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UBC Theses and Dissertations

An analysis of the effects of oncogenes and growth factors on rat adrenal cortex cells MacAuley, Iain Alasdair Somerled 1987

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of  An A n a l y s i s o f t h e E f f e c t s Oncogenes and Growth F a c t o r s on Rat A d r e n a l C o r t e x C e l l s by  Iain B.Sc,  A l a s d a i r Somerled MacAuley U n i v e r s i t y of C a l g a r y , 1981  Thesis Submitted i n P a r t i a l F u l f i l l m e n t of t h e R e q u i r e m e n t s f o r t h e D e g r e e of D o c t o r of P h i l o s o p h y in The  F a c u l t y of Graduate S t u d i e s Department o f M i c r o b i o l o g y U n i v e r s i t y of B r i t i s h Columbia  We  a c c e p t t h i s t h e s i s as c o n f o r m i n g to the r e q u i r e d standards  University  of  British  January cj  Iain  Alasdair  Columbia  1987 Somerled  MacAuley  In  presenting  degree  this  thesis in partial fulfilment of the  for  of  department  this thesis for or  by  his  or  scholarly her  I further agree that permission for  purposes  advanced  representatives.  permission.  Department The University of British Colurnl 1956 Main Mall Vancouver, Canada V6T 1Y3  extensive  may be granted by the head of It  is  understood  that  publication of this thesis for financial gain shall not be allowed without  DE-6(3/81)  an  at the University of British Columbia, I agree that the Library shall make it  freely available for reference and study. copying  requirements  copying  my or  my written  ii Abstract  The  process  been examined  of oncogenic  i n an a t t e m p t  transformation  to d e f i n e  mechanisms o f c a r c i n o g e n e s i s . infected  r a t adrenal  process  (Auersperg  transformation 1983b). express  cortex  a partially  (Calderwood infected  significant ras  nonestablished  cortex  phenotype  cells  I t could  than  either  initially further  phenotype o f Ki-MSV-  p r o g r e s s i o n to  i n the absence of of the  be e x p r e s s e d  i n these  of transformation.  to transform  primary  o f t h e c o - i n t r o d u c t i o n o f myc of adrenal  the a d r e n a l oncogene  cultures i n i t i a l l y  phenotypes  a s s o c i a t e d with  of  transformed  only  at.,  i n d i c a t e d t h a t an  be d e m o n s t r a t e d  to transform  a fully  occur  of  et  and a f t e r  of the e x p r e s s i o n  cooperate  transformation  infected  could  i n the absence  ras and myc  cells  i n d i c a t e d that  a c t i v a t e d ras gene c o u l d  MSV-induced  efficiently  cortex  (Land  Examination  These r e s u l t s  the e f f e c t  cooperate  1984).  cells  fibroblasts  examined.  cells  phenotype  changes i n the l e v e l  oncogenically  t o be a m u l t i s t e p  to a h i g h l y transformed  oncogene.  Since  adrenal  transformed  a h i g h l y transformed  viral  nonestablished  and A u e r s p e r g ,  adrenal  i n Ki-MSV-  et a l . , 1 9 8 1 ) , as does t h e p r o c e s s  i n other  progress  appears  has  molecular  Transformation  cells  The K i - M S V - i n f e c t e d  passaging  the  in v i t r o  cortex  that  express  was  myc and ras  cortex  alone,  cells  on K i -  cells  more  but t h a t the some o f t h e  transformation.  The  p h e n o t y p e , as m o n i t o r e d  appearance by g r o w t h i n  iii soft  agar,  was  infection.  An  not  expressed  analysis  the  activated  oncogenes i n i m m o r t a l i z e d i n the  Transformation cellular  by  of  The  Ki-MSV-infected  with  the  reduced  27000  These  step  two  growth r a t e  to  cortex  with  a  further  suppression  cells  to  cells.  require  the  cultures  appeared  transformed  of  cultures  was  from  correlated  of  a novel  ras-related  by  s r c and  myc  transformation  entirely of  the  culture  conditions.  src  ras  adrenal  lines cortex  oncogenes appeared  suppression.  to be  and  induced  pathway o f  cellular  appear  appears  emergence o f  expression  after  protein  Mr.  examined.  to  myc  cell  adrenal  rat adrenal  Transformation  two  and  associated  change r e s u l t i n g i n a l o s s o f  oncogene a c t i o n . the  phenotypes  coinfected  ras  passages  Ki-MSV/MMCV-infected  that  suppressed  the  several  indicated  be  some o f  of  until  i n the  cortex  The  free  of  cells  ability  transformation  cells  provides  of  be  to  the  as  did  early  not  the  modified  cooperate  support  in a  susceptible  influence  myc  further  not  phenotype  could  of  also  cooperate  was  transformed  transformed The  to  that  external  was  by with  passage for  mutistep  carcinogenesis. The in  the  effect  Y-1  of  o n c o g e n e s on  adrenocortical  steroidogenesis  tumour  the  virally  Y-1  cells  was  relatively  subtle.  stimulate  the  production  of  distinct  b o r n e o n c o g e n e s on  fashion.  cell  line.  g r o w t h and The  The  was  examined  effect  morphology of  oncogenes appear  fluorogenic  s t e r o i d s , each  of the to in a  A roles can  model of  be  the  of  transformation  oncogenes  evaluated.  transformation  for  the  two  potential  process  provides  an  be  derived  interaction  differences  the  study.  their  The  i l l u s t r a t e s and  and  can  in  the  combinations complexity  in v i t r o  of  model  in  which  with  the  pathways  of the  the c e l l  of  oncogenes transformation  system  for  further  V  Table  of Contents Page  Abstract L i s t of Tables L i s t of Figures L i s t of Abbreviations Dedication Acknowledgements  i i viii ix x i i xiv xv  CHAPTER 1 1.0  Introduction 1 1.1 Retroviruses 1 1.1.1 Classification 1 1.1.2 Structure 1 1.1.3 Replication 2 1.2 Oncogenic R e t r o v i r u s e s 3 1.2.1 A c u t e l y Oncogenic R e t r o v i r u s e s 4 1.2.2 O r i g i n o f v-oncs 5 1.2.3 Chronic/leukosis Viruses 5 1.3 O n c o g e n e s : O r i g i n s and F u n c t i o n s 6 1.3.1 R e t r o v i r a l Oncogenes 6 1.3.2 C e l l u l a r Oncogenes 8 1.3.3 9 1.3.4 myc 20 1.3-5 src 25 1.3.6 naf 28 1.4 T r a n s f o r m a t i o n : i n v i v o and i n v i t r o M o d e l s 30 1.5 T i s s u e C u l t u r e i n the Examination of Transformation 35 1.5.1 Establishment of C e l l Lines 35 1.5.2 R e g u l a t i o n o f C e l l Growth by Growth F a c t o r s 37 1.5.3 T r a n s f o r m a t i o n and Growth F a c t o r I n d e p e n d e n c e 3 9  .nas  CHAPTER 2 2.0  M a t e r i a l s and Methods 2.1 Cells 2.2 Viruses 2.3 I n f e c t i o u s Centre Assays 2.4 A n c h o r a g e I n d e p e n d e n t Growth 2.5 R a d i o l a b e l l i n g of C e l l s 2.6 Immunoprecipitation 2.7 Immune Complex K i n a s e R e a c t i o n s 2.8 SDS-polyacrylamide Gel E l e c t r o p h o r e s i s 2.9 Pulse L a b e l l i n g of Proteins 2.10 T r y p t i c P e p t i d e A n a l y s i s 2.11 S o u t h e r n H y b r i d i z a t i o n 2.12 HPLC A n a l y s i s o f S t e r o i d P r o d u c t s from t h e Y-1 A d r e n o c o r t i c a l C e l l Line  41 43 44 45 46 47 47 48 49 50 51 52  2.13  Radioimmunoassay o f C u l t u r e S u p e r n a t a n t MSV/MMCV T r a n s f o r m e d Rat A d r e n a l C o r t e x for Steroid Production..  of K i Cells 53  CHAPTER 3 3.0  Expression of V i r a l p 2 1 • D u r i n g A c q u i s i t i o n of a T r a n s f o r m e d P h e n o t y p e by Rat A d r e n a l C o r t e x C e l l s I n f e c t e d w i t h Ki-MSV r a s  3.1 3.2 3.3 3.4  Introduction Expression of V i r a l p 2 1 i n the Passages Immediately F o l l o w i n g I n f e c t i o n Viral p21 E x p r e s s i o n i n P a r t i a l l y and F u l l y Transformed Adrenal Cortex C e l l s Discussion  55  r a s  59  r s  66 72  CHAPTER 4 4.0  T r a n s f o r m a t i o n o f Rat A d r e n a l C o r t e x C e l l s by r a s and myc: E v i d e n c e f o r a R e q u i r e m e n t f o r a F u r t h e r C e l l u l a r Change 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8  Introduction 75 I n d u c t i o n o f F o c u s F o r m a t i o n and M o r p h o l o g i c a l T r a n s f o r m a t i o n by Ki-MSV and MMCV 80 The E x p r e s s i o n o f a T r a n s f o r m e d M o r p h o l o g y and Growth by K i - M S V / M M C V - i n f e c t e d A d r e n o c o r t i c a l C u l t u r e s R e q u i r e s a H i g h C o n c e n t r a t i o n o f Serum. 88 A d d i t i o n o f a P u r i f i e d Growth F a c t o r c a n R e p l a c e the R e q u i r e m e n t f o r a H i g h Serum S u p p l e m e n t C o n c e n t r a t i o n i n Ki-MSV/MMCV-infected C e l l s 93 A c q u i s i t i o n o f Serum I n d e p e n d e n t Growth and A n c h o r a g e I n d e p e n d e n t Growth 96 I s o l a t i o n o f T r a n s f o r m e d L i n e s from Ki-MSV/MMCVinfected Cultures 98 C h a r a c t e r i z a t i o n of the Transformed A d r e n o c o r t i c a l L i n e s f o r t h e P r e s e n c e o f t h e V i r a l Oncogenes and t h e i r P r o d u c t s , and S t e r o i d o g e n i c A b i l i t y 103 Discussion 114  CHAPTER 5 5.0  myc and s r c C o o p e r a t e i n t h e i n v i t r o T r a n s f o r m a t i o n o f E a r l y P a s s a g e Rat A d r e n a l C o r t e x Cells 5.1  Introduction:  119  vii  5.2 5-3 5.4 5.5  M o r p h o l o g i c a l A l t e r a t i o n s i n Response t o S u p e r i n f e c t i o n by t h e s r c C o n t a i n i n g R e t r o v i r u s , 2-1 A n c h o r a g e I n d e p e n d e n t Growth C h a r a c t e r i z a t i o n of Three Transformed L i n e s P r e s e n c e o f t h e V i r a l Oncogenes Discussion  121 125 f o r the 126 138  CHAPTER 6 6.0  E f f e c t s o f V i r a l Oncogenes on t h e S t e r o i d o g e n i c o f t h e Y-1 Mouse A d r e n o c o r t i c a l Tumour C e l l s 6.1 6.2 6.3 6.4  Ability  Introduction 142 The E f f e c t s o f R e t r o v i r a l l y Borne Oncogenes on the M o r p h o l o g y and Growth o f t h e Y-1 C e l l L i n e . . 146 A n a l y s i s o f S t e r o i d P r o d u c t i o n by t h e R e t r o v i r a l l y I n f e c t e d and U n i n f e c t e d Y-1 L i n e s 150 Discussion 161  CHAPTER 7 7.0  A 27000 Mr P r o t e i n S t r u c t u r a l l y R e l a t e d t o p21 - - - i s E x p r e s s e d i n Human and Rat P r i m a r y T i s s u e C u l t u r e C e l l s r  7.1 7.2 7.3 7.4 7.5 7.6  Introduction S u r v e y o f p27 E x p r e s s i o n F r a c t i o n a t i o n o f the L a b e l l e d L y s a t e Expressing C e l l Line T r y p t i c P e p t i d e Mapping o f p27 Pulse-chase L a b e l l i n g o f p27 Discussion  a  s  165 167 o f a p27 168 175 178 184  CHAPTER 8 8.0  Summary.  References  1 89 197  viii  List  Table  3.1  T a b l e 4.1  Table  Table  Table  4.2  4.3  [ S] I n c o r p o r a t i o n i n t o p21^ - - i n Ki-MSVi n f e c t e d and U n i n f e c t e d A d r e n a l C o r t e x Cells  65  F o c u s F o r m a t i o n i n Rat A d r e n a l C o r t e x C e l l s a f t e r I n f e c t i o n with Acutely Oncogenic R e t r o v i r u s e s  83  I n f e c t i o u s Centre Assay i n f e c t e d Adrenal Cortex  85  3 5  a  s  o f Ki-MSV/MMCVCells  Number o f P a s s a g e s Between I n f e c t i o n and E x p r e s s i o n o f Serum I n d e p e n d e n c e and A n c h o r a g e I n d e p e n d e n t Growth.  97  C o n v e r s i o n o f P r e g n e n o l o n e t o an Intermediate of the S t e r o i d o g e n i c Pathway, P r o g e s t e r o n e , by Ki-MSV/MMCVt r a n s f o r m e d Rat A d r e n a l C o r t e x C e l l Lines.  113  T a b l e 5.1  F o c u s F o r m a t i o n I n d u c e d by 2-1 i n P r e v i o u s l y I n f e c t e d Adrenal Cortex  124  Table  A n c h o r a g e I n d e p e n d e n t Growth o f 2-1/MMCVi n f e c t e d Adrenal Cortex C e l l s  129  Growth o f 2 - 1 / M M C V - i n f e c t e d A d r e n a l Cortex C e l l s i n the Presence o f D i f f e r e n t Serum S u p p l e m e n t s  132  I n h i b i t i o n o f DNA S y n t h e s i s by F o r s k o l i n i n t h e Y-1 and Y - 1 Lines  149  Measurement o f S t e r o i d P r o d u c t i o n from Y-1 C e l l s and V i r u s I n f e c t e d D e r i v a t i v e s by A c i d I n d u c e d F l u o r e s c e n c e  152  A n a l y s i s o f the R e l a t i v e Production of the S t e r o i d P r o d u c t s o f the V i r a l l y I n f e c t e d Y-1 C e l l L i n e s  159  Expression  171  Table  Table  4.4  of Tables  5.2 5.3  6.1  Cells  m H c v  Table  Table  Table  6.2  6.3  7.1  o f p27 i n Rat and Human  Cells  ix  List  of Figures  E a r l y P a s s a g e s o f K i M S V - i n f e c t e d Rat A d r e n a l C o r t e x C e l l s Grown w i t h 25% F e t a l B o v i n e o r 3% Horse Serum S u p p l e m e n t s  61  A n a l y s i s o f p21^ - - E x p r e s s i o n i n KiMSVi n f e c t e d and U n i n f e c t e d Rat A d r e n a l Cortex C e l l s  64  M o r p h o l o g y o f K i - M S V - i n f e c t e d Rat A d r e n a l C o r t e x C e l l s a t E a r l y and L a t e Passages  68  E x p r e s s i o n o f V i r a l p21 - - i n E a r l y and Late Passage C u l t u r e s o f K i - M S V - i n f e c t e d Adrenal Cortex C e l l s  70  F i g u r e 4.1  Diagram  o f MMCV  78  F i g u r e 4.2  F o c u s F o r m a t i o n i n E a r l y P a s s a g e Rat Adrenal Cortex C e l l s I n f e c t e d with Ki-MSV, MMCV o r B o t h  82  Overgrowth o f I n f e c t e d A d r e n a l Cortex C u l t u r e s by M o r p h o l o g i c a l l y T r a n s f o r m e d Cells  87  Serum S e n s i t i v i t y o f t h e T r a n s f o r m e d M o r p h o l o g y I n d u c e d by ras. and myc i n Rat Adrenal Cortex C e l l s  90  Serum Dependence o f t h e E x p r e s s i o n o f t h e A l t e r e d M o r p h o l o g y i n Ki-MSV/MMCVi n f e c t e d Rat A d r e n a l C o r t e x C e l l s  92  F i g u r e 3•1  Figure 3.2  Figure 3.3  Figure 3.4  Figure  4 .3  F i g u r e 4.4  Figure  Figure  4 .5  4 .6  a  r  Figure 4.8 Figure 4.9  of the P r o v i r a l  a  s  form  M o r p h o l o g i c a l Response o f Ki-MSV/MMCVi n f e c t e d Adrenal Cortex C e l l s to Purified  Figure 4.7  s  Growth F a c t o r s  A n c h o r a g e I n d e p e n d e n t Growth o f Ki-MSV/MMCV-infected A d r e n a l Cortex C e l l s Formation o f a Spontaneous Focus i n t h e P r e s e n c e o f 5% HS. A n a l y s i s o f p21 - -£ E x p r e s s i o n i n Ki-MSV/MMCV-infected Rat A d r e n a l Cortex C e l l s . r  95 100 102  a  105  X  F i g u r e 4 10  D e m o n s t r a t i o n o f t h e P r e s e n c e o f v-myc i n T r a n s f o r m e d C e l l L i n e s D e r i v e d from the K i - M S V / M M C V - i n f e c t e d Rat A d r e n a l Cortex C u l t u r e .  1 07  Expression of p57 SZ : i Transformed C e l l L i n e s D e r i v e d f r o m Ki-MSV/MMCVi n f e c t e d Rat A d r e n a l C o r t e x C u l t u r e s .  110  C e l l M o r p h o l o g y i n MMCV- o r Ki-MSVi n f e c t e d Rat A d r e n a l C o r t e x C e l l s S u p e r i n f e c t e d with the s r c Containing R e t r o v i r u s 2-1 .  123  C o l o n y F o r m a t i o n i n S o f t Agar by t h e 2-1s u p e r i n f e c t e d Adrenal Cortex C u l t u r e s .  128  Growth Rate o f 2 - 1 / M M C V - i n f e c t e d A d r e n a l Cortex C e l l s i n the Presence o f High or Low Serum S u p p l e m e n t s .  131  Examination o f Cloned C e l l Lines Derived from t h e 2 - 1 / M M C V - i n f e c t e d A d r e n a l Cortex C u l t u r e s f o r E l e v a t e d pSO^ ^ Kinase Activity.  135  S o u t h e r n A n a l y s i s o f t h e 2-1/MMCVi n f e c t e d Adrenal Cortex C e l l Lines f o r the P r e s e n c e o f v-myc.  137  S t e r o i d o g e n i c Pathway o f Y-1 A d r e n o c o r t i c a l Tumour C e l l s .  144  Figure 6  Morphology  148  Figure 6  Demonstration P r o d u c t s from Cell Lines.  F i g u r e 4 11  F i g u r e 5.1  Figure  5.2  F i g u r e 5.3  Figure  5.4  v _  c  n  1  F i g u r e 5.5  Figure  Figure  6. 1  6.4  Figure 7 Figure 7  o f Y-1 and y _ - |  0  M M C V  Lines.  of Comigration of Steroid t h e Y-1 and Y-1MMCV 155  Comparison o f the S t e r o i d Produced b by t h e Y-1, Y - 1 C e l l Lines  Products and Y - 1  m m c v  K i _ M S V  158  S u r v e y o f Rat P r i m a r y C u l t u r e s f o r t h e E x p r e s s i o n o f na_s.-related P r o t e i n s .  170  Rate-zonal F r a c t i o n a t i o n of L S ] m e t h i o n i n e L a b e l l e d Rat Lung F i b r o b l a s t Lysate.  174  J  Figure 7  Tryptic  Figure 7 and  Peptide  methionine p21 . J i a s  Maps o f p27 and p21 - c  Pulse-chase  a  §  177  o f p27 1 80  xi  Figure  Figure  7.5  8.1  Examination in a Ki-MSV  o f p 2 7 a n d p 2 i f f .43 Expression Transformed C e l l Line  Transformation Adrenocortical  Pathways Cells  of  Early  183  Passage 196  List  of Abbreviations  A  adenine  ACTH  adrenocorticotrophic  ATP  adenosine 5'-triphosphate  C  cytosine  Ci  Curie  cpm  counts per minute  dCTP  deoxycytosine  DMEM  Dulbecco's modified  DNA  deoxyribonucleic  EDTA  d i s o d i u m e t h y l e n e d i a m i n et e t r a a c e t i c  EF-Tu  elongation  EGF  e p i d e r m a l growth  FBS  fetal  FGF  fibroblast  FSV  Fujinami  xg  times g r a v i t y  GDP  guanosine 5'-diphosphate  GTP  guanosine 5'-triphosphate  Ha-MSV  Harvey murine sarcoma  HPLC  high  HS  h o r s e serum  IGF-II  insulin-like  Kb  kilobase  Kd  kilodalton  Ki-MSV  Kirsten  LTR's  long  hormone  5'-triphosphate Eagle's  medium  acid ac  factor-Tu factor  b o v i n e serum growth  sarcoma  factor virus  virus  performance l i q u i d  growth  factor  m u r i n e sarcoma  terminal  chromatography  repeats  two  virus  xiii  uCi  micro-Curie  MEM  minimal  mM  millimolar  Mo-MLV  Moloney murine leukemia  Mo-MSV  M o l o n e y m u r i n e sarcoma  Mr  relative  NaDOC  sodium  NRK  normal r a t k i d n e y  PAGE  polyacrylamide gel  PBS  phosphate buffered  PDGF  platelet-derived  Pi  inorganic  PIP ES  1 , 4-piperazinediethanesulfonic  R Rig  rabbit  RIPA  radioimmunoprecipitation  RNA  ribonucleic  RNaseH  ribonuclease H  RSV  Rous s a r c o m a  S  sulphur  SDS  sodium d o d e c y l  sulphate  SHE  Syrian  fibroblasts  TCA  trichloroacetic  TGF  tumour g r o w t h  TP A  tetradecanoylphobol-acetate  w/  weight  V  Eagle's  medium  virus virus  mobility  deoxycholate  electrophoresis saline  growth  factor  phosphate  anti-rat  immunoglobulin assay  acid  virus  hamster  acid  factor  t o volume  acid  Acknowl edg ements  I  would  supervisory Keith  Humphries  this  I  express  for their  and  and  also  like  Auersperg,  enthusiasm supplied my  so  this  t o thank  a l l the  work and  my  f o r s h a r i n g her  c r i t i c i s m and  Ian M c C l a r e n  Judith Black,  help  for their  and help  and  many t a n g i b l e and Dennis Dixon express  my  friendship, typed a l o n e.  my  and  Horvath.  a scientist:  always  twice served  such  my  on  and  such  a  Ehleen  Hinze  and  e n c o r a g ement; C r a i g Siemens t h a t was  given  Biochemistry  freely;  Finally  I would  irascibility  and  departments  especially:  to Kathy Dobinson  p a t i e n c e with with  who  encouragement have a l w a y s h e l p e d  indebtedness  thesis  people  humour  f o r being  i n t a n g i b l e ways, Dan  done.  has  has  receptive listener;  members o f t h e M i c r o b i o l o g y and whose f r i e n d s h i p  good  Weinmaster,  for their  critical  e x p e r t i s e and  Weeks, who  committee at s h o r t n o t i c e with  and  other  system,  apt  friend  and  s u p e r v i s o r , whose  g r o w t h as  a c c u r a t e and  Gerry  and  work t o be  Gerry  supportive  and  Tony Pawson, my  Dr.  Dr.  J u l i a Levy  my  the s u c c e s s f u l completion  freely;  enthusiasm;  a p p r e c i a t i o n to  considerable efforts  t o l e r a n c e allowed  would  N.  sincerest  George Spiegelman,  t o Dr.  have c o n t r i b u t e d t o t h i s Dr.  my  t h a t have underpinned  thesis,  patience  to  committee Drs.  evaluations of  like  Vera like  i n so  Webb, to  f o r her  unfailing  and  having  c a r e f o r l o v e and  for  friendship  I would l i k e t o thank my p a r e n t s and f a m i l y whose l o v e and s u p p o r t s t a n d s w i t h me e v e r y day o f my l i f e .  1  CHAPTER 1  INTRODUCTION 1.1  Retroviruses  1.1.1  Classification  The  retroviruses include  RNA  genome and  can  be  a RNA-dependent  subdivided  i n t o three  O n c o v i r u s e s , which closely  derived  family  i ) the  the o n c o g e n i c v i r u s e s  work i n t h i s subfamily,  and  other  v i r u s ; and i i i )  t h e s i s uses  viruses  the O n c o v i r u s e s ( f o r a  e t a l . , 1985).  genome o f a r e p l i c a t i o n  oncoviruses The  encodes  contains  three  gag gene e n c o d e s  p o l codes f o r r e v e r s e  DNA  polymerase.  and  the c o d i n g  structural repeats  These regions  genes  the group  promoters,  r e t r o v i r u s of the  present  g a g , p o l and  associated  genes  antigen,  i n the v i r a l  t r a n s c r i p t a s e or t h e are arranged  contain  e n h a n c e r s and  between  two  RNA-dependent  5'-gag-pol-env-3'  long  as s i g n a l s f o r the r e p l i c a t i o n  The  terminal  t h e e l e m e n t s o f the  terminators  env  membrane  are not u s u a l l y o v e r l a p p e d .  are l o c a t e d  (LTR's) which  competent  s t r u c t u r a l genes  the e n v e l o p e p r o t e i n s  and  well  subfamilies:  This  a  Structure The  env.  The  from t h e f i r s t see Weiss  1.1.2  polymerase.  o r s l o w v i r u s e s , s u c h as v i s n a  Spumaviruses.  review  includes  DNA  which c o n t a i n  r e l a t e d n o n o n c o g e n i c r e t r o v i r u s e s , i i ) the  Lentiviruses, the  the v i r u s e s  viral  o f t r a n s c r i p t i o n as ( s e e Varmus  and  2  Swanstrom  i n Weiss  retroviral  genome  The 1985)  virion  bound  (Panganiban  particle  i s composed  nucleocapsid.  e_t a_l. , 1985 ) and  The  t o t h e gag  proteins,  two  strands  messenger RNA 3  1  can be used  particle  contains  and  the p o l p r o d u c t .  i s a 60  are attached  they c o n t a i n  The  purified  to d i r e c t  products d e f i n i n g  in vitro  t h e genomic  responsible  membrane and receptors 1.1.3  initiating  i s t a k e n up  released both  into  by  required  the c e l l  as w e l l  and  ends.  1  Each  m e t h y l a t e d cap  the v i r i o n o f gag  i n the v i r i o n  particles  and  a  particles and  gag-pol  sense.  The  membrane  and  of the n u c l e o c a p s i d  w i t h the  to the  cellular  integration.  linear  The  and  bound  to the c e l l  surface,  the v i r a l  cytoplasm.  o f the v i r a l into  double  or c i r c u l a r  junction  between  contents are  Reverse  DNA-dependent DNA  for replication  as e i t h e r  has  as a RNaseH a c t i v i t y ,  i s i n i t i a l l y copied  exist  i n which  infection.  particle  the c e l l  RNA-dependent  activities  RNA  genomic  eukaryotic  as p o s i t i v e  of the v i r a l viral  5  RNA  Replication Once t h e v i r a l  it  from  RNA  for association  binding  a 5*  synthesis  env gene p r o d u c t s a r e embedded are  their  and  The  dimer,  resembles  i n that  RNA  t o 70S  near  molecules c l o s e l y  polyA t a i l .  et a l . ,  t h e genomic  the  RNA  ( f o r a r e v i e w see Weiss  nucleocapsid  virus  t h e two  1984).  envelope  of each  of  Temin,  of the  o f a membrane-derived  RNA  RNA  and  integration  transcriptase  has  polymerase a l l of which  genome.  The  s t r a n d e d DNA,  are  genomic which  can  molecules before t h e two  LTR's a p p e a r s  to  3  act  as t h e s i g n a l  genome i n t o 1984)  to allow  the host  cell  where t h e v i r a l  Virions  bud from  infection  (Panganiban  membrane  so t h a t  result in cell  form o f the p r o v i r u s  to daughter  Most p r o v i r u s e s  cells  integrate  The i n t e g r a t i o n  as a p a r t  expressed  conditions.  site  superinfection  by v i r u s e s  that  o f t h e env p r o d u c t  saturation  o f the c e l l  to  retroviruses  combined genes,  with  1.2  The classes in  vitro  o f most  genome. the host  becomes a c l o n a l The  provirus  environmental inhibits The  to r e s u l t i n a  r e s u l t i n g i n a block  receptors.  of the v i r u s e s  t h e use o f oncogene  to  does n o t e x t e n d These  properties,  to express  foreign  containing  the e f f e c t s of s p e c i f i c  of cultured  stably  oncogenes  cells.  Retroviruses  oncogenic based  into  analysis.  appears  use o t h e r  to e l u c i d a t e  Oncogenic  of the host  to s u p e r i n f e c t i o n  the a b i l i t y  the phenotypes  i s usually  use t h e same r e c e p t o r .  receptors  block  that  has a l l o w e d  retroviruses on  This  retroviral  i n f e c t i o n by a r e t r o v i r u s  expression  reinfection.  expressed.  lysis.  of the p r o v i r u s  l a r g e l y independently Productive  and Temin,  e s s e n t i a l l y randomly  marker and c a n be mapped by S o u t h e r n is  of the p r o v i r a l  genes can be a c t i v e l y  the c e l l  integrated  transmitted  DNA.  genome  does n o t u s u a l l y  The  integration  retroviruses  can be s u b d i v i d e d  on t h e i r r e l a t i v e a b i l i t i e s  and t h e r a p i d i t y w i t h w h i c h  animals.  The a c u t e l y  transform  cells  oncogenic  in vitro  they  to transform form  retroviruses  and c a u s e  tumours  i n t o two cells  tumours i n  can r a p i d l y  i n animals with a  4  short be  latent  able  to  requires 1.2.1  that and  period.  transform  a  long  Acutely  are  required  v i t r o ,  oncs) , but  do  not  rather  are  are  not  nonconditional  found  to  be  mutants  in  the  derived  sequences,  connection  between  the  sequences v i r a l As  a  able  genes  to  the  genes  c e l l u l a r  a  gene  lost be  in  virus.  of  of  the  oncogenes  (v-  (c-oncs  of  conditional  to  the  v-onc  by  the  a  the  some new  requires  that  trans  by  RSV  are  a  or  c e l l u l a r  is  the  novel  the  and  retrovirus  by  of  clear  the of  or  and  a c t i v i t y  mapped  naturally  except  oncogenic  genes  inactivated  All  vivo  c e l l u l a r  replication  in  in_  sequences,  acquiring  but  sequences  v i r a l  replacement  supplied  retroviruses  helper  to  any  demonstrating  oncogenic  or  from  have  autonomously  retrovirus.  transforming require  for  acutely  replicate  competent  results  required  result  structural the  usually  appear  c e l l s  v i r a l  transforming  presence  process  of  poorly  called  studies  retroviruses  The  contain  the  normal  c e l l u l a r l y  transformation.  in  derived  from  Genetic  oncogenic  not  tumourigenesis  transformation  sequences,  derived  proto-oncogenes).  acutely  rapid  appear  and  retroviruses  for  These  vitro  does  Retroviruses  oncogenic  but  retroviruses.  in  subclass  period.  Oncogenic  acutely  other  c e l l s  latent  The  in  The  of  the  sequences. no  longer  the  acquisition  of  replication  occurring, defective  acutely and  5  1.2.2  O r i g i n of v-oncs Although  Huebner and had  been  the i d e a  Todaro  derived  of oncogenes  i n 1969,  from f i n e  transformation-defective pncs r e m a i n e d genomic  DNA  uninfected the  v-onc  that  and  and  animals, contained o f RSV.  1985).  distinct,  (see  c-onc  c-oncs are h i g h l y  sequences  that  Slamon and  c-onc  derived  and  development  C l i n e , 1984).  They  p r o d u c t s may required  be  involved  to allow  from  v-oncs demonstrated from c e l l u l a r  genes were  t h e y were  s t r u c t u r a l gene.  conserved across  Varmus i n Weiss  the  derived  homologues t o w h i c h  r e s p o n s e s t o exogenous  functions  o f t h e v-  were r e l a t e d t o  species  e_t a_l. , 1 985 ).  o f some o f the c - o n c s a p p e a r s t o be  through growth  of  A l t h o u g h many o f t h e v - o n c s examined  B i s h o p and  cellular  the o r i g i n  cells,  Examination of other  t h e y a l l had  expression  existence  studies  mammalian  more c l o s e l y r e l a t e d t h a n t o a v i r a l The  by  s u b s e q u e n t l y shown t h a t  a l l v - o n c s were a p p a r e n t l y  (Bishop,  proposed  evidence of t h e i r  mutants,  I t was  of both avian  been  s t r u c t u r e mapping  viral  obscure.  had  (Muller  barriers  The regulated  e t a l . , 1982,  seem t o be a b l e  1983;  to modify  s i g n a l s , i n d i c a t i n g that  the  i n important regulatory  normal  cellular  growth  and  differentiation. 1.2.3  Chronic/leukosis The  r e m a i n d e r o f the. o n c o g e n i c r e t r o v i r u s e s do n o t  transform after  Viruses  cells  long  replication  i n c u l t u r e and  latent periods. competent  and  only  These contain  c a u s e tumours  viruses only  in_ v i v o  are u s u a l l y  the normal  retroviral  6  genes to  ( W e i s s et a l . , 1 9 8 5 ) .  activate  virus  The  usually  transcription  al.,  that  provided also  be  from t h e c e l l u l a r signals  i n c-onc  involved  in  Retroviral  cellular  usually  sequence;  and  signals  (Payne been  chromosome Yang  e t al_. , 1 9 8 5 ) , the c-oncs  in addition  viral  LTR.  results  and could  i ) truncation  point  involved  c-onc  The  changes  o f the  gene;  sequence  m u t a t i o n s i n the  by a gene.  i n a c t i v a t i o n o f the  i n t h e genes  o f the gene p r o d u c t s .  from  o f t h e s e genes  i n a number o f  t o the o v e r e x p r e s s i o n  s t r u c t u r a l changes  t o be d e r i v e d  of transduction  iii),  gene,  been  after  o f the normal  mutations are u s u a l l y  have  providing  These mechanisms have  a l l appear  include:  These  function  by  Functions  process  replacement of part  The  activate  regulatory  evidence that  oncogenes  The  the r e t r o v i r u s  different  can  The  carcinogenesis.  t h e gene w h i c h may  ii),  normal  Oncogenes  retroviral genes.  able  et a l . , 1 9 8 1 ; Neel et  1984;  some o f t h e e a r l i e s t  The  into  normal  et a_l. ,  and  gene e i t h e r  (Hayward  activation  (Hayday  t o be  to t r a n s f o r m a t i o n .  n e a r a c-onc  O n c o g e n e s - O r i g i n s and  1.3.1  leads  1 9 8 2 ; Hann e t a l . , 1 9 8 3 ) .  translocation  in  integrates  promoter  implicated  appear  use a number o f mechanisms t o  1 9 8 2 ) o r by d i s r u p t i n g  e_t__al.,  1.3  viruses  the d y s r e g u l a t i o n  stronger  viruses  c - o n c s d i r e c t l y by a l t e r i n g t h e i r  regulation. achieve  These  o f the gene by  a f f e c t the normal  effects  most c l e a r l y d e m o n s t r a t e d w i t h  the  o f the  v-src  and  changes v-ras.  v  The  v-src  product  the  sites  responsible e_t a l . ,  product  (Iba  1985).  Work w i t h  r a s gene i s n o t of  a v-ras  Many o f demonstrate that  an  (Hunter  and  these  the  EGF  other  growth  fms  inherent  The  the  unknown, but  they  of growth  which  are  they  factor  to be  receptor al. ,  which  1983;  genes  activity.  t o be  kinase  fall  domain  into and  a truncated  a  this erbB.  version  Downward  thought  t o be  altered versions  activity.  t o be  involved  v-sis  the  i t s effect  i s a tyrosine kinase et.al.,  1983).  in  in  oncogene p r o d u c t  is  the  derived  through  (Niman,  have to  are  the cells  platelet  of  of  f a c t o r M-CSF.  oncogene p r o d u c t s  differentiation The  that  c-fms i s t h o u g h t growth  of  et a l . ,  et a l . , 1 9 8 5 )  (Sherr  thought  to e x e r t  The  a l l demonstrate  that  tyrosine kinase  and  isolated  1984;  a homologue o f  Waterfield  c-  mutations  al.,  et  expressed.  (PDGF) and  of a  overexpression  homology i n the  for a hemopoietic  are  of  Courtneidge,  f g r , f p s / f e s , a b l , fms  are  other  regulation  thought  ros,  tyrosine kinase  the  as  have been  The  1985).  ros  1984;  overexpression  tyrosine kinases  factor receptors  r o l e s of  c-src  tyrosine kinase  (Ullrich  receptor  the  several activating  that  of s t r u c t u r a l  and  r e g u l a t i o n of  transforming  genes erbB i s thought  and  encode  intrinsic  receptor  loss  1984).  oncogenes  s r c , yes,  the  shown t h a t  contains  the  a c t i v a t e d by  ejb a l . ,  Bolen  potently  Cooper,  1984)  an  has  Wilkie,  encode  degree  Of  1984;  ras  as  the  certain  group are  for negative  gene which  ( S p a n d i d o s and  genes  is constitutively  7  the  1984;  growth PDGF R o b b i n s et_  8  The  oncogene  include, myb  myc,  that  are l o c a t e d  f o s and p 5 3 .  myb,  The  Klempnauer  possible  DNA  e t a_l. , 1 9 8 4 ) , a l t h o u g h  The r o l e s  although  of these  i t appears that  specific myc  cooperate  (Donner e_t a l . , i t has n o t y e t been  can r e n d e r  cells  1 9 8 6 ; Balk  (Stern  and  a group o f oncogenes t h a t  p53  define  i n transforming  e_t a l . ,  nonestablished are l o c a t e d  defined  group.  family,  the s e r i n e / t h r e o n i n e  The genes  o f the g e n e s  the  transforming  the  families  homologues, described,  in this  cells  f o r example  process  Neel et a l . ,  mil/raf,  genes  erbA and  initially although  that  cells,  described  as  i n some o f  do n o t have  have a l s o  mos.  viral  been  (Taparowsky e t a l . , 1 9 8 3 ) .  o f the v i r a l  by t h e p o o r l y  gene t h a t was  the r a s  o n c o g e n e s were o r i g i n a l l y  by i n t e g r a t i o n  cellular  group i n c l u d e  Oncogenes  as homologues  Transformation  N-ras  with  loosely  g r o u p were  cellular  can  to a  kinases  b u t can t r a n s f o r m  The c e l l u l a r  occur  in this  et a l . ,  i n the c y t o p l a s m .  gene o f a r e t r o v i r u s ,  related  Cellular  described  hypersensitive  growth f a c t o r s  The r e m a i n d e r o f t h e o n c o g e n e s b e l o n g  1.3.2  and  oncogenes are not understood  myc  o n c o g e n e s whose p r o d u c t s  Most  o f t h e myc  t o d e m o n s t r a t e any s e q u e n c e s p e c i f i c i t y f o r  binding.  1985).  i n the n u c l e u s  products  o n c o g e n e s have been shown t o b i n d  1982;  to  products  first  oncogenic  retroviruses  can be o n c o g e n i c a l l y f o r c-myc  1982), w h i c h had a l r e a d y  and  oncogenes.  o f the n o n t r a n s f o r m i n g  described  isolated  virus  activated.  (Hayward been  seems t o  near a This  et a l . ,  identified  1981; as the  9  cellular 1982).  homologue o f Further  It not  has  viral  isolated  from  activity  et _ a l . ,  or  tumours i s o l a t e d This  search  f o r an  implicates  due  in in vitro  1984),  homologous t o v - o n c s  1984).  isolate  humans or a n i m a l s  directly  other  this  lck  have had  indirectly  assays,  that  of  including  neu  ( V o r o n o v a e t a l . , 1985)  and  Many o f the  are  c-oncs  a c t i v a t e d forms with  conferred  considerable  formation  N-  associated formed  (Slamon  o f o n c o g e n e s as  i n the  that  spontaneously  work has  are  tumours  to a c t i v a t i o n  animals  oncogenes  c-oncs  formed  humans or  the  (Westaway e_t  mechanism.  from  understanding  e_t a l . ,  cellular  ras  novel  spontaneously  (Schwab e t a l . , 1 9 8 3 a ) .  either  revealed  a c t i v a t e d by  been p o s s i b l e to  from  (Schecter myc  be  homologues  transforming  has  (Vennstrom  ( N i l s e n e t a l . , 1 985 ) and  t h a t can  1986),  v-myc oncogene  examination  oncogenes, c-erbB al.,  the  et a l . ,  validity  on  the  i t directly  of  tumours i n  vivo. 1.3.3  Ras The  mammalian  approximately al.,  1979)  The  21,000 d a l t o n s  that  (Willingham  r a s genes encode p o l y p e p t i d e s  are  a s s o c i a t e d with  e t a l . , 1980)  r a s genes c o m p r i s e  at  least  and  two  the  r a s gene  includes  three  (Young  and  bind  e_t a l . , 1979; the GTP  plasma  ( S h i h e t a l . , 1980).  a f a m i l y o f genes o f which  H a - r a s - 2 and  family belongs  a number o f o t h e r  Ki-ras-1 .  to a gene genes w i t h  S h i h e_t  membrane  a c t i v e members; H a - r a s - 1 , K i - r a s - 2  pseudogenes,  of  and  I t seems  superfamily some  there  that  structural  are  N-ras, that  10  homology or w i t h homology. the  The  that  membrane by  anchors  Localization  and  f u n c t i o n a l (GTP  mammalian r a s gene p r o d u c t  cytoplasmic  chain  structural  i t to  i n the  binding)  i s associated  a c o v a l e n t l y attached  the  membrane  (Sefton  membrane i s a p p a r e n t l y  with  fatty  acid  e t a l . , 1982).  important  for  the  p S S  activity  o f p21  in  acid binding  fatty  function  as  , as  measured  mutagenesis appears  by  of  the  to r e s u l t  transforming  residue  involved  in a loss  activity  of  (Willumsen  et  a l . , 1984). The high  r a s gene p r o d u c t s  affintiy  specificity The  ras  GTP  that  GDP  and  for nucleotide  to c l e a v e  (McGrath et  has  adenylate  the  and,  by  regulatory  then  p21  I Q  .  p21  r a s genes a r e  1981)  1983).  and  to  produce  p21  i s thought  r a s  This  a c t i v a t i o n of  in binding  of  the  GTP  bound  in a l l eukaryotic  including single cell Pawson e t  mammals  I t would  hydrolyze  to  and GTP  would  .  D e f e o - J o n e s e t a l . , 1983),  Weinberg, al.,  r a s  phosphate  h y d r o l y s i s of  present  have been examined  (Reymond _e_t aJL. , 1984; 1984;  The  to  pathway, i n w h i c h a c t i v a t i o n  pathway r e s u l t s  i n a c t i v a t e the The  that  of  a  high  ability  hormonal  analogy,  of  very  Sweet e t _ a l . , 1 9 8 4 ) .  with  to a s i g n a l t r a n s d u c t i o n  p r o t e i n s with  ( S h i h e t a_l. , 1 9 8 0 ) .  terminal  a l . , 1984;  been a s s o c i a t e d  cyclase  activation  and  intrinsic  belong the  binding  binding  a l s o have an  appears  activity  GTP  for guanosine n u c l e o t i d e s  products  Pi  are  a l . , 1985; insects  (Ellis  a l s o appear  et that  organisms  Powers e t a l . ,  (Shilo  a^l. ,  species  1981;  and Shimizu  r a s genes a r e  very  et  11  widely  expressed,  translated  in a l l cells  in  Barbacid,  to  be r e g u l a t e d  is  relatively  ras  throughout  small.  the c e l l  i s a s s o c i a t e d with liver  appears  transcription, (Muller seen  et  (  R  important  e  y  m  o  n  to note  The  appears  to d i v i d e .  and  growth  induced and this  Kirsten spectrum  i n both 1986),  in cells implies a  i n the c o n t r o l of  differentiation.  by m u r i n e 1967).  murine  Iti s  1985).  expression  f o r t h e r a s gene p r o d u c t s  as t h e t r a n s f o r m i n g  process,  discoideum  ( S e g a l and S h i l o ,  This widespread  r a s genes were f i r s t  Mayer,  r e g u l a t i o n c a n a l s o be  t o be i n c r e a s e d  genes o f m u r i n e r e t r o v i r u s e s . isolated  The  small  t h a t t h e r a s genes a r e e x p r e s s e d  expression  cell  1983).  i s relatively  Pawson e t a l . ,  1984;  although  role  ,  e m b r y o g e n e s i s and  of the D i c t y o s t e l i u m  and n o n d i v i d i n g c e l l s  fundamental  of ras  s t i m u l a t i o n of  et a l .  throughout  dividing  stimulated  (Goyette  Developmental  et a l . ,  d  c y c l e but the v a r i a t i o n  the e a r l y  t h e change  1983).  i n the e x p r e s s i o n  ras  appears  t o be some r e g u l a t i o n o f t h e  although  al.,  (reviewed  o f t h e r a s genes  An i n c r e a s e i n t h e l e v e l  i n a regenerating  there  t r a n s c r i b e d and  w h i c h have been examined  genes a r e a l s o e x p r e s s e d  again  p 2 3  are apparently  The e x p r e s s i o n  1986).  transcription growth  as t h e y  leukemia  described  as t h e t r a n s f o r m i n g  The v i r u s e s were filtrate  from  solid  v i r u s e s (Harvey,  initially tumours  1 9 6 4 ;Kirsten  The two v i r u s e s t h a t were d e r i v e d  Harvey murine  sarcoma v i r u s  sarcoma v i r u s  o f tumours i n c l u d i n g  (Ha-MSV) and  (Ki-MSV), both carcinomas,  from  c a u s e a wide  s a r c o m a s and  12  leukemias. that  Both  viruses  i s d e r i v e d from  Kirsten  c o n t a i n a unique  the c e l l u l a r  ( c - K i - ) r a s genes  (Ellis  Harvey  but r e l a t e d  gene  (c-Ha-) r a s o r  e t a l . , 1980; A n d e r s e n  et  a l . , 1981 ) . These because  two genes have a t t r a c t e d  t h e y were t h e f i r s t  associated of c e l l  for  actively  cells  oncogenes  w i t h human t u m o u r s .  and  lines  considerable attention  d e r i v e d from  t o be  directly  The genomes o f human human  tumours were  t r a n s f o r m i n g genes by t r a n s f e c t i o n  and s e l e c t i o n  f o r transformed  cells  examined  into  The g e n e s t h a t  were d e r i v e d from  experiments  that  t e c h n i q u e were a c t i v a t e d  the c - r a s genes Pulciani 1982).  I t i s of p a r t i c u l a r contained s p e c i f i c  transfection this  detectable This and  and  provided  forms o f  point  that  t h e r a s genes  m u t a t i o n s , and  using the p u r i f i e d  assay  the f i r s t  et a l . ,  gene  indicated  (Seeburg  direct  link  being  e t a l . , 1984). between a c-onc  cancer. evidence  f o r the involvement  has been d e r i v e d from  papillomas induced  carcinogens.  an  importance  i n the t r a n s f e c t i o n  tumourigenesis  chemicals  e_t a_l. , 1983; T a b i n  change was r e s p o n s i b l e f o r t h e r a s genes  Further in  experiments  evidence human  the i n i t i a l  (Der e t a l . , 1982; G o l d f a r b e t a l . , 1982;  e t a l . , 1982; S h i m i z u  isolated  that  this  NIH 3T3  which c o n t a i n e d  human DNA.  used  tumours  that  activating  Treatment  by t r e a t m e n t  o f t h e r a s genes  analysis  of rodents  of rodents with  some  with mutagenic  c a n a c t as c a r c i n o g e n s , a p p e a r s mutation  o f tumours  i n one o f t h e r a s genes  to r e s u l t i n i n the c e l l s  13  that  form  al.,  1986;  as  the  tumour  cells  which  DNA  from  NIH  3T3  form  these cells  correspond  of  the  changing  and  1986).  used,  previously 1984).  The  carcinogenesis  In  Oncogenic amplification activating first ras  1982).  expressed  the  could this  into  initiated, activated  Balmain  et_ a _ l . ,  ras 1984;  a c t i v a t i n g mutations that  t o be  would  early  o f the  be  appear expected  events.  The  H a - r a s gene  ( Z a r b l e_t a _ l . , 1 985 ;  i n oncogenic  a c t i v a t i o n (Fasano  et  link  of tumourigenesis  vivo.  The  work a l s o  initiation  normal  implies  (Barbacid  that  cellular  coding  to occur  activated  also  genes r a t h e r  region  3T3  cells  ten  the  ras  1986). occur than  o f the g e n e s .  by by  It  normal  an was  c-Ha-  (Papageorge et a l . ,  the n o r m a l r a s gene had  gene.  activation  that  e_t al_. ,  o v e r e x p r e s s i o n o f the  t r a n s f o r m NIH  and  r o l e o f r a s genes i n  at l e v e l s approximately  oncogenically  transfection  genes  implied  o f the  I s o l a t i o n of  contain  o r 61  act  preneoplastic  a c t i v a t i o n o f c - r a s genes can  demonstrated  For  1983;  12  chemicals  r e g i o n s o f the r a s  l e s i o n i n the  allele  also  the  genes a c t i n tumour  the  Sukumar e t  two  close  Ha-ras s u p p o r t s  produce  l i m i t e d areas  1986),  implicated  can  appear  e i t h e r amino a c i d et a_l. ,  mutagenic  of changes  and  i n very  The  a n a l y s i s by  The  types  1983;  Pragnell,  the mouse s k i n .  that  Pragnell,  t o the  occur  Quintanilla  of  and  papilloma c e l l s  chemicals  mutations  and on  indicated  e_t a l . ,  to  al.,  papillomas  (Balmain  Quintanilla  papillomas  has  and  1985).  of oncogenesis  preneoplastic genes  g_t _ a l . ,  Zarbl  initiators  (Balmain  fold  Intermediate  higher  to  be  than  l e v e l s of  an  14  expression  will  result  transformation  (Winter  adrenocortical  tumour  amplified not  Winter  line  e_t a_l. ,  potential  1 985 ).  1986).  I n t h e Y-1 allele i s  but the a m p l i f i e d  lines  derived  mutation  from  does  George e t  tumours i t  i s amplified  (Pulciani  These r e s u l t s  mechanism by w h i c h  allele  e_t a l . , 1983;  one o f t h e r a s a l l e l e s  an a c t i v a t i n g  levels of  the c - K i - r a s  (Schwab  I n some c e l l  that  contains  cell  t o be mutated  1985).  appears  and P e r u c h o ,  and o v e r e x p r e s s e d ,  appear  al.,  i n intermediate  and a l s o  e_t a l . ,  1985;  i n d i c a t e another  the r a s proto-oncogene  c a n be  activated. The because  activating they  isolated  on  tumours  sparked  regulatory  EF-Tu.  As s e q u e n c e  transducin et  i n vivo.  The b i n d i n g  binding  demonstrate  regions  two r e g i o n s  The s p e c i f i c i t y interest  interest o f genes  o f the  as t o t h e i r  effect  o f GTP was r e m i n i s c e n t  o f GTP  p r o t e i n s , t h e G p r o t e i n s , t r a n s d u c i n and information  on t h e o t h e r  GTP b i n d i n g  p r o t e i n s became a v a i l a b l e i t was p o s s i b l e t o some s e q u e n c e (Jurnak,  1985;  homology between  that  the three  1985).  areas  susceptible to a c t i v a t i n g  indicating  that  the areas  functional  importance  p21  Leberman and E g n e r ,  a l . , 1985 ; Tanabe e_t a l . ,  interesting  within  considerable  ras activity.  regulatory  of r a s are of great  are concentrated  from  mutations  mutations  r a s  ,  EF-Tu and  1984;  Lochrie  It i s particularly  o f homology mutations  span  the three  i n ras  o f homology a r e a l s o a r e a s o f  (McCormick  e t a l . , 1985).  15  by  The  ability  of  using  protein  expressed  protein  p21  i n a background  (McGrath et j Q . , s o u r c e s has  confirmed  synthesized  Manne e t  a_l. , 1 9 8 5 ) .  ras  of  the  that  as  had  of  GTP  evidence  presence  through  was  bound  (Der  et  physical  their  The  ( M c G r a t h e_t a l . , 1984;  the  reduction  activated  regulatory  indicated change to  that  in rate  1984).  a broad  GTP  has  not  (Der  12  and  could  codon This  these  altered has  61  would  hold  p21 the  imply in  r a s  normal  proteins. 61  has  c o r r e l a t i o n between  the  the  at  codon  ability  of  a_l. , 1 986 ).  been done f o r codon  of  nucleotides  significantly  stimulating  and et  for  et.  i n GTPase a c t i v i t y  s i m i l a r r e s u l t s may  implications  a l . , 1985 ; Der  o f mutants  hydrolysis  that  Examination  GTP  affinity  ras  simple  1984;  ( M c G r a t h ejb  et_ a l . , 1 9 8 6 ) .  i s no  analysis  The  al.,  hydrolyze  codon  longer,  ras  indicated  were not  range  cells  that  to  involved  3T3  seems l i k e l y  (Gibbs et  in  gene p r o d u c t s  hydrolysis  NIH  transform  similar  i n GTP  there of  activity  other  of a c t i v a t i o n -  Manne e t  both  Der  pathway t h a t of  ras  reduction  conformation  Analysis  activities  hydrolyzed.  binding  properties  a_l. , 1 986 ).  the  the  a v a r i e t y of  this  cells  and  ability  been shown f o r m u t a n t s a t  that  of  demonstrated  provide  from  a cycle  activated  a reduced  a l . , 1986), a l t h o u g h other  to  was  These r e s u l t s made i t p r o b a b l e  a l . , 1984 ; G i b b s ejt a l . , 1984  and  GTP  contaminating  i n mammalian  oncogenically  they  free  the  p r o d u c t s moved  inactivation  in bacteria  1984), and  proteins  the  to h y d r o l y z e  r a s  hold  12  Although  a  mutants i t  (Seeburg  r e s u l t s are  a mutant  not  et a l . ,  clear  but  16  they  seem to  hydrolysis has  may  not  interact  with  reflected  the  i n the  ras  examined and  ranging  Kaziro,  from  1985; Axel,  p21  proteins  r a s  yeast  RAS  GTP  mutations.  of  GTP  and  to  this  hydrolysis  It  affect  affect its ability i t regulates,  in rate  yeast  1985) and  to  a l . , 1985). yeast  RAS  1984).  I t has  I t has  to man.  is  (Srivasta  functionally, proteins  correspond  ras  to  but  i t appears  that  and  that  sea  the  able  to  although  there  f o r the  Kataoka  mutations  significant  that  (Kataoka  activity  mammalian  This has  s t r u c t u r a l conservation  in  activating  i n S_. c e r e v i s i a e  the  be  normal  a_l. ,  stimulate  e_t a l . , 1985 ).  to  et  the  Fukui  mammalian  that  have  been  squirt  a l . , 1985;  the  cyclase  correspond  not  to  i n yeast  so  (Beckner  for  genes a l s o  adenylate  have  genes a p p e a r  been d e m o n s t r a t e d  been d e m o n s t r a t e d  are  i t implies  The  that  e_t a l . , 1983;  to  (DeFeo-Jones et  that  modulate  and  functional  a l . , 1984)  growth r e g u l a t i o n  products  a l . , 1985)  (DeFeo-Jones  i t i s possible  i n mammalian  on  in a l l species  functionally substitute  genes  effects  activity  present  gene p r o d u c t s  mutations  as  that  Powers e t  conserved,  ras  proteins  the  of  a c t i v a t i n g m u t a t i o n s may  directly  change  highly  RAS  the  and  genes a r e  (Madaule and  the  a l t e r a t i o n in rate  a l . , 1985) . The  et  the  a d i r e c t e f f e c t of  that  conformation  r a s  that  be  been s u g g e s t e d  p21  et  indicate  G  the  (Toda e_t proteins  mammalian  and  adenylate  pombe  cyclase  result is interesting been  considerable  between  the  yeast  RAS  17  and  mammalian r a s p r o d u c t s ,  they  are a c t i v e It  of  has  emerged  and  yeast  (Gallwitz  1985)  that  apparently  that  growth t o be  cellular  from  the G  proteins,  EF-Tu, t h e r e a p p e a r  t o be  o t h e r genes b o t h  et a l . , 19 8 3 ) and  mammals  their  by  GTP  m e c h a n i s m ( s ) by  products  interact  as  a biochemical l e v e l ,  with  cell  of complexity  remains  of  binding proteins i s quite high.  which  the a c t i v a t e d  a r e a b l e to t r a n s f o r m c e l l s  some o f t h e a c t i v a t i n g  Axel,  Whether  the ras products  level  in  but  gene f a m i l i e s .  will  a level  the p o t e n t i a l  regulation  The  new  products  fundamental but  ( M a d a u l e and  t o t h e r a s f a m i l y o f genes  represent d i s t i n c t  a t as seen,  t h e r a s gene f a m i l y i s a s u b g r o u p Apart  are related  t h e s e genes and  i n which  have d i v e r g e d .  a supergene f a m i l y .  transducin  t h e r e g u l a t o r y pathways  ras  gene  i s unclear.  Although  c h a n g e s i n r a s have been a n a l y z e d  the lack  at  of knowledge of p r o t e i n s with  r* 3. s w h i c h p21 of The  interacts  has  prevented  any  real  t h e mechanisms i n v o l v e d i n r a s m e d i a t e d transformed  appears  t o be  phenotype induced  the r e s u l t  (Devouge et a l . , 1 9 8 2 ) . studied growth  i n some d e t a i l factors  The  early  by  of complex One  understanding  transformation.  the ras  oncogenes  biochemical  of the changes  i s the i n d u c t i o n  changes  that  to ras t r a n s f o r m a t i o n .  realization  that  t r a n s f o r m a t i o n by  the a c u t e l y oncogenic  retroviruses  of c e l l s  t o grow w i t h  a reduced  proposal  that  cells  resulted  serum  been  of t r a n s f o r m i n g  i n response  transformed  has  some of  i n the  supplement  could support  ability  led to  their  own  the  18  growth was  by  a u t o c r i n e process  postulated that  required for a  an  transformed  to s t i m u l a t e t h e i r  exogenous s i g n a l s .  transformed  with  growth  factors  transformed  v-mos  growth been et  a l . ,  DeLarco  been  1978).  from  virally  in stimulation  of  polypeptides involved  appears  e_t _ a l . ,  Todaro,  the transformed  1982)  1980; and  can  component,  added  to normal  sequenced  transformed  to act through Carpenter  be  (Derynck  in cells  which TGF-oC  EGF 1983 ;  i t i s a b l e to induce  factors  induced  the  et a l . ,  by  have  (Marquardt  phenotype.  phenotype i n normal c e l l s  aspects  when added  in  et a l . , 1 9 8 4 ) .  e x p r e s s i o n of  the  et a l . ,  1 9 8 6 ).  The  TGF-/3 , o f t e n a c t s t o i n h i b i t  growth  when  TGF-oi p o l y p e p t i d e p r e c u r s o r other  and  the  cells  a r e two  Transformation  and  Todaro,  cells  There  the absence of o t h e r growth  to  and  described for  19 8 3 )  and  et a l . ,  f o r ras  et a _ l . ,  (Todaro and  specific  (Anzano  EGF  induce  them  (Ozanne  t r a n s f o r m a t i o n of normal  fully  need  p r o d u c t i o n of t r a n s f o r m i n g  were a c t i v e  to produce the to  the  treating  cells  o f c o n d i t i o n e d medium  that  factors  to t r a n s i e n t l y by  It  1980).  obviating  cells  initially  (DeLarco  morphological  1984).  related  receptor  of  cells  purified  interact is  by  components  and  growth,  The  Todaro,  could produce  transformed  (TGF's) i s not having  transformed  own  1982).  transformation  The  cells  phenotype i n normal  et a l . ,  Kaplan  and  It is possible  medium c o n d i t i o n e d by  1980;  (Sporn  cells  m o d u l a t e t h e number  alone of  enhance t h e a c t i v i t y  (Rosenthal  ( M a s u i _et a_l. ,  EGF  r e c e p t o r s on  o f TGF-c< ( A s s o i a n  1 986 ) the  but  i s able  treated  et a l . ,  cells  1 984 ;  19 Massague,  1 9 8 5 ) . I t has  substitute  f o r TGF-^/  stimulating  cell  Expression differentiated  p r o v e n t o be  i n inducing  growth  (Assoian  of a c t i v a t e d phenotype  PC12  i n the i n d u c t i o n  phenotype The  PC12  (Bar-Sagi line  genes,  neuronal cell  even  phenotype  cells  activities not  from  This  normal  with  cells  infection  uninfected  calcium  associated infected  with  with  line  forms  arrest.  of the  In  other  of the a c t i v a t e d of the  cells  differentiated  ras  normal  express enzymatic  embryonic  derived  cells  from the  by e x p r e s s i n g  viruses  genes  differentiated  Ha-MSV o r Ki-MSV t h e c e l l s  r e s p o n d t o the tumour p r o m o t e r  (Yuspa et_  r e s p o n d t o an  by e n t e r i n g two  are  derived  of d i f f e r e n t i a t i o n  keratinocytes  and  adult  In k e r a t i n o c y tes  by r a s c o n t a i n i n g  concentration  the f u l l y  as t h e  1985).  can be s e e n i n r a t a d r e n a l  et, a l . , i n p r e s s ) .  The  and  of the  cycle  of aspects  associated  with  r e s p o n d t o TPA  calcium  cell  the r e s p o n s e to i n d u c e r s  1985).  The  differentiated  t r a n s f o r m e d by Ki-MSV, w h i c h  normally  (Wiebe  increased and  o f a more  the i n t r o d u c t i o n  t h e mouse s k i n ,  alters al.,  including  phenotype.  associated  cortex  types.  m o d i f y the  F e r a m i s c o , 1 985 ; Noda e t a l . ,  i n the l o s s  differentiated  and  t h e pheochromocytoma  t h e y e x p r e s s many a s p e c t s  results  cortex  can a l s o  i n response to a c t i v a t e d  t y p e s examined  genes  into  to  1984).  i s neuroectodermal i n o r i g i n  differentiate, ras  and  et a l . ,  o f many c e l l  o f r a s genes  f o r EGF  transformation  r a s genes  introduction results  possible  growth  arrest  normally  phenotype. arrested  When  i n high  by r e g r e s s i o n  to a  less  d i f f e r e n t i a t e d phenotype.  expression  o f oncogenic  differentiated  to r e p l i c a t e  1985;  Papageorge  that to  of c e l l s ,  as w e l l  e_t a _ l . , 1 986 ). I t i s a l s o  exogenous s i g n a l s  that  as t h e a b i l i t y o f  e t a_l. , 1 9 8 4 ; M u l c a h y  (Feramisco  the r a s products  indicate  r a s genes c a n m o d u l a t e t h e  phenotype  cells  These r e s u l t s  are able  to modify  or to obviate  clearly  cellular  t h e need  et a l . , indicated  responses  f o r such  signals. Myc  1.3.4  The  myc oncogene was o r i g i n a l l y  transforming (Mellon virus  gene o f t h e a v i a n  fibroblastic  leukemias  a_l. , 1980; E n r i e t t o  to  t h e myc gene  in  SDS-PAGE  cells  that  also  in vitro  d i r e c t l y implicates  transformation.  been a s s o c i a t e d the chicken  cells  with  by a v i a n  near  1982).  Activation  of bursal  virus.  (Hay ward  e t a l . , 1981; N e e l e t al_. , 19 82; e t a l . , 1982) .  to transform  c-myc  gene  lymphomas  The l e u k o s i s  t h e c-myc gene and t h e p r e s e n c e to modulate  product  and Hayman,  o f the avian  the i n d u c t i o n leukosis  mapped  t h e myc gene i n v i r a l l y  appears  Fung  o f t h e myc  both  (Ramsay  Mutations  o f the v i r u s  enhancer  1982;  The MC29  in vitro  (Ramsay e t a l . , 1980; E n r i e t t o  induced  integrates  and Hayman,  a l t e r the a b i l i t y  This  in  avian  a f f e c t the m o b i l i t y  1982).  has  e t a l . , 1981).  v i r u s , MC29  and sarcomas and can t r a n s f o r m  and h e m a t o p o i e t i c  et  as t h e  myelocytomatosis  e_t a _ l . , 1978; V e n n s t r o m  causes  described  virus  of the v i r a l  t r a n s c r i p t i o n o f t h e c-myc Payne e t a l . ,  gene  As  with  other  retroviruses, that  is  highly  (Vennstrom also now  three  v-myc  a_l. ,  to  1984)  and  overexpression associated  plasmacytomas  1982;  both  the  and  and  animal  e_t  and  resulting  gene  expression.  Nau  The  number e_t  a l . ,  oncogenes (Schwab  of  1986)  in  It myc  the  with  amplification highly  tumour  e_t a _ l . ,  associated  N-myc  and  is  can  1985 ). lung  that  The  of  increase  are  Lee  and  been  mouse  of  the  and  tumours  et  et  a l . ,  (Crews  1982).  et In  chromosomal normal  or  regulation  lines.* (Lee  et  cooperate  primary of et  when  of  inappropriate be  Amplification  to  1983;  to  (Nau  genes  there  have  In  shown  apparently  myc  gene  Amplification  genes  the  been  c e l l  carcinomas  and  largely  transformation.  has  a l . ,  the  the  The  which  et  Taub  alter  transformation  transformed  genes  gene  of  c-myc  increased  derived  appears  appears  in  kingdom  association  1982;  to  animal  tumours.  tumours  thought  c e l l u l a r  1982).  L-myc  of  transforming a  1985).  formation  human  the  _al. ,  involving  al_. ,  are  a  strong  with  the  (Schwab  N-  a  from  family  c-,  rearrangements  in  N-myc  rearrangements  Shen-ong  gene  a l . ,  is  l o c i  et  et  human  animal  c-myc  a  acutely  derived  (Nau  the  there  immunoglobulin a l . ,  Crews  c-myc,  of  with  chromosomal  1982;  L-myc  is  in  throughout  constitute  members  found  gene  conserved  et  appear  a l . ,  the  oncogenes  amplified a l . ,  with  c e l l  1984;  other  cultures  the  L-myc  gene  a_l. ,  1985)  and  the  c e l l s  is  become  metastatic.  overexpression responsible transcript  for and  or  misregulation  their protein  of  involvement products  of  the in the  normal  c-myc  normal  cell  level  gene growth  o f gene  appears  the  rate  perturbed  by with  of the normal growth.  by a v a r i e t y  1984). cells  1984; B l a n c h a r d  This  stimulation  to traverse  growing  cells  cell  cycle  The  overexpression to higher  1 984 ) .  no  densities, from  appears  a and that  in  e t a l . , 1985; Normal  myc  (Schwab e t  appears  of growth growth  factors  expression  not appear  cycle  (Greenberg  i n c-myc  although  of  synchronized  the growth line  during  e t a l . , 1985).  to enable  the parental  required f o r  expression  e t a l . , 1985; Hann appears  et a l . ,  t o be  as a n a l y s i s  t o be  arrested  e_t a l . , 1 9 8 5 ; M u l l e r  o f c-myc  significantly  gene  including  change  normal  (Bentley  can vary  transformation  c-myc  does  (Thompson  mRNA  the  the chromosomal  o f c-myc  the c e l l  showed  the  change  of agents  I t also  (Blanchard  Stimulation  i n the s t i m u l a t i o n  Ziff,  at both The  in  The  and t o c o n t a i n  e t a l . , 1985).  Expression  results  grow  changes  e t a l . , 1 985 ) .  cells  and  o f t h e c-myc  associated  to c e l l  regulated  suppressed  e t a l . , 1986).  1986; Yang  related  t o be  c a n be  1985; P i e c h a c z y k  rearrangements  et a l . , 1985).  regulated  that  to environmental  c a n be  half-lives  posttranscriptional level.  1 9 8 6 ; Remmers  regulation  20 m i n u t e  (Eisenman  appears  terminator  e_t a l . ,  al. ,  and  of degradation  response Dony  in vitro  t o be n e g a t i v e l y  transcription Groudine,  approximately  expression  transcriptional gene  have  the c e l l s rate  (Keath  to  does n o t et a l . ,  The  cell  tumourigenic high  lines  transformed  phenotype  efficiency  and form et a l . ,  (Keath  by c-myc d e m o n s t r a t e colonies in soft  1 9 8 4 ; Vennstrom  agar a t  e_t a l . ,  1984).  The t u m o u r i g e n i c  require  o n l y o v e r e x p r e s s i o n o f t h e n o r m a l myc genes  al.,  I t i s not c l e a r  1985).  coding  of overexpression  S t a n t o n _et _ a l . ,  The results  i n some s p e c i f i c  1984).  The i n t r o d u c t i o n the s e n s i t i v i t y factors  of r e l a t i v e l y  anchorage  independent  requirements hematopoietic  f o r PDGF  of the c e l l s  Rapp e t a l . ,  1985).  relatively  o f a myc  c a n be enhanced  cells  1 986 ).  The NRK  c-myc gene r e s p o n d  to the  of growth  factor  i n a v i a n and murine i n response  to the  myc c o n s t r u c t s ( B a l k e t a l . , directly  responsiveness  subtle morphological  introduction  (Armelin et a l . ,  Abrogation  The r e s u l t s  factor  line  to the e f f e c t s o f  ( S t e r n ejb a l . ,  and mesenchymal c e l l s  i n growth  i n the c e l l  o f an exogenous c-myc i n NRK  be seen  of oncogenic  cells  need  growth.  can a l s o  alterations  low c o n c e n t r a t i o n s o f EGF by  introduction  genes  1986;  by c-myc o v e r e x p r e s s i o n  e x p r e s s i n g the t r a n s f e c t e d  addition  (Lee e t  o f c-myc i n t h e NIH 3 T 3 c e l l  The e f f e c t  exogenous g r o w t h  to  transformation i n  (Patschinsky et a l . ,  phenotypic  t o be a d e c r e a s e d  increases  appears  1984).  appears  cells  to induce  t r a n s f o r m a t i o n induced  used.  lines  i f any c h a n g e s i n t h e myc  sequence a r e s u f f i c i e n t  the absence  lines  conversion of c e l l  a  oncogene  implicate  of c e l l s .  changes t h a t into  by t r e a t m e n t  1985;  t h e myc  The  result  from t h e  r a t embryo  fibroblast  of the c e l l s  with the  24 tumour p r o m o t e r  t e t r a d e c a n o y l p h o r b o l - a c e t a t e (TPA)  et  I t would  a l . , 1985).  myc  genes can  cells  result  The  the a b i l i t y products  59,000 and  62,000 and  64,000  products al.,  indicated  that not  a l l cells.  1985; of  myc  Lacy  appear  t o be  introduction  of  modifying to  the r e s p o n s e  c-myc p r o d u c t s The  products  that  artefact  have been an  provide  Although  to induce  1985).  The cell  division  cell  of  types,  appear  implying  f o r most, i f  (Smeland  a  genes i s i t is  e t a_l. ,  highly  phenotype  some p h e n o t y p i c  changes  o f an  myc  1 986 ; Symonds e t _ a l . , 1 986 ) .  has  e x p r e s s i o n or o v e r e x p r e s s i o n  incompatible with  oncogenic  c-myc  (Donner e t  ( G o u s t i n e t a ^ . , 1985),  The  are  genes  e x p r e s s i o n o f the myc  stationary  the  work  myc  functions necessary  cell  of  r e p o r t s on  indicated  i n most  of  have Mr's  the n u c l e a r m a t r i x  Hancock,  or  respond.  might  and  i s not  differentiated,  changes i n t h e r e s p o n s e  further  e t a_l. , 1 986 ).  genes  products  et a l . , 1985), but  with mitogenesis  sufficient  c-  initial had  or i n d u c i b l e  products  associated not  this  (Evan  expressed the  The  o f the n u c l e u s  that  o f the  (Hann e_t _ a l . , 19 8 3 ) .  Eisenman  preparation be  the murine  were a s s o c i a t e d w i t h  1982;  the  a v i a n c-myc genes have Mr's  i n the n u c l e u s .  fractionation  either  o f the c e l l  o f the  57,000 and  localized  that  i n profound  to e x t e r n a l s i g n a l s ,  enhancing  to  appear  (Connan  as  although  the r e s u l t  (Endo and  there of  the  Nadal-Ginard,  do  25  1.3.5  Src The  s r c gene i s t h e t r a n s f o r m i n g  virus  (RSV).  t o be  isolated  occurring, competent  RSV  and  studies  appears c-src,  (Takeya al.,  with p60  of the  that  1970).  contained  Hybridization  poorly  from t h e n o r m a l  appear  Johnson s r c  t o be r e l a t e d t o v i r a l  et a l . , 1985).  The  o f 60,000 and  membrane  i s a p h o s p h o p r o t e i n i n which group  modification  1980).  p60  s r c  of t y r o s i n e , ( H u n t e r and  has been  gene  oncogenicity Coussens et  p r o d u c t of the s r c associated  e t aj^. , 1984).  the phosphate a relatively  Sefton,  residues  The  i s linked  rare  1 980 ; C o l l e t t  shown t o c o n t a i n  to p h o s p h o r y l a t e t y r o s i n e  cellular  is tightly  (Sefton  function  i n the p r i m a r y  1 9 8 3 ; I b a e t a_l. , 1984;  has an Mr  oncogenic  transforming  a r e a number o f changes  Hanafusa,  that  did not.  derived  that  responsible n o t an  a sequence  genome w h i l e  of  RSV  i t was  t o be d i r e c t l y  the c y t o p l a s m i c  ability  (Martin,  t o RSV  mapping  t h e u n i q u e gene  demonstrated  RSV  is replication  t h e RSV  and  cellular  naturally  encodes  p60  s r c  Genetic  mutants  sarcoma  oncogenic r e t r o v i r u s  that  the h y d r o x y l  al.,  that  related  s r c gene  1985;  gene,  and  although there  sequence  and  identified  to the c e l l u l a r  retroviruses The  i s the only  function  indicated  hybridized  1911)  e t a l . , 1985).  function  viral  acutely  nonconditional  transformation  essential  the f i r s t  oncogenic r e t r o v i r u s that  ( i n Weiss  transforming  to  (Rous,  acutely  conditional  for  was  gene o f Rous  in  an  ejb  intrinsic  specific  26 s e q u e n c e s and c e l l u l a r e_t _ a l . ,  Cooper  1984; Collett  autophosphorylate vitro  substrates  under  ( f o r a review  and E r i k s o n , 1982) and t o  some c o n d i t i o n s b o t h  s e e H u n t e r and C o o p e r ,  A number o f o t h e r  i n v i v o and i n 1985).  o n c o g e n e s have been d e s c r i b e d  show c o n s i d e r a b l e homology genes a l s o  ( P a t s c h i n s k y e t a l . , 1982;  to s r c .  The p r o d u c t s  that  of these  display  an i n t r i n s i c  tyrosine kinase  These genes a p p e a r  to r e p r e s e n t  a superfamily of regulatory  genes e n c o d i n g  tyrosine kinases.  The a b i l i t y t o  phosphorylate  tyrosine i s also  growth  r e c e p t o r s and a p p e a r s  factor  transduction the  viral  EGF  receptor  associated with  and m i t o g e n e s i s .  fps product, (Hunter  and C o o p e r ,  Rosen,  1983) i s a s s o c i a t e d w i t h  Phosphorylation of  p60  c - s r c  activity and  e t a_l. ,  on c e l l  activation  their  phenotype.  i n vivo tyrosine acceptor  (Courtneidge, of t h i s  receptor  1 9 8 4 ; S t a d t m a u e r and  enhancement o f  has been shown t o i n h i b i t  dephosphorylation  the  1984),  1981) o r i n s u l i n  and e f f e c t s  a t t h e major  in vitro  oncogenic  Petruzelli  activity  t o be i n v o l v e d i n s i g n a l  (Weinmaster e t a l . ,  et _al. ,  kinase  many o f t h e  Tyrosine phosphorylation of  (Rosen  tyrosine  1983 ;  P130  activity.  i t styrosine kinase  1985; B o l e n  site  site  et a l . ,  1984)  i s associated with  of the c - s r c product  by polyoma  middle  T antigen. There activation  has been l i t t l e  presented  o f t h e endogenous c - s r c gene w i t h  tumours, a l t h o u g h in  evidence  two tumours  there  (reviewed  i s some e v i d e n c e  to l i n k mammalian  of overexpression  i n Slamon e t a l . , 1984 ; D e l o r b e e_t  a l . , 1980). c-src  of  responsive  to  Dolberg  al.,  et  can  tumours by  external 1984)  require  1985)  and  nature  would  of  (Iba  et  as  of  The  increase  great  be  as  p60 ~ v  There  .  phosphorylate 1985).  mutations well The  transformation  to  seen  et  induce  alter  enhancing  the  the  by  of  multistep  that  substrate  T  to can  be lead  phenotype,  but is  cells  the  transformed  phenotype  These r e s u l t s certain  more e f f i c i e n t l y p60  v _ s r c  substrates the  to  content  transformed  phosphorylate  key than  does (Johnson  et  activating  specificity  of  p60  s r c  activity.  l e v e l s of  a stationary,  the  c-src  to  of  also  et a l . ,  appear  1985).  that  in  al_. , 1 9 8 3 a ) .  al.,  enzymatic  of high  (Gilmer  et  in transformation  src  phenomenon has  phosphotyrosine  a fully  of  1979; by  polyoma m i d d l e  in v-src  These r e s u l t s i m p l y  with  This  transformed  a d i f f e r e n t spectrum  expression  incompatible  al.,  in v-src  i s some e v i d e n c e  in v-src  as  et  overexpression  i s able  s r c  p60 "  s r c  (Bissell  Land  in cellular  that  involved  c  somewhat  by  mutations  e t a l . , 1985a; J o h n s o n that  be  system  induced  appearance  to  e_t a l . , 1 9 8 5 ) .  corroborated  a partially  substrates  as  the  remains unable  imply  al.,  to  a l . , 1982;  complex.  i f the  c-src  the  to  that  i n a mammalian  e f f e c t s of  expression  nearly  implying  transformation  relatively  even  o f genes r e l a t e d  to  appears  conditions  appear  (Rassoullzadegan The  RSV  a d d i t i o n a l steps.  been d e m o n s t r a t e d  the  family  ( C a s n e l l i e e_t a l . , 1 983 ; V o r o n o v a  induction  vivo  the  show a c t i v a t i n g changes r e l a t e d  tumours The  O t h e r members o f  p60  s r c  activity  highly differentiated  is  not  28 phenotype.  The  presence  of high  has  been shown i n some n e u r a l  the  cells  et  have s t o p p e d  a l . , 1985;  v-src  gene i n t o  formation the  Iba  and  the  differentiation of  PC12  pathway  v - s r c i n most o t h e r  normal d i f f e r e n t i a t e d  line  of other  normally  of p 6 0  and  i s expressed  introduction  can  induce  (Alema e t a_l. systems  ,  functions  including  this  to be  (Adkins  the  neurite  1985a).  appears  Fults  of  functions  a s s o c i a t e d with  after  1985 ;  The  neural  activity  c _ s r c  (Brugge §_t al_. ,  1985b).  cell  expression  growth a r r e s t  tissues  dividing  al.,  et  levels  The  loss  effect  of  the  e t a l . , 1984;  Alema  et _ a l . , 1 9 8 5 b ) . Transformation reduction (Adkins other  of  the  serum r e q u i r e m e n t s  oncogenes.  shown t h a t v - s r c can division  i n the  (Durkin  product be  and  i s seen  in a  for cellular  of t h i s  s t i m u l a t e a complete absence  Whitfield, either  cell  o f any  1984). division  phenotype  effects.  i s determined  changes i n the  by  the  growth  round  of  ability  and  Whether  process  exogenous  The  involved in terminal d i f f e r e n t i a t i o n of p o t e n t i a l  significant  for transformation  Examination  apparent  to i n d u c e  spectrum  by  v-src results  e_t a l . , 1984), as  viral  signal  by  by has  cell mitogenic of  the  transformation  by  or  i n d i c a t e s a wide the  resultant  substrates phosphorylated  pathways a f f e c t e d  src  the  or  s u b s t r a t e s i s not  clear. 1.3.6  Raf v-raf  oncogenic  i s the murine  active  transforming  retrovirus  3611  (Rapp  gene o f the  acutely  ejb a l . , 1 983a , 1 9 8 3 b  ) .  29 v-raf  i s derived  a l . , 1983a). another  from  retrovirus  oncogenes, MH2.  do n o t a p p e a r a l . , 1984).  as  serine/threonine  raf/mil and  t h a t encode  The r a f / m i l  e t a l . , 1984).  products  threonine  that  The c e l l u l a r  almost  products  The  expressed  the r a f / m i l  and t h e c o n s e r v a t i o n  to a fundamental r o l e  oncogenes t h a t  c a n be gene  to p r e v i o u s l y  to induce  (Fukui  that  a wide  range  e t al^. , 1985;  t h e r a f genes c a n be  i n n a t u r a l l y o c c u r r i n g tumours.  the r e g u l a t i o n o f c e l l  I t i s of great  o f t h e c - r a f gene has been  some human tumours  §_t a_l. , 1 985 ) i n d i c a t i n g  involved  points  with  clearly  kinases.  tumours and an a c t i v a t e d f o r m  Shimizu  in  although  v - r a f / m i l oncogenes a r e a b l e  associated  protein  pathways and gene  1982) .  to correspond  serine/threonine  of serine  o f s e r i n e and t h r e o n i n e  transformation,  are not thought  described  of  with  to a c t  in_ v i t r o  and has been  ( f o r a review  s e e Cohen,  (Sutrave  appear  a l l o f the c e l l u l a r  expression  associated  themselves  Phosphorylation  has been d e t e c t e d  phosphorylation  kinases,  s u b s t r a t e ( s ) of the  i n the r e g u l a t i o n of m e t a b o l i c  that  show some  the t y r o s i n e  implicated  interest  r e l a t e d to  and a u t o p h o s p h o r y l a t e  a r e n o t known.  comprises  phosphorylation  genes  oncogene p r o d u c t s  kinases  (Rapp e_t  v - m i l , of the avian  t o be t y r o s i n e k i n a s e s  et  (Moelling  t o be c l o s e l y  The r a f / m i l  homology t o t h e o n c o g e n e s but  oncogene, c - r a f  The r a f genes a p p e a r  of the v i r a l  oncogenic  a cellular  c-raf i s widely  of the r a f / m i l  f o r the products  growth.  have been d e s c r i b e d  As w i t h  sequences o f t h e genes  the other  c - r a f appears  t o be  representative be  able  o f a l a r g e r gene f a m i l y  to e f f e c t  transformation  that  of c e l l s  a l s o appears to  ( H u l e i h e l ejt a l . ,  1986). 1.4 T r a n s f o r m a t i o n - i n The  vivo  examination  of transformed  of probing  the g e n e t i c  regulation  o f growth  define  t h e number  the  earliest  appearance in  relation  questions  the nature  references  both  a s i n g l e step  therein).  many e s t a b l i s h e d appears  the  derived  formation  required examining Klein,  cell  to involve  Evidence  i n vivo  at l e a s t  of evidence and i n v i t r o  lines  changes  or e p i g e n e t i c .  had i n d i c a t e d could  that  appear to  e t a_l. , 1986, and  by r e t r o v i r a l l y  borne  of  oncogenes  a s i n g l e , a c t i v a t i n g change.  epidemiological  studies  two c h a n g e s i n t h e c e l l s .  the appearance  this  One o f  was whether t h e  indicated  o f many tumours was a p r o t r a c t e d  1985) and l u n g  supported  growth.  a s i n g l e or m u l t i p l e  (Sigal  in cell  In p a r t i c u l a r the t r a n s f o r m a t i o n  only  from  steps  of oncogenic  and i f t h e changes were g e n e t i c  transformation only  o f the  of the a c t i v a t i n g  to normal c e l l  t o be posed  a means  The a t t e m p t t o  a n a l y s i s of the process  Some o f t h e e a r l y l i n e s  require  basis  of the r e g u l a t o r y  o f tumours r e q u i r e d  the c e l l s  provides  and d i f f e r e n t i a t i o n .  to e l u c i d a t e  c h a n g e s and t h e i r  Models  cells  and b i o c h e m i c a l  and n a t u r e  g r o w t h has prompted transformation  and i n v i t r o  conclusion.  (Farber  that  Evidence  o f B u r k i t t ' s lymphoma  cancer  process  that  ( K l e i n and  and Cameron, 1980)  Studies carcinogens  on  the  also  pointed  tumourigenesis. carcinogenic promoters. capable  chemicals The  i n c r e a s e the  into  but  t o be  two  but  With  are  inefficiently  phenotype.  The  of tumourigenic to i n i t i a t e  the d e m o n s t r a t i o n  that  Abelson-MLV  (Whitlock  or a v a r i e t y  and  incompletely  1985)  can  transformed  to  act of  and  pass  transformed  phenotype.  activated al., It one  through  state  I t has  either  Calderwood,  r e q u i r e d f o r the  cellular  by  and  cell  line  by  et a l . ,  Ki-MSV  1984)  (Auersperg  o r Ha-MSV  further  cellular completely  o f some t r a n s f o r m e d  human tumours i n d i c a t e d  in a single  cells  e x p r e s s i o n of the  oncogene c o u l d be  been  an i n t e r m e d i a t e ,  that  Examination  appear  t o d e f i n e an i n  1 9 8 1 ; Whitlock  types  changes a r e  d e r i v e d from  process.  Witte,  of c e l l  et _ a l . , 1 9 8 1 ; A u e r s p e r g e_t a_l. ,  this  important  t r a n s f o r m a t i o n of lymphoid  than  are  converted  carcinogenesis could  that  et  that  tumourigenesis  demonstrated  single  and  conversion  mirrored  lines  separate  tumour p r o m o t e r s  that  (Yuspa  to  initiators  system  1983)  of  to c r e a t e c e l l s  a m u l t i - s t e p p r o c e s s , i t was  vitro  possible  groups:  are unable  chemical  s t e p pathway  appear  efficiency  cells,  tumours by  et a l . , 1982) .  (Hecker  be  proven  initiators  tumourigenic  initiated  to  I t has  of  t o a two  of tumourigenesis  a highly to  induction  altered  (Murray  that and  cell  more than  presumably  e t a_l. ,  1983 ;  Taya  1 984 ) .  has  proven  oncogene  possible i n the  t o show a r e q u i r e m e n t  t r a n s f o r m a t i o n o f many  f o r more primary  a  cells  (Land  et a l . ,  1983a,1983b;  Ruley,  1983).  of  o n c o g e n e s have been d e f i n e d on t h e b a s i s  to  cooperate.  These  two g r o u p s  o n c o g e n e s c-myc and c - r a s .  composed  of p53 ,  in  addition  located  t o myc.  and  polyoma m i d d l e  are  located  are  able to cooperate  but  not those These  that in  vivo.  t o have  primary  Wilkie,  t h e members  genes.  elimination  o f the background  can  apparently suppress  primary  cells  communication though located  group group  model  effect  appear  r a s induced  1 986 ).  multiple to the  that the  c a n be r e l i e v e d  cells  (Spandidos  implying that  I t would  oncogene  (Mehta e_t_ j a l . ,  i n p a r t by  not transformed  by r a s  the normal  cells  transformation i n  I t also  to c e l l - c e l l  appears  as  f o r two o n c o g e n e s whose p r o d u c t s a r e  i n the nucleus (Jenuwein  genes  r a s gene t o  i s n o t known, b u t may be r e l a t e d  i t i s possible  some c e l l s  1986),  The means by w h i c h  1986).  raf  with carcinogenesis  gene c a n have a s i m i l a r  f o r a second  et a l . ,  in vitro  i f i t i s overexpressed  e_t a_l. ,  of multiple  of these  of the other  f o r an a c t i v a t e d  requirement  (Land  contains ras,  The o n c o g e n e s o f one  strong correlations  1 9 8 4 ; Land  group i s  group.  cultures  changes i n a s i n g l e activation  group  have p r o v i d e d a u s e f u l  I t i s possible  transform and  o f t h e same  results  appears  The f i r s t  T and a l l o f t h e p r o d u c t s  with  ability  o f a l l o f t h e s e genes a r e  The s e c o n d  i n the cytoplasm.  of t h e i r  T a n t i g e n and a d e n o v i r u s EIA  The p r o d u c t s  i n the n u c l e u s .  groups  a r e e x e m p l i f i e d by two  cellular  polyoma l a r g e  Two  to cooperate  et a l . ,  1985).  i n the t r a n s f o r m a t i o n of These r e s u l t s  present  exceptions  t o the m a j o r i t y o f the  transformation the  of primary  cells  r e g u l a t o r y pathways need  types  and  ability  that  to e x p r e s s  oncogenes appear  elements  The  e_t al_. ,  (Koufos  tumourigenic isolate  phenotype  activated  appearance  o f the  genome, w h i c h a r e suppressor  e t al_. , The  supported somatic  ( G u e r t s van Stanbridge to are  implies  the  and  loss  although the  Wilm's the  i t i s possible  tumours.  of s p e c i f i c  involved in loss  The  e t a_l. ,  1983;  Wilm's  p a r t s of  the  of a  e x p r e s s i o n of o n c o g e n i c i t y Hansen e t a l . ,  of oncogenesis  s u p p r e s s i o n o f the  et a l . ,  that  retinoblastoma  have i n d i c a t e d  hybrids involving  the  otherwise  a recessive  t u m o u r s , r e t i n o b l a s t o m a and  K e s s e l e t a_l.  suppress  1986);  (Cavenee 1985;  1 985 ) .  the  cell  ,  that  r e c e s s i v e nature by  transforming  1985 )  from  the  phenotype.  et a l .  t o be  i s recessive,  that  in a l l cell  and  presumably  Godbout  suggest  Hansen e t a l . , 1 9 8 5 )  r a s genes  two  function  a l . , 1983;  appear  on  profoundly affect  t o a c t as d o m i n a n t  tumour, i s a s s o c i a t e d w i t h  Koufos  identical  s t u d i e s of h e r i t a b l e  (Cavenee e t a l . , 1 9 8 3 ;  et  be  u n d e r many c o n d i t i o n s ( S i g a l  phenomenon.  to  not  but  a transformed  however, t r a n s f o r m a t i o n can  tumour  in vitro,  e x t e r n a l c o n d i t i o n s can  of c e l l s  The  observations  1982).  effects  capable at l e a s t  ,  The  Klinger, ability  of a c t i v a t e d  of being  phenotype i n  transformed  cells  1980,1982,  of the normal  cells  oncogenes i n c e l l s  transformed  p a r t of the  further  transformed  n o r m a l and  1981;  is  ability  by  the  that  oncogene  of normal c e l l s  to  resist  transformation  activity  o f an oncogene  The in  SHE  induction  cells  (Oshimura again  of  associated  with  appear  cell  (Auersperg  oncogenes derived  of  suppression  e t al_. , 1 985 ).  The  lines  are d e r i v e d ,  o f oncogene  A u e r s p e r g and  Calderwood,  extensively  transgenic  e t a^L. , 1984;  a variety  to be c l o n a l ,  or t i s s u e mice  have  specific  (Brinster  form o n l y  that  some o n c o g e n e s  of t i s s u e s .  The  suggesting  that  placed  the  under  p r o m o t e r s and  e_t al_. , 1984; In t h e i n which  expressed,  are able  tumours  Tumours  can c o n t a i n  i n the t i s s u e s  m i g h t be e x p e c t e d t o be  f o r the  cellular  been  Adams e t a l . , 1 9 8 5 ) .  m i c e , tumours  demonstrating  tissues  Oncogenes  1984).  examined  for a review).  of d i f f e r e n t  also  activity  et^ a l . > 1984  to c r e a t e  i n many  would  ( s e e Slamon  of i n d u c i b l e  ability  but not i n the p r i m a r y c u l t u r e s  have been  oncogene.  15  transformed  forms o f  t h e oncogene  in  of the l o s s  suppression  1981;  from a v a r i e t y  transgenic  of the  myc  material i s  or a c t i v a t e d  the c o n t r o l  Stewart  of g e n e t i c  of a l t e r e d  same a c t i v a t e d  used  loss  t o p r o d u c e autonomous g r o w t h  this  Tumour t i s s u e s  This  by r a s and  monosomy f o r chromosome  (Thomassen  lines,  et a l . ,  appearance  t o s u p p r e s s the  1985).  the e x p r e s s i o n  t h e permanent  to r e f l e c t  with  the e f f e c t s  oncogenes  from w h i c h  (Knudson,  1985).  somewhat v a r i a b l e  immortalized  i n an a b i l i t y  o f an o n c o g e n i c p h e n o t y p e  although  certain  rest  i s correlated  et a l . ,  phenotype, are  may  that  to cause arise  tumours  also  appear  complete t r a n s f o r m a t i o n i s  35  infrequent,  and  carcinogenesis The  basis  f o r the  renders 1986;  al.,  of  primary  expression the  cells  show an those this  the  Balk  sensitivity  ras  enhanced  induces  mechanism  either  could  associated  with  these  does n o t  primary be  can  1.5  Tissue  occur  as  Culture  been this  of  the  production  autocrine  facilitates cells.  (Stern  et in  of  some o f  in vitro  model d e r i v e d  that  with appears  (Pruss  of  to  et a l . ,  transformation  TGF-ocstimulation of  from  of  TGF-oc  transformation  of  not  associated of  that  of  The  production  than  are  suppression  to  clear  oncogenes  oncogenes  by  growth.  Transformation  Lines transformation  using  observation Many o f  I t i s not  cell  TGF-y3,  expected  combinations  Examination  Cell  be  the  f o r the  the  TGF-cC and  stimulation  alone.  to o t h e r  work e x a m i n i n g  performed  transforming  to  absence  of  EGF  Since  induced  i n the  various  expected.  1 986 ), s u g g e s t i n g  i n the  into  to  the  obscure.  a l s o be  account  i n ras  c-myc  oncogenes might  apply  Finally,  rather  in  increase  oncogene  as  oncogenes  a similar  cooperating  Establishment Most  has  by  is still  production.  McKay e_t a l . ,  ras  1.5.1  TGF  cells.  dispensable  1978;  both  oncogenes  transformation  of  and  the  response  expressing  cooperating  nature  two  more r e s p o n s i v e  a l . , 1985),  et  of  overexpressed  cells  expressing  data  cells  to TGF-06might of  multistep  cooperation  i n t r o d u c t i o n o f an  lines  the  in vivo.  transformation The  supporting  the  cells  and  by  oncogenes  in tissue culture,  manipulation  experiments  use  of  the  fibroblastic and  cells,  functions  established  although  have a l s o  in culture  from  migrate  culture  conditions.  Other  culture  by  of  the  the  a variety  adrenal  The  presumed  as  cells  cell  can  be  established  derived  by  (Barrett  et  can  be  explants  the  cells  grow r a p i d l y  under  will  types  can  established cells  al_. , 1984;  culture  normal  tissue  in  cells  in essentially a_l. ,  are  cells  show r e s p o n s e s  the  established  ( S l a v i n s k i et  i n primary  cultures  be  as  Steroidogenic  reflect  traumatizing  different origins  Fibroblasts  techniques.  t o most a c c u r a t e l y  evoked  and  fibroblastic  a l t h o u g h most p r i m a r y those  tissues  tissue  cortex  same f a s h i o n  1974).  of  with  been u s e d .  readily  from  out  cells  in  vivo,  similar  from which  to  they  C o l l i n s e_t al_. , 1985;  are  Jaye  et  a l . , 1985). The  cells  partially arrest  determined  growth  diluted  grow t o  by  and  by  established  in culture  and  and  culture. will or  survive  die  cells  (Merz and  being Ross,  culture  at  least  conditions, until  majority  of  have r e l a t i v e l y  the  the  and  cells  cells  a small of  l i n e s and  percentage and  do  appear  appears  to  able  r e p l i c a t e and  to  1969).  r e s u l t from  are  limited l i f e  crisis not  then  that  a f t e r a c h a r a c t e r i s t i c length  t h i s period  Crisis  density,  quiescent  The  many s p e c i e s  permanent c e l l  again. of  In  the  remain  passaging.  senesce  a specific  become  an  to  of  spans,  of  the  time  in  cells  immortalized suffer  crisis  insufficient  grow to  are  number  confluence  Immortalized represent growth that and in  an  culture,  distinguish  the  in vivo.  The  transformation immortalized  cell  a number o f t h a t are  i s the  change.  ease  lines.  Some c e l l  to r e q u i r e o n l y appear  effects  show s i m i l a r  requirements  for cooperating  i s of i n t e r e s t  Smith  and  be  1.5.2  suppressed  Smith,  correlated hybrids  with  The  on  suppression  and  of C e l l  grown  but  show a p p r o x i m a t e l y  the  same low  of immortalized  cells  from  the  hybrids  can  (Pereiracan  be  seen i n  Factors culture is  Normal c e l l  lines  medium t o s u p p o r t cells  do  growth  not.  The  tumours a l s o r e q u i r e serum  (McCulloch  establishment derived  Growth  many t r a n s f o r m e d  f o r growth i n v i t r o  as  cells.  factors.  from many s p o n t a n e o u s  be  e t al_. , 1 986 ).  known i f t h i s  in tissue  i n the  a  oncogenes  transformation  Growth by  a serum s u p p l e m e n t  of  oncogene  immortality  cell  of  transformed  serum g r o w t h  replication,  factors  the  (Franza  i t i s not  growth of normal c e l l s  dependent  cells  i n somatic  to  of a s i n g l e  that c e l l u l a r  1983), but  of normal  Regulation  require  to n o t e  immortalized  Transformation  lines  cells  to  of  transforming  i n primary  adapted  of these d i f f e r e n c e s  relative  o f t e n appears  they  phenotypes  not  t o the  apparently  and  lines  t h a t are  most i m p o r t a n t  lines  have been d e s c r i b e d It  of c e l l s  of e s t a b l i s h e d c e l l  activating  resistant  u s e f u l in that  express  them from  current context  single  although  homogeneous p o o l  in tissue  cells  and  cells,  et_ a_l. , 1974)  and  r a t e of s u c c e s s f u l  lines  same t i s s u e .  i n c u l t u r e as The  serum  normal  growth  factors  are polypeptide  daltons.  They  receptors  specific  regulatory  appear  platelets five the  effects  initiating arrested  of c e l l  and f r o m p l a s m a and  cells  These  growth  replication.  PDGF o r FGF o n l y  synthesis  or c e l l  restriction Treatment  point  i n the  serum  starved,  restriction  Cells  that  have  division  with  o r competence  stimulation  o f DNA  been  treated  t o DNA  DNA  synthesis.  e i t h e r EGF o r I G F - I I ,  factors, will  synthesis.  to r e s u l t  10 t o 12  but a r r e s t at a second  4 to 8 hours b e f o r e  progression  t o a c t as  growth  point  do n o t u s u a l l y p r o g r e s s  o f the c e l l s  appear  role  TGF-^3  f a c t o r s c a n be  PDGF and FGF a p p e a r  to pass the f i r s t  DNA  responsible for  EGF, PDGF, I G F - I I ,  1982).  a  1984).  t o be l a r g e l y  f a c t o r s and s t i m u l a t e  hours before  serum  o f serum  division.  surface  b o t h from t h e r u p t u r e o f  i n t o two g r o u p s b a s e d on t h e i r  stimulation  with  and Westermark,  o f serum:  et a l . ,  to c e l l  f a c t o r and a c t i v a t i n g  f a c t o r s are derived  and FGF ( P l e d g e r divided  (Heldin  f a c t o r s appear  mitogenic  from 5,000 t o 28,000  t o a c t by b i n d i n g  i n the p r e p a r a t i o n  specific  ranging  f o r the growth  pathway  The g r o w t h  chains  then r e s u l t  The m i t o g e n i c  from t h e c o o p e r a t i o n  i n the  e f f e c t s of of these  two  groups o f growth f a c t o r s . 1.5.3  Transformation The  results serum cell  and Growth  transformation  of c e l l s  in either a partial  produce growth  Independence  by many o f t h e o n c o g e n e s  or complete  r e q u i r e m e n t s f o r growth. lines  Factor  l o s s of the normal  Some o f t h e tumour  f a c t o r s that  appear  derived  t o be i n v o l v e d  39  in  an a u t o c r i n e  1985;  Scott  stimulation  of c e l l  growth  e t a l . , 1985; T o d a r o e t a l . , 1980; van K e s s e l e t  <|1. , 1985; van Z o e l e n  e t a l . , 1 985 ).  Expression  factors  can r e s u l t i n t h e t u m o u r i g e n i c  cells.  The i n t r o d u c t i o n  cell  line  (Rosenthal  can t r a n s f o r m  expression  et _al.,  cells.  i n vivo  1986), o r e x p r e s s i o n t o PDGF  as w e l l  oncogene  and c-myc i n human p l a c e n t a l  et  a l . , 1985).  expression this  It  regulation vitro.  i n vivo  (Jefferson in  clear  tissue  alter  culture  tumourigenicity. most c e l l s  saturation  cells  the response  e t a_l. , 1984a,  suggesting a cells  to demonstrate the  The i n t r o d u c t i o n  f o r growth  o f d i f f e r e n t types can to growth  To s t u d y  correlate  independent growth.  serum  factors  transformation  to i d e n t i f y the  with  the a c q u i s i t i o n o f  of activated  oncogenes  r e s u l t s i n changes i n m o r p h o l o g y , reduced  of  d i f f e r e n t from t h o s e i n  i t has been n e c e s s a r y  density,  (Goustin  ( L e e e t a_l_. , 1 985 ) .  of c e l l s  1984b).  that  that  the c - s i s  the requirements  are s i g n i f i c a n t l y  changes seen i n v i t r o  into  that  e_t a l . ,  l i n k i n g the e x p r e s s i o n  The i n t e r a c t i o n between  significantly  cells  t o embryonic growth  i s becoming  of the s i s  with  o f both  been p o s s i b l e  o f TGF-oC i n embryos  ar a t  I t has been  o f growth i n these  I t has a l s o  growth f a c t o r  indicate  as i n v i t r o .  to demonstrate the e x p r e s s i o n  control  o f normal  (Besmer  c a n be a s s o c i a t e d  possible  normal a u t o c r i n e  conversion  These d a t a  o f growth f a c t o r s  oncogenesis  o f growth  o f t h e gene f o r TGF-oc i n t o  oncogene, w h i c h i s c l o s e l y r e l a t e d 1983)  (Reeve e_t a l . ,  increased  r e q u i r e m e n t s and a n c h o r a g e  I t has n o t been p o s s i b l e  t o show a  complete  correlation  tumourigenicity, strongest F i d l e r ,  of  It  the  to  would  animal.  parameter  of  to  of  and  serum  highly  to  1979; note  to  be  that for  growth  tumour  the  lack  c e l l s  of  to  independence  transformation  in  the  e_t  independence  although  in  an  grow is  it  is  and  and  (Auersperg  anchorage  and  the  coincident  c e l l s  the  Cifone  cortex why  Anchorage  reflect  Shin,  shows  phenotype  despite  allow  independence  with  tumourigenic  tumourigenicity, grow  phenotypes  requirements  appears  clear  these  interest  adrenal  not  with  a b i l i t y  a  rat  is  correlated  in  is  reduced  with  MSV-infected  substrate  (Kahn  independence  correlated  the  It  any  anchorage  correlation  acquisition  1986).  but  1980).  anchorage  of  K i -  a l . , should  thought  be that  appropriate inappropriately used  v i t r o .  as  a  41  CHAPTER 2 MATERIALS AND 2.1  Cells Establishment  Two  of primary  t o t h r e e month  dislocation  and t h e a d r e n a l g l a n d s  minced.  and  grown as s e p a r a t e  The a d r e n a l c e l l s  experiments. tissue  culture  was  filled  with  bovine  cell  The minced  the  fetal  cervical  aseptically maintained  a l l the  were a l l o w e d  modified  serum and p e n i c i l l i n  (DMEM + 25% F B S ) .  incubated  a t 37°C, 5% C 0  2  to a t t a c h to  Eagles  the d i s h 25%  medium w i t h  (1OOU/ml)/streptomycin  The t i s s u e  e x p l a n t s were  then  i n a h u m i d i f i e d atmosphere.  10 t o 14 d a y s f i b r o b l a s t i c  trypsinized  by  f o r 30 t o 60 m i n u t e s b e f o r e  Dulbecco's  to c o n f l u e n c e  cultures.  e a c h r a t were  throughout  adrenals  dish  were k i l l e d were removed  from  lines  (50ug/ml)  grown  adrenocortical cell  old Fischer rats  and  then  METHODS  cells  i n t h e 60 mm  from  After  the a d r e n a l glands  dishes.  The c e l l s  and f r o z e n i n DMEM + 2 0% FBS + 10?  had  were dimethyl  sulphoxide. The  r a t lung  fibroblasts  and r a t embryo  were e s t a b l i s h e d by e s s e n t i a l l y described  were from  Established  r a t and mouse c e l l  1981) was  (Topp,  were  stored frozen i n l i q u i d  grown  when t h e c e l l s  experiments.  as t h a t  The r a t embryo  F i s c h e r r a t embryos.  line  expanded  t h e same p r o c e s s  f o r the a d r e n a l c o r t e x c e l l s .  fibroblasts  fibroblasts  lines.  The r a t 2  i n DMEM + "\0% FBS. nitrogen  The  cells  ( 1 N ) and thawed and 2  were r e q u i r e d f o r a s s a y s  The F S V - t r a n s f o r m e d  cell  rat 2 cells  or  resulted  from  of the r a t 2 c e l l  transfection the  avian,  virus line  acutely  oncogenic  (FSV) ( c o u r t e s y was  derived  line  with a p r o v i r a l  retrovirus  o f G. W e i n m a s t e r ) .  from  culture  grew o u t a f t e r  passaging the i n f e c t e d  The cultures passed  (Harrison  strain grown  through  established and  showed  derived  as t h e f u l l y  o u t from  a normal  from  strain  serum i n d e p e n d e n t  culture  and t h e l i n e The c e l l s  fibroblastic A cells  ( f r o m N.  the c e l l s  was  from  had  permanently  were c o n t a c t  morphology.  by Ki-MSV  and a n c h o r a g e  that  (by N. A u e r s p e r g )  r a t adrenals u n t i l  in culture.  r a t ovarian  1981 ) .  were d e r i v e d  senescence  cell  transformed l i n e  and A u e r s p e r g ,  A cells  sarcoma  The T-ROG  a Ki-MSV-infected Fischer  granulosa.cell  Auersperg)  Fujinami  clone of  A2  Strain  infection  independent  inhibited  and  growth  was  showed and  was  tumourigenic. The from  NIH 3 T 3 c e l l  mouse embryo  line  fibroblasts  was  o b t a i n e d from  was  o b t a i n e d by i n f e c t i o n  of  ATCC.  Ki-MSV and i s o l a t i o n  ( f r o m D. The  ATCC .  line  ( T o d a r o and Green,  The Ki-MSV n o n p r o d u c e r o f NIH 3 T 3 c e l l s  derived 1 9 6 3 ) and  NIH 3 T 3  w i t h a low  of a focus of nonproducing  line  titre  cells  Lowy). calu-1  adenocarcinoma, obtained  i s an e s t a b l i s h e d  line,  derived  containing  from  a human l u n g  an a c t i v a t e d  f r o m ATCC and m a i n t a i n e d  c - r a s oncogene,  as recommended  by t h e  was  43  2.2  Viruses The  four a c u t e l y oncogenic  experiments 3611  were Ki-MSV, 3611,  arenatural  Mayer,  NIH 3T3 c e l l  nonproducing  of the r e p l i c a t i o n  leukemia  virus  1984)).  The c e l l s  transfection into  an Amicon  in  aliquots  in  t h e same way.  line  with  competent  transformed  a genomic  (Miller  through  a t 4 hour  The s u p e r n a t a n t  Both  NIH 3T3 c e l l  line  on r a t 2 c e l l s .  ug/ml p o l y b r e n e polybrene.  and t h e n  Rat 2 c e l l s ,  were t r e a t e d  with  was c o n c e n t r a t e d and t h e n s t o r e d  serially  was o b t a i n e d  diluted  0.5 ml o f t h e v i r u s  The p l a t e s  were f i l l e d  as  f o r the u n i n f e c t e d c e l l s  from a The  by f o c u s  s t o c k was b r o u g h t  approximately  37^C.  harvested  ( c o u r t e s y o f U.R. R a p p ) .  The v i r u s  at  after  intervals  t h e 3611 and MMCV were  The 3611(Mo-MLV) v i r u s  murine  and Verma,  2 passages  c o n c e n t r a t o r t o 40 t o 50 f o l d a t -80°C.  c l o n e , pMLV-  h e l p e r v i r u s Moloney  was h a r v e s t e d  serum.  ( K i r s t e n and  o f Ki-MSV(Mo-MLV)  o f t h e Ki-MSV and 3611 were d e t e r m i n e d  formation  were  t h e Ki-MSV  were expanded  and v i r u s  in  producing  Stocks  i n these  Ki-MSV and  tumours  (Mo-MLV) ( f r o m D. M i l l e r  DMEM + 1$ c a l f  titres  d e r i v e d from  by t r a n s f e c t i n g  used  MMCV and 2-1.  1 967 ; Rapp e_t a l . , 1 9 8 3 a ) .  were o b t a i n e d  48,  isolates  retroviruses  to 4  i n DMEM + 4 ug/ml  40 t o 60% c o n f l u e n t , dilutions  f o r 1 hour  w i t h medium and i n c u b a t e d  f o r 5 t o 7 days when t h e f o c i  counted. MMCV i s a r e p l i c a t i o n  retrovirus  defective,  constructed i n v i t r o  transforming  (Vennstrom  e t a l . , 1984)  and  44 provided from  i n an NIH 3T3 p r o d u c e r  Ha-MSV LTR's and a Mo-MLV  splice  site.  The a v i a n  and  in  48 h o u r s  assays  The r a t 2 c e l l s the c e l l s  i n a 60 mm  dilution  formation  a 3 ' splice  acceptor  The t i t r e  with  and s o f t  site  was  independent  were t r y p s i n i z e d  dish.  from t h e  v e r s i o n of the  were i n f e c t e d  growth MMCV and  agar  from each v i r u s After  w h i c h gave between  was d e t e r m i n e d  r e g i o n and  This  gag s e q u e n c e s .  were s e t up a t 10^ c e l l s  concentration virus  retrovirus.  by t h e i n d u c t i o n o f a n c h o r a g e  rat2 cells.  after  5* n o n c o d i n g  as i t c o n t a i n s  i s free of r e t r o v i r a l  determined  The v i r u s was d e r i v e d  v-myc oncogene was c l o n e d  OK 10 a c u t e l y t r a n s f o r m i n g oncogene was c h o s e n  line.  12 t o 14 days t h e  15 and 30$  and t h e v i r u s  colony  concentration  calculated. The  2-1 v i r u s i s a c o n s t r u c t e d  retrovirus  that  sarcoma v i r u s . (Anderson  contains  and S c o l n i c k ,  4070.  titre  1983) and v e r y v i r u s stock  I n f e c t i o n was c a r r i e d  MSV and t h e i n f e c t e d  cells  murine  v - s r c gene from  The v i r u s was c o n s t r u c t e d  Humphries as h i g h helper  the a v i a n  oncogenic  Rous  by S. A n d e r s o n  k i n d l y provided  using  the amphotropic  o u t as d e s c r i b e d  were m a i n t a i n e d  by K.  for K i -  in a level C  facility. 2.3  I n f e c t i o u s Centre The  Assays  number o f a d r e n a l  Ki-MSV o r 2-1 was e s t i m a t e d The  adrenal  passage  cortex  i n which  cells  they  cortex  cells  infected  by an i n f e c t i o u s  were t r y p s i n i z e d  by e i t h e r  centre  assay.  a t t h e end o f t h e  were i n f e c t e d , c o u n t e d  and p l a t e d a t  10  , 10- and 10  cells  3  uninfected used The  rat 2 cells.  as a p o s i t i v e cells  covered FBS.  foci  the  infected  o f focus  (Mo-MLV)-infected  formation,  rat 2 cells  o f focus  the Ki-MSV-infected  to  the e f f i c i e n c y  centre rat  assay  2 and a d r e n a l  inhibit seen 2.4  cell  between  penicillin sterile horse agar  layer  or c a l f contained  0.35$ a g a r o s e ,  water  assay. with  assay  or equal  cortex  did not The i n f e c t i o u s  Ki-MSV-infected  treated with  mitomycin  C to  d i f f e r e n c e was cells.  Growth was composed  o f 1X DMEM p l u s  5X MEM v i t a m i n s , 0.6$ a g a r o s e ,  t o volume and e i t h e r  25% FBS o r 5%  serum as was r e q u i r e d f o r t h e a s s a y . 1X DMEM p l u s a n t i b i o t i c s , sterile  that the  centre  C t r e a t e d and u n t r e a t e d  and s t r e p t o m y c i n ,  distilled  adrenal  and no s i g n i f i c a n t  the mitomycin  of infected  was g r e a t e r than  been p e r f o r m e d  replication  t h e Ki-MSV  I t was assumed  i n this  cortex c e l l s  bottom agar  from  adrenal cortex c e l l s  Anchorage Independent The  was c o r r e c t e d f o r t h e  of the Ki-MSV-infected  has a l s o  formed by  i n the i n f e c t i o u s  a d e t e c t a b l e response  and then  i n DMEM p l u s 10$  determined  rat 2 cells  The M M C V - i n f e c t e d  produce  efficiency.  and t h e p e r c e n t a g e  formation  were  f o r 10 t o 12 days and  was e s t i m a t e d .  by  cells.  formation  The number o f f o c i  cortex c e l l s  cortex c e l l s  efficiency  incubated  counted.  adrenal  rat 2 cells  t o a t t a c h f o r 4 t o 6 hours  were then  were t h e n  efficiency  f o r focus  an o v e r l a y o f 0.6$ a g a r o s e  The c e l l s  the  monolayers o f  Ki-MSV-transformed  control  were a l l o w e d  with  adrenal  on s u b c o n f l u e n t  d i s t i l l e d water  The t o p  5X MEM v i t a m i n s ,  t o volume, 25$ FBS,  5% h o r s e factors  or c a l f  serum, and exogenous p u r i f i e d  as n e c e s s a r y .  1 u \ 3 x 10^ o r 1 0  growth  cells  5  i n 0.1 t o  0.2  ml were s u s p e n d e d  i n 2.5 t o 3.0 ml o f t h e t o p a g a r mix  for  each  The s o f t  plus  antibiotics  factors 2.5  60 mm d i s h .  Radiolabelling  until  to  once w i t h  FBS  ice cold  0.7  phosphate  then  After  i n phosphate  MgCl , 2  Y13-259  labelling  with  (carrier  immunoprecipitations  pH 7.5, 100 mM N a C l ,  [pH 7 . 4 ] ) .  p21  other  a t 4°C,  supernatant  was r e c o v e r e d  cells once  5 mM KC1,  The c e l l s volume).  were For  monoclonal  was 100 mM N a C l ,  5 mM  X-100, 0.5% w/v  b u f f e r used f o r a n t i b o d i e s was 10 mM  Tris-  1 mM EDTA, 1% v / v NP40, 0.5% w/v  NaDOC, 0.1$ w/v SDS, 1 mM l e u p e p t i n . centrifuged  r a s  1$ v / v T r i t o n  The l y s i s with  140 mM N a C l ,  b u f f e r used  (pH 7 . 5 ) ,  were washed  buffer (final  the a n t i  the l y s i s  1mM l e u p e p t i n .  antiserum  incubated 8  DMEM w i t h 3%  free  the c e l l s  (25 mM T r i s ,  1 ml o f l y s i s  5mM T r i s - H C l  were  i n 10 t o 25% FBS and 3% HS f o r  Tris-saline  into  and t h e n  2  4  scraped  antibody  HCl  saline  The c e l l s  300-500 u C i o f ^ P - o r t h o p h o s p h a t e  immunoprecipitations  SDS,  free  mM N a H P 0 , 5.5 mM D - g l u c o s e , 2  f o r 2 t o 3 weeks.  and grown i n DMEM + serum as r e q u i r e d  incubated  i n 3% HS.  grown  growth  of Cells  ICN P h a r m a c e u t i c a l s )  for cells  w i t h DMEM  exogenous  were 50 t o 80% c o n f l u e n t .  12 h o u r s w i t h  free,  were c o v e r e d  or without  and i n c u b a t e d  were s e e d e d  the c e l l s  washed  in  and serum w i t h  as i n d i c a t e d  Cells  agars  The l y s a t e was  35,000 x g f o r 30 m i n u t e s and i n c u b a t e d  or frozen overnight.  Cells  with  and t h e the a p p r o p r i a t e  were l a b e l l e d  with  47 []methionine were washed  i n a s i m i l a r manner e x c e p t  with  T r i s - s a l i n e and  then  [^S]methionine  (800-1,000 Ci/mmol;  methionine  DMEM w i t h  lysed 2.6  and  free  treated  clarified  to  5 u l of  by  the  the  aureus  cell  with  with  150  uCi  Corp.) i n The  were i n c u b a t e d  m i n u t e s and  Cowan I  rabbit  cells  (IgG  50  sorb,  a n t i - r a t IgG  cell  i n the  lysate. f o r 15  the  buffer.  lysis  lysis  The  were  The  cell  Tris-HCl  (pH  8.0),  material  was  released  37^C  frozen  lysates  f o r 10  the  v/v  by  NaCl,  NP40, 1$  washed  washed  NP40.  incubation the  S_. a u r e u s and  adding  to in a  5 to 6 times  with  of  i n 1M  The  and  pelleted  NaCl,  10  mM  immunoprecipitated  i n SDS  supernatant  g e l sample collected  buffer and  Reaction  immune complex k i n a s e  were l y s e d  then  aureus  use.  Kinase  cultures 1$  were then  m i n u t e s and  Immune Complex  and  The  before  was  or f o r  o f S_.  centrifugation  3 times  60  Staphyloccus  immunoprecipitations  0.1$  a t -80°C u n t i l  For  seconds  of  suspension  by  directed  for a further  (Roc R i g ) .  buffer  i c e with 1  Enzyme C e n t e r )  immune complex  t o 20  labelled  then  The  u l o f a 10$  on  e s s e n t i a l l y as  suspension  a u r e u s + Woe R i g were p r e p a r e d  microfuge  2.7  lysates  u l o f a 10$  r a t monoclonals  resuspension  at  or 3 $ HS.  cells  above.  f o r 60  50  strain  coated  the  Amersham  appropriate antibody  supplier  minutes with  S.  incubated  the  Immunoprecipitation The  the  as  3 $ FBS  that  i n 1 ml  reaction  of modified  NaDOC, 0.1$  SDS,  10  mM  100  mm  RIPA b u f f e r  dish (0.15  NaPhosphate pH  M 7.0,  2 mM  EDTA, 14 mM  NaF)  a t 4°C.  the  for  The  clarified  anti-p60  lysate  monoclonal  s r c  30  were c o l l e c t e d  by  30  t h e n washed  0.1  M NaCl,  the  immunoprecipitates  10 mM  7.0,  for  2.8  (Sigma)  volume o f two  5  0.001$ bromphenol using  by  Laemmli  stacking  and  10.5$  p60  s r c  )  or  separating  gel.  acrylamide  and  The  The  0.8$  running buffer  For  1 0  u  acid  a 10$  the  S.  i n 10  C i [y-  3 2  P]  treated  pad  once i n  reactions  u l o f 20  mM  ATP  (-3000  rabbit  muscle  and  incubated at  were s t o p p e d  by  adding  g e l sample  pH  6.8,  10$  by  30 C U  an buffer.  The  v/v  was  g e l and  g e l s used  either  v _ m y c  heating at  (2$ SDS,  5$  2-  glycerol,  performed  discontinuous buffer  (for p 5 7  a  system  as  4.5$  a 12.5$  (for p 2 1  r a s  ) polyacrylamide  acrylamide stock s o l u t i o n  was  with  sucrose  the k i n a s e  Electrophoresis  (w/v)  of  RotMIg c o a t e d  g e l sample b u f f e r  (1970).  polyacrylamide  mis  e t a l . , 1983)  for electrophoresis  an S D S - p o l y a c r y l a m i d e  described  o  Tris-HCl  blue).  0.1  Electrophoresis  i n SDS  10 mM  through  c o n c e n t r a t e d SDS  Samples were p r e p a r e d 100°C f o r 3 m i n u t e s  t  ug  Gel  mM  immunoprecipitates  e t a l . , 1984),  fold  and  i n t h e m o d i f i e d RIPA and  reactions  SDS-polyacrylamide  mercaptoethanol,  incubated with  7.0.  2 >  10  (Cooper The  (Lipsich  The  50  as a b o v e .  were r e s u s p e n d e d  MnCl  leupeptin  immunoprecipitated  minutes.  PIPES pH  10 mM  10 m i n u t e s .  equal  and  three times  Ci/mmol, Amersham) and enolase  clarified  centrifugation  and  1 mM  a n t i b o d y 327  minutes  for a further  PIPES pH  was  s u p e r n a t a n t was  a further  aureus  2-mercaptoethanol,  N,N -bis-methylene 1  0.025 M T r i s ,  0.192  was  29.2$  acrylamide. M glycine  and  w/v  49  0.1$  SDS  (pH 8 . 3 ) .  electrophoresed The  gels  were  The d e n a t u r e d  a t c o n s t a n t power  then  fixed  blue, 7 . 5 $ acetic  distilled  water,  in r  [  to l o c a t e  distilled  water.  and 4 5 $ m e t h a n o l  acid  m o l e c u l a r weight  The g e l s  S]methionine  The  were  were  then  gel drier,  markers  (Sigma).  and 4 5 $  methanol  labelled  with gels  dried  screen  For  plus  5$  FBS  methionine  with  which  the c e l l s  were  removed  prior  that  course  Hoefer  (Kodak) a t P l u s ) was  -80°C. used  material.  and r e p l a c e d  incorporation essentially  to l a b e l l i n g .  as  incubated. had been  described  methionine.  f o r 1 hour.  this  medium u n t i l  harvest.  this  had been  The c e l l s 10 mM  to i n h i b i t  3 5  free  s]medium  as  described The medium  was  DMEM p l u s 10$ FBS and were m a i n t a i n e d i n  methionine  greater  [  For the p u l s e - c h a s e  starved  by prewarmed  unlabelled  The  to the m e t h i o n i n e  methionine  shown  on a  i n c u b a t e d i n DMEM-methionine  directly  the c e l l s  film  England  instructions.  paper  Lightning  labelled  had been  were p u l s e d w i t h  10 mM  were  f o r 4 hours  experiments  then  and t i m e  added  (New  Proteins  the c e l l s  was  to XAR-5  (Dupont,  of  the c e l l s  except  3MM f i l t e r  -labelled  pulse-chase  experiments above  onto  containing  Pulse L a b e l l i n g  w i t h En-'Hance  the manufacturer's  and e x p o s e d  intensifying  2.9  treated  Corp.) f o l l o w i n g  gels  slab  in  in  3  Nuclear  An  acid  of m a t e r i a l  35 J  per g e l .  o v e r n i g h t i n Coomassie  were d e s t a i n e d i n 7 . 5 $ a c e t i c  gels  were  of 3 to 4 watts  and s t a i n e d  brilliant  The  proteins  than  was  chosen  9 5 $ of the  as  50 incorporation the  same  2.10  The  Peptide  backing  p e p t i d e mapping  (50  p a p e r removed mM  4  overnight.  f r a g m e n t s washed bovine  at  boiled  4^c  once  i n 2 ml  centrifugation twice  was  made 20$  overnight.  washed  at  The  was  added  15000xg  f o r 30  in ethanol.  The  dried  (100  u l formic acid,  performic  acid  (30$  H o  i n an  dilution  2  ice slurry.  i n 3 ml  and  98$  The  reaction  distilled  oxidized protein  was  pellet 25  water  the  digested with  tosylamido-2-phenyl)ethyl chloromethyl trypsin hours  (Worthington)  at 37°C.  distilled two  water  more t i m e s  sample  was  i n 0.5  ml  o f 50  The  d i g e s t was  and  lyophilized.  from  distilled  resuspended  i n 10  water  of  stored  pellet  oxidized in and  40  ul  [1:9])) for 2  sample  by  lyophilized.  of  L-(1-  ketone-treated mM  diluted The  the  terminated  5 ug  gel  ug  and  u l methanol  was  at  the 50  2-  by  was  formic acid  and  and  acid,  and  t o 5$  supernatants  recovered  minutes  buffer,  elution  buffer.  pooled  as  gel  shaken  collected  to the  was  The  of  then  in trichloroacetic  protein  2  5 ml  of e l u t i o n  acid  The  at  essentially  added, b r o u g h t  was  performic  hours  cells  in elution  f o r 5 minutes,  supernatant  gamma g l o b u l i n  w h i c h were t h e n  was  swollen  crushed.  SDS)  3  The  was  and  N H H C 0 , 0.1$ and  analysis  (Weinmaster e t a l . , 1 9 8 4 ) .  a p p r o p r i a t e band  mercaptoethanol 37°C  t o the  Analysis  described  o f the  buffer  when added  S  tryptic  previously  the  [35 ] methionine  time.  Tryptic  slice  of  NH^HCOg f o r 6 t o 8  t o 3 ml  with  sample was  and  the  lyophilized  digested  protein  u l of e l e c t r o p h o r e s i s b u f f e r  and  5 u l was s p o t t e d  plate  ( E . Merck  2.1 b u f f e r  onto a 20 x 20 cm t h i n - l a y e r  Lab).  (water:88$  The sample was e l e c t r o p h o r e s e d formic  volume) a t 1,000 v o l t s electrophoresis perpendicular  The  plate  exposed 2.11  Southern  precipitated.  loading  for  was p r e p a r e d  buffer  e_t a l .  with  BamHI or  either  and e t h a n o l  (w/v) a g a r o s e  The g e l was t h e n  (1.5 M N a C l ,  blotting  as d e s c r i b e d  t h e membrane  washed  lines  i n Maniatis  i n w a t e r and  Tris-borate-EDTA  submarine g e l .  t h e g e l was t r e a t e d  with  0.25 N HC1  i n 0.5 M NaOH, 1.5 M N a C l f o r covered  0.5 M T r i s - H C l  2  exactly  cell  concentrated  15 m i n u t e s and d e n a t u r e d  capillary  E n h a n c e and  and t h e samples were  i n a 0.75$  N a E D T A ) f o r 30 m i n u t e s .  to  described  phenol extracted  was added  electrophoresis  solution  from  was d i g e s t e d  volume o f t w o - f o l d  30 m i n u t e s .  with  The p r e c i p i t a t e was r e d i s s o l v e d  electrophoresed After  i n N-  at -80°C.  by t h e p r o c e d u r e  (BRL-Gibco),  equal  and c h r o m a t o g r a p h e d  (75:15:60:50, by v o l u m e ) .  and s p r a y e d  The p u r i f i e d DNA  Hindlll  = 90:2:8 by  Hybridization  genomic DNA  (1982).  an  film  i n pH  After  was a i r d r i e d  acid:water:pyridine  was a i r d r i e d  essentially  acid  f o r 45 m i n u t e s .  the p l a t e  t o XAR-5  The  acid:acetic  to the d i r e c t i o n of e l e c t r o p h o r e s i s  butanol:acetic  cellulose  The DNA  with  neutralizing  pH 7.2, 0.001 M  was t h e n  transferred  t o Hybond-N (Amersham) n y l o n by t h e m a n u f a c t u r e r .  by U.V.  irradiation.  membranes  The DNA  was  The membrane  was  i n 0.1X SSC (1X SSC: 0.15 M N a C l ,  0.015 M  by  fixed  Na^itrate),  0.5$ SDS, a t 65 C f o r 60 m i n u t e s  prehybridized Denhardt's  i n 6X SSC, 5X D e n h a r d t ' s  solution:  2$  2% p o l y v i n y l  Ficoll,  sonicated, hours.  (w/v) b o v i n e  pyrollidone),  denatured  salmon  fragment  from  pMC38 ( a g i f t  s o l u t i o n (100X  0.5$ SDS, and 20 ug/ml a t 65°C f o r 4 t o 8  was p r e p a r e d  from  o f J.M. B i s h o p ,  with and  by n i c k t r a n s l a t i o n  [ c * ' P ] dCTP then  separated  chromatography with  24 h o u r s  on Sephadex  exposed  t o XAR-5  The  steroid  Millipore) infected  from  Y-1  was p r e p a r e d The  Corp.)  denatured  and t h e h y b r i d i z a t i o n  minutes,  twice  once i n 2X i n 2X SSC,  and once i n 0.1X was t h e n  w i t h an i n t e n s i f y i n g Products  SSC a t  a i r d r i e d , and s c r e e n a t -80°C.  from  t h e Y-1  Line  p r o d u c t s were p u r i f i e d  on a SepPak  (Waters-  t h e medium o f t h e p a r e n t a l and v i r u s  cell  lines  by w a s h i n g  medium was t h e n  washed  was  was t h e n washed  minutes  of Steroid  Cell  Nuclear  as t h e p r e h y b r i d i z a t i o n f o r  The membrane  film  2.12 HPLC A n a l y s i s Adrenocortical  for thirty  for thirty  f o r 10 m i n u t e s .  England  The probe  sperm DNA  The membrane  0.1$ SDS a t 65°C  0.5$ SDS a t 65°C 65^C  G-50.  i n t h e same b u f f e r  a t 65°C.  fragment  u n i n c o r p o r a t e d n u c l e o t i d e s by  t h e s o n i c a t e d salmon  was p e r f o r m e d  SSC,  from  The  et  ( M a n i a t i s e t a l . , 1982)  (3,000 Ci/mmol, New  32  a 1.5 kb  (Vennstrom  a l . , 1981)) c o n t a i n i n g t h e MC29 v-mvc gene. was l a b e l l e d  2% (w/v)  serum a l b u m i n ,  sperm DNA  The a v i a n v-myc probe  and t h e n  again with  (Ramirez  e t a l . , 1982).The  with dichloromethane  passed  over  column  and t h e n  t h e column d r o p w i s e  PBS. The s t e r o i d s  were t h e n  eluted  PBS.  and wth  dichloromethane drying  and d r i e d  the s t e r o i d s  under  a stream  were r e s u s p e n d e d  of nitrogen. After  i n either  methanol or  acetonitrile.  The HPLC was p e r f o r m e d  on a Waters C^g  reverse  10 um bead  a t room t e m p e r a t u r e a t  phase,  a flow rate  size  column  o f 1 ml p e r m i n u t e .  The sample was l o a d e d  t o 30 u l volume a t 30$ a c e t o n i t r i l e was  r u n a t 30$ a c e t o n i t r i l e  steroids  eluted  acetonitrile steroids on  i n water  spectrophotometric  (100  diluted  The t r a c e r  New E n g l a n d  i n ethanol.  NaCl,  0.46 g / 1 N a H P 0 , 4  Dextran  coated  Norit-A  charcoal (Fisher  just  prior  to use.  o f Ki-MSV/MMCV  for Steroid  Production  a n t i - p r o g e s t e r o n e - 1 1 (X progesterone  C o r p ) and t h e c o l d  The s t a n d a r d s dilutions 2  Chemicals)  volume  Ki-MSV/MMCV  The c e l l s  i n PBSG 1|  (7.94  1g/l  g/1  gelatin).  by m i x i n g  0.625 g  and 0.0625 g D e x t r a n T-  PBSG.  transformed  i n DMEM w i t h  Sigma  were made from t h e  1.01 g / 1 Na HP0 ,  c h a r c o a l was p r e p a r e d  i n 100 mis f i n a l  overnight.  A l l s o l v e n t s were o f  was [ 1 , 2 , 6 , 7 - ^ ]  Nuclear  by s e r i a l 2  a 35 mm d i s h  and i n t e g r a t e d  ( 4 - p r e g n e n - 3 , 2 0 - d i o n e ) was o b t a i n e d from  progesterone  The  and f i l t e r e d  was a r a b b i t  stock  70  The e l u t i o n o f  Integrator.  Cells  and t h e  gradient of  25 m i n u t e s .  Rat A d r e n a l C o r t e x  uCi/mmol,  f o r 15 m i n u t e s  o f C u l t u r e Supernatant  a n t i b o d y used  progesterone and  grade  (Miles-Yeda).  The column  a t 254 nm and r e c o r d e d  3390A  Radioimmunoassay  The BSA  over  was m o n i t o r e d  Transformed  i n water  i n a 30 t o 70$ l i n e a r  a Hewlett-Packard  2.13  i n water.  i n 15  cell  lines  were s e e d e d  10$ FBS and a l l o w e d  to attach  were i n c u b a t e d f o r 1 hour  i n DMEM  into  without  serum.  The c e l l s  were  then  x 10~9 g p r e g n e n o l o n e  ( 3 mis f i n a l  37^C.  centrifuged  The medium was  microfuge  standard  were mixed  approximately incubated then was  antibody,  2500 cpm  f o r 5 minutes  chilled added.  stored  progesterone  i n a 400 u l f i n a l  After  tracer,  coated  supernatants  with  scintillation  and were  charcoal  10 m i n u t e s t h e s a m p l e s were c e n t r i f u g e d a t and t h e s u p e r n a t a n t  were mixed  (Amersham)  sample  The s a m p l e s  2500 x g f o r 15 m i n u t e s  fluid  and  The  volume w i t h  f o r 1 hour.  on i c e and 200 u l o f d e x t r a n  in a  at -20°C.  tracer  of the p r o g e s t e r o n e  a t room t e m p e r a t u r e  i n DMEM w i t h 5  volume) f o r 4 h o u r s a t  a t 4°C and t h e s u p e r n a t a n t  anti-progesterone or  incubated  3 mis o f ACS  and t h e c o u n t s  counting  decanted. scintillation  were d e t e r m i n e d  by  The  CHAPTER 3 3.0  Expression  Transformed  of  Viral  P h e n o t y p e by  p21 Rat  r a s  during  Adrenal  A c q u i s i t i o n of  Cortex  w i t h K i r s t e n M u r i n e Sarcoma 3.1 The sought  analysis  to d e f i n e  carcinogenesis. multistep 1.4  of  the  transformation  the  molecular  mechanisms i n v o l v e d  oncogenesis has  Introduction)  requirement vitro.  INTRODUCTION  oncogenic  there  retroviruses  can  changes and  to  epigenetic  changes.  by  murine leukemia  Abelson  1981)  or  an  a l . , 1984) on  for in  Infection  produces  Abelson  also  cellular avian  genes  cells  (Whitlock  that  a highly apparently  between  et  Alema  behind  retrovirally  independent phenotype  a c t i v a t i o n of  Transformation  et  et  infected  of  shows  borne oncogenes  et a l . , 1985b; Kahn  or  dependence  cultures  also  in  cells  (Spooncer  involves  the  Witte,  tumourigenic  et a_l. , 1 9 8 3 ) -  (see  genetic  and  v i r u s become serum  a  transforming  have a r e d u c e d The  be  process  the  (Whitlock  from d i f f e r e n t i a t e d t i s s u e s  cooperativity a l . , 1984:  that  by  the  complementing  factors.  acquire  a stepwise fashion  cells  to  effort  reasons  murine r e t r o v i r u s  murine leukemia  g r o w t h and  appears  mimic  has  in  of m u r i n e bone marrow  virus  cultures  exogenous serum g r o w t h  with  involve  src-containing  vivo  e l u c i d a t e the  of p r i m a r y  appear  in  in vitro  been c o n s i d e r a b l e  to  for multiple  Transformation  Infected  Virus  of  Since  process  Cells  a  (Adkins  a l . , 1984).  Most  studies  oncogenes  have  on t h e a c t i o n  employed  cell  resemble malignant c e l l s (Sherbert, arise  1982).  in adult  oncogenes, from it  In c o n t r a s t  t h e ras genes,  adrenal  that  cortex,  have been  used  transformation types.  a model  i n Barbacid,  mechanisms o f (Jenuwien  Normal  steroidogenic, (Slavinski  on c e l l  rat adrenal  explants  i n 25%  intermediates  transformation  et_ a_l. , 1985 ).  cells  fetal  cortex  of the r a t  morphology  epithelial  and  capacity.  The c e l l s  stem  of the a d r e n a l  cells  be more h i g h l y d i f f e r e n t i a t e d .  from  grow a c t i v e l y ,  pathway.  and  cells adopt  pregnenolone to In t h e p r e s e n c e  grow s l o w l y ,  i n FBS  The  effect  hormone r e s p o n s i v e  e x p r e s s a more complex  cortex  by Ki-MSV  (FBS) as  can m e t a b o l i z e  cultured  cell  can be grown  These c e l l s  these  i n the  differentiation.  cells  of the s t e r o i d o g e n i c (HS)  cells  b o v i n e serum  and  epithelium,  to i n v e s t i g a t e the and  tumours  1986), but  of e p i t h e l i a l  adrenocorticotrophic  of 3% h o r s e serum  cells  growth  cortex  et a_l. , 1974).  a fibroblastic  with  of oncogenes  development  system i n which  of a r a s oncogene  tissue  cell  Some  (reviewed  I n f e c t i o n of r a t a d r e n a l  presents  cells..  a steroid-secreting glandular  and  tumours  associated  the r o l e  which  characteristics  have been  t o r e s e m b l e stem  t o examine  cells,  most s p o n t a n e o u s  for a l l cells  appear  cellular  or embryonic  epithelial  the molecular  are not i d e n t i c a l  or  i n some o f t h e i r  a v a r i e t y of t i s s u e s  Cultures  lines  differentiated  appears that  of v i r a l  appear  steroidogenic resemble those  fibroblastic  i n HS  appear t o  i n t e r c o n v e r s i o n of t h e  two  cell  types  intermediates  in culture  suggests  i n 25% FBS f o c i  Auersperg into  infected cases,  become serum  transformed.  adrenal c e l l s  prevent  (Auersperg  cortex  cells  infected  with  Culturing  appear  the level  and t h e c u l t u r e s  phenotype  et  a l . , 1981).  f o r two r e a s o n s :  w i t h Ki-MSV; and  regulation  by serum  the level  adrenal  cells  transformation  of v i r a l  of d i f f e r e n t i a t i o n  has been some e v i d e n c e  p21  r a s  c o u l d be c o r r e l a t e d  that the  can be c o r r e l a t e d ( W i n t e r and  to determine  i n t h e K i - M S V - i n f e c t ed with  their  or was c o n s t a n t . The amount  during  cells  factors  o f e x p r e s s i o n o f t h e r a s oncogene  whether  f o r adrenal  adrenal cortex  t o modulate t h e s t a t e There  first,  second,  1986), and s o i t was o f i n t e r e s t  expressed  passaging i n  and c a n , i n some  of t r a n s f o r m a t i o n of f i b r o b l a s t s  Perucho,  cultures i n  o f t h e Ki-MSV-  1984; A u e r s p e r g  of t h e Ki-MSV-infected  of u n i n f e c t e d c e l l s . degree  t o t h e slow-  further  independent  a r e of i n t e r e s t  subject to environmental  which a l s o  a r e passaged  a multistep transformation process  transformation is  With  i n 3% HS d e l a y s  and C a l d e r w o o d ,  indicate  revert  appearance of the transformed  These r e s u l t s they  et al_. , 1977;  morphology, w h i l e s i b l i n g foci.  i n vivo.  w i t h Ki-MSV and  When t h e s e c u l t u r e s  25% FBS c o n t i n u e t o e x p r e s s  become o v e r t l y  represent  pathway  (Auersperg  a l l of t h e c e l l s  epithelial  25% FBS t h e f o c i  are infected  appear  et a l . , 1981).  3% HS, u s u a l l y  growing,  they  o f t h e same d i f f e r e n t i a t i o n  When a d r e n a l c o r t e x c u l t u r e s cultured  that  degree of  of p 2 1  t r a n s f o r m a t i o n was u n c h a n g e d ,  r a s  suggesting  58 that allow  additional  changes  must  occur  e x p r e s s i o n of a t r a n s f o r m e d  i n the i n f e c t e d phenotype.  cells  to  R ESULTS  3.2  Expression  Adrenal  Cortex  Infection. end  of V i r a l p 2 1  Rat  of t h e  described  Cells  Ki-MSV-inf ected labelled  with  cellular  p21  r a s  the p 2 1  r a s  into  methionine present. only  the v i r a l  p21  used of  3  2  Pi  from  treated with  3  2  the v i r a l  p21  FBS  ( F i g . 3.1),  a l l passages  1/2  transformed.  had  but  after  3 and  The  a normal  being  S]-  p21  r a s  which  labels  the specific  that then  because alters  a c t as  a  e_t a_l. , 1982 ).  viral  infection  m a j o r i t y of  the  fibroblastic  particularly  infection  3 5  occurs  some t r a n s f o r m e d  of t h e c e l l s a p p e a r e d The  5 after  or 3% HS.  FBS  assayed,  f i v e passages  2,  of  The  s u s t a i n e d a mutation  passages  [  under  r a s  and  incorporated  Pi,  ( S h i h e_t a l . , 1982a; T s u c h i d a  i n 25%  cells  morphology  and  into  the  viral  with  amount  t o c o d e f o r a t h r e o n i n e w h i c h can  were c u l t u r e d i n 25%  1/4  the r e l a t i v e  ( P a p a g e o r g e _et . a l . , 1982).  r a s gene has  Cells  FBS,  of l a b e l  to d e t e c t a b l e l e v e l s  r a s  c u l t u r e d as  both  the c u l t u r e s incubated  reflect  the  c e l l s were m e t a b o l i c a l l y  amount  relative  at  expression  r a s  .  phosphoacc eptor  at  cortex  C u l t u r e s were a l s o  incorporation  infected  examine p 2 1  which l a b e l s  should  conditions  59  To  3 5  The  Following  K i - M S V ( K i - M L V ) and  [ S ] methionine,  the v i r a l  codon  2.  adrenal  from  Rat  c o r t e x c e l l s were i n f e c t e d  passage with  i n chapter  in Ki-MSV-infected  i n the Passages Immediately  adrenal  first  r a s  (panel  cells  appeared  apparent  in  25%  E) where between  morphologically  c u l t u r e s i n 3% HS  showed  a more  flattened  F i g u r e 3.1. E a r l y passages of KiMSV-infected r a t a d r e n a l c o r t e x c e l l s grown w i t h 25% f e t a l b o v i n e o r 3% h o r s e serum supplements. A r a t a d r e n a l c o r t e x p r i m a r y c u l t u r e was p r e p a r e d and i n f e c t e d w i t h K i M S V ( K i M L V ) a t p a s s a g e one a t m . o . i . ' s o f 1-3 as p r e v i o u s l y d e s c r i b e d . The c e l l s were grown i n 25% FBS and p a s s a g e d i n t o 25% FBS o r 3% HS a t t h e b e g i n n i n g o f each o f the passages noted below. The KiMSVi n f e c t e d a d r e n a l c o r t e x c e l l s were p l a t e d i n 10 mm w e l l s a t p a s s a g e 2 (A and B ) , p a s s a g e 3 (C and D) and p a s s a g e 5 (E and F ) , and c u l t u r e d i n 25% FBS (A,C,E) o r 3% HS ( B , D , F ) . C e l l s were t h e n c u l t u r e d f o r 4-17 days p r i o r t o photomicroscopic examination. P h o t o m i c r o g r a p h s a r e a t 100X magnification.  epithelial no  m o r p h o l o g y , grew more s l o w l y  morphologically  examined  for v i r a l  [ ^S]methionine lysates,  3  normalized  anti-p21  1982),  p21  or  3  the  transformed  Pi  2  and  normal  doublet  with  5  expressed  cells 3.2,  as  rat  labelled band  immunoprecipitating  antibody  Y13-259  1 and  (Shih  with  cell with  et a l . ,  adrenal  level  cortex  a cellular  c e l l s from  of p 2 1  than  r a s  Immunoprecipitation  c e l l s showed with  the  of  r a s  The  [  3 5  passages the  the  S]-  2,  3  uninfected band 3  p r e s e n c e of a  t h e more s l o w l y  cells  p21  t h e more i n t e n s e l y l a b e l l e d  5).  comigrated  (Furth  et a l . , 1982b).  K i - M S V - i n f e c t ed  by  or  serum.  9)  a higher  infected  that  labelling  of u n i n f e c t e d  lane  indicated  lanes  by  3 5  methionine l a b e l l e d and  few  T h e s e c u l t u r e s were  [ S ]methionin e revealed  ( F i g . 3.2,  contained  for TCA-precipitable radioactivity,  Immunoprecipitation labelled  cells.  , expression  monoclonal  r a s  or w i t h  r a s  and  2  (Fig.  Pi  specific  migrating  r35 L  S]methionine l a b e l l e d  band  is  characteristic  phosphorylated  (Papageorge identified produced adrenal  p21  r a s  et al., by  the  1982).  in a l l three  early  cells.  were e x c i s e d  labelled  scintillation  counting  r a s  ,  3 and ras  7)  specifically  with  3  2  p a s s a g e c u l t u r e s of  or  the  Pi,  was  the  infected  p h e n o t y p e of  [ S]methionine labelled 3 5  t h e g e l and  (Table  3.1).  subjected The  as  product  serum c o n d i t i o n s , r e g a r d l e s s  The  from  viral v  d e g r e e of t r a n s f o r m a t i o n cortex  lanes  Ki-MSV p 2 1 "  i t s a b i l i t y t o be  c e l l s under b o t h  relative adrenal  of  ( F i g . 3.2,  amount  of the  bands  to of  the  viral  of  F i g u r e 3.2 A n a l y s i s of p 2 1 expression i n KiMSV-infected and u n i n f e c t e d r a t a d r e n a l c o r t e x c e l l s . Immediately f o l l o w i n g p h o t o g r a p h y t h e c u l t u r e s shown i n f i g u r e 1 were l a b e l l e d w i t h [ s ] m e t h i o n i n e ( l a n e s 1, 2, 5, 6, 9 , 10) o r with P i ( l a n e s 3 , 4, 7, 8) i n 0 . 3 ml D u l b e c c o ' s m o d i f i e d E a g l e ' s medium l a c k i n g m e t h i o n i n e or p h o s p h a t e r e s p e c t i v e l y and s u p p l e m e n t e d w i t h 5$ FBS f o r t h e c e l l s p r e v i o u s l y grown i n 25$ FBS ( l a n e s 1-4,9,10) or 3$ HS ( l a n e s 5-8) f o r t h e c u l t u r e s grown i n 3$ HS. The l a b e l l e d c e l l s were l y s e d and c l a r i f i e d as p r e v i o u s l y d e s c r i b e d , n o r m a l i z e d f o r TCA p r e c i p i t a b l e r a d i o a c t i v i t y , immunoprecipitated with a n t i p21 m o n o c l o n a l a n t i b o d y Y13-259 ( l a n e s 1, 3 , 5, 7, 9) o r n o r m a l r a t serum ( l a n e s 2, 4, 6, 8, 1 0 ) . The i m m u n o p r e c i p i t a t e s were a n a l y z e d on a 12.5$ SDS-PAGE as d e s c r i b e d i n M a t e r i a l s and M e t h o d s . r a s  3 5  3  r a s  2  64  65 TABLE 3. 1  [  3 5  S]  Passage number  2  a  3  30  3  a  b  b  2b Blank  Incorporation into p 2 1 Uninfected Adrenal  r a s  Serum supplements  Ki-MSV infection  i n Ki-MSV-infected Cortex C e l l s  and  [ S]counts counts/minute per minute corrected for i n band total incorpora 3 5  FBS  +  675  HS  +  681  FBS  -  295  FBS  +  936  -  HS  +  474  573  FBS  -  280  -  FBS  +  1 201  1201  HS  +  238  954  FBS  +  81 1  1286  FBS  -  403  403  _  _  58  58  T a b l e 3. 1 The a r e a s o f t h e g e l s shown i n F i g u r e s 3.2 and 3-5 c o r r e s p o n d i n g t o p21 were e x c i s e d and p l a c e d i n PCS s c i n t i l l a t i o n f l u i d (Amersham). The g e l s l i c e s were then c o u n t e d f o r t ^ S ] m e t h i o n i n e . The l y s a t e f o r t h e i m m u n o p r e c i p i t a t i o n s shown i n F i g u r e 3.5 f o r t h e c e l l s i n HS a t p a s s a g e 30 o r FBS a t p a s s a g e 3 c o n t a i n e d fewer c o u n t s than t h e c e l l s i n FBS a t p a s s a g e 30 and so t h e c o u n t s d e r i v e d were c o r r e c t e d to account f o r t h i s . L ^ S ] c o u n t s i n p21 on t h e g e l shown i n F i g u r e 3.2. [ S ] counts i n p 2 1 on t h e g e l shown i n F i g u r e 3.4. r a s  3  3 5  r a s  p21  was  conditions within  approximately at  each  passage.  experiments  consistent  t h e same i n b o t h  (6  There  experiments  b i a s i n the l e v e l  the c u l t u r e  c o n d i t i o n s was  culture  were some on  of p 2 1  noted.  differences  3 cultures), r a s  but  production  Another  band  between  (M r  27,000) was  also  specifically  Y13-259 a n t i - p 2 1 [  3  S]methionine  labelled  discussed  further  3.3  p21  Viral  Transformed  r a 3  transformed determine  Rat  correlated  examined  a t p a s s a g e t h r e e , two  e x p r e s s i o n , an  stage  few  transformed  ( F i g . 3-3  c u l t u r e had  become f u l l y  growth  ( F i g . 3.3  3.3  were r a d i o l a b e l l e d  anti-p21 level  labelling  lysates  and  was  is  Fully  Cortex  Cells.  As  a r e passaged  Calderwood,  any  in  the  A and with  r a s  essentially  after  The  and  serum  [ S]methionine  antibody  was with  morphologically  cultures  3 5  To  level  infection  a t p a s s a g e 30,  were s u b j e c t e d t o  p21  change i n the  appeared  again  B).  1984).  transformed  passages  transformed  monoclonal  of v i r a l  and  adrenal cortex culture  cells  C ) , and  for  The  and  with  p21  with  Adrenal  (Auersperg  of v i r a l  cell  the  7.  i f the p r o g r e s s i o n to a f u l l y  Ki-MSV, a t w h i c h  by  the c u l t u r e g r a d u a l l y acquires a  phenotype  r a s  "  the  ( F i g u r e 3.2)  adrenal cortex c e l l s  FBS  p h e n o t y p e c o u l d be  labelled  in  Expression i n P a r t i a l l y  Ki-MSV-infected  of 25%  antibody  cultures  i n chapter  the Ki-MSV-infected presence  immunoprecipitated  monoclonal  r a s  no  when  the  independent  shown i n F i g u r e or  3  2  Pi  and  the  immunoprecipitation  as d e s c r i b e d b e f o r e .  e x p r e s s i o n as  r e v e a l e d by  t h e same i n a l l t h r e e  3  2  Pi  cultures  F i g u r e 3-3. Morphology of K i M S V - i n f e c t e d r a t a d r e n a l c o r t e x c e l l s a t e a r l y and l a t e p a s s a g e s ( 1 0 0 X ) . Rat a d r e n a l c o r t e x c e l l s i n f e c t e d w i t h KiMSV a t t h e f i r s t p a s s a g e were c a r r i e d u n t i l p a s s a g e 30, by which t i m e t h e c e l l s had a c q u i r e d a f u l l y t r a n s f o r m e d p h e n o t y p e and were s e r u m - i n d e p e n d e n t f o r growth. The K i M S V - i n f e c t e d c e l l s a t p a s s a g e 30 (29 p a s s a g e s a f t e r i n f e c t i o n ) were c u l t u r e d i n t h e p r e s e n c e o f 25% FBS (A) o r 3% HS ( B ) . The c e l l s used i n t h i s e x p e r i m e n t were from t h e same o r i g i n a l c u l t u r e shown i n F i g u r e 1. An a l i q u o t o f t h e same K i M S V - i n f e c t e d a d r e n a l c o r t e x c e l l s f r o z e n i n t h e f i r s t p a s s a g e a f t e r i n f e c t i o n was thawed, grown i n 25% FBS and examined a t p a s s a g e 3 (C) a t t h e same time as t h e c u l t u r e s i n p a s s a g e 30.  68  Figure 3 . 4 . E x p r e s s i o n of v i r a l p 2 1 i n e a r l y and l a t e passage c u l t u r e s of K i M S V - i n f e c t e d a d r e n a l c o r t e x c e l l s . The f o l l o w i n g c u l t u r e s ( i l l u s t r a t e d i n F i g u r e 3 . 4 ) were analyzed f o r p 2 1 : KiMSV-infected rat adrenal cortex c e l l s ( T r A ) a t p a s s a g e 30 ( l a n e s 1 - 6 ) or p a s s a g e 3 ( l a n e s 7 - 9 ) c u l t u r e d i n 25$ FBS ( l a n e s 1 - 4 , 7 - 9 ) o r 3$ HS ( l a n e s 5 and 6). U n i n f e c t e d a d r e n a l c o r t e x c e l l s (AC) were a l s o a n a l y z e d ( l a n e s 10 and 1 1 ) . Immediately f o l l o w i n g photography, c e l l s were l a b e l l e d w i t h ^ Pi ( l a n e s 1, 2, 5, 7 ) or [ S ] m e t h i o n i n e ( l a n e s 3, 4 , 6 , 8 - 1 1 ) . C e l l s were l y s e d , and a l i q u o t s c o n t a i n i n g e q u a l T C A - i n s o l u b l e r a d i o a c t i v i t y were immunoprecipitated with a n t i - p 2 1 Y13-259 a n t i b o d y ( l a n e s 1> 3 » 5-8, 10) or w i t h n o r m a l r a t serum ( l a n e s 2, 4 , 9, 11); the l y s a t e f o r l a n e 6 c o n t a i n e d o n l y 20$ o f the TCAi n s o l u b l e [ - " S i m e t h i o n i n e c o u n t s per m i n u t e compared to other [ 3 s ] m e t h i o n i n e - l a b e l l e d l y s a t e s . The immunoprecipitates were a n a l y z e d by e l e c t r o p h o r e s i s on a 12.5$ S D S - p o l y a c r y l a m i d e g e l as d e t a i l e d i n M a t e r i a l s and Methods. r a s  r a s  2  3 5  r a s  5  TrA  passage •< P-30 serum FBS HS  p-3 • FBS  AC  1 -2 3 4 5 6 7 8 9 1011 - 205 - 97 - 68 - 45 •30 - 24  (Fig.  3.4 l a n e s  was d e t e r m i n e d  1, 5 and 7 ) . The t o t a l  by [ 3 5  synthesis  t h e same i n b o t h  infected  adrenal  relative  rates  lanes  the early  cortex  of t  3 5  cells  3-4  bands. cells gene.  lane  r a s  p21  r a s  The t o t a l  five-fold  10) as q u a n t i t a t e d  These r e s u l t s adrenal  level  greater  suggested  cortex  cells  r a s  .  5  s ] -  cells  of excised  infected  of the v i r a l r a s  that progression  by enhanced  3  cortex  adrenal  i n virus  to the f u l l y  [  the l e v e l of  by t h e c o u n t i n g r a s  of  adrenal  than  uninfected  of p 2 1  p h e n o t y p e was n o t accompanied p21  f o r the  level  i n Ki-MSV-inf ected  was p r e s u m a b l y due t o t h e e x p r e s s i o n  infected  viral  p a s s a g e Ki-MSV-  when n o r m a l i z e d  i n corresponding  The i n c r e a s e d  and a p p e a r e d t o  S ] m e t h i o n i n e i n c o r p o r a t i o n ( F i g . 3-4  was a p p r o x i m a t e l y  endogenous p 2 1 (Fig.  cells  and l a t e  3, 6 and 8 and t a b l e 3 - 1 ) .  methionine l a b e l l e d  r a s  s ]  methionine l a b e l l i n g be  of p 2 1  o f Ki-MSV-  transformed production  of  72  3.4 The  data  presented  Ki-MSV-encoded the  acute  cortex  p21  here suggest  i s not  r a s  transformation  cells.  DISCUSSION  of  itself  of f r e s h l y  E a r l y passage c e l l s  expressing  levels  transformed  cell  of v i r a l lines  p21  can  infected  their  r e v e r t to a slow-growing, normal  the  p r e s e n c e o f HS  transformed  foci.  high  of v i r a l  levels  epithelial,  The  reverted  p21  of v i r a l  transforming adrenal FBS.  Auersperg,  p21  which r a s  r a s  ,  but  1981)  full  rather  of Ki-MSV p 2 1  previous  to  that  level the  expression  that  the  of  p r e s e n c e of a f a c t o r ( s ) i n  that  (Auersperg Harrison  a growth  and  and  factor(s) in  cooperates  with  formation.  many p a s s a g e s  suggesting  that  i n e a r l y passage  r a s  functionally  transformation  synthesize  normal  limited  i n FBS of p21  of a d r e n a l  was  not .  cortex  apparently  Both  cultures accompanied  e a r l y and Y*  cells  of  express  appear  the  e_t a l . , 1981;  from HS  phenotype i n  apparently  experiments  i t seems l i k e l y  and  sensitivity  continue  affect  Ki-MSV  appearance  I t would  r e q u i r e s the  by  i s absent  increased  does n o t  1984 ; A u e r s p e r g  i n focus  The after  cells  suggested  Calderwood,  FBS  activity  cortex  As  p21  serum  despite their  r a s  induce  adrenal  with  epithelial  cells  to  of  of Ki-MSV-  initial  s t a t i o n a r y phenotype.  medium serum s u p p l e m e n t expression  the  expression  sufficient  typical  r a s  retain  after  the  cultured rat  and  even  that  similar the  amounts of Ki-MSV p21  development  of serum  late  by  passage  3. S ,  insensitivity  in  late  passage c e l l s  expression overtly or  of t h e  r e q u i r e s a step viral  transformed  ras product.  p h e n o t y p e may  e p i g e n e t i c changes r e s u l t i n g  cooperating factors  or  cellular loss  phenotype.  of  gene, t h e  the  differentiation  of a v i a n  accompanied  by  subcellular the  (Myrdal p21  and  expression infected  an  be  cell.  which This  events,  different infected  can  be  can  v - m v  ^  transforming  of t h e  i n r a t kidney  cell.  altered  A  accompanies cells viral  variation  in  phenotypes  here suggest dependent  relieved  that  on  of  the  by  the a c t i o n  environmental  differentiated be  p h e n o t y p e of as  alteration  of v i r a l  in a variety  t i s s u e s have been cells  ester i s  apparently  simple  the  yet of a  still  p21  r a s  .  of primary  i n v e s t i g a t e d and  respond  to  The cell  the  unknown cellular  i n a f u n c t i o n a l complementation  activity  oncogenes  growth  transformed  Thus, a c h a n g e i n  i n v o l v i n g the  result  r a s  i n the  reported  d e p e n d e n c e can  presumably  ras  r a s  a  transformed  phorbol p45  an  cells.  i n f l u e n c e the  transforming  viral  1985).  cortex  p21  gene, w h i c h can the  of p 2 1  important  observations  the  virus  the cytoplasm  more s u b t l e t h a n  oncogenic  factors  to  by  of t h e  of t r a n s f o r m a t i o n  Auersperg,  may  of  to  genetic  of tumour  to suppress  induced  redistribution  adrenal  The of  the nucleus  activity  r a s  in activation  a redistribution  progression  progression  involve cellular  myeloblastosis  t o macrophages  from  i n a d d i t i o n to  et _al . (1984) have shown t h a t  myeloblasts  protein  This  expression  ability  Klempnauer  or s t e p s  of  effects  of  types  from  some Ki-MSV-  exogenous s i g n a l s  by  differentiating cannot types  (Yuspa  et _ a l . , 1985 ).  of c e l l s  addition Yoakum  may r e q u i r e  t o v - r as  Spandidos  and W i l k i e ,  cells,  contain  o v e r expr ess ed  but  19 8 4 ) .  and t h a t  acquisition  adrenocortical  cells  increase  results  further  activating  contribute  may be t r a n s f o r m e d (Land  r a s genes  (Winter  transformed  that  i n some a d r e n a l  the a m p l i f i c a t i o n of the K i - r a s  increased derived  expression  cell  line  1986),  p h e n o t y p e by t h e  gene.  These  for several  to give  full  the c e l l u l a r cortex  iiivivo  Ki-ras  tumours and i s s u p p o r t e d by  2 gene and i t s g r e a t l y  i n t h e Y-1 a d r e n o c o r t i c a l  (Schwab  lines  to c o r r e l a t e with a  cells  progression  o f some  and P e r u c h o ,  of the v-ras  The p o s s i b i l i t y  that  transform  variants  does n o t a p p e a r  to malignant  by r a s  et al.. , 1986;  r a s genes c a n  i n normal primary  gene may be a c t i v a t e d  some  et aJL. , 1985 ;  demonstrate the requirement  transformation.  that  I t has been d e m o n s t r a t e d  i n expression  steps  others  oncogene i n  (Rhim  tumourigenic  of a h i g h l y  further  both  conditions  ov er expr ess ed , a c t i v a t e d  primary  appear  a cooperating  f o r transformation  specific  1985), w h i l e  I t would  et _ a l . , 1985), b u t o t h e r s  a l o n e under  highly  ( P i e r c e and A a r o n s o n ,  et a l . , 1983).  tumour  CHAPTER 4 4.0 T r a n s f o r m a t i o n o f Rat A d r e n a l C o r t e x C e l l s by r„as. and mvc: E v i d e n c e f o r t h e R e q u i r e m e n t f o r a F u r t h e r C e l l u l a r Chang e 4.1  The  results  indicated with  the expression  demonstrated  transformation  systems  cells  that  when  to cooperate  either  cortex  examining  there  c a n be e x c e p t i o n s  The myc  in  vivo  require  (Jenuwein  hematopoietic  c e l l s _in v i t r o  et j a l . ,  requirement  1 986), described  could  n o t (Land  et  t o be  a v a r i e t y of  I t would  change.  f o r a f u r t h e r step  ras to r e s u l t i n  although these  r e p r e s e n t a t i v e o f two appear  that  o r r a s and myc  ( O s h i m u r a _et a_l. , 1985 ; Thomassen a further cellular  oncogenes  et a l . , 1985) and  by r a s a l o n e  a requirement  Vogt  and c e l l u l a r  et a l . , 1984 ),  c l a s s e s of oncogenes. of c e l l s  I t has been  oncogene appears  o n c o g e n e s have come t o be c o n s i d e r e d  transformation  cooperate  e a r l y passage,  oncogene a l o n e  et E Q . , 1 983 ; T a y a  (Murray  cooperating  cells.  ras i n transforming  cells  to  had  r a s oncogene i n t h e  o f an o n c o g e n i c  1 983 ).  with  chapter  changes  some v i r a l  the a c t i v i t y  a l . , 1 983a; R u l e y , able  for cellular  of the v i r a l  i n other  complement  i n the previous  of the adrenal  nonimmor t a l i z ed can  described  a requirement  transformation  INTRODUCTION  together  et al_. , 1 985 ) may  There a l s o appears  t o be  i n the transformation of  (Stevenson  and V o l s k y ,  1986 ;  b u t t h e r e has n o t been a s i m i l a r for fibroblasts.  The from  an  useful still  adrenal  cortex  cells,  a d u l t , hormone p r o d u c i n g system  of  f o r the  interest  as  that  of  adrenal  cortex  cellular  cells  associated highly  the  this  gene i n a r e l a t i v e l y  allows  containing  an  avian  retrovirus  construct  (see F i g u r e 4.1). the  same h e l p e r  B.  the  Productive reinfection  has  two  oncogenes  infection by  the  by  been  agar  by  closely  and  a  et a_l. , 1 986 ).  efficient  gene; and  introduced  manner. and  The  Mayer,  1967),  MMCV(Mo-MLV),  into  a  murine  Vennstrom) (Vennstrom  et a l . ,  MMCV were p s e u d o t y p e d  Moloney murine leukemia v i r u s  (Mo-MLV), n e c e s s i t a t i n g c o i n f e c t i o n by introduce  phenotype  i n t r o d u c t i o n of a f o r e i g n  Ki-MSV and  virus,  the  The  transformed  v-myc gene c l o n e d (from  in  to d e f i n e a f u r t h e r  were: Ki-MSV(Mo-MLV) ( K i r s t e n a c t i v a t e d v-ras  as  expression  e x p e r i m e n t s were  the  g e n t l e and  an  with  alterations  (Auersperg  i n these  the  The  to a l l o w  t o grow i n s o f t  containing  1984)  changes.  for transformation.  independent  of  morphological  adrenocortical cells  o n c o g e n e s used  used  been used  phenotype  r e t r o v i r u s e s as  viruses  has  ability  tumourigenic  The by  with  related  morphological  of a serum  t h e Ki-MSV- i n f e c t e d  to both  a It is  concentration  serum s u p p l e m e n t  change r e q u i r e d  acquisition  lead  growth  f o r a high  the Ki-MSV-induced  can  represent  of t r a n s f o r m a t i o n .  a change i n the  f u n c t i o n a l and  requirement  epithelium,  examination  medium serum s u p p l e m e n t well  b e c a u s e of t h e i r d e r i v a t i o n  into  the  the  two  virus.  to  adrenocortical cells.  retroviruses results  same h e l p e r  viruses  Each  i n a block line  of  to  F i g u r e 4.1. D i a g r a m o f t h e p r o v i r a l form o f MMCV ( t a k e n from Vennstrom e_t aJL. , 1 984 ). The p r o v i r a l s t r u c t u r e o f MMCV i s diagrammed showing t h e r e s t r i c t i o n s i t e s u s e d . The 2.5 Kb BamHI f r a g m e n t d e r i v e d from t h e OK-10 v i r u s i s indicated. The r e m a i n d e r o f t h e v i r a l genome i s d e r i v e d from Ha-MSV, Mo-MLV and pBR322. The s i n g l e H i n d l l l s i t e i s shown. The p r o v i r u s i s 5.6 Kb i n l e n g t h and i s shown approximately to s c a l e .  Hind III EBamM indlll LTR  figure 4.1  OK-TO fragment  BamM  I  v-myc  I  LTR  adrenocortical from  the  adrenal  described then  maintained  In ras  chosen is  as  the  thought  it  the  point  phenotype.  a s s o c i a t i o n of s o f t appears  strongly 1979;  t o be  the  and  would  be  of the  t o form  Fidler,  previously Each l i n e  that  adrenal  expression  was  any  cortex  colonies in soft  for transformation the  explant  apparent. cells  agar  in vitro  as  this  There i s c o n s i d e r a b l e precedent a g a r g r o w t h and  tumourigenicity 1980).  was  of a h i g h l y  phenotype i n v i t r o  associated with  Cifone  r a t by  s e p a r a t e l y so  lines  ability  a tissue  et a_l. , 1974).  transformation  the end  of a s i n g l e  studied  to represent  tumourigenic the  and  between  myc  e s t a b l i s h e d as  (Slavinski  examining and  was  glands  methods  variability  by  cells  transformation that  for and  i s most  (Kahn and  Shin,  R ESULTS 4.2  Induction  Transformation cell  o f Focus Formation  by Ki-MSV and MMCV.  l i n e s were i n f e c t e d  a l o n e , MMCV a l o n e morphological infections  and M o r p h o l o g i c a l  The r a t a d r e n a l  i n second  passage with  o r Ki-MSV and MMCV t o g e t h e r .  responses  a r e shown  cortex  Ki-MSV The  of thec e l l s  to the various  i n F i g u r e 4.2.  As c a n be seen  i n the  photographs both  Ki-MSV and MMCV were a b l e t o p r o d u c e  of  by t h e m s e l v e s .  altered  cells  cells  t h a t had a more  cells,  being  changes  e p i t h e l i o i d morphology  more p o l y g o n a l  i n shape.  elevated l e v e l s  Ki-MSV  infection resulted  showed  an e l o n g a t e d , induced  o f c-myc  much s m a l l e r t h a n  4.1  and 4 . 2 ) .  t h e number  of c e l l s  that resulted  more p r o n o u n c e d  morphological  the c e l l s  infected  the c e l l s  w i t h Ki-MSV  a g r e a t e r number  foci  cells  w i t h Ki-MSV a l o n e . and MMCV r e s u l t e d  of foci  than  t h a t were formed  either  induced  (Table  Ki-MSV  infection  relatively s i m i l a r but  when compared t o  The c o - i n f e c t i o n o f i n the formation of  ( T a b l e 4.1) and c e l l s  and r e f r a c t i l e  that  infected  showed  alterations  T h e r e was some v a r i a b i l i t y of  from  of f o c i  alterations  The K i - M S V / M M C V - i n f e c t e d  more r o u n d e d  ( S t e r n _et a l . , 1 986 ) .  morphology c h a r a c t e r i s t i c o f  were s m a l l and t h e m o r p h o l o g i c a l subtle.  line  i n t h e appearance of c e l l s  refractile  The f o c i  t h e normal  This i s s i m i l a r to  t r a n s f o r m a t i o n , b u t t h e number  was  produced  than  t h a t h a v e been d e s c r i b e d i n a NRK c e l l  expressing  v-ras  MMCV-infection  foci  virus  between l i n e s  t h a t were alone. i n t h e number  i n t h e Ki-MSV/MMCV-infected  F i g u r e 4.2. F o c u s f o r m a t i o n i n e a r l y p a s s a g e r a t a d r e n a l c o r t e x c e l l s i n f e c t e d w i t h Ki-MSV, MMCV o r b o t h . R a t a d r e n a l c o r t e x c e l l s were thawed and p a s s a g e d t w i c e i n 25$ FBS b e f o r e i n f e c t i o n . The c e l l s were seeded a t 1X10^ and i n f e c t e d when 50$ c o n f l u e n t w i t h Ki-MSV ( A ) , MMCV (B) o r K i MSV/MMCV ( C ) a t m . o . i . ' s o f 2-4 i n a 60mm d i s h . The c e l l s were t h e n m a i n t a i n e d i n 25$ FBS u n t i l e x a m i n a t i o n . The photomicrographs (100X) were t a k e n 7 d a y s a f t e r i n f e c t i o n .  TABLE 4.1  F o c u s F o r m a t i o n i n Rat A d r e n a l C o r t e x C e l l s a f t e r I n f e c t i o n with A c u t e l y Oncogenic R e t r o v i r u s e s Cell Lin e  Number  of Foci/60  Ki-MSV/MMCV  mm  Dish  Ki-MSV  MMCV.  A  25 .5  0  - (10)  B  5.5  0  -  (2)  C  12  0  -  (3)  E  1.01  6  -  (5)  F  15.5  1  -  (0)  Rat a d r e n a l c o r t e x c e l l s were i n f e c t e d ( a s d e s c r i b e d i n t h e l e g e n d t o F i g u r e 4.2) i n second p a s s a g e w i t h e i t h e r Ki-MSV, MMCV o r b o t h . T h e Ki-MSV/MMCV i n f e c t i o n s were done i n d u p l i c a t e w h i l e each o f t h e s i n g l e i n f e c t i o n s was done on a s i n g l e p l a t e of c e l l s . F o c i a p p e a r e d 3 t o 5 days a f t e r i n f e c t i o n and were c o u n t e d 5 t o 7 days a f t e r i n f e c t i o n . F o c i o f m o r p h o l o g i c a l l y a l t e r e d c e l l s were formed i n t h e c u l t u r e s i n f e c t e d w i t h MMCV a l o n e , b u t t h e m o r p h o l o g y was d i s t i n c t from t h a t seen i n t h e c u l t u r e s i n f e c t e d w i t h K i MSV/MMCV o r Ki-MSV a l o n e . The number o f f o c i formed i n t h e M M C V - i n f e c t e d c u l t u r e i s shown i n b r a c k e t s t o i n d i c a t e t h e a b i l i t y of t h e c e l l s t o respond t o t h i s v i r u s .  cultures of  (Table  4 . 1 ) , and t h e t r a n s f o r m a t i o n  t h e R EF 52 c e l l  line,  which  o n c o g e n e s , was c o n s i d e r a b l y basis  t o be c o i n f e c t e d  number  of c e l l s  determined number  relative titres  the  rat-2  cell  coinfected As  infected  was t h e n  c a n be seen  (Table  formation  was l o w e s t .  coexpression  required  The a c t u a l predicted  (Table  It  likely  t o be  from  number  of f o c i  was 5-10X  t h e estimated  of f o c i  cells  presumably  that t h e  between t h e arises  from  i n t h e assumptions does n o t a p p e a r  that  their  number  to result i n  The d i s c r e p a n c y  and i n a c c u r a c i e s  of co-infected  focus  4.1), which suggested  i n t h i s system, although  strictly  d e t e r m i n e d on  i n which  number  f o r c a l c u l a t i o n s , but there  l a r g e number foci  errors  by u s i n g  4.1 and 4.2) t h e e f f i c i e n c y o f  transformation.  and a c t u a l  experimental  of c e l l s  o f myc and r a s was s u f f i c i e n t  morphological predicted  cells  stocks  i n thecultures  t h e number  of c o - i n f e c t e d  and t h e p r o b a b l e  calculated.  was l o w e s t  than  assay  w i t h MMCV was e s t i m a t e d  The number  infection  higher  cells  by Ki-MSV i n each l i n e was  o f t h e two v i r u s  line.  To a n a l y z e t h e  by Ki-MSV and MMCV was e s t i m a t e d .  infected  the  cooperating  of adrenocortical  by an i n f e c t i o u s c e n t r e  of c e l l s  requires  more e f f i c i e n t .  o f t h e s e r e s u l t s t h e number  likely The  also  by Ki-MSV/MMCV  t o be a  a r e u n a b l e t o form  p r e s e n c e c a n n o t be  ruled out. c a n be seen  adrenocortical uninfected  cells  cells,  i n Figure  4.3 t h a t  thev i r a l l y  infected  had a s e l e c t i v e a d v a n t a g e o v e r t h e  as t h e c u l t u r e s  showed an i n c r e a s i n g  85 TABLE 4 .2 Infectious Adrenal  Centre  Culture  A  B  Rat  2  Assay o f K i - M S V / M M C V - i n f e c t e d A d r e n a l Cortex C e l l s  Virus Infection  % Cells Infected w i t h Ki-MSV  K/M  0.04  K  0.02  K/M  0.005  K  0.005  K/M  0.005  K  0.03  K/M  0.12  K  0.11  K  25.0  E s t i m a t e d Number of F o c i / 1 0 Cells 6  2  1  20  K= Ki-MSV M= MMCV C e l l s from t h e e x p e r i m e n t shown i n T a b l e 4.1 were t a k e n at t h e end o f t h e p a s s a g e i n which t h e y were i n f e c t e d and r e p l a t e d i n d u p l i c a t e on an i n d i c a t o r l i n e , Rat 2, a t 1 0 , 10-* and 10 c e l l s . The c e l l s were a l l o w e d t o a t t a c h f o r 8 t o 10 h o u r s and then t h e medium was removed and r e p l a c e d w i t h a 0.6% a g a r o v e r l a y . A p o s i t i v e c o n t r o l o f Rat 2 c e l l s i n f e c t e d w i t h Ki-MSV as d e s c r i b e d f o r i n f e c t i o n o f t h e a d r e n a l c o r t e x c e l l s ( T a b l e 4.1) was i n c l u d e d t o m o n i t o r t h e e f f i c i e n c y of t h e assay. The f o c i were c o u n t e d 13 t o 14 days a f t e r p l a t i n g t h e c e l l s . Adrenal cortex c e l l s i n f e c t e d w i t h MMCV a l o n e d i d n o t form any d e t e c t a b l e f o c i i n t h i s assay. The e s t i m a t e d number o f c e l l s c o i n f e c t e d was c a l c u l a t e d by m u l t i p l y i n g t h e p r o b a b i l i t y o f a c e l l b e i n g i n f e c t e d by a s i n g l e v i r u s by i t s e l f and t h e n m u l t i p l y i n g by 1 0 as t h e number o f c e l l s i n a d i s h a t t h e t i m e o f i n f e c t i o n . This assumes t h a t t h e p r e s e n c e o f one v i r u s does n o t i n t e r f e r e w i t h t h e i n f e c t i o n o f t h e o t h e r and t h a t t h e t i t r e s o f t h e two v i r u s e s a r e t h e same. 2  6  F i g u r e 4.3. O v e r g r o w t h of i n f e c t e d a d r e n a l c o r t e x c u l t u r e s by m o r p h o l o g i c a l l y t r a n s f o r m e d c e l l s . The c u l t u r e s shown i n F i g u r e 4.2 were p a s s a g e d and m a i n t a i n e d i n 25% FBS. The p h o t o m i c r o g r a p h s (100X) shown h e r e were t a k e n i n t h e s e c o n d p a s s a g e a f t e r i n f e c t i o n . A) K i - M S V - i n f e c t e d adrenal cortex c e l l s ; B) MMCV-inf e c t ed a d r e n a l c o r t e x c e l l s ; C) K i MSV/MMCV-infected a d r e n a l c o r t e x c e l l s . The morphological t r a n s f o r m a t i o n seen i n t h e s i n g l y i n f e c t e d c u l t u r e s (A,B) was n o t r e f l e c t e d i n t h e a b i l i t y o f t h e c e l l s t o grow i n s o f t a g a r i n t h e c o u r s e of t h i s experoment which was the p a r a m e t e r used t o d e f i n e a f u l l y t r a n s f o r m e d phenotype.  87  number  of m o r p h o l o g i c a l l y  passaging. unlikely helper 4.3  The i n c r e a s e  virus  results  The E x p r e s s i o n  Concentration  expression after  cells they Since with  required  o f serum  Cultures  work  Requires  by Ki-MSV had shown morphology  supplement  and g r o w t h  I f t h e K i - M S V - i n f ect ed  o f myc  into  requirement  (1 t o 5%  HS)  morphology.  has been  associated  f o r growth  et al.. , 1985), t h e c o i n f e c t e d t o grow and e x p r e s s a  i n t h e p r e s e n c e o f a low serum  d e m o n s t r a t e d i n F i g u r e 4.4 still  required  supplement t o express  m o r p h o l o g y and g r o w t h a s s o c i a t e d  infected  cells  f o r the a b i l i t y  MSV/MMCV-inf ect ed l i n e s  similar  early  (5% HS).  As i s c l e a r l y  serum  that the  (10 t o 25% FBS) i n t h e medium  e i a l . , 1 984 ; K e a t h  morphology  a High  the presence of a high  i n t h e serum  were examined  requirement lines.  by  on t h e t r a n s f o r m a t i o n  to a normal, e p i t h e l i a l - l i k e  a reduction  of t h e  to s u p e r i n f e c t i o n .  Previous  cells  the i n t r o d u c t i o n  transformed  high  as e x p r e s s i o n  and C a l d e r w o o d , 1 9 8 4 ) .  reverted  further  cells i s  were grown i n t h e p r e s e n c e o f low serum  (Armelin lines  infected  Adrenocortical  of the transformed  concentration  with  o f a T r a n s f o r m e d M o r p h o l o g y and Growth  cortex  infection  (Auersperg  spread  i n a block  o f Serum.  r a t adrenal  cells  in virally  t o o c c u r by v i r u s  Ki-MSV/MMCV-infected  of  altered  f o r serum  Addition  was  theK i -  the presence of a  the a l t e r a t i o n s i n with also  co-infection. seen i n both  o f FBS t o 25% r e s u l t e d  appearance of t h e morphology  characteristic  A singly  i n the  of c e l l s  that  F i g u r e 4.4. Serum s e n s i t i v i t y o f t h e t r a n s f o r m e d m o r p h o l o g y i n d u c e d by r a s and myc i n r a t a d r e n a l c o r t e x c e l l s . Three p a s s a g e s a f t e r i n f e c t i o n t h e c u l t u r e s shown i n F i g u r e 4.3 i n f e c t e d w i t h Ki-MSV and MMCV were p l a t e d i n DMEM w i t h 25% FBS and a f t e r 24 h o u r s were s h i f t e d i n t o DMEM w i t h 3% HS on t h e a p p r o p r i a t e p l a t e s and t h e p h o t o m i c r o g r a p h s (40X) were t a k e n 7 d a y s l a t e r a f t e r b e i n g m a i n t a i n e d i n e i t h e r 25% FBS (A) o r 3% HS ( B ) . The c e l l s i n t h e c o i n f e c t e d c u l t u r e s examined d i s p l a y e d a t r a n s f o r m e d m o r p h o l o g y t h a t was d i s t i n g u i s h a b l e from t h e m o r p h o l o g i e s o f t h e s i n g l y i n f e c t e d c u l t u r es.  90  F i g u r e 4.5 Serum dependence o f t h e e x p r e s s i o n o f t h e a l t e r e d m o r p h o l o g y i n Ki-MSV/MMCV-infected r a t adrenal cortex c e l l s . Two p a s s a g e s a f t e r i n f e c t i o n t h e Ki-MSV/MMCVi n f e c t e d a d r e n a l c o r t e x c e l l s were p l a t e d i n DMEM w i t h 25% FBS and a f t e r 24 h o u r s a l l c u l t u r e s were s h i f t e d i n t o DMEM w i t h 3% HS. P h o t o m i c r o g r a p h s (100X) were t a k e n a f t e r one week ( 1 , 3) and t h e a r e a s p h o t o g r a p h e d were marked. The medium was t h e n made up t o 25% FBS and a f t e r a f u r t h e r s e v e n days t h e same a r e a s were a g a i n p h o t o g r a p h e d (2,4 respectively).  were grown i n 25% FBS, d e s p i t e HS ( F i g u r e than  4.5),  that  t h e p r e s e n c e o f any f a c t o r s  morphologically the  indicating  t h e a b s e n c e o f FBS r a t h e r i n HS l e d t o a  normal phenotype,  K i - M S V - i n f e c t ed  p r e s e n c e o f 5%  the continued  as i s a l s o  adrenocortical  cells  t h e case f o r  (Auersperg  et a l . ,  1981). 4.4  Addition  Requirement  o f a P u r i f i e d Growth F a c t o r  f o r a H i g h Serum Supplement  MSV/MMCV-infected adrenocortical serum  cells  or  calf  as  sub-culturing  suggested growth  that  theactive  confluence.  infected  and m a i n t a i n e d  t h e s e growth  FBS.  factors  effects  including  factors  question  they  had r e a c h e d  (EGF, PDGF, FGF and  (see legend  EGF a p p e a r e d  seen  to F i g u r e 4.6).  t o be a b l e t o  i n response to the a d d i t i o n of  none o f t h e g r o w t h EGF a p p e a r e d  These r e s u l t s  might  this  be t h e  r e s p o n s e i n t h e Ki-MS V/MMC V - i n f e c t ed  o f FBS on t h e c e l l s  MMCV a l o n e . activity  i n 5% HS u n t i l  only  s i m i l a r to that  combination  result  o f serum might  t o address  combinations  Interestingly,  This  reversion  i n t h e p r e s e n c e o f 5% HS e i t h e r  added  induce a morphological cultures  o f FBS (1 t o 5%)  w i t h Ki-MSV, MMCV o r Ki-MSV/MMCV  P u r i f i e d growth  a l o n e or i n v a r i o u s  5% HS.  component  I n an a t t e m p t  I F G - I I ) were t h e n  Of  into  in Ki-  t h e Ki-MSV/MMCV-infected  i n low c o n c e n t r a t i o n s  of thec e l l s  cultures  were p l a t e d  Culturing  Concentration  (_< 10$) g a v e a s i m i l a r m o r p h o l o g i c a l  factors.  directly  Cells.  can Replace t h e  factors  t o be a b l e  alone or i n  t o mimic t h e  infected  with  either  suggested  that  EGF o r an E G F - l i k e  be t h e a c t i v e  component  Ki-MSV or  o f FBS t h a t was  F i g u r e 4.6. M o r p h o l o g i c a l r e s p o n s e o f K i - M S V / M M C V - i n f e c t e d a d r e n a l c o r t e x c e l l s t o p u r i f i e d growth f a c t o r s . K i MSV/MMCV-infected a d r e n a l c o r t e x c e l l s t h r e e p a s s a g e s a f t e r i n f e c t i o n , b e f o r e c e l l s were c l o n e d , were p l a t e d i n DMEM w i t h 25$ FBS and a f t e r 24 h o u r s were s h i f t e d i n t o DMEM + 5% HS and m a i n t a i n e d i n low serum f o r two weeks. At t h e end o f two weeks PDGF (10 ng/ml f i n a l ) , EGF (0.5 nM f i n a l ) and I G F I I (10 nM f i n a l ) were added t o t h e c e l l s i n DMEM + 5% HS. One d i s h had f r e s h DMEM w i t h 5% HS added and a n o t h e r was s h i f t e d t o DMEM w i t h 25$ FBS and f r e s h medium was added a t the same t i m e as f u r t h e r g r o w t h f a c t o r a d d i t i o n s . Fresh medium c o n t a i n i n g g r o w t h f a c t o r s a t 1/2 t h e i n i t i a l c o n c e n t r a t i o n was added a t 24 and 48 h o u r s a f t e r t h e i n i t i a l t r e a t m e n t and a g a i n a t 1/10 t h e i n i t i a l c o n c e n t r a t i o n s i x days a f t e r t h e f i r s t g r o w t h f a c t o r a d d i t i o n . The p h o t o m i c r o g r a p h s were t a k e n two weeks a f t e r t h e f i n a l a d d i t i o n o f growth f a c t o r s . A l l p o s s i b l e combinations of t h e t h r e e g r o w t h f a c t o r s have been t r i e d and a representative s e l e c t i o n o f p h o t o g r a p h s a r e shown EGF ( A ) , 25$ FBS ( B ) , PDGF + I G F - I I ( C ) , I G F - I I ( D ) , 5$ HS ( E ) and PDGF ( F ) . FGF has a l s o been t r i e d b u t f a i l e d t o e l i c i t a s i g n i f i c a n t r e s p o n s e e i t h e r a l o n e and w i t h PDGF o r I G F - I I . The r e s u l t s a r e n o t shown.  95  required  f o r r a s and  transformation 4. 5  vitro  parameter  not  agar u n t i l  possible  coincident  for  colony  be serum  the next passage.  in  soft  r a s and  immediate  myc  anchorage  The  fourth  was  positive  cells and  low  delay  i n the appearance  passage  were a b l e t o e x p r e s s a l l o f t h e  serum  the c e l l s  change  was  with a transformed phenotype.  d i d not s t i m u l a t e became serum  not  tested  formation  i n d e p e n d e n t , even  to  growth  colonies  as  anchorage  indicated  i n the adrenocortical The  growth  required  before  characteristics The  addition  i n t h e p r e s e n c e of h i g h colony  growth,  independent growth.  the c e l l s  factors  of  agar  of anchorage independent cellular  four  i t appeared  to r e s u l t  some f u r t h e r  growth  was  as soon  suggested  exogenous  of the  These r e s u l t s  of t h e e a r l y  i n anchorage  maintain a  for soft  serum  were n o t s u f f i c i e n t  as r e f l e c t e d  in soft  were a b l e t o form  expressed.  transformation  c e l l s did  independent line  the i n  fully  expression  i n t h e passage i n which  cells  associated  of a  In t h r e e  to demonstrate that  agar i n both h i g h growth  t o grow and  i n low serum.  The  was  t o form c o l o n i e s  were a b l e  i n d e p e n d e n t , b u t was  in  that  ability  ( T a b l e 4.3).  formation  independent  independent growth  r e q u i r e m e n t s and  was  Anchorage  The Ki-MSV/MMCV-infected  morphology  serum  Growth and  to monitor the expression  significant  i t was  reduced  used  the c u l t u r e s  transformed lines  Anchorage  phenotype.  show any  cells.  o f Serum I n d e p e n d e n t  Growth.  transformed  induced morphological  of the a d r e n o c o r t i c a l  Acquisition  Independent  myc  in soft though  or  low  agar EGF  of  before  was  97  TABLE 4.3 Number  o f P a s s a g e s Between I n f e c t i o n and E x p r e s s i o n o f Serum I n d e p e n d e n c e and A n c h o r a g e I n d e p e n d e n t Growth  Line Serum I n d e p e n d e n c e  Anchorage  Independence  A  5  6«  C  4  4  E  4  4  F  6  6  Serum i n d e p e n d e n c e was a s s a y e d a t each p a s s a g e a f t e r i n f e c t i o n by s w i t c h i n g t h e medium from 25% FBS t o 5% HS a f t e r t h e c e l l s had a t t a c h e d . Serum i n d e p e n d e n c e o f t h e K i MSV/MMC V - i n f ect ed a d r e n a l c o r t e x c e l l s was a s s e s s e d by g r o w i n g t h e c e l l s i n 3-5% HS and e x a m i n i n g t h e c e l l u l a r m o r p h o l o g y and g r o w t h p a t t e r n . At t h e end o f each p a s s a g e 1fj5 c e l l s were p l a t e d i n s o f t a g a r i n a 60 mm d i s h and c u l t u r e d f o r 2 t o 3 weeks. The e x p r e s s i o n o f a n c h o r a g e i n d e p e n d e n t g r o w t h was t a k e n as t h e f i r s t p a s s a g e i n w h i c h any c o l o n i e s were d e t e c t a b l e by m i c r o s c o p i c e x a m i n a t i o n . I n each c u l t u r e t h e r e were no c o u n t a b l e c o l o n i e s u n , t i l t h e p a s s a g e i n d i c a t e d i n t h e t a b l e , a t which t i m e g r e a t e r t h a n 150 c o l o n i e s / 1 0 ^ c e l l s were d e t e c t a b l e u n d e r t h e c o l o n y c o u n t e r . When t h e c o l o n i e s were r e a d i l y v i s i b l e t h e y were c o u n t e d u s i n g a c o l o n y c o u n t e r . The a d r e n a l c o r t e x c u l t u r e s i n f e c t e d w i t h e i t h e r Ki-MSV o r MMCV d i d n o t form c o l o n i e s i n s o f t agar d u r i n g t h e c o u r s e of t h e experiment. "growth  i n soft  agar n o t c h e c k e d  a t p a s s a g e 5.  sufficient the s i n g l y during 4.6  to e l i c i t infected  the course  Isolation  Infected  a transformed  lines  formed  of these  any c o l o n i e s  experiments  of Transformed  Cultures.  To d e t e r m i n e  lines  cultures. viral  were i s o l a t e d  These l i n e s  oncogenes,  requirements transformed to  from  i n soft  agar  Ki-MSV/MMCV  the origin  of the  for transformation,  the transformed,  were examined  or t h e i r  Neither of  (Figure 4 . 7 ) .  L i n e s from  s u s c e p t i b l e c e l l s and r e q u i r e m e n t s clonal  morphology.  co-infected  f o r the presence  p r o d u c t s , which s h o u l d  of the  r e f l e c t the  f o r i n i t i a t i o n and m a i n t e n a n c e o f t h e state.  The l i n e s  were a s s a y e d  f o r the a b i l i t y  m e t a b o l i z e a p r e c u r s o r o f t h e s t e r o i d o g e n i c pathway t o  intermediates, derived  from  which  indicated  whether  the adrenal cortex  the c e l l s  (Auersperg  were  et a l . , 1 9 7 7 ;  Wiebe et a l . , i n p r e s s ) . The  first  population the s o f t The  monolayers serum.  of transformed  agar  second  method used  assays  to i s o l a t e  cloned  lines  c e l l s was t o i s o l a t e  and grow t h e l i n e s  method was based  from  on t h e a p p e a r a n c e  o f Ki-MSV/MMCV-infected  colonies  had been m a i n t a i n e d  (Figure 4 . 8 ) .  of f o c i i n  c e l l s maintained  i n t h e f o c i and l i n e s  cells  isolated.  Spontaneous  i n low  after the  i n 5% HS f o r 2 t o 4 weeks  Cloning cylinders  cells  from  the colonies.  These f o c i arose a p p a r e n t l y spontaneously  monolayers  from t h e  were used  to i s o l a t e the  were grown d i r e c t l y f o c i also  from t h e  arose i n the cultures  F i g u r e 4.7. A n c h o r a g e i n d e p e n d e n t g r o w t h o f Ki-MSV/MMCVinfected adrenal cortex c e l l s . The i n f e c t e d a d r e n a l c o r t e x c e l l s were a s s a y e d f o r a n c h o r a g e i n d e p e n d e n t g r o w t h by s u s p e n d i n g 10^ c e l l s i n 3 mis o f 0.35$ a g a r o s e i n DMEM w i t h e i t h e r 25$ FBS o r 5$ HS. The c e l l s were i n c u b a t e d f o r 2 t o 3 weeks and t h e c o l o n i e s c o u n t e d and p h o t o g r a p h e d ( 4 0 X ) . A) K i - M S V / M M C V - i n f e c t e d c e l l s ; B) K i - M S V - i n f e c t e d c e l l s ; and C) M M C V - i n f e c t e d cells. A l l photomicrographs are of s o f t a g a r a s s a y s done i n t h e p r e s e n c e o f 5$ HS, b u t t h e s u b s t i t u t i o n o f 25$ FBS d i d n o t a l t e r t h e r e s u l t s . The s o f t a g a r s from t h e s i n g l y i n f e c t e d c u l t u r e s (b and c ) c o n t a i n s i n g l e c e l l s o r s m a l l a g g r e g a t e s ( a p p r o x i m a t e l y 4-10 c e l l s ) v i s i b l e i n phase c o n t r a s t as l i g h t a r e a s on t h e d a r k background. The K i - M S V / M M C V - i n f e c t e d c u l t u r e shows s i x l a r g e c o l o n i e s i n a d d i t i o n to the s i n g l e c e l l s .  F i g u r e 4.8. F o r m a t i o n o f a s p o n t a n e o u s f o c u s i n t h e p r e s e n c e o f 5% HS. The K i - M S V / M M C V - i n f e c t e d r a t a d r e n a l c o r t e x c e l l s were p l a t e d i n DMEM w i t h 25% FBS and 24 h o u r s l a t e r t h e medium was changed t o DMEM w i t h 5% HS. A f t e r t h r e e t o f o u r weeks f o c i a r o s e on some p l a t e s and r a p i d l y overgrew the c u l t u r e . The p h o t o m i c r o g r a p h (100X) i n t h e f i g u r e was t a k e n f o u r weeks a f t e r t h e c u l t u r e was p l a t e d .  102  infected be  w i t h Ki-MSV a l o n e , but no  isolated  4.7  from  t h e s e spontaneous  Characterization  Lines  the  and  adrenal cortex  both the  r a s and  myc.  cells The  method  al.,  oncogenes.  lysates  the p r e s e n c e of  were examined f o r The  p21  after  cell  lines  by  r a s  labelling  This  in_ v i v o  provides a simple  change a l l o w s t h e p 2 1  a c c e p t o r , where t h e p 2 1  lines  Sixteen have a l s o  Southern uncloned,  examined results  of t h e l i n e s been p r o b e d  blotting.  liberates  the  v-myc gene from  the  avian  p21  not  r a s  4.9).  from  the avian  et a_l_. , 1981).  a l l contained the avian  Of  both  lines  kilobase  the integrated  (Shih  of t h e  f o r t h e p r e s e n c e o f v-myc  a 2.5  to act  f o r t h e e x p r e s s i o n o f v-  genomic DNA  v-myc gene from  (Vennstrom  examined  The  examined  transformed a d r e n o c o r t i c a l  BamHI, w h i c h  Nineteen  contained the v i r a l shown i n F i g u r e  v - r a s  does  c - r a s  1982a; T s u c h i d a et a l . , 1982).  (illustrative  MC29  lines  et a l . , 1982).  amino a c i d  as a p h o s p h a t e  twenty  t r a n s f o r m a t i o n of  to r e q u i r e  cell  their  f o r d e m o n s t r a t i n g the presence of the v - r a s product  as a s p e c i f i c  ras  of c e l l  P i (Papageorge  J  viral  Rapid  f o r t h e p r e s e n c e of v i r a l  immunopr e c i p i t a t i o n with  appeared  Adrenocortical  Oncogenes and  Ability.  isolated  p r e s e n c e o f t h e two  were examined  et  of t h e V i r a l  Steroidogenic  could  foci.  of the Transformed  f o r the Presence  Products  transformed l i n e s  the cloned  was  fragment  provirus,  by  digested  with  containing  and  probed  myelocytomatosis the s i x t e e n  and  with  virus,  lines  v-myc gene seen  as a 2.5Kb.  F i g u r e 4.9. A n a l y s i s o f p 2 1 e x p r e s s i o n i n Ki-MSV/MMCVinfected r a t adrenal cortex c e l l s . The t r a n s f o r m e d c e l l l i n e s from t h e K i - M S V / M M C V - i n f e c t e d r a t a d r e n a l c o r t e x c u l t u r e s were p l a t e d i n 35 mm w e l l s i n DMEM w i t h 10$ FBS and a l l o w e d t o a t t a c h f o r 24 h o u r s . The c e l l s were t h e n l a b e l l e d w i t h 300 u C i o f P i i n DMEM l a c k i n g p h o s p h a t e and supplemented w i t h 2$ FBS f o r t w e l v e h o u r s . The l a b e l l e d c e l l s were t h e n washed, l y s e d and c l a r i f i e d as d e s c r i b e d and TCA p r e c i p i t a b l e c o u n t s d e t e r m i n e d . As a l l t h e l y s a t e s f e l l w i t h i n a t w o - f o l d r a n g e t h e y were i m m u n o p r e c i p i t a t e d w i t h o u t f u r t h e r adjustment. The l y s a t e s were i m m u n o p r e c i p i t a t e d with the K i - r a s s p e c i f i c a n t i - p 2 1 monoclonal antibody Y13-259 ( l a n e s d-2 and d-5) o r w i t h o u t an a n t i - r a s a n t i b o d y ( l a n e - A b ) w h i c h a c t s as a n e g a t i v e c o n t r o l . The i m m u n o p r e c i p i t a t e s were washed and a n a l y z e d by e l e c t r o p h o r e s i s t h r o u g h a 12.5$ S D S - p o l y a c r y l a m i d e g e l . G e l s were s t a i n e d f o r m o l e c u l a r w e i g h t m a r k e r s , d r i e d and exposed t o XAR-5 f i l m f o r 5 days a t room t e m p e r a t u r e . r a s  3  2  r a s  F i g u r e 4.10. D e m o n s t r a t i o n o f the p r e s e n c e o f v-myc i n t r a n s f o r m e d c e l l l i n e s d e r i v e d from the K i - M S V / M M C V - i n f e c t e d rat adrenal cortex c u l t u r e . Genomic DNA was i s o l a t e d from 10, 100 mm d i s h e s as d e s c r i b e d . The r e s t r i c t i o n enzyme d i g e s t i o n s were done e s s e n t i a l l y as d i r e c t e d by the m a n u f a c t u r e r u s i n g 10 ug o f DNA from e a c h l i n e , a t h r e e f o l d e x c e s s o f enzyme and i n c u b a t e d f o r 2 h o u r s . The enzyme r e a c t i o n was then e x t r a c t e d once w i t h p h e n o l - c h l o r o f o r m and ethanol p r e c i p i t a t e d . The p r e c i p i t a t e d DNA was r e s u s p e n d e d i n 1X TBE and e l e c t r o p h o r e s e d on a 0.75$ a g a r o s e g e l . The d i g e s t e d DNA was t r a n s f e r r e d t o a n y l o n membrane, Hybond-N (Amersham) and p r o b e d as d i r e c t e d by the m a n u f a c t u r e r . The DNA was p r o b e d w i t h a v-myc c o n t a i n i n g f r a g m e n t d e r i v e d from the m o l e c u l a r c l o n e o f the MC29 v i r u s , pMC38, w h i c h i s s p e c i f i c f o r the a v i a n myc gene c o n t a i n e d i n the MMCV under the c o n d i t i o n s used ( s e e F i g u r e 5.5, Vennstrom e t a l . , 1984). The DNA's i n the l a n e s marked B were d i g e s t e d w i t h BamHI w h i c h s h o u l d r e l e a s e a 2.5 Kb f r a g m e n t from the i n t e g r a t e d p r o v i r u s t h a t c o n t a i n s the v-myc g e n e . The DNA's i n the l a n e s marked H were c l e a v e d w i t h H i n d l l l w h i c h c u t s once i n the p r o v i r u s o u t s i d e t h e v-myc gene to g i v e a f r a g m e n t g r e a t e r t h a n 3.5 Kb. The 2.5 Kb BamHI band i s i n d i c a t e d as v-myc on the r i g h t . The bands i n the H i n d l l l d i g e s t e d DNA's a r e i n d i c a t e d by a r r o w h e a d s b e s i d e e a c h l a n e . A C _ D N A d e r i v e d from K i - M S V / M M C V - i n f e c t e d , u n c l o n e d l i n e E a f t e r t h e c u l t u r e had become serum i n d e p e n d e n t f o r g r o w t h . A l l o t h e r l a n e s a r e o f DNA d e r i v e d from l i n e s i s o l a t e d as f o c i from low serum ( e - 4 / L and e - 1 b / L ) , or c o l o n i e s i n s o f t a g a r ( e - d 1 ,1 2 ,1 4 ) . e  km  107  108  band  marked  shown  i n the f i g u r e  in Figure  4.10).  as  Five  v-myc lines  M S V / M M C V - i n f e c t ed  adrenocortical  for  the product  e x p r e s s i o n of  immunopr e c i p i t a t i o n . avian  v-myc p r o d u c t  mammalian  c-myc p r o d u c t  as  PAGF: ( H a n n _et a _ l . , 1983 ) . lines  probed  examined  and  strongly  oncogenes  The  than  results  independent  adrenocortical epigenetic  can  from  be  once  using  The  a r e most  results of  appeared  fully  by  SDS-  i n a l l 21  o f 20 of  lines  both  more  of serum  and  anchorage  Ki-MSV/MMCV-infected that was  some f u r t h e r necessary  genetic  to  allow  then  the  serum  s h o u l d have a r i s e n  from  the  clonal  number  of c e l l s The which  4.1).  that  clearly  can  had  integration only  cuts  Retroviral  p r o c e s s and  the uncloned  the i n i t i a l  the  were t h e c a s e  (Figure  random  the presence  from  Mr's  o f v-myc  the  oncogene a l o n e .  HindllJ  essentially  t h e DMA  presence  suggested  of a s m a l l  mapped  by  distinguishable  the appearance  I f this  examined  expressed  in transformation  i n the  culture  chosen  results  the K i -  were a l s o  have d i f f e r e n t  that  transformed phenotype.  provirus  from  The  change i n the c e l l s  independent expansion  lines  they  results  cells  transformation.  from  e x p r e s s i o n o f v - r a s i n 19  either  growth  cells  4.11),  indicates  in a cell  efficiently  fully  the  derived  o f t h e v-myc g e n e  A l l five (Figure  (representative  the  by  transformed.  by  In  each  of  o f MMCV  i s an marker.  the pattern  passaging u n t i l  the  integrated  a clonal  Ki-MSV/MMCV-inf ected  infection  site  integration  a c t as  illustrated  acquired  or  lines the  in  derived culture  the three uncloned  109  F i g u r e 4.11. E x p r e s s i o n of 57V-mye transformed cell l i n e s d e r i v e d from Ki-MSV/MMCV-infected r a t adrenal cortex cultures. F i v e l i n e s i s o l a t e d as c l o n e s f r o m s o f t a g a r were p l a t e d i n 60 mm d i s h e s a n d l a b e l l e d w i t h 2 5 0 u C i o f [ 3 5 ] _ m e t h i o n i n e f o r 2 hours e s s e n t i a l l y as d e s c r i b e d i n M a t e r i a l s and M e t h o d s . The c e l l s w e r e l y s e d i n a RIPA b u f f e r (1$ T r i t o n X - 1 0 0 , 0.5$ NaDOC, 0.1$ S D S , 100 mM N a C l , 20 mM T r i s H C l ( p H 7 . 5 ) , 1 mM E D T A ) , c l e a r e d a n d a l i q u o t s c o n t a i n i n g equal TCA-insoluble r a d i o a c t i v i t y were i m m u n o p r e c i p i t a t e d w i t h an a n t i - m y c r a b b i t p o l y c l o n a l a n t i b o d y ( l a n e s 2) ( a g i f t o f R. E i s e n m a n ) o r n o r m a l r a b b i t s e r u m ( l a n e s 1 ) . T h e i m m u n o p r e c i p i t a t e s were washed as recommended b y R. E i s e n m a n ( d e s c r i b e d i n M a t e r i a l s and M e t h o d s ) and s e p a r a t e d on a 1 0 . 5 $ S D S - p o l y a c r y l a m i d e g e l . T h e g e l was t r e a t e d with E n h a n c e (New E n g l a n d N u c l e a r C o r p . ) a s s u g g e s t e d b y t h e s u p p l i e r , d r i e d and e x p o s e d a t -80°C. The a v i a n v-myc p r o d u c t i s i n d i c a t e d as i s t h e endogenous r a t c-myc product. P  i  n  S  110  d-3 1 2  1  d-5 2  a  1  -1 2  a-7 1 2  .  205  '116 "97 .68  <p64 c-myc cp57 v-myc  •45  •30  lines  examined  (A, C and E, c e l l  example) a s i n g l e band the  genomic DNA  derived  from  apparent  4.10 l a n e s of  was p r e s e n t  (Figure  4.10, A C  these three  that  e-c11,12,l4,  c u l t u r e contained  parental  line  ).  clearly  This  not  complete,  but t h e d i f f e r e n t c e l l s  The o v e r g r o w t h was  i n t h e c u l t u r e as t h e i r i n t h e DNA f r o m  These data  support  apparently  presumably  the uncloned  the hypothesis  that  i n t h e Ki-MSV/MMCV-inf e c t ed  t h e r e s u l t o f a low f r e q u e n c y ,  that  represented  c h a r a c t e r i s t i c fragments  were n o t d e t e c t a b l e  as  that the  of a s i n g l e c l o n e  the culture.  cells  that the  from d i f f e r e n t  suggested  overgrown  independent  (Figure  indicated  a mixture of c e l l s  was l a r g e l y composed  strongly  i t was  The v a r i a t i o n i n s i z e  had  a minority  lines  f r a g m e n t , between t h e  lines  events.  digest of  When t h e c l o n e d  were examined  e-4/L,1b/L).  v-myc c o n t a i n i n g  infection/integration  R M  c u l t u r e was n o t c l o n a l  genomic DNA's o f t h e c l o n e d uncloned  E i s shown as an  i n the H i n d l l l  e  cultures  the parental  the H i n d l l l ,  line  genetic  lines. t h e serum  cultures  or  arose  epigenetic  event. The the  experiments  adult  Since  r a t adrenal  i t was p o s s i b l e  adrenal  cortex  transformation lines  given  marker  f a r have used  cortex that  derived  from  not derived  from t h e  and s u s c e p t i b l e t o  i n the original for their  cells  as a model f o r t r a n s f o r m a t i o n .  cells  were p r e s e n t  were a s s a y e d  specific  thus  cultures,  ability  for adrenocortical  p r e g n e n o l o n e as a p r e c u r s o r  eight  of the cloned  to produce s t e r o i d s , a  cells.  The c e l l s  of the s t e r o i d  were  biosynthetic  pathway and a s s a y e d  p r o g e s t e r o n e by a RIA. appeared results the  t o be p r o d u c i n g of s i x l i n e s  adrenocortical  indicating vitro  At l e a s t  that  differentiated  s i x of t h e eight  some s t e r o i d  intermediates  of lines (the  a r e shown i n T a b l e 4.4) and c o n f i r m e d  origin  o f some o f t h e t r a n s f o r m e d  t h e pathway  f o r the adrenocortical  model f o r c e l l s  f o r the production  of transformation cells  should  capable of expressing phenotype.  defined i n  be an  a highly  cells  applicable  113  TABLE 4.4 C o n v e r s i o n o f P r e g n e n o l o n e t o an I n t e r m e d i a t e o f t h e S t e r o i d o g e n i c Pathway, P r o g e s t e r o n e , by Ki-MSV/MMCVt r a n s f o r m e d Rat A d r e n a l C o r t e x C e l l L i n e s Standard Curve (ng u n l a b e l l e d prog e s t eron e/ml)  Counts-bound  Sample  Counts-bound  2.56  387 +  12  a-1  1462 + 263  1 .28  517 +  77  a-2  1785 +  62  0.64  832 + 1 22  c-2  1715 +  33  0.32  1 153 + 165  c-13  1015 +  43  0.16  1137 +  36  c-1 0  1780 +  47  0.08  1 890 +  58  c-14  1732 +  31  0.04  2028 + 108  0 .02  2108 +  nonspecific binding medium b l a n k  41  2 3 5 + 105 1836 + 32  The t r a n s f o r m e d l i n e s d e r i v e d from t h e Ki-MSV/MMCV-inf e c t e d a d r e n a l c o r t e x c u l t u r e s were p l a t e d i n 35mm d i s h e s and grown to 50-70? f i n a l d e n s i t y . The c e l l s were washed once i n DMEM and t h e n i n c u b a t e d i n DMEM f o r 45 m i n u t e s . The c e l l s were i n c u b a t e d i n 3 m l . DMEM w i t h 5x10~^M p r e g n e n o l o n e f o r f o u r h o u r s and t h e medium was spun i n a m i c r o f u g e f o r 5 m i n u t e s at 4 ° C . A s a m p l e o f t h e medium was r e t a i n e d and t r e a t e d i n p a r a l l e l w i t h t h e o t h e r samples t o a c t as a b l a n k c o n t r o l . The samples were p r o c e s s e d as d e s c r i b e d i n M a t e r i a l s and M e t h o d s . The s t a n d a r d c u r v e w i t h c o l d p r o g e s t e r o n e i s shown t o i n d i c a t e t h e s e n s i t i v i t y o f t h e a s s a y and t h e r e s u l t s f o r s i x o f t h e e i g h t samples p r o c e s s e d a r e p r e s e n t e d . The l i n e d e s i g n a t i o n s a r e t h e same as i n o t h e r f i g u r e s i n t h i s chapter. Each l i n e was a s s a y e d i n t r i p l i c a t e and t h e s t a n d a r d e r r o r was e s t i m a t e d .  4.8 DISCUSSION  The  data  presented  transformation  inability  colonies  i n soft  of  that  cells  appears  agar,  growth.  independent  growth  presence  a highly  transformed  The a c q u i s i t i o n i s correlated  anchorage  the loss  o f t h e serum  induced  the idea that  and serum  correlated, the  factors  was  to s t i m u l a t e anchorage independent  supports  that  of anchorage independent  o f 25% FBS or exogenous g r o w t h  cultures,  by K i -  interest  g r o w t h were a p p a r e n t l y c l o s e l y  infected  in  with  morphology  emergence o f a s i n g l e d o m i n a n t  frequency  change i n t h e  of anchorage  I t i s of p a r t i c u l a r  the a c q u i s i t i o n  insufficient  t o form  morphology,  f o r a fundamental  of the transformed  independent  cultures  The  even i n 25% FBS, d e s p i t e t h e p r e s e n c e  t h e need  MSV/MMCV i n f e c t i o n . although  three steps.  t o a l l o w r a s and myc t o i n d u c e  independent  dependency  in vitro  by t h e r a s and myc  to r e q u i r e at l e a s t  express  cells  that  o f Ki-MSV/MMCV-infected  to r e f l e c t  infected  indicate  of nonestablished c e l l s  oncogenes can appear initial  here  growth.  The  c l o n e i n t h e Ki-MSV/MMCV-  as r e f l e c t e d a further  i n the Southern event  that  blots,  occurs  a t a low  i s required f o r the progression of transformation  the adrenocortical  cells  r a t h e r than  t h e c u l t u r e by t h e K i - M S V / M M C V - i n f e c t e d  simple cells  overgrowth (Land  et a l . ,  1986) . The infection  efficient  formation  of f o c i  by Ki-MSV and MMCV c l e a r l y  i n response indicates  to co-  that  of  these  115  two  oncogenes  can  morphological when t h e growth  c o o p e r a t e and  alterations  cells  are  factor,  EGF.  phenotype induced the  dependence seen 1984).  alterations  by  pronounced  The  efficient  cells  infected The  morphological  enhance t h e myc  cultures  reduced  sufficient  to  p r o d u c e an  FBS  Ki-MSV/MMCV-infected  infected  these  cultures. able  to  f o r FBS  (EGF, in  but  clear the  FGF,  four  of  the  of  and  to  the  display  a  more  foci  formed  the  ability  in  cultures the  myc  v-ras.  It  serum r e q u i r e m e n t s  for  to  but  of  as  the not  of  EGF  growth  was  addition  of  i n Ki-MSV-  what f a c t o r ( s )  PDGF and  either  contrast  growth,  cells  transformed  morphological  s i z e and  effect similar  i s not  replace,  experiments  substitute  It  of  activity  m o r p h o l o g y and  exogenous  (Auersperg  r e l a t i v e to  the  the  i s reminiscent  i n the  transforming  further  the  cells  the  induce  an  Ki-MSV t h a t  alterations  altered  was  by  increased  of  i n the  with  i n sharp  w i t h Ki-MSV a l o n e i n d i c a t e s  that  or  induction  stands  to  transformation  serum d e p e n d e n c e o f  expression  myc  serum  i n K i - M S V - i n f e c t ed  phenotype.  oncogene to appears  in high  with  Ki-MSV/MMCV-infection  Ki-MSV/MMCV-infected  infected  sufficient  associated  co-infection  p e r c e n t a g e of  transformed  the  The  by  Calderwood,  low  grown  are  i n FBS used  in  I G F - I I ) were u n a b l e  to  singly  factors  v-  infected  c u l t u r es. T h e r e have been p r e v i o u s cellular myc  change i n a d d i t i o n  o n c o g e n e s may  Syrian  hamster  be  to  required  reports the to  embryo f i b r o b l a s t s  that  a  further  introduction  of  ras  and  produce tumourigenicity (Oshimura  e_t a l . , 1 985 ;  in  Thomassen  et _a_l. , 1 985 ).  This  cellular  change  i s associated  w i t h monosomy f o r chromosome 15 i n t h e tumours loss  of s u p p r e s s i o n  hybrids  between  associated  and  T h e r e i s as y e t no  in  described  the expression  cell  (Klinger  et a l . ,  1982).  the steps  transformed phenotype  The a d v a n t a g e o f t h e s y s t e m a tissue  by r a s and myc change,  of phenotypes  culture  that which  that  o f serum  and  model  appears to i s associated  have been  independent growth  expression  The  i s also  Stanbridge  of a h i g h l y  cellular  in  chromosomes  demonstration that  i t provides  serum  The  1985;  i s t h e same.  an a d d i t i o n a l  independence.  linked  with  anchorage  independent growth  a n c h o r a g e i n d e p e n d e n c e i n K i - M S V - i n f e c t ed r a t a d r e n a l  cortex  cells  phenotype  that  has been  formation  i s most c l o s e l y Shin,  expected growth would  associated  with  1979; that  in soft  agar i s t h e i n v i t r o  correlated  Cifone  with  and F i d l e r ,  the a c q u i s i t i o n  reflect  give rise  a similar  change  phenotype  tumourigenicity  1980). I t might  (Kahn  be  of anchorage independent •  i n t h e Ki-MSV/MMCV-infected  cellular  a highly tumourigenic  ( A u e r s p e r g jet a l . , 1 986 ).  Colony  and  clear  here i s that  tumourigenicity,  and  Bouck,  of t r a n s f o r m a t i o n  require with  systems  transformed c e l l s  of s p e c i f i c  i n the expression  t h e two  system  and  the loss  1980,1982; S t o l e r  involved  of the t r a n s f o r m e d phenotype  normal  with  formed.  increase  adrenocortical  cells  in tumourigenicity.  necessary i n the a d r e n o c o r t i c a l  t o a n c h o r a g e i n d e p e n d e n c e may  cells  be s i m i l a r  to  The to that  required hamster  to give r i s e embryonic  The of  to tumourigenicity i n the Syrian  fibroblasts.  serum d e p e n d e n c e f o r m o r p h o l o g i c a l t r a n s f o r m a t i o n  t h e Ki-MSV/MMCV-infected  transformation conditions. results  cells  of t h e c e l l s  Infection  indicates  c a n be m o d u l a t e d  of c e l l  lines  i n t h e p r o d u c t i o n o f t h e t r a n s f o r m i n g growth  TGF-y3 (Anzano  et a l . , 1983 ).  to  a transformed  phenotype i n c e l l  induce  absence of t h e v-ras et  a l . , 1 9 8 2 ; Kaplan  in  producing  oncogene  a transformed  e_t ^ 1 . ,  Roberts  (Marquardt  appears  1 9 8 4 ; Massague,  1982).  1 9 8 3 ; Todaro  The a b i l i t y  induction infected  et a_l. ,  cells  implies  oncogene t h e t r a n s f o r m e d  TGF-oc o r a r e n o t r e s p o n d i n g It  has been  shown  that  o v e r e x p r e s s i n g NRK c e l l s growth  independent cultures  growth  infected  1 983 ,  and  i n t h e Ki-MSV/MMCV-  are either  to the levels EGF t r e a t m e n t  o f t h e v-  not producing  produced. o f myc  i n anchorage  independent  EGF d i d n o t i n d u c e  or m o r p h o l o g i c a l w i t h MMCV a l o n e  Todaro,  f o r FBS i n t h e  d e s p i t e the presence cells  1 984 ;  i s related to  1 9 8 0 ; DeLarco  can r e s u l t  ( S t e r n et a l . , 1 9 8 6 ) .  enhancing  t h e EGF r e c e p t o r  alterations  that  cooperate  et a l . , 1 9 8 4 ) and  o f EGF t o s u b s t i t u t e  of morphological  TGF-/3  TGF-©c  1985).  through  i n the  et a_l. ,  (Assoian  et a l . , 1 9 8 4 ; D e r y n c k  lines  The TGF's  phenotype with  to exert i t s e f f e c t  (Carpenter,  T h e s e TGF's a r e a b l e  1983).  and Ozanne,  factor  et a l . , 1 9 8 0 ; K a p l a n  (Ozanne  t h e TGF-oc t r a n s f o r m i n g a c t i v i t y  ras  by e x t e r n a l  by Ha-MSV o r Ki-MSV  TGF-ocand  EGF  that  alterations  or w i t h  anchorage i n the  Ki-MSV.  These  results  imply  nonestablished line.  The  that there i s a r e s t r i c t i o n cells  which  acquisition  one  result  from  or b o t h  vitro  the  loss  oncogenes.  model s y s t e m  to  present  adrenocortical cells of s u p p r e s s i o n I t should  be  activity  i n t h e NRK  of anchorage independent  the Ki-MSV/MMCV-infected to  i s not  t o myc  of t h e  cell  growth i n  would  appear  activities  p o s s i b l e to use  e l u c i d a t e the nature  of t h e  anchorage independent growth  rat  expressing  cortex  cells  v-ras  and  of  this  in  cellular  change r e q u i r e d to a l l o w adrenal  in  v-myc.  in  the  5.0  myc  and  CHAPTER 5 src Cooperate i n the i n v i t r o Transformation E a r l y P a s s a g e Rat A d r e n o c o r t i c a l C e l l s 5.1  The  oncogenes  cooperate into  two  their  group  i n c l u d e s those  role  oncogenic Bolen  T  group  polyoma  Py  implying role,  this  T as  as  The  i n primary  t h a t an  but  cells.  LT  Cooperation  not  1985 ) and  v-src  cells  p60  c - s r c  of Py  r a s was The  1983)  examined  s r c and  the a b i l i t y  the  p21  to  other  ,  r a s  and  1983).  oncogene i s exert i t s  (Courtneidge, MT  1985;  to cooperate  play a  examined myc  has  chondroblasts  other  (Adkins  with  similar  i n mammalian  been shown i n  (Alema  et a l . ,  oncogenes i n a v i a n  et a_l. , 1984 ). 2-1  Using  the  (Anderson  of s r c to c o o p e r a t e  with  avian and myc  or  in rat adrenocortical cells.  murine r e t r o v i r u s  structural  p53,  been w e l l e s t a b l i s h e d ,  c o n t a i n i n g mammalian r e t r o v i r u s ,  Scolnick,  the  i t i s thought  been d i r e c t l y  between s r c and  hematopoietic  located in  s r c gene c o u l d  the t r a n s f o r m a t i o n of a v i a n  divided of  (PyLT),  c u l t u r e s has  between  be  the l o c a t i o n  a cooperating  ability  activated  has  can  to  et a l . , 1983a; R u l e y ,  activating  et a l . , 1984).  and  large T  (Py M T ) ( L a n d  by  with  able  l o c a t e d i n the cytoplasm,  interesting  effect  cells  i n c l u d e s those  of polyoma m i d d l e  particularly  myc  One  p62/64 myc,  polyoma m i d d l e The  t h a t have been shown t o be  that are correlated  products.  nucleus,  INTRODUCTION  i n t r a n s f o r m a t i o n of primary groups  of  elements  2-1  was  constructed  of the murine r e t r o v i r u s e s  from and  the v - s r c  gene from can  RSV.  be p a c k a g e d  competent the  murine r e t r o v i r u s e s .  amphotropic used  species  v i r u s uses  not prevent  gene i n t o c u l t u r e s  examine t h e a b i l i t y either  ras  Previous  (Gilmer with  helper  process  used  of thev-src  induce transformation  analysis  The v - s r c  to introduce  t h e v-  with  of t h e  cells. that  thev-src  which  showed  gene  of v-src  cells  action  that  conditions  shows an a b i l i t y  cooperation  The f o l l o w i n g  oncogene induced  of environmental  but s t i l l  by t h e  o f e a r l y p a s s a g e SHE c e l l s  ras  phenotype i n t h e a d r e n o c o r t i c a l  v-ras,  infection  gene t o c o o p e r a t e  work had d e m o n s t r a t e d  passage c e l l s .  from t h e  w i t h Ki-MSV and MMCV,  was used  rat adrenocortical  an i n v i t r o  previous  by 2-1 p a c k a g e d  myc ( O s h i m u r a _et a_l. , 1 985 ).  independent  i sdistinct  The  i n f e c t e d by e i t h e r Ki-MSV o r MMCV, t o  et al_. , 1985), u n l i k e  describe  that  o r myc i n t h e t r a n s f o r m a t i o n  nonestablished  alone could  e£ a l . , 1985).  v i r u s , so t h a t  superinfection This  v i r u s used was  i n c l u d i n g but not l i m i t e d to  a receptor  by t h e e c o t r o p i c  amphotrope 4070. src  RNA t h a t  i s capable of i n f e c t i n g a  ( f o r a r e v e i w s e e Weiss  Mo-MLV, t h e e c o t r o p i c  would  a genomic  The h e l p e r  v i r u s 4070, w h i c h  range o f host  m i c e and r a t s  one  can express  and r e p l i c a t e d by one o f t h e r e p l i c a t i o n  amphotropic  broad  by  The c o n s t r u c t  experiments  i n early  a transformed i s more  than  that  i n d u c e d by  to cooperate with  v-myc.  R ESULTS 5.2 M o r p h o l o g i c a l  A l t e r a t i o n s i n Response t o  Superinfection  by t h e s r c C o n t a i n i n g  adrenal  c u l t u r e s A,C,E and F, d e s c r i b e d  four,  cortex  were p a s s a g e d  or MMCV. titre  been  infected  stock  readily  o f 2-1.  with  2-1.  cells  greater  than  uninfected alone  i n either  ( F i g . 5.1).  adrenocortical  cells  that  Ki-MSV  cells  have a l s o  of t h e a d r e n o c o r t i c a l  cells  transformed  cells  that  (Table 5.1).  h a v e been  while  infected  t h e number  transformation The followed  virus,  The c e l l s  MMCV was  infected  The number  formed  indicating  which  treated  6  of c e l l s  infection  2-1 was i n s u f f i c i e n t  o f most n o n e s t a b l i s h e d  two p a s s a g e s .  that  1 to 2 x  was c o n s i d e r a b l y that  or  w i t h 2-1  c e n t r e assay  1% o f t h e 0.5-1X10  2-1 sup er i n f ec t ed a d r e n a l over  with  i n the  p e r d i s h was a p r o x i m a t e l y  of f o c i  (10-30 foci/60mm d i s h ) , containing  formed  the Ki-MSV-infected  t o an i n f e c t i o u s  approximately  c o u l d be  or MMCV-infected  previously infected i n either  that  t h e background o f  of f o c i  were p r o d u c t i v e l y i n f e c t e d .  should  cells  t h e Ki-MSV-  The number  formed  were s u b j e c t e d  indicated  the  adrenal  The i n f e c t i o n  d i s t i n g u i s h e d even a g a i n s t  cultures  src  by e i t h e r  s u p e r - i n f e c t e d with a  Uninfected  morphologically  transformed  1  infection  i n chapter  by t h e 2-1 v i r u s r e s u l t e d i n t h e a p p e a r a n c e o f f o c i o f  refractile,  10 *  after  T h e s e c u l t u r e s were t h e n  high  cells  twice  R e t r o v i r u s , 2-1. The  less  by t h e v-  to result i n  cells.  cortex  cells  As c a n be seen  were  i n F i g . 5.1,  c u l t u r e s t h a t had been p r e v i o u s l y i n f e c t e d  with  in  MMCV t h e  F i g u r e . 5.1. C e l l m o r p h o l o g y i n MMCV- o r K i - M S V - i n f e c t e d r a t adrenal cortex c e l l s s u p e r i n f e c t e d with the src c o n t a i n i n g r e t r o v i r u s 2-1. The c u l t u r e s i n f e c t e d w i t h e i t h e r Ki-MSV o r MMCV were p a s s a g e d t w i c e and t h e n s u p e r i n f e c t e d w i t h a h i g h t i t r e s t o c k o f 2-1 ( 1 0 f o c u s f o r m i n g u n i t s / m l ) . The c u l t u r e s were m a i n t a i n e d i n DMEM w i t h 25% FBS and p a s s a g e d twice before photomicrographs were t a k e n (200X, phase contrast). The p h o t o g r a p h o f t h e 2 - 1 / M M C V - i n f e c t e d c e l l s (A) i s r e p r e s e n t a t i v e o f t h e c u l t u r e , w h i l e t h e p h o t o g r a p h of t h e 2 - 1 / K i - M S V - i n f e c t e d c e l l s (B) was c h o s e n t o i n c l u d e one o f t h e f o c i i n t h e c u l t u r e as w e l l as t h e c e l l s e x p r e s s i n g a normal morphology. 6  123  TABLE 5.1 Cell  Line A  Virus Infections 2-1/Ki-MSV 2-1/MMCV  B  2-1/Ki-MSV 2-1/MMCV  Number  of F o c i 23 111 6 28  F o c u s f o r m a t i o n i n d u c e d by 2-1 i n p r e v i o u s l y i n f e c t e d adrenal cortex c e l l s . The Ki-MSV and M M C V - i n f e c t e d a d r e n a l c o r t e x c e l l s were p a s s a g e d t w i c e and t h e n sup e r i n f e c t ed w i t h h i g h t i t r e s t o c k s o f 2-1 e s s e n t i a l l y as d e s c r i b e d . The c e l l s were t h e n i n c u b a t e d f o r a f u r t h e r f i v e d a y s and t h e f o c i counted. The m o r p h o l o g y o f t h e f o c i i n d u c e d by 2-1 i s r e a d i l y d i s t i n g u i s h a b l e from t h a t i n d u c e d by Ki-MSV o r MMCV.  125  transformed than  cells  i n theculture  results  were seen  presumably cooperate  with  the h i g h l y  had  i n a l l four  each  infected cultures  other.  There  would  required  a h i g h serum s u p p l e m e n t  sensitivity after to  with  2-1 a l o n e were a l s o  superinfection.  following  independent  at t h e time  5.3  r a s induced  their  growth  Growth.  i n cultures  The c u l t u r e s  ability  2-1 o r  passage the a b i l i t y  i n medium  immediately that the  was l a r g e l y serum unlike  the effect  transformation.  MSV/MMCV has been c o r r e l a t e d growth.  demonstrated  of i t s appearance,  Anchorage Independent  independent  p h e n o t y p e a t each  by v - s r c  with  f o r serum  These r e s u l t s i n d i c a t e d  phenotype induced  of  tested  i n t h e passage  transformed  that  initially  m o r p h o l o g y and g r o w t h  supplement  infection.  cells  superinfected  The c u l t u r e s  a transformed  a low serum  only K i -  for the expression of a  The c e l l s  of the transformed  express  with  f o r t h e appearance of  c o i n f e c t i o n by Ki-MSV and MMCV  morphology.  with  infected  the m o r p h o l o g i c a l l y transformed  from  infected  oncogenes t o  f o r t r a n s f o r m a t i o n by r a s and myc  resulted  transformed  a s s a y e d , and  cells.  results described  shown t h a t  These  was no c o r r e s p o n d i n g  have a c c o u n t e d  transformed  w i t h Ki-MSV.  of t h e v i r a l  change i n t h e c u l t u r e s  o r MMCV t h a t  The  previously  reflected thea b i l i t i e s  morphological MSV  o v e r g r e w t h e c u l t u r e much more r a p i d l y  t o form  The a p p e a r a n c e o f serum  transformed  with  anchorage  superinfected colonies  with  by Ki-MSV o r K i independent  2-1 were t e s t e d  i n s o f t agar  two p a s s a g e s  for  after  infection.  cultures The  formed  As c a n be s e e n colonies  2-1/Ki-MSV-infected  lower the  efficiency  than  were a l s o  size  in  able  cultures,  smaller  t o form  colonies  of t h e c o l o n i e s  that  serum was much l o w e r low serum  (Figure  cells.  needed  cultured  for  there  grew  cell,  cell  and t h e with density  showing a of c e l l s  transformed  assayed.  by 2-1/MMCV  they  were  The g r o w t h r a t e o f  and low serum was 5.3 and T a b l e 5.3  of t h e growth  rate i n the the cells  actively. of Three Transformed  chapter,  Oncogenes .  to d i r e c t l y  or t h e i r  Lines  for the  As was d i s c u s s e d  implicate  o f t h e two oncogenes  i t was n e c e s s a r y  oncogenes  at a higher  i n Figure  slowing  Presence of the V i r a l  expression  colonies agars  to occur. i n high  cultures  c u l t u r e s grown i n low serum, a l t h o u g h  Characterization  previous  i n the K i -  i n low serum.  t h e medium i n w h i c h  growth  As w i t h  i n thesoft  the c e l l s  cultures  was a d i s t i n c t  that  at a  a t a lower  formed  t o t h e number  and as c a n be s e e n  transformed still  that  to condition optimal  that  5.2b and T a b l e 5.2),  implied  2-1/MMCV-infected examined  than  agar  were p l a t e d  resulted  decrease r e l a t i v e  These r e s u l t s still  cells  i n soft  5.2a).  colonies  t h e 2-1 sup e r i n f e c t ed  the percentage of c e l l s  nonlinear  5.4  formed  (Figure  t h e 2-1/MMCV-infected  t h e 2-1/MMC V - i n f e c t ed  density  low  cells  efficiency  appearance of anchorage independent growth  MSV/MMCV-inf e c t ed  If  at high  t h e 2-1/MMC V - i n f ec t ed  t h e need  for transformation  i n the  for coof a  to demonstrate t h e presence of t h e  products  in clonal cell  lines.  T h i s was  F i g u r e 5.2. C o l o n y f o r m a t i o n i n s o f t a g a r by t h e 2-1 superinfected adrenal cortex cultures. The Ki-MSV- and M M C V - i n f e c t e d c u l t u r e s s u p e r i n f e c t e d w i t h 2-1 were p a s s a g e d t w i c e and t h e n a s s a y e d f o r a n c h o r a g e i n d e p e n d e n t g r o w t h . The c e l l s were s u s p e n d e d a t 3 x 10^ o r 10 c e l l s i n 2 m i s o f 0.35$ a g a r o s e , DMEM and 5% c a l f serum e s s e n t i a l l y as d e s c r i b e d i n t h e M a t e r i a l s and Methods i n a 35 mm w e l l . The c e l l s were i n c u b a t e d f o r s i x t e e n days and p h o t o g r a p h s were t a k e n on an i n v e r t e d m i c r o s c o p e a t low power. A) 2-1/MMCVi n f e c t e d a d r e n a l c o r t e x c e l l s seeded a t 3 x 10^ c e l l s / w e l l . B) 2-1/MMCV-infected a d r e n a l c o r t e x c e l l s seeded a t 1 x 10 cells/well. C) 2 j - 1 / K i - M S V - i n f e c t e d a d r e n a l c o r t e x c e l l s s e e d e d a t 3 x 10^ c e l l s / w e l l . The p h o t o m i c r o g r a p h s (25X) a r e f r o m r e g u l a r o p t i c s and t h e c e l l s and c o l o n i e s a p p e a r as d a r k a r e a s on t h e l i g h t b a c k g r o u n d ( c o n t r a s t F i g u r e 4 . 7 ) .  TABLE  5.2  A n c h o r a g e I n d e p e n d e n t Growth o f 2-1/MMCV-inf e c t ed Adrenal Cortex C e l l s Cell  Line A  Number  of C e l l s  3 x 10* 10*  C  E  3 x 10*  Number  of Colonies 1 83 2 261  10*  21  3 x 10*  77  10*  5  t r a n s f o r m e d c u l t u r e s d e r i v e d from t h e 2 s u p e r i n f e c t i o n o f t h e MMCV-inf e c t ed a d r e n a l c o r t e x c e l l s were p l a t e d i n 0.35$ a g a r o s e w i t h 5% c a l f serum. The s o f t a g a r s were t h r e e mis f i n a l volume i n a 60 mm d i s h w i t h e i t h e r 3 x 10* o r 10* c e l l s i n each d i s h , as n o t e d . The number o f m a c r o s c o p i c c o l o n i e s were c o u n t e d a t t h e end o f t h r e e weeks as b e i n g r e p r e s e n t a t i v e o f t h e e f f i c i e n c y o f c o l o n y f o r m a t i o n and g r o w t h as seen by m i c r o s c o p i c examination.  F i g u r e 5.3- Growth r a t e o f 2 - 1 / M M C V - i n f e c t e d a d r e n a l c o r t e x c e l l s i n t h e p r e s e n c e o f h i g h o r low serum s u p p l e m e n t s . A l i n e o f 2 - 1 / M M C V - i n f e c t e d a d r e n a l c o r t e x c e l l s was p l a t e d i n 60 mm d i s h e s i n DMEM w i t h 10$ FBS and a l l o w e d t o a t t a c h overnight. A f t e r 12 h o u r s h a l f t h e p l a t e s were s h i f t e d i n t o DMEM w i t h 5$ c a l f serum. P l a t e s were h a r v e s t e d i n t r i p l i c a t e a t a p p r o x i m a t e l y t w e l v e hour i n t e r v a l s and t h e c e l l s counted. ( - X ) 2 - 1 / M M C V - i n f e c t e d c e l l s grown i n DMEM + 10$ FBS, (-#-) 2 - 1 / M M C V - i n f e c t e d c e l l s grown i n DMEM + 5$ c a l f serum. -  TABLE 5.3 Cell  Line A  E  Serum C o n c e n t r a t i o n  Number  of C e l l s  1 0$ FBS  7.35  +  1 .47  1 0$ CS  4.17  +  0.83  5$  CS  4 .50 + 0.90  2$  CS  3 .55 + 0.71  1 0$ FBS  3 .59 + 0 .40  10$  CS  2 .35 +  5$  CS  1 .86 + 0.21  2% CS  1.17  +  (x10 ) b  0.26  0.13  Growth o f 2 - 1 / M M C V - i n f e c t e d a d r e n a l c o r t e x c e l l s i n t h e p r e s e n c e o f d i f f e r e n t serum s u p p l e m e n t s . The 2-1/MMCVi n f e c t e d a d r e n a l c o r t e x c e l l s were p l a t e d i n 60 mm d i s h e s i n DMEM w i t h 10$ FBS and a l l o w e d t o a t t a c h f o r e i g h t h o u r s . A f t e r e i g h t h o u r s t h e c e l l s were s h i f t e d t o f r e s h medium w i t h t h e serum s u p p l e m e n t i n d i c a t e d i n t h e t a b l e , each serum c o n c e n t r a t i o n b e i n g a s s a y e d i n d u p l i c a t e on each l i n e . The c e l l s were i n c u b a t e d f o r 48 h o u r s , h a r v e s t e d and c o u n t e d . The e r r o r g i v e n i s t h e s t a n d a r d e r r o r e s t i m a t e d from t h e d u p l i c a t e samples.  particularly could  rapidly  important  with  transform  theadrenocortical  absence of another out  of s o f t  cultures poO  from  oncogene.  , as an i n d i c a t i o n o f t h e v-myc  of p 6 0  clearly  s r c  of v-src  oncogene.  relative  indicating  as i t a p p a r e n t l y cells  lines  i n the were c l o n e d  2-1/MMCV-infected  f o r t h e enhanced  t h e t h r e e 2-1/MMC V - i n f e c t ed  levels  Three  two d i f f e r e n t  and were a n a l y z e d  presence 5.4  agar  viral  t h e 2-1 v i r u s ,  expression of  e x p r e s s i o n , and t h e As c a n be seen  lines  i n Figure  a l l expressed  elevated  t o t h e Ki-MSV/MMCV-infected  t h e presence  of v-src  line,  i n the transformed  c ells. The Southern  presence blot  as d e s c r i b e d i n c h a p t e r  three transformed excise thev i r a l 4.1)  and compared  a Fischer  o f t h e v-myc o n c o g e n e was p r o b e d  lines  oncogene fragment to a l i n e  that  p r o b e o f t h e same s i z e ras  lines  as t h a t  i n t h e normal r a t - 2 c e l l  hybridization  i sspecific  will  (see F i g u r e  by r a s and myc,  cell  line,  rat-2.  ( F i g u r e 5.5 a-1, to the avian  i n the l i n e There  BamHI w h i c h  MMCV  transformed  hybridized  and myc ( F i g . 5.5 c - 1 6 ) .  fragment  from  r a t d e r i v e d untransformed  a fragment  The DNA's o f t h e  were d i g e s t e d w i t h  of t h e t h r e e 2 - 1 / M M C V - i n f e c t e d contained  4.  using a  and  Two e-3)  v-myc  t r a n s f o r m e d by  was no c o r r e s p o n d i n g  line  indicating  f o r t h e a v i a n myc gene.  that the  F i g u r e 5.4. E x a m i n a t i o n o f c l o n e d c e l l l i n e s d e r i v e d from the 2-1/MMCV-infected a d r e n a l c o r t e x c u l t u r e s f o r e l e v a t e d p60 kinase a c t i v i t y . The t h r e e c e l l l i n e s i s o l a t e d as s o f t agar c o l o n i e s were expanded and p l a t e d i n t o a 100 mm dishes. When t h e c e l l s were a p p r o x i m a t e l y 50$ c o n f l u e n t t h e c e l l s were l y s e d , c l a r i f i e d and s t o r e d a t -80°C u n t i l u s e . One o f t h e K i - M S V / M M C V - i n f e c t e d c e l l l i n e s grown o u t from a s o f t a g a r c o l o n y was t r e a t e d i n a s i m i l a r f a s h i o n f o r use as a non-src transformed c o n t r o l . The l y s a t e s were immunop r e c i p i t a t e d w i t h t h e a n t i - s r c a n t i b o d y 327. The i m m u n o p r e c i p i t a t e s were l a b e l l e d i n an in_ v i t r o k i n a s e r e a c t i o n i n t h e p r e s e n c e o f t h e exogenous s u b s t r a t e e n o l a s e and t h e n a n a l y z e d on a 12.5$ S D S - p o l y a c r y l a m i d e g e l . The g e l was s t a i n e d w i t h C o o m a s s i e B r i l l i a n t B l u e t o show t h e e n o l a s e , d r i e d and e x p o s e d t o f i l m w i t h a s c r e e n a t - 8 0 ° C . s r c  135  Ki-MSV  F i g u r e 5.5. S o u t h e r n a n a l y s i s o f the 2 - 1 / M M C V - i n f e c t e d a d r e n a l c o r t e x c e l l l i n e s f o r the p r e s e n c e o f v-myc. Genomic DNA was p r e p a r e d from the t h r e e c l o n e d 2-1/MMCVinfected adrenal cortex c e l l l i n e s . One o f t h e DNA samples from a K i - M S V / M M C V - i n f e c t e d l i n e , C - C 1 6 ( F i g u r e 4 . 1 0 ) , p r e v i o u s l y examined was i n c l u d e d f o r c o m p a r i s o n . Genomic DNA from the r a t - 2 c e l l l i n e was a l s o i n c l u d e d as a n e g a t i v e control. Ten m i c r o g r a m s o f e a c h o f the DNA s a m p l e s were d i g e s t e d w i t h BamHI and the f r a g m e n t s were s e p a r a t e d by e l e c t r o p h o r e s i s on a 0.75$ a g a r o s e g e l . T r a n s f e r and p r o b i n g o f the s a m p l e s was done as d e s c r i b e d above, and the b l o t was e x p o s e d t o f i l m w i t h a s c r e e n a t -80°C f o r s i x days.  MMCV a-l e-l e-3 2 1 /  Ki-MSV Rat c-16 -2  M M  +  C V  v-myc  5.5 DISCUSSION  The  results  presented  v-src  o n c o g e n e can r a p i d l y  serum  independent,  at  v-myc  efficiently adrenal P°0  cortex  to induce  with  cells.  that although  assayed  of f o c i  cells  It T, w h i c h can  and s i g n i f i c a n t l y  i t occurs  more  c u l t u r e by  The  rapid transformed  to transform  cooperate  the a d r e n o c o r t i c a l  efficiently.  has been appears  Land  blotting  the idea that v-src i s able to  but not v - r a s  cooperate  1984,  oncogene i s p r e s e n t  i n t h e Ki-MSV/2-1- or 2 - 1 - i n f e c t e d  supports  more  myc  d e t e c t a b l e frequency.  occurs  v-myc  passage  indicates  v-myc .need n o t be p r e s e n t  cells  with  i n the early  by S o u t h e r n  of t h e 2-1/MMCV-infected  cultures  cooperate  of e l e v a t e d l e v e l s of  The v i r a l  overgrowth than  able to  of t h e v - s r c gene i n t h e t r a n s f o r m a t i o n of  cortex  number  to  i t does so  characterized clearly  the v-src at a r e a d i l y  greater  growth  cells  t r a n s f o r m a t i o n more  The p r e s e n c e  two o f t h e t h r e e l i n e s  indicating  nonestablished  oncogene a l o n e  i n the three l i n e s  the adrenal in  either  cells.  the involvement  transform  v-src i s apparently  but not v - r a s than  demonstrate that while the  anchorage independent  a low e f f i c i e n c y .  with  here  p r e v i o u s l y demonstrated to transform  with  myc  cells  (Courtneidge,  et a_l. , 1 9 8 3 ) .  by a c t i v a t i n g 1985; B o l e n  The r e s u l t s t h a t myc  with  middle  p60 ~ c  s r c  ,  et a l . ,  polyoma  MT  provided  indirect  and s r c m i g h t  cooperate  in  i n t h e t r a n s f o r m a t i o n of n o n e s t a b l i s h e d ,  mammalian  vitro  evidence  t h a t polyoma  fibroblasts, the  first  but t h e r e s u l t s  direct  transformation avian  cells The  can  (Alema  rapidly  adrenal  1985b;  e t a_l.,  cells  e x p r e s s i o n of a reduced  the  passage  This  rapid  acquisition  agar  The v-myc  appears  The r a p i d  two p a s s a g e s  independent  superinfection.  independent  g r o w t h by  with the i n a b i l i t y  t o form  immediately  cells in  f o r growth i n  after  contrasts  colonies i n  following  infection.  i n d u c e d by 2-1 by i t s e l f ,  t o be r e l a t i v e l y  serum  unlike  independent.  t o f i t a two s t e p pathway.  a t h r e e s t e p pathway.  transformation presumably  thesis  i n the  fora  and a n c h o r a g e  cultures  and v-myc  cells This  by v - s r c stands i n  t o t r a n s f o r m a t i o n by v - r a s and v-myc w h i c h  follow  appears  need  transformation of the adrenocortical  contrast to  appeared  1984).  v-src  serum d e p e n d e n c y  cultures  transformed phenotype  that  i n the superinfected  of anchorage  i n t h e passages  Ki-MSV, a l s o  and  assayed  t h e Ki-MSV/MMCV-infected  soft The  when f i r s t  change.  superinfection  2-1/MMCV-infected  the of  after  been shown i n  et a_l. ,  Adkins  t h e apparent  cellular  i s reflected  chapter are  transformed phenotype  without  the  growth  has a l s o  presented here suggest  independent  transformation  i n this  Cooperation i n  and v-myc  induce a highly  cortex  further,  demonstration.  by v - s r c  results  described  of early  not subject  to inhibit and Oshimura  Stevenson  passage  This cells  to the c e l l u l a r  implies  that  by v - s r c  and v-myc i s  suppression that  t r a n s f o r m a t i o n by v - r a s and v-myc e_t a l . ,  and V o l s k y , 1986;  1985; Vogt  appears  Thomassen  g_fc a l . ,  et a l . , 1 9 8 6 ) .  The  (this 1985;  difference src  i n t h e pathways  and myc r e l a t i v e  pathways  of t r a n s f o r m a t i o n  of t u m o u r i g e n i c i t y  1985;  Gilmer  v-src  and myc does n o t a p p e a r  et a l . , 1985).  2-1/MMCV-infected  independent  f o r growth.  change i n t h e c e l l u l a r cells drop  Induction  also  considerably  density  i n low serum.  transformed  reduced.  soft  when  maintained  formation  the c e l l s  These r e s u l t s  but t h e e f f e c t  agar p r o v i d e s  of c e l l  agar  were a s s a y e d that  was  a t low  the c e l l s  are considerably serum d e p e n d e n c e i s n o t  density  some i n d i c a t i o n  i n low  show some serum  the requirements  The b a s i s o f t h e r e s i d u a l  t h e r e was a  i n soft  imply  by s r c and myc c a n s t i l l  dependence, a l t h o u g h  clear,  of colony  serum  o f t h e 2-1/MMCV-infected  a low serum s u p p l e m e n t ,  reduced  does.  t h e r e was no a p p a r e n t  morphology  The e f f i c i e n c y  o f t u m o u r i g e n i c i t y by  by r a s and myc  i n t h e growth r a t e of t h e c e l l s  serum.  et a l . ,  c u l t u r e s were n o t e n t i r e l y  Although  when c u l t u r e d w i t h  (Oshimura  t o r e q u i r e any f u r t h e r  change, but t r a n s f o r m a t i o n  The  by v-  t o r a s and myc c o r r e l a t e w i t h t h e  of development  cellular  in. v i t r o  on c o l o n y  formation i n  t h a t an a u t o c r i n e  mechanism may be i n v o l v e d . ^ *v~src  cell  poO  appears  t o be a b l e t o i n i t i a t e  division  in cell  lines  external serum and  while  lines  f a c t o r s to e f f i c i e n t l y  W h i t f i e l d , 19 84,  serum the  factors,  i n the apparent  1986).  f a c t o r s of t h e v - s r c  v-ras  expressing  line  expressing  a full  a b s e n c e o f any  v-ras  traverse the c e l l The r e l a t i v e  transformed  round of  r e q u i r e some cycle  (Durkin  independence  cell  line  from  r e l a t i v e to  i n the i n d u c t i o n of c e l l  division  does  seem t o p a r a l l e l  v-ras cells,  and  v-src  although  mechanisms may independent monolayer.  the d i f f e r e n c e s i n the a c t i o n of the  oncogenes  i n transforming  the adrenal  i t i s n o t known what common u n d e r l i e t h e two  growth  and  cell  phenotypes,  division  cortex  regulatory anchorage  i n a serum  deprived  CHAPTER 6 6.0  E f f e c t s o f V i r a l Oncogenes on t h e S t e r o i d o g e n i c o f Mouse A d r e n o c o r t i c a l Tumour C e l l s . Y-1. 6.1 The  formation  frequently  glucocorticoid 1986).  (Mulrow  INTRODUCTION  o f tumours i n t h e human a d r e n a l  associated  with  overproduction  s t e r o i d s , with  attendant  The e x p r e s s i o n  phenotype i s o f t e n  of  of a c e l l  references  therein).  differentiated proliferation  cells  The e x p r e s s i o n  of the c e l l s  normal d i f f e r e n t i a t e d Klein,  1985, 1 9 8 6 ) .  inhibit  The  of v i r a l  the loss 1 9 8 6 and  oncogenes i n  i n an i n c r e a s e i n  (Bishop,  The a b i l i t y  would  of v-ras  and v - s r c t o  differentiation appear  of  1984; K l e i n and  of t h e  t o be an e x c e p t i o n  phenotype of these c e l l s  (Bar-Sagi  1985; Noda et a l . , 1985; Alema  mouse a d r e n o c o r t i c a l tumour c e l l  p r o d u c e s some s t e r o i d end p r o d u c t s respond  to adrenocorticotrophic  inducer  of s t e r o i d o g e n e s i s ,  (Kowal and F i e d l e r , 1 9 6 8 ) . represents  with  but  a r o l e f o r t h e oncogenes i n m o d u l a t i n g t h e  differentiated Feramisco,  problems  and a l o s s o f some a s p e c t s  g r o w t h and s t i m u l a t e  underlines  metabolic  (Weir and S c o t t ,  to r e s u l t  function  pheochromocytoma c e l l s  o f some o f t h e  associated  to d i v i d e  appears  cortex i s  of a t e r m i n a l l y  differentiated the a b i l i t y  Ability  a truncated  pathway. , The Y-1  line  et a l . , 1 9 8 5 a ) . l i n e , Y-1,  ( F i g u r e 6.1) and c a n  hormone (ACTH), a n o r m a l  by i n c r e a s i n g s t e r o i d The Y-1 s t e r o i d o g e n i c  version contains  and  of the normal,  production pathway  i n vivo  an a m p l i f i e d and  F i g u r e 6.1. S t e r o i d o g e n i c pathway o f Y-1 a d r e n o c o r t i c a l tumour c e l l s ( t a k e n from Kowal and F i e d l e r , 1 9 6 8 ) . The pathway i s shown from t h e f i r s t i n t e r m e d i a t e o f t h e s t e r o i d o g e n i c pathway, p r e g n e n o l o n e . Pregnenolone i s d e r i v e d f r o m c h o l e s t e r o l by a s i n g l e s t e p m e d i a t e d by cytochrome P - 4 5 0 s i d e c h a i n c l e a v a g e . The major s e c r e t e d p r o d u c t s i n d e c r e a s i n g o r d e r a r e 11/3 - h y d r o x y p r o g e s t e r o n e , 11fi - h y d r o x y - 2 0 o C - d i h y d r o p r o g e s t e r o n e and 11 k e t o - 2 0 c £ d i h y d r o p r o g e s t e r o n e . P r o g e s t e r o n e and 20<X d i h y d r o p r o g e s t e r o n e a r e a l s o d e t e c t a b l e as minor p r o d u c t s .  PREGNENOLONE  »  PROGESTERONE  -> 20^-DIHYDROPROGESTERONE  PROGESTERONE  20cf-DIHYDROPREGNENOLONE  -> 1 1 ^,20^-DIHYDROPROGESTERONE  11  KETO,20#-DIHYDROPROGESTERONE  L i s t of the secreted products that are detected at 254nm in decreasing order of h y d r o p h i l i c i t y . llketo,20(*-progesterone >ll/2,20<s£-dihydroprogesterone >1^5-dihydroproges> terone 2Oar-di hydroprogesterone > progesterone  ov er expr ess ed c - K i - r a s the  transformation  2 gene t h a t  of the c e l l s  regulation  of s t e r o i d o g e n e s i s  (Schimmer,  1980)  characterized. useful the  target  expression  (Schwab  has been  and t h e m e t a b o l i c The Y-1  to study  cell  i s presumably i n v o l v e d  line  the e f f e c t  et a l . , 1 9 8 3 ) . extensively  pathway seemed  and  of the d i f f e r e n t i a t e d  The  studied  intermediates  to represent  of s p e c i f i c  in  a  o n c o g e n e s on  phenotype.  RESULTS 6 .2  The  Morphology Y-1  cells  E f f e c t s o f R e t r o v i r a l l y B o r n e Oncogenes  and  Growth  were i n f e c t e d  Ki-MSV, c o n t a i n i n g expresses All  o f t h e Y-1  Infection  retroviruses  (Rapp  infected  Y-1  cells  morphologically  o f t h e Y-1  to inducers  as t h e u n i n f e c t e d  by  the avian  or 3611  which  change  (Figure  the a b i l i t y  cells  1983b).  by a Mo-MLV  i n the  6.2).  The  to respond  of s t e r o i d o g e n e s i s  Y-1  v-myc,  the oncogenic  overt  cells  also retained  parental  et a l . , 1983a,  cells  d i d n o t r e s u l t i n any  morphology  The  used were p a c k a g e d  o f t h e Y-1  cellular  fashion  oncogene,  oncogene  the oncogenic v i r u s e s  helper.  Line.  by MMCV, c o n t a i n i n g  the v-ras  the v - r a f  Cell  on t h e  i n t h e same  (Kowal and F i e d l e r ,  1968) . The Y _ - ) M M C V Y-1  cells  for rate  g r o w t h r a t e was in  t h e two  the  growth  cells  C  E  i ±  L  n  e  of growth  w  a  s  compared  T h e r e was  o f t h e two  cell  no  lines.  inducers  of s t e r o i d o g e n e s i s ,  forskolin,  inhibits  their  and Weidman,  cell  line  forskolin was  and  1977;  growth  and  determined to assess  The Y - 1 ^ ^ l i n e  The  accumulation difference in  T r e a t m e n t o f t h e Y-1 s u c h as ACTH or  (Weidman and  Gill,  1976;  The  Y-1  d e r i v a t i v e were t r e a t e d  the incorporation the a b i l i t y  to r e l i e v e t h e f o r s k o l i n induced 6.1).  parental  M o r i w a k i et a l . , 1 9 8 2 ) .  t h e MMCV-infected  overnight,  protein  significant  with  Gill  to the  under n o r m a l c o n d i t i o n s .  d e t e r m i n e d by f o l l o w i n g  cultures. rate  L  of [ H] - t h y m i d i n e 3  of the v i r a l  growth  with  inhibition  oncogene (Table  a p p e a r e d t o be l e s s s u s c e p t i b l e t o  F i g u r e 6.2. M o r p h o l o g y o f Y-1 and y - 1 - c e l l s . Y-1 cells were i n f e c t e d w i t h a h i g h t i t r e s t o c k o f MMCV ( 2 X 1 0 colony f o r m i n g u n i t s / m l ) as d e s c r i b e d i n M e t h o d s . A f t e r two p a s s a g e s t h e c e l l s were p h o t o g r a p h e d on a L e i t z i n v e r t e d m i c r o s c o p e w i t h phase c o n t r a s t . Both c u l t u r e s had been m a i n t a i n e d and p a s s a g e d i d e n t i c a l l y a f t e r the i n f e c t i o n and r e p r e s e n t a t i v e a r e a s were c h o s e n . 6  148  TABL E 6. 1  Inhibition  Cell  Line  o f DNA  S y n t h e s i s by F o r s k o l i n L i n es  [ H ] t h y m i d i n e Uptake in Untreated C e l l s 3  Y-1  24,733  Y-1 MMCV  43 ,225 +  i n t h e Y-1  [ H ] t h y m i d i n e Uptake in C e l l s Treated with Forskolin 3  + 2458 316  and  Y-1  M M C V  ^Inhibition  7,300 + 866  70?  21 ,300 + 3 ,593  51?  Y-1 and Y - 1 c e l l s were s e e d e d i n t o a 96 w e l l t r a y a t 1 t o 2 x 10 c e l l s p e r w e l l and a l l o w e d t o a t t a c h and grow f o r 2 d a y s . F o r s k o l i n ,was t h e n added t o t h e medium t o 5 x 1 0 ~ M and t h e c e l l s were i n c u b a t e d f o r 14 h o u r s . One u C i o f [ ^ H ] t h y m i d i n e was t h e n added t o each w e l l and t h e c e l l s were t h e n i n c u b a t e d f o r a f u r t h e r six hours. The i n c o r p o r a t i o n o f [ H ] - t h y m i d i n e i n t o TCA p r e c i p i t a b l e m a t e r i a l was d e t e r m i n e d . Each datum i s an a v e r a g e o f t h r e e d e t e r m i n a t i o n s and t h e e r r o r s a r e g i v e n as s t a n d a r d d eviations. H n u v  5  3  the  f o r s k o l i n mediated  than in  t h e Y-1 c e l l s .  inhibition  of  -thymidine  uptake  The d i f f e r e n c e between Y-1 and  thymidine uptake,  although  small,  was  Y-1  m  m  c  v  apparently  significant.  6.3  Analysis  of S t e r o i d  Infected  and U n i n f e c t e d  products  o f t h e Y-1 c e l l  Production Y-1 l i n e s . line  the  of the accumulation  medium.  retroviruses cell  line  secreted  in  were compared  to the uninfected,  and n o r m a l i z e d  each c u l t u r e .  production  infected  cell  line  2 to 3 fold  material To  the  further  characterize  the parental  Infection  cells  For these  the effect  adenylate cyclase  experiments activity,  o f t h e Y-1  of f l u o r o g e n i c  to the uninfected  t o an i n d u c e r  cell  retroviruses resulted i n  i n the production  6.2) r e l a t i v e  then  d i f f e r e n c e i n the  between  Y-1 l i n e .  was  amounts o f p r o t e i n  cell  the response of  of s t e r o i d o g e n e s i s forskolin,  was used  line.  of the i n t r o d u c e d  on t h e p h e n o t y p e o f t h e Y-1 c e l l s ,  infected  analyzed. of  increase  The  t h e medium, d r i e d and  The f l u o r e s c e n c e  any o f t h e o n c o g e n i c  (Table  oncogenes  from  material  and t h e Mo-MLV-inf e c t ed  p a r e n t a l Y-1  w i t h Mo-MLV a l o n e .  T h e r e was no d e t e c t a b l e  of f l u o r o g e n i c  with  the oncogenic  f o r the r e l a t i v e  line  a  a quantitative  i n f e c t e d with  i n 100$ e t h a n o l .  a t low pH  of the s t e r o i d products i n  s t e r o i d s were e x t r a c t e d  measured  The major s t e r o i d  The Y-1 l i n e s  and Y-1 c e l l s  resuspended  the R e t r o v i r a l l y  are fluorescent  (Kowal and F i e d l e r , 1968) a l l o w i n g measurement  from  a potent  was inducer  to induce s t e r o i d  T a b l e 6 .2 C e l l s t o be a s s a y e d f o r s t e r o i d s were washed t w i c e i n c u l t u r e medium and i n c u b a t e d i n f r e s h medium w i t h f o r s k o l i n added i f n e c e s s a r y f o r two h o u r s . The medium was h a r v e s t e d and c e n t r i f u g e d a t 1600 x g f o r 10 m i n u t e s . The c l e a r e d medium was t h e n e x t r a c t e d w i t h one volume o f d i c h l o r o m e t h a n e . The d i c h l o r o - methane was t h e n e x t r a c t e d w i t h s u l p h u r i c acid:ethanol (65:35) and a f t e r 30 m i n u t e s t h e f l u o r e s c e n c e was measured on a P e r k i n - E l m e r 650-10S f l u o r e s c e n c e s p e c t r o p h o t o m e t e r w i t h e x c i t a t i o n a t 470 nm and d e t e c t i o n a t 525 nm. Each p o i n t i s from measurements i n t r i p l i c a t e and t h e amount o f f l u o r o g e n i c m a t e r i a l was n o r m a l i z e d t o t h e amount o f c e l l u l a r p r o t e i n i n t h e d i s h e s as d e t e r m i n e d u s i n g a B i o R a d p r o t e i n assay. The c u l t u r e medium was a l s o a s s a y e d and t h i s v a l u e has been s u b t r a c t e d from t h e f l u o r e s c e n c e . A) Comparison of t h e p r o d u c t i o n o f f l u o r o g e n i c s t e r o i d s by each of t h e v i r a l l y i n f e c t e d Y-1 c e l l l i n e s . B) C o n - c e n t r a t i o n c u r v e o f t h e e f f e c t o f f o r s k o l i n on t h e p r o d u c t i o n o f f l u o r o g e n i c s t e r o i d s by t h e Y-1 and y - 1 cell lines. M M c v  Measurement of S t e r o i d P r o d u c t i o n from Y-1 C e l l s and V i r u s I n f e c t e d D e r i v a t i v e s by A c i d I n d u c e d F l u o r e s c e n c e A) Cell Line  Relative Fluorescence  Y-1  Fold Increase R e l a t i v e t o U n t r e a t e d Y-1 C e l l s  1.1  Y-1  M M C V  3.1  2.2  ^  Y-1  3 6 1 1  2.8  2.0  |  3.6  2.6  -j*  Y-1  K i  B)  Forskolin concentration (M)  Relative Y-1  Fluorescence  Fold  Increase  y_-|MMCV  5.5  12.0  2.2  8  6.4  14.3  2.2  10-7  6.6  20.0  3.0  ,-6 10"  10.6  29.4  2.8  10 -5  14.4  43.2  3.0  10"  production  were o b t a i n e d responded steroids line. line  similar  ACTH.  remained  cell  (Table  production  enhanced line.  line  by f o r s k o l i n  3 fold  resulting relative  with  steroids  D'Agostino  products  there  t h e same i n a l l  Y-1  cell  and f o r s k o l i n  i n the production of untreated  for steroids detects a l l  derived  t h e assay  and examined by t h e Y-1  from  identical  cell  interfering  separately the  cell  line  p h a s e HPLC.  are readily  p h a s e column  line  The t h r e e were w e l l  background  t h e Mo-MLV-inf e c t ed Y-1 to the pattern  The s t e r o i d s p r o d u c e d  reflects a  by r e v e r s e  e£ a l . , 1 9 8 4 ) .  o f t h e Y-1  was l i t t l e  essentially line.  assay  by HPLC on a C^g r e v e r s e  1982;  pattern  concentrations  To examine t h e p r o d u c t s  were i s o l a t e d  separable  and  i n the  to the uninfected,  a 3keto,4A-structure,  s t e r o i d s produced  steroid  than  to theuninfected  increase  steroids relative  of products.  al.,  cell  line.  steroids  The  Y-1  i n a maintenance of t h e  of the i n f e c t i o n  Because t h e f l u o r e s c e n c e  pool  higher  at a l l forskolin  was a 7 t o 8 f o l d  fluorogenic  i n the uninfected  was a p p r o x i m a t e l y  lines,  The n e t r e s u l t  treatment  of fluorogenic  The s t i m u l a t i o n o f s t e r o i d  steroidogenesis  cell  seen  output  lines  o f f l u o r o g e n i c s t e r o i d s i n t h e Y-I^MCV  6.2B).  infected cell  results  Each o f t h e i n f e c t e d Y-1  approximately  Y-1  but s i m i l a r  by an i n c r e a s e d  to that  The p r o d u c t i o n  examined  Y-1  using  to forskolin  uninfected  the  et a l . , 1 9 8 2 ) ,  (Moriwaki  from  by t h e y - 1  ( R a m i r e z et major separated  ( F i g . 6.3).  The  l i n e was  the uninfected  M M C V  line  showed a  Y-1  F i g u r e 6.3. Demonstration of comigration of s t e r o i d p r o d u c t s d e r i v e d from t h e Y-1 and y_iMMCV i i . The s t e r o i d p r o d u c t s s e c r e t e d by t h e Y-1 and Y-1^ ^ cell lines were p u r i f i e d f r o m t h e c u l t u r e medium, a f t e r a 24 hour i n c u b a t i o n , on a SepPak (Waters S c i e n t i f i c ) as d e s c r i b e d . The e l u t e d p r o d u c t s were r e s u s p e n d e d i n a c e t o n i t r i l e f o r i n j e c t i o n i n t o t h e HPLC. The column was a C^g r e v e r s e phase column o f 10 um bead s i z e (3-9 mm x 30 mm) (uBondapak, Waters S c i e n t i f i c ) . The s t e r o i d s were l o a d e d a t 30? a c e t o n i t r i l e i n water and e l u t e d i n a l i n e a r g r a d i e n t o f 30 to 70? a c e t o n i t r i l e i n w a t e r . The e l u t i o n o f t h e s t e r o i d s was m o n i t o r e d a t 254 nm and r e c o r d e d by a H e w l e t t - P a c k a r d 3390A I n t e g r a t o r . E a c h o f t h e s a m p l e s from t h e Y-1 and _ MMCV s e p a r a t e l y (Y-1 and Y - 1 ) and c  e  l  l  n  e  s  M(  M M C V  y  1  then (Y-1  c  +  e  l  l  l  i  n  e  s  w  a  g  mixed.,and r e r u n Y-I^CV)  r  u  n  under  t h e same  conditions  pattern  that  was  (Figure 6 . 3 ) .  very s i m i l a r  The  integration  r e l a t i v e amounts i n each both  Y-1  the  6.3).  cells  M M C V  Mixing  to that  t h e Y-1  o f t h e two  the uninfected  samples  products did comigrate, c l e a r l y  Y-1  that  t h e same f o r cells the  steroid  indicating  that  the  secreted  the  uninfected  1 has  t h e Y-1^MCV  n  c e  been i d e n t i f i e d  1 y}-2  4 are thought  from  Examination  differences steroid  cells  when compared  The of  integration the s t e r o i d s  two  infected  Y-1361 1 the  a  n  d  by  secreted  lines  and  Y^Ki-MSV  Y-1 by  shift  their  expected  l  i  n  K i  s  s  h  o  w  steroids, (peak  1)  t h e Ki-MSV-  "  Y-1  e  Y-13611  an  Y-1  between  line.  enhanced  The c  e  i i  produced  Y-1  amounts the  Both  p r o d u c t i o n of 20 OC -  was  line  s  cells,  11yC3-dihydroprog e s t e r o n e  to the u n i n f e c t e d  i n the s t e r o i d s  and  6.4).  the r e l a t i v e  p r o g e s t e r o n e and  and  Y-1  (Figure  significantly  d  cells.  and  M S V  o f t h e samples  varied  e  by  the u n i n f e c t e d  the u n i n f e c t e d  2 ) , when compared This  standard.  significant  of t h e peaks shows t h a t  d i h y d r o p r o g est erone  6.3).  produced  the  i n the traces  more h y d r o p h o b i c  (peak  on  to the u n i n f e c t e d  products secreted  be seen  based  r e v e a l e d some  were t h e same as t h o s e s e c r e t e d as can  20c6-  t h e column.  of t h e s t e r o i d s  361 1 - i n f e c t ed Y-1  j  11 k e t o - 2 0 t f -  d i h y d r opr og e s t eron e r e s p e c t i v e l y , of e l u t i o n  a n c  1 1 ^ - d i h y d r o p r o g e s t eron e,  t o be  00C-dihydroprog e s t eron e and  order  s  as  d i h y d r o p r o g e s t e r o n e by c o m i g r a t i o n w i t h a p u r i f i e d 3 and  (Table  that  p r o d u c t s were t h e same f o r b o t h  Peaks 2,  the  confirmed  end  Peak  line  o f t h e peaks showed  peak were e s s e n t i a l l y  and  cells.  from  (Table  particularly  F i g u r e 6.4. C o m p a r i s o n o f the s t e r o i d p r o d u c t s p r o d u c e d by the Y-1, y_<|MMCV _ Ki-MSV l i n e s , . The steroid p r o d u c t s o f the Y-1, y - 1 and Y - 1 c e l l l i n e s were p r e p a r e d and a n a l y z e d as d e s c r i b e d i n the l e g e n d o f f i g u r e 6.3. The t r a c e s shown a r e o f e a c h sample r u n i n d i v i d u a l l y and the c o m i g r a t i n g peaks a r e i n d i c a t e d (1-4). Acetonitrile c o n c e n t r a t i o n i n c r e a s e s from l e f t to r i g h t . a  n  d  Y  1  c  M M C V  e  l  l  K l _ M b V  159  TABLE  6.3  A n a l y s i s o f t h e R e l a t i v e P r o d u c t i o n of t h e S t e r o i d P r o d u c t s o f t h e V i r a l l y i n f e c t e d Y-1 C e l l L i n e s Cell  Line  Peak 1  (p ere ent o f t h e sum o f p eaks 2  3  4  1 , 2, 3 and  1 + 2  3 +  Y-1  2.5  69 .6  16.8  11.3  72 .1  27.9  Y.-iMMCV  7.4  65.0  15.4  12.2  72.4  27 .6  17.5  71.6  5.3  5.5  89 • 1  10.9  11.9  69.0  9.6  9.4  80 .9  19.1  Y_iKi-MSV Y-13611  The e l u t i o n o f s t e r o i d s was m o n i t o r e d by a H e w l e t t P a c k a r d HP 3390 A I n t e g r a t o r . The t r a c e s as shown i n F i g u r e 6.4 were automatically integrated. T r a c e s i n which a l l t h e peaks were shown w i t h o u t t r u n c a t i o n were used and t h e a r e a s o f t h e peaks 1 t o 4 were summed. The p e r c e n t a g e o f each peak o f t h e t o t a l was d et ermin ed.  noticeable all  of  the  i n the Y - 1 2 to 3 f o l d  fluorogenic accumulation  increase  s t e r o i d s could o f peaks  1 and  11y3-dihydroprogesterone) . infection resulted  by  3611  and  in a further  pathway t h a n had  line  K l _ M S V  be 2 The  where i t would  i n the  accounted  production f o r by  the  increased  changes i n r e s p o n s e  truncation occurred  of the  that  these  and  to oncogenes  steroidogenic  i n t h e Y-1  that  of  (20oC - d i h y d r o p r o g e s t eron e  Ki-MSV i n d i c a t e d  already  appear  cells.  6.4  The  e f f e c t s of  o n c o g e n e s on Lefevre almost  the  t h e Y-1  simian  cell  et a l . ( 1 9 8 1 ) .  adenovirus  line  the  cells  p r e g n e n o l o n e and acutely  viral  were p r e v i o u s l y  I n f e c t i o n by  were a b l e  respond  oncogenic  SA-7  of t h e  synthesis  elevated  was  to  to convert ACTH.  The  steroidogenic  induced  by  forskolin.  1 cells  to  inducers  fluorogenic  the  lines  that  are  regulatory  to  the  of the  responsiveness  i s composed induced  pathways  by  an  ability,  i n f e c t e d by  Y-1 of  the  rate  same e x t e n t cells the  o f s t e r o i d s and  as  infected  Y-  the  not  infection,  for steroidogenesis  of  was  i n d i c a t e s that  retroviral  the  produce  pathway, but  of s t e r o i d o g e n e s i s  material  contaminants  The  in  by  c h o l e s t e r o l to  to approximately  s e e n when s t e r o i d p r o d u c t i o n  examined  resulted  r e t r o v i r u s e s continued  intermediates  that  SA-7  complete l o s s of normal s t e r o i d o g e n i c  although  that  DISCUSSION  and  remain  intact. The  i n t r o d u c t i o n of  enhance t h e the  Y-1  cells  fluorogenic steroid is  no  t h e Y-1 ras  expression as  cells  of  i n the  contain  (Schwab  The  the an  v-myc oncogene a p p e a r s  the  reflected  metabolites  2 gene  of  steroids.  truncation  the  Y-1  differentiated  i n the  increased  elevated y-1  M M C V  steroidogenic  amplified  and  et a l . , 1 983 ) t h e  enhance s t e r o i d p r o d u c t i o n ,  phenotype output  production cells  suggesting  to  of  of a l l t h e  implies  that  pathway.  overexpressed  effect that  of  of  there  Although c-Ki-  v-myc was  to  the i n t e r a c t i o n  162  of  the  myc  oncogene with  o n c o g e n e can  result  differentiated  the  i n the  increased  phenotype i n the  the  l o s s of d i f f e r e n t i a t i o n  The  overexpression  increase  the  of  the  I t would  expression In  of  the  y_i MCV  i i  the  a b s e n c e of  M  slight  relief  forskolin cells  by  are  n  Gill, the  e  v-myc  The  expressing  changes  expressed  Infection  M M C V  of  by  the  viruses  I t i s not  Y-1  (Land  of  the  1977).  r a t e and  replication  in  a  caused  by  hematopoietic  results in distinct (Symonds  transformed  In  occurs  and  et a l . ,  normal d i f f e r e n t i a t e d myc  et a l . ,  i n growth  Weidman,  of a v i a n  phenotype  of  increased  in steroidogenesis  cells  relative understood  s t e r o i d s , but  mechanisms i n v o l v e d  induction  differentiation.  and  viruses  can  enhance  Gill  with  cells  are  Ki-MSV  production why  produce a s i m i l a r i n c r e a s e  fluorogenic  cells  a reduction  of DNA  than  phenotype as  is  the  cells.  a l t e r a t i o n s i n the  steroids.  the  to  induction  infection  the  rather  the  change i n growth  in cell of  to  and  a  transformation.  with  inhibition  Many a s p e c t s  still  1976;  of  factors  i s able  the  proto-  line,  with  cells  growth  myc  increase  of t h e  the  of growth  a detectable  case i n the y - 1  in  that  aspects  treatment.  reproducible 1986).  of  i s associated  (Weidman and  cell  o n c o g e n e i n NRK  adrenocortical cells  steroidogenesis rate  myc  g r o w t h by  appear  other  Y-1  ras  expression  associated  susceptibility  anchorage independent 1986).  o v e r expr ess ed  i t would  distinct.  of  a l l the  or  3611  the  resulted  secreted  oncogenic  i n the  production  appear  that  of  the  B o t h Ki-MSV and  3611  appear  to r e s u l t i n a f u r t h e r  steroidogenic  pathway, so  o n c o g e n e s seem t o p h e n o t y p e of means by the  the  which  the  that  steps  of  of  the  the  It  has  efficient Wilkie, the  c-ras  1984).  The  provides  r a s  activated ras  and  Y-1  cells  further  of  (Noda  press).  cells  Y-1  and  that  Y-1  uninfected  here that  that  the  distinct first  in  few  distinct  that  v-ras  the  (Spandidos c-Ki-ras by  can  and  2 so  the  actions  distinct. they  i s more  that  viral  of Work  with  modulate  v-raf:  i n the  to respond  this  to  I t would  v-ras  however, as  Y-13611  can  are  the  ras  appear  result in  changes  distinguishable  the  infected  reflects  culture.  The  the  lines  (Mo-MLV).  from were  presence  titres  5  virus  and  from  of  were s i m i l a r (3611=6X10 ; Ki-MSV=10^)  same h e l p e r  of  e_t a l . , 1 985 ; Wiebe et a l . ,  that  i t i s p o s s i b l e that cells  respond  induced  be  d e d i f f er e n t i a t i o n .  s t e r o i d production  virus stocks  b o t h used  indicated  cells  r e s u l t s presented  cloned  genes can  appear  by  is  even t h e  cells  evidence that  The  those induced  the  i n t e r a c t with  cells  overexpress  Yuspa  the  two  the  demonstrated  et a_l. , 1985;  o n c o g e n e s by  Y-1  v-raf  d i f f e r e n t i a t e d p h e n o t y p e i n many t y p e s  raf  the  and  phenotype.  normal ras  the  v-ras  products  in transforming  oncogenes has  expression  not  v-raf  pathway can  been p r e v i o u s l y  than  the  the d i f f e r e n t i a t e d  a l t e r a t i o n s in s t e r o i d production  p21  in  and  steroidogenic  the  of  These r e s u l t s i n d i c a t e that  v-myc p r o d u c t ,  ways t o c h a n g e s i n c e l l  in  curtail  cells. v-ras  both  d i f f e r e n t i a t e d p h e n o t y p e of  from  the  that  further Y-1  truncation  Assuming  of  the and  that  the  introduction of  fluorogenic  viruses that  steroids  infected  the cultures  same e x t e n t . directly the  o f t h e two oncogenes  with  stimulated  t o t h e same e x t e n t  the production and t h a t  t h e same e f f i c i e n c y i t would  examined  were p r o b a b l y  infected  population  appear to the  The e f f i c i e n c y o f i n f e c t i o n was n o t measured  and t h e p e r c e n t  of c e l l s  infected  i s n o t known, b u t  d i s t i n c t i v e e f f e c t s i n each o f t h e i n f e c t e d  indicates  t h e two  the e f f e c t s that of i n f e c t e d  would  cells.  cultures  be s e e n i n a p u r e  CHAPTER 7 7.0 A 27000 Mr P r o t e i n S t r u c t u r a l l y R e l a t e d t o p 2 1 is E x p r e s s e d i n Human and Rat P r i m a r y T i s s u e C u l t u r e C e l l s r a s  7.1 In early  the  experiments  specifically  the  e a r l y passages  reported  family least  (Shih  three  N-ras , and including  Weinberg,  (Powers  already  1981)  d i s c o i d eum The  single  not  been  The  regulation.  frequent  i n the  role  ras  their  1986).  animal  such  et a l . , 1983)  and  weights  the ras  Pawson  these  family  could  expression organisms in  r a s - r e l a t e d genes stems  widespread Although  from  expression  the r o l e  a variety  from ras  of  i n normal c e l l s  of t h e  as  et  of  o c c u r e n c e and  tumours  and  organisms  a s s o c i a t i o n of o n c o g e n i c a l l y a c t i v a t e d  human and  at  m e l a n o g a s t er  f o r r a s gene p r o d u c t s  and  ras  species  r a s gene f a m i l y i n s u c h d i v e r s e  cellular  Interest  The  of  in  r a s  to c o n t a i n  et a_l. , 1984;  of m o l e c u l a r  Mr  cells  i n other  cell  DeFeo-Jones  (Reymond  diversity  a fundamental  Barbacid,  had  et al. , 1 979 ).  as D r o s o p h i l a  and  et a l . , 1984;  members o f t h e  and  of  been d e m o n s t r a t e d  i n d i c a t e d that products  genes w i t h  o f 27,000  band  Ki-MSV-infection  a w i d e r a n g e of s i z e s .  tissues  This  et a l . , 1979 ; Young  suggests  the  band  i n a d d i t i o n to p 2 1  infection.  i n v e r t e b r a t e s such  a l . , 1985).  of  Ki-MSV a n o v e l  of  f u n c t i o n a l genes i n mammals, H a - r a s , K i - r a s  D i c t y o s t elium  cover  after  transformation  t o have r e p r e s e n t a t i v e genes  and  proteins  the  immunoprecipitated  of genes has  (Shilo  by  associated with  previously  yeast  examining  passage r a t c e l l s  was  INTRODUCTION  r a s gene  (see  products  are  not  understood,  there  evidence to suggest regulation  of  of p u r i f i e d  arrested  i n low  division  (Feramisco  an  anti-p21  et  a l . , 1985).  and  serum can  antibody  r a s  In  required  yeast  f o r the  i n D i c t y o s t elium  Egner,  a l . , 1 985 ), and superfamily proteins. cells  novel  Gs,  synthesis  of  appears  t o be  that  any  27,000 Mr  polypeptide  to  stimuli  sporulation of  the  ras  highest  are  r e l a t e d to the  1 984 ; L o c h r i e  and  gene  in dividing  GTP  that  the  complexity  within  binding  appears  i s widely  binding  (Jurnak,  1985;  t o be  expressed  be  the  regulatory  o f genes  related polypeptides protein  GTP  et a l . , 1985 ; Tanabe et  a gene f a m i l y  encoding  EF-Tu  E l u c i d a t i o n of r a s gene f u n c t i o n  and  of  (Mulcahy  appear  environmental  expression  and  microinjection  gene p r o d u c t s  transducin,  represent  o f genes  requires  proteins  cells  et a l . , 1985).  proteins  Leberman and  3T3  cell division  initiation  The  r a s gene p r o d u c t s  regulatory  DNA  inhibit  the  proliferation.  i n t o NIH  r a s  RAS  circumstantial  to modify  and  in response to  products  The  able  stimulate  the  e_t a_l. , 1 9 8 4 ) .  (Pawson  p21  can  (Kataoka  cells  are  et a l . , 1984), w h i l e  c e l l growth  t o be  they  cell differentiation  Microinjection  regulate  that  i s considerable  i n mammalian encoding  determined.  a ras-related i n normal  cells.  the The  ras  R ESULTS  7.2  Survey  o f p27  E x p r e s s i o n . The e x p r e s s i o n  proteins  i n primary  examined  by immunopr e c i p i t a t i o n  cell 259  lysates  c u l t u r e s o f r a t and human c e l l s  the a n t i - p 2 1  ( F u r t h et a l . , 1982).  conserved is  with  of [ 3 5 s ]  able to p r e c i p i t a t e  m e  This antibody  thionine  recognizes  cells  epithelium, granulosa  and o t h e r  cells  fibroblastic  cells  confluent  or human p r i m a r y and t h e n  lysates  r a t ovarian surface cells,  the ovarian  as w e l l as  cells  from  and  other  organs.  c u l t u r e s were grown u n t i l  metabolically labelled  or p a s s a g e d  35  D.  and m u s c l e f a s c i a  of d i f f e r e n t i a t e d  C s] m e t h i o n i n e of c e l l  origin,  the lung  r a s  The c u l t u r e s examined  hormone s e c r e t i n g  from  p21  ( D e F e o - J o n e s et  and a d r e n a l c o r t e x c e l l s ,  complex m i x t u r e s Rat  r a s genes  et a_l. , 1 985 ).  of e p i t h e l i a l  Y13-  a highly  o f mammalian  as w e l l as t h e p r o t e i n s encoded by _S_. c er e v i s i a e ,  included  labelled  w i t h i n t h e r a s p r o t e i n s and  a l l known forms  d i s c o i d eum and D_. m e l a n o g a s t e r  was  monoclonal antibody  r a s  a n t i g e n i c determinant  a l . , 1983; Pawson  of r a s  f o r 16 h o u r s  once b e f o r e l a b e l l i n g .  c o n t a i n i n g e q u i v a l e n t TCA  nearly with  Aliquots  precipitabl e-[35  cpm were i m m u n o p r e c i p i t a t e d rat  monoclonal antibody.  cells was  transformed  labelled  positive  w i t h Y13-259 A line  by Ki-MSV  of r a t ovarian  irrelevant granulosa  ( H a r r i s o n and A u e r s p e r g ,  and i m m u n o p r e c i p i t a t e d  control  or an  in parallel  f o r the immunoprecipitation.  1981)  as a Cellular  s ]  p21  was s p e c i f i c a l l y  r a s  culture  cells,  result  extends  expression of  and c e l l  immunoprecipitated lines  t h e number o f t y p e s  of p 2 1  r a s  .  figure  demonstrated 7.1).  examined  of v i r a l  another  cultures,  b u t n o t from  f o rthe  protein  p21  cells ( l a n e 1,  r a s  w i t h an  a p p r o x i m a t e Mr o f 27,000 was i m m u n o p r e c i p i t a t e d primary  This  immunoprecipitation  r a s  r a t ovarian granulosa  expression  In a d d i t i o n  a l l primary  ( f i g u r e 7.1).  of c e l l s  The a n t i - p 2 1  t h e Ki-MSV t r a n s f o r m e d  readily  examined  from  from  a l l the  the Ki-MSV-transformed  cell  l i n e. This expressed  newly i d e n t i f i e d as p 2 1  ( F i g u r e 7-1 lines  such  r a s  protein  i n primary  between Although  and t h e human  culture  lines  calu-1  f o r a complete  expressed  such lung  list).  i n t h e degree of l a b e l l i n g  the d i f f e r e n t  t o drop  the i n t e n s i t y  with  to incorporate  [35  cell  l i n e , but  as mouse NIH 3T3 adenocarcinoma The r e a s o n of p 2 1  was used  r a s  line  f o rthe and p27  clear.  i n each  of the l a b e l  the decrease  culture  i n some  c u l t u r e s i s not e n t i r e l y  t h e same number o f c o u n t s  immunoprecipitation appeared  tissue  as r a t - 1 and a r a t a d r e n a l c o r t e x c e l l  ( s e e t a b l e 7.1 difference  t o be as w i d e l y  r a t and human  and T a b l e 7.1) and was  was n o t d e t e c t a b l e i n o t h e r fibroblasts  appeared  precipitated  i n the a b i l i t y  of the  s ]  methionine. 7.3 F r a c t i o n a t i o n Cell  Line.  of t h e L a b e l l e d L y s a t e  o f a p27  Expressing  S e v e r a l p o s s i b l e explanations f o r the co-  immunoprecipitation  o f p27 and p 2 1  r a s  present  themselves.  F i g u r e 7.1 Survey of r a t primary c u l t u r e s f o r t h e expression of r a s r e l a t e d p r o t e i n s . Primary c u l t u r e s from r a t t i s s u e s were e s t a b l i s h e d and grown as d e s c r i b e d i n M a t e r i a l s and M e t h o d s . The c e l l s from one 60mm d i s h were l a b e l l e d o v e r n i g h t a t t h e end o f f i r s t p a s s a g e w i t h 100 u C i methionine. The l a b e l l e d c e l l s were l y s e d , c l a r i f i e d and 1 . 5 x 1 0 TCA p r e c i p i t a b l e C.P.M.'s were immunoprecipitated with the a n t i - p 2 1 m o n o c l o n a l Y13-259. The p u r i f i e d p r o d u c t s were s e p a r a t e d on a 12.5? SDSp o l y a c r y l a m i d e g e l , E n H a n c e d and exposed t o XAR-5 f i l m a t -80°C. P a n e l A: A K i - M S V - t r a n s f o r m e d r a t o v a r i a n g r a n u l o s a c e l l l i n e was t r e a t e d i n p a r a l l e l , l a n e 1. The r a t p r i m a r y c u l t u r e s examined were: l a n e 2 - o v a r i a n g r a n u l o s a c e l l s ; l a n e 3 - l u n g f i b r o b l a s t s ; l a n e 4 - a d r e n a l c o r t e x parenchyma; l a n e 5 - m u s c l e f a s c i a ; l a n e 6 - k i d n e y s t r o m a ; P a n e l B: l a n e 1 - a d u l t lung f i b r o b l a s t s ; l a n e 2 - p r e a d u l t (prepubescent) lung f i b r o b l a s t s ; l a n e 3 - a d u l t a d r e n a l c o r t e x parenchyma; and lane 4-preadult adrenal cortex. a = a n t i - p 2 1 Y13-259, b=negative c o n t r o l , n o n s p e c i f i c r a t monoclonal antibody. 7  r a s  3  r a s  1 2 3 4 a b a b a b a b  27 p21c-ras  P  tp27 ip21e r a s  Table 7.1 Expression of p27 in some Rodent and Human Cell  Cultures.  C e l l s Examined  Origin of Cultures  p27 Expression  Rat-1  NT, Immortal  +  FSV-transformed Rat-1  fps-transformed  +  Strain A  NT, Immortal  +  Ki-MSV-trans formed Strain A  ras-transformed  +/-  NIH3T3  NT, Immortal  Ki-MSV-transformed NIH3T3  ras-transformed  Calu-1  ras-transformed human carcinoma  Rat embryo fibroblasts  NT, primary culture  Rat hepatocytes  •NT, primary culture  +  Human foreskin fibroblasts  NT, primary culture  +  C e l l s were l a b e l l e d and immunoprecipitated with Y13-259 anti-p21 as described in the legend to Figure 7.1 and Materials and Methods and analyzed by SDS-PAGE.  NT- nontransformed c u l t u r e s .  Calu-1  is a c e l l l i n e i s o l a t e d from a human lung adenocarcinoma that contains as activated ras gene that i s thought to be involved in transformation of the c e l l s . Materials and Methods.  The other l i n e s are described in  p27 is  might  share  recognized  an a n t i g e n i c d e t e r m i n a n t  by t h e Y13-259 a n t i b o d y ;  distinct  but r e l a t e d  of p 2 1  .  r a s  gene p r o d u c t  Another p o s s i b i l i t y p21  with  p21  i f s o , p27 m i g h t  or a h i g h l y m o d i f i e d  i n an i m m u n o p r e c i p i t a b 1 e  with  Increasing  t h e c o n c e n t r a t i o n o f SDS i n t h e c e l l  immunoprecipitation not  appear  making To  i t less  above w i t h  containing  antibodies  ( f i g u r e 7.2).  immunoprecipitation suggesting  sedimented  relative  monomolecular is  were t h e n  w i t h Y13-259  The g r a d i e n t was made up t o 0.5? w/v or c o n t r o l  o f 0.1? DOC  not i n t e r f e r e with  t h e p27 and p 2 1  r a s  the analysis  proteins  t o s i z e m a r k e r s as t h o u g h  by t h e a n t i - p 2 1  interaction  labelled  and t h e c l a r i f i e d  they  s p e c i e s ; t h e r e f o r e , i t seems u n l i k e l y  precipitated  noncovalent  /p27  i n a buffer  o f p27 i n t h e p r e s e n c e  Both  ,  I t was p o s s i b l e t o d e m o n s t r a t e t h e  t h a t DOC would  ( d a t a n o t shown).  r a s  by s i z e u s i n g c e n t r i f u g a t i o n on  and t h e f r a c t i o n s  and i m m u n o p r e c i p i t a t e d  r a s  p a s s a g e were  5 t o 20? w/v s u c r o s e g r a d i e n t .  fractionated SDS  i n early  of a p 2 1  0.1? w/v sodium d e o x y c h o l a t e , fractionated  l y s i s and  a r e i n a complex.  r a s  of formation  fibroblasts  complex.  o f p27 and p 2 1  [35s]methionine and l y s e d  l y s a t e was t h e n a linear  yield  t h a t p27 and p 2 1  explore the p o s s i b i l i t y  form  0? t o 0.1? or 0.5? w/v d i d  the r e l a t i v e  likely  complex, r a t l u n g as  b u f f e r s from  to a f f e c t  be a  i s t h a t p27 might be  associated  r a s  which  r a s  with  r a s  p21  r a s  antibody .  were t h a t p27  by v i r t u e  of a  F i g u r e 7.2 R a t e - z o n a l f r a c t i o n a t i o n o f [ 3 5 ] m e t h i o n i n e l a b e l l e d r a t lung f i b r o b l a s t l y s a t e . Rat l u n g f i b r o b l a s t s i n t h i r d p a s s a g e were l a b e l l e d o v e r n i g h t w i t h [ 3 5 ] m e t h i o n i n e and l y s e d i n b u f f e r c o n t a i n i n g 0.1$ DOC as t h e o n l y d e t e r g e n t . 10° c.p.m. o f t h e c l a r i f i e d l y s a t e was f r a c t i o n a t e d on a 5-20$ w/v l i n e a r s u c r o s e g r a d i e n t a t 50000rpm f o r 18 h o u r s i n a Beckman SW 50.1 r o t o r . The f r a c t i o n s were made up t o 0.5$ SDS and i m m u n o p r e c i p i t a t e d as d e s c r i b e d above. A p a r a l l e l g r a d i e n t with m o l e c u l a r weight m a r k e r s was r u n and f r a c t i o n a t e d i d e n t i c a l l y and t h e f r a c t i o n s a n a l y z e d on a 12.5$ S D S - p o l y a c r y l a m i d e g e l and t h e g e l o f t h e g r a d i e n t was marked t o i n d i c a t e t h e f r a c t i o n t h a t c o r r e s p o n d e d t o t h e most i n t e n s e s t a i n i n g o f t h e m a r k e r . a=anti p 2 1 m o n o c l o n a l Y13-259; b = n e g a t i v e c o n t r o l . S  S  r a s  175  7.4 T r y p t i c  Peptide  Mapping  appear  t o be any complex  seemed  probable  proteins  that  o f p27.  formed  between p27 and p 2 1  was due t o t h e p r e s e n c e o f a common homology w i t h  proteins  were compared  by t r y p t i c  proteins  were i s o l a t e d  from  and  fibroblasts, subjected  tryptic first  eluted  dimension  using  molecular  weight  hydrophobicity very  tryptic  comigrating these  making p21  r a s  i tunlikely  that  that  sequence  i n the f i r s t  i n t h e second  r a s  of t h e  d i m e n s i o n and d i m e n s i o n , and  sequence.  showed  are distinct  The  three  i n F i g u r e 7.3).  they  power  Apart p21  r a s  from were  p r o t e i n s and  p27 i s a h i g h l y m o d i f i e d  three comigrating  were o b s e r v e d r a s  primary  form o f  .  The  p21  (arrowed  process  t h e maps o f p27 and c e l l u l a r  indicating  trypsin  i n t h e second  This  t o c h a n g e s i n amino a c i d  peptides  with  and a f r a c t i o n a l  maps o f p27 and p 2 1  three spots  different,  of t h e i r  of charges  f o r separation  peptide  Both  a n a l y s i s of the r e s u l t i n g  by c h r o m a t o g r a p h y  f o r separation  sensitive  mapping.  e l e c t r o p h o r e s i s a t pH 2.1 i n t h e  on t h e b a s i s  on number  To  t h e two  r a s  a g e l , digested  ( s e e M a t e r i a l s and M e t h o d s ) .  depending  o f t h e two  3 5  from  followed  compares p e p t i d e s  , i t  epitope.  p21  peptide  r a s  [ S] methionine-labelled r a t  t o two d i m e n s i o n a l  peptides  dimension  is  there d i d not  the co-immunoprecipitation  examine p27 f o r s t r u c t u r a l  lung  Since  ,  distinct  in tryptic  r a t p27.  identified  i n F i g u r e 7.3  d i g e s t s o f Ki-MSV and Ha-MSV  which a r e s i m i l a r from  peptides  to the c e l l u l a r  p21  The c o - o r d i n a t e l y p o o r  r a s  viral  and  y i e l d of  F i g u r e 7.3 T r y p t i c p e p t i d e maps o f p27 and p 2 1 . Immunoprecipitated p27 and p 2 1 were e l u t e d from a g e l f r a g m e n t , d i g e s t e d w i t h t r y p s i n and a n a l y z e d by two d i m e n s i o n a l t r y p t i c p e p t i d e mapping. The d i g e s t e d p r o t e i n s were s p o t t e d a t t h e o r i g i n ( O r ) and e l e c t r ophor es ed (anode on t h e l e f t ) a t 1000V f o r 45 m i n u t e s and then c h r o m a t o g r a p h e d (bottom t o t o p ) . The p l a t e s were s p r a y e d w i t h E n H a n c e (NEN) and exposed t o XAR-5 f i l m a t - 8 0 ° C . The t r y p t i c p e p t i d e s were a n a l y z e d b o t h s e p a r a t e l y and mixed as n o t e d i n t h e f i g u r e , and a c a r t o o n o f t h e mixed p e p t i d e s i s drawn i n t h e lower r i g h t f r a m e . The empty c i r c l e s i n d i c a t e t h e u n i q u e p27 p e p t i d e s , t h e c i r c l e s w i t h v e r t i c a l h a t c h i n g i n d i c a t e the unique p 2 1 p e p t i d e s , and t h e s o l i d circles r e p r e s e n t t h e p e p t i d e s i n common between p27 and p 2 1 . T h e s e s h a r e d p e p t i d e s a r e a l s o arrowed i n t h e a u t o r a d i o g r a m s of t h e p e p t i d e maps. r d  r a s  3  r a s  r a s  177  conserved  spots  differently results and  modified  r a s  when t h e  were done u s i n g  dimension  comigration  ( d a t a not  the r a t lung  t h a t they  might  peptide.  tryptic  p e p t i d e maps o f  shown) s u g g e s t i n g was  not  has  revealed  and  p21  r a s  .  The  peptides  have been shown t o c o m i g r a t e cysteine-labelled 7.5 p21  p21  r a s  are s u f f i c i e n t l y  proteins primary  a r e not  with  distinct  To  further,  fibroblasts  pulse-chase  experiments  rates  of s y n t h e s i s and  cells  were p u l s e - l a b e l l e d  labelled label the  within f i f t e e n  during  an  excess  were d e g r a d e d  the  of  7.3d  in Fig. (  maps o f p27  to suggest  that  forms  these  both  p27  minutes  with  and  r a s  in  same  of p27 and  and  and  p21  r a s  continued  relative  p21  r a s  to  and  S  of n o n - r a d i o a c t i v e methionine The  p27  .  When  became accumulate  (Figure  [35 ] methionine  rates.  p27  which suggested  synthesized  of a  time-course  to c h a r a c t e r i z e the  degradation  at d i f f e r e n t  and  of t h e  were l a b e l l e d  i n c u b a t i o n times  were p u l s e d  1b from  between p 2 1  p r o t e i n s were i n d e p e n d e n t l y  When c e l l s with  longer  and  between maps  i n v e s t i g a t e the p o s s i b i l i t y  relationship  and  The  modified  precursor-product r a t lung  isolated  a further  peptides  of p27.  differently  gene p r o d u c t .  1a  Tryptic  .  Pulse-Chase L a b e l l i n g r a s  marked  the  the  proteins  3 5  p27  in  fortuitous.  c y s t e i n e c o n t a i n i n g p e p t i d e which c o m i g r a t e s p27  8.9  that  [ S ] c y s t e i n e - l a b e l l ed  fibroblasts  be Similar  e l e c t r o p h o r e s i s a t pH  shown i n f i g u r e 7.3  p e p t i d e a n a l y s i s of from  b suggests  forms o f a s i n g l e  were o b t a i n e d  p21  first  a and  then  that 7.4). chased  the p r o t e i n s  appeared  to  have  F i g u r e 7.4 [ S ] m e t h i o n i n e p u l s e - c h a s e o f p27 and p 2 1 . A 60mm d i s h o f r a t l u n g f i b r o b l a s t s were p u l s e d w i t h 100 u C i of [ S ] m e t h i o n i n e f o r 0.25 h o u r s ( l a n e 1 ) , 0.5 h o u r s ( l a n e 2 ) , 1 hour ( l a n e 3 ) , 4.5 h o u r s ( l a n e 4 ) , 10 h o u r s ( l a n e 5 ) , and 24 h o u r s ( l a n e 6 ) . In l a n e 1 a l l o f t h e l y s a t e was i m m u n o p r e c i p i t a t e d , i n l a n e s 2 and 3 a h a l f o f t h e l y s a t e was used i n each i m m u n o p r e c i p i t a t i o n and i n l a n e s 4-6 one t e n t h o f t h e l y s a t e was u s e d . a = a n t i - r a s m o n o c l o n a l Y13-259, b=negative c o n t r o l . B-Rat l u n g f i b r o b l a s t s were p u l s e d f o r one hour w i t h 100 u C i [ ^ S ] m e t h i o n i n e and c h a s e d w i t h 10mM u n l a b e l l e d m e t h i o n i n e f o r 3 h o u r s ( l a n e 1 ) , 7 h o u r s ( l a n e 2 ) , 11 h o u r s ( l a n e 3 ) and 2 3 h o u r s ( l a n e 4 ) . L a n e 5 i s an i m m u n o p r e c i p i t a t i o n o f r a t l u n g f i b r o b l a s t s l a b e l l e d f o r 24 h o u r s as d e s c r i b e d a b o v e . a = a n t i - r a s m o n o c l o n a l Y13-259, b = n e g a t i v e c o n t r o l . 3 5  3 5  3  r a s  B  1 2 3 4 5^ a ba a a a b U  205  *•-  205 II -- 116  TI6 97  - 97 - 66  66  - 45  - 29  p27- p21*f 29  18  - 24 f  .  f  - 18  a h a l f - l i f e of 6 to 8 hours,  while p 2 1  approximately  14  as  disappearance  of i m m u n o p r e c i p i t a b l e  material.  turnover  rate  o f p27  approximately  rapid  as  that  of p 2 1  clear  product-precursor relationship  t o 16  hours  appeared and  r a s  r a s  estimated  t o be  t h e r e d i d not  had by  a h a l f - l i f e of the Thus  appear  between  the  twice t o be  the  as any  two  prot eins. In  figure  7.1  normal r a t o v a r i a n g r a n u l o s a  Ki-MSV t r a n s f o r m e d  derivative  immunoprecipitated  under  readily  n o r m a l and  in  3-5)  an  cells,  but  expressed  ( f i g u r e 7.5)  increase ras  gave s i m i l a r  in p 2 1  r a s  NRK  line  and  cells  and is  apparent  in  comparing m o r p h o l o g i c a l l y  i n w h i c h p27  line but  when compared e x p r e s s i o n due  a  w h i l e p27  i t i s not  a p p a r e n t l y not  Similar  were l a b e l l e d  rat adrenal cortex c e l l s  results  a reduced  gene i s a p p a r e n t .  line  and  c o n d i t i o n s and  Experiments  In a r a t f i b r o b l a s t i c  derivative p27  cells.  Ki-MSV t r a n s f o r m e d  the normal  cells.  of  similar  d e t e c t a b l e i n the normal  the transformed  figure  cell  cells  i n the  the  was  (see  expressed  transformed  Ki-MSV-transformed  still  d e t e c t a b l e amount  to the p a r e n t a l l i n e .  The  to the presence  v-  results  i t s Ki-MSV-transformed  have been  of t h e  obtained i n  derivative.  F i g u r e 7.5 E x a m i n a t i o n o f p27 and p 2 1 expression i n a Ki-MSV t r a n s f o r m e d c e l l l i n e . An a d r e n a l c o r t e x c e l l l i n e , s t r a i n A ( l a n e b ) , and a Ki-MSV t r a n s f o r m e d s u b l i n e ( l a n e c ) were l a b e l l e d f o r 16 h o u r s w i t h 150uCi o f [ ^ S ] m e t h i o n i n e. The c e l l s were l y s e d and immunopr e c i p i t a t ed as d e s c r i b e d a b o v e w i t h 1.5x10^ TCA p r e c i p i t a b l e c.p.m. b e i n g used i n each i m m u n o p r e c i p i t a t i o n . Lanes b and c were from immunoprecipitates using the a n t i - p 2 1 monoclonal antibody Y13-259. Lane a i s a p a r a l l e l i m m u n o p r e c i p i t a t e u s i n g s t r a i n A l y s a t e w i t h o u t a n t i b o d y . The i m m u n o p r e c i p i t a t e s were a n a l y z e d on a 12.5$ S D S - p o l y a c r y l a m i d e g e l , E n h a n c e d , d r i e d and a u t o r a d i o g r a p h e d . r e l s  3  r a s  7.6  The  27,000 d a l t o n p r o t e i n  antigenically product  DISCUSSION  and  structurally  of a d i s t i n c t  gene.  d e s c r i b e d here related  T h i s new  expressed  i n a wide v a r i e t y  o f r a t and  cultures,  as  some c e l l  examined animals  and  two  o f mammalian  be  described presence  from  There  indicates  surviving  the animal, in  cells  and  that  o n l y 24  which  e x p r e s s i o n of  as  has  been  essentially  do  the not  after  the p r o b a b i l i t y  that  vivo. appear  and  expressed  the  t o be  any  direct  e x p r e s s i o n of p27  in terminally  at l e v e l s  or p 2 1  comparable to those  been d e s c r i b e d i n mammalian and  correlation  i t appears  r a s f  as  differentiated,  e x p r e s s i o n o f r a s gene p r o d u c t s  invertebrate cells,  in a l l  be s y n t h e s i z e d  to 36 hours  strengthens  and  most  e_t a l . , 1984), b u t  cells.  has  reflect  The  cells  cells  adult  endodermal,  i t may  are cultured,  tissue  represent  should  i n the animal.  the  cells  immature and  nondividing The  The  be  is  human p r i m a r y lines.  to  but  p27,  e x p r e s s i o n of p27  i n rat hepatocytes,  growth  are both  The  ,  r a s  protein,  The  proteins (Barrett  does n o t  between c e l l  cells  when c e l l s  f o r other  i s expressed  they  cells.  induced  grow i n c u l t u r e ,  p27  species.  a l l cells,  o f p27  isolation  embryonic,  to p 2 1  l i n e a g e s , mesodermal and  cultures  most, i f n o t may  and  of d i f f e r e n t i a t e d  these primary  p27  ,  mammalian  embryonic  many t y p e s  in  r a s  were d e r i v e d from  different  types  is p21  appears  dividing  in nondividing  as w e l l  that  in  as  r a s p r o t e i n s have  regulatory  f u n c t i o n s i n both  (Segal  Shilo,  and  1986).  growing  I t does n o t  expression  i s required  for c e l l  apparently  absent  some c e l l  p27  i s present  rapidly  and  from  i n these  so  i s not  conditions  used  expression  of p 2 1  distinct  cells  p27  cellular  p21  r a s  over  a limited  tryptic  be  p21  distinct  from  Y13-259 a n t i b o d y  p21  r a s  crossreactive  .  encoded  1983), a 23  a l . , 1985), and The r a s  r a s  against  proteins  p21  more  labelling  closely  of  both  are  an  by  S^  r a s  21  and  27  kd  major c o m i g r a t i n g i s a l s o present  stability.  epitope  to The  formed between  i s broadly  p r o t e i n s , and t h e 34  c e r e v i s i a e r a s genes  kd .D . d i s c o i d e u m  by  appears  sequence conserved  recognizes  to  defined  otherwise  i n s e q u e n c e and  eukaryotic ras p21  related  region minimally  This monoclonal antibody  t o mammalian  and  over  independence  p e p t i d e s , but  o f amino a c i d  addition  al.,  i t is  It i s p o s s i b l e that  the  presumably r e c o g n i z e s  the region  and  p27  f u r t h e r i n d i c a t e s t h a t they  protein i s apparently  the co-migrating  p27  that  as  i s turned  under  cells  proteins.  The  within  p27  appear  lines.  T h i s apparent and  r a s  arrested  viability  but  detected  here.  and  and  in 39  kd  ( D e F e o - J o n e s ^st  r a s p r o t e i n (Pawson et,  p r o t e i n s in_D. tryptic  peptide  in tryptic  melanogast er. o f mammalian  p27  p e p t i d e maps o f  [35 S ] m e t h i o n i n e - l a b e l l ed D. proteins. sequences tryptic  A comparison showed  peptide  d i s c o i d eum  and  S.  cerevisiae  of t h e r e l e v a n t d e r i v e d  that only a s i n g l e is sufficiently  amino  acid  methionine-containing  h i g h l y conserved  ras  in a l l  three ras (Powers  p r o t e i n s to account  et al.. , 1984;  a l . , 1984; peptide, with  69  residues  a l l identified RAS  and  S C 1  RAS  products  S C 2  amino a c i d  which  have a n e g l i g i b l e  should  peptide t o be  i n t h e mapping  a likely and  r a s  region  of  the ras  of the and  threonine proteins  et  p21  p r o t e i n s and  r a s  This  r a s  conserved  show a  of  single,  glutamate,  the m o b i l i t y of pentapeptide  comigrating  the  seems  spot  of  1986).  and  b in figure i n p27  immediately  oncogenic  by  activation  et a l . , 1984)  the monoclonal  of v i r a l  p21  participate peptide  i n GTP  that  appears  t o be  amino-terminal  to comigrate  this  that i s labelled  t h a t does n o t  Y13-259  between  p27  region  with  the  of  p21  both  with  in  pentapeptide and  that  identified  comigrate  the peptide  to the  is  h y d r o l y s i s (McCormick  [ ^ ^ S ] m e t h i o n i n e (43-68,  7.4)  some  transforming  r a s  suggesting  (59-  of  and  I t i s a l s o noteworthy  i s phosphorylated,  The  conserved  i s c l o s e t o amino a c i d s  p r o t e i n (Fasano  a t r e s i d u e 59  a l . . 1985).  thought  on  p21  exception  i s located within a highly  Aaronson,  p r o t e i n might  peptides  which  f o r t h e major  epitope recognized  [3^s]cysteine and  the  is  et  acid  o f mammalian  effect  procedure.  implicated i n the  transforming  the  amino  c h a n g e of a s p a r t a t e t o  candidate  peptide  that are  (Lacal  Reymond  p27.  This  part  t o 73  five  ras p r o t e i n s with  conservative  63)  This  2  yeast  p21  comigration  et a l . , 1 9 8 3 ;  DeFeo-Jones et a_l. , 1 9 8 2 ) .  comprising  observed  the sequence NH _asp-gln-tyr-met-arg-COOH  between the  Tsuchida  f o r the  .  p21  as  1a  any r a s  that i s This  peptide  is  the only  tryptic  peptide  c y s t e i n e and m e t h i o n i n e terminal  peptides  p21  derived Recent may  described other  w h i c h would  r a s  from  a known  ( M a d a u l e and A x e l ,  extensive.  r a s  that  which c o n t a i n s  antibody.  analogous  extensive  1985).  With  i t would  of adenyl represent  is distinct  to bind  involved  than  proteins  i a o  another  the a n t i - p 2 1 genes s u c h  homology  t o known  r a s  r a s  r a s gene previously  f a m i l i e s o f GTPthat the  will  p r o v e t o be  p27 i s r e l a t e d t o simply  share  recognized  appear  or  by t h e Y13-  t o be f u n c t i o n a l l y  mediate receptor c o n t r o l l e d (Gilman,  GTP b i n d i n g  1984).  regulatory  monoclonal antibody.  as N-myc and R - f o s  homology  A number o f  w h i c h have  have been  Thus  protein  but r e t a i n s s u f f i c i e n t  proto-oncogenes  a  i n nucleotide binding  cyclase activity  from p 2 1  cellular  that  splicing.  t h e d i s c o v e r y of  appear  the epitope  to the G proteins  modulation p27 m i g h t  presumably  The p 2 1  exon o f p27 i s p r o b a b l y  o v e r much o f i t s s e q u e n c e o r i t may  hydrolysis  The two  t h e mammalian  I t i s p o s s i b l e that  site  that  that  t h e r a s genes b e l o n g  functional  259  i n d i c a t e that  comprise other  proteins  t o which  t h e second  The amino  procedure to  a s i n g l e peptide.  more  both  et a _ l . , 1 9 8 2 ) .  r a s gene by a l t e r n a t e  f i n d i n g s suggest  regulatory  to contain  r a s  i n t h e mapping  within  be c o n s i d e r a b l y  superfamily quite  from  encoded  related proteins  binding  p21  one s p o t  a r e both  mammalian  family  (Tsuchida  g i n c a n be m o d i f i e d  g i v e more t h a n  not  in p21  limited  isolated  et a l . , 1 9 8 3 ; L e e et a l . , 1984; ) and i t seems l i k e l y  (Schwab that  p27  i s the product  o f a gene t h a t  the normal c e l l u l a r It by  appears  The  transformation  i n a decrease  loss  or r e d u c t i o n  separate  experiments.  decrease  that  o f t h e R EF  analogous s i t u a t i o n gene p r o d u c t  one.  by an  this  by an a c t i v a t e d Ha-  r a s  of t h e  I f p27 i n t e r a c t s this  expression  i s down r e g u l a t e d  The  i n the i n t e r a c t i o n  i n expression  et a l . , 1 9 8 6 ) .  i n which  this  i s obscure.  line  i n a decrease  (Franza  r a s  52 c e l l  i n four  either  synthesis.  o f p27, b u t what r o l e  same r e g u l a t o r y pathway as p 2 1  activated  that underlies  transformed  i n transformation  oncogene r e s u l t s  normal p 2 1 the  in cells  p27  expression.  was o b s e r v e d  or d e c r e a s e d  to the r e d u c t i o n  Transformation ras  o f p27  r a s gene i m p l i e s a s p e c i f i c i t y  may p l a y  to  expressing  i s n o t known, b u t may be  o f p27 e x p r e s s i o n  leads  decrease  or l o s s  The mechanism  p r o t e i n turnover  oncogenic  of c e l l s  o f p27 e x p r e s s i o n  c h a n g e i n p27 p r o d u c t i o n increased  related  r a s genes.  that  Ki-MSV r e s u l t s  i s similarly  with  may r e p r e s e n t  of a normal  by a r e l a t e d ,  an  cellular  one og e n i c a l l y  CHAPTER 8  SUMMARY  The  results  adrenal  cortex  derived  from  previous  on  cells  work  work  on  lines  transformation  described  on  cell  cells  lines  of t h e  e a r l y passage  h e r e complement  immortalized  primary  many i m m o r t a l i z e d shown t o be  the  cell  lines  in vitro.  by  the  and  that r e q u i r e m u l t i p l e steps,  In permanent  the  p a s s a g e c u l t u r e s of t h e  adrenal  ras  Ki-MSV and  immediately  c h a n g e was  growth  necessary  transformed The  low  4.2)  efficiency  and  The  cellular  of  the  p h e n o t y p e may  cell oncogenes  The  early  coinfected  with  anchorage  that a further of a  that  the r o l e to  reducing  fully  the c e l l s  (Stern  of a  formation  cells  by  (Table  4.1  mophologically  r e q u i r e some p r e d i s p o s i t i o n o f  o f myc  earlier  in transformation  activity  threshold et al.,  seems t o s u b s t i t u t e f o r  E x t r a p o l a t i o n of  enhance t h e the  focus  cortex  expression  i n t r o d u c t i o n o f myc  o n c o g e n e s was g e n e by  expression  been  myc  demonstrate  of i n d u c t i o n of  predisposition.  suggested  cells  indicating  of the r a t a d r e n a l  implies that  transformed cell.  to allow  of  phenotype.  Ki-MSV-infection and  ( T a b l e 4.3)  cortex  the  Transformation  cooperate to produce complete t r a n s f o r m a t i o n .  independent  much o f  a c t i v a t e d o n c o g e n e s has  a s i n g l e h i t phenomenon.  MMCV d i d n o t  models  of the  The  the  results  by  the  two  a c t i v a t e d ras  for transformation  1986).  the  different  by  TGF's  growth  factor  requirements  transformation  f o r expression of morphological  of t h e Ki-MSV-infected  phenotypic  o f t h e myc g e n e r e s u l t s  changes t h a t  requirements  include a loss  f o r some serum  to the K i -  (Figure 4 . 6 ) indicates  MSV/MMCV-inf e c t ed a d r e n a l c e l l s the i n t r o d u c t i o n  relative  factors,  that  i n complex  or r e d u c t i o n i n t h e  b u t n o t serum  i n d ep end enc e. The myc  requirement  for a third  step  t o complement  i n t r a n s f o r m a t i o n of nonimmortalized  a l s o been d e s c r i b e d f o r h e m a t o p o i e t i c Volsky, The  cultures  following cultures  Vogt  1986;  1986;  cells  Schwartz  coinfection.  et a l . ,  with  (Schwartz  before the c e l l s  that  could  The c u l t u r e s  alterations  shortly  to demonstrate  after  1986).  transformed  t h e emergence o f a s p e c i f i c  et a l . , 1 9 8 6 ) i n t h e t r a n s f o r m e d a further  express  phenotype.  and  immediately  The a p p e a r a n c e o f h i g h l y  was c o r r e l a t e d  suggesting  i n v i t r o has  (Stevenson  d i d n o t show autonomous g r o w t h  clone of c e l l s culture,  et a l . ,  cells  r a s and  c h a n g e was r e q u i r e d  a highly  d i d show some infection,  transformed phenotypic  b u t i t was n o t p o s s i b l e  any c h a n g e s i n t h e g r o w t h  factor  r e q u i r ements. The infected  morphological fibroblasts  t r a n s f o r m a t i o n o f t h e Ki-MSV/MMCV-  i n response  t o h i g h c o n c e n t r a t i o n s of  serum  or EGF s t a n d s  i n contrast to the i n a b i l i t y  serum  o r EGF t o s t i m u l a t e a n c h o r a g e i n d e p e n d e n t  serum  independence of the h i g h l y  presumably not s u f f i c i e n t  transformed  in itself  to r e s u l t  of either growth.  The  cultures i s i n anchorage  independence,  a l t h o u g h the e x p r e s s i o n of the phenotypes  is  l i n k ed. The  i n c o m p l e t e t r a n s f o r m a t i o n of e a r l y  MSV/MMCV-inf e c t ed a d r e n a l c e l l s inhibition be  lost  of t h e a c t i v i t i e s  when t h e c e l l s  growth.  The  to  form  colonies  cells,  in soft  but  even  exogenous  functions  myc  are suppressed The  Syrian  association  hamster  chromosome 15  suggests that t h e s e two  EGF,  indicates  reduction  of s u p p r e s s i o n (Oshimura  myc  oncogenes r e q u i r e s  and  can  for  transformation,  further  cellular  change,  oncogenes r e s u l t e d independent  and  and  i s exempt  serum  t o be  t h e need  appearance  independent  action  growth.  myc  cells.  of  sufficient  for a  as t h e p r e s e n c e o f t h e  i n the rapid  the  p r e v e n t s r a s and  appears  from  I t has  1 985 ) and i t  t r a n s f o r m i n g the adrenal cortex myc  or  suppress  Sager,  inhibition  o f s r c and  lines.  1985).  from  combination  passage  a loss  that  The  the  of a transformed  seems l i k e l y readily  a similar  that  w i t h monosomy f o r  et al.,  cells  (Craig  cells  of t u m o u r i g e n i c i t y i n  the induction  by  o f some o n c o g e n e p r o d u c t s  of t h e s e  in early  by r a s and  normal  passage  to immortalized c e l l  phenotype  that  early  i n t h e p r e s e n c e of h i g h  of the i n d u c t i o n  embryo c e l l s  been d e m o n s t r a t e d  i n the  oncogenes  relative  seems t o  independent f o r  the i n a b i l i t y  serum c o n c e n t r a t i o n s and  cells  an  t o a l l o w t h e e x p r e s s i o n of  phenotype  agar  o f t h e r a s and  from  o f t h e oncogenes t h a t  f o r EGF  altered  Ki-MSV/MMCV-infected  result  become a n c h o r a g e  requirement  the m o r p h o l o g i c a l l y  may  passage K i -  two  anchorage  The t r a n s f o r m i n g  ability cells  of v - s r c a l o n e  appears  conditions  than  t h a t of v - r a s by  independently  of  differentiated  analysis  to  v-ras  and  three  oncogenes i n t o  two  based  on  effect  of the  the a b i l i t y  and  suggested  Y-1  EGF  The  to  of the  of the  show s u c h  to  o f c-myc can  also  t h a t t h e f u n c t i o n o f myc  i s to  those in  oncogenes  cellular  enhance  be  expression  lines.  t o have r e d u c e d  an  distinction.  i t might  o f t h e p h e n o t y p e of c e l l appear  cut  enhance  these  represent  that a l l the  a clear  in  oncogenes  oncogenes t o c o o p e r a t e work does n o t  Y-  the  as r e f l e c t e d  that are s i m i l a r  clear  the  myc  steroidogenesis separates  i t i s not  MSV/MMCV-inf e c t ed c e l l s  done i n  the  seem t o a l t e r  ( S t e r n et a l . , 1986), and  of s e v e r a l a s p e c t s  was  production  the d i v i s i o n  groups  g r o u p s would  Overexpression to  on  in  phenotype,  transformation, this  survey  t h e two  response  v-raf  Although  on  from  determined  cell  oncogene p r o d u c t s .  enhance t h e s t e r o i d  production.  exhaustive  the  p r o v i d e some c l u e s as  of t h e d i f f e r e n t i a t e d  cell  of  seems t o be the  the  suppression  susceptibility  of t h e changes  of t h e  w h i l e both  their  that  adrenocortical cells  based  primary  indicating  or v - s r c w i t h  p h e n o t y p e would  oncogene appears  steroid  t h e Y-1  of a c t i o n  expression  environmental  s u s c e p t i b l e to The  cortex  myc.  t h e hope t h a t an  1 cells,  v-ras.  of v - r a s  work w i t h  mechanism  of  pathway t o s u p p r e s s i o n  the i n t e r a c t i o n  The  passage adrenal  alone,  v-src i s less  t r a n s f o r m a t i o n by  transformation by  early  t o be more i n d e p e n d e n t  transformation than  i n the  serum  The  Ki-  requirements relative that  to either  i n these  factor than ras  f o r expression  cells  oncogene.  supports  of t h e c e l l  that  the role  i s not c l e a r  The r o l e  o f TGF-C6  as enhancement of other  phenotypic  p21  r a s  alterations  o f myc i n c o o p e r a t i o n  w i t h v-  as i t was n o t p o s s i b l e t o d i s s e c t t h e  phenotype of t h e t r a n s f o r m i n g detect  o f t h e v-  (McKay ^ t a l . , 1986)  may n o t be as s i m p l e  of r a s .  effect  o f myc i n c o o p e r a t i v e  as w e l l as myc i n d u c e d  independent  fashion  t h a t TGF-oc p r o d u c t i o n i s  b u t may i n v o l v e enhancement  functions,  t h e growth  i n a more complex  f o r ras transformation  transformation  c u l t u r e s , implying  of the transforming  The s u g g e s t i o n  the idea  activity  infected  morphology  t h e myc may a c t t o a l t e r  enhancement  dispensable  src  of the s i n g l y  requirements  simple  o f an a l t e r e d  any d i f f e r e n c e s between  cells  in sufficient  d e t a i l to  t h e 2-1/MMC V - i n f e c t ed or 2-1  i n f e c t ed c e l l s . The a s s o c i a t i o n o f v - r a s early  passage c e l l s  activity p27  with  induced  the loss  t r a n s f o r m a t i o n of  of suppression  o f t h e o n c o g e n e s and t h e r e d u c e d  r a s - r e l a t ed p r o t e i n i s c u r i o u s .  of t h e  expression  Without  of the  any k n o w l e d g e  of t h e f u n c t i o n o f p27, i t i s n o t p o s s i b l e t o know i f t h e reduced  expression  coincidental  o f p27 i s d i r e c t l y  to expression  of t r a n s f o r m a t i o n .  The model o f t h e t r a n s f o r m a t i o n in  Figure  8.1.  requirements highly  The most  f o r at l e a s t  transformed  i n v o l v e d or  important  pathway i s diagrammed  features  arethe  one f u r t h e r c h a n g e t o r e s u l t  phenotype a f t e r  in a  the c o i n t r o d u c t i o n of ras  and  myc,  as  compared  transformation differences  by  to the  s r c or  two  step  s r c and  i n the process  that  somewhat d i f f e r e n t  The  useful with  system  pathway.  cells  to these  i n which  transformation  by  to the  The  with  ability  of t r a n s f o r m a t i o n  d i f f er ent. one og en es s u g g e s t s  adrenocortical  myc.  pathway seen  by  of  d i f f e r e n c e s should  examine t h e c h a n g e s various  oncogenes.  detect  the  each o n c o g e n e may sensitivity  to  follow a  the provide  associated  a  F i g u r e 8.1 D i a g r a m a t i c r e p r e s e n t a t i o n o f t h e pathways a s s o c i a t e d with oncogenic t r a n s f o r m a t i o n of e a r l y passage adrenocortical cells. The oncogenes used i n t h e e x p e r i m e n t s are i n d i c a t e d . The boxes i n t h e d i a g r a m r e p r e s e n t u n c h a r a c t e r i z e d s t e p s i n t h e t r a n s f o r m a t i o n pathway t h a t have been d e f i n e d by a v a r i e t y o f e x p e r i m e n t s including those described i n t h i s t h e s i s . The p a r t i a l l y transformed p h e n o t y p e i s d e f i n e d as t h e s t a g e a t which t h e c e l l s a r e m o r p h o l o g i c a l l y t r a n s f o r m e d , b u t serum and a n c h o r a g e dependent f o r growth. The c e l l s a r e c o n s i d e r e d t o be f u l l y t r a n s f o r m e d when t h e y become a n c h o r a g e i n d e p e n d e n t .  ras partially — transformed  ras  +  myc -fully transformed  src  src  H  +-  myc  REFER ENC ES  Adams,J.M., H a r r i s , J.W., P i n k e r t , C.A., Corcoran, L.M., A l e x a n d e r , W.S., C o r y , S., P a l m i t e r , R.D. and B r i n s t e r , R.L. ( 1 9 8 5 ) . 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A s s o i a n , R.K., K o m o r i y a , A., M e y e r s , C.A., M i l l e r , D.M. and S p o r n , M.B. (1983). T r a n s f o r m i n g growth f a c t o r in human p l a t e l e t s : i d e n t i f i c a t i o n o f a major s t o r a g e s i t e , p u r i f i c a t i o n and c h a r a c t e r i z a t i o n . J . B i o l . Chem. 258:7155-7160. A u e r s p e r g , N. and C a l d e r w o o d , G.A. ( 1 9 8 4 ) . Development o f serum i n d e p e n d e n c e i n K i r s t e n m u r i n e sarcoma v i r u s i n f e c t e d r a t a d r e n a l c e l l s . C a r c i n o g e n e s i s 5_: 175-181. A u e r s p e r g , N., Hudson, J.B., Goddard, E.G. and K l e m e n t , V. (1977). T r a n s f o r m a t i o n of c u l t u r e d r a t a d r e n o c o r t i c a l c e l l s by K i r s t e n m u r i n e sarcoma v i r u s ( K i - M S V ) . I n t . J . C a n c e r 19:81-89A u e r s p e r g , N. S i e m e n s , C.H., K r y s t a l , G. and M y r d a l , S.E. ( 1 9 8 6 ) . 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