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Response to ACTH and dibutyryl cyclic AMP by nucleated and enucleated adrenocortical tumor cells Chen, Lydia May 1975

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RESPONSE TO ACTH AND DIBUTYRYL CYCLIC AMP BY NUCLEATED AND ENUCLEATED ADRENOCORTICAL TUMOR CELLS by LYDIA MAY CHEN B.Sc, The University of B r i t i s h Columbia, 1972 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Zoology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA July , 1975 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requirements fo r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e fo r reference and study. I f u r t h e r agree tha t permiss ion fo r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y .purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . It i s understood that copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed without my w r i t t e n p e r m i s s i o n . Department of lLo 0 LP Y The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date \Sst A-vuiLUut . IHS ABSTRACT The objective of th i s study was to determine conclusively whether or not the nucleus i s required for the c y c l i c AMP-mediated steroidogenic response of adrenocortical c e l l s to adrenocorticotropic hormone (ACTH). Enucleated c e l l s are i d e a l for studying various aspects of c e l l u -l a r metabolism without the added complexity of superimposed t r a n s c r i p t i o n a l control. Cytochalasin B can trigger the enucleation of several types of cultured c e l l s . Therefore, by modifying a procedure which has been described i n the l i t e r a t u r e , cultured adrenocortical c e l l s were enu-cleated with cytochalasin B, and the response of these enucleated c e l l s to ACTH and dibutyryl c y c l i c AMP was investigated. The type of adrenocortical c e l l s chosen for t h i s study was the Y - l functional mouse adrenocortical tumor c e l l l i n e . However, i t was found that the Y - l c e l l s could not be enucleated e f f i c i e n t l y , thus, during the course of t h i s study, a subline denoted Y-l-L c e l l s was selected from the Y - l c e l l l i n e . These c e l l s were l a t e r passaged i n isogeneic animals. Tumor #2 c e l l s were tumor c e l l s a r i s i n g from a LAF^ mouse which had received a subcutaneous inoculum of Y-l-L c e l l s . Similar to the Y - l c e l l s , Y-l-L c e l l s and Tumor #2 c e l l s did not require ACTH for growth or maintenance of a basal s t e r o i d output, but would respond to added ACTH or dibutyryl c y c l i c AMP by a change i n morphology and by an increase i n steroidogenesis. In contrast to the Y - l c e l l s , Y-l-L c e l l s and Tumor //2 c e l l s could be e f f i c i e n t l y enucleated by the enucleation procedures developed and used. The morphology of Y-l-L c e l l s and Tumor i i i i i #2 c e l l s i n control medium, i n medium containing ACTH, and i n medium containing dibutyryl c y c l i c AMP was characterized by l i g h t and electron microscopy, and the steroid outputs of these c e l l s under various incubation conditions were characterized by standard biochemical methods. Enucleated Y-l-L c e l l s and Tumor #2 c e l l s were viable as shown by dye exclusion for a minimum of 60 hours, and synthesized proteins as indicated by 3H-leucine incorporation into acid-insoluble material for at least 48 hours. Enucleated adrenocortical c e l l s responded to either ACTH (10 mU/ml) or dibutyryl c y c l i c AMP (1 mM) by a change f rom f l a t to rounded c e l l shape, and by increased steroidogenesis as determined by spectrofluorometric assay of the culture medium. The steroidogenic response of enucleated c e l l s during the f i r s t three hours after enu-cleation was comparable i n magnitude to that of the nucleated c e l l s , and persisted i n diminishing degrees for an additional 9 hours. On the other hand, the morphologic change can be induced by ACTH even at 33 hours following enucleation. The results of t h i s study show that the nucleus i s not required for the expression of the acute effects of ACTH, and that the cytoplasmic components necessary for c e l l "rounding" and steroidogenesis are stable for at least 36 and 12 hours respectively. ACKNOWLEDGMENTS I wish to express my sincere appreciation to: Dr. N. Auersperg, Department of Zoology and Cancer Research Centre, for her concern, guidance and encouragement throughout t h i s investigation. Dr. R. L. Noble, Director of the Cancer Research Centre, for the opportunities and f a c i l i t i e s to carry out th i s research. Drs. A. B. Acton and J . D. Berger, Department of Zoology, for t h e i r interest i n this i n v e s t i g a t i o n , and for t h e i r review of th i s manu-s c r i p t . Dr. A. F. Burton, Department of Biochemistry, for h i s c r i t i c a l comments and he l p f u l suggestions. Dr. J. B. Hudson, Department of Medical Microbiology, for h i s consideration and use of the S o r v a l l RC 2B centrifuge. Dr. L. Kraintz, Department of Oral Biology, for the use of the P h i l l i p s 300 electron microscope. Dr. M. J. Hollenberg, Department of Anatomy, for the use of the Cambridge Stereoscan electron microscope. Mr. W. Siep, Cancer Research Centre, for h i s s k i l l e d technical help. Mrs. E. A. S l a v i n s k i , Mrs. D. McClellan and Mrs. L. Lee for th e i r competent help and advice. During the tenure of this i n v e s t i g a t i o n , the author was the recipient of a McLean Fraser Memorial Fellowship and a bursary from the National Research Council of Canada. This research was supported by grants from the National Cancer I n s t i t u t e of Canada to Dr. N. Auersperg. i v TABLE OF CONTENTS Page ABSTRACT i i ACKNOWLEDGMENTS i v TABLE OF CONTENTS . . . . . . . . v LIST OF TABLES v i i i LIST OF FIGURES x INTRODUCTION 1 MATERIALS AND METHODS 7 I. The Adrenocortical Cells Used for the Study of ACTH Action 7 I I . Tissue Culture 1 3 I I I . Preparation of ACTH, Dibutyryl Cyclic AMP and Cytochalasin B 14 IV. Microscopy 15 V. Steroid Determination 18 VI. Enucleation Procedure 2 1 VII. Chromosome Preparation 23 V I I I . C e l l V i a b i l i t y 24 IX. Protein Determination 24 X. Autoradiography 25 RESULTS . . . . . . . . . . . . . . 27 I. Morphological and Biochemical Responses of Intact (Nucleated) Y - l , Y-l-L and Tumor #2 Cells to ACTH and Dibutyryl C y c l i c AMP 27 v v i Page 1. Morphological responses - l i g h t microscopy 27 2. Steroidogenic responses 32 3. I d e n t i f i c a t i o n of endogenous ste r o i d production during a 12-hour incubation 37 4. 1 1 +C-Pregnenolone metabolism of Y-l-L and Tumor #2 Cells 39 5. Electron microscopy of Y-l-L C e l l s 41 I I . Enucleation Procedure 53 I I I . Effects of DMSO, Cytochalasin B and Enucleation Procedure Treatments (without actual enucleation) upon Y-l-L and Tumor #2 Cells 56 1. Effects upon steroidogenesis 56 2. Effects upon morphology . . 60 IV. The Metabolic A c t i v i t i e s and the Responses of Enucleated Y-l-L Cells and Tumor #2 Cells to ACTH and Dibutyryl C y c l i c AMP 64 1. V i a b i l i t y . . 64 2. Protein synthetic a c t i v i t y 67 3. Morphologic responses to ACTH and dibutyryl c y c l i c AMP . 67 4. Steroidogenic responses to ACTH and dibutyry l c y c l i c AMP 70 DISCUSSION 79 I. The Adrenocortical Cells Used i n th i s Study 79 I I . The Morphologic Effect of ACTH 84 I I I . Enucleation Procedure 87 IV. Effects of DMSO and Cytochalasin B upon Y-l-L Cell s and Tumor #2 Cells 90 V. Mechanism of ACTH Action 92 VI. General Outlook 97 SUMMARY . . . 99 BIBLIOGRAPHY 102 v i i Page APPENDIX I. Preparation of Cells for Transmission Electron Microscopy 110 I I . I d e n t i f i c a t i o n of 20a Dihydroprogesterone by Acetylation P r i o r to R e c r y s t a l l i z a t i o n . . . . . . . . I l l I I I . Preparation of Lowry's Reagent 113 LIST OF TABLES Table Page 1 Characteristics of Y - l , Y-l-L Cells and Tumor #2 Cells 12 2 Comparison of Steroidogenic Responses to ACTH and Dibutyryl C y c l i c AMP between Y - l Cells (ATCC CCL 79) and Y-l-L Cells ' . . 35 3 Steroidogenic Responses to ACTH and Dibutyryl Cy c l i c AMP by Y-l-L Cells and Tumor #2 Cells a f t e r S e r i a l Subculture . . . . 36 4 Acetylation of 20oH Dihydroprogesterone Produced Endogenously by Y-l-L Cells with 3H-Acetic Anhydride, and R e c r y s t a l l i z a t i o n of 20a Dihydroprogesterone-3H-Acetate to constant 3H:11+C Ratio i n the Presence of 11+C-20a Dihydroprogesterone- 3H-Acetate 42 5 Radiochemical I d e n t i f i c a t i o n of 11+C-20a Dihydro-progesterone Produced by Tumor #2 Cells 43 6 Effects of DMSO, Cytochalasin B, and Enucleation Procedure Treatment upon the Steroidogenic Responses of Y-l-L Cells to ACTH and Dibutyryl C y c l i c AMP . . . 58 7 Effects of DMSO, Cytochalasin B, and Enucleation Procedure Treatments upon the Steroidogenic Responses of Tumor #2 Cells to ACTH and Dibutyryl C y c l i c AMP . . 58 8 Effects of DMSO, Ethanol, Cytochalasin B Dissolved i n DMSO, Cytochalasin B Dissolved i n Ethanol, and Enu-cleation Procedure Treatments upon the Steroidogenic Responses of Tumor #2 Cells to ACTH and Dibutyryl C y c l i c AMP 59 9 Radiochemical I d e n t i f i c a t i o n of ll+C-20a Dihydro-progesterone Produced by Enucleated Tumor #2 Cells . . 73 10 Steroidogenic Responses to ACTH and Dibutyryl C y c l i c AMP by Enucleated Y-l-L Cel l s 75 11 Steroidogenic Responses to ACTH and Dibutyryl C y c l i c AMP by Enucleated Tumor #2 Cells . . . . . . 77 v i i i i x Table Page 12 Steroidogenic Responses to ACTH and Dibutyryl Cy c l i c AMP by Nucleated and Enucleated Y-l-L Cells and Tumor #2 C e l l s : Steroid Output i n 12 Hours . . . . 78 13 Effects of Actinomycin D Upon Adrenocortical Steroidogenesis: Summary of Results Reported i n the Literature 93 LIST OF FIGURES Figure Page 1 - 2 Responses to ACTH and Dibutyryl C y c l i c AMP by Y - l Cells (Biocult) and Y-l-L Cells . . . . . . . . . 8 3 Summary of Enucleation Procedure: Coverslip Method . . 22 4 Summary of Enucleation Procedure: 6 cm P e t r i Dish Method 22 5 - 6 Response to ACTH by Y-l-L Cells 28 7 - 8 Histology of Tumor #2 and the Response of Tumor #2 C e l l s i n Culture to ACTH and Dibutyryl C y c l i c AMP . . 30 9 The Endogenous Steroid Products and l l +C-Pregnenolone Metabolites of Cells During 12 Hours of Incubation . . 38 1 0 - 1 3 Ultrastructure of Y-l-L Cells i n s i t u 44 1 4 - 1 8 Ultrastructure of Y-l-L C e l l s i n s i t u : Response to ACTH 47 1 9 - 2 0 Y-l-L Cells Following Treatment with Trypsin and Trypsin together with Versene 5 1 2 1 Experimental Design: Steroidogenic Responses to ACTH and Dibutyryl C y c l i c AMP by Enucleation Procedure Treatment Cells and Enucleated Cells . . 57 2 2 - 2 6 Effects of ACTH, Dibutyryl C y c l i c AMP, DMSO, and Cytochalasin B upon the Morphology of Tumor #2 C e l l s . 6 1 2 7 - 2 8 V i a b i l i t y and Protein Synthetic A c t i v i t y of Enucleated Y-l-L C e l l s 65 2 9 - 3 0 Responses to ACTH by Nucleated and Enucleated Tumor #2 Cells 68 3 1 - 3 3 Morphologic Responses to ACTH and Dibutyryl C y c l i c AMP by Enucleated Y-l-L Cell s 71 , 34 Steroid Pathway i n Adrenal 0Tumor C e l l Cultures . . . . 83 x INTRODUCTION Normal c e l l u l a r metabolism i s a result of complex nucleo-cytoplasmic interactions. Many approaches have been used to define more precisely the r e l a t i v e roles of the nucleus and cytoplasm. The c l a s s i c studies involving the comparison between the a c t i v i t i e s of nuc-leate and anucleate fragments of eggs and s i n g l e - c e l l e d organisms have shown that the expression of the genetic information present i n the nucleus i s determined by the events which occur i n the cytoplasm (e.g. Haemmerling, 1963). These studies also showed that to a variable extent and for a variable period of time, the anucleate cytoplasm w i l l r e t a i n the properties of the cytoplasm i n the i n t a c t c e l l . I t i s for t h i s reason that the anucleate state has provided the opportunities for the studies concerning the morphogenetic and metabolic capacities of the cytoplasm i n the absence of direct nuclear control. To date, the two most frequently employed methods of i n i t i a t i n g the anucleate condition have been shown to have drawbacks (Keck, 1969). Physical enucleation by microdissection i s technically d i f f i c u l t and becomes tedious when large numbers of c e l l s are required for biochemical analysis. Chemical "enucleation" by drug-induced i n h i b i t i o n of RNA synthesis, thereby simulating physical enucleation, may produce dras t i c side effects which are unrelated to the cessation of RNA synthesis (Ivarie et a l . , 1974), hence, results obtained i n these studies without other evidence are equivocal. 1 2 Currently there i s a novel method for the production of anucleate populations of c e l l s (Carter, 1967; 1972). Cytochalasin B, a fungal metabolite, i s capable of inducing the protrusion of nuclei when added to c e l l s i n monolayer cultures. Within minutes of cytochalasin treatment, the nucleus becomes displaced from the main cytoplasmic mass, and i n time, the nucleus becomes attached to the rest of the c e l l by a mere filament of cytoplasm. The mechanism of this curious phenomenon remains to be explained. However, the property of nuclear protrusion has been exploited (Prescott et a l . , 1972; Wright and Hayflick, 1972; F o l l e t , 19 74): by subsequent centrifugation, the thin strand of cytoplasm uniting the nucleus and the cytoplasm i s e a s i l y severed and a population of c e l l s with protruded n u c l e i may thus be converted to a population of t o t a l l y anucleate c e l l s . Furthermore, the apparent lack of effect on c e l l u l a r metabolism following the removal of cytochalasin B i s of impor-tance (Carter, 1967; Ivarie et a l . , 1974). Therefore, the anucleate c e l l s produced by this method are p o t e n t i a l l y useful for the study of various aspects of the functional capacities of the cytoplasm i n the absence of immediate nuclear control. The objective of th i s study was to use cytochalasin B-mediated enucleation to determine whether or not new RNA synthesis i s required for the steroidogenic response of adrenocortical c e l l s to ACTH. By applying t h i s enucleation technique, a study of the role of the cytoplasm i n the metabolism of Y - l c e l l s , the functional murine adrenocortical tumor l i n e c e l l s was undertaken. Y - l c e l l s respond to ACTH morpholo-g i c a l l y and biochemically. Immediately upon addition of ACTH or dibutyry l 3 c y c l i c AMP, the c h a r a c t e r i s t i c a l l y flattened, e p i t h e l i a l - l i k e c e l l s "round up," that i s , they transform into spherical c e l l s . Concomitantly, there i s an increase i n steroidogenesis (Yasumura eit a l . , 1966b). In recent years, there have been s i g n i f i c a n t advances i n the understanding of the mechanism of action of ACTH i n regulating the steroidogenic a c t i v i t y of the adrenal cortex ( G i l l , 1972). Nevertheless, while i t i s w e l l established that c y c l i c AMP i s the i n t r a c e l l u l a r medi-ator of ACTH action, the precise mechanism by which c y c l i c AMP induces steriodogenesis remains obscure. Studies involving the effect of various metabolic i n h i b i t o r s upon ACTH action indicate that protein synthesis i s essential for the capacity of adrenocortical c e l l s to respond to ACTH (Garren et a l . , 19 71). The requirement for RNA synthesis i s un-resolved. Previously, actinomycin D has been used to c l a r i f y the role of RNA synthesis i n the acute steroidogenic response of adrenocortical c e l l s to ACTH. However, a survey of the l i t e r a t u r e reveals that con-f l i c t i n g results were reported. I t i s now evident that actinomycin D i s toxic and can produce a myriad of side e f f e c t s . Thus, the p o s s i b i l i t y of ambiguities due to drug usage i n these studies can not be ruled out. Enucleation by cytochalasin B treatment ensured the complete removal of the genome. As a r e s u l t , conclusive evidence pertaining to whether or not direct nuclear a c t i v i t y i s required for the expression of ACTH effects was obtained by studying the morphological and steroido-genic response of enucleated adrenocortical c e l l s to ACTH. I n i t i a l l y , the enucleation procedure used was the method described by Prescott ejt a l . (1972). With this method, only small amounts of enu-cleated c e l l s could be produced. Greater amounts of enucleated c e l l s 4 were required for the accurate analysis of t h e i r s t e r o i d output. There-fore, a modified enucleation procedure was developed as part of this study, which resulted i n a ten-fold increase i n the number of enucleated c e l l s produced. Furthermore, the o r i g i n a l Y - l c e l l s chosen for t h i s study could not be enucleated e f f i c i e n t l y , because, at the high c e n t r i -fugal forces necessary for enucleation, c e l l loss due to detachment from the substratum was great. To overcome this problem, a more adhe-sive subline, denoted as Y-l-L, was selected from the Y - l c e l l l i n e . Y-l-L c e l l s were l a t e r passaged i n isogeneic animals. Tumor #2 c e l l s were tumor c e l l s a r i s i n g from a LAF^ mouse which had received a sub-cutaneous inoculum of Y-l-L c e l l s . Both Y-l-L c e l l s and Tumor #2 c e l l s adhered more tenaciously to th e i r substratum than did the o r i g i n a l Y - l l i n e and as a r e s u l t , the loss of c e l l s during the enucleation of Y-l-L c e l l s and Tumor #2 c e l l s was comparatively much less. Because Y-l-L c e l l s and Tumor #2 c e l l s could be consistently enucleated with high e f f i c i e n c y , these were the adrenocortical c e l l s used for the study of ACTH action. In recent years, many b i o l o g i c a l effects of cytochalasin B have been described (Aldrich, 1975). Although i t has been established that many cytochalasin B-induced effects are reversible within 10-15 minutes after the removal of the drug (Carter, 1967; Spooner, 1973), several experiments were also performed to determine whether or not the cyto-chalasin B treatment during the enucleation procedure had any effect upon the subsequent morphology and steriodogenic a c t i v i t i e s of Y-l-L c e l l s and Tumor #2 c e l l s . 5 The ultrastructure of the rat adrenal cortex under normal and experimental conditions has been described (e.g. Sabatini et a l . , 1962; Rhodin, 1971) and modulation i n the ultrastructure of the rat adrenal cortex 10 minutes a f t e r the administration of ACTH has been reported (Rhodin, 1971). These changes include the depletion of glycogen and l i p i d droplets, and changes i n the structure and s p a t i a l arrangements of mitochondria, Golgi bodies and smooth endoplasmic reticulum. Such rapid alterations i n fine structure upon ACTH stimulation suggest that u l t r a s t r u c t u r a l studies may be of great value i n determining whether or not enucleated Y-l-L c e l l s and Tumor #2 c e l l s are functional. Further-more, i t has been recognized that the goal of preserving the adrenal cortex i n an unstressed, normal state for u l t r a s t r u c t u r a l studies i s unattainable, considering the stresses involved i n obtaining the tissue. Less trauma i s involved i n f i x i n g c e l l s i n tissue culture. Therefore, the u l t r a s t r u c t u r a l analysis of nucleated Y-l-L c e l l s at various times after ACTH administration was undertaken to test the theories l i n k i n g hormone-induced u l t r a s t r u c t u r a l changes causally with the acute increase i n steroidogenesis, and also to elucidate the mechanisms involved i n cort i c o s t e r o i d secretion. The term used to describe the morphological response of Y - l c e l l s to ACTH, "rounding up" i s an ambiguous one. In tissue culture, trypsin and versene are routinely used i n the subculture procedure. The reaction of c e l l s to t r y p s i n i z a t i o n i s also referred to as "rounding up." Therefore, the morphologic responses of Y-l-L c e l l s to trypsin and trypsin together with versene, were also investigated by l i g h t and electron microscopy, and compared with the morphological response of these c e l l s to ACTH. In summary, this study consists of the development of an enu-cleation procedure which was capable of producing s u f f i c i e n t quantities of enucleated c e l l s for biochemical analyses, the production of a subline of adrenocortical c e l l s which could be e f f i c i e n t l y enucleated, and the characterization of the morphologic and steroidogenic responses of nucle-ated and enucleated Y-l-L c e l l s and Tumor #2 p e l l s to ACTH and dibutyry l c y c l i c AMP. MATERIALS AND METHODS I. The Adrenocortical Cells Used for the Study of ACTH Action The adrenocortical c e l l s used i n this study were Y - l c e l l s pur-chased from American Type Culture Collection (ATCC CCL79), Y-l-L c e l l s , and Tumor #2 c e l l s . During the 'course of this study, i t was established that while Y - l c e l l s (ATCC CCL79) could not be e f f i c i e n t l y enucleated, 95-98% enu-cleat i o n e f f i c i e n c y was consistently achieved with Y-l-L c e l l s and Tumor #2 c e l l s . Therefore, Y-l-L c e l l s and Tumor #2 c e l l s were the adreno-c o r t i c a l c e l l s used predominantly i n this study. The Y - l c e l l s (ATCC CCL79) were used i n i t i a l l y for control purposes when the Y-l-L c e l l s and Tumor #2 c e l l s were being characterized. The Y-l-L c e l l s were selected from the Y - l c e l l l i n e which was purchased from Biocult (BCL 721). Although the prescribed culturing procedure was followed, the Y - l c e l l s from Biocult were heterogeneous i n morphology, and did not respond i n the manner described i n the l i t e r a -ture, to ACTH or dibutyry l c y c l i c AMP. While the majority of the Y - l c e l l s were e p i t h e l i a l - l i k e i n morphology, there were also rounded c e l l s and f i b r o b l a s t - l i k e c e l l s (Fig. l a ) . The morphologic responses of these Y - l c e l l s to ACTH and dibutyry l c y c l i c AMP were inconsistent. Approxi-mately half the population of c e l l s became rounded i n the presence of 10 mU ACTH/ml of medium or 1 mM dibutyry l c y c l i c AMP, and i t could 7 8 Plate I Responses to ACTH and Dibutyryl C y c l i c AMP by Y - l Cells (Biocult) and  Y-l-L C e l l s . Methanol fixation', Giemsa sta i n i n g , x350. Figs. l a - c . Y - l c e l l s (Biocult) i n culture, (a) i n control medium, (b) 3 hours i n medium containing 10 mU ACTH/ml, (c) 3 hours i n medium containing 1 mM dibutyryl c y c l i c AMP. Note the heterogeneity i n c e l l morphology and responsiveness to ACTH and dibutyryl c y c l i c AMP. Figs. 2a-c. Y-l-L- c e l l s i n culture. (a) i n control medium, (b) 3 hours i n medium containing 10 mU ACTH/ml, (c) 3 hours i n medium containing 1 mM dibutyry l c y c l i c AMP. The morphology of Y-l-L c e l l s i n control medium and the morphological response of these c e l l s to ACTH and dibutyryl c y c l i c AMP were more uniform than the Y - l c e l l s ( B i o c u l t ) . « l a . VT.. | « # 5 a 10 not be determined whether those "rounded" c e l l s were the i n i t i a l l y f l a t or already rounded c e l l s (Figs, lb and c ) . During media changes, and during s e r i a l subculture, a difference was noticed between the e p i t h e l i a l - l i k e c e l l s and the other c e l l s . The rounded c e l l s were loosely attached to the P e t r i dish and could be e a s i l y detached by squirting with a pipette. During s e r i a l subculture, the rounded c e l l s and f i b r o b l a s t - l i k e c e l l s were ea s i l y dispersed within 10 minutes by incubation i n 0.12% trypsin i n Ca^ + and Mg^+ free Hanks' balanced s a l t s o l u t i o n , whereas the e p i t h e l i a l - l i k e c e l l s s t i l l remained attached to the p l a s t i c substratum. By repeated s e l e c t i v e culturing of c e l l s which remained a f t e r 10 minutes of t r y p s i n i z a t i o n during the subculture procedure, and by repeated s q u i r t i n g off of the rounded c e l l s which appeared, a population i n which 100% of the c e l l s were f l a t and e p i t h e l i a l - l i k e was obtained (Fig. 2a). These morphologically homogeneous c e l l s also responded uni-formly to ACTH and dibutyryl c y c l i c AMP (Figs. 2b and c). This subline of c e l l s was denoted as Y-l-L c e l l s . Similar to the parent l i n e , Y-l-L c e l l s also had a basal s t e r o i d output i n the absence of ACTH, and responded to added ACTH or dib u t y r y l c y c l i c AMP by increased steroidogenesis. The major s t e r o i d products of Y-l-L c e l l s were i d e n t i f i e d by thin layer chromatography of media extracts on s i l i c a gel to be 20a dihydroprogesterone, HB OH, 20a dihydroprogesterone and progesterone, which were i d e n t i c a l to those produced by the parent Y - l c e l l l i n e . However, i n comparison with the Y - l c e l l s , Y-l-L c e l l s had a much lower s t e r o i d output under the various incubation conditions. 11 The amount of steroid output also diminished a f t e r s e r i a l subcultures. Y-l-L c e l l s were used for several experiments, but the use of these c e l l s was discontinued due to the d i f f i c u l t y of accurate measurement of the low s t e r o i d output. I t has been reported i n the l i t e r a t u r e that cultured tumor c e l l s which have been p e r i o d i c a l l y passaged i n isogeneic animals following passages i n v i t r o had an enhanced a b i l i t y to grow and function i n culture subsequently (Buonassisi e_t a l . , 1962) . This phenomenon has been ob-served for Y - l c e l l s as w e l l (Buonassisi et a l . , 1962; Kowal, 1970b). Therefore, by using Buonassisi's animal passage method, four tumor c e l l l i nes were generated from Y-l-L c e l l s . Tumor #2 c e l l s were derived from a LAF^ mouse which had received a subcutaneous inoculum of 10 6 10th passage Y-l-L c e l l s . Tumor #2 c e l l s were used for subsequent experiments because they were most s i m i l a r to the Y-l-L c e l l s i n terms of morphology, generation time, growth pattern (Table I) and steroidogenic a c t i v i t y . Tumor #2 c e l l s i n i t i a l l y produced steroids at a high rate, but t h e i r steroidogenic a c t i v i t y also diminished a f t e r s e r i a l subculture. This phenomenon i s quite common and has been reported by several laboratories that work with the Y - l c e l l s . Several culture samples of Tumor 42 c e l l s were frozen and preserved i n l i q u i d nitrogen a f t e r the second passage i n v i t r o . These frozen samples were p e r i o d i c a l l y re-established i n culture for experiments. This policy provided cultures with r e l a t i v e l y s i m i l a r l e v els of s t e r o i d output for the remainder of the experiments. 12 TABLE I. Characteristics of Y - l , Y-l-L and Tumor #2 Cells Property Y - l (ATCC CCL 79) Y-l-L Tumor #2 Generation time (on p l a s t i c ) Chromosomes Modal Number Frequency Growth On glass On p l a s t i c Morphology On glass On p l a s t i c Growth pattern On glass On p l a s t i c 40-50 hours 40-42 75% poor, c e l l s s e t t l e d and grew slowly c e l l s s e t t l e d quickly and grew more quickly heterogeneous e p i t h e l i a l - l i k e monolayer c e l l s p i l e d up, became rounded and shed into the medium 30-40 hours 40-42 24-36 hours 40-42 95% c e l l s s e t t l e d quickly and grew w e l l 95% c e l l s s e t t l e d quickly and grew w e l l same as on glass same as on glass e p i t h e l i a l - l i k e e p i t h e l i a l - l i k e monolayer c e l l s p i l e d up extensively, be-came more fibro-b l a s t - l i k e ep i t h e l i a l - 1 i k e e p i t h e l i a l - l i k e monolayer c e l l s p i l e d up extensively, be-came more f i b r o -b l a s t - l i k e 13 I I . Tissue Culture Cells stocks were maintained at 37°C i n 250 ml p l a s t i c tissue culture flasks (Falcon P l a s t i c s , Oxnard, C a l i f . ) on Ham's nutrient medium, F-10, supplemented with 15% horse serum, 2.5% f e t a l c a l f serum, 100 units/ml of p e n i c i l l i n , and 100 yg/ml of streptomycin (Microcan Research Ltd., Calgary, A l t a . ) . The medium was changed twice weekly, and the c e l l s were subcultured every 8-10 days by diss o c i a t i o n i n 0.12% c r y s t a l -l i n e trypsin i n C a 2 + and Mg 2 + free Hanks' balanced s a l t solution (Microcan Research Ltd.). Culture conditions were modified as required to f a c i l i t a t e various studies: for the l i g h t and scanning electron microscopy of i n s i t u c e l l s , and for the quantitation and i d e n t i f i c a t i o n of st e r o i d production, the c e l l s were grown on 6 cm diameter P e t r i dishes at 37°C i n a water-saturated atmosphere of 5% CO2 i n a i r ; for the u l t r a s t r u c t u r a l analysis of i n s i t u c e l l s , the c e l l s were grown on Leighton tubes (Bellco, Vineland, N.J.). Growth characteristics of Y-l-L c e l l s and Tumor #2 c e l l s were the same on glass and on p l a s t i c . For the enucleation experiments, the c e l l s were grown on either round glass coverslips, or on sulphuric acid-treated 6 cm diameter P e t r i dishes (see Enucleation Procedure). Four tumor li n e s were started by subcutaneous i n j e c t i o n of 10 6 10th passage Y-l-L c e l l s into 6-8 week old LAF^ male mice (Jackson Labora-t o r i e s , Bar Harbour, Maine). The tumors were excised when they reached the s i z e of 2 x 1/2 x 1/2 cm (2-4 weeks). Portions of each tumor were fixed and processed for l i g h t microscopy, and other portions were cultured. Portions suitable for culture were minced f i n e l y with scissors i n trypsin 14 for 10 minutes at room temperature. The trypsin solution was decanted after centrifugation. The c e l l s were washed twice with complete medium and 4-5 x 10 5 c e l l s were plated i n 75 ml p l a s t i c tissue culture f l a s k s . I I I . Preparation of ACTH, Dibutyryl c y c l i c AMP and Cytochalasin B 1. Porcine adrenocorticotropic hormone Grade I (150 IU/mg) was purchased from Sigma Chemical Company, St. Louis, Missouri. A stock solution of 1 IU ACTH/ml of s t e r i l e s a l i n e was made and stored i n 1 ml aliquots at -20°C. For experiments, ACTH was diluted i n nutrient medium to give a f i n a l concentration of 10 mU/ml; th i s medium was used within 1 hour of preparation. 2. N 60 2' dibutyryl adenosine 3'5' c y c l i c monophosphoric acid Grade 11 was also purchased from Sigma. Dibutyryl c y c l i c AMP was dissolved d i r e c t l y i n nutrient medium to give a 1 mM solution. 3. I n i t i a l l y , the cytochalasin B used was a g i f t from Dr. S. B. Carter, Imperial Chemical Industries, Macclesfield, Cheshire. However, most of the cytochalasin B used during the course of this study was purchased from Aldr i c h Chemical Company, Millwaukee, Wisconsin. A stock solution was prepared i n DMSO (Fisher Scien-t i f i c Co., Vancouver, B.C.) to give a concentration of 1 mg/ml and stored at 4°C. For experiments, the stock solution was diluted i n aqueous medium to give a f i n a l cytochalasin B con-centration of 10 yg/ml, and a DMSO concentration of 1%. 15 IV. Microscopy 1. Light microscopy Cultures i n s i t u were either fixed i n 95% alcohol and stained with 1% toluidine blue (Fisher S c i e n t i f i c Co.), or fixed i n methanol and stained with Giemsa (Davenport, 1970) (Fisher S c i e n t i f i c Co.). Portions of tumors a r i s i n g i n mice following the i n j e c t i o n of Y-l-L c e l l s were fixed i n Bourns', dehydrated through a graded series of alcohol solutions, and embedded i n wax according to standard h i s t o -l o g i c a l procedure. Sections which were approximately 8 microns-thick were cut, and observed under the l i g h t microscope following haematoxylin and eosin staining ( C u l l i n g , 1963). Materials prepared for electron microscopy were also examined by l i g h t microscopy. Epon sections, 0.5-1.0 micron-thick, were cut with glass knives on a Reichert ultramicrotome. The sections were trans-ferred to glass s l i d e s with a fine wire loop, heat-fixed onto the s l i d e s , and stained with 1% to l u i d i n e blue i n 1% sodium borate (Pease, 1964). 2. Electron microscopy The ultrastructures of both Y-l-L c e l l s and Tumor #2 c e l l s were studied. a) Transmission electron microscopy of Y-l-L c e l l s Y-l-L c e l l s were grown to near-confluency on Leighton tubes. Following incubation i n one of the following: ( i ) Basal medium ( i . e . Ham's F-10 supplemented with serum and a n t i -b i o t i c s ) 16 ( i i ) Medium containing 10 mU ACTH/ml for 5 minutes ( i i i ) Medium containing 10 mU ACTH/ml for 30 minutes (iv) Medium containing 10 mU ACTH/ml for 1 hour (v) Medium containing 10 mU ACTH/ml for 3 hours (vi) Medium containing 10 mU ACTH/ml for 24 hours ( v i i ) Medium containing 1 mM di b u t y r y l c y c l i c AMP for 3 hours, the i n s i t u cultures were washed gently with Hanks' balanced s a l t solu-t i o n , fixed i n cold 2.5% glutaraldehyde i n Millonig's buffer ( M i l l o n i g , 1962; Pease, 1964) at pH 7.4 for 1 hour, washed twice with buffer, post-fixed i n cold 1% osmium tetroxide i n Millonig's buffer for 30 minutes, and again washed twice with buffer. The cultures were dehydrated through a graded series of ethanol and propylene oxide, and i n f i l t r a t e d with Epon according to routine procedure (Appendix I ) . Then, the Leighton tubes were completely f i l l e d with Epon, and following polymerization, rapidly transferred from 56°C to ice water, thereby breaking the tubes, and releasing the blocks of Epon. Appropriate areas of the embedded cultures were selected by l i g h t microscopy, cut out and mounted on blocks for sectioning. Thin sections were cut with a Du Pont diamond knife on a Reichert ultramicrotome, mounted on copper gr i d s , stained with uranyl acetate (K and K Lab, Inc., Plainview, N.Y.) (Watson, 1958) and lead c i t r a t e (Fisher S c i e n t i f i c Co.) (Venable and Coggeshall, 1965), and examined with either a Hitachi HS-7S electron microscope or a P h i l l i p s 300 electron microscope. The ultrastructure of Y-l-L c e l l s following incubation i n either (i ) 0.12% Trypsin i n C a 2 + and-Mg 2 + free Hanks' balanced s a l t solution for 10 minutes, or ( i i ) 0.04% Versene and 0.12% Trypsin i n Ca 2 _ r and Mg2"*" free Hanks' balanced s a l t solution for 10 minutes, was also investigated. The detached c e l l s were pelleted by centrifuga-t i o n , then fixed and processed as described above, with the addition of gentle centrifugation following each step to concentrate the c e l l s . The c e l l s were embedded i n p l a s t i c capsules. The material was also sectioned and stained as described above. b) Transmission electron microscopy of Tumor #2 Cell s Tumor #2 c e l l s were grown to near-confluency i n 6 cm diameter P e t r i dishes. Following incubation i n one of the following: (i ) Basal medium ( i i ) Medium containing 10 mU ACTH/ml for 3 hours ( i i i ) Medium containing 1 mM di b u t y r y l c y c l i c AMP for 3 hours (iv) Medium containing 1% DMSO for 45 minutes (v) Medium containing 10 yg cytochalasin B/ml for 45 minutes (vi) Medium containing 10 yg cytochalasin B/ml and 10 mU ACTH/ml for 3 hours ( v i i ) Basal medium for 3 hours, following preincubation i n 10 yg cyto-chalasin B/ml for 45 minutes, and two washes with Hanks' balanced s a l t solution. ( v i i i ) Medium containing 10 mU ACTH/ml for 3 hours, following preincu-bation i n 10 yg cytochalasin B/ml for 45 minutes, and two washes with Hanks' balanced s a l t s o l u t i o n , the cultures were washed gently with Hanks' balanced s a l t s o l u t i o n , and fixed as described previously. The c e l l s were dehydrated through 18 a graded series of alcohol. When the c e l l s were i n the second change of absolute alcohol, 75% of the c e l l s were scraped off with a rubber policeman. These c e l l s were collected, p e l l e t e d , embedded, sectioned, stained and examined also as described previously. The 25% of the c e l l s remaining were a i r - d r i e d , and stored i n a dessicator. These c e l l s were l a t e r prepared for scanning electron microscopy. c) Scanning electron microscopy of Tumor #2 Cell s To f a c i l i t a t e handling and manipulation, the areas of P e t r i dishes with the fixed and a i r - d r i e d c e l l s were cut out and mounted on aluminium specimen stubs with S i l v e r Dag mounting medium. To render o the specimen conductive, the c e l l s were coated with a 150 A-thick layer of gold i n a vacuum evaporator. The surface topography of the c e l l s was examined i n a Cambridge Stereoscan Scanning Electron Microscope. V. Steroid Determination 1. Quantitation The assay of st e r o i d output by the u t i l i z a t i o n of the fluorescent properties of 20a hydroxylated derivatives of progesterone i n ethanolic-sulphuric acid was determined as described by Kowal (Kowal and F i e d l e r , 1968) . Each P e t r i dish of c e l l s was incubated with 2-3 ml of medium for either 3 hours or 12 hours. At the end of the incubation period, the medium was removed and extracted with 10 ml of spectrophotometry grade methylene chloride (Fisher S c i e n t i f i c Co.). The solvent phase was pipetted out and f i l t e r e d into another extraction tube. Two 19 m i l l i l l t r e s of 65% sulphuric acid (reagent grade, Fisher S c i e n t i f i c Co.) i n absolute ethanol which had been mixed on ice 45 minutes previously, were added and the tube was shaken vigorously for 30 seconds. The fluores-cence of the acid extract was read 45 minutes l a t e r at 535 my with an excitation wavelength of 470 my i n an Aminco-Bowman spectrofluorometer. A comparable aliquot of medium incubated without c e l l s and ex-tracted i n the same manner, served as a'blank. 20a dihydroprogesterone, 50 ng, 100 ng, or 1 yg (Steraloids Inc., Pauling, N.Y.) were added to medium and extracted. These served as steroid standards. The st e r o i d outputs of Y - l , Y-l-L and Tumor #2 c e l l s were quantitated as nanograms of 20a dihydroprogesterone. 2. I d e n t i f i c a t i o n The endogenous steroids produced by the adrenocortical c e l l s during 12 hours of incubation were tentatively i d e n t i f i e d by comparing t h e i r m o b i l i t i e s on thin-layer chromatograms with s t e r o i d standards (Steraloids Inc., Sigma). The methylene chloride extracts of medium pooled from 4 P e t r i dishes of c e l l s were taken down to dryness under a i r , taken up i n 0.1 ml of ethanol and chromatographed on s i l i c a gel plates (Kodak Eastman, Rochester, N.Y.) using benzene-acetone (120:30, v:v). The major steroids were i d e n t i f i e d by t h e i r absorption i n the u l t r a v i o l e t . Additional incubations were performed with ^C-pregnenolone (50-60 mCi/mM) (New England Nuclear, Boston, Mass.). Ten microcuries of ^C-pregnenolone was dissolved i n 0.5 ml of absolute ethanol. Approxi-mately 100,000-200,000 dpm of 1£*C-pregnenolone were added to the culture 20 medium. Following 12 hours of incubation, the medium was removed, ex-tracted with methylene chloride, taken to dryness, redissolved and chroma-tographed i n two dimensions on s i l i c a gel plates using benzene:acetone (120:30, v:v), and benzene:acetone (100:50, v:v) with 10 yg each of non-radioactive standards of pregnenolone, 20a dihydropregnenolone, proges-terone, and 20a dihydroprogesterone. Autoradiography of the chromatogram were prepared as described by J e l l i n c k and Goudy (1967). The radioactive areas on the s i l i c a gel plate were scraped o f f , eluted with ethanol, f i l t e r e d , evaporated to dryness, redissolved and rechromatographed on s i l i c a gel plates using benzene:acetone (120:30, v:v), The radioactive areas corresponding to the UV-absorbing spots of progesterone and 20a dihydroprogesterone, the radioactive areas corresponding to pregnenolone and 20a dihydropregnenolone, which were sprayed with phosphomolybdic acid to detect A5-3B hydroxy steroids, and the radioactive areas corres-ponding to the Rf's of 11B0H progesterone, 11B0H, 20a dihydroprogesterone, and 11 keto, 20a dihydroprogesterone were scraped off and eluted with ethanol. The amount of r a d i o a c t i v i t y present i n each metabolite was determined by counting aliquots i n toluene (Fisher S c i e n t i f i c Co.) con-taining 2,5-diphenyl oxazole (4 g / l i t r e ) and p-bis-[2-(5-phenyloxezolyl)]-benzene (50 mg/litre) (Fisher S c i e n t i f i c Co.) using a Nuclear Chicago Unilux I I I or a Packard Tri-Carb Model 334 l i q u i d s c i n t i l l a t i o n counter. 20a dihydroprogesterone was p o s i t i v e l y i d e n t i f i e d by either r e c r y s t a l l i z a t i o n from ethanol:water to constant s p e c i f i c a c t i v i t y with non-radioactive c a r r i e r 20a dihydroprogesterone, or by acetylation with 3H-acetic anhydride (400 mCi/mM) (New England Nuclear) p r i o r to 21 r e c r y s t a l l i z a t i o n from ethanol:water to constant 3H: 1 4C r a t i o with non-radioactive 20cx dihydroprogesterone acetate (Appendix II) . The amount of st e r o i d present i n the crystals was measured by standard techniques for gas chromatography on a Tracor MT220 gas chromatograph. VI. Enucleation Procedure I n i t i a l l y , c e l l s were enucleated by a modification of the tech-nique described by Prescott (Prescott et a l . , 1972). S t e r i l e 18 mm round glass coverslips (Corning, Corning, N.Y.) were placed i n 35 mm diameter P e t r i dishes. 2 - 4 x 10 5 c e l l s i n 0.5 ml of medium were added and allowed to attach. Two hours l a t e r a further 1.5 ml of medium was added and the c e l l s were allowed to grow for 24-48 hours. The cover-s l i p s with the c e l l s were incubated i n serum-free medium for 1 hour p r i o r to enucleation. C e l l enucleation was achieved by centrifugation of coverslips with the monolayer of c e l l s facing the bottom of the 30 ml centrifuge tube (Corning) with 5 ml serum-free medium containing 10 yg cytochalasin B/ml at 7710 g for 40 minutes i n a S o r v a l l RC 2B centrifuge using the s s 3 4 rotor which had been prewarmed to 37°C. After centrifugation, the coverslips with c e l l s were washed twice with Hanks' balanced s a l t solution, then transferred into fresh medium with serum (Fig. 3). The enucleation e f f i c i e n c y was checked p e r i o d i c a l l y by f i x i n g the c e l l s a fter enucleation and staining with Giemsa. Routinely, popu-lations i n which at least 95% of the c e l l s were enucleated were consistently produced. 22 2 - 4 x 10 5 c e l l s 24-48 hours i n nutrient medium 1 hour i n serum-free medium So r v a l l RC 2B ss34 rotor 37°C 7710 g for 40 minutes 10 yg CB/ml serum-free medium Wash 2X with BSS Fig. 3. Summary of Enucleati Incubation i n fresh medium with serum Procedure: Coverslip Method 10 6 c e l l s 3 mm 24-48 hours i n nutrient medium 58 mm So r v a l l RC 2B GSA rotor 37°C 10800 g for 40 minutes 10 yg CB/ml serum-free medium ..j 1 hour i n serum-free medium Wash 2X with BSS Incubation i n fresh medium with serum Fig. 4. Summary of Enucleation Procedure: 6 cm P e t r i Dish Method 23 During the course of this study, the Prescott method was further modified to f a c i l i t a t e the enucleation of c e l l s grown i n 6 cm diameter P e t r i dishes. 6 cm diameter P e t r i dishes were treated with concentrated reagent grade sulphuric acid for 15 minutes. This was followed by thorough r i n s i n g i n running water for 24 hours, then s t e r i l i z a t i o n by alcohol and UV-irradiation. 10 6 c e l l s were seeded and allowed to attach and grow for 24-48 hours i n these P e t r i dishes containing 3 ml of nut-rient medium. One hour p r i o r to enucleation, the nutrient medium was removed and replaced with serum-free medium. Cells were incubated i n this serum-free medium for 1 hour. C e l l enucleation was achieved by centrifugation of P e t r i dishes containing the monolayer of c e l l s with the c e l l s facing the.bottom of the 250 ml nalgene centrifuge tube (Fisher S c i e n t i f i c Co.) with 100 ml of serum-free medium containing 10 Vg cyto-chalasin B/ml. The P e t r i dish was held i n place by placing a 5 cm piece of 58 mm diameter p l a s t i c a c r y l i c tubing with a 3 mm-thick w a l l (Universal P l a s t i c s Ltd., Burnaby, B.C.) on top of the inverted P e t r i dish. The c e l l s were centrifuged at 10,800 g for 40 minutes i n a S o r v a l l RC 2B centrifuge using the GSA rotor which had been prewarmed to 37°C. After centrifugation, the P e t r i dishes were scanned with an inverted microscope and any area which contained fragments of nucleated c e l l s was c a r e f u l l y scraped off with a rubber policeman. The remaining enucleate c e l l s were then incubated with fresh medium (Fig. 4). VII. Chromosome Preparations Cells were seeded onto 20 mm square coverslips (Corning) i n 35 mm P e t r i dishes covered with 2 ml of nutrient medium. In order to 24 obtain well-spread metaphase plates, the c e l l s were used before they reached 75% confluency. When a great number of c e l l d i v isions were detected (by observation with an inverted microscope), 0.2 ml of a 0.01% solution of colchicine (BDH Chemicals, Poole, Dorset, England) was added for 3-4 hours. The coverslips were then transferred to P e t r i dishes containing 1% sodium c i t r a t e solution for 20 minutes. The hypotonic treatment caused c e l l swelling; thus, chromosomes which were w e l l spread out and separated were produced. The c e l l s were then fixed with acid alcohol, washed with d i s t i l l e d water, and a i r - d r i e d . When dried, the c e l l s were stained for 5 minutes with 2% aceto-orcein, dehydrated through 95% alcohol, absolute alcohol, butanol, butanol-xylol, x y l o l and mounted on glass sl i d e s with Permount (Fisher S c i e n t i f i c Co.). VIII. C e l l V i a b i l i t y C e l l v i a b i l i t y was determined by the method of Yip and Auersperg (19 72). Nucleated and enucleated c e l l s were fixed i n 2.5% glutaraldehyde i n Millonig's buffer ( M i l l o n i g , 1962; Pease, 1964) at pH 7.4 for 1 hour at 4°C. The fixed c e l l s were washed with Millonig's buffer, then stored at 4°C i n buffer for up to 1 week. Exclusion of 0.03% a l c i a n blue (Fisher S c i e n t i f i c Co.) i n buffer for 10 minutes at room temperature by these glutaraldehyde-fixed c e l l s was used as an in d i c a t i o n of c e l l v i a b i l i t y . IX. Protein Determination The amount of c e l l protein i n each culture of c e l l s was deter-mined by the method of Oyama and Eagle (1956). Cultures were washed 25 gently with Hanks' balanced s a l t solution and a i r - d r i e d . The c e l l s were dissolved i n Lowry's reagent (Appendix I I I ) , and reacted with a phenol (Folin-Ciocalteau) reagent (Fisher S c i e n t i f i c Co.). The o p t i c a l density of this solution was read 30 minutes l a t e r at 660 mp using a Coleman Junior spectrophotometer. A mixture of 1 ml of water and Lowry's reagent which reacted with the phenol reagent served as a blank. Crys-t a l l i n e bovine serum albumin (100 yg/ml of water) was dissolved i n Lowry's reagent and reacted with the phenol reagent. This served as a protein standard. The amount of protein per culture was quantitated as bovine serum albumin equivalent. X. Autoradiography 3H-leucine (30-50 Ci/mM) (New England Nuclear) was dilut e d to a concentration of 10 uCi/ml of nutrient medium. Nucleated c e l l s grown on coverslips and enucleated c e l l s on coverslips at varying time int e r v a l s following enucleation were incubated i n medium containing 3H-leucine for 3 hours. Excess 3H-leucine was removed by dipping the coverslips containing the c e l l s i n 3 changes of Hanks' balanced s a l t solution. The c e l l s were then fi x e d i n methanol, extracted with 5% cold t r i c h l o r o a c e t i c acid (Fisher S c i e n t i f i c Co.), rinsed with d i s t i l l e d water, and a i r - d r i e d . To f a c i l i t a t e handling, the coverslips, with the c e l l side up, were mounted on glass s l i d e s with melted p a r a f f i n . The s l i d e s were coated with Kodak NTB3 emulsion (Kodak Eastman) at 43°C, allowed to dry, and then stored at 4°C i n l i g h t - t i g h t boxes for 3 days. 26 The autoradiograms were processed i n D-19 developer, fixed and rinsed under running water. The c e l l s were then stained with Giemsa, di f f e r e n t i a t e d with acetone, dehydrated through a graded alcohol s e r i e s , and mounted i n Permount by placing another covers l i p over the exposed c e l l s . RESULTS I. Morphological and Biochemical Responses of Intact (Nucleated)  Y - l , Y-l-L and Tumor #2 Cells to ACTH and Dibutyryl C y c l i c AMP 1. Morphological responses - l i g h t microscopy a) Y - l c e l l s (ATCC CCL 79) The morphology of Y - l c e l l s i n control medium, and medium containing either ACTH or dibutyr y l c y c l i c AMP, were i d e n t i c a l to those reported i n the l i t e r a t u r e (Yasumura et a l . , 1966a; Kowal et a l . , 1974). b) Y-l-L c e l l s Y-l-L c e l l s changed markedly from a stretched and flattened to a rounded-up shape i n the presence of either ACTH or di b u t y r y l c y c l i c AMP (Figs. 2a-c). In the presence of 10 mU ACTH/ml, the c e l l rounding started within a few minutes, attained a maximum i n 1-2 hours and re-verted gradually to a flattened morphology 12-15 hours a f t e r ACTH adminis-t r a t i o n . This response i s shown most dramatically by the t o l u i d i n e blue-stained epon-embedded cultures (Figs. 5a-e). A l l sections were cut perpendicular to the substratum. In t e r e s t i n g l y , although l i v e c u l -tures appeared as monolayers with l i g h t microscopy, up to 6 overlapping layers of Y-l-L c e l l s were found by examining perpendicularly-cut p l a s t i c sections of confluent cultures f i x e d and embedded i n s i t u (Figs. 6a and b). In the presence of ACTH, the c e l l s i n direct contact with the medium were rounded up, while most deeper c e l l s i n the s t r a t i f i e d layer assumed a more or less ovoid c e l l shape (Fig. 6b), Plate I I Response to ACTH by Y-l-L C e l l s . Epon-embedded sections, toluidine blue s t a i n i n g , x750. Figs. 5a-e. Y-l-L c e l l s . (a) i n control medium, (b) 5 minutes i n medium containing 10 mU ACTH/ml, (c) 1 hour i n medium containing 10 mU ACTH/ml, (d) 3 hours i n medium containing 10 mU ACTH/ml, and (e) 24 hours i n medium containing 10 mU ACTH/ml. Maximum c e l l rounding was attained at 1-2 hours. Figs. 6a and b. Multilayered Y-l-L c e l l s . (a) i n control medium; c e l l s i n a l l layers were flattened and appeared squamous-like i n long i t u d i n a l sections, (b) 3 hours i n medium containing 10 mU ACTH/ml; c e l l s i n the s u p e r f i c i a l layer appeared most rounded i n morphology. 29 Plate I I I Histology of Tumor ill and the Response of Tumor #2 Cells i n Culture to ACTH and Dibutyryl C y c l i c AMP. Figs. 7a and b. Bouins' f i x a t i o n , hematoxylin and eosin staining. Histology of Tumor #2. (a) low magnification, x250, (b) some areas resemble t y p i c a l l y carcinomatous tiss u e , x700. Figs. 8a-c. Ethanol f i x a t i o n , t o luidine blue s t a i n i n g , x350. Tumor #2 c e l l s i n culture. (a) i n control medium, (b) 3 hours i n medium containing 10 mU ACTH/ml, (c) 3 hours i n medium containing 1 mM dibutyry l c y c l i c AMP. The morphology of these c e l l s was s i m i l a r to that of the Y-l-L c e l l s . 31 32 The morphological response of Y-l-L c e l l s to 1 mM dibutyryl c y c l i c AMP proceeded at a slower rate. C e l l rounding was only v i s i b l e 1 hour after the addition of dib u t y r y l c y c l i c AMP, and maximum rounding was attained i n 2-3 hours. 18-24 hours after treatment with the c y c l i c nucleotide, the normal flattened morphology was resumed. c) Tumor #2 Cells H i s t o l o g i c a l l y , the tumors derived from the inocula of Y-l-L c e l l s into mice were pleomorphic adrenocortical carcinomas. Areas ranging from anaplastic tissue to t y p i c a l l y carcinomatous tissue were observed (Figs. 7a and b). The morphology of cultured Tumor #2 c e l l s i n control medium and i n medium containing either ACTH or dib u t y r y l c y c l i c AMP i s shown i n Figs. 8a-c. The morphologic response of these c e l l s to ACTH and d i -butyryl c y c l i c AMP was comparable to that of the Y-l-L c e l l s . 2. Steroidogenic responses a) Introduction I n i t i a l l y , A4-3 keto s t e r o i d output of the Y - l c e l l s and Y-l-L c e l l s was assayed by determining the c h a r a c t e r i s t i c u l t r a v i o l e t absorption at 242 my by the st e r o i d products present i n the methylene chloride extract of medium (Buonassisi eit a l . , 1962) . Because of the r e l a t i v e i n s e n s i t i v i t y of this method (accurate to 0.3 yg of C o r t i s o l standard), and the high blanks obtained from extracing medium containing large quantities of serum, the accurate measurement of steroids required the accurate preparation of p a r a l l e l medium blanks, and the measurement of at least 0.5 yg of st e r o i d . 33 By a s l i g h t modification of this method - washing the methylene chloride extract with either water or 0.1 M NaOH - s u f f i c i e n t i n t e r -f e r i n g substances were removed to permit the quantitation of steroids produced by 1 mg c e l l p r o t e i n / P e t r i dish of c e l l s i n 3 hours. An increase i n i n absorbency of incubation medium from c e l l s treated with ACTH could be seen, but the increase was usually too small to permit precise quanti-tation. A substantially more sensitive analysis of st e r o i d output was developed by Kowal (Kowal and F i e d l e r , 1968), and the spectrofluorometric assay described i n Materials and Methods was used i n this study. The merits of this assay are: the fluorescence of 20a hydroxylated deriva-tives of progesterone i n ethanolic sulphuric acid i s intense enough to permit the precise quantitation of 15 ng of 20a dihydroprogesterone, a 200-fold increase over the lowest l e v e l of the UV assay; the fluores-cence of medium blanks i s low; at most i t i s equivalent to 5-10 ng of 20a dihydroprogesterone, and the maximum fluorescence i s stable 45-90 minutes a f t e r extraction of the steroids into ethanolic sulphuric acid. Y - l c e l l s produce 20a dihydroprogesterone and 11B0H, 20a dihydro-progesterone. Both steroids fluoresce under the conditions described, and i t has been determined that the fluorescence of 11B0H, 20a dihydro-progesterone i s approximately 1.6 times greater than the i n t e n s i t y of 20a dihydroprogesterone. In order to quantitate the steroid output precisely, the exact proportion of the two steroids would have to be known i n each incubation. Y-l-L c e l l s and Tumor #2 c e l l s produce minute quantities of 1130H, 20a dihydroprogesterone (see Results section I 3a and c) i n r e l a t i o n to 20a dihydroprogesterone. To simplify matters, 34 the steroids have been quantitated as nanograms of 20a dihydroproges-terone . b) Y - l Cells (ATCC CCL 79) Steroid output of Y - l c e l l s under various conditions i s tabu-lated i n Table 2. Steroidogenesis was determined on cultures just p r i o r to reaching confluency, when each P e t r i dish of c e l l s contained approxi-mately 1 mg of c e l l protein. Cells were incubated for 3 hours i n 3 ml of either control medium, medium containing 10 mU ACTH/ml, or medium containing 1 mM dibutyryl c y c l i c AMP. Y - l c e l l s produced steroids equiva-lent to 2 yg of 20a dihydroprogesterone i n control medium i n 3 hours. In the presence of either ACTH or dibutyry l c y c l i c AMP, the steroid output was increased 1.5-fold. These values were comparable to those values which had been reported i n the l i t e r a t u r e for Y - l c e l l s . c) Y-l-L Cel l s In comparison with the Y - l c e l l s , Y-l-L c e l l s had a much lower steroid output under the same conditions (Table 2). In addition, the amount of ste r o i d output also diminished a f t e r routine s e r i a l sub-cultures (Table 3). Y-l-L c e l l s were used for several experiments, but the use of these c e l l s was discontinued due to the d i f f i c u l t y of accurate measurement of the low ste r o i d output. d) Tumor #2 Cells Tumor #2 c e l l s i n i t i a l l y produced steroids at a high rate (Table 3) which was comparable i n magnitude to that of Y - l c e l l s (ATCC CCL 79). Nevertheless, the steroidogenic a c t i v i t y of these c e l l s under TABLE 2. Comparison of Steroidogenic Responses to ACTH and Dibutyryl C y c l i c AMP Y - l Cells (ATCC CCL 79) and Y-l-L Cells (ng s t e r o i d / P e t r i dish/3 hours) 3 Y - l (ATCC CCL 79) Y-l-L Basal 2050 160 10 mU ACTH/ml 3170 300 1 mM dbcAMP 3020 225 Each point represents the average steroid output of 3 P e t r i dishes of c e l l s . The var i a t i o n was less than 10% within each group. 36 TABLE 3. Steroidogenic Responses to ACTH and Dibutyryl C y c l i c AMP by Y-l-L Cells and Tumor //2 Cells after S e r i a l Subculture Y-l-L Tumor #2 Passage Number Basal ACTH dbcAMP Basal ACTH dbcAMP (ng s t e r o i d / P e t r i dish/3 hours) Primary - - - 1090 19 30 1790 2 160 - 300 225 390 880 640 3 - - - 340 610 560 4 125 290 250 240 420 370 5 80 190 155 200 350 310 6 75 220 150 200 320 320 8 70 120 90 - - -10 60 100 - 180 280 290 12 b 50 120 100 — Each point represents the average steroid output of 3 P e t r i dishes of c e l l s . The v a r i a t i o n was less than 10% within each group. ng s t e r o i d / P e t r i dish/12 hours. 37 various conditions also diminished after s e r i a l subculture. This pheno-menon i s quite common and has been reported by several laboratories which work with the Y - l c e l l l i n e . Between the t h i r d and s i x t h culture passages, the steroidogenic a c t i v i t i e s of Tumor #2 c e l l s were quite stable. A l l subsequent experiments were performed on cultures between these passages. Cultures after the s i x t h passage were terminated, and culture samples frozen after the second passage were re-established for experiments. 3. I d e n t i f i c a t i o n of the endogenous steroids produced during a  12-hour incubation a) Y - l (ATCC CCL 79) Thin layer chromatography of the endogenous steroid production of Y - l c e l l s i n either control medium or medium containing 10 mU ACTH/ml revealed that there were 5 UV-absorbing spots which were i d e n t i c a l to those described by Kowal (Kowal and F i e d l e r , 1968). Although there were no q u a l i t a t i v e differences i n the steroids produced i n either control medium or medium with ACTH, the UV-absorbing spots of the l a t t e r incu-bation seemed more intense. The 2 major UV-absorbing spots had the mobility i d e n t i c a l to that of ste r o i d standards 20a dihydroprogesterone and llgOH, 20a dihydroprogesterone. The 3 minor UV-absorbing spots corresponded to those of progesterone, 1130H progesterone and 11 keto, 20a dihydroprogesterone (Fig. 9). b) Y-l-L C e l l s The analysis of the endogenous steroid production of Y-l-L c e l l s (3rd passage) revealed that the steroids elaborated were i d e n t i c a l OQ 3 n> M 0 &• O oo O (t) Hi 0 o o c n> co M H w to rt n> u n c o oo "it) t-t M O to (X PC O O rt C CO H CO pi o Hi 3 0 O O I S- ^  a* n fu (D rt 00 H- 0 O fD 0 0 O O 0 a> f? rt s-O fD CO Y - l (Kowal and Fi e d l e r , 1968) Y - l (ATCC CCL 79) Y-l-L (3P) Y-l-L (10P) Tumor //2 (5P) ll*C-Pregnenolone Metabolism Y-l-L (8P), Tumor #2 (5P) Steroid Standards o t, CO rt O 0* rt CO < CO T3 0* O CO o i -S n> o H PJ v: H cr rt fa ro o CO m <o o i-l H- Cu D- ^< • H' 0 00 < I & en O 3-H-0 00 CO 13 O rt CO n cr • n O w § (D rt 0 O N 00 CD 1-1 0 CU •• 0* > H-o o fD rt C/i O 0 CO fD rt ft> ~ 3 ho O O c CO fl> PJ CO 0 a 9 t n o i i o A A H A t—' U) O O O O 1/1 1 1 1 1 N3 N> to -O O O o o 8-3 3^ 9 o @ • o ® 8e o H' M ho ho •d fD o M o M O i-l I-l TO H "CD P TO P fD O ro rt o 5*! o 00 00 0 H- a (0 a a a 0 fD a- O »# rt H- fD CO CO O o 0* 0" 0 rt •• CD hO «* H ><! o fD rt O PL O a. 1—1 i-t (D P N> 00 i-i o O H O o fD o 0 0 O a P T> CO •tf fD fD 0 H" H rt n ro 0 * a ro ft> o 00 H 00 & 0* 0 O fD H ^ n> 0 CO O P. 0 fl> rt •a i-l o fD H o M i-l o >d o o 00 n 0 0 fD o fD fD CO 00 rt ro fD CO i-l rt o n> 0 n (t> one 39 to those of the Y - l c e l l s (ATCC CCL 79); however, a quantitative d i f -ference was noted. The UV-absorbing spot corresponding to that of proges-terone was much more intense, while the other 4 spots were smaller and less d i s t i n c t . Corresponding to the low st e r o i d output by 10th passage Y-l-L c e l l s , only 3 UV-absorbing spots were v i s i b l e on the developed thin-layer chromatogram. The prominent spot near the solvent front corresponded to that of progesterone; following that were those of 20a dihydroprogesterone and a very f a i n t l y v i s i b l e spot corresponding to that of llgOH, 20a dihydroprogesterone (Fig. 9). c) Tumor #2 Cells The endogenous ste r o i d products of Tumor #2 c e l l s during a 12-hour incubation period i n nutrient medium were i d e n t i c a l to those of 10th passage Y-l-L c e l l s : progesterone, 20a dihydroprogesterone and a trace of 11B0H, 20a dihydroprogesterone (Fig. 9). 4. * ^ -pregnenolone metabolism by Y-l-L Cells and Tumor #2 Cells a) General The metabolism of l l fC-pregnenolone during 12 hours of incu-bation i n nutrient medium by Y-l-L c e l l s (8th passage) and Tumor #2 c e l l s (5th passage) was very s i m i l a r . Within 12 hours, approximately 80-90% of the 1^-pregnenolone was metabolized: 10-20% into 20a dihydro-pregnenolone, 30-40% into progesterone, 10-20% into 20a dihydroproges-terone, and trace amounts into 1130H progesterone, 1130H, 20a dihydro-progesterone and 11 keto, 20a dihydroprogesterone (Fig. 9). 40 b) P o s i t i v e i d e n t i f i c a t i o n of 20g dihydroprogesterone produced  by Y-l-L Cells Since the assay of steroid output was quantitated as equiva-lent nanograms of 20a dihydroprogesterone, an attempt was made to pos i -t i v e l y i d e n t i f y the 20a dihydroprogesterone endogenously produced by the Y-l-L c e l l s . The intent was to acetylate the 20a dihydroprogesterone produced endogenously by Y-l-L c e l l s , to acetylate t h i s product wi th 3H -acetic anhydride, and to r e c r y s t a l l i z e the resultant 20a dihydroprogesterone-3H-acetate i n the presence of authentic 20a dihydroprogesterone acetate. Approximately 1 ug of 20a dihydroprogesterone produced by Y-l-L c e l l s endogenously was iso l a t e d . The result of the acetylation experiment i s presented i n Table 4. ^C^Oa dihydroprogesterone was added i n the experiment to monitor the recovery. The 3H:ll+C r a t i o of the 3rd and 4th crystals of each Standard 1, Standard 2 and sample were constant (within 5% of each other). However, the 3H:llfC r a t i o of the sample was not greater than that of the standards. Two explanations can be given: one i s that during the procedure, an error was made such that either more than 0.5 yg of standard 20a -dihydroprogesterone was used, or that there was a loss of sample during the process. Another explanation i s that the 1 4C-20a dihydroproges-terone- 3H-acetate standard used contained so much s i n g l y - l a b e l l e d 20a -dihydroprogesterone- 3H-acetate that this obscured the amounts present i n the sample and the standards. In view of the fact that 1I+C-20a dihydro-progesterone from the chromatogram of 1 ^ -pregnenolone metabolism of 41 Y-l-L c e l l s contained 10 yg of cold c a r r i e r , and that the acetylated product was equally divided and added to each of the sample and the two standards to monitor recovery, this explanation seems more feasible. Although the goal of this exercise was not achieved, i t could be concluded unequivocally that the 1 L fC-pregnenolone metabolite produced by Y-l-L c e l l s was ll+C-20a dihydroprogesterone. This compound could be acetylated, and the acetylated product c o - c r y s t a l l i z e d with standard 20a dihydroprogesterone-acetate. Since Y-l-L c e l l s could metabolize minute quantities of added pregnenolone into 20a dihydroprogesterone, there i s no reason to suspect that Y-l-L c e l l s should not produce t h i s compound endogenously, unless the substrate was absent. c) P o s i t i v e i d e n t i f i c a t i o n of 1'tC-20a dihydroprogesterone  produced by Tumor 7/2 Ce l l s ll*C-20a dihydroprogesterone metabolized from l l +C-pregnenolone by the Tumor #2 c e l l s was p o s i t i v e l y i d e n t i f i e d by c o - c r y s t a l l i z i n g to constant s p e c i f i c a c t i v i t y with added authentic 20a dihydroproges-terone. The r e c r y s t a l l i z a t i o n data are presented i n Table 5 . 5 . Electron microscopy of Y-l-L Cel l s a) Ultrastructure of Y-l-L Cells i n s i t u A tangential section through Y-l-L c e l l s i n culture i s shown i n Fig. 10. The fact that these c e l l s did not resemble the characteris-t i c rodent adrenocortical c e l l s i n vivo i s immediately apparent. The nucleus of the Y-l-L c e l l was large and mostly euchromatic; a prominent nucleolus was often v i s i b l e . In contrast to the elaborate network of 42 TABLE 4. Acetylation of 20a Dihydroprogesterone Produced Endogenously by Y-l-L Cells with 3H-Acetic Anhydride, and R e c r y s t a l l i z a t i o n of 20a Dihydroprogesterone-3H-Acetate to Constant 3H:1^C Ratio i n the Presence of 1 4C-dihydroprogesterbne- 3H-Acetate 3H a 1 4 C 3 3H: 1 4C Mean + 5% Standard 1 XL 3 10,733 60 XLi+ 9,827 53 178.88 185.42 182.15 + 9.11 Standard 2 XL 3 12,636 42 XLu 11,173 36 300.86 310.36 305.11 + 15.28 Sample XL 3 X L 4 14,185 11,132 61 49 232.54 227.18 229.86 + 11.49 Expressed as dpm/min. k The standards used were two 0.5 yg aliquots of authentic 20a dihydroprogesterone which were treated i n the same manner as the 20a dihydroprogesterone produced by the Y-l-L c e l l s (Sample). c The amount of sample used was approximately equivalent to 1 yg of 20a dihydroprogesterone. 43 TABLE 5. Radiochemical I d e n t i f i c a t i o n of 1 4C-20a Dihydro-progesterone Produced by Tumor #2 C e l l s 3 Condition Crystals (dpm/mg) Mother Liquor (dpm/mg) Basal XL X XL 2 XL 3 3,882 5,331 5,309 5,258 ML! ML2 ML3 MLi+ 19,696 6,141 5,488 5,248 ACTH XL! XL 2 XL 3 XL 4 4,965 4,303 4,270 4,245 MLi ML2 ML3 M L 4 10,222 5,005 4,516 4,273 a Six 6 cm P e t r i dishes of Tumor #2 Cells were incubated i n either control medium or medium with ACTH (10 mU/ml) containing 1 1 +C-pregnenolone (200,000 dpm/culture). After 12 hours, the medium was removed and medium from 3 P e t r i dishes under each condition were pooled. The i s o l a t i o n of 20a dihydroprogesterone has been described i n Materials and Methods. 11+C-20a dihydroprogesterone was r e c r y s t a l l i z e d from ethanol water with 10 mg of cold c a r r i e r . k Expressed as dpm/mg of authentic s t e r o i d . 44 Plate IV Ultrastructure of the Y-l-L Cells in_ s i t u . Uranyl acetate and lead c i t r a t e staining. Fi g . 10. Tangential section through Y-l-L c e l l s i n control medium, x28,000. Fig. 11. Y-l-L c e l l s 24 hours i n medium containing 10 mU ACTH/ml. These c e l l s have resumed t h e i r normal flattened morphology and were i d e n t i c a l to c e l l s i n control medium. Section cut perpendicular'to the substratum, x8,000. Fig. 12. Y-l-L c e l l i n control medium. The side facing the medium i s on the righ t . Microtubules were found to be scattered throughout the c e l l while the microfilaments were found to be near the surfaces of the c e l l and running p a r a l l e l to the substratum. Mitochondria with s h e l f - l i k e cristae i s also shown, xl9,000. Fig. 13. Golgi apparatus present i n a Y-l-L c e l l i n control medium, x30,000. Abbreviations: b.v. budding v i r u s ; g. g o l g i ; m. mitochondria; mf. micro-filaments; mt. microtubules; m.w. membranous whorls; p.v. pinocytotic v e s i c l e s ; v.p. virus p a r t i c l e s . 45 46 smooth endoplasmic reticulum, the c h a r a c t e r i s t i c mitochondria with v e s i -cular c r i s t a e , and the abundance of glycogen and l i p i d droplets present i n the cytoplasm of the c e l l s i n the fa s c i c u l a t a and r e t i c u l a r i s zones, the Y-l-L cytoplasm contained p r o f i l e s of long, granular endoplasmic reticulum and mitochondria with s h e l f - l i k e cristae (Figs. 12 and 13). In some of the c e l l s , an abundance of mitochondria was present (Fig. 18). Glycogen p a r t i c l e s were absent and l i p i d droplets were rarely observed i n Y-l-L c e l l s . Well-developed Golgi bodies with 3-4 p a r a l l e l arrays of cisternae (Figs. 10 and 13), lysosomes with homogeneous dense content, membranous whorls, and microtubules were often discernible. Cytoplasmic o filaments approximately 40 A i n diameter were present near the plasma membrane and oriented p a r a l l e l to the substratum (Fig. 10). Pinocytotic vesicles were also present near the plasma membrane, and pinocytotic invaginations were observed on the c e l l surface (Fig. 11). Typical C-type p a r t i c l e s as free e x t r a c e l l u l a r virus and t h e i r membrane-budding precursors were also observed. Adjacent c e l l s attached s o l e l y by i n t e r -o mediate junctions with an i n t e r c e l l u l a r space of about 200 A. Otherwise, large i n t e r c e l l u l a r spaces 1-2 microns i n diameter extended i n a l l direc-tions around the c e l l s . Large membrane whorls and virus p a r t i c l e s were present i n these spaces (Fig. 10). The ultrastructure of approximately 50 Y-l-L c e l l s following incubation i n medium containing ACTH for 5 minutes, 30 minutes, 1 hour, 3 hours and 24 hours was examined. The c h a r a c t e r i s t i c u l t r a s t r u c t u r a l changes i n response to ACTH reported i n the l i t e r a t u r e , namely, the presence of pinocytotic vesicles and the penetration of mitochondria 47 Plate V Ultrastructure of Y-l-L Cells i n s i t u : Response to ACTH. Uranyl acetate and lead c i t r a t e staining. Figs. 14 and 15. Y-l-L c e l l s following incubation i n medium containing 10 mU ACTH/ml for 1 hour. Cells were rounded i n morphology. Note the swollen Golgi bodies, x9,500. Fig. 16. Dilated Golgi apparatus i n a Y-l-L c e l l following incubation i n medium containing 10 mU ACTH/ml for 3 hours, x20,000. Fig. 17. Y-l-L c e l l s following incubation i n medium containing 10 mU ACTH/ml f o r 3 hours. Two of the ch a r a c t e r i s t i c normal responses to ACTH, namely, the presence of coated v e s i c l e s , and the penetration of mitochondria into l i p i d droplets to form myelin-structures were also observed i n Y-l-L c e l l s , xl8,000. Fig . 18. Y-l-L c e l l s following incubation i n medium containing 10 mU ACTH/ml for 5 minutes. Abundance of mitochondria within c e l l , x8,700. Abbreviations: c.v. coated v e s i c l e s ; g. g o l g i ; p.v. pinocytotic v e s i c l e s ; z.a. zonular adherens; 48 49 into l i p i d droplets to form myelin-like structures were observed i n Y-l-L c e l l s following 30 minutes, 1 hour, and 3 hours of incubation with ACTH (Fig. 17). In the same cultures, the d i l a t i o n of Golgi c i s -ternae, and the increase i n size of i n t e r c e l l u l a r spaces were consis-tently seen (Figs. 14-16). Swollen endoplasmic reticulum and coated vesicles were observed i n a few c e l l s , and there appeared to be fewer cytoplasmic microtubules i n a great number of ACTH-treated c e l l s observed. Although increased steroidogenesis i n response to ACTH occurs within minutes upon the addition of ACTH, the ultrastructure of Y-l-L c e l l s following 5 minutes' incubation with ACTH was i d e n t i c a l to that of c e l l s i n control medium. Y-l-L c e l l s , following 24 hours' incubation i n medium containing ACTH, were also comparable to those i n control medium. Electron microscopy of monolayers cut perpendicular to the sub-stratum revealed that Y-l-L c e l l s were exceedingly f l a t and t h i n , re-sembling squamous c e l l s i n control medium and i n medium containing ACTH for 24 hours (Fig. 11). Monolayer Y-l-L c e l l s i n medium containing ACTH for 5 minutes, 30 minutes, 1 hour and 3 hours assumed i r r e g u l a r l y round or rhomboidal outlines with large i n t e r c e l l u l a r spaces (Figs. 14 and 15). In sections of multilayered Y-l-L c e l l s , only the s u p e r f i c i a l layer of c e l l s was rounded. Long, but thin surface f o l d s , approximately o 800-1000A i n thickness, were frequently observed on the c e l l surface which was exposed to the medium of the rounded c e l l s (Fig. 14). From this study, no correlation between the ultrastructure and the acute steroidogenic response of Y-l-L c e l l s could be made. The 50 mechanism of c o r t i c o s t e r o i d secretion at the u l t r a s t r u c t u r a l l e v e l could not be distinguished and remains enigmatic. c) Ultrastructure of Y-l-L Cells following incubations i n  trypsin and versene Toluidine blue-stained thick sections of Y-l-L c e l l s following 10 minutes of incubation i n either t r y p s i n , or trypsin and versene, are shown i n Figs. 19a and 20a. The outlines of c e l l s appeared "fuzzy." At the l e v e l of resolution of the electron microscope, i r r e g u l a r con-volutions at the c e l l surface exposed to the medium were observed. The surface folds of c e l l s exposed to trypsin only were comparatively longer and more slender (Fig. 19b) than those exposed to both trypsin and ver-sene (Fig. 20b). The surface folds of these c e l l s were club-shaped, resembling pseudopodia. The results of this study show that the term used to describe the morphological response of Y - l c e l l s to ACTH, "rounding up," i s an ambiguous one. In tissue culture, trypsin and/or versene i s routinely used i n the subculture procedure. The reaction of c e l l s to t r y p s i n i z a t i o n i s also referred to as "rounding up." I t i s evident that the ultrastructure of rounded Y-l-L c e l l s i n the presence of ACTH was not the same as that of rounded Y-l-L c e l l s i n the presence of trypsin or trypsin and versene: the abundance of pinocytotic vesicles and the complex folds of the plasma membrane, as w e l l as the persistence of organelles (mitochondria) i n the c o r t i c a l cytoplasm i n c e l l s responding to ACTH suggested an active exchange of materials with the environment; any evidence of such a c t i v i t y was lacking i n the trypsin and versene treated c e l l s . 51 Plate VI Y-l-L Cells Following Treatment with Trypsin and Trypsin together with Versene. Figs. 19a and 20a. Epon-embedded sections, toluidine blue-staining, xl,100. Y-l-L c e l l s following 10 minutes incubation i n (a) 0.12% t r y p s i n , (b) 0.12% trypsin and 0.04% versene. Figs. 19b and 20b. Uranyl acetate and lead c i t r a t e staining. Ultrastructure of the surface of Y-l-L c e l l s following 10 minutes of incubation i n (a) 0.12% tr y p s i n , xl8,000, (b) 0.12% trypsin and 0.04% versene, x20,000. The surface folds of c e l l s exposed to trypsin only were comparatively longer and more slender (Fig. 19b) than those exposed to both trypsin and versene (Fig. 20b). The surface folds of these c e l l s were club-shaped, resembling pseudopodia. 52 53 I I I . Enucleation Procedure Nuclear protrusion occurred when cytochalasin B was added to Y - l c e l l s (ATCC CCL 79) i n monolayer cultures. However, only one out of several thousand c e l l s became spontaneously enucleated as a result of cytochalasin B treatment alone. When the enucleation procedure described by Prescott and his colleagues was adopted (Prescott et a l . , 1972), populations i n which 10-20% of the Y - l c e l l s were anucleate were produced. B a s i c a l l y t h i s procedure involved growing Y - l c e l l s on round glass coverslips and cen-t r i f u g i n g the coverslips of c e l l s with the c e l l s facing downwards i n centrifuge tubes f i l l e d with medium containing cytochalasin B. Many attempts were made to increase the enucleation e f f i c i e n c y ; these i n -cluded alterations either s i n g l y , or i n combinations of: timing of addition of cytochalasin B, concentration of cytochalasin B, c e n t r i f u g a l force and centrifugation time. At best, up to 40% enucleation was achieved. A major drawback of this technique was that c e l l loss due to detachment from the coverslip during centrifugation was great. At high c e n t r i f u g a l forces (> 5,000 g), the amount of c e l l loss was severe, while at lower centrifugal forces, the proportion of c e l l s which became enucleated was low. Following centrifugation at 8,000 g for 30 minutes, approximately 25% of the c e l l s o r i g i n a l l y present remained on the coverslips. An analysis of the c e l l s detached from the coverslips and pelleted at the bottom of the centrifuge tube was made, and a higher percentage of enu-cleated c e l l s was found. Y - l c e l l s appeared to adhere more tenaciously to p l a s t i c sub-stratum, especially to etched p l a s t i c surfaces (surfaces treated with 54 sulphuric acid or permanganate). When Y - l c e l l s were enucleated on p l a s t i c coverslips which had been treated with sulphuric acid for 15 minutes, c e l l loss during centrifugation was decreased. Optimal enu-cleation of Y - l c e l l s was 50% when Y - l c e l l s grown on sulphuric acid-treated p l a s t i c coverslips were centrifuged i n medium containing 10 yg cytochalasin B/ml at 7,710 g for 30 minutes i n a So r v a l l RC 2B c e n t r i -fuge using the ss34 rotor. With this method, the morphologic response of enucleated Y - l c e l l s to ACTH could be e a s i l y determined. Nevertheless, the steroido-genic response of enucleated Y - l c e l l s to the trophic hormone would be impossible to assess as i t could not be determined conclusively whether the steroids were produced by the enucleated or the nucleated Y - l c e l l s . In contrast to the Y - l c e l l s , the subline Y-l-L c e l l s were readily enucleated. The success i n enucleation of the Y-l-L c e l l s was attributed to the increased adhesion of Y-l-L c e l l s to the substratum. Loss of c e l l s due to the high centri f u g a l forces used during the enucleation procedure was much less i n comparison with the Y - l c e l l s . By a s l i g h t increase i n centrifugation time, populations i n which 95-98% of the Y-l-L c e l l s were anucleate were routinely produced by centrifuging the monolayer of c e l l s grown on glass coverslips i n medium containing 10 yg cytochalasin B/ml at 7,710 g for 40 minutes i n a S o r v a l l RC 2B centrifuge using the ss34 rotor. By a s l i g h t modification of the method, c e l l loss was further minimized. By pre-incubation of Y-l-L c e l l s i n serum-free medium, and by subsequent centrifugation i n serum-free medium containing cytochalasin 55 B, s i g n i f i c a n t l y more c e l l s which were enucleated remained on the cover-s l i p s . Thus, the enucleation e f f i c i e n c y was increased. Since v i r t u a l l y uniform enucleation was achieved with Y-l-L c e l l s , t his subline was used for the study of the response of enucleated adrenocortical c e l l s to ACTH and dibutyryl c y c l i c AMP. Relatively large numbers of enucleate c e l l s were required for biochemical analyses; therefore the 18 mm diameter round coverslip method proved to be quite tedious, as only small amounts (200-300 yg) of anu-cleate c e l l s could be produced during each 40-minute centrifugation run. During the course of this study, the Prescott method was further modified to f a c i l i t a t e the enucleation of c e l l s grown on 6 cm diameter P e t r i dishes (see Materials and Methods). 95-98% enucleation was achieved when Y-l-L c e l l s grown on sulphuric acid-treated P e t r i dishes were pre-incubated i n serum-free medium, and centrifuged i n serum-free medium containing 10 yg cytochalasin B/ml at 10,800 g for 35 minutes i n a RC 2B centrifuge using the GSA rotor. As a r e s u l t , a ten-fold increase i n amount (i.e.'2-3 mg) of anucleate c e l l s could be produced i n the same amount of time. Tumor #2 c e l l s , s i m i l a r to the Y-l-L c e l l s , were readily enu-cleated with i d e n t i c a l e f f i c i e n c y under the same conditions. Enucleated Y-l-L c e l l s and Tumor #2 c e l l s resumed t h e i r e p i -t h e l i a l - l i k e morphology within 15 minutes following the removal of cyto-chalasin B, two changes of Hanks' balanced s a l t s olution, and incubation i n fresh nutrient medium. By using phase optics, any c e l l retaining i t s nucleus could be eas i l y i d e n t i f i e d . Routinely, enucleated cultures were 56 randomly selected for the determination of enucleation e f f i c i e n c y . The proportion of nucleated c e l l s counted per 200-300 c e l l s was consistently found to be less than 5%. I I I . Effects of DMSO, Cytochalasin B and Enucleation Procedure Treatments (without actual enucleation) Upon Y-l-L and Tumor  #2 Cells 1. Effect upon steroidogenesis The protocol which was used to determine whether or not, i n the absence of actual enucleation, cytochalasin B or the centrifugation used during the enucleation procedure had any effect upon the subsequent steroidogenic a c t i v i t i e s of the Y-l-L c e l l s and Tumor #2 c e l l s i s i l l u s -trated i n Fig. 21. Results of three experiments on the effects of DMSO, cytochalasin B and enucleation procedure treatments upon the steroidogenesis of Y-l-L c e l l s and Tumor #2 c e l l s are presented i n Tables 6-8. In the presence of 10 yg cytochalasin B/ml, as w e l l as 10 y l DMSO/ml, the steroidogenic responses of both Y-l-L c e l l s and Tumor #2 ce l l s were usually decreased. Nevertheless, the steroidogenic a c t i v i t i e s of Y-l-L c e l l s and Tumor #2 c e l l s following the removal of cytochalasin B, or following centrifugation, were comparable to those of the control c e l l s (Tables 6-8). Thus, the lack of effects upon steroidogenesis following the removal of treatments used during the enucleation procedure was v e r i f i e d . In subsequent experiments, i n which the metabolic a c t i -v i t i e s of enucleated and nucleated c e l l s were compared, the nucleated c e l l s which served as controls received i d e n t i c a l treatments as the 57 Fig. 21. Experimental Design: Steroidogenic Responses to ACTH and Dibutyryl Cyclic AMP by Enucleation Procedure Treatment Cells and Enucleated C e l l s . 0 10 _ L 11 12 Hours Control DMSO ETOH CB (DMSO) CB (ETOH) EPT (CB) EPT (cent) 0-3 3-6 6-9 9-12 Legends: ent I S . F I F I s . r i e t\ I S . F J E rl I S . F 4 E DMSO ETOH CB (DMSO) CB (ETOH) EPT (CB) EPT (cent) S.F. E Incubation i n control medium = Basal medium containing 10 mU ACTH/ml = dbcAMP medium containing 1 mM dibutyryl c y c l i c AMP = dbcAMP At the end of this incubation period, the media were removed and the amounts of steroids produced were assayed. 1% DMSO was also added to the incubation medium. 1% ETOH was also added to the incubation medium. 10 yg cytochalasin B (dissolved i n DMSO)/ ml was also added to the incubation medium. 10 yg cytochalasin B (dissolved i n ETOH)/ml was also added to the incubation medium. Enucleation Procedure Treatment: the steroidogenic a c t i v i t i e s of the c e l l s were determined following incubation i n media containing CB. Enucleation Procedure Treatment: the steroidogenic a c t i v i t i e s of the c e l l s were determined following centrifugation i n serum-free medium. 1 hour preincubation i n serum-free medium. Enucleation - cytochalasin B treatment + centrifugation. 2 washes i n Hanks' balanced s a l t solution Incubation i n fresh medium. TABLE 6. Effects of DMSO, Cytochalasin B and Enucleation Procedure Treatment on the Steroidogenic Responses of Y-l-L Cells to ACTH and Dibutyryl Cyclic AMPa ng s t e r o i d / P e t r i dish/3 hour Basal ACTH dbcAMP Control DMSO Cytochalasin B EPT (CB) 80 + 3 65 + 3 80 + 0 100 + 2 190 + 15 115 + 3 145 + 0 255 + 6 155 + 8 9 0 + 5 125 + 16 205 + 9 TABLE 7. Effects of DMSO, Cytochalasin B and Enucleation Treatments on the Steroidogenic Responses of Tumor #2 Cells to ACTH and Dibutyryl C y c l i c AMPa ng s t e r o i d / P e t r i dish/3 hour° Basal ACTH dbcAMP Control 390 + 10 880 + 35 655 + 35 DMSO 350 + 11 595 + 26 455 + 18 Cytochalasin B 350 + 11 645 + 30 480 + 22 EPT (CB) 430 + 10 935 + 37 685 + 12 EPT (cent) 405 + 18 815 + 48 710 + 73 See Fig. 21 for incubation protocol. k Each point represents the average steroid output + S.E. of 3 determinations. Each point represents the average ste r o i d output + S.E. of 5 determinations. 59 TABLE 8. Effects of DMSO, Ethanol, Cytochalasin B Dissolved i n DMSO, Cytochalasin B Dissolved i n Ethanol, and Enucleation Procedure Treatments on the Steroido-genic Responses of Tumor #2 Cells to ACTH and Dibutyryl Cy c l i c AMPa ng s t e r o i d / P e t r i dish/3 hour Basal ACTH dbcAMP Control 188 + 1 290 + 20 236 + 17 DMSO 185 + 3 242 + 5 213 + 5 Ethanol 199 + 9 278 + 4 259 + 3 Cytochalasin B (DMSO) 186 + 2 248 + 6 215 + 5 Cytochalasin B (Ethanol) 185 + 3 250 + 8 212 + 7 EPT (CB) 190 + 5 283 + 4 250 + 8 EPT (cent) 194 + 10 288 + 6 245 + 4 See Fig. 21 for incubation protocol. k Each point represents the average s t e r o i d output + S.E. of 5 determinations. 60 c e l l s which were enucleated - the only difference was that the c e n t r i -fugation step was omitted i n the former case. These nucleated control c e l l s were designated as Enucleation Procedure Treatment Control (EPT Control). To elucidate whether the effect of 10 pg cytochalasin B/ml upon steroidogenesis was due sol e l y to cytochalasin B, or to both the fungal metabolite and i t s solvent DMSO, ethanol was used as a substitute solvent. Addition of 10 P i ethanol/ml had no effect upon the steroidogenesis of Tumor #2 c e l l s . At the concentration of 10 pg cytochalasin B/ml i n either 1% of DMSO or 1% of ethanol/ml of medium, the steroidogenic responses by Tumor #2 c e l l s to ACTH and dibutyryl c y c l i c AMP were de-pressed (Table 8). From these r e s u l t s , i t was determined that although cytochalasin B and DMSO both had effects upon steroidogenesis, i t appeared that these effects were not additive. 2. Effect upon morphology Since the three-dimensional configuration of the adrenocortical tumor c e l l s was the major change i n morphology i n response to ACTH and dibutyryl c y c l i c AMP (Figs. 5a-e), the effects of DMSO and cytochalasin B upon the morphology of Tumor #2 c e l l s were investigated by scanning electron microscopy. Tumor #2 c e l l s i n control medium, 3 hours i n medium containing ACTH, and 3 hours i n medium containing dibutyryl c y c l i c AMP are shown i n Figs. 22-24. The projection of knobby protuberances (zeiosis) was v i s i b l e along the c e l l membranes of Tumor #2 c e l l s within minutes of addition of 10 yg of cytochalasin B/ml. The "blebbing" movements were followed 61 Plate VII Effects of ACTH, Dibutyryl Cyclic AMP, DMSO, and Cytochalasin B upon  the Morphology of Tumor #2 Ce l l s . Glutaraldehyde f i x a t i o n , scanning electron microscopy.* Fig. 22. Tumor #2 c e l l s i n control medium, xl,200. Fig. 23. Tumor #2 c e l l s following 3 hours incubation i n medium containing 10 mU ACTH/ml, xl,200. Fig. 24. Tumor #2 c e l l s following 3 hours incubation i n medium containing 1 mM dibutyryl c y c l i c AMP, xl,200. Fig. 25. Tumor #2 c e l l s following 3 hours incubation i n medium containing 10 yg cytochalasin B/ml, xl,200. Figs. 26a and b. Tumor #2 c e l l s following 3 hours incubation i n medium containing 1% DMSO. (a) xl,200. (b) transmission electron microscopy. The most prominent feature of c e l l s treated with DMSO was the d i l a t i o n of the endoplasmic reticulum, xl5,000. *Except for Fig. 26b. Abbreviations: e.r. endoplasmic reticulum. 62 63 by the retraction of the c e l l , which was apparent 15 minutes following cytochalasin treatment: l o c a l regions along the o r i g i n a l margins of the f l a t , e p i t h e l i a l - l i k e c e l l remained anchored to the substratum while the intervening c e l l membrane and cytoplasm pulled towards the centre of the c e l l . This resulted i n the retracted c e l l s shown i n F i g . 25 with processes of varying shapes, lengths and widths. Tumor #2 c e l l s incubated i n medium containing either ACTH or dibutyryl c y c l i c AMP to-gether with 10 yg of cytochalasin B/ml were s i m i l a r i n morphology to those c e l l s incubated s o l e l y with cytochalasin B. Cells incubated i n the presence of 1% DMSO were extremely f l a t -tened (Fig. 26a) i n comparison with c e l l s i n control medium (Fig. 22). Transmission electron microscopy of c e l l s i n 1% DMSO revealed that the rough endoplasmic reticulum of these c e l l s was dil a t e d (Fig. 26b). Cells incubated i n the presence of either ACTH or dibutyry l c y c l i c AMP together with 1% DMSO (not shown) were s i m i l a r to the c e l l s which were incubated i n either ACTH or dibutyryl c y c l i c AMP only; that i s , the c e l l s were also rounded. The surface of the rounded c e l l s was quite smooth, and the rounded c e l l was attached to the substratum by a few thin processes. Both DMSO and cytochalasin B effects upon morphology were rapidly reversible. In this study, Tumor #2 c e l l s were incubated i n medium containing either DMSO or cytochalasin for 3 hours. The normal morpho-logy of the c e l l s was resumed within 15 minutes following the removal of medium containing DMSO or cytochalasin, and replacement with fresh nutrient medium. 64 IV. The Metabolic A c t i v i t i e s and the Responses of Enucleated Y-l-L C e l l s and Tumor #2 Cells to ACTH and Dibutyryl C y c l i c  AMP. 1. V i a b i l i t y The majority of the enucleated Y-l-L c e l l s and enucleated Tumor #2 c e l l s remained e p i t h e l i a l - l i k e for 24 hours after enucleation. At increasing time i n t e r v a l s following enucleation, the number of c e l l s which lysed or retracted from the substratum and floated i n the medium increased. At the same time, while some enucleated c e l l s retained t h e i r c h a r a c t e r i s t i c e p i t h e l i a l - l i k e c e l l shape, other enucleated c e l l s were contracted and r e f r a c t i l e under phase optics. Up to 24 hours following enucleation, 90% of the enucleated c e l l s resisted a l c i a n blue dye uptake. Approximately 5% of the enu-cleated c e l l s were stained intensely and completely with a l c i a n blue; these c e l l s were, on the whole, much smaller than the unstained c e l l s . Another 5% of the enucleated c e l l s were stained p a r t i a l l y , i . e . only a small portion of the c e l l membrane and cytoplasm were stained with alcian blue. At increasing time in t e r v a l s following enucleation, the proportion of stained enucleated c e l l s increased. At 60 hours a f t e r enucleation, 40% of the enucleated c e l l s were stained with a l c i a n blue. However, c e l l death was actually greater than that indicated by t h i s method, because, i n addition, the majority of the non-viable c e l l s had detached from the substratum. At 60 hours after enucleation, only 25% of the enucleated c e l l s present i n i t i a l l y remained attached to the cover-s l i p s . Enucleated Y-l-L c e l l s at 48 hours and 60 hours following enu-cleation are shown i n Figs. 27a and b. Most of the enucleated c e l l s 65 Plate VIII V i a b i l i t y and Protein Synthetic A c t i v i t y of Enucleated Y-l-L C e l l s . Methanol Fixation, Giemsa staining. Figs. 27a and b. Enucleated Y-l-L c e l l s at (a) 48 hours, (b) 60 hours after enucleation. Some enucleated c e l l s s t i l l retained t h e i r e p i t h e l i a l - l i k e morphology at 60 hours after enucleation, x750. Fig. 28. Autoradiographs of enucleated Y-l-L c e l l s incubated i n medium containing 10 uCi 3H-leucine/ml for 3 hours at 0-3, 21-24, and 45-48 hours following enucleation. Arrows point to a single nucleated c e l l i n each f i e l d , xl,200. 66 67 had l o s t t h e i r c h a r a c t e r i s t i c e p i t h e l i a l - l i k e c e l l shape. However, the majority of the c e l l s present s t i l l excluded a l c i a n blue dye uptake. 2. Protein synthetic a c t i v i t y The amount of H-leucine incorporated into acid-insoluble material was compared between enucleated and nucleated Y-l-L c e l l s at various times following enucleation. Some of the results are shown i n Fig. 28. According to grain counts, the rate of protein synthesis i n enucleate c e l l s was the same as i n nucleate c e l l s between 0-3 hours following enu-cleation. Enucleate c e l l s were s t i l l capable of incorporating 3H-leucine into acid-insoluble material at 45 hours following enucleation. However, this occurred at a greatly reduced rate i n comparison with the nucleate c e l l s . These results indicated that enucleated c e l l s were metabolically active. 3. Morphologic responses to ACTH and d i b u t y r y l c y c l i c AMP Fig. 29 shows a monolayer of nucleated Tumor #2 c e l l s , while Fig. 30a shows a t y p i c a l enucleated monolayer of Tumor #2 c e l l s at one hour a f t e r enucleation. The arrows point to the few nucleated c e l l s remaining a f t e r the enucleation procedure. An enucleated monolayer of Tumor #2 c e l l s following 1 hour of incubation i n medium containing 10 mU ACTH/ml i s i l l u s t r a t e d i n Fig. 30b. The morphologic response of enucleate Tumor #2 cytoplasm to i t s t i s s u e - s p e c i f i c hormone was i d e n t i c a l to that of the nucleate c e l l s (Fig. 29b). Within minutes of ACTH addition, the enucleated c e l l retracted from the substratum and became attached to the P e t r i dish by a few prominent processes, while the rest of the Plate IX Responses to ACTH by Nucleated and Enucleated Tumor #2 C e l l s . Phase contrast microscopy, x350. Figs. 29a and b. Nucleated Tumor #2 c e l l s i n culture. (a) i n control medium, (b) i n medium containing 10 mU ACTH/ml for 1 hour. Figs. 30a and b. Tumor #2 c e l l s at one hour after enucleation i n (a) control medium, (b) i n medium containing 10 mU ACTH/ml. Arrows point at the few nuc-leated c e l l s remaining after the enucleation procedure. The morpho-log i c response of the enucleated c e l l s to ACTH was comparable to that of the nucleated c e l l s . 69 70 cytoplast became quite r e f r a c t i l e under phase contrast microscopy. Enu-cleated Y-l-L c e l l s and Tumor #2 c e l l s also responded to dibutyryl c y c l i c AMP, and the response was i d e n t i c a l to that of the nucleated c e l l s . The "cell-rounding" was v i s i b l e after 1 hour of incubation i n medium containing the c y c l i c nucleotide, and attained a maximum "rounding" i n 2-3 hours. Enucleated Y-l-L c e l l s and enucleated Tumor #2 c e l l s responded morphologically to ACTH and dibutyryl c y c l i c AMP up to 33 hours after enucleation. Enucleated Y-l-L cytoplasms at 12, 24 and 36 hours after enucleation are shown i n Figs. 31a, 32a, and 33a. The morphology of simil a r c e l l s which had been incubated i n medium containing 10 mU ACTH/ml for 3 hours i s shown i n Figs. 31b, 32b, and 33c. Following 3 hours of incubation i n 1 mM dibutyryl c y c l i c AMP, the morphology of enucleated Y-l-L c e l l s was comparable to that of c e l l s which had been incubated i n medium containing ACTH (Figs. 31c, 32c and 33c). After 40 hours following enucleation, the enucleated c e l l s were no longer uniformly e p i t h e l i a l - l i k e , and i t could not be unequivocally determined whether or not these c e l l s responded morphologically to ACTH or di b u t y r y l c y c l i c AMP. 4. Steroidogenic responses to ACTH and dibutyryl c y c l i c AMP a) I d e n t i f i c a t i o n of 20a dihydroprogesterone ll*C-20a dihydroprogesterone produced by enucleated Tumor #2 c e l l s was p o s i t i v e l y i d e n t i f i e d by r e c r y s t a l l i z a t i o n to constant speci-f i c a c t i v i t y i n the presence of' added c a r r i e r s teroid (Table 9). Plate X Morphologic Responses to ACTH and Dibutyryl C y c l i c AMP by Enucleated  Y-l-L C e l l s . Methanol f i x a t i o n , Giemsa st a i n i n g , xl,000. Figs. 31a-c. Enucleated Y-l-L c e l l s i n culture: 12 hours after enucleation, (a) i n control medium, (b) 3 hours i n medium containing 10 mU ACTH/ml pri o r to f i x a t i o n , (c) 3 hours i n medium containing 1 mM dibutyry l c y c l i c AMP p r i o r to f i x a t i o n . Figs. 32a-c. Enucleated Y-l-L c e l l s i n culture: 24 hours after enucleation, (a), (b) and (c) are the same as that for Fig. 31. Figs. 33a-c. Enucleated Y-l-L c e l l s i n culture: 36 hours after enucleation, (a), (b) and (c) are the same as that for Fig. 31. 73 TABLE 9 . Radiochemical I d e n t i f i c a t i o n of 1 4C - 2 0 a Dihydro-progesterone Produced by Enucleated Tumor #2 C e l l s 3 Condition Crystals (dpm/mg)b Mother Liquor (dpm/mg)*3 Basal a XLi 2,993 ML2 9,051 X L 2 2,852 ML2 3,091 XL 3 2,854 ML3 3,111 X L 4 2,854 MLi* 2,837 Basal b XL X 2,366 MLi 9,863 XL 2 2,361 ML2 2,585 XL 3 2,181 ML3 2,148 XLk 2,180 M I 4 2,137 ACTH a XL! 7,525 MLi 12,685 XL 2 4,958 ML2 11,736 XL 3 4,919 ML3 5,464 . XL 4 4,716 MLtt 4 ,777 ACTH b XLi 605 MLi 3,506 XL 2 607 ML2 802 XL 3 554 ML3 599 XL it 560 ML+ 587 Twenty-•four 6 cm P e t r i dishes of enucleated Tumor #2 c e l l s were incubated i n either control medium or medium with ACTH (10 mU/ml) containing l t +C-pregnenolone (approximately 150,000 dpm/culture) following enucleation. After 12 hours the medium was removed and media from 4 groups of 6 P e t r i dishes each of enucleated c e l l s under the same con-d i t i o n were pooled. The i s o l a t i o n of l l tC-20a dihydroprogesterone has been described i n Materials and Methods. l t +C-20a dihydroprogesterone was r e c r y s t a l l i z e d from ethanolrwater with 10 mg of cold c a r r i e r . k Expressed as dpm/mg of authentic s t e r o i d . 74 b) Steroid output The protocol that was employed to determine the amounts of steroids produced by enucleated Y-l-L c e l l s i n either control medium, medium containing ACTH, or medium containing dibutyryl c y c l i c AMP at various time intervals after enucleation i s also i l l u s t r a t e d i n Fig. 21. Three general observations can be made from the results which are sum-marized i n Table 10: enucleated Y-l-L c e l l s were capable of producing steroids; at 0-3 hours following enucleation, enucleated Y-l-L c e l l s responded to ACTH and dibutyryl c y c l i c AMP by increased steroidogenesis which was comparable i n magnitude to the nucleated c e l l s ; and, at i n -creasing time intervals following enucleation, the amounts of steroids produced under a l l three conditions diminished. Upon examining the results more cl o s e l y , i t i s apparent that the low levels of steroids were assayed spectrofluorometrically at the l i m i t of the Aminco Bowman spectrofluorometer (10-15 ng 20a dihydro-progesterone) . When the enucleation procedure was adapted to f a c i l i t a t e the enucleation of larger quantities of c e l l s , the aforementioned study was repeated. The results of one of these experiments are summarized i n Table 11 and the protocol employed to study the ste r o i d output of enucleated Tumor #2 c e l l s was analogous to that outlined previously (Fig. 21). Enucleated Tumor #2 c e l l s , s i m i l a r to the enucleated Y-l-L c e l l s responded to ACTH and dibutyryl c y c l i c AMP by increased steroidogenesis. At 0-3 hours after enucleation, the increase i n ste r o i d output was approximately equivalent to that of nucleated c e l l s . At increasing 75 TABLE 10. Steroidogenic Responses to ACTH and Dibutyryl Cy c l i c AMP by Enucleated Y-l-L Cel l s Basal ACTH dbcAMP (ng steroid/100 yg protein/3 h o u r ) 3 Control' 5 15 + 0. 5 30 + 3. o d .22 + 1.5 d EPT Control 1 5 26 + 2. 3 47 + 6. 5 d 33 + 2.3 d 0-3 hours following enucleation 0 22 + 2. 2 40 + 1. 3 d 33 + 2.4 e 3-6 hours following enucleation 0 20 + 1. 1 23 + 1. 6 24 + 0.8 6-9 hours following enucleation 0 17 + 0. 6 20 + 0. 7 e 22 + 3.3 9-12 hours following enucleation 0 15 + 0. 8 18 + 0 22 + 1.5 d 3-hour incubations were carried out i n t r i p l i c a t e . Each point represents the average st e r o i d output/100 yg protein + S.E. k The steroidogenic a c t i v i t y of the nucleated c e l l s and the enucleation procedure treated nucleated c e l l s were determined from incu-bation medium pooled from 2 coverslips of c e l l s . Six coverslips of nucleated c e l l s were incubated/group. Incubation medium of 4 coverslips of enucleated c e l l s was pooled for each determination. Twelve coverslips of enucleated c e l l s were incubated/group. d S t a t i s t i c a l l y d i f f e r e n t from basal production during the same time i n t e r v a l . One-tailed t te s t , s i g n i f i c a n t at the 0.01 l e v e l of s i g n i -ficance. S i g n i f i c a n t at the 0.05 l e v e l of signi f i c a n c e . 76 time in t e r v a l s after enucleation, the increase i n ste r o i d output i n response to the trophic hormone or to the c y c l i c nucleotide diminished. However, i t i s apparent from the results presented i n Table 11 that increased steroidogenesis by enucleated Tumor #2 c e l l s i n response to dibutyryl c y c l i c AMP persi s t s longer than that i n response to ACTH. Also evident i s that enucleated Tumor #2 c e l l s are s t i l l capable of producing steroids i n control medium at 21-24 hours after enucleation. The s t e r o i d output i n 12 hours by enucleated Y-l-L c e l l s and enucleated Tumor #2 c e l l s under various conditions i s tabulated i n Table 12. I t i s evident that the 12 hour-steroid output of the enucleated c e l l s i n the presence of either ACTH or dibutyryl c y c l i c AMP was also higher than that of enucleated c e l l s incubated i n control medium (Table 12). In comparison with the nucleated EPT c e l l s , the steroidogenesis of the enucleated c e l l s was lower under a l l three incubation conditions. These results were consistent with the e a r l i e r results obtained with 3 hour-incubations of enucleated c e l l s at various time i n t e r v a l s following enucleation (Tables 10 and 11). That i s , the progressively diminished steroidogenic a c t i v i t y of enucleated c e l l s at increasing 3-hourly i n t e r v a l s a f t e r enucleation i s reflected i n the o v e r a l l decrease i n 12 hour-steroid output of enucleated c e l l s as compared with the nucleated EPT c e l l s . 77 TABLE 11. Steroidogenic Responses to ACTH and Dibutyryl Cyclic AMP by Enucleated Tumor #2 Cells Hours after EPT or Enucle-ation Nucleated (EPT Co n t r o l ) 3 Enucleated*3 Basal ACTH dbcAMP Basal ACTH dbcAMP (ng steroid/mg protein/3 hour)° 0-3 265 + 14 425 + 20 d 385 + 16 d 225 + 6 270 + 4 d 295 + 6 d 3-6 230 + 6 425 + 2 d 345 + 8 d 220 + 10 245 + 6 250 + 4 e 6-9 235 + 3 425 + 2 d 365 + 3 d 210 + 9 235 + 11 245 + 7 e 9-12 230 + 6 420 + i o d ~ 360 + i o d 210 + 5 210 + 10 235 + 9 e 12-15 245 + 8 415 + 3d' 375 + 9 d 205 + 8 205 + 10 210 + 9 15-18 - - - 195 + 4 18-21 - - - - 180 + 13 21-24 240 + 3 - - 165 + 13 3 Incubation medium from one 6 cm P e t r i dish of Tumor #2 Cells was used for each determination. Five P e t r i dishes of nucleated c e l l s were incubated/group. k Incubation medium pooled from two 6 cm P e t r i dishes of enucleated Tumor #2 C e l l s was used for each determination. Ten P e t r i dishes of enu-cleated c e l l s were incubated/group. c Each point represents the average st e r o i d output + S.E. of 5 determinations. d S t a t i s t i c a l l y different from basal production during the same time i n t e r v a l . One-tailed t t e s t , s i g n i f i c a n t at the 0.01 l e v e l of s i g n i -ficance. S i g n i f i c a n t at the 0.05 l e v e l of signif i c a n c e . 78 TABLE 12. Steroidogenic Responses to ACTH and Dibutyryl Cy c l i c AMP by Nucleated and Enucleated Y-l-L Cells and Tumor ill C e l l s : Steroid Output i n 12 Hours Y-l-L Tumor #2 Nucleated 3 Enucleated Nucleated 0 Enucleated (ng steroid/100 yg protein/ (ng steroid/1 mg protein/ 12 hour) e 12 hour) f Basal 125 + 8 95 + 5 595 + 33 560 + 42 10 mU ACTH/ml 370 + 29 8 140 + 10 8 725 + 5 g 645 + 42 1 mM dbcAMP 400 + 16 s 150 + 13 s 990 + 14 8 740 + 51 h Incubation medium of 2 coverslips of nucleated c e l l s was pooled for each determination. Six coverslips of nucleated c e l l s were incubated/ group. 1J Incubation of 4 coverslips of enucleated c e l l s was pooled for each determination. Twelve coverslips of enucleated c e l l s were incubated/ group. c Incubation medium from one 6 cm P e t r i dish of nucleated c e l l s was used for each determination. Six P e t r i dishes of nucleated c e l l s were incubated/group. d Incubation medium pooled from two 6 cm P e t r i dishes of enu-cleated c e l l s was used for each determination. Twelve P e t r i dishes of enucleated c e l l s were incubated/group. Each point represents the average s t e r o i d output + S.E. of 3 determinations. ^ Each point represents the average ste r o i d output + S.E. of 6 determinations. S t a t i s t i c a l l y different from basal production during the same time i n t e r v a l . One-tailed t t e s t , s i g n i f i c a n t at the 0.01 l e v e l of significance. S i g n i f i c a n t at the 0.05 l e v e l of sig n i f i c a n c e . DISCUSSION I. The Adrenocortical Cells Used i n This Study The increasing heterogeneity of the adrenocortical tumor c e l l s and the decrease i n t h e i r t i s s u e - s p e c i f i c function following prolonged periods i n s e r i a l culture have been reported by several laboratories that work with Y - l c e l l s (Yasumura, 1968; Kowal, 1970b, Schimmer, 1974). Although the causative factors of these changes s t i l l remain to be e l u c i -dated, two lines of approach have been used to maintain the d i f f e r e n -t i a t e d function of these tumor c e l l s . Both of the approaches were modelled after the enrichment culture methods used i n microbiology. One was based upon the notion that s e l e c t i v e overgrowth by stromal c e l l s was the most serious obstacle to the culturing of functionally d i f f e r e n t i a t e d c e l l s ; i n addition, the culture environment may not be adequate for the f u l l phenotypic expression of the specialized c e l l s . In t h i s method, adrenocortical tumor c e l l s were p e r i o d i c a l l y passaged i n isogeneic mice following passages i n v i t r o . The animal acted as a s e l e c t i v e device which permitted the growth of only those tumor c e l l s which withstood culture conditions, while c e l l types of non-malignant o r i g i n were e l i -minated. At the same time, the i n vivo environment provided the proper conditions^ necessary for the f u l l expression of the spe c i a l i z e d function of the adrenocortical tumor c e l l s . Culture-derived tumors were found to have an enhanced a b i l i t y to grow and function i n culture subsequently (Buonassisi e^ a l . , 1962; Kowal, 1970b). The second approach was based 79 80 upon the idea that c e l l s i n v i t r o were also susceptible to a l t e r a t i o n s since change i s one of the c h a r a c t e r i s t i c properties of l i v i n g things. Thus, by the procedure of systematic cloning and re- c l o n i n g , the propagation of f u n c t i o n a l c e l l s could be continued while the culture of non-functional or l e s s f u n c t i o n a l c e l l s could be terminated. Clonal l i n e s of varying f u n c t i o n a l a c t i v i t i e s have been established from the adr e n o c o r t i c a l tumor c e l l s (Yasumura, 1968; Schimmer, 1969). By using these methods i n combination with routine culture tech-niques, f u n c t i o n a l adrenocortical tumor c e l l s have been maintained and have provided the opportunities f o r many experiments of importance i n c e l l biology and endocrinology. With only a few exceptions, the propagation of c e l l s i n v i t r o requires the presence of macromolecules i n the culture medium whose structure and function have not yet been defined. Serum has been used as the constituent i n cul t u r e medium which provides these molecules. Much progress has been made i n de f i n i n g chemically the n u t r i t i o n a l r e q uire-ments f o r both the growth and function of the Y - l c l o n a l l i n e (Yasumura eJL a l . , 1966a; Cuprak and Sato, 1968; Lammi and Cuprak, 1974; Wishnow and F e i s t , 19 74). By v i r t u e of the resemblance i n physiology between the adreno-c o r t i c a l tumor c e l l s and the normal adrenal cortex i n v i v o , these ACTH-responsive, corticosteroid-producing c e l l s have served as a u s e f u l model for the s i t e of action of ACTH (Schimmer e_t a l . , 1969), and f o r the study of the acute and prolonged e f f e c t s of ACTH upon a v a r i e t y of meta-b o l i c pathways i n the adrenal c e l l (Pierson, 1967; Kowal, 1969a,b,c, 81 1970a,b, 1973; Kowal and Fie d l e r , 1968, 1969; Kowal et a l . , 1970). The mechanism of hormonal activation of the adenylate cyclase system has been the subject of much attention. The adenylate cyclase system of Y - l c e l l s has been characterized (Tauton et a l . , 1969) , and mutants of Y - l c e l l s with d i f f e r i n g degrees of competence i n the pathway of ACTH-stimulated steroidogenesis have been used for investigating the mechanisms of coupling between hormone interaction with i t s receptor and the a c t i -vation of adenylate cyclase (Schimmer, 1969, 1972; Rodbell et a l . , 1974). Recently, evidence has accumulated that cholera enterotoxin i s capable of stimulating the adenylate cyclcase enzyme of a variety of c e l l s , including adrenocortical tumor c e l l s (Donta et al., 19 73; Wolff et a l . , 19 73). The response of these c e l l s to cholera toxin has been charac-te r i z e d (Wolff et a l . , 19 73), and compared with the ACTH effects (Kowal et a l . , 19 74) i n order to gain further insights into the mechanism of hormone action upon adenylate cyclase (Bennett et a l . , 19 75). The c e l l s of the Y - l l i n e were the adrenocortical tumor c e l l s of choice to use i n this study on the response of enucleated c e l l s to ACTH since these clones have been well-characterized (Yasumura et a l . , 1966a), and are commercially available. In addition, a 3 to 4-fold increase i n steroidogenesis i n response to ACTH has been reported for these c e l l s ; thus, the functional a c t i v i t y of the enucleated c e l l s could be c l e a r l y determined. Nevertheless, i t was found d i f f i c u l t to enucleate the Y - l c e l l s (ATCC CCL 79) e f f i c i e n t l y . In contrast, Y-l-L c e l l s were readily enucleated. Thus, Y-l-L c e l l s , and subsequently Tumor #2 c e l l s , were the adrenocortical c e l l s used i n this study i n spite of th e i r com-paratively low functional a c t i v i t y . 82 Although the Y-l-L c e l l s were non-clonal c e l l s , these c e l l s were s u f f i c i e n t l y homogeneous, as indicated by t h e i r uniformity i n morpho-logy, and by t h e i r s i m i l a r i t y i n chromosome number (Table I ) . The pro-gressively lower s t e r o i d output under various incubation conditions (Table I I I ) , and the absence of 1130H progesterone, together with less e a s i l y detectable amounts of 1130H, 20a dihydroprogesterone and 20a dihydro-progesterone i n l a t e passage Y-l-L c e l l s , suggested that functionally less d i f f e r e n t i a t e d c e l l s were selected from the o r i g i n a l Y - l c e l l s pur-chased from B i o c u l t , and that further dedifferentiation occurred following i n creasing culture passage. Furthermore, the low steroid output assayed by spectrofluorometry may be a result of the i n a b i l i t y of less d i f f e r e n -t i a t e d Y-l-L c e l l s to convert progesterone into flurogenic metabolites. The steroids which required the 113 hydroxylation reaction were i n i t i a l l y q uantitatively less detectable i n early passage Y-l-L c e l l s , and sub-sequently v i r t u a l l y absent i n l a t e passage Y-l-L c e l l s by chromatographic analysis (Fig. 9). These steroids were furthest along i n the s t e r o i d pathway (Fig. 34) proposed for the adrenocortical tumor c e l l s (Pierson, 1967; Kowal and F i e d l e r , 1 9 6 8 ) . I t has been reported that long-term culture of these adrenocortical tumor c e l l s resulted i n a decline i n 113 hydroxylase a c t i v i t y while primary culture of tumors generated i n the animals from these c e l l s have high levels of this enzyme a c t i v i t y (Kowal, 1 9 6 9 ; Kowal et a l . , 1970). Interestingly, lowered capacity for 113 hydroxylation has also been described for the S n e l l adrenocortical carcinoma 494 (Kimmel et a l . , 1974). The 113 hydroxylation occurs pre-dominantly i n the adrenal cortex; thus, the decrease i n 113 hydroxylation Cholesterol 11/3-Hydroxyprogesterone 11/3-Hydroxy -20a-d ihydro-progesterone 11 Keto-20d-dihydroprogesterone Fig. 34. Steroid Pathway i n Adrenal Tumor C e l l Cultures Taken from Kowal (1970b). The major ste r o i d products of the Y - l c e l l l i n e are underlined. The major fluorogenic s t e r o i d product of Y - l - L c e l l s and Tumor #2 c e l l s was found to be 20a dihydroprogesterone. 84 may r e f l e c t a general decline i n the t i s s u e - s p e c i f i c a c t i v i t y of these tumor c e l l s . The i n i t i a l high steroid output by the primary cultures of Tumor #2 c e l l s may be attributable to the fulfilment of the n u t r i t i o n a l require-ments necessary for the f u l l phenotypic expression of thse c e l l s during preceding passage i n vivo. The decline i n steroid output following s e r i a l subculture may be due to the inadequacy of the culture environ-ment. In view of the absence of ACTH i n the control basal culture medium used, and i n l i g h t of the findings that ACTH i s required for the main-tenance of both the structure and function of the normal adrenal cortex i n vivo ( G i l l , 1972), the aforementioned explanations seem reasonable. I I . The Effect of ACTH Upon Morphology ACTH has been shown to have pronounced effects upon the u l t r a -structure of the adrenal gland. I t i s required for the morphological d i f f e r e n t i a t i o n of the t y p i c a l zona f a s c i c u l a t a mitochondrion (Kahri, 1968).- Acute ultras t r u c t u r a l changes i n the zona f a s c i c u l a t a c e l l s within 10 minutes of ACTH administration have been described (Rhodin, 1971). Furthermore, the hypertrophy and hyperplasia of smooth endoplasmic r e t i -culum and mitochondria due to prolonged ACTH treatment have also been reported (Nussdorfer e_t al., 19 71). As a r e s u l t , many i n t r i g u i n g hypo-theses have been proposed to correlate the structure with the functional a c t i v i t i e s of adrenocortical c e l l s . Recently, results from several laboratories have cast grave doubts on these theories l i n k i n g ACTH-induced u l t r a s t r u c t u r a l changes causally with ACTH-induced steroidogenesis. I t has been established 85 that although r e l a t i v e l y few u l t r a s t r u c t u r a l changes occurred i n the adrenal cortex during the f i r s t day after hypophysectomy, the steroido-genic responsiveness of the gland was almost abolished (Tait e_t _ a l . , 1967; Idelman, 1970). S i m i l a r l y , i t has been demonstrated that ACTH-induced steroidogenesis s t i l l proceeded i n superfused adrenal glands i n which the c e l l s had undergone gross s t r u c t u r a l alterations and con-tained no detectable intact mitochondria (Kuo and Tchen, 1973). Moreover, i t has been shown that the ACTH-induced morphological d i f f e r e n t i a t i o n of f e t a l adrenocortical c e l l s occurred p r i o r to the a b i l i t y of these c e l l s to respond steroidogenically to ACTH (Idelman, 19 70). Thus, these results suggested that although ACTH i s capable of inducing both u l t r a -s t r u c t u r a l effects and steroidogenesis, the mechanism of action upon these effects may be different (Kuo and Tchen, 1973). Results obtained from the present study of the ultrastructure of Y-l-L c e l l s i n control medium and i n medium containing ACTH for various time int e r v a l s indicated that acute u l t r a s t r u c t u r a l changes were not apparent i n Y-l-L c e l l s following 5 minutes of ACTH administration. The significance of these results i s dubious i n view of the fact that Y-l-L c e l l s are tumor c e l l s . The ultrastructure of these tumor c e l l s was different from those of normal adrenocortical c e l l s i n vivo. Thus, i t i s conceivable that the lack of u l t r a s t r u c t u r a l changes of these c e l l s i n the presence of ACTH might not r e f l e c t the true a c t i v i t i e s of normal adrenocortical c e l l s . Nevertheless, the most s t r i k i n g effect of ACTH or dibutyryl c y c l i c AMP upon the morphology of Y - l c e l l s , Y-l-L c e l l s and Tumor #2 86 c e l l s could not be ignored. I t i s evident that " c e l l rounding" i n response to ACTH was u l t r a s t r u c t u r a l l y different from that of " c e l l rounding" i n response to t r y p s i n i z a t i o n . In both "rounding" responses, the surface area to c e l l volume r a t i o was apparently maintained. In response to tr y p s i n , or trypsin together with versene, the surface of the rounded Y-l-L c e l l s was greatly convoluted. Long and t h i n surface folds were observed on the rounded c e l l s i n response to ACTH. There-fore, the surface area to volume r a t i o was maintained i n a l l three observed cases. Whether or not the "rounding" response also occurs i n adrenocortical c e l l s i n vivo i s not known, and the significance of th i s response i s not clear. The mechanism of co r t i c o s t e r o i d secretion remains obscure. The c e l l u l a r organelles which may act as a vehicle for the export of s t e r o i d into the e x t r a c e l l u l a r f l u i d have yet to be i d e n t i f i e d . I t i s now apparent that c o r t i c o s t e r o i d secretion i s accompanied by release of c y c l i c AMP (Kowal and R a i l , 1972; Espiner, et a l . , 1974), and secretion of protein (Rubin et^ a l . , 19 74). In l i g h t of the evidence which sug-gested that the action of ACTH i s not associated with measurable changes i n membrane permeability (Jaanus, 1971), the mechanism of transport of these substances becomes even more elusive. More extensive studies w i l l be required to elucidate the mechanism of action upon the morpho-logy of adrenocortical c e l l s , and upon the mechanism of co r t i c o s t e r o i d secretion. 87 I I I . Enucleation Procedure The precise mechanism by which cytochalasin B induces nuclear protrusion and nuclear extrusion remains enigmatic. In an early attempt to explain this phenomenon, Carter proposed that the boundary tension between the plasma and nuclear membranes might be lowered by the adsorp-tion of cytochalasin B on the i n t e r n a l and external surfaces of these membranes respectively. As a r e s u l t , the chances of stable areas of adhesion formed between these membranes might increase. Carter sug-gested that this could explain the observed "wrapping" of the c e l l mem-brane around the nucleus u n t i l i t i s v i r t u a l l y excluded (Carter, 1967). Recently, the l o c a l i z a t i o n of 3H cytochalasin B predominantly by c e l l u l a r membrane systems, both at the c e l l surface and i n t r a c e l l u l a r l y , has been reported (Lin and Spudich, 1974; Mayhew e_t al, , 1974). In addition, i t has been speculated that the binding of cytochalasin B occurs i n the l i p i d phase of membranes (Mayhew et a l . , 1974). Altered membrane functions and properties could be produced by a l l o s t e r i c changes i n proteins due to the presence of cytochalasin B i n the v i c i n i t y ; thus, Carter's o r i g i n a l proposal seems credible. I t i s evident from the l i t e r a t u r e that the success i n e f f i c i e n t enucleation of a p a r t i c u l a r c e l l type i s dependent upon two major c e l l u l a r factors: the i n t r i n s i c property of s u s c e p t i b i l i t y to cytochalasin B-induced enucleation (Poste, 1972), and the adhesiveness of c e l l s to the i r substratum; hence, the a b i l i t y to r e s i s t detachment from the sub-stratum at the high centrifugal forces used i n the enucleation procedure. Efforts i n improving the enucleation e f f i c i e n c y of c e l l s have been 88 directed towards the modification of the substratum to render i t more adhesive to c e l l s (Prescott, 1972), the rationale being that the chances of cytochalasin B-induced enucleation would be increased by v i r t u e of the prolonged and sustained adhesion of the c e l l s to the substratum. There was circumstantial evidence which suggested that Y-l-L c e l l s adhered more tenaciously onto thei r substratum i n comparison with the Y - l c e l l s (ATCC CCL 79). A s i g n i f i c a n t l y greater proportion of Y-l-L c e l l s remained attached to the coverslips following the i d e n t i c a l enucleation procedure for both c e l l types. In addition, i n view of the selection procedure used to i n i t i a t e the Y-l-L subline, the notion that c e l l s with increased adhesion to t h e i r substratum were selected f o r , seemed reasonable. Therefore, the a b i l i t y to consistently and uniformly enucleate populations of Y-l-L c e l l s and the tumor c e l l s derived from these c e l l s , could be p a r t i a l l y attributed to the adhesive properties of these c e l l s . In studies on the mechanism of c e l l u l a r adhesion, i t was found that neutrophils (Garvin, 1968), neuroblastoma c e l l s (Schubert et a l . , 1971) and BHK fibroblasts (Maroudas, 1975) a l l adhered more rapidly onto various substrata i n the absence of serum. Although no evidence has been obtained as yet to explain the mechanism of i n h i b i t i o n by serum, i t has been suggested that serum proteins might weaken the i n t e r a c t i o n between the c e l l surface and the substratum (Schubert et a l . , 1971). Maroudas also proposed that the forces which bind c e l l s to the substratum would also bind serum competitively (Maroudas, 19 75). Along the same l i n e s , the s i g n i f i c a n t reduction i n c e l l u l a r binding of 3H cytochalasin 89 B by ELD ascites carcinoma c e l l s i n the presence of serum was also ten-t a t i v e l y explained by the competition of serum factors with cytochalasin B for c e l l u l a r binding s i t e s (Mayhew et a l . , 1974). Thus, the observed s i g n i f i c a n t increase i n the enucleation e f f i c i e n c y of Y-l-L c e l l s and Tumor #2 c e l l s when the c e l l s were preineubated i n serum-free medium pr i o r to the enucleation by centrifugation i n serum-free medium con-taining cytochalasin B may be explained by the increased adhesion of c e l l s to the substratum, and the more e f f i c i e n t c e l l u l a r binding of cytochalasin B, and therefore increased enucleation i n the absence of serum. I t i s apparent that the amount of enucleated c e l l s obtainable by the two enucleation methods used i n th i s study was l i m i t e d by the t o t a l surface area which could be centrifuged. A new preparative method has been described for obtaining greater samples of enucleated c e l l s . Wigler and Weinstein demonstrated that L c e l l s were enucleated with high e f f i c i e n c y by centrifugation i n F i c o l l density gradients containing cytochalasin B (Wigler and Weinstein, 1975). Although a scale-up of the numbers of c e l l s that were enucleated was achieved, the authors did agree that the procedure might be more traumatic for the c e l l s . The potential usefulness of this method i s indicated. Nevertheless, further investigations into the side effects due to the method i t s e l f are re-quired, p r i o r to i t s adoption for obtaining quantitative amounts of cytoplasts or karyoplasts necessary for biochemical studies. 90 IV. Effects of DMSO and Cytochalasin B upon Y-l-L Cells and Tumor #2  Cells Cytochalasin B and i t s commonly-used solvent, DMSO, both had effects upon the morphology and steroidogenesis of Y-l-L c e l l s and Tumor #2 c e l l s . However, most importantly, i t was established that effects of these substances during the enucleation procedure were rapidly rever-s i b l e following t h e i r removal, and have no apparent effects upon the subsequent morphology and steroidogenic a c t i v i t i e s of Y-l-L c e l l s and Tumor #2 c e l l s . Although i t was established from this study that steroidogenesis of Y-l-L c e l l s and Tumor #2 c e l l s was depressed i n the presence of either 10 yg cytochalasin B/ml or 1% DMSO, i t was not determined whether the lowered steroid output assayed by spectrofluorometry of the incubation media extracts under various incubation conditions was due to the i n -h i b i t i o n of ste r o i d synthesis or ste r o i d secretion by these agents. I t has been reported that a variety of agents which disrupted microtubules stimulated steroidogenesis of Y - l c e l l s while D 2 O , an agent which s t a b i l i z e d microtubules, prevented the stimulation of Y - l steroido-genesis by ACTH or dibutyryl c y c l i c AMP (Temple et a l . , 1972; Temple -and Wolff, 1973). I t was proposed that microtubules r e s t r i c t e d the transport of cholesterol to the mitochondrion which i s the major s i t e of hormone synthesis, thus steroidogenesis was li m i t e d i n the presence of D 2 O while steroidogenesis was enhanced i n the presence of antimicro-tubular agents. This hypothesis was highly speculative, and was offered only for the want of a better explanation of findings that the mode of stimulation of steroidogenesis by antimicrotubular agents was unlike 91 ACTH-stimulated steroidogenesis i n many respects (Temple and Wolff, 1973). Recently, i t has been reported that DMSO s t a b i l i z e d the tubule form of microtubules (Dulak and C r i s t , 1974): i n the presence of DMSO, s i g n i f i c a n t l y more microtubules were observed electron microscopically, i n addition, the rate of tubulin polymerization was enhanced, and i n -creased concentration of colchicine was necessary to prevent tubulin polymerization. In l i g h t of these findings, i t i s conceivable that the DMSO used i n this study s t a b i l i z e d the tubule form of microtubules, thus r e s t r i c t i n g the transport of cholesterol and thereby l i m i t i n g steroidogenesis. The exceedingly flattened c e l l s i n the presence of 1% DMSO which was observed by scanning electron microscopy (Fig. 26a) might be an ind i c a t i o n of the effect of DMSO upon the microtubular cyto-skeleton. By transmission electron microscopy, pronounced swollen endo-plasmic reticulum was observed i n c e l l s treated with DMSO (Fig. 26b). This effect has been reported i n tendon c e l l s (Streaton and Grim, 1974), although the significance of this effect i s not clear., The endoplasmic reticulum i s also the s i t e of several steps i n the pathway of st e r o i d synthesis. Therefore, the decreased steroidogenesis i n the presence of DMSO might be due to the altered functional a c t i v i t i e s of the or-ganelles required for steroidogenesis. A voluminous l i t e r a t u r e ( A l d r i c h , 1975) has arisen on the use of cytochalasin B since the f i r s t report by Carter (1967) on the b i o -l o g i c a l effects of this drug. Although the primary mode of action of cytochalasin B has not been ascertained, i t has been proposed that the wide array of phenotypic effects when cytochalasin B i s applied to c e l l s 92 and tissues may be due to i t s action at the l e v e l of the plasma membrane to cause rearrangements and alterations of the proteins embedded i n the membrane l i p i d . I t has been shown that cytochalasin B i n h i b i t e d the release of thyroxine from the thyroid gland after the stimulation by thyroid stimulating hormone (Williams and Wolff, 1971), and also i n h i b i t e d the release of a-amylase from the parotid gland after stimu-l a t i o n by epinephrine (Butcher and Goldman, 1972). In addition, the i n h i b i t i o n of the induction of tyrosine aminotransferase a c t i v i t y by i n s u l i n or C o r t i s o l i n the presence of cytochalasin B has also been reported (Butcher and Perdue, 1973). Thus, i t i s conceivable that the effects of cytochalasin B upon the steroidogenesis of Y-l-L c e l l s and Tumor #2 c e l l s may be due to either the i n h i b i t i o n of s t e r o i d synthesis or release. In view of the great variety of effects of both DMSO and cyto-chalasin B, further work upon the effects of DMSO and cytochalasin B on the steps involved i n steroidogenesis i s e s s e n t i a l before any sub-s t a n t i a l explanation can be given. V. Mechanism of ACTH Action The a n t i b i o t i c , actinomycin D, i s a potent i n h i b i t o r of DNA-dependent RNA synthesis. Numerous investigations designed to define whether or not RNA synthesis i s required i n the acute steroidogenic response to ACTH have u t i l i z e d actinomycin D i n vivo and i n v i t r o . The results of most of these studies are summarized i n Table 13. In the different i n vivo and i n v i t r o systems examined, actinomycin D has TABLE 13 EFFECTS OF ACTINOMYCIN D UPON ADRENOCORTICAL STEROIDOGENESIS ! SUMMARY OF RESULTS REPORTED IN THE LITERATURE SYSTEM USED ACTIXOWfTO: D DOSACF. ROUTE OF ADMINTS--DUHATION OK ANALYSIS 1 EXPERIMENT ANALYSIS OF STEROIDOGENESIS RESISTS (COMPARED TO CONTROLS  WITHOUT ACTINOMYCIN D) REFERENCES IS VIVO:BASAL Intact Guinea pigs Intact Guinea pigs Intact Cuinea pigs Estradiol-treated ovarlectomized rats Iotact rats Intact Guinea pigs Intact Guinea pigs IN VIVOrACTH* Hypophysectomlzed . rats . Hypophysectomlzed : rats I Hypophysectoaized I rats Kypophysectoolzed ra ta . Hypophysectomlzed , rats Intact Cuinea Pigs . l : l V7TH0:BA.SAL . P-at adrenal, f rag-aents Rat adrenal .frag-ments . Cent adrenal • s l ices, Y - l mouse tumor, ce l l s Trypaln-dissociated . rat adrenal ce l l s single dose 3ug/100g single dose 7.5ug/100g 3ug/100g/day/7 days 375-500ug/rat 50pg/100ug/15.5 hours single dose lOug/lOOg single dose lOpg/lOOg single dose 25ug/100g single dose 27-75ug/100g single dose 25-75ug/100g s ingle dose 5Oug/100g s ingle dose lmg/rac s ingle dose lOug/lOOg . fCollagcnase-disso-4/ elated rat adrenal ^ C i l l s 10" 5-10"''M lOOug/nl 20ug/ml 20J g/ml 2-10 vM 1-50 VH 5 IP 2 IP IP IP IP IP I* sc3 IP or SC IP SC IP IP da l ly da i ly dal ly da l ly dal ly da i ly every 30nin 12 daya Plasma 170H cort icoids 12 days Plasma 170H cort icoids 32 days Plasma 170H cort icoids 4 hours Plasma corticosterone 21 hours Plasaa corticosterone 5 days Urinary 17011 corticoids 5 days Plasma hydrocortisone . 24 hours Corticosterone content of adrenal 26 hours Corticosterone content of adrenal 24 hours Plasma corticosterone 24 hours Plasma corticosterone 8 hours Adrenal vein corticosterone 5 days Plasma hydrocortisone 2.5 hours Corticosterone 3 hours Corticosterone 1.5 hours - Cor t i so l and Corticosterone 3 hours 20a dihydroprogesterone 2 hours Corticosterone 1 hour Corr.icosterone Increased for 2 days Increased for 7 days Prolonged increase Increased S l ight ly Increased Increased during f i r s t 3 days Increased during f i r s c 3 days Planelles J_t a l . 1952 Planelles et a l . 1962 Planelles et a l . 1962 Lippe and Szebo. 1965 Oyama and Dallgcoa, 1967 .' Bransome, 1969 Bransome, 1969 ACTH-induced Increase unaffected Vernikos-Danellis at 30 min. prevented at 24 hours and Ha l l , 1962 Unaffected Decreased Unaffected Unaffected Diminished response at 1 day Increased response 3-5 days S l ight ly decreased Unaffected Unaffected Unaffected Abolished Decreased 45-70X Hialhe Voloss et^  a l . 1966 Hlalhe Voloss et a l . 1966 Mialhe Voloss et a l . 1966 Key et a l . 1966 Bransome, 1969 Ferguson and Merita, 1964 Halkerston et. a l . 1965 Farese, 1966 • Kowal, 1970a Shanu, 1974 Schulster, 1974 IN VTiTt:ACTH CAMP , ' Rat adrenal f rag -ments Rat adrenal f rag-ments . Cov adrenal s l i ces Y - l mouse tumor ce l l s Superfused rat adrenal glands Collagenaae-disso-clated rat adrenal ce l l s ; . l O ' S - l O - " M lOOug/ml 20ug/ml 20ug/ml l-50uM Legends: every 30mln every 30min 2.5 hours 3 hours 1.5 hours 3 hours 8 hours 1 hour Corticosterone Corticosterone Cort i so l and Corticosterone 20a dihydroprogesterone Corticosterone Corticosterone Unless otherwise stated, analysis of steroid output was carried out at the end of the experiment. IF • Intraperitoneal . SC - Subcutaneous ACTd was administered 15min-l hour pr ior to termination of experiment. Molecular Weight of Actinomycin D - approximately 1200. Decreased s l i gh t ly Unaffected Inhibited Unaffected Ferguson and Korlta, 1964 aalkerston et a l . 1965 Farese, 1966 Kowal, 1970 Increased rate of steroid output M e l t . f , p b - u r and I c b « 0 , 1972 delayed onset of refractory period ACTH- and cyc l i c AMP-stimulated levels decreased by 53-641; Schulster, 1974 48-642 respectively 94 been found not only to increase, but also to decrease or else not affect the basal l e v e l of steroidogenesis. Furthermore, the steroidogenic response to ACTH has been observed to be elevated, prevented, or un-affected by actinomycin D. Two previous investigations which were undertaken by Bransome (1969) and Schulster (19 74) to c l a r i f y the relationship between RNA synthesis and ACTH-stimulated steroidogenesis are noteworthy. Bransome conducted a comprehensive i n vivo study on the effects of actinomycin D upon the morphology, steroidogenesis, DNA synthesis, RNA synthesis and protein synthesis of the guinea pig adrenal. Schulster systemati-c a l l y studied the responses of collagenase-dispersed adrenal c e l l sus-pensions exposed to a range of actinomycin D, ACTH and c y c l i c AMP con-centrations. From these two studies, i t was determined that the acute steroidogenic action of ACTH does not require newly synthesized RNA, and that some of the e a r l i e r discrepancies concerning actinomycin D and ACTH action might be the result of differences i n experimental design, i n dosage of actinomycin D used, of differences i n c e l l s examined, or i n the timing of actinomycin D administration and subsequent observation of i t s e f f e c t s . The t o x i c i t y and multiple side effects of actinomycin D have been described. Thus, the p o s s i b i l i t y of ambiguities introduced by drug usage i n experiments employing actinomycin D i s conceivable. Although the studies reported by Bransome (1969) and Schulster (1974) were thorough, i t i s now quite obvious that an effect of actinomycin D upon adrenocor-t i c a l functions cannot unequivocably be assumed to be sol e l y and d i r e c t l y 95 related to interference with the t r a n s c r i p t i o n a l stage of protein syn-thesis (Mostafapour and Tchen, 1972). In this study, the effects of ACTH upon anucleate adrenocortical c e l l s were analyzed. Thus, the p o s s i b i l i t y of unpredictable side effects of actinomycin D was eliminated. Enucleated Y-l-L c e l l s and Tumor #2 c e l l s did respond to either ACTH or dibutyryl c y c l i c AMP. Thus, i t could be concluded from this study that the nucleus, and therefore new RNA synthesis, i s d e f i n i t e l y not necessary for the capacity of Y-l-L c e l l s and Tumor #2 c e l l s to respond to ACTH or dibutyryl c y c l i c AMP. The basal, ACTH-stimulated and dibuty r y l c y c l i c AMP-stimulated steroid outputs of enucleated Y-l-L c e l l s were i d e n t i c a l to those of the EPT control Y-l-L c e l l s during the f i r s t three hours post-enucleation. This i s consistent with Kowal's observation that the basal and ACTH-stimulated steroid production of Y - l c e l l s was unaffected by 20 pg of actinomycin D/ml (Kowal, 1970a). Although the ste r o i d output of enucleated Tumor #2 c e l l s was s l i g h t l y lower than those of t h e i r nucleated counterparts, the increases i n steroidogenesis i n response to ACTH or dibutyryl c y c l i c AMP were com-parable to the EPT control Tumor #2 c e l l s during the f i r s t three hours after enucleation. Previous i n vivo studies u t i l i z i n g actinomycin D have indicated that the synthesis of protein(s) believed to mediate the steroidogenic effects of ACTH depended on RNA(s) that was (were) stable for at least 8 hours (Ney et a l . , 1966), while i n v i t r o studies u t i l i z i n g actinomycin D have estimated the h a l f - l i f e of any RNA(s) involved i n the ACTH effect 96 to be at least 70 minutes (Schulster, 1974). Experiments on the adrenal steroidogenic capacity of hypophysectomized animals have indicated that the decay i n corticosteroid biosynthesis by the adrenals was a function of time post-hypophysectomy. The h a l f - l i f e of th i s decline has been estimated to be 6 to 7 hours. I t was also suggested that the decay i n steroidogenic capacity may be correlated with or due to the decay of the messenger RNA for the steroidogenic protein (Mostafapour and Tchen, 1973). Due to the r e l a t i v e l y long incubation periods used i n this study, and i n view of the lack of information upon the i d e n t i t y of the RNA i n -volved i n ACTH action, the h a l f - l i f e of the factor involved i n stimulated steroidogenesis could not be measured. Nevertheless, i t can be con-cluded from this study that the cytoplasmic component required for i n -creased steroidogenesis i n response to ACTH i s r e l a t i v e l y stable. The o r i g i n of a basal s t e r o i d output i n the absence of added ACTH by the adrenocortical tumor c e l l s i s not clear. There i s no evidence to suggest that there are separate regulatory mechanisms for basal and stimulated steroidogenesis. Thus, i t i s quite reasonable that the basal steroid output was also decreased at increasing time i n t e r v a l s following enucleation due to the decay of the cytoplasmic components necessary for steroidogenesis. While the steroidogenic responses of enucleated adrenocortical c e l l s to ACTH and dib u t y r y l c y c l i c AMP were abolished 12 hours following enucleation, the morphological responses to ACTH and dibutyryl c y c l i c AMP were inducible up to 36 hours following enucleation. There i s 97 increasing evidence that the effects of ACTH upon the morphology and steroidogenic a c t i v i t y of the adrenal cortex i n vivo may not, of neces-s i t y , be i n t e r r e l a t e d (Kuo and Tchen, 19 73). Moreover, i t has been demonstrated that Y - l c e l l s responded morphologically to ACTH under conditions i n which steroid synthesis was i n h i b i t e d (Kowal, 1969c). Thus, i t i s plausible that although morphological changes and increased steroidogenesis are both effects due to ACTH, the mechanism of ACTH action upon these a c t i v i t i e s may be different and may not be causally related. From this study, i t was determined that the cytoplasmic com-ponents necessary for c e l l "rounding" are stable for at least 36 hours. VI. General Outlook In t h i s study, i t was conclusively established that the nucleus, hence nuclear RNA synthesis, i s not required i n the acute steroidogenic response of adrenocortical c e l l s to ACTH, since enucleated c e l l s responded to ACTH both morphologically and steroidogenically. The roles of the nucleus and cytoplasm i n a variety of c e l l u l a r a c t i v i t i e s have also been ascertained by studying the metabolic c a p a b i l i t i e s of a variety of enu-cleated c e l l types (e.g. Prescott et a l . , 1971; Goldman et a l . , 1973; M i l l e r and Ruddle, 1974; Rechsteiner and Catanzarite, 1974; Ivarie et a l . , 19 74). whatever the precise mechanism of cytochalasin B-induced enu-cleation, i t i s now evident that the prediction that enucleation of mammalian c e l l s by this drug could provide a p o t e n t i a l l y useful t o o l i n c e l l biology (Carter, 1967) has been re a l i z e d . The t o t a l evaluation of the role of the cytoplasm i n the intact c e l l cannot be obtained by the studies on the cytoplasm i s o l a t e d from 98 the nucleus. Obviously, the presence of the nucleus provides the neces-sary conditions for the complete functioning of the cytoplasm. The converse i s also true. Were this not so, the evolution of such an or-ganization would be enigmatic. Studies involving nuclear transplantation have revealed that the cytoplasm also plays an important role i n the regulation of nuclear a c t i v i t y (Gurdon, 19 70). Although the genetic information present i n the nucleus of a l l c e l l s i n a m u l t i c e l l u l a r organism i s equivalent, the expression of this information varies i n different c e l l types and i s dependent upon the cytoplasm i n which the nucleus resides. Such studies have been l i m i t e d to very few c e l l types, as micromanipulation becomes technically formidable when the c e l l s are small. Recently, methods for the fusion of unlike c e l l s by Sendai virus (Harris, 1970) and l y s o l e t h i c i n (Keay et a l . , 1972) have been described. I t has been postulated that the nuclear transplantation can be achieved by combining the methods involved with cytochalasin B-induced formation of cytoplasts and karyoplasts together with methods involved with c e l l fusion. Thus, i t i s conceivable that the introduction of n u c l e i into heterologous cytoplasms i n the manner described, could provide a unique experimental system for the study of the metabolic and genetic regulation i n mammalian c e l l s (Poste and Reeve, 1972). SUMMARY 1. Previously, actinomycin D has been used to c l a r i f y the role of RNA synthesis i n the acute steroidogenic response of adrenocortical c e l l s to ACTH. However, a survey of the l i t e r a t u r e revealed that c o n f l i c t i n g results were reported. I t i s now evident that actino-mycin D i s tox i c and can produce a myriad of side e f f e c t s . Thus, the p o s s i b i l i t i e s of ambiguities due to the drug usage i n these studies could not be ruled out. The purpose of this study was to use an alternate approach to determine conclusively whether new RNA synthesis i s required i n the response to ACTH. The re-sponses to ACTH by adrenocortical c e l l s which had been enucleated by cytochalasin B treatment were investigated. 2. The functional murine adrenocortical tumor c e l l l i n e , the Y - l c e l l s , were the adrenocortical c e l l s chosen for t h i s study. I t was found during the course of this study that Y - l c e l l s could not be e f f i c i e n t l y enucleated by the enucleation procedures developed and used. In contrast, Y-l-L c e l l s and Tumor #2 c e l l s were enu-cleated consistently and e f f i c i e n t l y , thus, these c e l l s were used for the study of ACTH action. 3. Y-l-L c e l l s were selected from the Y - l c e l l l i n e . Tumor #2 c e l l s were derived from an isogeneic mouse which had received an inoculum of Y-l-L c e l l s . The success i n enucleation of these c e l l s was attributed to the property of increased adhesion to the substratum - 99 100 of these c e l l s . The morphologic and steroidogenic responses of these c e l l s to ACTH and dibutyryl c y c l i c AMP were characterized. Similar to the Y - l c e l l s , Y-l-L c e l l s and Tumor #2 c e l l s did not require the presence of ACTH for the maintenance of a basal s t e r o i d output, but responded to added ACTH or dibutyry l c y c l i c AMP by a change from f l a t to rounded c e l l shape, and by increased steroido-genesis . In comparison with the Y - l c e l l s , the steroid output of Y-l-L c e l l s and Tumor #2 c e l l s under various incubation conditions was r e l a t i v e l y lower. In addition, the ste r o i d production by these c e l l s was further diminished with increasing culture passage. Evidence which suggested that Y-l-L c e l l s were less d i f f e r e n t i a t e d than the parent Y - l c e l l l i n e , and that ded i f f e r e n t i a t i o n also occurred with i n -creasing culture passage was presented and discussed. The ultrastructure of the Y-l-L c e l l s was different than that of adrenocortical c e l l s i n vivo. No correlation could be made between the ultrastructure and the functional a c t i v i t y of these c e l l s . Although i t i s evident that " c e l l rounding" i n response to ACTH was not i d e n t i c a l to " c e l l rounding" i n response to trypsin or trypsin and versene i n terms of ult r a s t r u c t u r e , the significance and mechanism of this morphologic response i s not clear. I t was established that the treatments used during the enucleation procedure did.not have any effect on the subsequent morphology and steroidogenic a c t i v i t i e s of Y-l-L c e l l s and Tumor #2 c e l l s . During the course of this study, two enucleation methods were de-veloped. These procedures were modelled a f t e r the method described 101 by Prescott ejt a l . (1972). Populations i n which 95-98% of the Y-l-L c e l l s or Tumor #2 c e l l s were anucleate were routinely produced by these methods. 9. Some enucleated c e l l s were viable for at least 60 hours, and were metabolically active for 48 hours, as indicated by dye exclusion and incorporation of 3H-leucine into acid-insoluble material. 10. Enucleated Y-l-L c e l l s and Tumor #2 c e l l s responded to either ACTH or dibuty r y l c y c l i c AMP morphologically and steroidogenically. Thus i t was conclusively established that the nucleus i s not re-quired for the expression of these acute effects of ACTH and dib u t y r y l c y c l i c AMP. 11. 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In V i t r o 7: 323-329. APPENDIX I Preparation of Cells for Transmission Electron Microscopy A. Dehydration and Embedding 1. 50% ETOH, 5 minutes 2. 70% ETOH, 5 minutes 3. 90% ETOH, 5 minutes 4. 95% ETOH, 5 minutes 5. 100% ETOH, 5 minutes 6. 100% ETOH, 5 minutes 7. 100% ETOH: Propylene Oxide (1:1), 10 minutes 8. Propylene Oxide, 10 minutes 9. Propylene Oxide, 10 minutes 10. Propylene Oxide:Epon (1:1), 1 hour 11. Propylene Oxide:Epon (1:3), 1 hour 12. Epon, 1 hour 13. Fresh Epon, 24 hours at 37°C, then overnight at 56°C. B. Epon Solution Stock sol u t i o n A: Stock solution B: Working solution: Epon 812 Resin 62 ml Dodecenyl Succinyl Anhydride (DDSA) 100 ml Epon 812 Resin 100 ml Nadic Methyl Anhydride (NMA) 89 ml Mix solution A and B (2:3), then add 2% by volume,the accelerator dimethylamino-methyl phenol (DMP-30-Tris) 110 APPENDIX I I Id e n t i f i c a t i o n of 20a Dihydroprogesterone by Acetylation P r i o r to  Re c r y s t a l l i z a t i o n A. Preparation of Carrier 20a Dihydroprogesterone-Acetate 1. 30 mg 20a dihydroprogesterone standard + 3 ml acetic anhydride + 3 ml pyridine at 37°C for 2 hours. 2. 0.5 ml of ethanol was added to stop the reaction. 3. The reaction mixture was extracted with 20 ml of methylene chloride. 4. The methylene chloride extract was washed with 1.0 ml of water. 5. The solvent phase was removed and evaporated under nitrogen. 6. Assuming that there was 100% acetylation, the product remaining was 100% 20a dihydroprogesterone-acetate. B. Preparation of JH-Acetic Anhydride 3H-acetic anhydride (400 uCi/mM) was purchased from New England Nuclear and diluted to 40 uCi/mM i n 12% benzene. C. Acetylation Experiment 1. The standards used were two 0.5 yg aliquots of authentic 20a dihydro-progesterone. The sample 20a dihydroprogesterone was obtained from the thin layer chromatogram of the endogenous ste r o i d production during 12 hours of incubation of ten 6 cm diameter P e t r i dishes of 8th passage Y-l-L c e l l s . The amount of 20a dihydroprogesterone i s o l a t e d from the chromatogram was equivalent to approximately 1 yg of steroid. 2. ll+C-20a dihydroprogesterone was used to monitor the recovery of 20a dihydroprogesterone during t h i s experiment. ll*C-20a dihydro-progesterone was not commercially available, therefore, the C-20a dihydroprogesterone used was obtained from the chromatographic i s o l a t i o n of 1 ^ -pregnenolone metabolites of Y-l-L c e l l s . The areas on the chromatogram containing 11+C-20a dihydroprogesterone was scraped o f f , eluted with ethanol, f i l t e r e d , evaporated, and then redissolved i n 1 ml of methanol. I l l 112 3. 0.3 ml of 1 4C-20a dihydroprogesterone was added to each of the following 3 tubes: (a) Standard 1, which contained 0.5 yg of authentic 20a dihydro-progesterone. (b) Standard 2, which contained 0.5 yg of authentic 20a dihydro-progesterone. (c) Sample, which contained approximately 1.0 yg of 20a dihydro-progesterone which was produced by Y-l-L c e l l s . 4. The materials i n each of the 3 tubes were evaporated under nitrogen. 5. 30 y l of pyridine and 1.5 y l of 3H-acetic anhydride was added to each of the 3 tubes. 6. After 24 hours, the reaction was stopped by the addition of 0.5 ml of ethanol. 7. Each of Standard 1, Standard 2, and Sampled was extracted with 5 ml of carbon tetrachloride. 8. The carbon tetrachloride extract was washed with 0.5 ml of water. The water phase was then pipetted o f f , and the solvent was evaporated under nitrogen. 9. The material remaining i n each of the three tubes was chromatographed i n benzene:acetone (120:30, v:v) on s i l i c a gel plates together with 10 yg each of authentic 20a dihydroprogesterone and prepared 20a dihydroprogesterone-acetate. 10. Autoradiograms of the chromatograms were prepared. 11. For each of Standard 1, Standard 2, and Sample, there was one major exposed area on the X-ray f i l m . This corresponded to the R^ of 20a dihydroproges terone-acetate. 12. This area on the s i l i c a gel plate was scraped o f f , eluted with ethanol, f i l t e r e d , and then evaporated separately for each of Standard 1, Standard 2, and Sample. 13. Each of Standard 1, Standard 2, and Sample was r e c r y s t a l l i z e d i n the presence of 10 mg of prepared 20a dihydroprogesterone-acetate. 14. An aliquot of the 3rd and 4th crystals of each of Standard 1, Standard 2, and Sample were counted, and' the lt+C:3H r a t i o of these crystals was established. APPENDIX I I I Preparation of Lowry's Reagent Stock solution A: 2% Na 2C0 3 i n 0.1 M NaOH Stock solution B: 2% NA K tartarate Stock solution B: 1% CuS04-5H20 Working solution: (Lowry's reagent): 100A:1B:1C - use within 2 hours 113 

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