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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 . S c , The U n i v e r s i t y 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 t h i s t h e s i s as conforming t o the required standard  THE UNIVERSITY OF BRITISH COLUMBIA J u l y , 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  an advanced degree at the U n i v e r s i t y of B r i t i s h C o l u m b i a , I agree 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 for  I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e  r e f e r e n c e and copying o f t h i s  of  this thesis for  written  It  i s understood that c o p y i n g or  thesis  Department of  lLo 0 LP  Y  The U n i v e r s i t y o f B r i t i s h Columbia  2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5  Date  \Sst  A-vuiLUut  .  IHS  or  publication  f i n a n c i a l g a i n s h a l l not be a l l o w e d without my  permission.  that  study.  f o r s c h o l a r l y .purposes may be g r a n t e d by the Head o f my Department by h i s r e p r e s e n t a t i v e s .  for  ABSTRACT  The o b j e c t i v e of t h i s study was t o determine c o n c l u s i v e l y whether or not the nucleus i s required f o r the c y c l i c AMP-mediated s t e r o i d o g e n i c response of a d r e n o c o r t i c a l c e l l s to a d r e n o c o r t i c o t r o p i c hormone (ACTH). Enucleated c e l l s are i d e a l f o r studying various aspects of c e l l u l a r metabolism without the added complexity control.  of superimposed t r a n s c r i p t i o n a l  Cytochalasin B can t r i g g e r the enucleation of s e v e r a l types  of c u l t u r e d 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 , c u l t u r e d a d r e n o c o r t i c a l c e l l s were enucleated w i t h c y t o c h a l a s i n B, and the response of these enucleated  cells  to ACTH and d i b u t y r y l c y c l i c AMP was i n v e s t i g a t e d . The type of a d r e n o c o r t i c a l c e l l s chosen f o r t h i s study was the Y - l f u n c t i o n a l mouse a d r e n o c o r t i c a l tumor c e l l l i n e . found that the Y - l c e l l s could not be enucleated  However, i t was  e f f i c i e n t l y , thus,  during  the course of t h i s study, a s u b l i n e denoted Y - l - L c e l l s was s e l e c t e d from the Y - l c e l l l i n e . animals.  These c e l l s were l a t e r passaged i n isogeneic  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 .  S i m i l a r 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 d i d not require ACTH f o r growth o r maintenance of a b a s a l s t e r o i d output, but would respond t o added ACTH or d i b u t y r y l c y c l i c AMP by a change i n morphology and by an increase i n steroidogenesis.  I n 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 ii  iii #2 c e l l s i n c o n t r o l  medium, i n medium containing ACTH, and i n medium  containing d i b u t y r y l c y c l i c AMP was characterized by l i g h t and e l e c t r o n microscopy, and the s t e r o i d 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 v i a b l e as shown by dye exclusion f o r a minimum of 60 hours, and synthesized proteins as i n d i c a t e d by H-leucine 3  at l e a s t 48 hours.  incorporation into acid-insoluble material f o r  Enucleated a d r e n o c o r t i c a l c e l l s responded to e i t h e r  ACTH (10 mU/ml) or d i b u t y r y l 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 c u l t u r e medium. response of enucleated  The steroidogenic  c e l l s during the f i r s t three hours a f t e r enu-  c l e a t i o n was comparable i n magnitude to that of the nucleated  cells,  and p e r s i s t e d i n diminishing degrees f o r an a d d i t i o n a l 9 hours.  On the  other hand, the morphologic change can be induced by ACTH even at 33 hours f o l l o w i n g enucleation. The r e s u l t s of t h i s study show that the nucleus i s not required f o r the expression of the acute e f f e c t s of ACTH, and that the cytoplasmic components necessary f o r c e l l "rounding" and steroidogenesis are s t a b l e f o r a t l e a s t 36 and 12 hours r e s p e c t i v e l y .  ACKNOWLEDGMENTS  I wish t o express my sincere appreciation t o : Dr. N. Auersperg, Department of Zoology and Cancer Research Centre, f o r her concern, guidance and encouragement throughout t h i s investigation. Dr. R. L. Noble, D i r e c t o r of the Cancer Research Centre, f o r the opportunities and f a c i l i t i e s to carry out t h i s research. Drs.  A. B. Acton and J . D. Berger, Department of Zoology, f o r  t h e i r i n t e r e s t i n t h i s i n v e s t i g a t i o n , and f o r t h e i r review of t h i s manuscript. Dr. A. F. Burton, Department of Biochemistry, f o r h i s c r i t i c a l comments and h e l p f u l suggestions. Dr. J . B. Hudson, Department of Medical Microbiology, f o r h i s consideration and use of the S o r v a l l RC 2B c e n t r i f u g e . Dr. L. K r a i n t z , Department of O r a l B i o l o g y , f o r the use of the P h i l l i p s 300 e l e c t r o n microscope. Dr. M. J . Hollenberg, Department of Anatomy, f o r the use o f the Cambridge Stereoscan e l e c t r o n microscope. Mr. W. Siep, Cancer Research Centre, f o r h i s s k i l l e d t e c h n i c a l help. Mrs. E. A. S l a v i n s k i , Mrs. D. M c C l e l l a n and Mrs. L. Lee f o r t h e i r competent help and advice. During the tenure of t h i s i n v e s t i g a t i o n , the author was the r e c i p i e n t of a McLean Fraser Memorial Fellowship and a bursary from the N a t i o n a l Research C o u n c i l of Canada.  This research was supported by grants from  the N a t i o n a l Cancer I n s t i t u t e of Canada t o Dr. N. Auersperg. iv  TABLE OF CONTENTS  Page ABSTRACT  i i  ACKNOWLEDGMENTS TABLE OF CONTENTS  iv .  . . . . . . .  LIST OF TABLES  v viii  LIST OF FIGURES  x  INTRODUCTION  1  MATERIALS AND METHODS  7  I. II. III.  The A d r e n o c o r t i c a l C e l l s Used f o r the Study of ACTH Action  13  Tissue Culture Preparation of ACTH, D i b u t y r y l Cyclic AMP and Cytochalasin B  IV. V. VI. VII. VIII. IX. X.  14  Microscopy  15  S t e r o i d Determination  18  Enucleation Procedure  21  Chromosome P r e p a r a t i o n  23  Cell Viability  24  P r o t e i n Determination  24  Autoradiography  25  RESULTS I.  7  . . . . . . . . . . . . . . Morphological and Biochemical Responses of I n t a c t (Nucleated) Y - l , Y - l - L and Tumor #2 C e l l s to ACTH and D i b u t y r y l C y c l i c AMP v  27  27  vi Page 1. Morphological responses - l i g h t microscopy 2. Steroidogenic responses 3. I d e n t i f i c a t i o n of endogenous s t e r o i d production during a 12-hour incubation 4. C-Pregnenolone metabolism of Y - l - L and Tumor #2 C e l l s 5. E l e c t r o n microscopy of Y - l - L C e l l s  27 32  39 41  Enucleation Procedure  53  E f f e c t s o f DMSO, Cytochalasin B and Enucleation Procedure Treatments (without a c t u a l enucleation) upon Y - l - L and Tumor #2 C e l l s  56  1. E f f e c t s upon steroidogenesis 2. E f f e c t s upon morphology . .  56 60  37  11+  II. III.  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 C e l l s to ACTH and D i b u t y r y l C y c l i c AMP 1. 2. 3. 4.  Viability . . 64 Protein synthetic a c t i v i t y 67 Morphologic responses to ACTH and d i b u t y r y l c y c l i c AMP . 67 Steroidogenic responses t o ACTH and d i b u t y r y l c y c l i c AMP 70  DISCUSSION I. II. III. IV.  79  The A d r e n o c o r t i c a l C e l l s Used i n t h i s Study  79  The Morphologic E f f e c t of ACTH  84  Enucleation Procedure  87  E f f e c t s of DMSO and Cytochalasin B upon Y - l - L C e l l s and Tumor #2 C e l l s  V. VI. SUMMARY  64  90  Mechanism of ACTH A c t i o n  92  General Outlook  97  . . .  99  BIBLIOGRAPHY  102  vii Page APPENDIX I. II. III.  Preparation of C e l l s f o r Transmission E l e c t r o n Microscopy I d e n t i f i c a t i o n of 2 0 a Dihydroprogesterone A c e t y l a t i o n P r i o r to R e c r y s t a l l i z a t i o n Preparation of Lowry's Reagent  by . . . . . . . .  110 I l l 113  LIST OF TABLES  Table 1 2  3  4  Page C h a r a c t e r i s t i c s of Y - l , Y - l - L C e l l s and Tumor #2 Cells Comparison of Steroidogenic Responses to ACTH and D i b u t y r y l C y c l i c AMP between Y - l C e l l s (ATCC CCL 79) and Y - l - L C e l l s '. . Steroidogenic Responses to ACTH and D i b u t y r y l C y c l i c AMP by Y - l - L C e l l s and Tumor #2 C e l l s a f t e r S e r i a l Subculture .... A c e t y l a t i o n of 20oH Dihydroprogesterone Produced Endogenously by Y - l - L C e l l s w i t h H-Acetic Anhydride, and R e c r y s t a l l i z a t i o n of 20a DihydroprogesteroneH-Acetate to constant H: C Ratio i n the Presence of C-20a Dihydroprogesterone- H-Acetate  12  35  36  3  3  3  11+  11+  3  42  5  Radiochemical I d e n t i f i c a t i o n of C-20a Dihydroprogesterone Produced by Tumor #2 C e l l s  43  6  E f f e c t s of DMSO, Cytochalasin B, and Enucleation Procedure Treatment upon the Steroidogenic Responses of Y - l - L C e l l s to ACTH and D i b u t y r y l C y c l i c AMP . . .  58  E f f e c t s of DMSO, Cytochalasin B, and Enucleation Procedure Treatments upon the Steroidogenic Responses of Tumor #2 C e l l s to ACTH and D i b u t y r y l C y c l i c AMP . .  58  E f f e c t s of DMSO, Ethanol, Cytochalasin B Dissolved i n DMSO, Cytochalasin B Dissolved i n Ethanol, and Enuc l e a t i o n Procedure Treatments upon the Steroidogenic Responses of Tumor #2 C e l l s to ACTH and D i b u t y r y l C y c l i c AMP  59  Radiochemical I d e n t i f i c a t i o n of C-20a Dihydroprogesterone Produced by Enucleated Tumor #2 C e l l s . .  73  Steroidogenic Responses to ACTH and D i b u t y r y l C y c l i c AMP by Enucleated Y - l - L C e l l s  75  Steroidogenic Responses to ACTH and D i b u t y r y l C y c l i c AMP by Enucleated Tumor #2 C e l l s . . . . . .  77  7  8  9 10 11  11+  ll+  viii  ix Table 12  13  Page Steroidogenic Responses to ACTH and D i b u t y r y l C y c l i c AMP by Nucleated and Enucleated Y - l - L C e l l s and Tumor #2 C e l l s : S t e r o i d Output i n 12 Hours . . . .  78  E f f e c t s of Actinomycin D Upon A d r e n o c o r t i c a l Steroidogenesis: Summary of Results Reported i n the L i t e r a t u r e  93  LIST OF FIGURES  Figure 1-2  Page Responses to ACTH and D i b u t y r y l C y c l i c AMP by Y - l C e l l s ( B i o c u l t ) and Y - l - L C e l l s  8  Method . .  22  3  Summary of Enucleation Procedure: Coverslip  4  Summary of Enucleation Procedure: 6 cm P e t r i Dish Method  22  5-6  Response to ACTH by Y - l - L C e l l s  28  7-8  H i s t o l o g y of Tumor #2 and the Response of Tumor #2  9 10-13 14-18 19-20 21  22-26  27-28  29-30  31-33 34  . . . . . . . . .  C e l l s i n Culture t o ACTH and D i b u t y r y l C y c l i c AMP . . The Endogenous S t e r o i d Products and C-Pregnenolone Metabolites of C e l l s During 12 Hours of Incubation . . U l t r a s t r u c t u r e of Y - l - L C e l l s i n s i t u U l t r a s t r u c t u r e of Y - l - L C e l l s i n s i t u : Response t o ACTH  30  47  Y - l - L C e l l s Following Treatment w i t h Trypsin and Trypsin together w i t h Versene  51  Experimental Design: Steroidogenic Responses t o ACTH and D i b u t y r y l C y c l i c AMP by Enucleation Procedure Treatment C e l l s and Enucleated C e l l s . .  57  E f f e c t s of ACTH, D i b u t y r y l C y c l i c AMP, DMSO, and Cytochalasin B upon the Morphology of Tumor #2 C e l l s .  61  V i a b i l i t y and P r o t e i n Synthetic A c t i v i t y of Enucleated Y-l-L C e l l s  65  Responses to ACTH by Nucleated and Enucleated Tumor #2 C e l l s  68  Morphologic Responses to ACTH and D i b u t y r y l AMP by Enucleated Y - l - L C e l l s  71 ,  ll+  Cyclic  S t e r o i d Pathway i n Adrenal Tumor C e l l Cultures . . . . 0  x  38 44  83  INTRODUCTION  Normal c e l l u l a r metabolism i s a r e s u l t of complex nucleocytoplasmic i n t e r a c t i o n s . Many approaches have been used to define more p r e c i s e l y the r e l a t i v e r o l e s of the nucleus and cytoplasm.  The  c l a s s i c studies i n v o l v i n g the comparison between the a c t i v i t i e s of nucl e a t e 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 a l s o showed that to a v a r i a b l e extent  and f o r a v a r i a b l e p e r i o d of time, the anucleate cytoplasm w i l l r e t a i n the p r o p e r t i e s of the cytoplasm i n the i n t a c t c e l l .  I t i s for this  reason that the anucleate s t a t e has provided the o p p o r t u n i t i e s f o r the studies concerning the morphogenetic and metabolic c a p a c i t i e s of the cytoplasm i n the absence of d i r e c t nuclear c o n t r o l . To date, the two most frequently employed methods of i n i t i a t i n g the anucleate c o n d i t i o n have been shown to have drawbacks (Keck, 1969). P h y s i c a l enucleation by m i c r o d i s s e c t i o n i s t e c h n i c a l l y d i f f i c u l t and becomes tedious when l a r g e numbers of c e l l s are required f o r biochemical analysis.  Chemical "enucleation" by drug-induced i n h i b i t i o n of RNA  s y n t h e s i s , thereby s i m u l a t i n g p h y s i c a l enucleation, may produce d r a s t i c side e f f e c t s which are unrelated to the cessation of RNA synthesis ( I v a r i e et a l . , 1974), hence, r e s u l t s obtained i n these studies without evidence are equivocal. 1  other  2 Currently there i s a novel method f o r 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 p r o t r u s i o n of n u c l e i when added to c e l l s i n monolayer c u l t u r e s .  Within minutes of c y t o c h a l a s i n treatment,  the nucleus becomes displaced from the main cytoplasmic mass, and i n time, the nucleus becomes attached to the r e s t of the c e l l by a mere filament of cytoplasm. to be explained.  The mechanism of t h i s curious phenomenon remains  However, the property of nuclear p r o t r u s i o n has been  e x p l o i t e d (Prescott et a l . , 1972; Wright and H a y f l i c k , 1972; F o l l e t , 19 74):  by subsequent c e n t r i f u g a t i o n , the t h i n strand of cytoplasm u n i t i n g  the nucleus and the cytoplasm i s e a s i l y severed and a population of c e l l s w i t h protruded n u c l e i may thus be converted t o a population of t o t a l l y anucleate  cells.  Furthermore, the apparent l a c k of e f f e c t on  c e l l u l a r metabolism f o l l o w i n g the removal of c y t o c h a l a s i n B i s of importance (Carter, 1967; I v a r i e et a l . , 1974).  Therefore,  the anucleate  c e l l s produced by t h i s method are p o t e n t i a l l y u s e f u l f o r the study of various aspects of the f u n c t i o n a l c a p a c i t i e s of the cytoplasm i n the absence of immediate nuclear c o n t r o l . The o b j e c t i v e of t h i s study was t o use c y t o c h a l a s i n B-mediated enucleation to determine whether or not new RNA synthesis i s required f o r the steroidogenic response of a d r e n o c o r t i c a l c e l l s to ACTH. applying t h i s enucleation technique,  By  a study of the r o l e of the cytoplasm  i n the metabolism of Y - l c e l l s , the f u n c t i o n a l murine a d r e n o c o r t i c a l tumor l i n e c e l l s was undertaken. g i c a l l y and biochemically.  Y - l c e l l s respond to ACTH morpholo-  Immediately upon a d d i t i o n of ACTH or d i b u t y r y 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 f l a t t e n e d , 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 i n t o s p h e r i c a l c e l l s . there i s an increase i n steroidogenesis  Concomitantly,  (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 a c t i o n of ACTH i n r e g u l a t i n g 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 e s t a b l i s h e d that c y c l i c AMP i s the i n t r a c e l l u l a r mediator of ACTH a c t i o n , the p r e c i s e mechanism by which c y c l i c AMP induces steriodogenesis remains obscure.  Studies i n v o l v i n g the e f f e c t of various  metabolic i n h i b i t o r s upon ACTH a c t i o n i n d i c a t e that p r o t e i n synthesis i s e s s e n t i a l f o r the capacity of a d r e n o c o r t i c a l c e l l s to respond to ACTH (Garren et a l . , 19 71). resolved.  The requirement f o r RNA synthesis i s un-  P r e v i o u s l y , actinomycin  D has been used to c l a r i f y the r o l e  of RNA synthesis i n the acute steroidogenic response of a d r e n o c o r t i c a l 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 r e s u l t s were reported.  I t i s now evident that actinomycin D  i s t o x 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 y of ambiguities due to drug usage i n these studies can not be r u l e d out. Enucleation by cytochalasin B treatment ensured the complete removal of the genome. As a r e s u l t , conclusive evidence p e r t a i n i n g to whether o r not d i r e c t nuclear a c t i v i t y i s required f o r the expression of ACTH e f f e c t s was obtained by studying the morphological genic response of enucleated  and s t e r o i d o -  a d r e n o c o r t i c a l 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 t h i s method, only small amounts of enu-  cleated c e l l s could be produced.  Greater amounts of enucleated  cells  4 were required f o r the accurate a n a l y s i s of t h e i r s t e r o i d output.  There-  f o r e , a modified enucleation procedure was developed as part of t h i s study, which r e s u l t e d i n a t e n - f o l d 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 f o r 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 f u g a l forces necessary f o r enucleation, c e l l loss due to detachment from the substratum was great.  To overcome t h i s problem, a more adhe-  sive s u b l i n e , denoted as Y - l - L , was s e l e c t e d 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 subcutaneous 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 t h e i r substratum than d i d 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 l e s s .  Because Y - l - L  c e l l s and Tumor #2 c e l l s could be c o n s i s t e n t l y enucleated w i t h high e f f i c i e n c y , these were the a d r e n o c o r t i c a l c e l l s used f o r the study of ACTH a c t i o n . In recent years, many b i o l o g i c a l e f f e c t s of c y t o c h a l a s i n B have been described ( A l d r i c h , 1975).  Although i t has been e s t a b l i s h e d that  many c y t o c h a l a s i n B-induced e f f e c t s are r e v e r s i b l e w i t h i n 10-15 minutes a f t e r the removal of the drug (Carter, 1967; Spooner, 1973), s e v e r a l experiments were a l s o performed to determine whether or not the cytoc h a l a s i n B treatment during the enucleation procedure had any e f f e c t 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 u l t r a s t r u c t u r e of the r a t adrenal cortex under normal and experimental  conditions has been described  (e.g. S a b a t i n i et a l . , 1962;  Rhodin, 1971) and modulation i n the u l t r a s t r u c t u r e of the r a t adrenal cortex 10 minutes a f t e r the a d m i n i s t r a t i o n of ACTH has been reported (Rhodin, 1971).  These changes include the depletion of glycogen and  l i p i d d r o p l e t s , and changes i n the s t r u c t u r e and s p a t i a l arrangements of mitochondria,  G o l g i bodies and smooth endoplasmic reticulum.  Such  r a p i d a l t e r a t i o n s i n f i n e s t r u c t u r e upon ACTH s t i m u l a t i o n 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 o r not enucleated Y - l - L c e l l s and Tumor #2 c e l l s are f u n c t i o n a l . more, i t has been recognized  Further-  that the goal of preserving the adrenal  cortex i n an unstressed, normal s t a t e f o r 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 t i s s u e . Less trauma i s i n v o l v e d i n f i x i n g c e l l s i n t i s s u e c u l t u r e .  Therefore,  the u l t r a s t r u c t u r a l a n a l y s i s of nucleated Y - l - L c e l l s at various times a f t e r ACTH a d m i n i s t r a t i o n was undertaken t o t e s t 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 c a u s a l l y w i t h the acute increase i n steroidogenesis, and also to e l u c i d a t e the mechanisms involved i n corticosteroid 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. I n t i s s u e c u l t u r e , t r y p s i n and versene are r o u t i n e l y used i n the subculture procedure. The r e a c t i o n of c e l l s to t r y p s i n i z a t i o n i s a l s o r e f e r r e d to as "rounding up."  Therefore,  the morphologic responses of Y-l-L c e l l s t o t r y p s i n and  t r y p s i n together with versene, were a l s o i n v e s t i g a t e d by l i g h t and e l e c t r o n  microscopy, and compared w i t h the morphological response of these c e l l s to ACTH. In summary, t h i s study c o n s i s t s of the development of an enuc l e a t i o n procedure which was capable of producing s u f f i c i e n t  quantities  of enucleated c e l l s f o r biochemical analyses, the production of a s u b l i n e of a d r e n o c o r t i c a l c e l l s which could be e f f i c i e n t l y enucleated, and the c h a r a c t e r i z a t i o n of the morphologic and s t e r o i d o g e n i c responses of nucleated and enucleated Y - l - L c e l l s and Tumor #2 p e l l s to ACTH and d i b u t y r y l c y c l i c AMP.  MATERIALS AND METHODS  I.  The A d r e n o c o r t i c a l C e l l s Used f o r the Study of ACTH A c t i o n The a d r e n o c o r t i c a l c e l l s used i n t h i s study were Y - l c e l l s pur-  chased from American Type Culture C o l l e c t i o n (ATCC CCL79), Y - l - L c e l l s , and Tumor #2 c e l l s . During the 'course of t h i s study, i t was e s t a b l i s h e d that w h i l e 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% enuc l e a t i o n e f f i c i e n c y was c o n s i s t e n t l y achieved w i t h 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 t h i s study.  The Y - l c e l l s (ATCC  CCL79) were used i n i t i a l l y f o r c o n t r o l purposes when the Y - l - L c e l l s and Tumor #2 c e l l s were being c h a r a c t e r i z e d . The Y - l - L c e l l s were s e l e c t e d from the Y - l c e l l l i n e which was purchased from B i o c u l t (BCL 721). Although the p r e s c r i b e d c u l t u r i n g procedure was followed, the Y - l c e l l s from B i o c u l t were heterogeneous i n morphology, and d i d not respond i n the manner described i n the l i t e r a ture, to ACTH or d i b u t y r y 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 a l s o 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 ( F i g . l a ) .  The morphologic responses of these  Y - l c e l l s to ACTH and d i b u t y r y l c y c l i c AMP were i n c o n s i s t e n t .  Approxi-  mately h a l f the population of c e l l s became rounded i n the presence of 10 mU ACTH/ml of medium or 1 mM d i b u t y r y l c y c l i c AMP, and i t could 7  8  Plate I Responses to ACTH and D i b u t y r y l C y c l i c AMP by Y - l C e l l s ( B i o c u l t ) and Y-l-L C e l l s . Methanol fixation', Giemsa s t a i n i n g , x350. Figs. l a - c . Y - l c e l l s ( B i o c u l t ) i n culture, (a) i n c o n t r o l medium, (b) 3 hours i n medium containing 10 mU ACTH/ml, (c) 3 hours i n medium containing 1 mM d i b u t y r y l c y c l i c AMP. Note the heterogeneity i n c e l l morphology and responsiveness to ACTH and d i b u t y r y l c y c l i c AMP. F i g s . 2a-c. Y-l-L- c e l l s i n c u l t u r e . (a) i n c o n t r o l medium, (b) 3 hours i n medium containing 10 mU ACTH/ml, (c) 3 hours i n medium containing 1 mM d i b u t y r y l c y c l i c AMP. The morphology of Y - l - L c e l l s i n c o n t r o l medium and the morphological response of these c e l l s to ACTH and d i b u t y r y l 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 ) .  «la.  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  flat  or already rounded c e l l s ( F i g s , l b and c ) . During media changes, and during s e r i a l subculture, a d i f f e r e n c e was n o t i c e d 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 l o o s e l y attached to the P e t r i d i s h and could be e a s i l y detached by s q u i r t i n g w i t h a p i p e t t e .  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 e a s i l y dispersed w i t h i n 10 minutes by incubation i n 0.12% t r y p s i n i n C a ^ 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 c u l t u r i n g 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 o f f 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 a l s o responded u n i -  formly to ACTH and d i b u t y r y l c y c l i c AMP (Figs. 2b and c ) .  This s u b l i n e  of c e l l s was denoted as Y - l - L c e l l s . S i m i l a r to the parent l i n e , Y - l - L c e l l s a l s o had a b a s a l s t e r o i d output i n the absence of ACTH, and responded t o added ACTH or d i b 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 t h i n l a y e r chromatography of media e x t r a c t s on s i l i c a g e l to be 20a dihydroprogesterone, H B 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 line.  However, i n comparison w i t h 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 c o n d i t i o n s .  11 The amount of s t e r o i d 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 f o r s e v e r a l 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 c u l t u r e d 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 f o l l o w i n g passages i n v i t r o had an enhanced a b i l i t y to grow and f u n c t i o n i n c u l t u r e subsequently (Buonassisi e_t a l . , 1962) . This phenomenon has been observed f o r 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 n e s 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 passage Y-l-L c e l l s .  6  10th  Tumor #2 c e l l s were used f o r 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 s t e r o i d s at a high r a t e , but t h e i r steroidogenic a c t i v i t y a l s o diminished a f t e r s e r i a l subculture.  This  phenomenon i s quite common and has been reported by s e v e r a l l a b o r a t o r i e s that work with the Y - l c e l l s .  Several c u l t u r e 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 in vitro.  These frozen samples were p e r i o d i c a l l y r e - e s t a b l i s h e d i n  c u l t u r e f o r experiments.  This p o l i c y provided cultures with  relatively  s i m i l a r l e v e l s of s t e r o i d output f o r the remainder of the experiments.  12 TABLE I . C h a r a c t e r i s t i c s of Y - l , Y - l - L and Tumor #2 C e l l s  Property Generation time (on p l a s t i c )  Y - l (ATCC CCL 79)  Y-l-L  40-50 hours  30-40 hours  Tumor #2  24-36 hours  Chromosomes Modal Number Frequency  40-42  40-42  40-42  75%  95%  95%  Growth On glass  poor, c e l l s s e t t l e d and grew slowly  cells settled q u i c k l y and grew well  cells settled q u i c k l y and grew well  On p l a s t i c  cells settled q u i c k l y and grew more q u i c k l y  same as on glass  same as on glass  On glass  heterogeneous  epithelial-like  ep i t h e l i a l - 1 i k e  On p l a s t i c  epithelial-like  epithelial-like  epithelial-like  On glass  monolayer  monolayer  monolayer  On p l a s t i c  c e l l s p i l e d up, became rounded and shed i n t o the medium  c e l l s p i l e d up e x t e n s i v e l y , became more f i b r o blast-like  c e l l s p i l e d up e x t e n s i v e l y , became more f i b r o blast-like  Morphology  Growth p a t t e r n  13 II.  Tissue Culture C e l l s stocks were maintained at 37°C i n 250 ml p l a s t i c t i s s u e  c u l t u r e f l a s k s (Falcon P l a s t i c s , Oxnard, C a l i f . ) on Ham's n u t r i e n t medium, F-10,  supplemented w i t h 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 L t d . , Calgary, A l t a . ) .  (Microcan Research  The medium was changed twice weekly, and the  c e l l s were subcultured every 8-10 days by d i s s o c i a t i o n i n 0.12% c r y s t a l line trypsin i n C a  2 +  and M g  2+  f r e e Hanks' balanced s a l t s o l u t i o n (Microcan  Research L t d . ) . Culture conditions were modified as required to f a c i l i t a t e various studies:  f o r the l i g h t and scanning e l e c t r o n microscopy of i n s i t u  c e l l s , and f o r the q u a n t i t a t i o n and i d e n t i f i c a t i o n of s t 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 watersaturated atmosphere of 5% CO2 i n a i r ; f o r the u l t r a s t r u c t u r a l a n a l y s i s of i n s i t u c e l l s , the c e l l s were grown on Leighton tubes ( B e l l c o , Vineland, N.J.).  Growth c h a r a c t e r i s t i c s 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 e i t h e r round glass c o v e r s l i p s , or on s u l p h u r i c a c i d treated 6 cm diameter P e t r i dishes (see Enucleation  Procedure).  Four tumor l i n e s were s t a r t e d by subcutaneous i n j e c t i o n of 10  6  10th passage Y - l - L c e l l s i n t o 6-8 week o l d LAF^ male mice (Jackson Laborat 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). f i x e d and processed  P o r t i o n s o f each tumor were  f o r l i g h t microscopy, and other p o r t i o n s were c u l t u r e d .  Portions s u i t a b l e f o r c u l t u r e were minced f i n e l y with  scissors i n trypsin  14 f o r 10 minutes a t room temperature. after centrifugation.  The t r y p s i n s o l u t i o n was decanted  The c e l l s were washed twice w i t h complete medium  and 4-5 x 1 0 c e l l s were p l a t e d i n 75 ml p l a s t i c t i s s u e c u l t u r e f l a s k s . 5  III. 1.  Preparation of ACTH, D i b u t y r y l c y c l i c AMP and Cytochalasin B Porcine a d r e n o c o r t i c o t r o p i c hormone Grade I (150 IU/mg) was purchased from Sigma Chemical Company, S t . L o u i s , M i s s o u r i . A stock s o l u t i o n of 1 IU ACTH/ml o f s t e r i l e s a l i n e was made and stored i n 1 ml a l i q u o t s at -20°C.  For experiments, ACTH was  d i l u t e d i n n u t r i e n t medium to give a f i n a l concentration of 10 mU/ml; t h i s medium was used w i t h i n 1 hour of preparation. 2.  N 0 ' d i b u t y r y l adenosine 3'5' c y c l i c monophosphoric a c i d Grade 6  2  11 was also purchased from Sigma.  D i b u t y r y l c y c l i c AMP was  d i s s o l v e d d i r e c t l y i n n u t r i e n t medium to give a 1 mM s o l u t i o n . 3.  I n i t i a l l y , the c y t o c h a l a s i n B used was a g i f t from Dr. S. B. Carter, Imperial Chemical I n d u s t r i e s , M a c c l e s f i e l d , Cheshire. However, most of the c y t o c h a l a s i n B used during the course of t h i s study was purchased from A l d r i c h Chemical Company, Millwaukee, Wisconsin.  A stock s o l u t i o n was prepared i n DMSO ( F i s h e r Scien-  t i f i c Co., Vancouver, B.C.) to give a concentration of 1 mg/ml and stored a t 4°C.  For experiments, the stock s o l u t i o n was  d i l u t e d i n aqueous medium to give a f i n a l c y t o c h a l a s i n B conc e n t r a t i o n of 10 yg/ml, and a DMSO concentration of 1%.  15 IV.  Microscopy 1. L i g h t microscopy Cultures i n s i t u were e i t h e r f i x e d i n 95% a l c o h o l and s t a i n e d  with 1% t o l u i d i n e blue (Fisher S c i e n t i f i c Co.), or f i x e d i n methanol and  s t a i n e d 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 f o l l o w i n g the i n j e c t i o n of  Y-l-L c e l l s were f i x e d i n Bourns', dehydrated through a graded s e r i e s of a l c o h o l s o l u t i o n s , 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 f o l l o w i n g haematoxylin and eosin s t a i n i n g ( C u l l i n g , 1963). M a t e r i a l s prepared f o r e l e c t r o n microscopy were also examined by l i g h t microscopy.  Epon s e c t i o n s , 0.5-1.0 micron-thick, were cut  with glass knives on a Reichert ultramicrotome.  The sections were t r a n s -  f e r r e d to glass s l i d e s w i t h a f i n e wire loop, h e a t - f i x e d onto the s l i d e s , and s t a i n e d w i t h 1% t o l u i d i n e blue i n 1% sodium borate (Pease, 1964). 2. E l e c t r o n microscopy The u l t r a s t r u c t u r e s of both Y - l - L c e l l s and Tumor #2 c e l l s were studied. a) Transmission e l e c t r o n 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 f o l l o w i n g : (i)  Basal medium ( i . e . Ham's F-10 supplemented with serum and a n t i biotics)  16 (ii)  Medium containing 10 mU ACTH/ml f o r 5 minutes  ( i i i ) Medium containing 10 mU ACTH/ml f o r 30 minutes (iv)  Medium containing 10 mU ACTH/ml f o r 1 hour  (v)  Medium containing 10 mU ACTH/ml f o r 3 hours  (vi)  Medium containing 10 mU ACTH/ml f o r 24 hours  ( v i i ) Medium containing 1 mM d i b u t y r y l c y c l i c AMP f o r 3 hours, the i n s i t u cultures were washed gently w i t h Hanks' balanced s a l t s o l u t i o n , f i x e d i n cold 2.5% glutaraldehyde  i n Millonig's buffer (Millonig,  1962; Pease, 1964) a t pH 7.4 f o r 1 hour, washed twice with b u f f e r , postf i x e d i n c o l d 1% osmium t e t r o x i d e i n M i l l o n i g ' s b u f f e r f o r 30 minutes, and again washed twice w i t h b u f f e r .  The cultures were dehydrated through  a graded s e r i e s of ethanol and propylene oxide, and i n f i l t r a t e d w i t h Epon according  to routine procedure (Appendix I ) .  Then, the Leighton  tubes were completely f i l l e d w i t h Epon, and f o l l o w i n g  polymerization,  r a p i d l y t r a n s f e r r e d from 56°C to i c e water, thereby breaking and r e l e a s i n g the blocks of Epon.  Appropriate  the tubes,  areas of the embedded  cultures were s e l e c t e d by l i g h t microscopy, cut out and mounted on blocks for sectioning.  Thin sections were cut w i t h a Du Pont diamond k n i f e on  a Reichert ultramicrotome, mounted on copper g r i d s , stained with  uranyl  acetate (K and K Lab, Inc., P l a i n v i e w , N.Y.) (Watson, 1958) and lead c i t r a t e ( F i s h e r S c i e n t i f i c Co.) (Venable and Coggeshall,  1965), and  examined with e i t h e r a H i t a c h i HS-7S e l e c t r o n microscope or a P h i l l i p s 300 e l e c t r o n microscope. The u l t r a s t r u c t u r e of Y - l - L c e l l s f o l l o w i n g incubation i n e i t h e r (i)  0.12% Trypsin i n C a  2 +  f o r 10 minutes, or  and-Mg  2+  free Hanks' balanced s a l t s o l u t i o n  (ii)  0.04% Versene and 0.12% Trypsin i n C a  and Mg"*" free Hanks'  2_r  2  balanced s a l t s o l u t i o n f o r 10 minutes, was a l s o i n v e s t i g a t e d .  The detached c e l l s were p e l l e t e d by c e n t r i f u g a -  t i o n , then f i x e d and processed as described above, with the a d d i t i o n of gentle c e n t r i f u g a t i o n f o l l o w i n g each step to concentrate The c e l l s were embedded i n p l a s t i c capsules.  the c e l l s .  The m a t e r i a l was a l s o  sectioned and s t a i n e d as described above. b) Transmission e l e c t r o n microscopy of Tumor #2 C e l l 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 f o l l o w i n g :  (i)  Basal medium  (ii)  Medium containing 10 mU ACTH/ml f o r 3 hours  (iii)  Medium containing 1 mM d i b u t y r y l c y c l i c AMP f o r 3 hours  (iv)  Medium containing 1% DMSO f o r 45 minutes  (v)  Medium containing 10 yg c y t o c h a l a s i n B/ml f o r 45 minutes  (vi)  Medium containing 10 yg c y t o c h a l a s i n B/ml and 10 mU ACTH/ml f o r 3 hours  (vii)  Basal medium f o r 3 hours, f o l l o w i n g preincubation i n 10 yg cytoc h a l a s i n B/ml f o r 45 minutes, and two washes w i t h Hanks' balanced salt solution.  ( v i i i ) Medium containing 10 mU ACTH/ml f o r 3 hours, f o l l o w i n g preincub a t i o n i n 10 yg c y t o c h a l a s i n B/ml f o r 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 w i t h Hanks' balanced s a l t s o l u t i o n , and f i x e d as described p r e v i o u s l y .  The c e l l s were dehydrated through  18 a graded s e r i e s of a l c o h o l .  When the c e l l s were i n the second change  of absolute a l c o h o l , 75% of the c e l l s were scraped o f f w i t h a rubber policeman.  These c e l l s were c o l l e c t e d , p e l l e t e d , embedded, s e c t i o n e d ,  s t a i n e d and examined a l s o as described p r e v i o u s l y .  The 25% of the c e l l s  remaining were a i r - d r i e d , and stored i n a d e s s i c a t o r .  These c e l l s were  l a t e r prepared f o r scanning e l e c t r o n microscopy. c) Scanning e l e c t r o n microscopy of Tumor #2 C e l l s To f a c i l i t a t e handling and manipulation, the areas of P e t r i dishes w i t h the f i x e d and a i r - d r i e d c e l l s were cut out and mounted on aluminium specimen stubs w i t h S i l v e r Dag mounting medium.  To render o  the specimen conductive, the c e l l s were coated w i t h a 150 A - t h i c k l a y e r 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 E l e c t r o n Microscope. V.  S t e r o i d Determination 1. Q u a n t i t a t i o n The assay of s t e r o i d output by the u t i l i z a t i o n of the f l u o r e s c e n t  p r o p e r t i e s of 20a hydroxylated d e r i v a t i v e s of progesterone i n e t h a n o l i c s u l p h u r i c a c i d 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 w i t h 2-3 ml of medium for e i t h e r 3 hours or 12 hours.  At the end of the incubation p e r i o d ,  the medium was removed and extracted w i t h 10 ml of spectrophotometry grade methylene c h l o r i d e (Fisher S c i e n t i f i c Co.).  The s o l v e n t phase  was p i p e t t e d out and f i l t e r e d i n t o another e x t r a c t i o n tube.  Two  19 m i l l i l l t r e s of 65% s u l p h u r i c a c i d (reagent grade, F i s h e r S c i e n t i f i c Co.) i n absolute ethanol which had been mixed on i c e 45 minutes p r e v i o u s l y , were added and the tube was shaken v i g o r o u s l y f o r 30 seconds.  The f l u o r e s -  cence of the a c i d e x t r a c t was read 45 minutes l a t e r at 535 my w i t h an e x c i t a t i o n wavelength of 470 my i n an Aminco-Bowman spectrofluorometer. A comparable a l i q u o t of medium incubated without c e l l s and ext r a c t e d i n the same manner, served as a'blank.  20a dihydroprogesterone,  50 ng, 100 ng, or 1 yg ( S t e r a l o i d s Inc., P a u l i n g , N.Y.) were added to medium and extracted.  These served as s t e r o i d standards.  The s t 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 s t e r o i d s produced by the a d r e n o c o r t i c a l c e l l s during 12 hours of incubation were t e n t a t i v e l y 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 t h i n - l a y e r chromatograms w i t h s t e r o i d standards ( S t e r a l o i d s I n c . , Sigma).  The methylene c h l o r i d e e x t r a c t s 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 g e l plates (Kodak Eastman, Rochester, N.Y.) using benzene-acetone (120:30, v:v).  The major s t e r o i d s were i d e n t i f i e d by t h e i r absorption i n the  ultraviolet. A d d i t i o n a l incubations were performed w i t h ^C-pregnenolone (50-60 mCi/mM) (New England Nuclear, Boston, Mass.). of  Ten microcuries  ^C-pregnenolone was d i s s o l v e d i n 0.5 ml of absolute ethanol.  Approxi-  mately 100,000-200,000 dpm of *C-pregnenolone were added to the c u l t u r e 1£  20 medium.  Following 12 hours of i n c u b a t i o n , the medium was removed, ex-  t r a c t e d w i t h methylene c h l o r i d e , taken to dryness, r e d i s s o l v e d and chromatographed i n two dimensions on s i l i c a g e l p l a t e s using benzene:acetone (120:30, v : v ) , and benzene:acetone (100:50, v:v) w i t h 10 yg each of nonr a d i o a c t i v e standards of pregnenolone, 20a dihydropregnenolone, progesterone, and 20a dihydroprogesterone.  Autoradiography of the chromatogram  were prepared as described by J e l l i n c k and Goudy (1967).  The r a d i o a c t i v e  areas on the s i l i c a g e l p l a t e were scraped o f f , eluted w i t h ethanol, f i l t e r e d , evaporated to dryness, r e d i s s o l v e d and rechromatographed s i l i c a g e l p l a t e s using benzene:acetone (120:30, v : v ) , areas corresponding to the UV-absorbing  on  The r a d i o a c t i v e  spots of progesterone and 20a  dihydroprogesterone, the r a d i o a c t i v e areas corresponding to pregnenolone and 20a dihydropregnenolone, which were sprayed w i t h phosphomolybdic a c i d to detect A5-3B hydroxy s t e r o i d s , and the r a d i o a c t i v e areas corresponding to the R 's f  of 11B0H progesterone, 11B0H, 20a dihydroprogesterone,  and 11 keto, 20a dihydroprogesterone were scraped o f f and e l u t e d w i t h 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 a l i q u o t s i n toluene (Fisher S c i e n t i f i c Co.)  con-  t a i n i n g 2,5-diphenyl oxazole (4 g / l i t r e ) and p - b i s - [ 2 - ( 5 - p h e n y l o x e z o l y l ) ] benzene (50 m g / l i t r e ) (Fisher S c i e n t i f i c Co.) using a Nuclear Chicago U n i l u x 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 e i t h e r 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 w i t h non-radioactive c a r r i e r 20a dihydroprogesterone, or by a c e t y l a t i o n w i t h 3  H - a c e t i c anhydride (400 mCi/mM) (New England Nuclear) p r i o r to  21 recrystallization  from ethanol:water  r a d i o a c t i v e 20cx dihydroprogesterone  to constant H: C r a t i o w i t h non3  14  acetate (Appendix I I ) .  The amount  of s t e r o i d present i n the c r y s t a l s was measured by standard  techniques  f o r 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 m o d i f i c a t i o n of the tech-  nique described by P r e s c o t t (Prescott et a l . , 1972). round glass c o v e r s l i p s (Corning, Corning, N.Y.)  S t e r i l e 18  were placed i n 35  mm mm  diameter P e t r i dishes.  2 - 4 x 10  and allowed to attach.  Two hours l a t e r a f u r t h e r 1.5 ml of medium was  5  c e l l s i n 0.5 ml of medium were added  added and the c e l l s were allowed to grow f o r 24-48 hours.  The  cover-  s l i p s w i t h the c e l l s were incubated i n serum-free medium f o r 1 hour p r i o r to enucleation.  C e l l enucleation was achieved by c e n t r i f u g a t i o n  of c o v e r s l i p s w i t h the monolayer of c e l l s f a c i n g the bottom of the 30 ml centrifuge tube (Corning) w i t h 5 ml serum-free medium containing 10 yg cytochalasin B/ml  at 7710 g f o r 40 minutes i n a S o r v a l l RC 2B c e n t r i f u g e  using the s s 3 4 r o t o r which had been prewarmed to 37°C.  After centrifugation,  the c o v e r s l i p s w i t h c e l l s were washed twice w i t h Hanks' balanced  salt  s o l u t i o n , then t r a n s f e r r e d i n t o f r e s h medium w i t h serum ( F i g . 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  the c e l l s a f t e r enucleation and s t a i n i n g w i t h Giemsa.  fixing  R o u t i n e l y , popu-  l a t i o n s i n which at l e a s t 95% of the c e l l s were enucleated were c o n s i s t e n t l y produced.  22 2 - 4 x 10  24-48 hours in nutrient medium  5  cells S o r v a l l RC 2B ss34 r o t o r 37°C 7710 g f o r 40 minutes 10 yg CB/ml serum-free medium  1 hour i n serum-free medium  Wash 2X w i t h BSS  Incubation i n f r e s h medium w i t h serum F i g . 3.  10  6  Summary of E n u c l e a t i  3 mm  cells  Procedure: Coverslip Method  58 mm  S o r v a l l RC 2B GSA r o t o r 37°C 10800 g f o r 40 minutes 10 yg CB/ml serum-free medium  24-48 hours i n n u t r i e n t medium  ..j  1 hour i n serum-free medium Wash 2X w i t h BSS  Incubation i n fresh medium w i t h serum F i g . 4.  Summary of Enucleation Procedure: 6 cm P e t r i Dish Method  23 During the course of t h i s study, the Prescott method was f u r t h e r 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 t r e a t e d w i t h  reagent grade s u l p h u r i c a c i d f o r 15 minutes.  concentrated  This was followed by  thorough r i n s i n g i n running water f o r 24 hours, then s t e r i l i z a t i o n by a l c o h o l and U V - i r r a d i a t i o n . 10 and grow  6  c e l l s were seeded and allowed t o attach  f o r 24-48 hours i n these P e t r i dishes containing 3 ml of nut-  r i e n t medium.  One hour p r i o r to enucleation, the n u t r i e n t medium was  removed and replaced w i t h serum-free medium. t h i s serum-free medium f o r 1 hour.  C e l l s were incubated i n  C e l l enucleation was achieved by  c e n t r i f u g a t i o n of P e t r i dishes containing the monolayer of c e l l s w i t h the c e l l s f a c i n g the.bottom of the 250 ml nalgene c e n t r i f u g e tube ( F i s h e r S c i e n t i f i c Co.) w i t h 100 ml of serum-free medium containing 10 Vg cytoc h a l a s i n B/ml.  The P e t r i dish was held i n place by p l a c i n g a 5 cm piece  of 58 mm diameter p l a s t i c a c r y l i c tubing w i t h a 3 mm-thick w a l l ( U n i v e r s a l P l a s t i c s L t d . , Burnaby, B.C.) on top of the i n v e r t e d P e t r i d i s h .  The  c e l l s were c e n t r i f u g e d a t 10,800 g f o r 40 minutes i n a S o r v a l l RC 2B centrifuge using the GSA r o t o r which had been prewarmed to 37°C.  After  c e n t r i f u g a t i o n , the P e t r i dishes were scanned w i t h an i n v e r t e d microscope and any area which contained fragments of nucleated c e l l s was scraped o f f w i t h a rubber policeman.  carefully  The remaining enucleate c e l l s  were then incubated w i t h fresh medium ( F i g . 4 ) .  VII.  Chromosome Preparations C e l l s were seeded onto 20 mm square c o v e r s l i p s (Corning) i n  35 mm P e t r i dishes covered w i t h 2 ml of n u t r i e n t medium.  In order to  24 obtain well-spread metaphase p l a t e s , 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 i s i o n s were  detected (by observation w i t h an i n v e r t e d microscope), 0.2 ml of a 0.01% s o l u t i o n of c o l c h i c i n e (BDH Chemicals, Poole, Dorset, England) was added for 3-4 hours.  The c o v e r s l i p s were then t r a n s f e r r e d to P e t r i dishes  containing 1% sodium c i t r a t e s o l u t i o n f o r 20 minutes.  The hypotonic  treatment caused c e l l s w e l l i n g ; thus, chromosomes which were w e l l spread out and separated were produced.  The c e l l s were then f i x e d w i t h a c i d  a l c o h o l , washed w i t h d i s t i l l e d water, and a i r - d r i e d .  When d r i e d , the  c e l l s were s t a i n e d f o r 5 minutes with 2% a c e t o - o r c e i n , dehydrated  through  95% a l c o h o l , absolute a l c o h o l , b u t a n o l , b u t a n o l - x y l o l , x y l o l and mounted on glass s l i d e s w i t h Permount (Fisher S c i e n t i f i c Co.).  VIII.  Cell Viability C e l l v i a b i l i t y was determined by the method of Y i p and Auersperg  (19 72).  Nucleated and enucleated c e l l s were f i x e d i n 2.5% glutaraldehyde  i n M i l l o n i g ' s b u f f e r ( M i l l o n i g , 1962; Pease, 1964) at pH 7.4 f o r 1 hour at 4°C. The f i x e d c e l l s were washed w i t h M i l l o n i g ' s b u f f e r , then s t o r e d at 4°C i n b u f f e r f o r up t o 1 week.  E x c l u s i o n of 0.03% a l c i a n blue (Fisher  S c i e n t i f i c Co.) i n b u f f e r f o r 10 minutes at room temperature by these glutaraldehyde-fixed c e l l s was used as an i n d i c a t i o n of c e l l v i a b i l i t y .  IX.  P r o t e i n Determination The amount of c e l l p r o t e i n i n each c u l t u r e of c e l l s was deter-  mined by the method of Oyama and Eagle (1956).  Cultures were washed  25 gently w i t h Hanks' balanced s a l t s o l u t i o n and a i r - d r i e d .  The  cells  were d i s s o l v e d i n Lowry's reagent (Appendix I I I ) , and reacted w i t h a phenol ( F o l i n - C i o c a l t e a u ) reagent (Fisher S c i e n t i f i c Co.).  The o p t i c a l  density of t h i s s o l u t i o n 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 w i t h the phenol reagent served as a blank.  Crys-  t a l l i n e bovine serum albumin (100 yg/ml of water) was d i s s o l v e d i n Lowry's reagent and reacted w i t h the phenol reagent. standard.  This served as a p r o t e i n  The amount of p r o t e i n per c u l t u r e was q u a n t i t a t e d as bovine  serum albumin e q u i v a l e n t .  X.  Autoradiography 3  H-leucine (30-50 Ci/mM) (New England Nuclear) was d i l u t e d to a  concentration of 10 uCi/ml of n u t r i e n t medium. Nucleated c e l l s grown on c o v e r s l i p s and enucleated c e l l s on c o v e r s l i p s at v a r y i n g time i n t e r v a l s f o l l o w i n g e n u c l e a t i o n were incubated i n medium containing H - l e u c i n e f o r 3 hours. 3  Excess H - l e u c i n e was 3  removed by dipping the c o v e r s l i p s containing the c e l l s i n 3 changes of Hanks' balanced s a l t s o l u t i o n .  The c e l l s were then f i x e d i n methanol,  extracted w i t h 5% c o l d t r i c h l o r o a c e t i c a c i d (Fisher S c i e n t i f i c Co.), r i n s e d w i t h 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 h a n d l i n g , the c o v e r s l i p s , w i t h the c e l l s i d e up, were mounted on glass s l i d e s w i t h 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 f o r 3 days.  26 The autoradiograms were processed i n D-19 rinsed under running water.  developer, f i x e d and  The c e l l s were then stained w i t h Giemsa,  d i f f e r e n t i a t e d w i t h acetone, dehydrated through a graded a l c o h o l s e r i e s , and mounted i n Permount by p l a c i n g another covers l i p over the exposed cells.  RESULTS  I.  Morphological and Biochemical Responses of I n t a c t (Nucleated) Y - l , Y - l - L and Tumor #2 C e l l s to ACTH and D i b u t y r y l 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 c o n t r o l medium, and medium containing e i t h e r ACTH or d i b u t y r 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 e t a l . , 1966a; Kowal e t a l . , 1974).  b) Y - l - L c e l l s Y-l-L  c e l l s changed markedly from a s t r e t c h e d and f l a t t e n e d  to a rounded-up shape i n the presence of e i t h e r ACTH or d i b u t y r y l c y c l i c AMP  (Figs. 2a-c).  In the presence of  s t a r t e d w i t h i n a few minutes,  10 mU ACTH/ml, the c e l l rounding  a t t a i n e d a maximum i n 1-2 hours and r e -  verted gradually to a f l a t t e n e d morphology tration.  12-15 hours a f t e r ACTH adminis-  This response i s shown most d r a m a t i c a l l y by the t o l u i d i n e  b l u e - s t a i n e d epon-embedded c u l t u r e s ( F i g s . 5a-e). cut perpendicular t o the substratum.  A l l sections were  I n t e r e s t i n g l y , although l i v e c u l -  tures appeared as monolayers w i t h l i g h t microscopy, up to 6 overlapping layers of Y - l - L c e l l s were found by examining p e r p e n d i c u l a r l y - c u t  plastic  sections of confluent c u l t u r e s 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 d i r e c t contact w i t h the medium  were rounded up, w h i l e most deeper c e l l s i n the s t r a t i f i e d l a y e r assumed a more or l e s s ovoid c e l l shape ( F i g . 6b),  Plate I I Response to ACTH by Y - l - L C e l l s . blue s t a i n i n g , x750.  Epon-embedded s e c t i o n s , t o l u i d i n e  Figs. 5a-e. Y-l-L c e l l s . (a) i n c o n t r o l 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. F i g s . 6a and b. M u l t i l a y e r e d Y - l - L c e l l s . (a) i n c o n t r o l medium; c e l l s i n a l l layers were f l a t t e n e d and appeared squamous-like i n l o n g i t u d i n a l s e c t i o n s , (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 C e l l s i n Culture to ACTH and D i b u t y r y l C y c l i c AMP. F i g s . 7a and b. Bouins' f i x a t i o n , hematoxylin and eosin s t a i n i n g . Histology of Tumor #2. (a) low m a g n i f i c a t i o n , x250, (b) some areas resemble t y p i c a l l y carcinomatous t i s s u e , x700. F i g s . 8a-c. Ethanol f i x a t i o n , t o l u i d i n e blue s t a i n i n g , x350. Tumor #2 c e l l s i n c u l t u r e . (a) i n c o n t r o l medium, (b) 3 hours i n medium containing 10 mU ACTH/ml, (c) 3 hours i n medium containing 1 mM d i b u t y r y 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 d i b u t y r y l c y c l i c AMP proceeded at a slower r a t e .  C e l l rounding was only v i s i b l e  1 hour a f t e r the a d d i t i o n of d i b u t y r y l c y c l i c AMP, and maximum rounding was a t t a i n e d i n 2-3 hours.  18-24 hours a f t e r treatment w i t h the c y c l i c  n u c l e o t i d e , the normal f l a t t e n e d morphology was resumed. c) Tumor #2 C e l l s H i s t o l o g i c a l l y , the tumors derived from the i n o c u l a of Y - l - L c e l l s i n t o mice were pleomorphic a d r e n o c o r t i c a l carcinomas.  Areas ranging  from a n a p l a s t i c t i s s u e to t y p i c a l l y carcinomatous t i s s u e were observed (Figs. 7a and b ) . The morphology  of c u l t u r e d Tumor #2 c e l l s i n c o n t r o l medium and  i n medium containing e i t h e r ACTH o r d i b u t y r y l c y c l i c AMP i s shown i n F i g s . 8a-c. The morphologic response of these c e l l s to ACTH and d i b u t y r y l c y c l i c AMP was comparable t o that of the Y - l - L c e l l s . 2. Steroidogenic responses a) I n t r o d u c t i o n 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 s t e r o i d products present i n the methylene c h l o r i d e e x t r a c t 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 t h i s method (accurate to 0.3 yg of C o r t i s o l standard), and the high blanks obtained from e x t r a c i n g medium containing large q u a n t i t i e s of serum, the accurate measurement of s t e r o i d s required the accurate preparation of p a r a l l e l medium b l a n k s , and the measurement of at l e a s t 0.5 yg of s t e r o i d .  33 By a s l i g h t m o d i f i c a t i o n of t h i s method - washing the methylene c h l o r i d e extract w i t h e i t h e r 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 q u a n t i t a t i o n of s t e r o i d s 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 i n c u b a t i o n medium from c e l l s treated w i t h ACTH could be seen, but the increase was u s u a l l y too small to permit p r e c i s e q u a n t i tation. A s u b s t a n t i a l l y more s e n s i t i v e a n a l y s i s of s t 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 M a t e r i a l s and Methods was used i n t h i s study. merits of t h i s assay are:  The  the fluorescence of 20a hydroxylated d e r i v a -  t i v e s of progesterone i n e t h a n o l i c s u l p h u r i c a c i d i s intense enough to permit the p r e c i s e q u a n t i t a t i o n of 15 ng of 20a dihydroprogesterone, a 200-fold increase over the lowest l e v e l of the UV assay; the f l u o r e s 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 s t a b l e 45-90 minutes a f t e r e x t r a c t i o n of the s t e r o i d s i n t o  ethanolic sulphuric acid.  Y - l c e l l s produce 20a dihydroprogesterone and 11B0H, 20a dihydroprogesterone.  Both s t e r o i d s f l u o r e s c e under the c o n d i t i o n s described,  and i t has been determined that the fluorescence of 11B0H, 20a dihydroprogesterone i s approximately 1.6 times greater than the i n t e n s i t y of 20a dihydroprogesterone.  In order to q u a n t i t a t e the s t e r o i d output  p r e c i s e l y , the exact p r o p o r t i o n of the two s t e r o i d s would have to be known i n each i n c u b a t i o n . Y - l - L c e l l s and Tumor #2 c e l l s produce minute q u a n t i t i e s of 1130H, 20a dihydroprogesterone (see Results s e c t i o n I 3a and c) i n r e l a t i o n to 20a dihydroprogesterone.  To s i m p l i f y matters,  34 the s t e r o i d s have been quantitated as nanograms of 20a dihydroprogesterone . b) Y - l C e l l s (ATCC CCL 79) S t e r o i d output of Y - l c e l l s under various conditions i s tabul a t e d i n Table 2.  Steroidogenesis was determined on c u l t u r e s j u s t p r i o r  to reaching confluency, when each P e t r i dish of c e l l s contained approximately 1 mg of c e l l p r o t e i n .  C e l l s were incubated f o r 3 hours i n 3 ml  of e i t h e r c o n t r o l medium, medium containing 10 mU ACTH/ml, or medium containing 1 mM d i b u t y r y l c y c l i c AMP.  Y - l c e l l s produced s t e r o i d s equiva-  l e n t to 2 yg of 20a dihydroprogesterone i n c o n t r o l medium i n 3 hours. In the presence of e i t h e r ACTH o r d i b u t y r y l c y c l i c AMP, the s t e r o i d 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 C e l l s In comparison w i t h 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 same conditions (Table 2).  In a d d i t i o n ,  the amount of s t e r o i d output a l s o diminished a f t e r routine s e r i a l subcultures (Table 3).  Y - l - L c e l l s were used f o r s e v e r a l 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. d) Tumor #2 C e l l s Tumor #2 c e l l s i n i t i a l l y produced s t e r o i d s 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 D i b u t y r y l C y c l i c AMP Y - l C e l l s (ATCC CCL 79) and Y-l-L C e l l s  (ng s t e r o i d / P e t r i dish/3 h o u r s ) Y - l (ATCC CCL 79)  3  Y-l-L  Basal  2050  160  10 mU ACTH/ml  3170  300  1 mM dbcAMP  3020  225  Each point represents the average s t e r o i d 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% w i t h i n each group.  36  TABLE 3.  Steroidogenic Responses to ACTH and D i b u t y r y l C y c l i c AMP by Y - l - L C e l l s and Tumor //2 C e l l s a f t e r S e r i a l Subculture  Y-l-L Passage Number  Basal  ACTH  Tumor #2 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  300  225  390  880  640  2  160  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  50  120  100  12  b  -  —  Each point represents the average s t e r o i d 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 l e s s than 10% w i t h i n each group. ng s t e r o i d / P e t r i dish/12  hours.  37 various conditions a l s o diminished a f t e r s e r i a l subculture.  This pheno-  menon i s quite common and has been reported by s e v e r a l l a b o r a t o r i e s which work w i t h the Y - l c e l l l i n e .  Between the t h i r d and s i x t h c u l t u r e  passages, the steroidogenic a c t i v i t i e s of Tumor #2 c e l l s were q u i t e stable.  A l l subsequent experiments were performed on c u l t u r e s between  these passages.  Cultures a f t e r the s i x t h passage were terminated, and  c u l t u r e samples frozen a f t e r the second passage were r e - e s t a b l i s h e d f o r experiments. 3. I d e n t i f i c a t i o n of the endogenous s t e r o i d s produced during a 12-hour incubation a) Y - l (ATCC CCL 79) Thin l a y e r chromatography of the endogenous s t e r o i d production of Y - l c e l l s i n e i t h e r c o n t r o l 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 d i f f e r e n c e s i n the s t e r o i d s produced i n e i t h e r c o n t r o l medium or medium w i t h ACTH, the UV-absorbing spots of the l a t t e r i n c u bation seemed more intense.  The 2 major UV-absorbing spots had the  m o b i l i t y i d e n t i c a l to that of s t e r o i d standards 20a and llgOH, 20a dihydroprogesterone.  The 3 minor UV-absorbing spots  corresponded to those of progesterone, 20a dihydroprogesterone  dihydroprogesterone  1130H progesterone  and 11 keto,  (Fig. 9).  b) Y - l - L C e l l s The a n a l y s i s of the endogenous s t e r o i d production of Y - l - L c e l l s (3rd passage) revealed that the s t e r o i d s elaborated were i d e n t i c a l  a  0  Y - l (Kowal and F i e d l e r , 1968)  Y - l (ATCC CCL 79) OQ  Y-l-L (3P) 3 n> M  0  Y - l - L (10P)  9  Tumor //2 (5P)  t  &• O  oo  O (t) Hi 0  o o c n> co M  H to u c  w rt n> n o  oo "it) t-t M O to (X  no  *C-Pregnenolone Metabolism Y-l-L (8P), Tumor #2 (5P) ll  PC O O rt C CO H CO pi  o  A  i H  A  O  1/1  A  1 N3  O  1  N>  O  O  3  i  8-3  o t—'  O  U) O  to  -O  1  o  3^9  1  o  Hi 0  O  O I  S- ^ a* n  fu (D rt 00 H- 0 O fD 0  o  S t e r o i d Standards  @ • o  ®  0  O O  o  0  a>  t, CO  rt  O 0 * rt CO  f? rt  T3 0*  s-  <  O CO CO  fD CO O  o iS n> o H PJ  v: H cr rt fa ro  o  CO  m <o •  8e  H'  0 00  I  & en O  3H-  0 00  CO 13 O rt CO  O  w § (D 0  rt O  N 00 CD 1-1 0 CU ••  0*  >  H-  o o fD rt O 0  fD  C/i  CO rt  ft> ~ 3 ho c O CO fl>  O PJ  CO  fD  TO ro 0  aC O ••  o o H  rt HO O CD rt (D H  O 0  ro  H' "CD  o a  »#  M M 5*! (0  rt  o  hO O  «*  aH"  P  P  0*  &  H O  N> O  a 0*  ^P.  i-l •a H o o >nd 00 fD o CO 00  rt fD i-l  ro CO rt  o n> 0 n (t> one  o i-l H- Cu D- ^<  <  n cr • n  ho  o P a 0*  PL  o T> H  ro  00 0  n> 0  o M o 0 fD  M  ho O  o a  a  00 0  0"  0  TO  P  H-  H O  ><!  fD CO rt ft>  o •tf n  O  fD CO rt fD i-l  00  H 0  fl>  a. i-i  o  00  o 0 fD  i-l fD  fD  o 1— o 0 1  fD  •d  I-l  O  00  fD CO rt fD i-t O 0  fD  39 to those of the Y - l c e l l s (ATCC CCL 79); however, a q u a n t i t a t i v e d i f ference was noted.  The UV-absorbing spot corresponding to that of proges-  terone was much more i n t e n s e , w h i l e the other 4 spots were s m a l l e r and less d i s t i n c t . Y-l-L  Corresponding to the low s t e r o i d output by 10th passage  c e l l s , only 3 UV-absorbing spots were v i s i b l e on the developed  t h i n - l a y e r chromatogram.  The prominent spot near the solvent f r o n t  corresponded to that of progesterone; f o l l o w i n g 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 ( F i g . 9 ) . c) Tumor #2 C e l l s The endogenous s t e r o i d products of Tumor #2 c e l l s during a 12-hour i n c u b a t i o n p e r i o d i n n u t r i e n t 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 ( F i g . 9 ) . 4. * ^-pregnenolone  metabolism by Y - l - L C e l l s and Tumor #2 C e l l s  a) General The metabolism of  llf  C-pregnenolone during 12 hours of i n c u -  b a t i o n i n n u t r i e n t 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 ^-pregnenolone was metabolized: 1  10-20% i n t o 20a dihydro-  pregnenolone, 30-40% i n t o progesterone, 10-20% i n t o 20a dihydroprogesterone, and trace amounts i n t o 1130H progesterone, 1130H, 20a dihydroprogesterone and 11 keto, 20a dihydroprogesterone ( F i g . 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 C e l l s Since the assay of s t e r o i d output was q u a n t i t a t e d as equival e n t nanograms of 20a dihydroprogesterone, an attempt was made to p o s 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 i n t e n t was to a c e t y l a t e the 20a dihydroprogesterone produced endogenously by Y - l - L c e l l s , to a c e t y l a t e t h i s product wi th H - a c e t i c 3  anhydride, and to r e c r y s t a l l i z e the r e s u l t a n t 20a dihydroprogesterone3  H-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 i s o l a t e d . The r e s u l t of the a c e t y l a t i o n experiment i s presented i n Table 4.  ^ C ^ O a dihydroprogesterone was added i n the experiment to monitor  the recovery.  The H: C r a t i o of the 3rd and 4th c r y s t a l s of each 3  ll+  Standard 1, Standard 2 and sample were constant (within 5% of each o t h e r ) . However, theH: C r a t i o of the sample was not greater than that of 3  llf  the standards. Two explanations can be given:  one i s that during the procedure,  an e r r o r was made such that e i t h e r more than 0.5 yg of standard 20a dihydroprogesterone was used, or that there was a l o s s of sample during the process.  Another explanation i s that the C-20a dihydroproges14  terone- H-acetate standard used contained so much s i n g l y - l a b e l l e d 20a 3  dihydroprogesterone- H-acetate that t h i s obscured the amounts present 3  i n the sample and the standards.  I n view of the fact that  progesterone from the chromatogram of ^-pregnenolone 1  1I+  C-20a dihydro-  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 a c e t y l a t e d product was equally divided and added to each of the sample and the two standards to monitor recovery, t h i s explanation seems more f e a s i b l e . Although the goal of t h i s e x e r c i s e was not achieved, i t could be concluded unequivocally that the C-pregnenolone metabolite produced 1Lf  by Y - l - L c e l l s was  ll+  C-20a dihydroprogesterone.  This compound could  be a c e t y l a t e d , and the a c e t y l a t e d product c o - c r y s t a l l i z e d w i t h standard 20a dihydroprogesterone-acetate. Since Y - l - L c e l l s could metabolize minute q u a n t i t i e s of added pregnenolone i n t o 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 ' C-20a dihydroprogesterone produced by Tumor 7/2 C e l l s 1  ll  t  *C-20a dihydroprogesterone metabolized from  ll+  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 w i t h added authentic 20a dihydroprogesterone.  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 . E l e c t r o n microscopy of Y - l - L C e l l s a) U l t r a s t r u c t u r e of Y - l - L C e l l s i n s i t u A t a n g e n t i a l s e c t i o n through Y - l - L c e l l s i n c u l t u r e i s shown i n F i g . 10.  The f a c t that these c e l l s d i d not resemble the c h a r a c t e r i s -  t i c rodent a d r e n o c o r t i c a l c e l l s i n v i v o 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. A c e t y l a t i o n of 20a Dihydroprogesterone Produced Endogenously by Y - l - L C e l l s w i t h H - A c e t i c Anhydride, and R e c r y s t a l l i z a t i o n of 20a DihydroprogesteroneH-Acetate to Constant H: ^C Ratio i n the Presence of C-dihydroprogesterbne- H-Acetate 3  3  3  14  3  3  Standard 1  Standard 2  Sample  1  H  a  1 4  C  3  3  H: C 14  XL 3  10,733  60  178.88  XLi+  9,827  53  185.42  XL 3  12,636  42  300.86  XLu  11,173  36  310.36  XL 3  14,185  61  232.54  XL4  11,132  49  227.18  Mean + 5%  182.15 + 9.11  305.11 + 15.28  229.86 + 11.49  Expressed as dpm/min. k The standards used were two 0.5 yg a l i q u o t s 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 t o 1 yg of 20a dihydroprogesterone.  43  TABLE 5.  Radiochemical I d e n t i f i c a t i o n of C-20a Dihydroprogesterone Produced by Tumor #2 C e l l s 14  3  Condition  C r y s t a l s (dpm/mg)  Basal  ACTH  Mother Liquor (dpm/mg)  XL  X  3,882  ML!  19,696  XL  2  5,331  ML  2  6,141  XL  3  5,309  ML  3  5,488  5,258  MLi+  5,248  XL!  4,965  MLi  10,222  XL  2  4,303  ML  2  5,005  XL  3  4,270  ML  3  4,516  XL  4  4,245  ML4  4,273  S i x 6 cm P e t r i dishes of Tumor #2 C e l l s were incubated i n e i t h e r c o n t r o l medium or medium w i t h ACTH (10 mU/ml) c o n t a i n i n g Cpregnenolone (200,000 dpm/culture). A f t e r 12 hours, the medium was removed and medium from 3 P e t r i dishes under each c o n d i t i o n were pooled. The i s o l a t i o n of 20a dihydroprogesterone has been described i n M a t e r i a l s and Methods. C-20a dihydroprogesterone was r e c r y s t a l l i z e d from ethanol water w i t h 10 mg of cold c a r r i e r . a  11+  11+  k Expressed as dpm/mg of authentic s t e r o i d .  44 P l a t e IV U l t r a s t r u c t u r e of the Y - l - L C e l l s in_ s i t u . citrate staining.  Uranyl acetate and lead  F i g . 10. Tangential s e c t i o n through Y - l - L c e l l s i n c o n t r o l medium, x28,000. F i g . 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 f l a t t e n e d morphology and were i d e n t i c a l to c e l l s i n c o n t r o l medium. Section cut perpendicular'to the substratum, x8,000. F i g . 12. Y-l-L c e l l i n c o n t r o l medium. The s i d e facing the medium i s on the r i g h t . Microtubules were found to be s c a t t e r e d 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 w i t h s h e l f - l i k e c r i s t a e i s a l s o shown, xl9,000. F i g . 13. G o l g i apparatus present i n a Y - l - L c e l l i n c o n t r o l medium, x30,000.  Abbreviations: b.v. budding v i r u s ; g. g o l g i ; m. mitochondria; mf. microf i l a m e n t s ; mt. microtubules; m.w. membranous whorls; p.v. p i n o c y t o t i c v e s i c l e s ; v.p. v i r u s 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 w i t h v e s i c u l a r 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 f a 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 w i t h s h e l f - l i k e c r i s t a e (Figs. 12 and 13). In some of the c e l l s , an abundance of mitochondria was present ( F i g . 18). Glycogen p a r t i c l e s were absent and l i p i d droplets were r a r e l y observed in Y-l-L c e l l s .  Well-developed G o l g i bodies w i t h 3-4 p a r a l l e l arrays  of c i s t e r n a e (Figs. 10 and 13), lysosomes w i t h homogeneous dense content, membranous whorls, and microtubules were often d i s c e r n i b l e .  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 ( F i g . 10).  Pinocytotic  v e s i c l e s were a l s o present near the plasma membrane, and p i n o c y t o t i c invaginations were observed on the c e l l surface ( F i g . 11). T y p i c a l Ctype p a r t i c l e s as free e x t r a c e l l u l a r v i r u s 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 j u n c t i o n s w i t h 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 d i r e c tions around the c e l l s .  Large membrane whorls and v i r u s p a r t i c l e s were  present i n these spaces ( F i g . 10). The u l t r a s t r u c t u r e of approximately 50 Y - l - L c e l l s f o l l o w i n g incubation i n medium containing ACTH f o r 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 p i n o c y t o t i c v e s i c l e s and the penetration of mitochondria  47 Plate V U l t r a s t r u c t u r e of Y - l - L C e l l s i n s i t u : Response to ACTH. and lead c i t r a t e s t a i n i n g .  Uranyl  acetate  Figs. 14 and 15. Y-l-L c e l l s f o l l o w i n g incubation i n medium containing 10 mU ACTH/ml for 1 hour. C e l l s were rounded i n morphology. Note the swollen G o l g i bodies, x9,500. Fig. 16. D i l a t e d Golgi apparatus i n a Y - l - L c e l l f o l l o w i n g incubation i n medium containing 10 mU ACTH/ml f o r 3 hours, x20,000. Fig. 17. Y-l-L c e l l s f o l l o w i n g incubation i n medium containing 10 mU ACTH/ml f o r 3 hours. Two of the c h 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 i n t o l i p i d droplets to form myelin-structures were a l s o observed i n Y-l-L c e l l s , xl8,000. F i g . 18. Y-l-L c e l l s f o l l o w i n g incubation i n medium containing 10 mU ACTH/ml f o r 5 minutes. Abundance of mitochondria w i t h i n 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. p i n o c y t o t i c v e s i c l e s ; z.a. zonular adherens;  48  49 i n t o l i p i d droplets to form m y e l i n - l i k e s t r u c t u r e s were observed i n Y-l-L c e l l s f o l l o w i n g 30 minutes, 1 hour, and 3 hours of incubation w i t h ACTH ( F i g . 17). In the same c u l t u r e s , the d i l a t i o n of G o l g i c i s ternae, and the increase i n s i z e of i n t e r c e l l u l a r spaces were c o n s i s t e n t l y seen (Figs. 14-16).  Swollen endoplasmic reticulum and coated  v e s i c l e s 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  w i t h i n minutes upon the a d d i t i o n of ACTH, the u l t r a s t r u c t u r e of Y - l - L c e l l s f o l l o w i n g 5 minutes' incubation w i t h ACTH was i d e n t i c a l to that of c e l l s i n c o n t r o l medium.  Y - l - L c e l l s , f o l l o w i n g 24 hours' incubation  i n medium containing ACTH, were also comparable to those i n c o n t r o l medium. E l e c t r o n microscopy of monolayers cut perpendicular to the substratum revealed that Y - l - L c e l l s were exceedingly f l a t and t h i n , r e sembling squamous c e l l s i n c o n t r o l medium and i n medium containing ACTH f o r 24 hours ( F i g . 11). Monolayer Y - l - L c e l l s i n medium containing ACTH f o r 5 minutes, 30 minutes, 1 hour and 3 hours assumed i r r e g u l a r l y round or rhomboidal o u t l i n e s w i t h large i n t e r c e l l u l a r spaces (Figs. 14 and 15). In sections of m u l t i l a y e r e d 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 t h i n 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 ( F i g . 14). From t h i s study, no c o r r e l a t i o n between the u l t r a s t r u c t u r e 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 s e c r e t i o n 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 d i s t i n g u i s h e d  and remains enigmatic.  c) U l t r a s t r u c t u r e of Y-l-L C e l l s f o l l o w i n g incubations i n t r y p s i n and versene Toluidine blue-stained t h i c k sections 10 minutes of incubation  of Y-l-L c e l l s  following  i n e i t h e r t r y p s i n , or t r y p s i n and versene, are  shown i n F i g s . 19a and 20a.  The o u t l i n e s of c e l l s appeared  "fuzzy."  At the l e v e l of r e s o l u t i o n of the e l e c t r o n microscope, i r r e g u l a r convolutions at the c e l l surface exposed to the medium were observed.  The  surface folds of c e l l s exposed to t r y p s i n only were comparatively longer and more slender ( F i g . 19b) sene ( F i g . 20b).  than those exposed to both t r y p s i n and  ver-  The surface f o l d s of these c e l l s were club-shaped,  resembling pseudopodia.  The  r e s u l t s of t h i s 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 t i s s u e c u l t u r e , t r y p s i n and/or versene  i s r o u t i n e l y 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 r e f e r r e d to as "rounding up."  I t i s evident  that the u l t r a s t r u c t u r e 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 t r y p s i n  or t r y p s i n and versene:  the abundance of p i n o c y t o t i c v e s i c l e s 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 a c t i v e exchange of materials w i t h the environment; any evidence of such a c t i v i t y was treated  cells.  l a c k i n g i n the t r y p s i n and versene  51  P l a t e VI Y - l - L C e l l s Following Treatment with Trypsin and Trypsin together w i t h Versene. Figs. 19a and 20a. Epon-embedded s e c t i o n s , t o l u i d i n e b l u e - s t a i n i n g , xl,100. Y-l-L c e l l s f o l l o w i n g 10 minutes incubation i n (a) 0.12% t r y p s i n , (b) 0.12% t r y p s i n and 0.04% versene. F i g s . 19b and 20b. Uranyl acetate and lead c i t r a t e s t a i n i n g . U l t r a s t r u c t u r e of the surface of Y - l - L c e l l s f o l l o w i n g 10 minutes of incubation i n (a) 0.12% t r y p s i n , xl8,000, (b) 0.12% t r y p s i n and 0.04% versene, x20,000. The surface folds of c e l l s exposed to t r y p s i n only were comparatively longer and more slender ( F i g . 19b) than those exposed to both t r y p s i n and versene ( F i g . 20b). The surface folds of these c e l l s were club-shaped, resembling pseudopodia.  52  53 III.  Enucleation Procedure Nuclear p r o t r u s i o n occurred when cytochalasin B was  Y - l c e l l s (ATCC CCL  79) i n monolayer c u l t u r e s .  added to  However, only one  of s e v e r a l thousand c e l l s became spontaneously enucleated  out  as a r e s u l t  of c y t o c h a l a s i n B treatment alone. When the enucleation procedure described by Prescott and h i s colleagues was  adopted (Prescott et a l . , 1972), populations  10-20% of the Y - l c e l l s were anucleate were produced.  i n which  Basically this  procedure involved growing Y - l c e l l s on round glass c o v e r s l i p s and  cen-  t r i f u g i n g the c o v e r s l i p s of c e l l s w i t h the c e l l s f a c i n g downwards i n centrifuge tubes f i l l e d w i t h 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 a l t e r a t i o n s e i t h e r s i n g l y , or i n combinations of:  timing of  a d d i t i o n of c y t o c h a l a s i n B, concentration of c y t o c h a l a s i n B, c e n t r i f u g a l force and c e n t r i f u g a t i o n time.  At best, up to 40% enucleation was  A major drawback of t h i s technique was  achieved.  that c e l l l o s s due to detachment  from the c o v e r s l i p during c e n t r i f u g a t i o n was  great.  forces (> 5,000 g ) , the amount of c e l l l o s s was  At high c e n t r i f u g a l  severe, while at lower  c e n t r i f u g a l f o r c e s , the proportion of c e l l s which became enucleated low.  was  Following c e n t r i f u g a t i o n at 8,000 g f o r 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 c o v e r s l i p s .  An  a n a l y s i s of the c e l l s detached from the c o v e r s l i p s and p e l l e t e d at the bottom of the centrifuge tube was made, and a higher percentage of enucleated 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 substratum, e s p e c i a l l y to etched p l a s t i c surfaces  (surfaces treated w i t h  54 s u l p h u r i c a c i d or permanganate).  When Y - l c e l l s were enucleated on  p l a s t i c c o v e r s l i p s which had been treated with s u l p h u r i c a c i d f o r 15 minutes, c e l l l o s s during c e n t r i f u g a t i o n was decreased.  Optimal enu-  c l e a t i o n of Y - l c e l l s was 50% when Y - l c e l l s grown on s u l p h u r i c a c i d treated p l a s t i c c o v e r s l i p s were centrifuged i n medium containing 10 yg cytochalasin B/ml at 7,710 g f o r 30 minutes i n a S o r v a l l RC 2B c e n t r i fuge using the ss34 r o t o r . With t h i s 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 s t e r o i d o -  genic response of enucleated Y - l c e l l s to the t r o p h i c hormone would be impossible  to assess as i t could not be determined c o n c l u s i v e l y whether  the s t e r o i d s 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 r e a d i l y enucleated.  The success i n enucleation  of the Y - l - L c e l l s was a t t r i b u t e d  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 c e n t r i f u g a l forces used during the enucleation procedure was much l e s s i n comparison with the Y - l c e l l s .  By a s l i g h t  increase i n c e n t r i f u g a t i o n time, populations i n which 95-98% of the Y-l-L c e l l s were anucleate were r o u t i n e l y produced by c e n t r i f u g i n g the monolayer of c e l l s grown on glass c o v e r s l i p s i n medium containing 10 yg cytochalasin B/ml at 7,710 g f o r 40 minutes i n a S o r v a l l RC 2B centrifuge using the ss34 r o t o r . By a s l i g h t m o d i f i c a t i o n of the method, c e l l l o s s was f u r t h e r minimized.  By pre-incubation  of Y - l - L c e l l s i n serum-free medium, and  by subsequent c e n t r i f u g a t i o n 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 coverslips.  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 w i t h Y - l - L  c e l l s , t h i s s u b l i n e was used f o r the study of the response of enucleated a d r e n o c o r t i c a l c e l l s to ACTH and d i b u t y r y l c y c l i c AMP. R e l a t i v e l y large numbers of enucleate c e l l s were required f o r biochemical analyses; therefore the 18 mm diameter round c o v e r s l i p method proved to be quite tedious, as only small amounts (200-300 yg) of anucleate c e l l s could be produced during each 40-minute c e n t r i f u g a t i o n run. During the course of t h i s study, the P r e s c o t t method was f u r t h e r 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 M a t e r i a l s and Methods).  95-98% enucleation was achieved  when Y - l - L c e l l s grown on s u l p h u r i c a c i d - t r e a t e d P e t r i dishes were p r e incubated i n serum-free medium, and centrifuged i n serum-free medium containing 10 yg c y t o c h a l a s i n B/ml at 10,800 g f o r 35 minutes i n a RC 2B centrifuge using the GSA r o t o r .  As a r e s u l t , a t e n - f o l d 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 r e a d i l y enucleated w i t h i d e n t i c a l e f f i c i e n c y under the same c o n d i t i o n s . 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 w i t h i n 15 minutes f o l l o w i n g the removal of cytoc h a l a s i n B, two changes of Hanks' balanced s a l t s o l u t i o n , and incubation i n fresh n u t r i e n t medium.  By using phase o p t i c s , any c e l l r e t a i n i n g i t s  nucleus could be e a s i l y i d e n t i f i e d .  Routinely, enucleated c u l t u r e s were  56 randomly s e l e c t e d f o r 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 c o n s i s t e n t l y found to be l e s s than 5%.  III.  E f f e c t s of DMSO, Cytochalasin B and Enucleation Procedure Treatments (without a c t u a l enucleation) Upon Y-l-L and Tumor #2 C e l l s 1. E f f e c t upon steroidogenesis The p r o t o c o l which was used to determine whether or not, i n the  absence of a c t u a l enucleation, c y t o c h a l a s i n B or the c e n t r i f u g a t i o n used during the enucleation procedure had any e f f e c t 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 t r a t e d i n F i g . 21. Results of three experiments on the e f f e c t s of DMSO, c y t o c h a l a s i n 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 c y t o c h a l a s i n 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 c e l l s were u s u a l l y 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 f o l l o w i n g the removal of c y t o c h a l a s i n B, o r f o l l o w i n g c e n t r i f u g a t i o n , were comparable to those of the c o n t r o l c e l l s (Tables 6-8). Thus, the l a c k of e f f e c t s upon steroidogenesis f o l l o w i n g the removal of treatments used during the enucleation procedure was v e r i f i e d .  I n subsequent experiments, i n which the metabolic  acti-  v i t i e s o f 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 F i g . 21.  Experimental Design: Steroidogenic Responses to ACTH and D i b u t y r y l Cyclic AMP by Enucleation Procedure Treatment C e l l s and Enucleated C e l l s . 0  10 _L  11  12 Hours  Control DMSO ETOH CB (DMSO) CB (ETOH) EPT (CB) EPT (cent)  ent  0-3  I  S  .  F  I  F  I s . r i e t\  3-6 6-9  I S . F J  9-12  I  S. F  rl  E  4  E  Legends:  Incubation i n c o n t r o l medium = Basal medium containing 10 mU ACTH/ml = dbcAMP medium containing 1 mM d i b u t y r y l c y c l i c AMP = dbcAMP At the end of t h i s incubation p e r i o d , the media were removed and the amounts of s t e r o i d s produced were assayed. DMSO  1% DMSO was a l s o added to the incubation medium.  ETOH  1% ETOH was also added to the incubation medium.  CB (DMSO)  10 yg c y t o c h a l a s i n B ( d i s s o l v e d i n DMSO)/ ml was also added to the incubation medium.  CB (ETOH)  10 yg c y t o c h a l a s i n B ( d i s s o l v e d i n ETOH)/ml was also added to the incubation medium.  EPT (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 f o l l o w i n g incubation i n media containing CB.  EPT (cent)  Enucleation Procedure Treatment: the steroidogenic a c t i v i t i e s of the c e l l s were determined f o l l o w i n g c e n t r i f u g a t i o n i n serum-free medium.  S.F.  1 hour preincubation i n serum-free medium.  E  Enucleation - c y t o c h a l a s i n B treatment + c e n t r i f u g a t i o n . 2 washes i n Hanks' balanced s a l t s o l u t i o n Incubation i n f r e s h medium.  TABLE 6. E f f e c t s of DMSO, Cytochalasin B and Enucleation Procedure Treatment on the Steroidogenic Responses of Y - l - L C e l l s to ACTH and D i b u t y r y l C y c l i c AMP a  ng s t e r o i d / P e t r i dish/3 hour Basal  ACTH  dbcAMP  Control  80 + 3  190 + 15  DMSO  65 + 3  115 +  Cytochalasin B  80 + 0  145 + 0  125 + 16  100 + 2  255 + 6  205 + 9  EPT (CB)  3  155 +  8  90+5  TABLE 7. E f f e c t s of DMSO, Cytochalasin B and Enucleation Treatments on the Steroidogenic Responses of Tumor #2 C e l l s to ACTH and D i b u t y r y l C y c l i c AMP a  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 F i g . 21 f o r incubation p r o t o c o l . k Each point represents the average s t e r o i d output + S.E. o f 3 determinations. Each p o i n t represents the average s t e r o i d output + S.E. of 5 determinations.  59  TABLE 8. E f f e c t s of DMSO, Ethanol, Cytochalasin B Dissolved i n DMSO, Cytochalasin B Dissolved i n Ethanol, and Enucleation Procedure Treatments on the Steroidogenic Responses of Tumor #2 C e l l s t o ACTH and D i b u t y r y l C y c l i c AMP a  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  194 + 10  288 +  6  245 +  4  (cent)  See F i g . 21 f o r incubation p r o t o c o l . 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 d i f f e r e n c e was that the c e n t r i -  fugation step was omitted i n the former case. c e l l s were designated  These nucleated c o n t r o l  as Enucleation Procedure Treatment C o n t r o l (EPT  Control). To e l u c i d a t e whether the e f f e c t of 10 pg c y t o c h a l a s i n B/ml upon steroidogenesis was due s o l e l y to c y t o c h a l a s i n B, or to both the fungal metabolite and i t s solvent DMSO, ethanol was used as a s u b s t i t u t e solvent. A d d i t i o n of 10 P i ethanol/ml had no e f f e c t upon the steroidogenesis of Tumor #2 c e l l s .  At the concentration of 10 pg c y t o c h a l a s i n B/ml i n  e i t h e r 1% of DMSO o r 1% of ethanol/ml of medium, the steroidogenic responses by Tumor #2 c e l l s to ACTH and d i b u t y r y l c y c l i c AMP were depressed (Table 8 ) . From these r e s u l t s , i t was determined that although c y t o c h a l a s i n B and DMSO both had e f f e c t s upon steroidogenesis, i t appeared that these e f f e c t s were not a d d i t i v e . 2. E f f e c t upon morphology Since the three-dimensional  c o n f i g u r a t i o n of the a d r e n o c o r t i c a l  tumor c e l l s was the major change i n morphology i n response to ACTH and d i b u t y r y l c y c l i c AMP ( F i g s . 5a-e), the e f f e c t s of DMSO and c y t o c h a l a s i n B upon the morphology of Tumor #2 c e l l s were i n v e s t i g a t e d by scanning e l e c t r o n microscopy.  Tumor #2 c e l l s i n c o n t r o l medium, 3 hours i n medium  containing ACTH, and 3 hours i n medium containing d i b u t y r y l c y c l i c AMP are shown i n F i g s . 22-24. The p r o j e c t i o n of knobby protuberances ( z e i o s i s ) was v i s i b l e along the c e l l membranes of Tumor #2 c e l l s w i t h i n minutes of a d d i t i o n of 10 yg of c y t o c h a l a s i n B/ml.  The "blebbing" movements were followed  61  Plate VII E f f e c t s of ACTH, D i b u t y r y l C y c l i c AMP, DMSO, and Cytochalasin B upon the Morphology of Tumor #2 C e l l s . Glutaraldehyde f i x a t i o n , scanning e l e c t r o n microscopy.* F i g . 22. Tumor #2 c e l l s i n c o n t r o l medium, xl,200. F i g . 23. Tumor #2 c e l l s f o l l o w i n g 3 hours incubation i n medium containing 10 mU ACTH/ml, xl,200. F i g . 24. Tumor #2 c e l l s f o l l o w i n g 3 hours incubation i n medium containing 1 mM d i b u t y r y l c y c l i c AMP, xl,200. F i g . 25. Tumor #2 c e l l s f o l l o w i n g 3 hours incubation i n medium containing 10 yg cytochalasin B/ml, xl,200. F i g s . 26a and b. Tumor #2 c e l l s f o l l o w i n g 3 hours incubation i n medium containing 1% DMSO. (a) xl,200. (b) transmission e l e c t r o n 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 f o r F i g . 26b. Abbreviations:  e.r. endoplasmic reticulum.  62  63 by the r e t r a c t i o n of the c e l l , which was apparent 15 minutes f o l l o w i n g 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 i n t e r v e n i n g c e l l membrane and cytoplasm p u l l e d towards the centre of the c e l l .  This r e s u l t e d i n the r e t r a c t e d 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 e i t h e r ACTH or d i b u t y r y l c y c l i c AMP t o gether w i t h 10 yg of c y t o c h a l a s i n B/ml were s i m i l a r i n morphology t o those c e l l s incubated s o l e l y with c y t o c h a l a s i n B. C e l l s incubated i n the presence of 1% DMSO were extremely f l a t tened ( F i g . 26a) i n comparison w i t h c e l l s i n c o n t r o l medium ( F i g . 22). Transmission e l e c t r o n 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 d i l a t e d ( F i g . 26b). C e l l s incubated i n the presence of e i t h e r ACTH o r d i b u t y r y l c y c l i c AMP together with 1% DMSO (not shown) were s i m i l a r t o the c e l l s which were incubated i n e i t h e r ACTH or d i b u t y r y l c y c l i c AMP only; that i s , the c e l l s were a l s o rounded.  The surface of the rounded c e l l s was q u i t e  smooth, and the rounded c e l l was attached t o the substratum by a few thin  processes. Both DMSO and c y t o c h a l a s i n B e f f e c t s upon morphology were r a p i d l y  reversible.  I n t h i s study, Tumor #2 c e l l s were incubated i n medium  containing e i t h e r DMSO or c y t o c h a l a s i n f o r 3 hours.  The normal morpho-  logy of the c e l l s was resumed w i t h i n 15 minutes f o l l o w i n g the removal of medium containing DMSO or c y t o c h a l a s i n , and replacement with f r e s h n u t r i e n t 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 C e l l s t o ACTH and D i b u t y r y l 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 f o r 24 hours a f t e r enucleation. At i n c r e a s i n g time i n t e r v a l s f o l l o w i n g enucleation, the number of c e l l s which l y s e d or r e t r a c t e d from the substratum and f l o a t e d 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 o p t i c s . Up t o 24 hours f o l l o w i n g enucleation, 90% of the enucleated c e l l s r e s i s t e d a l c i a n blue dye uptake.  Approximately 5% of the enu-  cleated c e l l s were s t a i n e d i n t e n s e l y and completely w i t h a l c i a n b l u e ; these c e l l s were, on the whole, much smaller than the unstained  cells.  Another 5% of the enucleated c e l l s were s t a i n e d p a r t i a l l y , i . e . only a small p o r t i o n of the c e l l membrane and cytoplasm were stained with a l c i a n blue.  At i n c r e a s i n g time i n t e r v a l s f o l l o w i n g e n u c l e a t i o n , the  proportion of s t a i n e d enucleated c e l l s increased.  At 60 hours a f t e r  e n u c l e a t i o n , 40% of the enucleated c e l l s were s t a i n e d w i t h a l c i a n b l u e . However, c e l l death was a c t u a l l y greater than that i n d i c a t e d by t h i s method, because, i n a d d i t i o n , the majority of the non-viable c e l l s had detached from the substratum. of the enucleated  At 60 hours a f t e r e n u c l e a t i o n , only 25%  c e l l s present i n i t i a l l y remained attached to the cover-  slips. Enucleated Y - l - L c e l l s at 48 hours and 60 hours f o l l o w i n g enuc l e a t i o n are shown i n F i g s . 27a and b.  Most of the enucleated  cells  65  Plate VIII V i a b i l i t y and P r o t e i n Synthetic A c t i v i t y of Enucleated Y - l - L C e l l s . Methanol F i x a t i o n , Giemsa s t a i n i n g . F i g s . 27a and b. Enucleated Y - l - L c e l l s at (a) 48 hours, (b) 60 hours a f t e r 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 a f t e r enucleation, x750. Fig. 28. Autoradiographs of enucleated Y - l - L c e l l s incubated i n medium containing 10 uCi H-leucine/ml f o r 3 hours at 0-3, 21-24, and 45-48 hours f o l l o w i n g enucleation. Arrows point to a s i n g l e nucleated c e l l i n each f i e l d , xl,200. 3  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. P r o t e i n s y n t h e t i c a c t i v i t y The amount of  H-leucine incorporated i n t o a c i d - i n s o l u b l e m a t e r i a l  was compared between enucleated and nucleated Y - l - L c e l l s at various times f o l l o w i n g enucleation.  Some of the r e s u l t s are shown i n F i g . 28.  According to g r a i n counts, the r a t e of p r o t e i n 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 f o l l o w i n g enucleation.  Enucleate c e l l s were s t i l l capable of i n c o r p o r a t i n g H - l e u c i n e 3  i n t o a c i d - i n s o l u b l e m a t e r i a l at 45 hours f o l l o w i n g enucleation.  However,  t h i s occurred at a g r e a t l y reduced rate i n comparison w i t h the nucleate cells.  These r e s u l t s i n d i c a t e d that enucleated c e l l s were m e t a b o l i c a l l y  active. 3. Morphologic responses to ACTH and d i b u t y r y l c y c l i c  AMP  F i g . 29 shows a monolayer of nucleated Tumor #2 c e l l s , while F i g . 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 f o l l o w i n g 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 F i g . 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 ( F i g . 29b).  Within minutes of ACTH a d d i t i o n ,  the enucleated c e l l r e t r a c t e d from the substratum and became attached to the P e t r i dish by a few prominent processes, while the r e s t of the  P l a t e 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 c u l t u r e . (a) i n c o n t r o l medium, (b) i n medium containing 10 mU ACTH/ml f o r 1 hour. F i g s . 30a and b. Tumor #2 c e l l s at one hour a f t e r enucleation i n (a) c o n t r o l medium, (b) i n medium containing 10 mU ACTH/ml. Arrows point at the few nucleated c e l l s remaining a f t e r the enucleation procedure. The morphol o g 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 c y t o p l a s t 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 d i b u t y r y l 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  cells.  The " c e l l - r o u n d i n g " was v i s i b l e a f t e r 1 hour of incubation i n medium containing the c y c l i c n u c l e o t i d e , and a t t a i n e d 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 d i b u t y r y l c y c l i c AMP up to 33 hours a f t e r enucleation.  Enucleated Y - l - L cytoplasms at 12, 24 and 36 hours a f t e r  enucleation are shown i n F i g s . 31a, 32a, and 33a. The morphology of s i m i l 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 F i g s . 31b, 32b, and 33c. Following 3 hours of incubation i n 1 mM d i b u t y r y l 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).  A f t e r 40 hours  f o l l o w i n g 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 t o ACTH or d i b u t y r y l c y c l i c AMP. 4. Steroidogenic responses to ACTH and d i b u t y r y l c y c l i c AMP a) I d e n t i f i c a t i o n of 20a ll  dihydroprogesterone  *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 s p e c i -  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 t e r o i d (Table 9 ) .  Plate X Morphologic Responses to ACTH and D i b u t y r y l 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 s t a i n i n g , xl,000. F i g s . 31a-c. Enucleated Y - l - L c e l l s i n c u l t u r e : 12 hours a f t e r enucleation, (a) i n c o n t r o l medium, (b) 3 hours i n medium containing 10 mU ACTH/ml p r i o r to f i x a t i o n , (c) 3 hours i n medium containing 1 mM d i b u t y r y l c y c l i c AMP p r i o r to f i x a t i o n . F i g s . 32a-c. Enucleated Y - l - L c e l l s i n c u l t u r e : 24 hours a f t e r enucleation, (a), (b) and (c) are the same as that f o r F i g . 31. F i g s . 33a-c. Enucleated Y - l - L c e l l s i n c u l t u r e : 36 hours a f t e r enucleation, (a), (b) and (c) are the same as that f o r F i g . 31.  73 TABLE 9 .  Condition  Radiochemical I d e n t i f i c a t i o n of C - 2 0 a Dihydroprogesterone Produced by Enucleated Tumor #2 C e l l s 14  C r y s t a l s (dpm/mg)  Mother Liquor (dpm/mg)*  b  Basal a  Basal b  ACTH a  2,993  ML  2  9,051  XL2  2,852  ML  2  3,091  XL 3  2,854  ML  3  3,111  XL4  2,854  MLi*  2,837  XL  X  2,366  MLi  9,863  XL  2  2,361  ML  2  2,585  XL  3  2,181  ML3  2,148  2,180  MI4  2,137  XL!  7,525  MLi  12,685  XL  2  4,958  ML  2  11,736  XL  3  4,919  ML3  5,464  4,716  MLtt  4,777  XLi  605  MLi  3,506  XL  607  ML  2  802  554  ML3  599  560  ML+  587  k  . XL ACTH b  3  XLi  XL  XL  2  3  XL it  4  3  Twenty-•four 6 cm P e t r i dishes of enucleated Tumor #2 c e l l s were incubated i n e i t h e r c o n t r o l medium o r medium w i t h ACTH (10 mU/ml) containing C-pregnenolone (approximately 150,000 dpm/culture) f o l l o w i n g enucleation. A f t e r 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 cond i t i o n were pooled. The i s o l a t i o n of C-20a dihydroprogesterone has been described i n M a t e r i a l s and Methods. C-20a dihydroprogesterone was r e c r y s t a l l i z e d from ethanolrwater w i t h 10 mg of cold c a r r i e r . lt+  llt  lt+  k Expressed as dpm/mg o f authentic s t e r o i d .  74 b) S t e r o i d output The p r o t o c o l that was employed to determine the amounts of s t e r o i d s produced by enucleated Y - l - L c e l l s i n e i t h e r c o n t r o l medium, medium containing ACTH, or medium containing d i b u t y r y l c y c l i c AMP at various time i n t e r v a l s a f t e r enucleation i s also i l l u s t r a t e d i n F i g . 21. Three general observations can be made from the r e s u l t s which are summarized i n Table 10: enucleated Y - l - L c e l l s were capable of producing s t e r o i d s ; at 0-3 hours f o l l o w i n g enucleation, enucleated Y - l - L c e l l s responded to ACTH and d i b u t y r y l 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 i n t e r v a l s f o l l o w i n g e n u c l e a t i o n , the amounts of s t e r o i d s produced under a l l three conditions  diminished.  Upon examining the r e s u l t s more c l o s e l y , i t i s apparent that the low l e v e l s of s t e r o i d s were assayed s p e c t r o f l u o r o m e t r i c a l l y 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 l a r g e r q u a n t i t i e s of c e l l s , the aforementioned study was repeated.  The r e s u l t s of one of these experiments are summarized  i n Table 11 and the p r o t o c o l employed t o study the s t e r o i d output of enucleated Tumor #2 c e l l s was analogous t o that o u t l i n e d p r e v i o u s l y (Fig. 21). Enucleated Tumor #2 c e l l s , s i m i l a r t o the enucleated Y - l - L c e l l s responded to ACTH and d i b u t y r y l c y c l i c AMP by increased steroidogenesis. At 0-3 hours a f t e r enucleation, the increase i n s t e r o i d output was approximately  equivalent to that of nucleated c e l l s .  At i n c r e a s i n g  75  TABLE 10.  Steroidogenic Responses to ACTH and D i b u t y r y l C y c l i c AMP by Enucleated Y - l - L C e l l s  Basal  ACTH  dbcAMP  (ng steroid/100 yg protein/3 h o u r ) Control'  5  EPT C o n t r o l  15  0-3 hours f o l l o w i n g e n u c l e a t i o n  0  3-6 hours f o l l o w i n g e n u c l e a t i o n  0  6-9 hours f o l l o w i n g e n u c l e a t i o n  0  9-12 hours f o l l o w i n g e n u c l e a t i o n  0  15 + 0. 5  30 + 3. o  d  26 + 2. 3  47 + 6. 5  22 + 2. 2  40 + 1. 3  20 + 1. 1  23 + 1. 6  17 + 0. 6  20 + 0. 7  15 + 0. 8  18 + 0  + 1.5  d  d  33 + 2.3  d  d  33 + 2.4  e  .22  3  24 + 0.8 e  3-hour incubations were c a r r i e d out i n t r i p l i c a t e . represents the average s t e r o i d output/100 yg p r o t e i n + S.E.  22 + 3.3 22 + 1.5  d  Each p o i n t  k The s t e r o i d o g e n i c a c t i v i t y of the nucleated c e l l s and the enucleation procedure t r e a t e d nucleated c e l l s were determined from i n c u b a t i o n medium pooled from 2 c o v e r s l i p s of c e l l s . S i x c o v e r s l i p s of nucleated c e l l s were incubated/group. Incubation medium of 4 c o v e r s l i p s of enucleated c e l l s was pooled f o r each determination. Twelve c o v e r s l i p s of enucleated c e l l s were incubated/group. S t a t i s t i c a l l y d i f f e r e n t from b a s a l 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. d  S i g n i f i c a n t at the 0.05  l e v e l of s i g n i f i c a n c e .  76 time i n t e r v a l s a f t e r enucleation, the increase i n s t e r o i d output i n response to the t r o p h i c hormone or t o the c y c l i c nucleotide  diminished.  However, i t i s apparent from the r e s u l t s presented i n Table 11 that increased steroidogenesis by enucleated Tumor #2 c e l l s i n response to d i b u t y r y l c y c l i c AMP p e r s i 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 s t e r o i d s i n c o n t r o l medium at 21-24 hours a f t e r 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 h o u r - s t e r o i d output of the enucleated c e l l s i n the presence of e i t h e r ACTH or d i b u t y r y l c y c l i c AMP was a l s o higher than that of enucleated c e l l s incubated i n c o n t r o l medium (Table 12).  I n comparison w i t h 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 i n c u b a t i o n c o n d i t i o n s . These r e s u l t s were consistent with the e a r l i e r r e s u l t s obtained w i t h 3 hour-incubations  of enucleated c e l l s at various time i n t e r v a l s f o l l o w i n g  enucleation (Tables 10 and 11).  That i s , the p r o g r e s s i v e l y diminished  steroidogenic a c t i v i t y of enucleated c e l l s at i n c r e a s i n g 3-hourly i n t e r v a l s a f t e r enucleation i s r e f l e c t e d i n the o v e r a l l decrease i n 12 h o u r - s t e r o i d output of enucleated c e l l s as compared w i t h the nucleated EPT c e l l s .  77  TABLE 11.  Hours after EPT or Enucleation  Steroidogenic Responses to ACTH and D i b u t y r y l C y c l i c AMP by Enucleated Tumor #2 C e l l s  Nucleated (EPT C o n t r o l ) Basal  ACTH  Enucleated*  3  dbcAMP  Basal  3  ACTH  dbcAMP  (ng steroid/mg protein/3 hour)° 0-3  265  + 14  425  + 20  d  385  + 16  d  225  +  6  270  +  4  3-6  230  +  6  425  +  2  d  345  +  8  d  220  + 10  245  +  6-9  235  +  3  425  +  2  d  365  +  3  d  210  +  9  9-12  230  +  6  420  + io ~  360  + io  d  210  +  12-15  245  +  8  415  + 3'  375  + 9  d  205  d  d  295  + 6  d  6  250  + 4  e  235  + 11  245  + 7  e  5  210  + 10  235  + 9  e  +  8  205  + 10  210  + 9  4  15-18  -  -  -  195  +  18-21 -  -  -  -  180  + 13  -  -  165  + 13  21-24  240  +  3  d  3  Incubation medium from one 6 cm P e t r i dish of Tumor #2 C e l l s was used f o r 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 f o r each determination. Ten P e t r i dishes of enucleated c e l l s were incubated/group. c  Each p o i n t represents the average s t e r o i d output + S.E. of 5 determinations. S t a t i s t i c a l l y d i f f e r e n t from b a s a l 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. d  S i g n i f i c a n t at the 0.05 l e v e l of s i g n i f i c a n c e .  78 TABLE 12. Steroidogenic Responses to ACTH and D i b u t y r y l C y c l i c AMP by Nucleated and Enucleated Y - l - L C e l l s and Tumor ill C e l l s : S t e r o i d Output i n 12 Hours  Y-l-L  Tumor #2  Nucleated Enucleated (ng steroid/100 yg p r o t e i n / 12 h o u r ) 3  e  Basal  125 +  10 mU ACTH/ml  370 + 2 9  1 mM dbcAMP  400 + 16  8 8  s  95 +  0  f  595 + 33  5  140 + 10  8  150 + 1 3  Nucleated Enucleated (ng s t e r o i d / 1 mg p r o t e i n / 12 h o u r )  s  725 +  560 + 42  5  990 + 14  8  g  645 + 42 740 + 5 1  h  Incubation medium of 2 c o v e r s l i p s of nucleated c e l l s was pooled for each determination. S i x c o v e r s l i p s of nucleated c e l l s were incubated/ group. 1J  Incubation of 4 c o v e r s l i p s of enucleated c e l l s was pooled f o r each determination. Twelve c o v e r s l i p s 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 f o r each determination. S i x P e t r i dishes of nucleated c e l l s were incubated/group. Incubation medium pooled from two 6 cm P e t r i dishes of enucleated c e l l s was used f o r each determination. Twelve P e t r i dishes of enucleated c e l l s were incubated/group. d  Each point represents the average s t e r o i d output + S.E. of 3 determinations. ^ Each point represents the average s t e r o i d output + S.E. of 6 determinations. S t a t i s t i c a l l y d i f f e r e n t from b a s a l 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. Significant  at the 0.05 l e v e l of s i g n i f i c a n c e .  DISCUSSION  I.  The A d r e n o c o r t i c a l C e l l s Used i n This Study The i n c r e a s i n g heterogeneity of the a d r e n o c o r t i c a l 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 f u n c t i o n f o l l o w i n g prolonged periods i n s e r i a l c u l t u r e have been reported by s e v e r a l l a b o r a t o r i e s that work w i t h 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 l i n e s of approach have been used to maintain the d i f f e r e n t i a t e d f u n c t i o n of these tumor c e l l s .  Both of the approaches were modelled  a f t e r the enrichment c u l t u r e 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 c u l t u r i n g of f u n c t i o n a l l y d i f f e r e n t i a t e d c e l l s ; i n a d d i t i o n , the c u l t u r e environment may not be adequate f o r the f u l l phenotypic expression of the s p e c i a l i z e d c e l l s .  In t h i s method,  a d r e n o c o r t i c a l tumor c e l l s were p e r i o d i c a l l y passaged i n isogeneic mice f o l l o w i n g 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 c u l t u r e c o n d i t i o n s , w h i l e 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 f o r the f u l l expression of the s p e c i a l i z e d f u n c t i o n of the a d r e n o c o r t i c a l tumor c e l l s .  Culture-derived tumors were found  to have an enhanced a b i l i t y t o grow and f u n c t i o n i n c u l t u r e subsequently (Buonassisi e^ a l . , 1962; Kowal, 1970b). 79  The second approach was based  80 upon the i d e a t h a t c e l l s  i n v i t r o were a l s o s u s c e p t i b l e  s i n c e change i s one o f the c h a r a c t e r i s t i c p r o p e r t i e s Thus, by t h e p r o c e d u r e o f s y s t e m a t i c of f u n c t i o n a l c e l l s  of l i v i n g  things.  c l o n i n g and r e - c l o n i n g , t h e p r o p a g a t i o n  c o u l d be c o n t i n u e d  or l e s s f u n c t i o n a l c e l l s  to a l t e r a t i o n s  w h i l e the c u l t u r e of  c o u l d be t e r m i n a t e d .  non-functional  C l o n a l l i n e s of v a r y i n g  f u n c t i o n a l a c t i v i t i e s have been e s t a b l i s h e d from the a d r e n o c o r t i c a l tumor c e l l s By  (Yasumura, 1968; Schimmer, 1969). using  these methods i n c o m b i n a t i o n w i t h r o u t i n e  culture  tech-  n i q u e s , f u n c t i o n a l a d r e n o c o r t i c a l tumor c e l l s have been m a i n t a i n e d and have p r o v i d e d c e l l biology  the o p p o r t u n i t i e s and  f o r many experiments o f importance i n  endocrinology.  With o n l y a few e x c e p t i o n s , requires  the p r o p a g a t i o n o f c e l l s  i n vitro  the p r e s e n c e o f macromolecules i n the c u l t u r e medium whose  s t r u c t u r e and f u n c t i o n have n o t y e t been d e f i n e d . as the c o n s t i t u e n t  i n c u l t u r e medium w h i c h p r o v i d e s  Much p r o g r e s s has been made i n d e f i n i n g c h e m i c a l l y  Serum has been used these molecules. the n u t r i t i o n a l  ments f o r b o t h t h e growth and f u n c t i o n o f t h e Y - l c l o n a l l i n e  require-  (Yasumura  eJL a l . , 1966a; Cuprak and S a t o , 1968; Lammi and Cuprak, 1974; Wishnow and  F e i s t , 19 74). By v i r t u e o f the resemblance i n p h y s i o l o g y  cortical  tumor c e l l s  responsive, for  and t h e normal a d r e n a l  corticosteroid-producing  between t h e adreno-  c o r t e x i n v i v o , these ACTH-  c e l l s have s e r v e d  as a u s e f u l model  the s i t e o f a c t i o n o f ACTH (Schimmer e_t a l . , 1969), and f o r the  study o f the acute and p r o l o n g e d e f f e c t s o f ACTH upon a v a r i e t y o f metab o l i c pathways i n the a d r e n a l  cell  ( P i e r s o n , 1967;  Kowal, 1969a,b,c,  81 1970a,b, 1973; Kowal and F i e d l e r , 1968, 1969; Kowal et a l . , 1970).  The  mechanism of hormonal a c t i v a t i o n of the adenylate cyclase system has been the subject of much a t t e n t i o n . 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 w i t h d i f f e r i n g degrees of competence i n the pathway of ACTHstimulated steroidogenesis have been used f o r i n v e s t i g a t i n g the mechanisms of coupling between hormone i n t e r a c t i o n w i t h i t s receptor and the a c t i v a t i o n of adenylate cyclase (Schimmer, 1969, 1972; Rodbell et a l . , 1974). Recently, evidence has accumulated that cholera enterotoxin i s capable of s t i m u l a t i n g the adenylate cyclcase enzyme of a v a r i e t y of c e l l s , i n c l u d i n g a d r e n o c o r t i c a l 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 t o x i n has been charac-  t e r i z e d (Wolff e t a l . , 19 73), and compared w i t h the ACTH e f f e c t s (Kowal et a l . , 19 74) i n order to gain f u r t h e r i n s i g h t s i n t o the mechanism of hormone a c t i o n 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 a d r e n o c o r t i c a l tumor c e l l s of choice to use i n t h i s study on the response of enucleated c e l l s to ACTH since these clones have been w e l l - c h a r a c t e r i z e d (Yasumura et a l . , 1966a), and are commercially  a v a i l a b l e . I n a d d i t i o n , a 3 to 4 - f o l d  increase i n steroidogenesis i n response t o ACTH has been reported f o r these c e l l s ; thus, the f u n c t i o n a l 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 . r e a d i l y enucleated.  I n c o n t r a s t , Y - l - L c e l l s were  Thus, Y - l - L c e l l s , and subsequently  Tumor #2 c e l l s ,  were the a d r e n o c o r t i c a l c e l l s used i n t h i s study i n s p i t e of t h e i r comp a r a t i v e l y low f u n c t i o n a l 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 i n d i c a t e d by t h e i r uniformity i n morphology, 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-  g r e s s i v e l y lower s t e r o i d output under various incubation conditions (Table I I I ) , and the absence of 1130H  progesterone, together w i t h l e s s  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 f u n c t i o n a l l y l e s s d i f f e r e n t i a t e d c e l l s were s e l e c t e d from the o r i g i n a l Y - l c e l l s purchased from B i o c u l t , and that f u r t h e r d e d i f f e r e n t i a t i o n occurred f o l l o w i n g i n creasing c u l t u r e passage. by spectrofluorometry  Furthermore, the low s t e r o i d output assayed  may be a r e s u l t of the i n a b i l i t y of l e s s 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 i n t o f l u r o g e n i c metabolites. The s t e r o i d s which required the 113 hydroxylation r e a c t i o n were i n i t i a l l y q u a n t i t a t i v e l y l e s s detectable i n e a r l y passage Y-l-L c e l l s , and subsequently 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 ( F i g . 9).  These s t e r o i d s were f u r t h e s t along i n the s t e r o i d  pathway ( F i g . 34) proposed f o r the a d r e n o c o r t i c a l 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  c u l t u r e of these a d r e n o c o r t i c a l tumor c e l l s r e s u l t e d i n a decline i n 113 hydroxylase a c t i v i t y while primary c u l t u r e of tumors generated i n the animals from these c e l l s have high l e v e l s of t h i s enzyme a c t i v i t y (Kowal, 1 9 6 9 ; Kowal et a l . , 1970).  I n t e r e s t i n g l y , lowered capacity f o r  113 hydroxylation has a l s o been described f o r the S n e l l a d r e n o c o r t i c a l 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 - 2 0 a - d i h y d r o progesterone  11 Keto-20d-dihydroprogesterone  F i g . 34. S t e r o i d Pathway i n Adrenal Tumor C e l l Cultures Taken from Kowal (1970b). l i n e are underlined.  The major s t e r o i d products of the Y - l c e l l  The major f l u o r o g e n i c 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 s t e r o i d output by the primary cultures of Tumor #2 c e l l s may be a t t r i b u t a b l e to the f u l f i l m e n t of the n u t r i t i o n a l r e q u i r e ments necessary f o r the f u l l phenotypic expression of thse c e l l s preceding passage i n v i v o .  during  The decline i n s t e r o i d output f o l l o w i n g  s e r i a l subculture may be due to the inadequacy of the c u l t u r e environment.  I n view of the absence of ACTH i n the c o n t r o l b a s a l c u l t u r e medium  used, and i n l i g h t of the f i n d i n g s that ACTH i s required f o r the maintenance of both the s t r u c t u r e and function of the normal adrenal i n vivo ( G i l l , 1972), the aforementioned explanations  II.  cortex  seem reasonable.  The E f f e c t of ACTH Upon Morphology ACTH has been shown to have pronounced  s t r u c t u r e of the adrenal gland.  e f f e c t s upon the u l t r a -  I t i s required f o r 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 ( K a h r i , 1968).- Acute u l t r a s 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 w i t h i n 10 minutes of ACTH a d m i n i s t r a t i o n have been described  (Rhodin, 1971).  Furthermore, the hypertrophy and h y p e r p l a s i a of smooth endoplasmic r e t i culum and mitochondria due t o 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 c o r r e l a t e the s t r u c t u r e w i t h the f u n c t i o n a l a c t i v i t i e s of a d r e n o c o r t i c a l c e l l s . Recently, r e s u l t s from s e v e r a l l a b o r a t o r i e s 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 c a u s a l l y with ACTH-induced steroidogenesis.  I t has been e s t a b l i s h e d  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 a f t e r hypophysectomy, the s t e r o i d o 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 a l t e r a t i o n s and contained no detectable i n t a c t 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 a d r e n o c o r t i c a l 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 t o respond s t e r o i d o g e n i c a l l y t o ACTH (Idelman, 19 70).  Thus, these  r e s u l t s 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 e f f e c t s and steroidogenesis, the mechanism of a c t i o n upon these e f f e c t s may be d i f f e r e n t (Kuo and Tchen, 1973). Results obtained from the present study of the u l t r a s t r u c t u r e of Y - l - L c e l l s i n c o n t r o l medium and i n medium c o n t a i n i n g ACTH f o r various time i n t e r v a l s i n d i c a t e d 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 f o l l o w i n g 5 minutes of ACTH a d m i n i s t r a t i o n . The s i g n i f i c a n c e of these r e s u l t s i s dubious i n view of the f a c t that Y - l - L c e l l s are tumor c e l l s .  The u l t r a s t r u c t u r e of these tumor c e l l s  was d i f f e r e n t from those of normal a d r e n o c o r t i c a l c e l l s i n v i v o .  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 e f f e c t of ACTH or d i b u t y r y l 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 d i f f e r e n t 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 t r y p s i n , or t r y p s i n together w i t h versene, the surface of the rounded Y - l - L c e l l s was g r e a t l y 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-  f o r e , the surface area t o volume r a t i o was maintained i n a l l three observed cases.  Whether or not the "rounding" response a l s o occurs i n  a d r e n o c o r t i c a l c e l l s i n v i v o i s not known, and the s i g n i f i c a n c e of t h i s response i s not c l e a r . The mechanism of c o r t i c o s t e r o i d s e c r e t i o n remains obscure.  The  c e l l u l a r organelles which may a c t as a v e h i c l e f o r the export of s t e r o i d i n t o 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 s e c r e t i o n i s accompanied by release of c y c l i c AMP (Kowal and R a i l , 1972; E s p i n e r , et a l . , 1974), and s e c r e t i o n of p r o t e i n (Rubin et^ a l . , 19 74).  I n l i g h t of the evidence which sug-  gested that the a c t i o n of ACTH i s not associated w i t h measurable changes i n membrane p e r m e a b i l i t y (Jaanus, 1971), the mechanism of transport of these substances becomes even more e l u s i v e .  More extensive s t u d i e s  w i l l be r e q u i r e d to e l u c i d a t e the mechanism of a c t i o n upon the morphology of a d r e n o c o r t i c a l c e l l s , and upon the mechanism of c o r t i c o s t e r o i d secretion.  87 III.  Enucleation Procedure The p r e c i s e mechanism by which c y t o c h a l a s i n B induces nuclear  p r o t r u s i o n and nuclear e x t r u s i o n remains enigmatic.  I n an e a r l y attempt  to e x p l a i n t h i s phenomenon, Carter proposed that the boundary tension between the plasma and nuclear membranes might be lowered by the adsorpt i o n of c y t o c h a l a s i n B on the i n t e r n a l and e x t e r n a l surfaces of these membranes r e s p e c t i v e l y .  As a r e s u l t , the chances of s t a b l e areas of  adhesion formed between these membranes might increase.  Carter sug-  gested that t h i s could e x p l a i n the observed "wrapping" of the c e l l membrane 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 H c y t o c h a l a s i n B predominantly by c e l l u l a r 3  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 ( L i n and Spudich, 1974; Mayhew e_t al, , 1974).  In a d d i t i o n ,  i t has been speculated that the b i n d i n g of c y t o c h a l a s i n B occurs i n the l i p i d phase of membranes (Mayhew et a l . , 1974).  A l t e r e d membrane  functions and properties could be produced by a l l o s t e r i c changes i n p r o t e i n s due to the presence of c y t o c h a l a s i n 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 c r e d i b l e . 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 c y t o c h a l a s i n B-  induced enucleation (Poste, 1972), and the adhesiveness of c e l l s to t h e i r substratum; hence, the a b i l i t y to r e s i s t detachment from the substratum at the high c e n t r i f u g a l forces used i n the enucleation procedure. E f f o r t s i n improving the enucleation e f f i c i e n c y of c e l l s have been  88 d i r e c t e d towards the m o d i f i c a t i o n of the substratum to render i t more adhesive to c e l l s ( P r e s c o t t , 1972), the r a t i o n a l e being that the chances of c y t o c h a l a s i n 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  c i r c u m s t a n t i a l evidence which suggested that Y - l - L  c e l l s adhered more tenaciously onto t h e i r substratum i n comparison w i t h the Y - l c e l l s (ATCC CCL 79).  A significantly  greater p r o p o r t i o n of  Y - l - L c e l l s remained attached to the c o v e r s l i p s f o l l o w i n g the enucleation procedure f o r both c e l l types.  identical  In a d d i t i o n , i n view of the  s e l e c t i o n procedure used to i n i t i a t e the Y-l-L s u b l i n e , the n o t i o n that c e l l s w i t h increased adhesion to t h e i r substratum were s e l e c t e d f o r , seemed reasonable.  Therefore,  enucleate populations  the a b i l i t y to c o n s i s t e n t l y and  uniformly  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 a t t r i b u t e d to the adhesive p r o p e r t i e s 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 n e u t r o p h i l s (Garvin, 1968), neuroblastoma c e l l s (Schubert et a l . , 1971)  and BHK  f i b r o b l a s t s (Maroudas, 1975)  a l l adhered more r a p i d l y  onto various substrata i n the absence of serum.  Although no evidence  has been obtained as yet to e x p l a i n 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 a l s o proposed that the forces which bind c e l l s to the substratum would a l s o bind serum competitively (Maroudas, 19 75). l i n e s , the s i g n i f i c a n t  Along the same  reduction i n c e l l u l a r binding of H 3  cytochalasin  89 B by ELD a s c i t e s carcinoma c e l l s i n the presence of serum was also tent a t i v e l y explained by the competition of serum f a c t o r s with c y t o c h a l a s i n B f o r 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  p r i o r to the enucleation by c e n t r i f u g a t i o n i n serum-free medium cont a i n i n g c y t o c h a l a s i n 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 c y t o c h a l a s i n 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 t h 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 p r e p a r a t i v e method  has been described f o r obtaining greater samples of enucleated  cells.  Wigler and Weinstein demonstrated that L c e l l s were enucleated w i t h high e f f i c i e n c y by c e n t r i f u g a t i o n 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 d i d agree that the procedure might be more traumatic f o r the c e l l s .  The  p o t e n t i a l usefulness of t h i s method i s i n d i c a t e d . Nevertheless, f u r t h e r i n v e s t i g a t i o n s i n t o the side e f f e c t s due to the method i t s e l f are r e quired, p r i o r to i t s adoption f o r o b t a i n i n g q u a n t i t a t i v e amounts of c y t o p l a s t s or karyoplasts necessary  f o r biochemical s t u d i e s .  90 IV.  E f f e c t s of DMSO and Cytochalasin B upon Y - l - L C e l l s and Tumor #2 Cells Cytochalasin B and i t s commonly-used solvent, DMSO, both had  e f f e c t s 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 e s t a b l i s h e d that e f f e c t s  of these substances during the enucleation procedure were r a p i d l y revers i b l e f o l l o w i n g t h e i r removal, and have no apparent e f f e c t s 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 e s t a b l i s h e d from t h i s 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 e i t h e r 10 yg c y t o c h a l a s i n B/ml  or 1% DMSO, i t was not determined whether the  lowered s t e r o i d output assayed by spectrofluorometry  of the incubation  media e x t r a c t s under various incubation conditions was due to the i n h i b i t i o n of s t e r o i d synthesis or s t e r o i d s e c r e t i o n by these agents. I t has been reported that a v a r i e t y 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 s t i m u l a t i o n of Y - l s t e r o i d o genesis by ACTH o r d i b u t y r y l c y c l i c AMP (Temple e t 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 c h o l e s t e r o l t o the mitochondrion which i s the major s i t e of hormone s y n t h e s i s , thus steroidogenesis was l i m i t e d i n the presence of D 2 O while steroidogenesis was enhanced i n the presence of antimicrotubular agents.  This hypothesis was h i g h l y s p e c u l a t i v e , and was o f f e r e d  only f o r the want of a b e t t e r explanation of f i n d i n g s that the mode of s t i m u l a t i o n of steroidogenesis by antimicrotubular agents was u n l i k e  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 e l e c t r o n m i c r o s c o p i c a l l y , i n a d d i t i o n , the rate of t u b u l i n polymerization was enhanced, and i n creased concentration of c o l c h i c i n e was necessary to prevent t u b u l i n polymerization.  I n l i g h t of these f i n d i n g s , i t i s conceivable that the  DMSO used i n t h i s 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 c h o l e s t e r o l and thereby steroidogenesis.  The exceedingly  limiting  f l a t t e n e d c e l l s i n the presence of  1% DMSO which was observed by scanning e l e c t r o n microscopy ( F i g . 26a) might be an i n d i c a t i o n of the e f f e c t of DMSO upon the microtubular skeleton.  cyto-  By transmission e l e c t r o n microscopy, pronounced swollen endo-  plasmic r e t i c u l u m was observed i n c e l l s t r e a t e d w i t h DMSO ( F i g . 26b). This e f f e c t has been reported i n tendon c e l l s (Streaton and Grim, 1974), although the s i g n i f i c a n c e of t h i s e f f e c t i s not clear., The endoplasmic reticulum i s a l s o the s i t e of s e v e r a l steps i n the pathway of s t e r o i d synthesis.  Therefore, the decreased steroidogenesis i n the presence  of DMSO might be due to the a l t e r e d f u n c t i o n a l a c t i v i t i e s of the o r ganelles required f o r steroidogenesis. A voluminous l i t e r a t u r e ( A l d r i c h , 1975) has a r i s e n on the use of c y t o c h a l a s i n B since the f i r s t report by Carter (1967) on the b i o l o g i c a l e f f e c t s of t h i s drug.  Although the primary mode of a c t i o n of  c y t o c h a l a s i n B has not been a s c e r t a i n e d , i t has been proposed that the wide array of phenotypic e f f e c t s when c y t o c h a l a s i n B i s applied t o c e l l s  92 and t i s s u e s may be due to i t s a c t i o n at the l e v e l of the plasma membrane to cause rearrangements and a l t e r a t i o n s of the proteins embedded i n the membrane l i p i d .  I t has been shown that c y t o c h a l a s i n B i n h i b i t e d  the release of thyroxine from the t h y r o i d gland a f t e r the s t i m u l a t i o n by t h y r o i d s t i m u l a t i n g hormone (Williams and Wolff, 1971), and a l s o i n h i b i t e d the release o f a-amylase from the p a r o t i d gland a f t e r stimul a t i o n by epinephrine  (Butcher and Goldman, 1972).  I n a d d i t i o n , the  i n h i b i t i o n of the i n d u c t i o n of t y r o s i n e aminotransferase a c t i v i t y by i n s u l i n o r C o r t i s o l i n the presence o f c y t o c h a l a s i n B has a l s o been reported (Butcher and Perdue, 1973).  Thus, i t i s conceivable that the  e f f e c t s of c y t o c h a l a s i n 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 e i t h e r 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 v a r i e t y of e f f e c t s of both DMSO and cytoc h a l a s i n B, f u r t h e r work upon the e f f e c t s of DMSO and c y t o c h a l a s i n B on the steps involved i n steroidogenesis i s e s s e n t i a l before any subs t a n t i a l explanation can be given.  V.  Mechanism of ACTH A c t i o n The a n t i b i o t i c , actinomycin  dependent RNA synthesis.  D, i s a potent i n h i b i t o r of DNA-  Numerous i n v e s t i g a t i o n s designed to define  whether o r 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 r e s u l t s of most of these studies are summarized i n Table 13.  In  the d i f f e r e n t 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  ACTIXOWfTO: D DOSACF.  SYSTEM USED  ROUTE OF ADMINTS--  ANALYSIS  DUHATION OK EXPERIMENT  1  !  SUMMARY OF RESULTS REPORTED IN THE LITERATURE  ANALYSIS OF STEROIDOGENESIS  RESISTS (COMPARED TO CONTROLS WITHOUT ACTINOMYCIN D)  REFERENCES  IS VIVO:BASAL Intact Guinea pigs  s i n g l e dose 3ug/100g  IP 2  dally  12 daya  Plasma 170H c o r t i c o i d s  Increased  for 2 days  P l a n e l l e s J_t a l . 1952  Intact Guinea pigs  single dose 7.5ug/100g  IP  daily  12 days  Plasma 170H c o r t i c o i d s  Increased f o r 7 days  P l a n e l l e s et a l . 1962  Intact Cuinea pigs  3ug/100g/day/7  IP  dally  32 days  Plasma 170H c o r t i c o i d s  Prolonged increase  P l a n e l l e s et a l . 1962  Estradiol-treated ovarlectomized rats  375-500ug/rat  Iotact rats  50pg/100ug/15.5  Intact Guinea pigs  s i n g l e dose  lOug/lOOg  Intact Guinea pigs  s i n g l e dose  lOpg/lOOg  Hypophysectomlzed . rats .  s i n g l e dose  25ug/100g  sc  Hypophysectomlzed : rats  s i n g l e dose  27-75ug/100g  Hypophysectoaized rats  s i n g l e dose  25-75ug/100g  Kypophysectoolzed ra ta  days  hours  IP  4 hours  Plasma corticosterone  Increased  IP  21 hours  Plasaa corticosterone  Slightly  Lippe and Szebo. 1965 Increased  Oyama and Dallgcoa, 1967  IP  dally  5 days  Urinary 17011 c o r t i c o i d s  Increased during f i r s t 3 days  Bransome, 1969  I*  dally  5 days  Plasma hydrocortisone .  Increased during f i r s c 3 days  Bransome, 1969  24 hours  Corticosterone content of adrenal  ACTH-induced Increase unaffected at 30 min. prevented at 24 hours  Vernikos-Danellis and H a l l , 1962  26 hours  Corticosterone content of adrenal  Unaffected  Hialhe Voloss et^ a l . 1966  IP  24 hours  Plasma corticosterone  Decreased  Hlalhe Voloss et a l . 1966  s i n g l e dose 5Oug/100g  SC  24 hours  Plasma corticosterone  Unaffected  Mialhe Voloss et a l . 1966  s i n g l e dose  IP  8 hours  Adrenal vein corticosterone  Unaffected  Key et a l . 1966  Plasma hydrocortisone  Diminished response at 1 day Increased response 3-5 days  Bransome, 1969  2.5 hours  Corticosterone  Slightly  Ferguson and Merita, 1964  3 hours  Corticosterone  Unaffected  Halkerston et. a l . 1965  Unaffected  Farese, 1966 •  IN VIVOrACTH*  I I  . Hypophysectomlzed , rats Intact Cuinea Pigs  lmg/rac  s i n g l e dose lOug/lOOg  3  IP or SC  IP  daily  5 days  . l : l V7TH0:BA.SAL . P-at adrenal, f r a g aents  10" -10"''M  Rat adrenal .fragments .  lOOug/nl  5  Cent adrenal • slices,  20ug/ml  Y - l mouse tumor, cells  20J g/ml  Trypaln-dissociated . r a t adrenal c e l l s . Collagcnase-disso4/ elated r a t adrenal ^Cills  5  1.5 hours every 30nin  - C o r t i s o l and Corticosterone  decreased  3 hours  20a dihydroprogesterone  Unaffected  Kowal, 1970a  2-10 vM  2 hours  Corticosterone  Abolished  S h a n u , 1974  1-50 VH  1 hour  Corr.icosterone  Decreased 45-70X  Schulster, 1974  f  IN VTiTt:ACTH CAMP , ' Rat adrenal f r a g ments  lO'S-lO " M -  2.5 hours  Corticosterone  Decreased s l i g h t l y  Ferguson and K o r l t a , 1964  Rat adrenal f r a g ments  lOOug/ml  3 hours  Corticosterone  Unaffected  aalkerston et a l . 1965  C o r t i s o l and Corticosterone  Inhibited  Farese, 1966 Kowal, 1970  . Cov adrenal s l i c e s Y - l mouse tumor cells  20ug/ml 20ug/ml  Superfused r a t adrenal glands Collagenaae-dissoclated rat adrenal cells .  l-50uM  1.5 hours every 30mln  3 hours  20a dihydroprogesterone  Unaffected  every 30min  8 hours  Corticosterone  Increased rate of s t e r o i d output delayed onset of r e f r a c t o r y period  1 hour  Corticosterone  ACTH- and c y c l i c AMP-stimulated l e v e l s decreased by 53-641; 48-642 respectively  ;  Legends: Unless otherwise s t a t e d , analysis of s t e r o i d output was c a r r i e d out at the end of the experiment. IF • I n t r a p e r i t o n e a l . SC -  Subcutaneous  ACTd was administered 15min-l hour p r i o r to termination of experiment. Molecular Weight of Actinomycin D - approximately 1200.  M e l t  . ,pbf  u r  and  Schulster, 1974  I  c  b  « , 0  1972  .'  94 been found not only to increase, but a l s o to decrease or e l s e not a f f e c t the b a s a l l e v e l of steroidogenesis.  Furthermore, the steroidogenic  response to ACTH has been observed to be elevated, prevented, or una f f e c t e d by actinomycin D. Two previous i n v e s t i g a t i o n s which were undertaken by Bransome (1969) and Schulster (19 74) to c l a r i f y the r e l a t i o n s h i p between RNA synthesis and ACTH-stimulated steroidogenesis are noteworthy.  Bransome  conducted a comprehensive i n vivo study on the e f f e c t s of actinomycin D upon the morphology, steroidogenesis, DNA s y n t h e s i s , RNA synthesis and p r o t e i n synthesis of the guinea p i g adrenal.  S c h u l s t e r systemati-  c a l l y s t u d i e d 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 concentrations.  From these two s t u d i e s , i t was determined that the acute  steroidogenic a c t i o n 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 a c t i o n might be the r e s u l t of d i f f e r e n c e s i n experimental  design,  i n dosage of actinomycin D used, of d i f f e r e n c e s i n c e l l s examined, or i n the timing of actinomycin D a d m i n i s t r a t i o n and subsequent observation of i t s e f f e c t s . The t o x i c i t y and m u l t i p l e side e f f e c t s 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 q u i t e obvious that an e f f e c t of actinomycin D upon adrenocort i c a l functions cannot unequivocably be assumed to be s o l e l y and d i r e c t l y  95 r e l a t e d to i n t e r f e r e n c e w i t h the t r a n s c r i p t i o n a l stage of p r o t e i n synthesis (Mostafapour and Tchen, 1972). In t h i s study, the e f f e c t s of ACTH upon anucleate a d r e n o c o r t i c a l c e l l s were analyzed.  Thus, the p o s s i b i l i t y of unpredictable s i d e e f f e c t s  of actinomycin D was e l i m i n a t e d . Enucleated Y - l - L c e l l s and Tumor #2 c e l l s d i d respond t o e i t h e r ACTH or d i b u t y r y l c y c l i c AMP.  Thus, i t could be concluded from t h i s  study that the nucleus, and therefore new RNA s y n t h e s i s , i s d e f i n i t e l y not necessary f o r the capacity of Y - l - L c e l l s and Tumor #2 c e l l s t o respond to ACTH or d i b u t y r y l c y c l i c AMP. and d i b u t y r y l c y c l i c AMP-stimulated  The b a s a l ,  ACTH-stimulated  s t e r o i d 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 c o n t r o l Y - l - L c e l l s during the f i r s t three hours post-enucleation.  This i s consistent w i t h Kowal's  observation that the b a s a l and ACTH-stimulated Y-l  s t e r o i d production of  c e l l s was unaffected by 20 pg of actinomycin D/ml (Kowal, 1970a).  Although the s t e 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 t o ACTH or d i b u t y r y l c y c l i c AMP were comparable to the EPT c o n t r o l Tumor #2 c e l l s during the f i r s t three hours after enucleation. Previous i n v i v o studies u t i l i z i n g actinomycin D have i n d i c a t e d that the synthesis of p r o t e i n ( s ) b e l i e v e d t o mediate the steroidogenic e f f e c t s of ACTH depended on RNA(s) that was (were) s t a b l e f o r at l e a s t 8 hours (Ney et a l . , 1966), w h i l e 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 o f any RNA(s) i n v o l v e d i n the ACTH e f f e c t  96 to be at l e a s t 70 minutes ( S c h u l s t e r , 1974).  Experiments on the adrenal  steroidogenic capacity of hypophysectomized animals have i n d i c a t e d that the decay i n c o r t i c o s t e r o i d b i o s y n t h e s i s by the adrenals was a f u n c t i o n of time post-hypophysectomy.  The h a l f - l i f e of t h i s d e c l i n e has been  estimated to be 6 to 7 hours.  I t was a l s o suggested that the decay  i n steroidogenic capacity may be c o r r e l a t e d w i t h or due to the decay of the messenger RNA f o r the steroidogenic p r o t e i n (Mostafapour and Tchen, 1973). Due to the r e l a t i v e l y long incubation periods used i n t h i s  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 a c t i o n , the h a l f - l i f e of the f a c t o r i n v o l v e d i n stimulated steroidogenesis could not be measured.  Nevertheless, i t can be con-  cluded from t h i s study that the cytoplasmic component required f o r i n creased steroidogenesis i n response t o ACTH i s r e l a t i v e l y s t a b l e . The o r i g i n of a b a s a l s t e r o i d output i n the absence of added ACTH by the a d r e n o c o r t i c a l tumor c e l l s i s not c l e a r .  There i s no evidence  to suggest that there are separate regulatory mechanisms f o r b a s a l and stimulated steroidogenesis.  Thus, i t i s q u i t e reasonable  that the b a s a l  s t e r o i d output was a l s o decreased at i n c r e a s i n g time i n t e r v a l s f o l l o w i n g enucleation due to the decay of the cytoplasmic components necessary for  steroidogenesis. While the steroidogenic responses of enucleated a d r e n o c o r t i c a l  c e l l s t o ACTH and d i b u t y r y l c y c l i c AMP were abolished 12 hours f o l l o w i n g enucleation, the morphological  responses to ACTH and d i b u t y r y l c y c l i c  AMP were i n d u c i b l e up to 36 hours f o l l o w i n g enucleation.  There i s  97 i n c r e a s i n g evidence that the e f f e c t s of ACTH upon the morphology and steroidogenic a c t i v i t y of the adrenal cortex i n v i v o may not, of necess 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 s t e r o i d synthesis was i n h i b i t e d (Kowal, 1969c). Thus, i t i s p l a u s i b l e that although morphological  changes and increased  steroidogenesis are both e f f e c t s due to ACTH, the mechanism of ACTH a c t i o n upon these a c t i v i t i e s may be d i f f e r e n t and may not be c a u s a l l y related.  From t h i s study, i t was determined that the cytoplasmic com-  ponents necessary f o r c e l l "rounding" are s t a b l e f o r at l e a s t 36 hours.  VI.  General Outlook In t h i s study, i t was c o n c l u s i v e l y e s t a b l i s h e d that the nucleus,  hence nuclear RNA s y n t h e s i s , i s not required i n the acute steroidogenic response of a d r e n o c o r t i c a l c e l l s to ACTH, since enucleated to ACTH both morphologically  and s t e r o i d o g e n i c a l l y .  c e l l s responded  The r o l e s of the  nucleus and cytoplasm i n a v a r i e t y of c e l l u l a r a c t i v i t i e s have a l s o been ascertained by studying the metabolic  c a p a b i l i t i e s of a v a r i e t y of enu-  cleated c e l l types (e.g. P r e s c o t t e t a l . , 1971; Goldman et a l . , 1973; M i l l e r and Ruddle, 1974; Rechsteiner 19 74).  and C a t a n z a r i t e , 1974; I v a r i e et a l . ,  whatever the p r e c i s e mechanism of c y t o c h a l a s i n B-induced enu-  c l e a t i o n , i t i s now evident that the p r e d i c t i o n that enucleation of mammalian c e l l s by t h i s drug could provide a p o t e n t i a l l y u s e f u l t o o l i n c e l l biology (Carter, 1967) has been r e a l i z e d . The t o t a l evaluation of the r o l e of the cytoplasm i n the i n t a c t 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 f o r the complete f u n c t i o n i n g of the cytoplasm. converse i s also t r u e .  The  Were t h i s not so, the e v o l u t i o n of such an or-  g a n i z a t i o n would be enigmatic. Studies i n v o l v i n g nuclear t r a n s p l a n t a t i o n have revealed that the cytoplasm a l s o plays an important r o l e i n the r e g u l a t i o n 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 e q u i v a l e n t , the expression of t h i s information v a r i e s i n d i f f e r e n t c e l l types and i s dependent upon the cytoplasm i n which the nucleus r e s i d e s . Such studies have been l i m i t e d to very few c e l l types, as micromanipulation becomes t e c h n i c a l l y formidable when the c e l l s are s m a l l . Recently, methods f o r the f u s i o n of u n l i k e c e l l s by Sendai v i r u s ( H a r r i s , 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 t r a n s p l a n t a t i o n can be achieved by combining the methods i n v o l v e d w i t h c y t o c h a l a s i n B-induced formation of c y t o p l a s t s and karyoplasts together w i t h methods i n v o l v e d w i t h c e l l fusion.  Thus, i t i s conceivable that the i n t r o d u c t i o n of n u c l e i i n t o  heterologous cytoplasms i n the manner described, could provide a unique experimental system f o r the study of the metabolic and genetic r e g u l a t i o n i n mammalian c e l l s (Poste and Reeve, 1972).  SUMMARY  1.  P r e v i o u s l y , actinomycin  D has been used to c l a r i f y the r o l e of  RNA synthesis i n the acute steroidogenic response of a d r e n o c o r t i c a l c e l l s to ACTH. conflicting  However, a survey of the l i t e r a t u r e revealed that  r e s u l t s were reported.  I t i s now evident that a c t i n o -  mycin D i s t o x i c and can produce a myriad of s i d e 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 r u l e d out.  The purpose of t h i s study was  to use an a l t e r n a t e approach to determine c o n c l u s i v e l y whether new RNA synthesis i s required i n the response to ACTH.  The r e -  sponses to ACTH by a d r e n o c o r t i c a l c e l l s which had been  enucleated  by c y t o c h a l a s i n B treatment were i n v e s t i g a t e d . 2.  The f u n c t i o n a l murine a d r e n o c o r t i c a l tumor c e l l l i n e , the Y - l c e l l s , were the a d r e n o c o r t i c a l c e l l s chosen f o r t h i s study. I t was found during the course of t h i s 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.  I n c o n t r a s t , Y - l - L c e l l s and Tumor #2 c e l l s were enu-  c l e a t e d c o n s i s t e n t l y and e f f i c i e n t l y , thus, these c e l l s were used f o r the study of ACTH a c t i o n . 3.  Y - l - L c e l l s were s e l e c t e d 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  a t t r i b u t e d 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 d i b u t y r y l c y c l i c AMP were  characterized.  S i m i l a r 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 f o r the maintenance of a b a s a l s t e r o i d output, but responded to added ACTH or d i b u t y r y 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 s t e r o i d o genesis . In comparison w i t h the Y - l c e l l s , the s t e r o i d output of Y-l-L and Tumor #2 c e l l s under various incubation lower.  cells  conditions was r e l a t i v e l y  I n a d d i t i o n , the s t e r o i d production by these c e l l s was  f u r t h e r diminished w i t h i n c r e a s i n g  c u l t u r e passage.  Evidence which  suggested that Y - l - L c e l l s were l e s s 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 d e d i f f e r e n t i a t i o n also occurred w i t h i n creasing  c u l t u r e passage was presented and discussed.  The u l t r a s t r u c t u r e of the Y-l-L c e l l s was d i f f e r e n t than that o f adrenocortical  c e l l s i n vivo.  No c o r r e l a t i o n could be made between  the u l t r a s t r u c t u r e and the f u n c t i o n a l 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 t o ACTH was not i d e n t i c a l t o " c e l l rounding" i n response t o t r y p s i n or t r y p s i n and versene i n terms of u l t r a s t r u c t u r e , the s i g n i f i c a n c e and mechanism of t h i s morphologic response i s not c l e a r . I t was e s t a b l i s h e d  that the treatments used during the enucleation  procedure did.not have any e f f e c t 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 t h i s study, two enucleation methods were developed.  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 r o u t i n e l y produced by these methods. 9.  Some enucleated c e l l s were v i a b l e f o r at l e a s t 60 hours, and were m e t a b o l i c a l l y a c t i v e f o r 48 hours, as i n d i c a t e d by dye e x c l u s i o n and i n c o r p o r a t i o n of H-leucine i n t o a c i d - i n s o l u b l e m a t e r i a l . 3  10.  Enucleated Y - l - L c e l l s and Tumor #2 c e l l s responded t o e i t h e r ACTH or d i b u t y r y l c y c l i c AMP morphologically  and s t e r o i d o g e n i c a l l y .  Thus i t was c o n c l u s i v e l y e s t a b l i s h e d that the nucleus i s not r e quired f o r the expression of these acute e f f e c t s of ACTH and d i b u t y r y l c y c l i c AMP. 11.  The steroidogenic c e l l s was  response of enucleated Y - l - L c e l l s and Tumor #2  comparable i n magnitude t o that of the EPT c o n t r o l c e l l s  during the f i r s t three hours and p e r s i s t e d i n diminishing f o r an a d d i t i o n a l 9 hours.  degrees  On the other hand, the morphologic  change was induced by ACTH or d i b u t y r y l c y c l i c AMP even at 33 hours f o l l o w i n g enucleation.  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Studies on the adrenoc o r t i c o t r o p i c hormone-activated adenyl cyclase of a f u n c t i o n a l adrenal tumor. J . B i o l . Chem. 244: 247-253. Temple, R., W i l l i a m s , J . A., Wilber, J . F., and W o l f f , J . (1972). C o l c h i c i n e and hormone s e c r e t i o n . Biochem. and Biophys. Res. Comm. 46: 1454-1461. , and Wolff, J . (1972). S t i m u l a t i o n of s t e r o i d s e c r e t i o n by a n t i m i c r o t u b u l a r agents. J . B i o l . Chem. 248: 2691-2698. Venable, J . H., and Coggeshall, R. (1965). A s i m p l i f i e d lead c i t r a t e s t a i n f o r use i n e l e c t r o n microscopy. J . C e l l B i o l . 25: 407-408. V e r n i k o s - D a n e l l i s , J . , and H a l l , M. (1965). I n h i b i t i o n of a d r e n o c o r t i c a l responsiveness to ACTH by actinomycin D i n v i v o . Nature 207: 766. Watson, M. L. (1958). S t a i n i n g of t i s s u e s e c t i o n s f o r e l e c t r o n microscopy w i t h heavy metals. J . Biophys. and Biochem. C y t o l . 4_: 475-479. Wigler, M. H., and Weinstein, J . B. (1975). A p r e p a r a t i v e method f o r obtaining enucleated mammalian c e l l s . Biochem. Biophys. Res. Comm. 63: 669-674. W i l l i a m s , J . A., and Wolff, J . (1971). Cytochalasin B i n h i b i t s t h y r o i d s e c r e t i o n . Biochem. Biophys. Res. Comm. 44: 422-425. Wishnow, R. M., and F e i s t , P. (1974). The e f f e c t of calcium concent r a t i o n on ACTH s t i m u l a t i o n of steroidogenesis i n mouse adrenal tumor c e l l s . J . C e l l . P h y s i o l . 83: 419-424. Wolff, J . , Temple, R., and Cook, G. H. (1973). S t i m u l a t i o n of s t e r o i d s e c r e t i o n i n adrenal tumor c e l l s by choleragen. Proc. N a t l . Acad. S c i . 70: 2741-2744. Wright, W. E. and H a y f l i c k , L. (1972). Formation of anucleate and multinucleate c e l l s i n normal and SV40 transformed WI-38 by cytoc h a l a s i n B. E x p t l . C e l l Res. 74: 187-194. Yasumura, Y. (1968). Retention of d i f f e r e n t i a t e d f u n c t i o n i n c l o n a l animal c e l l l i n e s p a r t i c u l a r l y hormone-secreting cultures. Am. Zool. 8: 285-305. , B u o n a s s i s i , V., and Sato, G. (1966a). C l o n a l a n a l y s i s of d i f f e r e n t i a t e d f u n c t i o n i n animal c e l l c u l t u r e s . Cancer Res. 26: 529-535.  109 Yasumura, Y., Tashjian, A. H., J r . , and Sato, G. H. (1966b). E s t a b l i s h ment of four f u n c t i o n a l , c l o n a l s t r a i n s of animal c e l l s i n c u l t u r e . Science 154: 1186-1189. Y i p , D. K., and Auersperg, N. (1972). The dye exclusion t e s t f o r c e l l v i a b i l i t y : persistence of d i f f e r e n t i a l s t a i n i n g f o l l o w i n g f i x a t i o n . In V i t r o 7: 323-329.  APPENDIX I  Preparation of C e l l s f o r Transmission E l e c t r o n Microscopy A. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. B.  Dehydration and Embedding 50% ETOH, 5 minutes 70% ETOH, 5 minutes 90% ETOH, 5 minutes 95% ETOH, 5 minutes 100% ETOH, 5 minutes 100% ETOH, 5 minutes 100% ETOH: Propylene Oxide (1:1), 10 minutes Propylene Oxide, 10 minutes Propylene Oxide, 10 minutes Propylene Oxide:Epon (1:1), 1 hour Propylene Oxide:Epon (1:3), 1 hour Epon, 1 hour Fresh Epon, 24 hours at 37°C, then overnight at 56°C. Epon S o l u t i o n  Stock s o l u t i o n A:  Epon 812 Resin Dodecenyl S u c c i n y l Anhydride (DDSA)  62 ml 100 ml  Stock s o l u t i o n B:  Epon 812 Resin Nadic Methyl Anhydride (NMA)  100 ml 89 ml  Working s o l u t i o n :  Mix s o l u t i o n A and B (2:3), then add 2% by volume,the a c c e l e r a t o r dimethylaminomethyl phenol (DMP-30-Tris)  110  APPENDIX I I  I d e n t i f i c a t i o n of 20a Dihydroprogesterone by A c e t y l a t i o n P r i o r to Recrystallization A.  Preparation of C a r r i e r 20a Dihydroprogesterone-Acetate  1.  30 mg 20a dihydroprogesterone standard + 3 ml a c e t i c anhydride + 3 ml p y r i d i n e at 37°C f o r 2 hours.  2.  0.5 ml of ethanol was added to stop the r e a c t i o n .  3.  The r e a c t i o n mixture was e x t r a c t e d w i t h 20 ml of methylene c h l o r i d e .  4.  The methylene c h l o r i d e e x t r a c t was washed w i t h 1.0 ml of water.  5.  The solvent phase was removed and evaporated under n i t r o g e n .  6.  Assuming that there was 100% a c e t y l a t i o n , the product remaining was 100% 20a dihydroprogesterone-acetate.  B.  Preparation of H - A c e t i c Anhydride J  H - a c e t i c anhydride (400 uCi/mM) was purchased from New England Nuclear and d i l u t e d to 40 uCi/mM i n 12% benzene. 3  C.  Acetylation  1.  The standards used were two 0.5 yg a l i q u o t s of authentic 20a dihydroprogesterone. The sample 20a dihydroprogesterone was obtained from the t h i n l a y e r chromatogram of the endogenous s t e 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 s t e r o i d .  2.  Experiment  C-20a dihydroprogesterone was used to monitor the recovery of 20a dihydroprogesterone during t h i s experiment. *C-20a dihydroprogesterone was not commercially a v a i l a b l e , t h e r e f o r e , the C20a dihydroprogesterone used was obtained from the chromatographic i s o l a t i o n of ^-pregnenolone metabolites of Y - l - L c e l l s . The areas on the chromatogram containing C-20a dihydroprogesterone was scraped o f f , e l u t e d w i t h ethanol, f i l t e r e d , evaporated, and then r e d i s s o l v e d i n 1 ml of methanol.  ll+  ll  1  11+  Ill  112 3.  0.3 ml of C-20a dihydroprogesterone was added to each f o l l o w i n g 3 tubes: (a) Standard 1, which contained 0.5 yg of authentic 20a progesterone. (b) Standard 2, which contained 0.5 yg of authentic 20a progesterone. (c) Sample, which contained approximately 1.0 yg of 20a progesterone which was produced by Y - l - L c e l l s . 14  of the dihydrodihydrodihydro-  4.  The m a t e r i a l s i n each of the 3 tubes were evaporated under n i t r o g e n .  5.  30 y l of p y r i d i n e and 1.5 y l of H - a c e t i c anhydride was added to each of the 3 tubes.  6.  A f t e r 24 hours, the r e a c t i o n was stopped by the a d d i t i o n of 0.5 ml of ethanol.  7.  Each of Standard 1, Standard 2, and Sampled was e x t r a c t e d w i t h 5 ml of carbon t e t r a c h l o r i d e .  8.  The carbon t e t r a c h l o r i d e e x t r a c t was washed w i t h 0.5 ml of water. The water phase was then p i p e t t e d o f f , and the solvent was evaporated under n i t r o g e n .  9.  The m a t e r i a l 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 g e l p l a t e s together w i t h 10 yg each of authentic 20a dihydroprogesterone and prepared 20a dihydroprogesterone-acetate.  3  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 g e l p l a t e was scraped o f f , eluted w i t h ethanol, f i l t e r e d , and then evaporated separately f o r each of Standard 1, Standard 2, and Sample.  13.  Each of Standard 1, Standard 2, and Sample was recrystallized i n the presence of 10 mg of prepared 20a dihydroprogesterone-acetate.  14.  An a l i q u o t of the 3rd and 4th c r y s t a l s of each of Standard 1, Standard 2, and Sample were counted, and' the C: H r a t i o of these c r y s t a l s was e s t a b l i s h e d . lt+  3  APPENDIX I I I  Preparation of Lowry's Reagent Stock s o l u t i o n A:  2% Na C0  Stock s o l u t i o n B:  2% NA K t a r t a r a t e  Stock s o l u t i o n B:  1% CuS0 -5H 0  Working s o l u t i o n :  (Lowry's reagent): 100A:1B:1C - use w i t h i n 2 hours  2  4  3  i n 0.1 M NaOH  2  113  

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