age when mammary glands are proliferative and have a high mitotic index, pos- sibly enhancing the response to prolactin. However, Lasfargues and Murray (l959) indicated a possible stimulatory effect of prolactin on mouse mammary epithelial cell proliferation jn_vit§9, even though they did not use quantitative measurements of mitotic activity. El-Darwish and Rivera (l970) observed that prolactin alone had a moderately stimulatory influence on DNA content during the first two days of cul- ture of mouse mammary glands. However, combination of insulin and prolactin resulted in higher stimulation than did either hormone alone. Juergens §t_al, (l965), reported that insulin and prolactin were 50% more active in promoting DNA synthesis than exclusively insulin- treated cultures (48 hours) from mammary tissue of pregnant mice. It appears, therefore, that prolactin has a stimulatory effect on the cell growth of rat mammary carcinoma and both mouse and rat mammary glands jg_vitro. The jn_vitro studies using normal and neoplastic rat mammary tissue parallel the results of a number of ig_vivo studies. Increased prolactin secretion results in enhanced growth of spontaneous and carcinogen-induced rat mammary tumors (Meites §t_al., l972). Known to 117 increase blood-prolactin levels in rats are various treatments, such as pituitary homografts (Chen §t__l,, 1970); median-eminence lesions (Welsch gt 21,, l970); and certain drugs (Lu gt al,, 1970). Such treatments are also reported to stimulate carcinogen-induced mammary tumor growth in intact female rats (Welsch gt_al,, l968, l969; Welsch and Meites, l970). Furthermore, the administration of prolactin for at least l5 days stimulated tumor growth of DMBA-induced rat mammary carcinomas, which had been regressing as the result of adrenalectomy and ovariectomy (Nagasawa and Yanai, 1970), or adrenalectomy, ovariec- tomy and hypophysectomy (Pearson §t__l,, 1969). In ovariectomized mammary tumor bearing rats, however, pituitary homografts (Welsch _t_al,, l968); median eminence lesions (Welsch gt_al,, l969); and reserpine (Welsch and Meites, l970), treatments which markedly increase prolactin secretion generally lack mammary tumor stimulatory effect. It appears, therefore, that the mammary tumor must be sensitized by ovarian hormones, prerequisite to the stimulatory activity of prolactin. The fact that animals from some of the present experiments were previ- ously ovariectomized and that their mammary tumors responded signifi- cantly to prolactin in_vitrg_(Tables 7-9, Figure 3) seems to indicate that ovarian hormones are not necessary for tumor growth in animals having sufficiently high levels of prolactin. However, it is possible that the mammary tumor cells were sufficiently sensitized by ovarian hormones before ovariectomy and prior to tumor removal for cultures. In support of this view, mammary tumor bearing rats which were ovariec- tomized and lesioned in the median eminence at the same time, responded with an initial increase in mammary tumor growth (for at least l0 days) ll8 followed by a decrease in tumor growth despite consistently high levels of blood prolactin (Welsch gt al,, 1968; Klaiber gt al,, 1969; Dao and Sinha, l972). In comparing the effects of prolactin and corticosterone, in combination, to cultures containing only corticosterone, cells cul- tured for 24 and 48 hours showed slight effect of prolactin, whereas cells cultured for l20 hours showed a greater stimulatory effect of this hormone. When cells were plated in either microplates or petri dishes in the presence of prolactin and corticosterone for 24 hours of cul- ture, the number of cells observed at termination of these cultures, was not significantly different from the number of cells cultured in media lacking prolactin (Tables 31, 32). There was, however, a sig- nificant increase in cell number in corticosterone plus prolactin- treated cultures in comparison with cultures containing corticosterone alone when unattached, dispersed carcinoma cells were analyzed (Table 34). Because this type of study (analysis of unattached cells) was only done once, conclusive evidence can not be drawn from this experiment. When dispersed cells of rat mammary carcinoma were cul- tured for 48 hours in the presence of prolactin and corticosterone (compared with corticosterone alone), the number of cells observed at the end of culture was significantly greater in only one of the three cultures (Table 33; Figure ll). Furthermore, studies of unattached dispersed cells of rat mammary carcinoma cultured for 48 hours show that the cell number was not significantly greater in the presence of the two hormones than in cultures lacking prolactin (Table 34). Cultures were further analyzedat five days of culture. Results show that the combination of prolactin and corticosterone increased 3.4 and l19 3.6 times the colony number and area, respectively, in comparison to cultures lacking prolactin (Tables 27-29). Therefore, these results indicated that the combination of prolactin and corticosterone initial- ly (24 and 48 hours of culture) had slight stimulatory effect upon cellular growth of rat mammary carcinoma. At five days of culture, however, results showed that prolactin plus corticosterone—treated cultures had a greater stimulatory effect on cell growth, when com— pared with cultures lacking prolactin. El-Darwish and Rivera (l970) also compared the effects of prolactin and corticosterone on cell division of mouse mammary tissue in 31339: They showed that these two hormones combined markedly enhanced DNA synthesis of mouse mammary tissue during the first three days of culture and that this effect is inhibited with time. These results are opposed to those obtained in these studies which showed that prolactin and corticosterone at five days of culture had greater stimulatory effect on cell growth, when compared with cultures lacking prolactin. This study also demonstrates that estradiol-I78 failed to stimu— late or reactivate in 11339 DNA synthesis in DMBA-induced rat mammary carcinoma cells obtained from ovariectomized rats. It was interesting to observe that estrogen could not reactivate growth of these tumors. The tumors at the time of culture were in a state of regression because of ovariectomy, and presumably, one could have expected at least some response to estrogen for the lack of this hormone i__vivo, at least in part, caused this regression. Therefore, these findings further empha- size the important role of other hormones in addition to ovarian hor- mones in inducing and maintaining the growth of rat mammary carcinomas. l20 However, the fact that these tumors were regressing after ovariectomy may indicate that estrogens have a role jn_vivg_that cannot be demon- strated ig_vitrg, at least under these experimental conditions, i.e., stimulating pituitary-prolactin secretion. Neither did tumors derived from intact rats and cultured jg_vitrg respond to physiological levels of estrogen with increased DNA synthesis (Welsch and Rivera, l972) which further supports this supposition. Thus, it appears that car- cinogen-induced rat mammary carcinoma, whether obtained from ovariec- tomized or intact tumor-bearing rats, does not respond with increased DNA synthesis after addition of estrogen to the culture media. One cannot exclude, however, the possibility that physiological levels of estrogen may stimulate other important processes and/or synergize with other hormones for enhancement of DNA synthesis. Other authors have also reported the lack of stimulatory effect of estrogen on rat mammary tumor growth (DNA synthesis) jg_vjtrg, Thus, Hollander (1970) observed that estrogen had no effect on H3 -thymidine incorporation into DNA of rat mammary tumors (MT/W9). Dao and Sinha (l972) confirmed the lack of effect of estrogen on DMBA-induced rat mammary tumor growth in 11329: Turkington and Hilf (l968) observed that physiological dose levels of estradiol-l7 were also totally ineffective in influencing DNA synthesis of rat mammary tumors (R3230AC). Estrogen has also been shown to have no effect on DNA synthesis of mouse mammary tumors, normal mammary epithelial cells (Turkington and Hilf, 1967) or rat mammary gland (Koyama gt _1,, l972) in culture. It is also possible that the failure of estrogen to stimulate mammary tumors jg_vitro, as observed in these studies, may reflect the dosage of the hormone added to the culture lZl media. It is common to use 0.0l pg per ml of medium as a "physiological" dose of estrogens jg_vitrg, This level is approximately lO-lOO times the level of estrogens found in the blood of most mammals (Esber gt_al,, l973). Taking into consideration that the amount of estrogens used in_!jt§g_could not as effectively reach the cells, because of a lack of a functional vascular bed, it is common to use higher amounts of hor- mones jg_vitro than the "physiological" levels found in_vivo. Welsch and Rivera (1972) used different doses of estrogens, i.e., l/lO, l/lOO, of that found in the blood, and they did not observe significant effects on DNA synthesis. There is no doubt that high levels of estrogen in- hibit DNA synthesis of mammary tissue in vitro in the rat (Welsch and Rivera, l972) and in mice (Turkington and Hilf, l968), as well as jn_vjvg_in the rat (Huggins §t_a1,, l959b), and human (Stoll, l969). It has also been suggested that estrogen can act directly on mammary tissue increasing specific activity of certain enzyme systems in 313:2 (Hollander gt 21,, 1958; Hilf gt_al,, 1966). These studies further analyzed the effects of prolactin and estradiol-l7B, in combination,i.e., is there a synergistic effect of these two hormones on DNA synthesis of DMBA-induced mammary carci- noma obtained from ovariectomized rats? Results of the present study show that combination of prolactin and estrogen increased 2.1 times the H3-thymidine incorporation into DNA in comparison to controls, whereas prolactin alone promoted a 2.7 times the concentration of the isotope into DNA (Tables 7-9; Figure 3). This difference, however, was not found to be significant. Therefore, these studies suggest 122 that under these experimental conditions there does not appear to be a synergism between these two hormones. The lack of demonstrable synergism between the two hormones does not rule out the possibility that synergism exists. The dosage used was perhaps not optimal for demonstrating the synergistic effect, i.e., it may have been too low or too high. It has been established that high doses of estrogen interfere with stimulatory effects of prolactin on cellular proliferav tion (Meites, l972; Welsch and Rivera, l972). Meites gt a1, (197l), provided jn_vivo evidence that large doses of estrogen directly in- hibited prolactin induced stimulation of DMBAvinduced rat mammary tumor growth. It is possible that large doses of estrogen inhibit the action of prolactin on mammary tumor growth by blocking binding of prolactin to receptor sites. This antagonistic effect has also been observed in normal mammary development, growth and lactation jg_vjvg (Meites and Nicoll, 1966), but the mechanism is still unknown. Furthermore, Welsch and Rivera (l972) demonstrated in_vitrg_that estrogen can also block the prolactin stimulatory effect on DNA synthesis of DMBA—induced rat mammary carcinoma. It is interesting to observe that although Welsch and Rivera (l972) used the same dosage of estrogen that was used in this study (0.01 mg Per ml) in combination with prolactin, as in this study, a significant effect of the steroid on the inhibitory effect of prolactin was observed. It is possible that tumors derived from ovariectomized rats, i.e., tumors not exposed to estrogen for a long period of time jn_vjyg, are more responsive to the combination of estrogen and prolactin than tumors derived from intact rats, i.e., tumors 123 continuously exposed to estrogen. Therefore, a synergism may exist between prolactin and estrogen (in 31359) in regressing tumor tissue as a result of ovariectomy. Although a synergism has not been un- equivocally observed ifl_vjt§9, synergism between these two hormones has been observed jn_vivo. Dao and Sinha (l972) showed a distinct synergism between ovarian hormones and prolactin in the growth of DMBA-induced rat mammary tumors 1 vivo. They reported that growth of carcinogen-induced rat mammary tumors could be reactivated in ovari- ectomized, median eminence-lesioned rats tn! ovarian grafts. Further- more, Nagasawa and Yanai (l97l) demonstrated jn_vj!9, that estrogen increased the mammary gland response of ovariectomized rats to the stimulatory effects of prolactin (from pituitary grafts). These studies also analyzed the effect of growth hormone on DNA synthesis in DMBA-induced rat mammary tumors obtained from intact rats. Results suggest that either ovine (Tables lO-l3; Figure 4) or human (Tables l4-l6; Figures 5, 6) growth hormone alone has either no effect or only a slight stimulatory effect on DNA synthesis in_vitro. In the ovine growth hormone studies.statistical evaluation of the four experiments combined showed no significant stimulatory effect of the hormone. However, when each experiment was analyzed separately, three of the four cultures responsed positively to the stimulatory effects of the hormone. In the human growth hormone studies, two of the three cultures responded positively to the stimulatory effects of the hormone. Therefore, these results suggest that ovine or human growth hormone may have some role in the stimulation of DNA synthesis. A firm interpreta- tion of these results in terms of whether either growth hormone had a 124 stimulatory effect, is extremely difficult. I am inclined to feel that the results of this study indicate a slight stimulation by growth hormone. This is strengthened by further analysis of the data, indi- cating no significant difference in DNA synthesis between either ovine or human growth hormone-treated cultures when compared with prolactin- treated cultures. Growth hormone probably has a slight stimulatory effect on the growth of rat mammary carcinoma jn_vitrg_but this effect is consistently less than that of prolactin. These conclusions are not, however, consistent with some 12.!ivg_experiments which demon- strated that administered growth hormone (bovine) did not affect mammary tumor growth in ovariectomized-adrenalectomized (Nagasawa and Yanai, 1970); nor ovariectomized-adrenalectomized-hypophysectomized (Pearson gt__l,, l969) rats bearing DMBA-induced mammary tumors (regressing for at least l5 days). However, Young (l96l) reported that growth hormone (bovine), in conjunction with ovarian hormones, induced mammary tumor formation in carcinogen-treated hypophysectomized rats. In vitro studies have also been contradictory. Hollander (l970) reported that growth hormone failed to increase H3-thymidine incorpora- tion into DNA of rat mammary tumors after 6-l2 days of culture. However, Turkington gt_gl, (1973) observed that growth hormone added to synthe- tic media augmented the rate at which epithelial cells from mouse ’ mammary glands initiated DNA synthesis. The discrepancy in these results may have resulted from the differences in timing of the experi- ments. Turkington gt 21, (1973), observed that growth hormone was stimulatory (DNA synthesis) during only the first 24 hours of culture, 125 since this hormone increased the number of epithelial cells which entered the S phase of the cell—cycle. Therefore, it can be suggested that growth hormone may have a role in mammary tumorigenesis, although a definitive conclusion can not be made at this point. Furthermore, one can not exclude the possibility that either ovine (Apostolakis, 1968) or human (Wilhelmi, 196l; Lee and Lew, l966; Apostolakis, l968) growth hormone was contaminated with prolactin, which might explain the slight stimulatory effect of this hormone. Unfortunately, auto— radiographic studies (Figure 6) did not provide additional insight into this problem. The results of the autoradiographic studies were identical to the isotope studies, i.e., human growth hormone did not significantly alter the number of cells having silver grains (engaged in DNA synthesis) when compared to either control or prolactinntreated cultures. Once again, growth hormone took an intermediate position, i.e., between control and prolactin-treated cultures in its effects on DNA synthesis of rat mammary carcinoma. These studies were important, however, in that they further demonstrated that epithelial cells and not connective tissue elements incorporated H3-thymidine, as indicated by the presence of silver grains only over epithelial cells (Figure 8). This provides further evidence that conneCtive tissue elements are not responsive to hormonal stimulation and the response that we are measur- ing (H3-thymidine incorporation into DNA) represents epithelial cell (carcinoma) activity and not non-cancerous connective tissue elements. Turkington (1968) using autoradiographic analysis, observed that hor- monal stimulation (growth hormone) increased the number of epithelial 126 cells of mouse mammary explants engaged in DNA synthesis jn_vitrg_and did not quantitatively influence connective tissue elements. This in- crease reached a maximum value at 24 hours of culture, suggesting a possible effect of growth hormone long before five days of culture. The effects of combined prolactin and human growth hormone on DNA synthesis was also analyzed in carcinogen-induced rat mammary carcinoma. Results of such studies show that combination of prolactin and human growth hormone under these experimental conditions markedly enhanced DNA synthesis after five days of culture (Tables l4-l6; Figures 5, 6). This hormonal combination was, however, as effective as prolactin alone in stimulating DNA synthesis. Combined prolactin and human growth hormone increased 6.1 times the H3 —thymidine incorpora- tion into DNA in comparison to control, in contrast to prolactin alone and human growth hormone alone, which increased 3.8 and 2.5 times, respectively. The stimulatory effect was also observed in autoradiographic studies. In the presence of these 2 hormones combined, the labelling index of mammary carcinoma cells was l6.5 times greater than the con- trols. In cultures treated with prolactin and human growth hormone alone, the labelling index was 5.6 and 2.7 times greater than the con- trols (Figure 6), as indicated by the presence of silver grains over the neoplastic cells. A striking parallelism observed between the labelling index and H3 -thymidine incorporation into chemically extracted DNA further corroborates the stimulatory effect ig_vitro of these two hormones. These results are in accord with an in_vitro study demon- strating that growth hormone magnified the effect of prolactin in 127 inducing epithelial cell growth of benign human hyperplastic mammary glands (Ceriani gt_al,, 1972). Furthermore, Meites (1965) showed an enhancement by growth hormone of prolactin-treated rat mammary growth 1 vivo. It appears, therefore, that there is a possible synergism between prolactin and human growth hormone in the growth of normal and neoplastic mammary tissue in vitro as well as in vivo. Since insulin has been extensively used in a number of culture studies using a variety of tissues, experiments were undertaken to determine whether insulin could influence the cell growth of rat mammary carcinoma. The studies were designed to determine whether insulin could influence the growth of rat mammary tumor cells in cultures con- taining l) corticosterone, or in cultures containing 2) corticosterone plus prolactin. In comparing the effects of insulin and corticosterone, in combination, to cultures containing only corticosterone, cells cul- tured for only 24 hours consistently showed no effect of insulin, whereas cell cultures for 48 and 120 hours showed a slight stimulatory effect of insulin. When cells were plated in either microplates or petri dishes in the presence of insulin and corticosterone for 24 hours of culture, the number of cells observed at termination of these cul- tures, was not significantly different from the number of cells cultured in media lacking insulin (Tables 3l, 32; Figure ll). These results sug- gest that insulin had no effect on cell growth after 24 hours of cul- ture. The results were further confirmed by studying unattached dispersed cells of rat mammary carcinoma. The cell number was once again not significantly different from that in cultures lacking insulin (Table 34). When dispersed cells of rat mammary carcinoma 128 were cultured for 48 hours in the presence of insulin and cortico- sterone (compared with corticosterone alone) the number of cells observed at the end of culture was significantly greater in only one of the three cultures (Table 33; Figure ll). There was, however, a significant increase in cell number in insulin plus corticosterone- treated cultures in comparison with cultures containing corticosterone alone when unattached, dispersed carcinoma cells were analyzed (Table 34). Because this type of study (analysis of unattached cells) was done only once, conclusive evidences cannot be drawn from this experi- ment. Further analysis of l20 hour—cultures also showed that when the combination of insulin and corticosterone was compared with cultures containing corticosterone alone, only one out of three of the cultures responded positively (stimulation) to the effects of insulin (Tables 27-29). These results suggest that the effects of insulin on the growth of rat mammary carcinoma cells cultured in media containing corticosterone were either absent (at 24 hours of culture) or slight (at 48 and l20 hours of culture). These results are generally in accord with El-Darwish and Rivera (l970) who reported a lack of a stimulatory effect of insulin upon DNA synthesis in corticosterone- containing cultures of mouse mammary tissue. One can conclude that corticosterone may interfere with the stimulatory effect of insulin; or, other hormonal factors may be deficient (corticosterone and/or prolactin). The next experiments were undertaken to determine whether insulin could stimulate cellular division of dispersed cells of DMBA-induced 129 rat mammary carcinoma in the presence of prolactin and corticosterone, after 24, 48 and l20 hours of culture. When dispersed cells were plated in microplates for 24 hours in the presence of insulin, pro- lactin, and corticosterone, the number of cells at the termination of culture was 2.4 times greater than the number of cells observed in media lacking insulin (Table 3l; Figure 9). When the cells were plated in petri dishes for 24 hours in the presence of all three hor- mones, the cell number was 2.8 times greater than the number of cells cultured in media lacking insulin (Table 32; Figures 9, ll). When the cells were plated in petri dishes for 48 hours, under similar experi- mental conditions, the cell number was 3.l times greater in the presence of the three hormones than cells cultured in media lacking insulin (Table 33; Figures 9, ll). Furthermore, studies of unattached dispersed cells of rat mammary carcinoma cultured for 24 and 48 hours show that the cell number was l.8 (24 hours) and 2.2 (48 hours) times greater in the presence of all three hormones than in cultures lacking insulin (Table 34). Results were further analyzed at five days of culture. The data show that the three hormones combined increased 2.8 and 5.3 times the colony number and area, respectively, in comparison to cultures lack- ing insulin (Tables 27-29; Figure 9). DNA analysis (H3-thymidine incorporation) of 5-day cultures of DMBA-induced rat mammary carcinoma showed that combination of insulin, prolactin, and corticosterone increased 2.7 times the H3-thymidine incorporation into DNA in compari- son to cultures lacking insulin (Tables 4-6; Figure 9). Therefore, 130 these studies indicate that a distinct and consistent synergism exists between insulin, prolactin and corticosterone, in enhancing cellular division of rat mammary carcinoma after 24, 48 and l20 hours of culture. This synergism was found when analyzing increases in either cell number, colony number or area, as well as H3-thymidine incorporation into DNA. To this writer's knowledge, the only report using a compar- able experimental design was that of El-Darwish and Rivera (l970), who showed that the hormonal combination of insulin, prolactin and cortico- sterone was more effective in stimulating DNA synthesis than were corticosterone plus prolactin-treated cultures of mouse mammary glands. Thus, in corticosterone and prolactinwtreated cultures, insulin appears to enhance cellular division of neoplastic as well as normal mammary tissue. It was also of interest to compare the stimulatory effects of insulin and prolactin upon cellular division of rat mammary carcinoma. The effects of either insulin or prolactin in the presence of cortico- sterone at 24, 48 and l20 hours of culture were initially investigated. Following these studies I compared the effects of these two hormones on rat mammary carcinoma cultured in media free of other hormones. Comparison of the effects of cultures containing insulin plus corticosterone, to cultures containing prolactin plus corticosterone and cultured for 24 hours in microplates (Table 31; Figure 9), or in petri dishes for 24 and 48 hours of culture (Tables 32, 33; Figures 9, ll), no significant difference at termination of cultures was indicated upon cell growth between the two hormonal combinations. This was con- sistent in all experiments. When dispersed cells of rat mammary 131 carcinoma were cultured for five days in the presence of either insulin or prolactin (media containing corticosterone) at termination of cultures the colony number was not found to be significantly dif- ferent between the two groups in two of the three experiments. In one experiment, prolactin showed a greater stimulatory effect than did insulin. The total area was found to be significantly larger in pro- lactin-treated cultures in two of the three experiments (Tables 27-29). The latter results were not totally in accord with the DNA analysis of 5-day cultures of explants of rat mammary carcinoma which showed no significant difference between insulin-treated or prolactin-treated cultures (in presence of corticosterone) (Tables 4-6; Figures 2 and 9). Therefore, these results demonstrated that at 24 and 48 hours of cul- ture there was no significant difference upon cellular division of rat mammary carcinoma between insulin and prolactin when corticosterone was in the media. At five days of culture, however, results suggest that prolactin may be slightly superior to insulin in stimulating cell division of rat mammary carcinoma. In part, similar findings have been reported by El-Darwish and Rivera (l970) who observed that the hormonal combination of insulin and corticosterone, when compared with prolactin and corticosterone treated cultures, was found to be insig- nificantly different when DNA synthesis of mouse mammary gland was determined after two days of culture. However, at three days, DNA synthesis of mouse mammary tissue in cultures containing insulin plus corticosterone was superior to that of cultures containing prolactin plus corticosterone. These results suggest that perhaps rat mammary 132 carcinoma has a greater dependency on prolactin (and lesser dependency on insulin) in a prolonged period of culture (five days), whereas the opposite appears to be true in the mouse. When dispersed cells of rat mammary carcinoma were cultured for five days in the presence of either insulin or prolactin but in the absence of corticosterone there was no significant difference in total number of colonies between these cultures; but in the prolactin-treated cultures the total area of colonies was significantly larger in two out of the three experiments (Tables 24-26). Colony area is a much more reliable criterion of cell growth than is colony number. Therefore, it appears that prolactin alone is superior to insulin alone, in stimulat- ing growth of rat mammary carcinoma in 5-day cultures. This is in accord with the results obtained from 5-day cultures containing cortico- sterone which, as previously described, showed that prolactin was superior to insulin in promoting cell growth. The lack of consistency in the 5-day culture experiments may be due to the inherent variability of tumors, some tumors being more fibrotic and having fewer epithelial cells than others. Even though attempts were not made to analyze quan- titatively this characteristic, it was observed, albeit subjectively, that tumors of predominantly connective tissue elements appeared to respond better to insulin, whereas those of predominantly epithelial cells appeared to respond better to prolactin. El-Darwish and Rivera (l970) appear to be the only investigators who compared the effects of prolactin and insulin on cell division of mouse mammary tissue jg_vjtrg, They showed that insulin alone had a marked effect in augmenting DNA 133 synthesis of mouse mammary tissue on the first day of culture and that this effect decreased with time. Prolactin alone had only a moderately stimulatory influence on DNA content during the first two days of culture, and on the third day was without effect. Their results are opposed to those obtained in these studies which showed that prolactin at five days of culture was superior to insulin in maintaining DNA synthesis of carcinogen-induced rat mammary carcinoma. It appears therefore that the carcinogen-induced rat mammary tumor is one of the few mammary tissues more readily responsive to prolactin than insulin, in contrast to most mammary tissues which appear to be more insulin-dependent. Several investigators have demonstrated the need for insulin in the growth of normal mouse mammary glands (Elias, l959, l96l; Stockdale et 21,, l966; Turkington, l968), rat mammary carcinomas (Turkington and Hilf, l968; Heuson, l969), and mouse mammary carcinoma (Turkington and Hilf, l968). Heuson and Legros (l97l) provide indica- tions that some carcinogen-induced rat mammary carcinomas are insulin dependent jn_vjt§9, DNA synthesis as well as DNA polymerase activity was shown to be markedly enhanced in 4-day culture of a few carcinogen- induced rat mammary carcinoma as a result of adding insulin to the media. Some rat mammary carcinomas are consistently independent of insulin for stimulation of DNA synthesis. The R323OAC, a transplant- able mammary carcinoma, appears to be an example of this type of tumor (Turkington and Hilf, 1968). The discrepancy between normal and neo- plastic mammary tissue on insulin sensitivity in_vitro suggests that 134 there is less of a need for the hormone jn_vjtrg_in neoplastic mammary tissue than in normal tissue. It is possible that mechanisms which are induced by insulin in normal mammary tissue or in some mammary carcinomas may become constitutive in the cells of other mammary carcinomas. Other experiments were performed to determine whether the combina- tion of insulin plus prolactin in corticosterone-containing media will stimulate cellular growth of rat mammary carcinoma at 24, 48 and l20 hours of culture. When dispersed cells of rat mammary carcinoma were cultured for 24 and 48 hours, the cell number at termination of cultures was increased in cultures containing prolactin plus insulin from 2.7 to 3.7 times that found in those cultures lacking these hormones (Tables 31-33; Figure ll). When dispersed cells were cultured for five days, the number of colonies and area of cultures were increased 8.2 and 2.l times, respectively, in cultures containing the two hormones when com- pared to cultures lacking these hormones (Tables 27-29). Studies of unattached dispersed cells of rat mammary carcinoma also indicated that in the combination of insulin and prolactin, in corticosterone- containing media, cell number was 3.6 and 2.6 times greater at 24 and 48 hours, respectively, than in cultures lacking insulin and prolactin (Table 34). These results demonstrate that insulin and prolactin, in the presence of corticosterone, significantly increase cell growth at 24, 48 and l20 hours of culture. Furthermore, the combination of these hormones stimulated an increase of cell numbers far greater than occurred in cultures containing single hormones, demonstrating a defini- tive synergism. This synergism has also been observed by El-Darwish 135 and Rivera (l970), who reported that insulin plus prolactin, in the presence of corticosterone, in 3— and 5-day cultures of midpregnant mouse mammary glands markedly enhanced DNA synthesis in these tissues. Since corticosterone has also been used in a number of ig_vitrg_ studies using different types of tissues, experiments were undertaken to determine the effects of this hormone on cell growth in dispersed cells of DMBA-induced rat mammary carcinoma. The studies were de- signed to determine l) whether corticosterone alone, 2) corticosterone plus insulin, or 3) corticosterone plus prolactin, were able to stimu- late cell growth of rat mammary carcinoma in_vitrg, When cells were cultured for 24 and 48 hours in the presence of corticosterone, the number of cells increased from l.4 to 3.2 times as many as found in cultures lacking hormones (Tables 3l-33; Figure ll). When dispersed cells were cultured for five days in the presence of corticosterone, the colony-number was found to be uninfluenced by the hormone in two of the three experiments, and was actually inhibited in the third experiment, in comparison to controls. Furthermore, the total colony area was significantly reduced in cultures containing corticosterone when compared with the controls in all three experiments performed (Tables 27-29). Therefore, these results indicate that corticosterone initially stimulates (24 and 48 hours) cellular growth of rat mammary carcinoma and ultimately inhibits (at five days) cell growth. Why short term cultures respond positively to corticosterone and negatively to prolonged cultures, can not be conclusively ascertained at this time. Apparently, rat mammary carcinoma grown ifl_vitro, under the 136 condition in this study, are more metabolically receptive to the stimu- latory effects of corticosterone, whereas cells in prolonged cultures lose such metabolic susceptability. On the other hand, corticosterone has been reported to be toxic to mammary carcinoma in_vivo (Stoll, l969), an effect which has been demonstrated in this study in vjt:g_ (5-day cultures). Several investigators have demonstrated the need for corticoids for normal mouse mammary tissue growth and differentiation (Stockdale _t_al,, 1966; Lockwood _t__l,, l967b). However, other reports have indicated that corticosterone alone did not either stimulate DNA syn- thesis or maintain mouse mammary glands during the first five days of culture (Rivera and Bern, l96l; El-Darwish and Rivera, l970). The next experiments were undertaken to determine whether corti- costerone plus insulin could stimulate cellular division of dispersed cells of rat mammary carcinoma at 24, 48 and 120 hours of culture. When dispersed cells were cultured for 24 and 48 hours, the cell number in cultures containing insulin plus corticosterone was increased from l.7 to 4.8 times that found in cultures lacking those hormones (Tables 3l-33; Figure ll). When dispersed cells were cultured for five days, the total number of colonies, in cultures containing corticosterone plus insulin, were fewer in two of the three experiments, and signifi— cantly larger in one of the three experiments, in comparison to controls (Tables 27-29). Therefore, these results demonstrated that at 24 and 48 hours of culture, corticosterone plus insulin stimulate cell growth, but these effects did not last for five days. At five days of Culture, 137 corticosterone plus insulin had no apparent effect (perhaps some slight inhibition) upon cell division of rat mammary carcinoma. The lack of consistency observed in the 5-day cultures (there was considerable variation among the three experiments) may be due to the variability of the tumors. Similar results have been previously reported by El-Darwish and Rivera (l970) who observed that insulin plus cortico- sterone after one and two days of culture of mouse mammary gland en- hanced DNA synthesis in these tissues. But they also found that these hormones inhibited DNA synthesis of mouse mammary tissue after five days of culture. It was of interest to determine whether corticosterone plus insulin could enhance rat mammary carcinoma cell division in cultures containing prolactin. When dispersed cells were cultured for five days in the presence of these three hormones, colony number and area were 2.9 and 3.0 times greater, respectively, than in cultures lacking corticosterone and insulin (Tables 24-26). Therefore, these results provide evidence that corticosterone and insulin in the presence of prolactin, markedly stimulated cell growth in 5-day cultures of rat mammary carcinoma, when compared with cultures containing prolactin alone. These findings emphasize the need for corticosterone and insulin for prolonged (5 day) stimulation of rat mammary carcinoma cells by prolactin. Other investigators have demonstrated the importance of corticosterone and insulin in prolactin-induced differentiation of mouse mammary tissue in_vit[9_(Lockwood et_al:, l967a; Turkington gt_al., l973). However, it appears that this triad of hormones is involved in 138 rat mammary carcinoma cell division, rather than differentiation as occurs in the normal mouse mammary gland. El-Darwish and Rivera (l970) also reported the need for these three hormones in cellular division of mouse mammary gland after five days of culture, in compari- son to cultures containing prolactin alone. It was also interesting to determine whether corticosterone plus prolactin were able to stimulate cell growth of rat mammary carcinoma after 24, 48 and 120 hours of culture. When dispersed cells were cul- tured for 24 and 48 hours of culture, cell numbers were increased in cultures containing these two hormones, from 1.8 to 3.8 times that found in cultures lacking hormones (Tables 31-33; Figure ll). When dispersed cells were cultured for five days in the presence of these two hormones, the number of colonies was found to be insignificantly different from the number of colonies observed in cultures lacking hormones (Tables 27-29). The total area of colonies however, was found to be smaller than the total area of colonies observed in the controls. These results demonstrate that corticosterone plus prolactin had a stimulatory effect on the cell growth of rat mammary carcinoma at 24 and 48 hours of culture. However, these two hormones had either no effect or an inhibitory effect on cell division of rat mammary carcinoma cells when compared to controls, at five days of culture. These find- ings indicate that mammary carcinoma cell proliferation occurs within a restricted period of time, perhaps the first 48 hours, in the presence of corticosterone and prolactin; but this effect does not persist (for five days). It is possible that corticosterone's capacity to initiate 139 cellular division is of finite duration, then it may enhance, or synergize with, other hormones, i.e., insulin, or insulin plus pro- lactin, to stimulate cell growth. Similar findings have been reported by El-Darwish and Rivera (1970) who observed that corticosterone plus prolactin had a stimulatory effect upon DNA synthesis of mouse mammary gland at 24 and 48 hours of culture. An inhibitory effect upon DNA synthesis in the presence of these hormones was reported after five days of culture, paralleling the results of the present study. Other experiments were designed to determine the effects of corticosterone plus prolactin upon cellular division of rat mammary carcinoma, but this time in the presence of insulin. When dispersed cells were cultured for five days in the presence of the triad of hor- mones, colony number and area were 3.3 and 2.8 times greater, respectively, than in cultures lacking corticosterone and prolactin (Tables 22-26). Therefore, these results clearly demonstrate that corticosterone plus prolactin, in the presence of insulin, markedly stimulates cell division in 5-day cultures. These results are in accord with the studies of Juergens gt_al, (1965), and El-Darwish and Rivera (1970) who showed that this triad of hormones was superior to cultures containing insulin alone in stimulating DNA synthesis of cultured mouse mammary gland. Once again, the triad of hormones, i.e., insulin, prolactin and corticosterone, was demonstrated to be efficacious in stimulating rat mammary carcinoma growth jn_vit§9, It appears that in comparing the triad of hormones upon cell growth (in 5-day cultures), with insulin alone, corticosterone alone, or prolactin alone, the 140 greatest difference was observed between the corticosteronevtreated cultures and the triad of hormones. The least difference was observed between the triad of hormones and either prolactin alone or insulin alone, the latter being comparable. These results provide further evidence that insulin alone is comparable to prolactin alone in stimu— lating growth of rat mammary carcinoma, while corticosterone alone is considerably less effective. In comparing the triad of hormones with either corticosterone plus insulin, or corticosterone plus prolactin, it was found that the triad of hormones was considerably more effective in stimulating cell growth than either of these combinations. Each combination, i.e., corticosterone plus prolactin and corticosterone plus insulin, was found to be comparable to each other in its effect on cell growth. These results are in accord with the preceding studies comparing the triad of hormones versus either insulin alone or prolactin alone. Since corticosterone, insulin or prolactin have been found to significantly modify growth of rat mammary carcinoma jg_vitro, experi- ments were performed to determine the combined effects of these hormones with media lacking hormones at 24, 48 and 120 hours of culture. When cells were cultured for 24 and 48 hours in the presence of the three hormones, the number of cells observed at termination of cultures, was increased 4.3 to 12.1 times that found in cultures lacking hormones (Tables 31-33; Figure 11). Studies of unattathed cells of rat mammary carcinoma indicate that in the presence of all three hormones, the cell number at termination of culture was 2.6 and 2.5 times greater at 24 and 141 48 hours, respectively, than in cultures lacking hormones (Table 34). When dispersed cells were cultured for 48 hours, only two studies showed marked colony formation, similar to that of 5-day cultures. This was the only time that colonies were formed in such a short period of time (two days). The colony number and area in the cultures con- taining all three hormones were 11.2 and 6.1 times greater than control cultures lacking hormones (Tables 17, 18). When dispersed cells were cultured for five days in the presence of the triad of hormones, the colony number and area at termination of cultures, was increased to 5.8 and 4.3 times, respectively, over those in cultures lacking hormones (Tables 19-21; 27-29). Once again, these results provide evidence that the combination of insulin, prolactin and corticosterone greatly influ- ence cell growth in comparison to controls (hormone free media) at 24, 48 and 120 hours of culture. Furthermore, these differences (three hormones combined versus no hormones) far exceeded the differences ob- served when the triad of hormones was compared with each hormone alone or any paired combination. A significant increase in cell growth at 24 and 48 hours was evident in 7 of the 8 tumors when cultured in the presence of the triad of hormones. It can be concluded that the triad of hormones used in these studies (insulin, corticosterone and pro- lactin) synergistically act to promote the growth of carcinogen-induced rat mammary carcinoma in X1339: Furthermore, although prolactin alone appears to be an efficacious growth stimulator of rat mammary carcinoma in_vjvg_($terenta1 gt_al,, 1963; Meites and Nicoll, 1966; Pearson §t_al,, 1969; Nagasawa and Yanai, 1970; Welsch gt_al,, 1970) it may be that 142 maximum activity of this hormone ig_vitrg requires participation of insulin and corticosterone. Serum has been used in many ifl_vitrg_studies; particularly those involving cell culture; in fact, the vast majority of cell cultures require the presence of serum for maintenance and growth (Rivera, 1971). It was interesting, therefore, to determine the influence of fetal calf serum on the growth of rat mammary carcinoma cells in cultures with and without hormones. At 24 and 48 hours of culture (unattached cells), fetal calf serum in the presence of the triad of hormones, increased 3.8 and 2.5 times, respectively, the number of cells in comparison to cultures containing the hormones but lacking serum (Table 35). Furthermore, under similar experimental conditions, but using media free of hormones, fetal calf serum increased the cell number 2.7 and 3.0 times at 24 and 48 hours of culture, respectively, when compared with cultures lacking serum. Cultures were further analyzed at five days. Results show that fetal calf serum, in the presence of all three hor- mones increased 5.4 and 1.4 times the colony number and area, reSpec- tively, in comparison to cultures lacking serum (Table 30). Furthermore, using media free of hormones, fetal calf serum increased colony number and area 3.6 and 4.1 times, respectively, in comparison to cultures lacking serum. Thus, these results indicate that fetal calf serum consistently stimulates cell growth of rat mammary carcinoma at 24, 48 and 120 hours of culture in either the presence or absence of hormones. Was fetal calf serum a more potent stimulator of cellular growth than the triad of hormones? It is apparent that at 24 and 48 hours of 143 culture, serum was significantly better than the triad of hormones at stimulating cellular growth; whereas at five days, the triad of hormones was considerably superior to serum. It is wellvknown that serum is an important additive to the maintenance and growth of cell cultures for reasons not completely understood. In endocrine- responsive tissues, i.e., rat mammary carcinoma, the hormones contained in the serum (Esber §t_al,, 1973) may be influential in enhancement of cellular growth. Other factors such as proteins may also be im— portant for growth of cells jg_vjtrg, Majumder and Turkington (1971) observed the existence of one or more protein factors in serum which stimulated epithelial cells of mouse mammary gland. They found that addition of fetal calf serum to the chemically defined medium induced a characteristic pattern of cell proliferation, indicated by several waves of DNA synthesis which continued for prolonged periods of culture. Further investigation is required to determine whether this character- istic pattern of cell proliferation is also present in cultured rat mammary carcinoma cells. The triad of hormones, plus fetal calf serum stimulated rat mam- mary carcinoma growth better than either the fetal calf serum or triad of hormones alone at 24, 48 and 120 hours of culture. A synergism between fetal calf serum and the triad of hormones is indicated in these studies. The triad of hormones markedly enhanced cellular growth in serum-containing media, i.e., the total number of colonies and area were 7.8 and 12.4 times, respectively, greater in the hormone—containing media than in the media containing only serum at five days of culture. 144 This difference was also significant at 24 and 48 hours of culture, but not as striking as at five days. Once again, the triad of hormones was shown to be an effective stimulator of cell growth, even in serum- containing media. Parenthetically, the triad of hormones plus serum, when compared to media lacking both hormones and serum, stimulated cell growth more than any other combination evaluated in these studies. It is clear, therefore, that carcinogen-induced rat mammary carcinoma cells are extremely responsive jn_vit§9_to hormonal combinations of prolactin, insulin and corticosterone, irregardless of the presence or absence of serum. Extensive ifl_vitrg studies using serum were not pursued because of the uncertainty regarding hormonal content of serum. Investigations involving the influences of hormones in development and growth of cells in culture, the use of a chemically defined media, free of hormones, is very important. Although many tissues can not survive in vitrg_without serum, the techniques described in this investigation permit study of the hormonal influence on rat mammary carcinoma cells, in a chemically defined (serum-free) media. SUMMARY The influence of ovine prolactin, estradiol—178, ovine and human growth hormone on the growth of DMBA-induced rat mammary carcinoma was studied i vitro (organ culture and cell culture) at 24, 48 and 120 hours. Secondary interest was given to insulin and corticosterone. In addition, these studies described a method of preparation, dissociation and culture of these tumor cells, a tumor—system which heretofore has not been successfully prepared for cell cultures. Cell growth was evaluated by 1) determining DNA synthesis of explants (120 hours of culture) and 2) growing diSpersed cells and determining changes in cell number (24 and 48 hours of culture) and colony number and area (120 hours of culture). These studies indicated that prolactin, in the presence of insu- lin and corticosterone, when compared to cultures containing insulin and corticosterone alone, significantly enhanced DNA synthesis in explants of rat mammary carcinoma (120 hours of culture) obtained from either intact or ovariectomized rats. Although prolactin consistently stimulated DNA synthesis of organ cultures, studies were undertaken to determine its effects on tumor cell growth. Prolactin in the presence of insulin and corticosterone, con- sistently increased cell growth of dispersed cells at 24, 48 and 120 hours of culture. Prolactin, in the presence of corticosterone alone, 145 146 initially (at 24 and 48 hours of culture) had slight stimulatory effect upon cell growth. At 120 hours of culture, however, prolactin and corticosterone had a greater stimulatory effect on cell growth than cultures containing corticosterone alone. In comparing the effects of prolactin and insulin, in corticosterone-containing media, there was no significant difference between these two hormones at 24 and 48 hours of culture. At 120 hours of culture, however, prolactin was slightly superior to insulin in stimulating cell growth. Prolactin alone was also slightly superior to insulin alone (in media lacking corticosterone) in stimulating cell growth after 120 hours of culture. Prolactin plus insulin, in the presence of corticosterone had a greater stimulatory effect on cell growth than did corticosterone alone at 24, 48 and 120 hours of culture. Similarly, prolactin plus corticosterone, in the presence of insulin, also had a greater stimulatory effect on cell growth than did insulin alone at 120 hours of culture. In comparing the effect of the triad of hormones (prolactin, insulin and corticosterone) upon cell growth (at 120 hours of culture) with the effect of prolactin alone, insulin alone, and corticosterone alone, the greatest difference observed was between corticosterone alone and the triad of hormones and the least between the triad of hormones and either prolactin alone or insulin alone. Comparison of the triad of hormones with either prolactin plus corticosterone, or insulin plus corticosterone, indi- cated that the triad of hormones was considerably more effective in stimulating cell growth than was either of the combinations at 24, 48 and 120 hours of culture. Each combination, i.e., prolactin plus 147 corticosterone, or insulin plus corticosterone, was found to be compar- able in effect on cellular growth. The triad of hormones was consis- tently superior in stimulating cell growth to media totally lacking hormones at 24, 48 and 120 hours of culture. Furthermore, these differences far exceeded the differences observed when the triad of hormones was compared with either each hormone alone or any other paired hormones. Thus, it appears that the three hormones used in these studies (prolactin, insulin and corticosterone) synergistically act to promote the growth of DMBA-induced rat mammary carcinoma ig_vitrg, Although prolactin alone has been found to be an efficacious growth stimulant in_vivg, it was demonstrated in these studies that maximal activity of this hormone jn_vjtrg_requires also the participation of insulin and corticosterone. These studies further demonstrated that estradiol-178 was ineffec- tive in stimulating or reactivating DNA synthesis of explants obtained from ovariectomized rats after 120 hours of culture (medium containing insulin and corticosterone). Estrogen plus prolactin, however, was as effective as prolactin alone in stimulating DNA synthesis in explants from ovariectomized rats after 120 hours of culture. Thus, it appears that estrogen may act through other mechanisms (i.e., via the pituitary), at least in part, in influencing the growth of rat mammary carcinoma. A synergism between estrogen and prolactin was not demonstrated in these studies. 148 The effect of growth hormone on DNA synthesis was also analyzed. Either ovine or human growth hormone alone slightly stimulated DNA synthesis after 120 hours of culture (medium containing insulin and corticosterone). Human growth hormone and prolactin combined, however, when compared with human growth hormone alone markedly enhanced DNA synthesis after 120 hours of culture (medium containing insulin and corticosterone). This hormonal combination, however, was no more effective than prolactin alone in stimulating DNA synthesis. A parallelism was observed between labeling index and H3-thymidine incorporation into DNA. Since insulin and corticosterone have been extensively used in a number of culture studies, using a variety of tissues, experiments were performed to determine the effects of these hormones on cell growth. Results indicate that insulin plus corticosterone when com— pared with corticosterone alone had either no effect (at 24 hours of culture), or a slight stimulatory effect (at 48 and 120 hours of culture) on cell growth. Insulin, in the presence of prolactin and corticosterone, when compared with prolactin plus corticosterone, was, however, consistently superior in enhancing cellular growth (at 24, 48 and 120 hours of culture) and DNA synthesis (at 120 hours of culture). Thus, these results indicate that insulin had greater stimulatory effect on cellular growth as long as prolactin was in the media. Corticosterone alone initially (at 24 and 48 hours of culture) stimulated cell growth and eventually (at 120 hours of culture) 149 inhibited it. Either corticosterone plus prolactin or corticosterone plus insulin, when compared with cultures lacking hormones, had a greater stimulatory effect on cell growth after 24 and 48 hours of culture. At 120 hours of culture, however, these hormonal combinations had no striking effect (and perhaps some slight inhibition) on cellular growth. Corticosterone plus insulin, in the presence of prolactin, had a greater stimulatory effect on cell growth than did prolactin alone after 120 hours of culture. Thus, these results indicate that either corticosterone alone, corticosterone plus prolactin, or cortico- sterone plus insulin, initially (at 24 and 48 hours of culture) stimu- lated cell growth; but these effects did not persist (for 120 hours of culture). Addition of prolactin to corticosterone plus insulin cul- tures significantly enhanced the stimulatory effects of corticosterone plus insulin on cell growth at 120 hours of culture. Studies were undertaken to determine the importance of serum (fetal calf) for growth of dispersed cells. Results indicate that fetal calf serum, either in cultures lacking or containing hormones (triad of hormones), consistently stimulated cell growth at 24, 48 and 120 hours of culture. Fetal calf serum plus the triad of hormones markedly stimulated cell growth when compared with either serum alone or the triad of hormones alone at 24, 48 and 120 hours of culture. Thus, a distinct synergism between the serum and the triad of hormones is indicated in these studies. The triad of hormones plus serum when compared to media lacking both hormones and serum, stimulated cell growth more than any other combination here evaluated. It is also 150 apparent that serum is not essential to the growth of these dispersed cells. These dispersed cells can effectively grow in a serum-free medium (Medium 199) as long as this medium contains the triad of hormones. APPENDICES 151 APPENDIX I (Tables 1, 2 and 3) DNA Synthesis in DMBA—Induced Rat Mammary Carcinoma at 12, 24, 48 and 96 Hours of Organ Culture Randomized Block Test (H3—Thymidine per pg DNA, cpm) T1 12 T3 T4 T Group I Group II Group III Group IV 12 hours 24 hours 48 hours 96 hours R] 26.4 78.8 53.8 174.9 333.8 R2 156.6 149.0 130.5 204.3 640.3 R3 80.5 52.1 69.8 489.6 701.0 263.5 279.9 254.1 868.8 1675.1 Dunnet's Test to, 0.01, 3, 6 = 3.8 T4 vs T1 = 7.6 T4 vs T2 = 7.4 T4 vs T3 = 7.7 Orthogonal Contrast Test F, 0.001, 1, 6 = 35.5 F, 0.05, 1, 6 = 5.9 T4 vs (T]+T2+T3) = 83.2 T] vs T2 = 0.04 T1 vs T3 = 0.01 T vs T = 0.1 2 3 152 APPENDIX II (Tables 4, 5 and 6) Effect of Prolactin and Insulin on DNA Synthesis in 5—Day Organ Cultures of DMBA-Induced Rat Mammary Carcinoma Randomized Block Test (H3vThymidine per ug DNA, cpm) T1 T2 T3 T Group I Group II Group III Insulin Prolactin Insulin + Prolactin R] 63.8 45.7 134.2 243.7 R2 170.1 126.2 227.0 523.2 R3 131.0 37.5 212.0 380.5 364.9 209.3 573.2 1147.4 Dunnet's Test tD, 0.05, 2, 5 = 2.6 tD, 0.01, 2, 5 = 3.6 T] vs T3 = 3.3 T2 vs T3 = 5.8 Orthogonal Contrast Test F, 0.01, 1, 4 = 21.2 F, 0.05, 1, 4 = 7.7 T3 vs (T]+T2) = 28.0 T vs T = 6.2 2 3 153 APPENDIX III (Tables 7, 8 and 9) Effect of Prolactin and Estrogen on DNA Synthesis in 5«Day Organ Cultures on DMBA—Induced Mammary Carcinoma of Ovariectomized Rats Randomized Block Test (H3-Thymidine per pg DNA, cpm) T1 T2 T3 T4 T Group I Group II Group III Group IV - Estrogen Prolactin Estrogen + Prolactin R1 23.6 9.0 37.1 28.7 99.3 R2 9.0 17.0 25.7 26.7 78.4 R3 11.4 8.2 57.4 40.1 117.2 44.0 34.2 120.2 95.5 294.9 Dunnet's Test t 0.05, 3, 6 = 2.5 0’ T1 vs T2 = 0.4 T1 vs T3 = 3.3 T1 vs T4 = 2.2 Orthogonal Contrast Test F9 0.05, I, 6 = 5.9 F, 0.01, 6 = 13.7 T1 vs (T2+T3+T4) = 4.3 T2 vs T3 = 13.8 T2 vs T4 = 6.9 T vs T = 1.1 3 4 154 APPENDIX IV (Tables 10, 11, 12 and 13) Effect of Prolactin and Ovine Growth Hormone on DNA Synthesis in 5-Day Organ Cultures of DMBA-Induced Rat Mammary Carcinoma Randomized Block Test (H3-Thymidine per pg DNA, cpm) T1 T2 13 T Group I Group II Group III - Ovine GH Prolactin R1 18.0 153.0 159.8 330.8 R2 20.9 53.6 119.7 194.2 R3 331.0 284.6 495.5 1111.1 R4 78.7 230.3 256.7 565.6 448.6 721.5 1031.7 2202.0 Dunnet's Test tD = 0.05, 2, 6 = 2.3 T1 vs T2 = 1.4 T1 vs T3 = 3.4 Orthogonal Contrast Test F, 0.05, 1, 6 = 5.9 F, 0.025, 1, 6 = 8.8 T vs (T2+T3) = 9.4 1 T vs T 2 3 = 3.7 (Tables 14, 15 and 16) 155 APPENDIX V Effect of Prolactin and Human Growth Hormone Alone, and in Combination,on DNA Synthesis in 5-Day Organ Cultures of DMBAhInduced Rat Mammary Carcinoma Randomized Block Test (H3-Thymidine per pg DNA, cpm) T1 T2 T3 T4 T Group I Group II Group III Group IV - Human GH Prolactin Human GH + Prolactin R] 24.9 5.3 33.7 86.3 150.2 R2 11.1 88.6 142.7 142.7 350.8 R3 3.9 8.7 18.1 40.9 40.9 39.9 102.6 152.3 247.1 541.9 Dunnet's Test 2 x , 0.05, 3, 6 = 2.5 x2, 0.01, 3, 6 = 3.8 11 vs T2 = 2.4 T1 vs T3 = 2.5 T1 vs T4 = 4.7 Orthogonal Contrast Test F, 0.05, 1, 6 = 5.9 F, 0.025, 1, 6 = 8.8 T] vs (T2+T3+T4) = 10.7 T2 vs T3 = 1.1 T2 vs T4 = 8.7 T3 vs T4 = 4.0 E C 156 APPENDIX VI (Figure 6) ffect of Prolactin and Human Growth Hormone Alone, and in ombination, on DNA Synthesis in 5-Day Organ Cultures of DMBA-Induced Rat Mammary Carcinoma Test: Two-way Analysis of Variance with Unegual Number (Fedever-Zelen Method to obtain SS) (Labeling Index) I_ §39g9_ Treatment Labeling Index T1 I - 4.9 T2 II Human Growth Hormone 12.8 T3 III Prolactin 27.4 14 IV Human Growth Hormone 81.9 + Prolactin F = 158.6 F, 0.001, 1, 67 = 11.4 Scheffé Test* F, 0.001, 67, 3 = 5.79 F, 0.05, 67, 3 = 2.88 T1 vs T2 = 0.9 (N.S.) 11 vs 13 = 2.4 T1 vs T4 = 5.9 T2 vs T3 = 0.9 (N.S.) 12 vs T4 = 4.3 T3 vs T4 = 2.8 continued 157 APPENDIX VI--continued Relationship between H3-thymidineper pg DNA and Labeling Index H3-Thymidineper pg DNA (cpm) A Prolactin = Y T2 + T4 ' Y T] + T3 = 78.7 A Human Growth Hormone = Y T3 + T4 - Y T] + 72 = 128.5 A P/A HGH = 0.6 Labeling Index A Prolactin = Y T2 + T4 - Y T] + T3 = 577.5 A Human Growth Hormone = Y T3 + T4 - y T] + T2 = 906.4 A P/A HGH = 0.6 158 APPENDIX VII (Tables 17 and 18) Effect of Combination of Insulin, Prolactin and Corticosterone on Growth of Collagenase Dispersed Cells of DMBA—Induced Rat Mammary Carcinoma after Two Days of Culture x2 Test (Total Number of Colonies) I, Qfggp_ Treatment Tumor #1 Tumor #2 D1 02 T] I - 9 0 9 T2 II Insulin 96 5 101 + Prolactin + Corticosterone 105 5 110 x2 = 0.4 x2, 0.05, 1 = 3.8 Treatment was independent upon density, therefore data was combined over densities and justification for combining them was the non-significance of the first step. x2 Results between Treatments and Densities x2, 0.005, 1 = 7.9 76.9 T1 vs T2 0 vs 0 1 90.0 2 The ratio of colonies observed from density to density was independent of treatment. The ratio of numbers of colonies observed from treatment to treatment was the same within sampling errors, for each density. Conversely, test was symmetric, ratio did not depend on density. 159 APPENDIX VIII (Tables 19, 20 and 21) Effect of Combination of Insulin, Prolactin and Corticosterone on Growth of Collagenase Dispersed Cells of DMBA-Induced Rat Mammary Carcinoma after Five Days of Culture x2 Test (Total Number of Colonies) I_ figggp. Treatment Tumor #1 Tumor #2 Tumor #3 D1 D2 03 T] I "' 7 2 0 9 T2 II Insulin 42 7 17 66 + Prolactin + Corticosterone 49 9 17 75 x2 = 3.5 x2, 0.005, 2 = 10.6 Treatment was independent of density. Therefore data was combined over densities and justification for combining them was the non-significance of the first step. x2 Results between Treatments and Densities x2, 0.005, 1 x2, 0.005, 2 = 10.6 7.9 57.0 T1 vs T2 0 vs D 1 2 35.8 160 APPENDIX IX (Tables 22 and 23) Effect of Combination of Insulin, Prolactin and Corticosterone on Growth of Collagenase Dispersed Cells of DMBAeInduced Rat Mammary Carcinoma after Five Days of Culture. Comparison with Cultures Containing Insulin Alone. x2 Test (Total Number of Colonies) I_ Group Treatment Tumor #1 Tumor #2 D1 02 T1 I Insulin 0 54 54 T2 II Insulin 37 186 223 + Prolactin + Corticosterone 37 240 277 x2 = 10.3 2 x , 0.005, 1 = 7.9 There was significant interaction, the ratio of colonies observed from density to density was influenced by the treatment. In other words, the ratio from treatment to treatment was dependent upon density. Data was not combined over densities due to the significance of the first step. x2 Results between Treatments x2, 0.005, 1 = 7.9 _ Tumor #1 Tumor #2 T1 VS T2 ' "37l0“' "7§T6“' 161 APPENDIX X (Tables 24, 25 and 26) Effect of Combination of Insulin, Prolactin and Corticosterone on Growth of Collagenase Dispersed Cells of DMBA-Induced Rat Mammary Carcinoma after Five Days of Culture. Comparison with Cultures Containing Insulin and Prolactin Alone x2 Test (Total Number of Colonies) I_ Group Treatment Tumor #1 Tumor #2 Tumor #3 D1 D2 D3 T] I Insulin 97 5 0 102 12 II Prolactin 85 13 2 100 T3 III Insulin 229 52 7 288 + Prolactin + Corticosterone 411 70 9 490 x2 = 14.0 2 x , 0.01, 4 = 13.2 There was significant interaction. The ratio of colonies observed from density to density was influenced by the treatment. In other words, the ratio from treatment to treatment was dependent upon densities. x2 Results between Treatments N x , 0.05, l = 3.8 x2, 0.01. 1 = 6.6 x2, 0.005, 1 = 7.9 Tumor #1 Tumor #2 Tumor #3 T1 vs T2 = 0.8 3.6 2.0 T1 vs T3 = 53.4 38.8 7.0 = 66.0 23.4 2.8 T vs T3 (Tables 27, 28 and 29) 162 APPENDIX XI Effect of Combination of Insulin, Prolactin and Corticosterone on Growth of Collagenase Dispersed Cells of DMBAvInduced Rat Mammary Carcinoma after Five Days of Culture x2 Test (Total Number of Colonies) I_ Group Treatment T] I -- T2 11 Corticosterone T3 III Corticosterone + Insulin T4 IV Corticosterone + Prolactin T5 V Corticosterone + Insulin + Prolactin x2 = 29.8 2 x , 0.005, 8 = 21.9 2 x Results between Treatments 2 x , 0.05, 1 = 3.8 —4 —+ —4 -+ -4 —4 —1 -4 -a -4 x2, x , 0. 0 VS VS VS VS VS VS VS VS VS VS .025, 1 = 5.0 005, 1 = 7.9 Tumor #1 -+ -1 -4 -4 -4 -4 —4 —4 -4 -4 UTU'l-bU‘I-bWU‘I-th 12. 5. 3. 47. 1. 26. 91. 19. 77. 26. mmmomboowtow Tumor #1 Tumor #2 Tumor #3 D1 ”2 D3 33 15 1 49 10 15 0 25 16 37 0 53 51 21 2 74 118 79 9 206 228 167 12 407 Tumor #2 Tumor #3 0.0 1.0 9.3 1.0 1.0 0.5 43.4 6.4 9.3 0.0 9.1 2.0 43.4 10.0 2.7 2.0 15.0 10.0 33.6 4.1 163 APPENDIX XII (TabIe 30) Effect of Fetal Calf Serum on Growth of Collagenase Dispersed Cells of DMBA-Induced Rat Mammary Carcinoma after Five Days of Culture x2 Test (Total Number of Colonies) I. §£222_ Treatment No Serum Serum SI 52 T] I -- 3 11 19 T2 11 Insulin 16 86 97 + Prolactin + Corticosterone 14 102 116 x2 = 3.0 2 x , 0.05, 1 = 3.8 Treatment was independent of the presence of serum. Therefore, these data were combined due to the non-significance of the first step. x2 Results between Treatments and Presence of Serum x2, 0.005, 1 = 7.9 T1 vs T2 31 vs S2 66.6 52.4 164 APPENDIX XIII (Table 31) Effect Of Insulin, Prolactin and Corticosterone on Growth of Collagenase DiSpersed Cells of DMBAeInduced Rat Mammary Carcinoma after 24 Hours of Culture 2 x Test (Average Number of Cells per Hell) I_ Group Treatment Tumor #1 Tumor #2 Tpmor #3 01 D2 D3 T] I -- 9.1 41.1 16.6 66.9 2 II Corticosterone 10.5 46.1 38.4 95.1 T3 III Corticosterone 8.4 66.2 41.5 116.1 + Insulin T4 IV Corticosterone 7.7 51.8 58.8 118.4 + Prolactin T5 V Corticosterone 28.7 140.5 116.5 285.7 + Insulin + Prolactin 64.4 345.7 271.8 1 682.2 x2 = 14.2 2 x , 0.05, 8 = 15.5 x2 Results between Treatments 2, 0.25, 1 = 3.8 x2, 0.005, 1 = 7.9 T] vs T2 = 4.9 T1 vs T3 = 13.2 T] vs T4 = 14.3 TI vs T5 =135.8 T2 vs T3 = 2.1 T2 vs T4 = 2.5 T2 vs T5 = 95.4 T3 vs T4 = 0.1 T3 vs T5 = 71.6 T4 vs 15 = 69.3 165 APPENDIX XIV (Table 32) Effect of Insulin, Prolactin and Corticosterone on Growth of Collagenase Dispersed Cells of DMBA-Induced Rat Mammary Carcinoma after 24 Hours of Culture x2 Test (Average Number of Cells per Petri Dish) 1_ Group Treatment Tumor #1 Tumor #2 Tumor #3 D1 ”2 D3 T] I -— 94.6 0.0 47.0 141.6 T2 II Corticosterone 173.5 87.0 125.0 385.5 T3 III Corticosterone 203.6 110.0 136.0 449.6 + Insulin T4 IV Corticosterone 165.8 85.0 115.3 366.1 + Prolactin T5 V Corticosterone 304.2 384.0 337.0 1025.2 + Insulin + Prolactin 941.7 666.0 760.3 2368.0 x2 = 181.3 x2, 0.005, 8 = 21.9 x2 Results between Treatments x2, 0.05, 1 = 3.8 x2, 0.005, 1 = 7.9 Tumor #1 Tumor #2 Tumor #3 T1 vs T2 = 23.9 87.0 35.4 11 vs T3 = 38.0 110.0 43.2 11 vs T4 = 20.0 95.0 28.0 T1 vs 15 = 110.0 384.0 220.0 12 vs T3 = 2.4 2.6 0.4 T2 vs T4 = 0.2 0.1 0.4 12 vs T5 = 34.0 186.0 96.0 T3 vs 14 = 3.7 3.2 1.8 T3 vs T5 = 19.6 156.0 84.0 T4 vs T5 = 41.6 190.0 110.0 Effect of Insulin, Prolactin and Corticosterone on Growth of 166 APPENDIX XV (Table 33) Collagenase Dispersed Cells of DMBA-Induced Rat Mammary Carcinoma after 48 Hours of Culture x2 Test (Average number of cells per Petri Dish) I. Gpppp_ Treatment 1] I -- T2 II Corticosterone T3 III Corticosterone + Insulin T4 IV Corticosterone + Prolact1n T5 V Corticosterone + Insulin + Prolactin x2 = 104.5 x2, 0.005, 8 = 21.9 x2 Results between Treatments x2, 0.05, 1 = 3.8 x2, 0.005, 1 = 7.9 Tumor #1 T1 vs 12 = 20.0 T1 vs T3 = 98.0 T] vs T4 = 66.0 T1 vs T5 = 230.0 T2 vs T3 = 21.0 T2 vs T4 = 9.1 T2 vs T5 = 76.8 13 vs T4 = 2.7 T3 vs T5 = 22.0 T4 vs T5 = 74.0 Tumor #1 Tumor #2 Tumor #3 ”2 D3 0.0 58.0 71.0 18.0 166.0 232.0 136.0 16.0 190.0 342.0 100.0 14.0 160.0 274.0 267.0 125.0 470.0 862.0 564.0 173.0 1004.0 1781.0 Ilflfiflllfli llflEflLifii 18.0 26.0 16.0 35.1 14.0 23.9 125.0 160.7 0.1 1.6 0.4 0.1 80.0 72.7 0.1 1.3 86.0 59.4 88.6 76.3 167 APPENDIX XVI (Table 34) Effect of Insulin, Prolactin and Corticosterone on Growth of Collagenase Dispersed Cells of DMBA-Induced Rat Mammary Carcinoma after 24 and 48 Hours of Culture x2 Test (Average Number of Unattached Cells per Petri Dish) 1_ Group Treatment 24 Hours 48 Hours T] I -- 85.3 52.0 137.0 T2 11 Corticosterone 60.0 49.0 109.0 T3 III Corticosterone 80.0 78.0 158.0 + Insulin T4 IV Corticosterone 118.6 59.0 177.6 + Prolactin T5 V Corticosterone 218.6 132.0 351.6 + Insulin + Prolactin 563.5 370.0 933.5 x2 = 9.2 2 x , 0.05, 4 = 9.4 Very nearly significant at the 95% level. It is likely that number of cells vary from treatment to treatment in a different manner at 24 and 48 hours. Therefore, one should examine treatments separately. x2 Results between Treatments 2 x , 0.05, 1 = 3.8 x2, 0.01, 1 = 6.6 x2, 0.005, 1 = 7.9 24 Hours: T1 vs TZ+T3+T4+T5 = 8.3 T +T vs T +1 = 82.0 2 3 4 5 12 vs T3 = 2.8 T4 vs T5 = 29.5 48 Hours: T] vs T2+T3+T4+T5 = 721.5 T2+T3 vs T4+T5 = 207.4 T2 vs T3 = 7.56 T4 vs T5 = 27.3 168 APPENDIX XVII (Table 35) Effect of Fetal Calf Serum on Growth of Collagenase DiSpersed Cells of DMBA- Induced Rat Mammary Carcinoma after 24 and 48 Hours of Culture x2 Test (Average Number of Unattached Cells per Petri Dish) I_ Group Treatment 24 Hours 48 Hours T] I -- 67.0 23.0 I 90.0 T2 II Fetal Calf Serum 183.0 70.0 253.0 T3 III Insulin 83.3 50.0 133.3 + Prolactin + Corticosterone 4 IV Insulin 320.0 127.0 447.0 + Prolactin + Corticosterone + Fetal Calf Serum 653.3 270.0 923.3 = 3.6 x2, 0.05, 3 = 7.8 Treatment was independent of the presence of serum and time, therefore these data were combined due to the nonvsignificance of the first step. x2 Results between Treatments x2, 0.005, 1 = 7.9 Tlv sT2 = 78.2 11 vs T3 = 8.3 T1 vs T4 =239.l T2 vs T3 = 37.3 12 vs T4 = 26.8 T3 vs T4 =170. 0 T1+T 2 vs T 3+T4 = 60.8 T1 +T3 vs T2+T4= 165.2 BIBLIOGRAPHY BIBLIOGRAPHY Apostolakis, M. 1968. 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