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

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An A n a l y s i s of the E f f e c t s of Oncogenes and Growth F a c t o r s on Rat Adrenal Cortex C e l l s by I a i n A l a s d a i r Somerled MacAuley B . S c , U n i v e r s i t y of Calgary, 1981 Thesis Submitted i n P a r t i a l F u l f i l l m e n t of the Requirements f o r the Degree of Doctor of Philosophy The F a c u l t y of Graduate Studies Department of 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 accept t h i s t h e s i s as conforming to the r e q u i r e d standards U n i v e r s i t y of B r i t i s h Columbia cj I a i n A l a s d a i r Somerled MacAuley i n January 1987 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. The University of British Colurnl 1956 Main Mall Vancouver, Canada Department V6T 1Y3 DE-6(3/81) i i A b s t r a c t The process of oncogenic t r a n s f o r m a t i o n in v i t r o has been examined i n an attempt to d e f i n e the molecular mechanisms of c a r c i n o g e n e s i s . Transformation i n Ki-MSV-i n f e c t e d r a t adrenal cortex c e l l s appears to be a m u l t i s t e p process (Auersperg et a l . , 1981), as does the process of t r a n s f o r m a t i o n i n other n o n e s t a b l i s h e d c e l l s (Land et at., 1983b). The Ki-MSV-infected a d r e n a l cortex c e l l s i n i t i a l l y express a p a r t i a l l y transformed phenotype and a f t e r f u r t h e r passaging progress to a h i g h l y transformed phenotype (Calderwood and Auersperg, 1984). Examination of Ki-MSV-i n f e c t e d adrenal cortex c e l l s i n d i c a t e d that p r o g r e s s i o n to a h i g h l y transformed phenotype could occur i n the absence of s i g n i f i c a n t changes i n the l e v e l of the ex p r e s s i o n of the v i r a l ras oncogene. These r e s u l t s i n d i c a t e d that an o n c o g e n i c a l l y a c t i v a t e d ras gene could be expressed i n these n o n e s t a b l i s h e d c e l l s i n the absence of t r a n s f o r m a t i o n . Since ras and myc cooperate to transform primary f i b r o b l a s t s the e f f e c t of the c o - i n t r o d u c t i o n of myc on K i -MSV-induced t r a n s f o r m a t i o n of adrenal cortex c e l l s was examined. I t could be demonstrated that myc and ras cooperate to transform the adrenal cortex c e l l s more e f f i c i e n t l y than e i t h e r oncogene alone, but that the i n f e c t e d c u l t u r e s i n i t i a l l y only express some of the phenotypes a s s o c i a t e d with t r a n s f o r m a t i o n . The appearance of a f u l l y transformed phenotype, as monitored by growth i n i i i s o f t agar, was not expressed u n t i l s e v e r a l passages a f t e r i n f e c t i o n . An a n a l y s i s of the Ki-MSV/MMCV-infected c u l t u r e s i n d i c a t e d that some of the phenotypes a s s o c i a t e d with a c t i v a t e d oncogenes i n immortalized c e l l l i n e s appeared to be suppressed i n the c o i n f e c t e d adrenal cortex c e l l s . T ransformation by ras and myc appears to r e q u i r e a f u r t h e r c e l l u l a r change r e s u l t i n g i n a l o s s of the suppression of oncogene a c t i o n . The emergence of transformed c u l t u r e s from the Ki-MSV-infected r a t adrenal cortex c e l l s was c o r r e l a t e d with the reduced e x p r e s s i o n of a novel r a s - r e l a t e d p r o t e i n of 2 7 0 0 0 Mr. Transformation induced by s r c and myc was a l s o examined. These two oncogenes appeared to cooperate i n a two step pathway of t r a n s f o r m a t i o n that was not s u s c e p t i b l e to c e l l u l a r s u p p r e s s i o n . The transformed phenotype d i d not appear to be e n t i r e l y f r e e of e x t e r n a l i n f l u e n c e as the growth r a t e of the transformed c e l l s could be modified by c u l t u r e c o n d i t i o n s . The a b i l i t y of myc to cooperate with s r c and ras i n the t r a n s f o r m a t i o n of the e a r l y passage adrenal cortex c e l l s provides f u r t h e r support f o r mutistep c a r c i n o g e n e s i s . The e f f e c t of oncogenes on s t e r o i d o g e n e s i s was examined i n the Y-1 a d r e n o c o r t i c a l tumour c e l l l i n e . The e f f e c t of the v i r a l l y borne oncogenes on growth and morphology of the Y-1 c e l l s was r e l a t i v e l y s u b t l e . The oncogenes appear to s t i m u l a t e the p r o d u c t i o n of f l u o r o g e n i c s t e r o i d s , each i n a d i s t i n c t f a s h i o n . A m o d e l o f t r a n s f o r m a t i o n c a n b e d e r i v e d i n w h i c h t h e r o l e s o f t h e o n c o g e n e s a n d t h e i r i n t e r a c t i o n w i t h t h e c e l l c a n b e e v a l u a t e d . T h e d i f f e r e n c e s i n t h e p a t h w a y s o f t r a n s f o r m a t i o n f o r t h e t w o c o m b i n a t i o n s o f o n c o g e n e s i l l u s t r a t e s t h e p o t e n t i a l c o m p l e x i t y o f t h e t r a n s f o r m a t i o n p r o c e s s a n d p r o v i d e s a n in v i t r o m o d e l s y s t e m f o r f u r t h e r s t u d y . V Table of Contents Page A b s t r a c t i i L i s t of Tables v i i i L i s t of F i g u r e s i x L i s t of A b b r e v i a t i o n s x i i D e d i c a t i o n x i v Acknowledgements xv CHAPTER 1 1.0 I n t r o d u c t i o n 1 1.1 R e t r o v i r u s e s 1 1.1.1 C l a s s i f i c a t i o n 1 1.1.2 S t r u c t u r e 1 1.1.3 R e p l i c a t i o n 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 of v-oncs 5 1.2.3 C h r o n i c / l e u k o s i s V i r u s e s 5 1 .3 Oncogenes: O r i g i n s and Functions 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 .nas 9 1.3.4 myc 20 1.3-5 s r c 25 1.3.6 naf 28 1.4 Tran s f o r m a t i o n : i n v i v o and i n v i t r o Models 30 1.5 Tis 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 Regula t i o n of C e l l Growth by Growth F a c t o r s 37 1.5.3 Transformation and Growth F a c t o r Independence39 CHAPTER 2 2.0 M a t e r i a l s and Methods 2.1 C e l l s 41 2.2 V i r u s e s 43 2.3 I n f e c t i o u s Centre Assays 44 2.4 Anchorage Independent Growth 45 2.5 R a d i o l a b e l l i n g of C e l l s 46 2.6 Immunoprecipitation 47 2.7 Immune Complex Kinase Reactions 47 2.8 SDS-polyacrylamide Gel E l e c t r o p h o r e s i s 48 2 .9 Pulse L a b e l l i n g of P r o t e i n s 49 2.10 T r y p t i c Peptide A n a l y s i s 50 2.11 Southern H y b r i d i z a t i o n 51 2.12 HPLC A n a l y s i s of S t e r o i d Products from the Y-1 A d r e n o c o r t i c a l C e l l Line 52 2 . 1 3 Radioimmunoassay of C u l t u r e Supernatant of K i -MSV/MMCV Transformed Rat Adrenal Cortex C e l l s f o r S t e r o i d P r o d u c t i o n . . 5 3 CHAPTER 3 3.0 Ex p r e s s i o n of V i r a l p 2 1 r a s • D u r i n g A c q u i s i t i o n of a Transformed Phenotype by Rat Adrenal Cortex C e l l s I n f e c t e d with Ki-MSV 3.1 I n t r o d u c t i o n 55 3.2 Ex p r e s s i o n of V i r a l p 2 1 r a s i n the Passages Immediately F o l l o w i n g I n f e c t i o n 59 3 . 3 V i r a l p21 r s 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 66 3.4 D i s c u s s i o n 72 CHAPTER 4 4.0 Transformation of Rat Adrenal Cortex C e l l s by ras and myc: Evidence f o r a Requirement f o r a Fu r t h e r C e l l u l a r Change 4.1 I n t r o d u c t i o n 75 4.2 In d u c t i o n of Focus Formation and Mo r p h o l o g i c a l Transformation by Ki-MSV and MMCV 80 4 . 3 The Ex p r e s s i o n of a Transformed Morphology and Growth by Ki-MSV/MMCV-infected A d r e n o c o r t i c a l C u l t u r e s Requires a High C o n c e n t r a t i o n of Serum. 88 4.4 A d d i t i o n of a P u r i f i e d Growth Fa c t o r can Replace the Requirement f o r a High Serum Supplement Co n c e n t r a t i o n i n Ki-MSV/MMCV-infected C e l l s 93 4.5 A c q u i s i t i o n of Serum Independent Growth and Anchorage Independent Growth 96 4.6 I s o l a t i o n of Transformed Lines from Ki-MSV/MMCV-i n f e c t e d C u l t u r e s 98 4.7 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 the Presence of the V i r a l Oncogenes and t h e i r Products, and S t e r o i d o g e n i c A b i l i t y 103 4.8 D i s c u s s i o n 114 CHAPTER 5 5.0 myc and s r c Cooperate i n the i n v i t r o Transformation of E a r l y Passage Rat Adrenal Cortex C e l l s 5.1 I n t r o d u c t i o n : 119 v i i 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 to S u p e r i n f e c t i o n by the s r c Conta i n i n g R e t r o v i r u s , 2-1 121 5 - 3 Anchorage Independent Growth 125 5.4 C h a r a c t e r i z a t i o n of Three Transformed Lines f o r the Presence of the V i r a l Oncogenes 126 5.5 D i s c u s s i o n 138 CHAPTER 6 6.0 E f f e c t s of V i r a l Oncogenes on the S t e r o i d o g e n i c A b i l i t y of the 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 I n t r o d u c t i o n 142 6.2 The E f f e c t s of R e t r o v i r a l l y Borne Oncogenes on the Morphology and Growth of the Y-1 C e l l L i n e . . 146 6 . 3 A n a l y s i s of S t e r o i d Production by the R e t r o v i r a l l y I n f e c t e d and Uninfected Y-1 Lines 150 6.4 D i s c u s s i o n 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 Related to p21r-a-s- i s Expressed i n Human and Rat Primary Tissue C u l t u r e C e l l s 7.1 I n t r o d u c t i o n 165 7.2 Survey of p27 Exp r e s s i o n 167 7 . 3 F r a c t i o n a t i o n of the L a b e l l e d Lysate of a p27 Expressing C e l l Line 168 7.4 T r y p t i c Peptide Mapping of p27 175 7.5 Pulse-chase L a b e l l i n g of p27 178 7.6 D i s c u s s i o n 184 CHAPTER 8 8.0 Summary. 1 89 References 197 v i i i L i s t of Tables Table 3.1 Table 4.1 Table 4.2 Table 4.3 Table 4.4 Table 5.1 Table 5.2 Table 5.3 Table 6.1 Table 6.2 Table 6.3 [ 3 5 S ] I n c o r p o r a t i o n i n t o p21^a-s- i n Ki-MSV-i n f e c t e d and Uninfected Adrenal Cortex C e l l s Focus Formation i n Rat Adrenal Cortex 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 I n f e c t i o u s Centre Assay of Ki-MSV/MMCV-i n f e c t e d Adrenal Cortex C e l l s Number of Passages Between I n f e c t i o n and Express i o n of Serum Independence and Anchorage Independent Growth. Conversion of Pregnenolone to an Intermediate of the S t e r o i d o g e n i c Pathway, Progesterone, by Ki-MSV/MMCV-transformed Rat Adrenal Cortex C e l l L i n e s . Focus Formation Induced 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 Anchorage Independent Growth of 2-1/MMCV-i n f e c t e d Adrenal Cortex C e l l s Growth of 2-1/MMCV-infected Adrenal Cortex C e l l s i n the Presence of D i f f e r e n t Serum Supplements I n h i b i t i o n of DNA Synthesis by F o r s k o l i n i n the Y-1 and Y - 1 m H c v Lines Measurement of S t e r o i d Production 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 Acid Induced Fluorescence A n a l y s i s of the R e l a t i v e Production of the S t e r o i d Products of the V i r a l l y I n f e c t e d Y-1 C e l l Lines 6 5 83 85 97 Table 7.1 Exp r e s s i o n of p27 i n Rat and Human C e l l s 113 124 129 132 149 152 159 171 i x L i s t of F i g u r e s F i g u r e 3•1 Fi g u r e 3.2 Fi g u r e 3.3 Figur e 3.4 Fig u r e 4.1 Figur e 4.2 Fi g u r e 4 .3 F i g u r e 4.4 Fig u r e 4 .5 Fig u r e 4 .6 Fi g u r e 4.7 Fig u r e 4.8 Fig u r e 4.9 E a r l y Passages of KiMSV-infected Rat Adrenal Cortex C e l l s Grown with 25% F e t a l Bovine or 3% Horse Serum Supplements 61 A n a l y s i s of p21^a-s- Expre s s i o n i n KiMSV-i n f e c t e d and Uninfected Rat Adrenal Cortex C e l l s 64 Morphology of Ki-MSV-infected Rat Adrenal Cortex C e l l s at E a r l y and Late Passages 68 Expr e s s i o n of V i r a l p21 r-a-s i n E a r l y and Late Passage C u l t u r e s of Ki-MSV-infected Adrenal Cortex C e l l s 70 Diagram of the P r o v i r a l form of MMCV 78 Focus Formation i n E a r l y Passage Rat Adrenal Cortex C e l l s I n f e c t e d with Ki-MSV, MMCV or Both 82 Overgrowth of I n f e c t e d Adrenal 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 Transformed C e l l s 87 Serum S e n s i t i v i t y of the Transformed Morphology Induced by ras. and myc i n Rat Adrenal Cortex C e l l s 90 Serum Dependence of the Express i o n of the A l t e r e d Morphology i n Ki-MSV/MMCV-i n f e c t e d Rat Adrenal Cortex C e l l s 92 Mor p h o l o g i c a l Response of Ki-MSV/MMCV-i n f e c t e d Adrenal Cortex C e l l s to P u r i f i e d Growth F a c t o r s 95 Anchorage Independent Growth of Ki-MSV/MMCV-infected Adrenal Cortex C e l l s 100 Formation of a Spontaneous Focus i n the Presence of 5% HS. 102 A n a l y s i s of p21r-a-£ E x p r e s s i o n i n Ki-MSV/MMCV-infected Rat Adrenal Cortex C e l l s . 105 X F i g u r e 4 Fi g u r e 4 10 Demonstration of the Presence of v-myc i n Transformed C e l l Lines Derived from the Ki-MSV/MMCV-infected Rat Adrenal Cortex C u l t u r e . 11 E x p r e s s i o n of p57 v _SZ c: i n Transformed C e l l Lines Derived from Ki-MSV/MMCV-i n f e c t e d Rat Adrenal Cortex C u l t u r e s . 1 07 110 F i g u r e 5.1 Fig u r e 5.2 Fig u r e 5.3 Fig u r e 5.4 Fig u r e 5.5 Fig u r e 6 . 1 F i g u r e 6 Fig u r e 6 C e l l Morphology i n MMCV- or Ki-MSV-i n f e c t e d Rat Adrenal Cortex C e l l s S u p e r i n f e c t e d with the s r c Contai n i n g R e t r o v i r u s 2-1 . Colony Formation i n S o f t Agar by the 2-1-s u p e r i n f e c t e d Adrenal Cortex C u l t u r e s . Growth Rate of 2-1/MMCV-infected Adrenal Cortex C e l l s i n the Presence of High or Low Serum Supplements. Examination of Cloned C e l l Lines Derived from the 2-1/MMCV-infected Adrenal Cortex C u l t u r e s f o r Elevated pSO^1^0-Kinase A c t i v i t y . Southern A n a l y s i s of the 2-1/MMCV-i n f e c t e d Adrenal Cortex C e l l Lines f o r the Presence of v-myc. S t e r o i d o g e n i c Pathway of Y-1 A d r e n o c o r t i c a l Tumour C e l l s . Morphology of Y-1 and y _ - | M M C V L i n e s . Demonstration of Comigration of S t e r o i d Products from the Y-1 and Y-1MMCV C e l l L i n e s . F i g u r e 6.4 Comparison of the S t e r o i d Products Produced b C e l l Lines y the Y-1, Y - 1 m m c v and Y - 1 K i _ M S V F i g u r e 7 Fig u r e 7 Fig u r e 7 Fig u r e 7 Survey of Rat Primary C u l t u r e s f o r the Expr e s s i o n of na_s.-related P r o t e i n s . Rate-zonal F r a c t i o n a t i o n of L J S]methionine L a b e l l e d Rat Lung F i b r o b l a s t Lysate. T r y p t i c Peptide Maps of p27 and p21c-a-§ methionine Pulse-chase of p27 and p 2 1 J i a s . 123 128 131 135 137 144 148 155 158 170 174 177 1 80 x i F i g u r e 7 . 5 E x a m i n a t i o n o f p 2 7 a n d p 2 i f f .43 E x p r e s s i o n i n a K i - M S V T r a n s f o r m e d C e l l L i n e 1 8 3 F i g u r e 8 . 1 T r a n s f o r m a t i o n P a t h w a y s o f E a r l y P a s s a g e A d r e n o c o r t i c a l C e l l s 1 9 6 L i s t of A b b r e v i a t i o n s A adenine ACTH a d r e n o c o r t i c o t r o p h i c hormone ATP adenosine 5 ' - t r i p h o s p h a t e C c y t o s i n e C i C u r i e cpm counts per minute dCTP deoxycytosine 5 ' - t r i p h o s p h a t e DMEM Dulbecco's modified Eagle's medium DNA d e o x y r i b o n u c l e i c a c i d EDTA disodium ethylene d iamin et etraac e t i c ac EF-Tu e l o n g a t i o n f a c t o r - T u EGF epidermal growth f a c t o r FBS f e t a l bovine serum FGF f i b r o b l a s t growth f a c t o r FSV Fujinami sarcoma v i r u s xg times g r a v i t y GDP guanosine 5'-diphosphate GTP guanosine 5 ' - t r i p h o s p h a t e Ha-MSV Harvey murine sarcoma v i r u s HPLC high performance l i q u i d chromatography HS horse serum IGF-II i n s u l i n - l i k e growth f a c t o r two Kb k i l o b a s e Kd k i l o d a l t o n Ki-MSV K i r s t e n murine sarcoma v i r u s LTR's long t e r m i n a l repeats x i i i uCi m i c r o - C u r i e MEM minimal Eagle's medium mM m i l l i m o l a r Mo-MLV Moloney murine leukemia v i r u s Mo-MSV Moloney murine sarcoma v i r u s Mr r e l a t i v e m o b i l i t y NaDOC sodium deoxycholate NRK normal r a t kidney PAGE p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s PBS phosphate b u f f e r e d s a l i n e PDGF p l a t e l e t - d e r i v e d growth f a c t o r P i i n o r g a n i c phosphate PIP ES 1 , 4 - p i p e r a z i n e d i e t h a n e s u l f o n i c a c i d R Rig r a b b i t a n t i - r a t immunoglobulin RIPA r a d i o i m m u n o p r e c i p i t a t i o n assay RNA r i b o n u c l e i c a c i d RNaseH r i b o n u c l e a s e H RSV Rous sarcoma v i r u s S sulphur SDS sodium dodecyl s u l p h a t e SHE S y r i a n hamster f i b r o b l a s t s TCA t r i c h l o r o a c e t i c a c i d TGF tumour growth f a c t o r TP A t e t r a d e c a n o y l p h o b o l - a c e t a t e w/ V weight to volume Acknowl edg ements I would l i k e to express my s i n c e r e s t a p p r e c i a t i o n to my s u p e r v i s o r y committee Drs. George Spiegelman, J u l i a Levy and K e i t h Humphries f o r t h e i r c o n s i d e r a b l e e f f o r t s and c r i t i c a l e v a l u a t i o n s t hat have underpinned the s u c c e s s f u l completion of t h i s t h e s i s , and to Dr. Tony Pawson, my s u p e r v i s o r , whose p a t i e n c e and t o l e r a n c e allowed t h i s work to be done. I would a l s o l i k e to thank a l l the other people who have c o n t r i b u t e d to t h i s work and my growth as a s c i e n t i s t : Dr. N. Auersperg, f o r s h a r i n g her system, e x p e r t i s e and enthusiasm so f r e e l y ; Dr. Gerry Weeks, who has always s u p p l i e d a c c u r a t e and apt c r i t i c i s m and has twice served on my committee at short n o t i c e with good humour and enthusiasm; Dr. Gerry Weinmaster, f o r being such a s u p p o r t i v e f r i e n d and r e c e p t i v e l i s t e n e r ; Ehleen Hinze and Ian McClaren f o r t h e i r help and encorag ement; C r a i g Siemens and J u d i t h Black, f o r t h e i r help that was given f r e e l y ; and members of the M i c r o b i o l o g y and Biochemistry departments whose f r i e n d s h i p and encouragement have always helped i n so many t a n g i b l e and i n t a n g i b l e ways, e s p e c i a l l y : Vera Webb, Dennis Dixon and Dan Horvath. F i n a l l y I would l i k e to express my indebtedness to Kathy Dobinson f o r her u n f a i l i n g f r i e n d s h i p , p a t i e n c e with my i r a s c i b i l i t y and f o r having typed my t h e s i s with such c a r e f o r l o v e and f r i e n d s h i p alon e. I would l i k e to thank my parents and f a m i l y whose l o v e and support stands with me every day of my l i f e . 1 CHAPTER 1 INTRODUCTION 1.1 R e t r o v i r u s e s 1.1.1 C l a s s i f i c a t i o n The r e t r o v i r u s e s i n c l u d e the v i r u s e s which c o n t a i n a RNA genome and a RNA-dependent DNA polymerase. This f a m i l y can be sub d i v i d e d i n t o three s u b f a m i l i e s : i ) the Oncoviruses, which i n c l u d e s the oncogenic v i r u s e s and other c l o s e l y r e l a t e d nononcogenic r e t r o v i r u s e s , i i ) the L e n t i v i r u s e s , or slow v i r u s e s , such as v i s n a v i r u s ; and i i i ) the Spumaviruses. The work i n t h i s t h e s i s uses v i r u s e s d e r i v e d from the f i r s t s ubfamily, the Oncoviruses ( f o r a review see Weiss et a l . , 1985). 1.1.2 S t r u c t u r e The genome of a r e p l i c a t i o n competent r e t r o v i r u s of the oncoviruses c o n t a i n s three s t r u c t u r a l genes gag, p o l and env. The gag gene encodes the group a s s o c i a t e d a n t i g e n , env encodes the envelope p r o t e i n s present i n the v i r a l membrane and p o l codes f o r reverse t r a n s c r i p t a s e or the RNA-dependent DNA polymerase. These genes are arranged 5'-gag-pol-env-3' and the coding regions are not u s u a l l y overlapped. The s t r u c t u r a l genes are l o c a t e d between two long t e r m i n a l repeats (LTR's) which c o n t a i n the elements of the v i r a l promoters, enhancers and t e r m i n a t o r s of t r a n s c r i p t i o n as w e l l as s i g n a l s f o r the r e p l i c a t i o n (see Varmus and 2 Swanstrom i n Weiss e_t a_l. , 1985 ) and i n t e g r a t i o n of the r e t r o v i r a l genome (Panganiban and Temin, 1984). The v i r i o n p a r t i c l e ( f o r a review see Weiss et a l . , 1985) i s composed of a membrane-derived envelope and n u c l e o c a p s i d . The n u c l e o c a p s i d c o n t a i n s the genomic RNA bound to the gag p r o t e i n s , and the p o l product. The genomic RNA of each v i r u s p a r t i c l e i s a 60 to 70S dimer, i n which the two RNA strands are attached near t h e i r 5 1 ends. Each of the two RNA molecules c l o s e l y resembles e u k a r y o t i c messenger RNA i n that they c o n t a i n a 5* methylated cap and a 3 1 polyA t a i l . The RNA p u r i f i e d from the v i r i o n p a r t i c l e s can be used to d i r e c t i n v i t r o s y n t h e s i s of gag and gag-pol products d e f i n i n g the genomic RNA as p o s i t i v e sense. The env gene products are embedded i n the v i r i o n membrane and are r e s p o n s i b l e f o r a s s o c i a t i o n of the n u c l e o c a p s i d with the membrane and b i n d i n g of the v i r a l p a r t i c l e s to the c e l l u l a r r e c e p t o r s i n i t i a t i n g v i r a l i n f e c t i o n . 1.1.3 R e p l i c a t i o n Once the v i r a l p a r t i c l e has bound to the c e l l s u r f a c e , i t i s taken up by the c e l l and the v i r a l contents are r e l e a s e d i n t o the c e l l cytoplasm. Reverse t r a n s c r i p t a s e has both RNA-dependent and DNA-dependent DNA polymerase a c t i v i t i e s as w e l l as a RNaseH a c t i v i t y , a l l of which are r e q u i r e d f o r r e p l i c a t i o n of the v i r a l genome. The genomic RNA i s i n i t i a l l y copied i n t o double stranded DNA, which can e x i s t as e i t h e r l i n e a r or c i r c u l a r molecules before i n t e g r a t i o n . The j u n c t i o n between the two LTR's appears to 3 act as the s i g n a l to allow i n t e g r a t i o n of the p r o v i r a l genome i n t o the host c e l l genome (Panganiban and Temin, 1984) where the v i r a l genes can be a c t i v e l y expressed. V i r i o n s bud from the c e l l membrane so that r e t r o v i r a l i n f e c t i o n does not u s u a l l y r e s u l t i n c e l l l y s i s . The i n t e g r a t e d form of the p r o v i r u s i s u s u a l l y s t a b l y t r a n s m i t t e d to daughter c e l l s as a part of the host genome. Most p r o v i r u s e s i n t e g r a t e e s s e n t i a l l y randomly i n t o the host DNA. The i n t e g r a t i o n s i t e of the p r o v i r u s becomes a c l o n a l marker and can be mapped by Southern a n a l y s i s . The p r o v i r u s i s expressed l a r g e l y independently of most environmental c o n d i t i o n s . P r o d u c t i v e i n f e c t i o n by a r e t r o v i r u s i n h i b i t s s u p e r i n f e c t i o n by v i r u s e s that use the same r e c e p t o r . The expr e s s i o n of the env product appears to r e s u l t i n a s a t u r a t i o n of the c e l l r e c e p t o r s r e s u l t i n g i n a block to r e i n f e c t i o n . T h i s block to s u p e r i n f e c t i o n does not extend to r e t r o v i r u s e s that use other r e c e p t o r s . These p r o p e r t i e s , combined with the a b i l i t y of the v i r u s e s to express f o r e i g n genes, has allowed the use of oncogene c o n t a i n i n g r e t r o v i r u s e s to e l u c i d a t e the e f f e c t s of s p e c i f i c oncogenes on the phenotypes of c u l t u r e d c e l l s . 1 . 2 Oncogenic R e t r o v i r u s e s The oncogenic r e t r o v i r u s e s can be subd i v i d e d i n t o two c l a s s e s based on t h e i r r e l a t i v e a b i l i t i e s to transform c e l l s i n v i t r o and the r a p i d i t y with which they form tumours i n animals. The a c u t e l y oncogenic r e t r o v i r u s e s can r a p i d l y transform c e l l s i n v i t r o and cause tumours i n animals with a 4 s h o r t l a t e n t p e r i o d . T h e o t h e r s u b c l a s s d o e s n o t a p p e a r t o b e a b l e t o t r a n s f o r m c e l l s i n v i t r o a n d t u m o u r i g e n e s i s r e q u i r e s a l o n g l a t e n t p e r i o d . 1 . 2 . 1 A c u t e l y O n c o g e n i c R e t r o v i r u s e s T h e a c u t e l y o n c o g e n i c r e t r o v i r u s e s c o n t a i n s e q u e n c e s t h a t a r e r e q u i r e d f o r r a p i d t r a n s f o r m a t i o n o f c e l l s in_ v i v o a n d i n v i t r o , b u t a r e n o t f o u n d i n t h e p o o r l y o n c o g e n i c r e t r o v i r u s e s . T h e s e s e q u e n c e s , c a l l e d v i r a l o n c o g e n e s ( v -o n c s ) , d o n o t a p p e a r t o b e d e r i v e d f r o m a n y v i r a l s e q u e n c e s , b u t r a t h e r a r e d e r i v e d f r o m n o r m a l c e l l u l a r g e n e s ( c - o n c s o r p r o t o - o n c o g e n e s ) . G e n e t i c s t u d i e s o f c o n d i t i o n a l a n d n o n c o n d i t i o n a l m u t a n t s i n t h e t r a n s f o r m i n g a c t i v i t y o f t h e a c u t e l y o n c o g e n i c r e t r o v i r u s e s h a v e m a p p e d t o t h e n o v e l o r c e l l u l a r l y d e r i v e d s e q u e n c e s , d e m o n s t r a t i n g a c l e a r c o n n e c t i o n b e t w e e n t h e p r e s e n c e o f t h e v - o n c a n d t r a n s f o r m a t i o n . T h e p r o c e s s o f a c q u i r i n g t h e c e l l u l a r s e q u e n c e s u s u a l l y r e s u l t s i n r e p l a c e m e n t o f s o m e o f t h e v i r a l g e n e s r e q u i r e d f o r r e p l i c a t i o n b y t h e n e w s e q u e n c e s . A s a r e s u l t t h e a c u t e l y o n c o g e n i c r e t r o v i r u s i s n o l o n g e r a b l e t o r e p l i c a t e a u t o n o m o u s l y b u t r e q u i r e s t h a t t h e s t r u c t u r a l g e n e s l o s t o r i n a c t i v a t e d b y t h e a c q u i s i t i o n o f t h e c e l l u l a r g e n e b e s u p p l i e d i n t r a n s b y a r e p l i c a t i o n c o m p e t e n t r e t r o v i r u s . A l l t h e n a t u r a l l y o c c u r r i n g , a c u t e l y t r a n s f o r m i n g r e t r o v i r u s e s e x c e p t R S V a r e d e f e c t i v e a n d r e q u i r e a h e l p e r v i r u s . 5 1.2.2 O r i g i n of v-oncs Although the idea of oncogenes had been proposed by Huebner and Todaro i n 1969, and evidence of t h e i r e x i s t e n c e had been d e r i v e d from f i n e s t r u c t u r e mapping s t u d i e s of t r a n s f o r m a t i o n - d e f e c t i v e v i r a l mutants, the o r i g i n of the v-pncs remained obscure. I t was subsequently shown that the genomic DNA of both avian and mammalian c e l l s , d e r i v e d from u n i n f e c t e d animals, contained sequences that were r e l a t e d to the v-onc of RSV. Examination of other v-oncs demonstrated that a l l v-oncs were appa r e n t l y d e r i v e d from c e l l u l a r genes (Bishop, 1985). Although many of the v-oncs examined were d i s t i n c t , they a l l had c-onc homologues to which they were more c l o s e l y r e l a t e d than to a v i r a l s t r u c t u r a l gene. The c-oncs are h i g h l y conserved across s p e c i e s b a r r i e r s (see Bishop and Varmus i n Weiss e_t a_l. , 1 985 ). The expre s s i o n of some of the c-oncs appears to be r e g u l a t e d through growth and development ( M u l l e r et a l . , 1982, 1983; Slamon and C l i n e , 1984). They seem to be able to modify c e l l u l a r responses to exogenous s i g n a l s , i n d i c a t i n g that the c-onc products may be i n v o l v e d i n important r e g u l a t o r y f u n c t i o n s r e q u i r e d to allow normal c e l l u l a r growth and d i f f e r e n t i a t i o n . 1.2.3 C h r o n i c / l e u k o s i s V i r u s e s The remainder of the. oncogenic r e t r o v i r u s e s do not transform c e l l s i n c u l t u r e and only cause tumours in_ v i v o a f t e r long l a t e n t p e r i o d s . These v i r u s e s are u s u a l l y r e p l i c a t i o n competent and c o n t a i n only the normal r e t r o v i r a l 6 genes (Weiss et a l . , 1 9 8 5 ) . These v i r u s e s appear to be able to a c t i v a t e c-oncs d i r e c t l y by a l t e r i n g t h e i r normal r e g u l a t i o n . The v i r u s e s use a number of mechanisms to achieve the d y s r e g u l a t i o n that leads to t r a n s f o r m a t i o n . The v i r u s u s u a l l y i n t e g r a t e s near a c-onc and can a c t i v a t e t r a n s c r i p t i o n from the c e l l u l a r gene e i t h e r by p r o v i d i n g s t r o n g e r promoter s i g n a l s (Hayward et a l . , 1 9 8 1 ; Neel et a l . , 1 9 8 2 ) or by d i s r u p t i n g normal r e g u l a t o r y s i g n a l s (Payne e_t__al., 1 9 8 2 ; Hann et a l . , 1 9 8 3 ) . These mechanisms have been i m p l i c a t e d i n c-onc a c t i v a t i o n a f t e r chromosome t r a n s l o c a t i o n (Hayday et a_l . , 1 9 8 4 ; Yang et al_. , 1 9 8 5 ) , and provided some of the e a r l i e s t evidence that the c-oncs could a l s o be i n v o l v e d i n c a r c i n o g e n e s i s . 1 . 3 Oncogenes-Origins and Functions  1 . 3 . 1 R e t r o v i r a l Oncogenes The r e t r o v i r a l oncogenes a l l appear to be d e r i v e d from c e l l u l a r genes. The process of t r a n s d u c t i o n of these genes i n t o the r e t r o v i r u s u s u a l l y r e s u l t s i n a number of changes i n the gene which may i n c l u d e : i ) t r u n c a t i o n of the gene; i i ) , replacement of p a r t of the normal c-onc sequence by a d i f f e r e n t sequence; and i i i ) , p o i n t mutations i n the gene. These mutations are u s u a l l y i n v o l v e d i n a c t i v a t i o n of the gene, i n a d d i t i o n to the ov e r e x p r e s s i o n of the gene by the v i r a l LTR. The s t r u c t u r a l changes i n the genes a f f e c t the normal f u n c t i o n of the gene products. The e f f e c t s of the changes have been most c l e a r l y demonstrated with v - s r c and v - r a s . v 7 The v - s r c product i s c o n s t i t u t i v e l y a c t i v a t e d by the l o s s of the s i t e s r e s p o n s i b l e f o r negative r e g u l a t i o n of the c - s r c product (Iba e_t a l . , 1 9 8 4 ; Bolen ejb a l . , 1 9 8 4 ; Courtneidge, 1 9 8 5 ) . Work with ras has shown that overexpression of a c-ras gene i s not as p o t e n t l y t r a n s f o r m i n g as o v e r e x p r e s s i o n of a v-ras gene which co n t a i n s s e v e r a l a c t i v a t i n g mutations (Spandidos and W i l k i e , 1 9 8 4 ) . Many of the oncogenes that have been i s o l a t e d demonstrate an i n t r i n s i c t y r o s i n e kinase a c t i v i t y . The genes that encode the t y r o s i n e kinases a l l demonstrate a c e r t a i n degree of s t r u c t u r a l homology i n the kinase domain (Hunter and Cooper, 1 9 8 5 ) . The genes that f a l l i n t o t h i s group are s r c , yes, r o s , f g r , f p s / f e s , a b l , fms and erbB. Of these genes erbB i s thought to be a truncated v e r s i o n of the EGF r e c e p t o r ( U l l r i c h et a l . , 1 9 8 4 ; Downward et a l . , 1 9 8 4 ) and fms and ros are thought to be a l t e r e d v e r s i o n s of other growth f a c t o r r e c e p t o r s (Sherr et a l . , 1 9 8 5 ) that have an i n h e r e n t t y r o s i n e kinase a c t i v i t y . c-fms i s thought to encode the r e c e p t o r f o r a hemopoietic growth f a c t o r M-CSF. The r o l e s of the other t y r o s i n e kinase oncogene products are unknown, but they are thought to be i n v o l v e d i n the r e g u l a t i o n of growth and d i f f e r e n t i a t i o n of the c e l l s i n which they are expressed. The v - s i s oncogene product i s thought to be a homologue of the p l a t e l e t d e r i v e d growth f a c t o r (PDGF) and to exert i t s e f f e c t through the PDGF re c e p t o r which i s a t y r o s i n e kinase (Niman, 1 9 8 4 ; Robbins et_ a l . , 1 9 8 3 ; W a t e r f i e l d e t . a l . , 1 9 8 3 ) . 8 The oncogene products that are l o c a t e d i n the nucleus i n c l u d e , myc, myb, fos and p 5 3 . The products of the myc and myb oncogenes have been shown to bind DNA (Donner e_t a l . , 1982; Klempnauer et a_l. , 1984), although i t has not yet been p o s s i b l e to demonstrate any sequence s p e c i f i c i t y f o r b i n d i n g . The r o l e s of these oncogenes are not understood although i t appears that myc can render c e l l s h y p e r s e n s i t i v e to s p e c i f i c growth f a c t o r s (Stern e_t a l . , 1 9 8 6 ; Balk et a l . , 1985). myc and p53 d e f i n e a group of oncogenes that can cooperate i n tr a n s f o r m i n g n o n e s t a b l i s h e d c e l l s with oncogenes whose products are l o c a t e d i n the cytoplasm. The remainder of the oncogenes belong to a l o o s e l y d e f i n e d group. The genes i n t h i s group i n c l u d e the ras f a m i l y , the s e r i n e / t h r e o n i n e k i nases m i l / r a f , erbA and mos. Most of the genes i n t h i s group were i n i t i a l l y d e s c r i b e d as the t r a n s f o r m i n g gene of a r e t r o v i r u s , although i n some of the f a m i l i e s r e l a t e d c e l l u l a r genes that do not have v i r a l homologues, but can transform c e l l s , have a l s o been d e s c r i b e d , f o r example N-ras (Taparowsky et al.,1983). 1.3.2 C e l l u l a r Oncogenes The c e l l u l a r oncogenes were o r i g i n a l l y i s o l a t e d and d e s c r i b e d as homologues of the v i r a l oncogenes. Transformation by the p o o r l y oncogenic r e t r o v i r u s e s seems to occur by i n t e g r a t i o n of the nontransforming v i r u s near a c e l l u l a r gene that can be o n c o g e n i c a l l y a c t i v a t e d . This process was f i r s t d e s c r i b e d f o r c-myc (Hayward et a l . , 1981; Neel et a l . , 1982), which had a l r e a d y been i d e n t i f i e d as the 9 c e l l u l a r homologue of the v-myc oncogene (Vennstrom e_t a l . , 1982). Fur t h e r examination has r e v e a l e d other c e l l u l a r oncogenes, c-erbB ( N i l s e n et a l . , 1 985 ) and ras (Westaway e_t a l . , 1 9 8 6 ) , that can be a c t i v a t e d by t h i s mechanism. I t has been p o s s i b l e to i s o l a t e novel c-oncs that are not v i r a l homologues from spontaneously formed tumours i s o l a t e d from humans or animals due to a c t i v a t i o n of t r a n s f o r m i n g a c t i v i t y i n i n v i t r o assays, i n c l u d i n g neu (Schecter et _ a l . , 1 9 8 4 ) , l c k (Voronova et a l . , 1985) and N-myc (Schwab et a l . , 1983a). Many of the c-oncs that are homologous to v-oncs have had a c t i v a t e d forms a s s o c i a t e d e i t h e r d i r e c t l y or i n d i r e c t l y with spontaneously formed tumours i s o l a t e d from humans or animals (Slamon et a l . , 1984). This work has c o n f e r r e d c o n s i d e r a b l e v a l i d i t y on the search f o r an understanding of oncogenes as i t d i r e c t l y i m p l i c a t e s the oncogenes i n the formation of tumours i n  v i v o . 1.3.3 Ras The mammalian ras genes encode p o l y p e p t i d e s of approximately 21,000 d a l t o n s (Young e_t a l . , 1979; Shih e_t a l . , 1 9 7 9 ) that are a s s o c i a t e d with the plasma membrane (Willingham et a l . , 1980) and bind GTP (Shih et a l . , 1980). The ras genes comprise a f a m i l y of genes of which there are at l e a s t three a c t i v e members; Ha-ras-1, K i - r a s - 2 and N-ras, and two pseudogenes, Ha-ras-2 and K i - r a s - 1 . I t seems that the ras gene f a m i l y belongs to a gene s u p e r f a m i l y that i n c l u d e s a number of other genes with some s t r u c t u r a l 10 homology or with s t r u c t u r a l and f u n c t i o n a l (GTP binding) homology. The mammalian ras gene product i s a s s o c i a t e d with the c y t o p l a s m i c membrane by a c o v a l e n t l y attached f a t t y a c i d chain that anchors i t to the membrane (Sefton et a l . , 1982). L o c a l i z a t i o n i n the membrane i s a p p a r e n t l y important f o r the p S S a c t i v i t y of p21 , as mutagenesis of the r e s i d u e i n v o l v e d i n f a t t y a c i d b i n d i n g appears to r e s u l t i n a l o s s of f u n c t i o n as measured by t r a n s f o r m i n g a c t i v i t y (Willumsen et a l . , 1984). The ras gene products are GTP b i n d i n g p r o t e i n s with a high a f f i n t i y f o r n u c l e o t i d e b i n d i n g and very high s p e c i f i c i t y f o r guanosine n u c l e o t i d e s (Shih et a_l. , 1980). The ras products a l s o have an i n t r i n s i c a b i l i t y to hydrolyze GTP that appears to c l e a v e the t e r m i n a l phosphate to produce GDP and P i (McGrath et a l . , 1984; Sweet et _ a l . , 1984). This a c t i v i t y has been a s s o c i a t e d with hormonal a c t i v a t i o n of adenylate c y c l a s e and, by analogy, p 2 1 r a s i s thought to belong to a s i g n a l t r a n s d u c t i o n pathway, i n which a c t i v a t i o n of the r e g u l a t o r y pathway r e s u l t s i n b i n d i n g of GTP and a c t i v a t i o n of p 2 1 I Q . The h y d r o l y s i s of the bound GTP would then i n a c t i v a t e the p 2 1 r a s . The ras genes are present i n a l l e u k a r y o t i c s p e c i e s that have been examined i n c l u d i n g s i n g l e c e l l organisms (Reymond _e_t aJL. , 1984; Pawson et a l . , 1985; Powers et a l . , 1984; Defeo-Jones et a l . , 1983), i n s e c t s ( S h i l o and Weinberg, 1981) and mammals ( E l l i s et a^l. , 1981; Shimizu et a l . , 1983). I t would a l s o appear that ras genes are very 11 widely expressed, as they are ap p a r e n t l y t r a n s c r i b e d and t r a n s l a t e d i n a l l c e l l s which have been examined (reviewed i n B a rbacid, 1 9 8 6 ) . The ex p r e s s i o n of the ras genes appears to be r e g u l a t e d throughout the c e l l c y c l e but the v a r i a t i o n i s r e l a t i v e l y s m a l l . An i n c r e a s e i n the l e v e l of ras t r a n s c r i p t i o n i s a s s o c i a t e d with the e a r l y s t i m u l a t i o n of growth i n a r e g e n e r a t i n g l i v e r (Goyette et a l . , 1 9 8 3 ) . The ras genes are a l s o expressed throughout embryogenesis and again there appears to be some r e g u l a t i o n of the t r a n s c r i p t i o n , although the change i s r e l a t i v e l y s m a l l (Mul l e r et a l . , 1 9 8 3 ) . Developmental r e g u l a t i o n can a l s o be seen i n the ex p r e s s i o n of the D i c t y o s t e l i u m discoideum  p 2 3 r a s ( R e y m o n d et a l . , 1 9 8 4 ; Pawson et a l . , 1 9 8 5 ) . I t i s important to note that the ras genes are expressed i n both d i v i d i n g and n o n d i v i d i n g c e l l s (Segal and S h i l o , 1 9 8 6 ) , although e x p r e s s i o n appears to be i n c r e a s e d i n c e l l s s t i m u l a t e d to d i v i d e . This widespread exp r e s s i o n i m p l i e s a fundamental r o l e f o r the ras gene products i n the c o n t r o l of c e l l growth and d i f f e r e n t i a t i o n . The ras genes were f i r s t d e s c r i b e d as the transfo r m i n g genes of murine r e t r o v i r u s e s . The v i r u s e s were i n i t i a l l y i s o l a t e d as the transfo r m i n g f i l t r a t e from s o l i d tumours induced by murine leukemia v i r u s e s (Harvey, 1 9 6 4 ; K i r s t e n and Mayer, 1 9 6 7 ) . The two v i r u s e s that were d e r i v e d from t h i s process, Harvey murine sarcoma v i r u s (Ha-MSV) and K i r s t e n murine sarcoma v i r u s (Ki-MSV), both cause a wide spectrum of tumours i n c l u d i n g carcinomas, sarcomas and 12 leukemias. Both v i r u s e s c o n t a i n a unique but r e l a t e d gene that i s d e r i v e d from the c e l l u l a r Harvey (c-Ha-) ras or K i r s t e n ( c - K i - ) ras genes ( E l l i s et a l . , 1980; Andersen et a l . , 1981 ) . These two genes have a t t r a c t e d c o n s i d e r a b l e a t t e n t i o n because they were the f i r s t oncogenes to be d i r e c t l y a s s o c i a t e d with human tumours. The genomes of human tumours and of c e l l l i n e s d e r i v e d from human tumours were examined f o r a c t i v e l y 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 i n t o NIH 3T3 c e l l s and s e l e c t i o n f o r transformed c e l l s which contained human DNA. The genes that were d e r i v e d from the i n i t i a l experiments that used t h i s technique were a c t i v a t e d forms of the c-ras genes (Der et a l . , 1982; Goldfarb et a l . , 1982; P u l c i a n i et a l . , 1982; Shimizu e_t a_l. , 1983; Tabin et a l . , 1982). I t i s of p a r t i c u l a r importance that the ras genes i s o l a t e d contained s p e c i f i c p o i n t mutations, and t r a n s f e c t i o n experiments using the p u r i f i e d gene i n d i c a t e d that t h i s change was r e s p o n s i b l e f o r the ras genes being d e t e c t a b l e i n the t r a n s f e c t i o n assay (Seeburg et a l . , 1984). This evidence provided the f i r s t d i r e c t l i n k between a c-onc and human cancer. F u r t h e r evidence f o r the involvement of the ras genes i n tumourigenesis has been d e r i v e d from a n a l y s i s of tumours and papillomas induced by treatment of rodents with c a r c i n o g e n s . Treatment of rodents with some mutagenic chemicals that can act as carcin o g e n s , appears to r e s u l t i n an a c t i v a t i n g mutation i n one of the ras genes i n the c e l l s 13 that form the tumour (Balmain and P r a g n e l l , 1 9 8 3 ; Sukumar et a l . , 1 9 8 6 ; Z a r b l g_t _ a l . , 1 9 8 5 ) . The mutagenic chemicals act as i n i t i a t o r s of oncogenesis and can produce p r e n e o p l a s t i c c e l l s which form papillomas on the mouse s k i n . I s o l a t i o n of DNA from these papillomas and a n a l y s i s by t r a n s f e c t i o n i n t o NIH 3T3 c e l l s has i n d i c a t e d that the i n i t i a t e d , p r e n e o p l a s t i c p a pilloma c e l l s a l s o c o n t a i n a c t i v a t e d ras genes (Balmain and P r a g n e l l , 1 9 8 3 ; Balmain et_ a_l . , 1984; Q u i n t a n i l l a e_t a l . , 1 9 8 6 ) . The a c t i v a t i n g mutations appear to correspond to the types of changes that would be expected of the chemicals used, and appear to be e a r l y events. The mutations occur i n very l i m i t e d areas of the Ha-ras gene changing e i t h e r amino a c i d 12 or 61 ( Z a r b l e_t a_l., 1 985 ; Q u i n t a n i l l a et a_l . , 1 9 8 6 ) , the two regions of the ras genes p r e v i o u s l y i m p l i c a t e d i n oncogenic a c t i v a t i o n (Fasano et  a l . , 1 9 8 4 ) . The c l o s e l i n k of tumourigenesis and a c t i v a t i o n of Ha-ras supports the i m p l i e d r o l e of ras genes i n c a r c i n o g e n e s i s In v i v o . The work a l s o i m p l i e s that the ras genes act i n tumour i n i t i a t i o n (Barbacid e_t al_. , 1 9 8 6 ) . Oncogenic a c t i v a t i o n of c-ras genes can a l s o occur by a m p l i f i c a t i o n of the normal c e l l u l a r genes r a t h e r than by an a c t i v a t i n g l e s i o n i n the coding r e g i o n of the genes. I t was f i r s t demonstrated that o v e r e x p r e s s i o n of the normal c-Ha-ras a l l e l e could transform NIH 3T3 c e l l s (Papageorge et a l . , 1 9 8 2 ) . For t h i s to occur the normal ras gene had to be expressed at l e v e l s approximately ten f o l d higher than an o n c o g e n i c a l l y a c t i v a t e d gene. Intermediate l e v e l s of 14 e x p r e s s i o n w i l l r e s u l t i n in t e r m e d i a t e l e v e l s of tr a n s f o r m a t i o n (Winter and Perucho, 1986). In the Y-1 a d r e n o c o r t i c a l tumour c e l l l i n e the c - K i - r a s a l l e l e i s a m p l i f i e d and overexpressed, but the a m p l i f i e d a l l e l e does not appear to be mutated (Schwab e_t a l . , 1983; George et a l . , 1985). In some c e l l l i n e s d e r i v e d from tumours i t appears that one of the ras a l l e l e s i s a m p l i f i e d and a l s o c o n t a i n s an a c t i v a t i n g mutation ( P u l c i a n i e_t a l . , 1985; Winter e_t a_l. , 1 985 ). These r e s u l t s i n d i c a t e another p o t e n t i a l mechanism by which the ras proto-oncogene can be a c t i v a t e d . The a c t i v a t i n g mutations of ras are of great i n t e r e s t because they are concentrated w i t h i n two regions of genes i s o l a t e d from tumours i n v i v o . The s p e c i f i c i t y of the mutations sparked c o n s i d e r a b l e i n t e r e s t as to t h e i r e f f e c t on ras a c t i v i t y . The bi n d i n g of GTP was re m i n i s c e n t of GTP bin d i n g r e g u l a t o r y p r o t e i n s , the G p r o t e i n s , t r a n s d u c i n and EF-Tu. As sequence i n f o r m a t i o n on the other GTP bi n d i n g r e g u l a t o r y 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 to demonstrate some sequence homology between p 2 1 r a s , EF-Tu and tr a n s d u c i n (Jurnak, 1985; Leberman and Egner, 1984; L o c h r i e et a l . , 1985 ; Tanabe e_t a l . , 1985). I t i s p a r t i c u l a r l y i n t e r e s t i n g that the three areas of homology span the three r e g i o n s s u s c e p t i b l e to a c t i v a t i n g mutations i n ras i n d i c a t i n g that the areas of homology are a l s o areas of f u n c t i o n a l importance (McCormick et a l . , 1985). 15 The a b i l i t y of p 2 1 r a s to hydrolyze GTP was demonstrated by using p r o t e i n expressed i n b a c t e r i a to provide the p r o t e i n i n a background f r e e of contaminating a c t i v i t i e s (McGrath et j Q . , 1984), and evidence from a v a r i e t y of other sources has confirmed the presence of t h i s a c t i v i t y i n ras p r o t e i n s s y n t h e s i z e d i n mammalian c e l l s (Gibbs et a l . , 1984; Manne et a_l. , 1985). These r e s u l t s made i t probable that the ras products moved through a c y c l e of a c t i v a t i o n -i n a c t i v a t i o n as GTP was bound and h y d r o l y z e d . Examination of the o n c o g e n i c a l l y a c t i v a t e d ras gene products i n d i c a t e d that they had a reduced a b i l i t y to hydrolyze GTP (McGrath ejb a l . , 1984 ; Gibbs ejt a l . , 1984 Manne et a l . , 1985 ; Der et. a l . , 1986), although t h e i r b i n d i n g a f f i n i t y f o r n u c l e o t i d e s and other p h y s i c a l p r o p e r t i e s were not s i g n i f i c a n t l y a l t e r e d (Der et a_l. , 1 986 ). The r e d u c t i o n i n GTPase a c t i v i t y has been shown f o r mutants at both codon 12 and codon 61 (McGrath e_t a l . , 1984; Der et_ a l . , 1986). This would imply that the r e d u c t i o n i n GTP h y d r o l y s i s could hold p 2 1 r a s i n the a c t i v a t e d conformation longer, s t i m u l a t i n g the normal r e g u l a t o r y pathway that i n v o l v e d ras p r o t e i n s . A n a l y s i s of a broad range of mutants at codon 61 has i n d i c a t e d that there i s no simple c o r r e l a t i o n between the change i n r a t e of GTP h y d r o l y s i s and the a b i l i t y of a mutant to transform NIH 3T3 c e l l s (Der et a_l. , 1 986 ). Although a s i m i l a r a n a l y s i s has not been done f o r codon 12 mutants i t seems l i k e l y t h a t s i m i l a r r e s u l t s may hold (Seeburg et a l . , 1984). The i m p l i c a t i o n s of these r e s u l t s are not c l e a r but 16 they seem to i n d i c a t e that the a l t e r a t i o n i n r a t e of GTP h y d r o l y s i s may not be a d i r e c t e f f e c t of the mutations. I t has been suggested that the a c t i v a t i n g mutations may a f f e c t p 2 1 r a s conformation and d i r e c t l y a f f e c t i t s a b i l i t y to i n t e r a c t with the p r o t e i n s that i t r e g u l a t e s , and t h i s i s r e f l e c t e d i n the change i n r a t e of GTP h y d r o l y s i s ( S r i v a s t a et a l . , 1985) . The ras genes are present i n a l l s p e c i e s that have been examined ranging from yeast (DeFeo-Jones e_t a l . , 1983; Fukui and K a z i r o , 1985; Powers et a l . , 1984) to sea s q u i r t (Madaule and A x e l , 1985) to man. The genes appear to be h i g h l y conserved, and i t i s p o s s i b l e f o r the mammalian p 2 1 r a s p r o t e i n s to f u n c t i o n a l l y s u b s t i t u t e f o r the normal yeast RAS gene products (DeFeo-Jones et a l . , 1985; Kataoka et a l . , 1985). I t has been demonstrated that mutations i n the yeast RAS genes that correspond to the a c t i v a t i n g mutations i n mammalian ras genes a l s o have s i g n i f i c a n t e f f e c t s on growth r e g u l a t i o n i n yeast (Kataoka et a_l. , 1984). I t has been demonstrated i n S_. c e r e v i s i a e that the RAS products modulate adenylate c y c l a s e a c t i v i t y (Toda e_t a l . , 1985) and so correspond to the mammalian G p r o t e i n s f u n c t i o n a l l y , but i t appears that the mammalian and pombe ras p r o t e i n s are not able to s t i m u l a t e adenylate c y c l a s e a c t i v i t y (Beckner e_t a l . , 1985 ). This r e s u l t i s i n t e r e s t i n g as i t i m p l i e s that although there has been c o n s i d e r a b l e f u n c t i o n a l and s t r u c t u r a l c o n s e r v a t i o n between the yeast RAS 17 and mammalian ras products, the r e g u l a t o r y pathways i n which they are a c t i v e have d i v e r g e d . I t has emerged that the ras gene f a m i l y i s a subgroup of a supergene f a m i l y . Apart from the G p r o t e i n s , t r a n s d u c i n and EF-Tu, t h e r e appear to be other genes both i n yeast ( G a l l w i t z et a l . , 19 8 3 ) and mammals (Madaule and Axel, 1985) that are r e l a t e d to the ras f a m i l y of genes but a p p a r e n t l y r e p r e s e n t d i s t i n c t new gene f a m i l i e s . Whether these genes and t h e i r products w i l l i n t e r a c t with c e l l growth at as fundamental a l e v e l as the ras products remains to be seen, but the p o t e n t i a l l e v e l of complexity of c e l l u l a r r e g u l a t i o n by GTP b i n d i n g p r o t e i n s i s q u i t e high. The mechanism(s) by which the a c t i v a t e d ras gene products are a b l e to transform c e l l s i s u n c l e a r . Although some of the a c t i v a t i n g changes i n ras have been analyzed at a b i o c h e m i c a l l e v e l , the lack of knowledge of p r o t e i n s with r* 3. s which p21 i n t e r a c t s has prevented any r e a l understanding of the mechanisms i n v o l v e d i n ras mediated t r a n s f o r m a t i o n . The transformed phenotype induced by the ras oncogenes appears to be the r e s u l t of complex b i o c h e m i c a l changes (Devouge et a l . , 1982). One of the changes that has been s t u d i e d i n some d e t a i l i s the i n d u c t i o n of t r a n s f o r m i n g growth f a c t o r s i n response to ras t r a n s f o r m a t i o n . The e a r l y r e a l i z a t i o n that t r a n s f o r m a t i o n by some of the a c u t e l y oncogenic r e t r o v i r u s e s r e s u l t e d i n the a b i l i t y of c e l l s to grow with a reduced serum supplement led to the proposal that transformed c e l l s could support t h e i r own 18 growth by an a u t o c r i n e process (Sporn and Todaro, 1 9 8 0 ) . I t was p o s t u l a t e d t h a t transformed c e l l s could produce f a c t o r s r e q u i r e d to s t i m u l a t e t h e i r own growth, o b v i a t i n g the need f o r exogenous s i g n a l s . I t i s p o s s i b l e to t r a n s i e n t l y induce a transformed phenotype i n normal c e l l s by t r e a t i n g them with medium c o n d i t i o n e d by transformed c e l l s (Ozanne et a l . , 1 9 8 0 ; Kaplan et a l . , 1 9 8 2 ) . The p r o d u c t i o n of t r a n s f o r m i n g growth f a c t o r s (TGF's) i s not s p e c i f i c f o r ras t r a n s f o r m a t i o n having been i n i t i a l l y d e s c r i b e d f o r c e l l s transformed by v-mos (DeLarco and Todaro, 1 9 7 8 ) . The components of c o n d i t i o n e d medium from v i r a l l y transformed c e l l s that were a c t i v e i n s t i m u l a t i o n of the growth 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 of normal c e l l s have been p u r i f i e d (Anzano et a_l . , 19 8 3) and sequenced (Marquardt et a l . , 1 9 8 4 ) . There are two p o l y p e p t i d e s i n v o l v e d which i n t e r a c t to produce the f u l l y transformed phenotype. TGF-oC i s r e l a t e d to EGF and appears to act through the EGF r e c e p t o r (Todaro e_t _ a l . , 1 9 8 0 ; Carpenter et a l . , 1 9 8 3 ; DeLarco and Todaro, 1 9 8 2 ) and i t i s a b l e to induce aspects of the transformed phenotype i n normal c e l l s when added i n the absence of other growth f a c t o r s (Derynck et a l . , 1 9 8 4 ) . Transformation can be induced i n c e l l s by expression of the TGF-oi polyp e p t i d e p r e c u r s o r (Rosenthal et a l . , 1 9 8 6 ). The other component, TGF-/3 , o f t e n acts to i n h i b i t growth when added to normal c e l l s alone (Masui _et a_l . , 1 9 8 6 ) but i s a b l e to modulate the number of EGF r e c e p t o r s on the t r e a t e d c e l l s and enhance the a c t i v i t y of TGF-c< (Assoian et a l . , 1 9 8 4 ; 19 Massague, 1985). I t has proven to be p o s s i b l e f o r EGF to s u b s t i t u t e f o r TGF-^/ i n i n d u c i n g t r a n s f o r m a t i o n and s t i m u l a t i n g c e l l growth (Assoian et a l . , 1984). Expression of a c t i v a t e d ras genes can a l s o modify the d i f f e r e n t i a t e d phenotype of many c e l l types. The i n t r o d u c t i o n of ras genes i n t o the pheochromocytoma l i n e PC12 r e s u l t s i n the i n d u c t i o n of a more d i f f e r e n t i a t e d phenotype (Bar-Sagi and Feramisco, 1 985 ; Noda et a l . , 1985). The PC12 l i n e i s neuroectodermal i n o r i g i n and as the c e l l s d i f f e r e n t i a t e , even i n response to a c t i v a t e d forms of the ras genes, they express many aspects of the d i f f e r e n t i a t e d neuronal phenotype i n c l u d i n g c e l l c y c l e a r r e s t . In other c e l l types examined the i n t r o d u c t i o n of the a c t i v a t e d ras genes r e s u l t s i n the l o s s of aspects of the normal d i f f e r e n t i a t e d phenotype. This can be seen i n r a t adr e n a l cortex c e l l s transformed by Ki-MSV, which express enzymatic a c t i v i t i e s normally a s s o c i a t e d with embryonic c e l l s and are not a s s o c i a t e d with normal c e l l s d e r i v e d from the a d u l t cortex (Wiebe et, a l . , i n p r e s s ) . In k e r a t i n o c y tes d e r i v e d from the mouse s k i n , i n f e c t i o n by ras c o n t a i n i n g v i r u s e s a l t e r s the response to inducers of d i f f e r e n t i a t i o n (Yuspa et_ a l . , 1985). The u n i n f e c t e d k e r a t i n o c y t e s respond to an i n c r e a s e d c alcium c o n c e n t r a t i o n by e n t e r i n g growth a r r e s t and respond to TPA by e x p r e s s i n g two genes normally a s s o c i a t e d with the f u l l y d i f f e r e n t i a t e d phenotype. When i n f e c t e d with Ha-MSV or Ki-MSV the c e l l s a r r e s t e d i n high calcium respond to the tumour promoter by r e g r e s s i o n to a l e s s d i f f e r e n t i a t e d phenotype. These r e s u l t s i n d i c a t e that e x p r e s s i o n of oncogenic ras genes can modulate the d i f f e r e n t i a t e d phenotype of c e l l s , as w e l l as the a b i l i t y of c e l l s to r e p l i c a t e (Feramisco et a_l. , 1 9 8 4 ; Mulcahy et a l . , 1985; Papageorge e_t a_l., 1 986 ). I t i s a l s o c l e a r l y i n d i c a t e d that the ras products are able to modify c e l l u l a r responses to exogenous s i g n a l s or to obvia t e the need f o r such s i g n a l s . 1 . 3 . 4 Myc The myc oncogene was o r i g i n a l l y d e s c r i b e d as the transform i n g gene of the avian myelocytomatosis v i r u s , MC29 (Mellon e_t a_l., 1978; Vennstrom et a l . , 1981). The MC29 v i r u s causes leukemias and sarcomas and can transform both f i b r o b l a s t i c and hematopoietic avian c e l l s i n v i t r o (Ramsay et a_l. , 1980; E n r i e t t o and Hayman, 1982). Mutations mapped to the myc gene that a f f e c t the m o b i l i t y of the myc product i n SDS-PAGE a l s o a l t e r the a b i l i t y of the v i r u s to transform c e l l s i n v i t r o (Ramsay et a l . , 1980; E n r i e t t o and Hayman, 1982). This d i r e c t l y i m p l i c a t e s the myc gene i n v i r a l l y induced t r a n s f o r m a t i o n . A c t i v a t i o n of the avian c-myc gene has been a s s o c i a t e d with the i n d u c t i o n of b u r s a l lymphomas i n the chicken by avian l e u k o s i s v i r u s . The l e u k o s i s v i r u s i n t e g r a t e s near the c-myc gene and the presence of the v i r a l enhancer appears to modulate t r a n s c r i p t i o n of the c-myc gene (Hay ward et a l . , 1981; Neel et al_. , 19 82; Payne et a l . , 1982; Fung et a l . , 1982) . A s w i t h o t h e r o n c o g e n e s f o u n d i n a c u t e l y t r a n s f o r m i n g r e t r o v i r u s e s , t h e v - m y c g e n e i s d e r i v e d f r o m a c e l l u l a r g e n e t h a t i s h i g h l y c o n s e r v e d t h r o u g h o u t t h e a n i m a l k i n g d o m ( V e n n s t r o m e t a _ l . , 1 9 8 2 ; C r e w s e t _ a l . , 1 9 8 2 ) . T h e m y c g e n e s a l s o a p p e a r t o c o n s t i t u t e a g e n e f a m i l y o f w h i c h t h e r e a r e now t h r e e m e m b e r s c - m y c , N - m y c ( S c h w a b e t a l . , 1 9 8 3 ; L e e e t a l . , 1 9 8 4 ) a n d L - m y c ( N a u e t a l . , 1 9 8 5 ) . A m p l i f i c a t i o n a n d o v e r e x p r e s s i o n o f t h e c - , N - a n d L - m y c g e n e s h a v e b e e n a s s o c i a t e d w i t h h u m a n a n d a n i m a l t u m o u r s . I n m o u s e p l a s m a c y t o m a s t h e r e i s a s t r o n g a s s o c i a t i o n o f t h e c h r o m o s o m a l r e a r r a n g e m e n t s i n v o l v i n g c - m y c a n d i m m u n o g l o b u l i n l o c i w i t h f o r m a t i o n o f t h e t u m o u r s ( C r e w s e t a l . , 1 9 8 2 ; S h e n - o n g e_t a l _ . , 1 9 8 2 ; T a u b e t a l . , 1 9 8 2 ) . I n b o t h t h e a n i m a l a n d h u m a n t u m o u r s t h e c h r o m o s o m a l r e a r r a n g e m e n t s a r e t h o u g h t t o a l t e r t h e n o r m a l r e g u l a t i o n o f t h e c - m y c g e n e r e s u l t i n g i n i n c r e a s e d o r i n a p p r o p r i a t e e x p r e s s i o n . T h e N - m y c g e n e h a s b e e n s h o w n t o b e a m p l i f i e d i n a n u m b e r o f t u m o u r d e r i v e d c e l l l i n e s . * ( L e e e t a l . , 1 9 8 4 ; N a u e_t a l . , 1 9 8 6 ) a n d c a n a p p a r e n t l y c o o p e r a t e w i t h o t h e r o n c o g e n e s i n t h e t r a n s f o r m a t i o n o f p r i m a r y c e l l c u l t u r e s ( S c h w a b e_t a _ l . , 1 9 8 5 ) . A m p l i f i c a t i o n o f t h e L - m y c g e n e i s a s s o c i a t e d w i t h l u n g c a r c i n o m a s ( N a u et a _ l . , 1 9 8 5 ) a n d a m p l i f i c a t i o n a p p e a r s t o i n c r e a s e w h e n t h e c e l l s b e c o m e h i g h l y t r a n s f o r m e d a n d m e t a s t a t i c . I t a p p e a r s t h a t o v e r e x p r e s s i o n o r m i s r e g u l a t i o n o f t h e m y c g e n e s i s l a r g e l y r e s p o n s i b l e f o r t h e i r i n v o l v e m e n t i n t r a n s f o r m a t i o n . T h e t r a n s c r i p t a n d p r o t e i n p r o d u c t s o f t h e n o r m a l c-myc gene have a p p r o x i m a t e l y 20 m i n u t e h a l f - l i v e s i n n o r m a l c e l l g r o w t h i n v i t r o ( E i s e n m a n e t a l . , 1985). The l e v e l o f gene e x p r e s s i o n a p p e a r s t o be r e g u l a t e d a t b o t h t h e t r a n s c r i p t i o n a l and p o s t t r a n s c r i p t i o n a l l e v e l . The n o r m a l gene a p p e a r s t o be n e g a t i v e l y r e g u l a t e d and t o c o n t a i n a t r a n s c r i p t i o n t e r m i n a t o r t h a t can be s u p p r e s s e d ( B e n t l e y and G r o u d i n e , 1986; Remmers e t a l . , 1 9 86). I t a l s o a p p e a r s t h a t t h e r a t e o f d e g r a d a t i o n o f t h e c-myc mRNA can v a r y i n r e s p o n s e t o e n v i r o n m e n t a l c h a n ges ( B l a n c h a r d e t a l . , 1985; Dony e_t a l . , 1985; P i e c h a c z y k e t a l . , 1985). Normal myc r e g u l a t i o n can be p e r t u r b e d by t h e chromosomal r e a r r a n g e m e n t s a s s o c i a t e d w i t h t r a n s f o r m a t i o n (Schwab e t a l . , 1986; Yang e t a l . , 1 985 ). E x p r e s s i o n o f the n o r m a l c-myc gene a p p e a r s t o be r e l a t e d t o c e l l g r o w t h . S t i m u l a t i o n o f g r o w t h a r r e s t e d c e l l s by a v a r i e t y o f a g e n t s i n c l u d i n g g r o w t h f a c t o r s r e s u l t s i n t h e s t i m u l a t i o n o f c-myc e x p r e s s i o n ( G r e e n b e r g and Z i f f , 1984; B l a n c h a r d e_t a l . , 1985; M u l l e r e t a l . , 1984). T h i s s t i m u l a t i o n does n o t a p p e a r t o be r e q u i r e d f o r c e l l s t o t r a v e r s e t h e c e l l c y c l e as a n a l y s i s o f s y n c h r o n i z e d g r o w i n g c e l l s showed no change i n c-myc e x p r e s s i o n d u r i n g t h e c e l l c y c l e (Thompson e t a l . , 1985; Hann e t a l . , 1 9 85). The o v e r e x p r e s s i o n o f c-myc a p p e a r s t o e n a b l e the c e l l s t o grow t o h i g h e r d e n s i t i e s , a l t h o u g h t h e g r o w t h r a t e does n o t change s i g n i f i c a n t l y from t h e p a r e n t a l l i n e ( K e a t h e t a l . , 1 984 ) . The c e l l l i n e s transformed by c-myc demonstrate a tumourigenic phenotype and form c o l o n i e s i n s o f t agar at high e f f i c i e n c y (Keath et a l . , 1 9 8 4 ; Vennstrom e_t a l . , 1 9 8 4 ) . The tumourigenic c o n v e r s i o n of c e l l l i n e s appears to r e q u i r e only o v e r e x p r e s s i o n of the normal myc genes (Lee et a l . , 1 9 8 5 ) . I t i s not c l e a r i f any changes i n the myc coding sequence are s u f f i c i e n t to induce t r a n s f o r m a t i o n i n the absence of ov e r e x p r e s s i o n (Patschinsky et a l . , 1 9 8 6 ; Stanton _et _ a l . , 1 9 8 4 ) . The t r a n s f o r m a t i o n induced by c-myc o v e r e x p r e s s i o n r e s u l t s i n some s p e c i f i c phenotypic a l t e r a t i o n s i n the c e l l l i n e s used. The e f f e c t of c-myc i n the NIH 3 T 3 c e l l l i n e appears to be a decreased need f o r PDGF (Armelin et a l . , 1 9 8 4 ) . The i n t r o d u c t i o n of an exogenous c-myc i n NRK c e l l s i n c r e a s e s the s e n s i t i v i t y of the c e l l s to the e f f e c t s of exogenous growth f a c t o r s (Stern ejb a l . , 1 9 8 6 ) . The NRK c e l l s e x p r e s s i n g the t r a n s f e c t e d c-myc gene respond to the a d d i t i o n of r e l a t i v e l y low c o n c e n t r a t i o n s of EGF by anchorage independent growth. Abrogation of growth f a c t o r requirements can a l s o be seen i n avian and murine hematopoietic and mesenchymal c e l l s i n response to the i n t r o d u c t i o n of oncogenic myc c o n s t r u c t s (Balk et a l . , 1 9 8 5 ; Rapp et a l . , 1 9 8 5 ) . The r e s u l t s d i r e c t l y i m p l i c a t e the myc genes i n growth f a c t o r responsiveness of c e l l s . The r e l a t i v e l y s u b t l e morphological changes that r e s u l t from the i n t r o d u c t i o n of a myc oncogene i n t o r a t embryo f i b r o b l a s t c e l l s can be enhanced by treatment of the c e l l s with the 24 tumour promoter 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) (Connan et a l . , 1985). I t would appear that the products of the c-myc genes can r e s u l t i n profound changes i n the response of c e l l s to e x t e r n a l s i g n a l s , e i t h e r modifying the response or enhancing the a b i l i t y of the c e l l to respond. The products of the avian c-myc genes have Mr's of 57,000 and 59,000 and the murine c-myc products have Mr's of 62,000 and 64,000 (Hann e_t _ a l . , 19 83) . The products are l o c a l i z e d i n the nucleus. The i n i t i a l r e p o r t s on f r a c t i o n a t i o n of the nucleus had i n d i c a t e d that the c-myc products were a s s o c i a t e d with the n u c l e a r matrix (Donner et a l . , 1982; Eisenman et a l . , 1985), but f u r t h e r work has i n d i c a t e d that t h i s might have been an a r t e f a c t of p r e p a r a t i o n (Evan and Hancock, 1985). The myc genes appear to be expressed or i n d u c i b l e i n most c e l l types, implying that the products provide f u n c t i o n s necessary f o r most, i f not a l l c e l l s . Although e x p r e s s i o n of the myc genes i s a s s o c i a t e d with mitogenesis (Goustin et a^. , 1985), i t i s not s u f f i c i e n t to induce c e l l d i v i s i o n (Smeland et a_l. , 1985; Lacy et a_l. , 1 986 ). The e x p r e s s i o n or overexpression of myc genes i s not incompatible with a h i g h l y d i f f e r e n t i a t e d , s t a t i o n a r y c e l l phenotype although there do appear to be some phenotypic changes as the r e s u l t of the i n t r o d u c t i o n of an oncogenic myc (Endo and Nadal-Ginard, 1 986 ; Symonds et _ a l . , 1 986 ) . 25 1 . 3.5 Src The s r c gene i s the tr a n s f o r m i n g gene of Rous sarcoma v i r u s (RSV). RSV was the f i r s t a c u t e l y oncogenic r e t r o v i r u s to be i s o l a t e d (Rous, 1911) and i s the only n a t u r a l l y o c c u r r i n g , a c u t e l y oncogenic r e t r o v i r u s that i s r e p l i c a t i o n competent ( i n Weiss et a l . , 1985). Genetic mapping of c o n d i t i o n a l and n o n c o n d i t i o n a l mutants of the RSV transfo r m i n g f u n c t i o n i d e n t i f i e d the unique gene r e s p o n s i b l e f o r t r a n s f o r m a t i o n and demonstrated that i t was not an e s s e n t i a l v i r a l f u n c t i o n ( M a r t i n , 1970). H y b r i d i z a t i o n s t u d i e s i n d i c a t e d that RSV contained a sequence that h y b r i d i z e d to the c e l l u l a r genome while p o o r l y oncogenic r e t r o v i r u s e s r e l a t e d to RSV d i d not. The s r c gene that encodes the RSV transforming f u n c t i o n appears to be d i r e c t l y d e r i v e d from the normal c e l l u l a r gene c - s r c , although there are a number of changes i n the primary sequence that appear to be r e l a t e d to v i r a l o n c o g e n i c i t y (Takeya and Hanafusa, 1 9 8 3 ; Iba et a_l. , 1984; Coussens et a l . , 1985; Johnson et a l . , 1985). The product of the s r c gene, p 6 0 s r c has an Mr of 60,000 and i s t i g h t l y a s s o c i a t e d with the cyt o p l a s m i c membrane (Sefton et aj^. , 1984). The p 6 0 s r c i s a phosphoprotein i n which the phosphate i s l i n k e d to the hydroxyl group of t y r o s i n e , a r e l a t i v e l y r a r e c e l l u l a r m o d i f i c a t i o n (Hunter and Se f t o n , 1 980 ; C o l l e t t ejb a l . , 1980). p 6 0 s r c has been shown to c o n t a i n an i n t r i n s i c a b i l i t y to phosphorylate t y r o s i n e r e s i d u e s i n s p e c i f i c 26 sequences and c e l l u l a r s u b s t r a t e s ( P a t s c h i n s k y et a l . , 1982; Cooper e_t _ a l . , 1 9 8 4 ; C o l l e t t and E r i k s o n , 1982) and to autophosphorylate under some c o n d i t i o n s both i n v i v o and i n  v i t r o ( f o r a review see Hunter and Cooper, 1 9 8 5 ) . A number of other oncogenes have been d e s c r i b e d that show c o n s i d e r a b l e homology to s r c . The products of these genes a l s o d i s p l a y an i n t r i n s i c t y r o s i n e kinase a c t i v i t y . These genes appear to rep r e s e n t a s u p e r f a m i l y of r e g u l a t o r y genes encoding t y r o s i n e k i n a s e s . The a b i l i t y to phosphorylate t y r o s i n e i s a l s o a s s o c i a t e d with many of the growth f a c t o r r e c e p t o r s and appears to be i n v o l v e d i n s i g n a l t r a n s d u c t i o n and mitogenesis. T y r o s i n e p h o s p h o r y l a t i o n of the v i r a l fps product, P130 (Weinmaster et a l . , 1 9 8 4 ) , the EGF r e c e p t o r (Hunter and Cooper, 1981) or i n s u l i n r e c e p t o r (Rosen et _ a l . , 1 9 8 3 ; P e t r u z e l l i et a_l . , 1 9 8 4 ; Stadtmauer and Rosen, 1983) i s a s s o c i a t e d with enhancement of t h e i r t y r o s i n e kinase a c t i v i t y and e f f e c t s on c e l l phenotype. P h o s p h o r y l a t i o n at the major i n vi v o t y r o s i n e acceptor s i t e of p 6 0 c - s r c has been shown to i n h i b i t i t s t y r o s i n e kinase a c t i v i t y i n v i t r o (Courtneidge, 1985; Bolen et a l . , 1 9 8 4 ) and d e p h o s p h o r y l a t i o n of t h i s s i t e i s a s s o c i a t e d with oncogenic a c t i v a t i o n of the c - s r c product by polyoma middle T a n t i g e n . There has been l i t t l e evidence presented to l i n k a c t i v a t i o n of the endogenous c - s r c gene with mammalian tumours, although there i s some evidence of ove r e x p r e s s i o n i n two tumours (reviewed i n Slamon et a l . , 1984 ; Delorbe e_t a l . , 1980). Other members of the f a m i l y of genes r e l a t e d to c - s r c show a c t i v a t i n g changes r e l a t e d to the appearance of tumours ( C a s n e l l i e e_t a l . , 1 983 ; Voronova e_t a l . , 1985). The i n d u c t i o n of tumours by RSV appears to be somewhat respons i v e to e x t e r n a l c o n d i t i o n s ( B i s s e l l et a l . , 1979; Dolberg et a l . , 1 9 8 4 ) implying that t r a n s f o r m a t i o n by s r c i n v i v o can r e q u i r e a d d i t i o n a l s t e p s . T h i s phenomenon has a l s o been demonstrated i n a mammalian system (Gilmer et a l . , 1985) and would appear to be c o r r o b o r a t e d by the m u l t i s t e p nature of t r a n s f o r m a t i o n induced by polyoma middle T (Rassoullzadegan et a l . , 1982; Land et al_. , 1983a). The e f f e c t s of the mutations i n v - s r c appear to be r e l a t i v e l y complex. The o v e r e x p r e s s i o n of c - s r c can lead to the e x p r e s s i o n of a p a r t i a l l y transformed phenotype, but even i f the i n c r e a s e i n c e l l u l a r phosphotyrosine content i s n e a r l y as great as that seen i n v - s r c transformed c e l l s the c - s r c remains unable to induce a f u l l y transformed phenotype (Iba et a l . , 1985a; Johnson et al., 1985). These r e s u l t s imply that p 6 0 v ~ s r c i s able to phosphorylate c e r t a i n key s u b s t r a t e s i n v o l v e d i n t r a n s f o r m a t i o n more e f f i c i e n t l y than p 6 0 c " s r c . There i s some evidence that p 6 0 v _ s r c does phosphorylate a d i f f e r e n t spectrum of s u b s t r a t e s (Johnson et a l . , 1985). These r e s u l t s imply that the a c t i v a t i n g mutations i n v - s r c a l t e r the s u b s t r a t e s p e c i f i c i t y of p 6 0 s r c as w e l l as enhancing the enzymatic a c t i v i t y . The e x p r e s s i o n of high l e v e l s of p 6 0 s r c a c t i v i t y i s not incompatible with a s t a t i o n a r y , h i g h l y d i f f e r e n t i a t e d 28 phenotype. The presence of high l e v e l s of p 6 0 c _ s r c a c t i v i t y has been shown i n some ne u r a l t i s s u e s and i s expressed a f t e r the c e l l s have stopped d i v i d i n g (Brugge §_t al_. , 1 9 8 5 ; F u l t s et a l . , 1985; Iba et a l . , 1 9 8 5 b ) . The i n t r o d u c t i o n of the v - s r c gene i n t o the PC12 c e l l l i n e can induce n e u r i t e formation and e x p r e s s i o n of other n e u r a l f u n c t i o n s i n c l u d i n g the growth a r r e s t normally a s s o c i a t e d with t h i s d i f f e r e n t i a t i o n pathway (Alema et a_l. , 1 9 8 5 a ) . The e f f e c t of v - s r c i n most other systems appears to be l o s s of the normal d i f f e r e n t i a t e d f u n c t i o n s (Adkins et a l . , 1984; Alema et _ a l . , 1 985b). Transformation by v - s r c r e s u l t s i n a s i g n i f i c a n t r e d u c t i o n of the serum requirements f o r c e l l u l a r growth (Adkins e_t a l . , 1984), as i s seen f o r t r a n s f o r m a t i o n by other v i r a l oncogenes. Examination of t h i s process has shown that v - s r c can s t i m u l a t e a complete round of c e l l d i v i s i o n i n the apparent absence of any exogenous mitogenic s i g n a l (Durkin and W h i t f i e l d , 1984). The a b i l i t y of the s r c product to induce e i t h e r c e l l d i v i s i o n and t r a n s f o r m a t i o n or be i n v o l v e d i n t e r m i n a l d i f f e r e n t i a t i o n i n d i c a t e s a wide spectrum of p o t e n t i a l e f f e c t s . Whether the r e s u l t a n t phenotype i s determined by the s u b s t r a t e s phosphorylated or by changes i n the pathways a f f e c t e d by the s u b s t r a t e s i s not c l e a r . 1.3.6 Raf v - r a f i s the a c t i v e t r a n s f o r m i n g gene of the a c u t e l y oncogenic murine r e t r o v i r u s 3611 (Rapp ejb a l . , 1 983a , 1 9 8 3 b ) . 29 v - r a f i s d e r i v e d from a c e l l u l a r oncogene, c - r a f (Rapp e_t a l . , 1983a). The r a f genes appear to be c l o s e l y r e l a t e d to another of the v i r a l oncogenes, v - m i l , of the avian oncogenic r e t r o v i r u s MH2. The r a f / m i l genes show some homology to the oncogenes that encode the t y r o s i n e k i n a s e s , but do not appear to be t y r o s i n e kinases themselves (Sutrave et a l . , 1984). The r a f / m i l oncogene products appear to act as s e r i n e / t h r e o n i n e kinases and autophosphorylate in_ v i t r o ( M o e l l i n g et a l . , 1984). The c e l l u l a r s u b s t r a t e ( s ) of the r a f / m i l products are not known. Ph o s p h o r y l a t i o n of s e r i n e and threonine comprises almost a l l of the c e l l u l a r p r o t e i n p h o s p h o r y l a t i o n that has been detected and has been c l e a r l y i m p l i c a t e d i n the r e g u l a t i o n of metabolic pathways and gene exp r e s s i o n ( f o r a review see Cohen, 1982) . I t i s of great i n t e r e s t that p h o s p h o r y l a t i o n of s e r i n e and threonine can be a s s o c i a t e d with t r a n s f o r m a t i o n , although the r a f / m i l gene products are not thought to correspond to p r e v i o u s l y d e s c r i b e d s e r i n e / t h r e o n i n e k i n a s e s . The v - r a f / m i l oncogenes are able to induce a wide range of tumours and an a c t i v a t e d form of the c - r a f gene has been a s s o c i a t e d with some human tumours (Fukui et al^. , 1985; Shimizu §_t a_l. , 1 985 ) i n d i c a t i n g that the r a f genes can be i n v o l v e d i n n a t u r a l l y o c c u r r i n g tumours. c - r a f i s widely expressed and the c o n s e r v a t i o n of the r a f / m i l sequences p o i n t s to a fundamental r o l e f o r the products of the genes i n the r e g u l a t i o n of c e l l growth. As with the other oncogenes that have been d e s c r i b e d c - r a f appears to be r e p r e s e n t a t i v e of a l a r g e r gene f a m i l y that a l s o appears to be able to e f f e c t t r a n s f o r m a t i o n of c e l l s ( 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 v i v o and i n v i t r o Models The examination of transformed c e l l s p r o v i d e s a means of probing the g e n e t i c and bio c h e m i c a l b a s i s of the r e g u l a t i o n of growth and d i f f e r e n t i a t i o n . The attempt to d e f i n e the number and nature of the r e g u l a t o r y steps i n c e l l growth has prompted a n a l y s i s of the process of oncogenic t r a n s f o r m a t i o n to e l u c i d a t e the nature of the a c t i v a t i n g changes and t h e i r r e l a t i o n to normal c e l l growth. One of the e a r l i e s t questions to be posed was whether the appearance of tumours r e q u i r e d a s i n g l e or m u l t i p l e changes i n the c e l l s and i f the changes were g e n e t i c or e p i g e n e t i c . Some of the e a r l y l i n e s of evidence had i n d i c a t e d that t r a n s f o r m a t i o n both i n viv o and i n v i t r o could appear to r e q u i r e only a s i n g l e step ( S i g a l et a_l. , 1986, and r e f e r e n c e s t h e r e i n ) . 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 of many e s t a b l i s h e d c e l l l i n e s by r e t r o v i r a l l y borne oncogenes appears to i n v o l v e only a s i n g l e , a c t i v a t i n g change. Evidence d e r i v e d from e p i d e m i o l o g i c a l s t u d i e s i n d i c a t e d that the formation of many tumours was a p r o t r a c t e d process that r e q u i r e d at l e a s t two changes i n the c e l l s . Evidence examining the appearance of B u r k i t t ' s lymphoma ( K l e i n and K l e i n , 1985) and lung cancer (Farber and Cameron, 1980) supported t h i s c o n c l u s i o n . Studies on the i n d u c t i o n of tumours by chemical carcinogens a l s o pointed to a two step pathway of tumourigenesis. I t has proven to be p o s s i b l e to separate c a r c i n o g e n i c chemicals i n t o two groups: i n i t i a t o r s and promoters. The i n i t i a t o r s appear to c r e a t e c e l l s that are capable of tumourigenesis but are i n e f f i c i e n t l y converted to a h i g h l y tumourigenic phenotype. The tumour promoters act to i n c r e a s e the e f f i c i e n c y of tumourigenic c o n v e r s i o n of i n i t i a t e d c e l l s , but are unable to i n i t i a t e tumourigenesis (Hecker et a l . , 1 9 8 2 ) . With the demonstration that c a r c i n o g e n e s i s could appear to be a m u l t i - s t e p process, i t was important to d e f i n e an i n v i t r o system that mirrored t h i s p rocess. I t has been demonstrated that t r a n s f o r m a t i o n of lymphoid c e l l s by Abelson-MLV (Whitlock and Witte, 1 9 8 1 ; Whitlock et a l . , 1 9 8 3 ) or a v a r i e t y of c e l l types by e i t h e r Ki-MSV (Auersperg et _ a l . , 1 9 8 1 ; Auersperg and Calderwood, 1 9 8 4 ) or Ha-MSV (Yuspa e_t a_l. , 1 9 8 5 ) can pass through an i n t e r m e d i a t e , i n c o m p l e t e l y transformed s t a t e and that f u r t h e r c e l l u l a r changes are r e q u i r e d f o r the e x p r e s s i o n of the completely transformed phenotype. Examination of some transformed c e l l l i n e s d e r i v e d from human tumours i n d i c a t e d that more than a s i n g l e c e l l u l a r oncogene could be a l t e r e d and presumably a c t i v a t e d i n a s i n g l e c e l l l i n e (Murray et a_l. , 1 9 8 3 ; Taya et a l . , 1 9 8 4 ) . I t has proven p o s s i b l e to show a requirement f o r more than one oncogene i n the t r a n s f o r m a t i o n of many primary c e l l s (Land et a l . , 1 9 8 3 a , 1 9 8 3 b ; Ruley, 1 9 8 3 ) . Two groups of oncogenes have been d e f i n e d on the b a s i s of t h e i r a b i l i t y to cooperate. These two groups are e x e m p l i f i e d by two c e l l u l a r oncogenes c-myc and c - r a s . The f i r s t group i s composed of p 5 3 , polyoma l a r g e T antigen and adenovirus EIA i n a d d i t i o n to myc. The products of a l l of these genes are l o c a t e d i n the nucleus. The second group c o n t a i n s r a s , r a f and polyoma middle T and a l l of the products of these genes are l o c a t e d i n the cytoplasm. The oncogenes of one group are able to cooperate with the members of the other group but not those of the same group. These r e s u l t s have provided a u s e f u l i n v i t r o model that appears to have strong c o r r e l a t i o n s with c a r c i n o g e n e s i s i n v i v o . I t i s p o s s i b l e f o r an a c t i v a t e d ras gene to transform primary c u l t u r e s i f i t i s overexpressed (Spandidos and W i l k i e , 1 9 8 4 ; Land e_t a_l . , 1 9 8 6 ) , implying that m u l t i p l e changes i n a s i n g l e gene can have a s i m i l a r e f f e c t to the a c t i v a t i o n of m u l t i p l e genes. I t would appear that the requirement f o r a second oncogene can be r e l i e v e d i n part by e l i m i n a t i o n of the background c e l l s not transformed by ras (Land et a l . , 1 9 8 6 ) . The means by which the normal c e l l s can a p p a r e n t l y suppress ras induced t r a n s f o r m a t i o n i n primary c e l l s i s not known, but may be r e l a t e d to c e l l - c e l l communication (Mehta e_t_ j a l . , 1 9 8 6 ) . I t a l s o appears as though i t i s p o s s i b l e f o r two oncogenes whose products are l o c a t e d i n the nucleus to cooperate i n the t r a n s f o r m a t i o n of some c e l l s (Jenuwein et a l . , 1 9 8 5 ) . These r e s u l t s present exceptions to the m a j o r i t y of the o b s e r v a t i o n s on t r a n s f o r m a t i o n of primary c e l l s i n v i t r o , but suggest that the r e g u l a t o r y pathways need not be i d e n t i c a l i n a l l c e l l types and that e x t e r n a l c o n d i t i o n s can profoundly a f f e c t the a b i l i t y of c e l l s to express a transformed phenotype. The oncogenes appear to act as dominant transf o r m i n g elements under many c o n d i t i o n s ( S i g a l et a l . , 1 9 8 6 ) ; however, t r a n s f o r m a t i o n can appear to be a r e c e s s i v e phenomenon. The s t u d i e s of h e r i t a b l e r e t i n o b l a s t o m a (Cavenee et a l . , 1 9 8 3 ; Hansen et a l . , 1 9 8 5 ) and Wilm's tumour (Koufos e_t al_. , 1 9 8 5 ) have i n d i c a t e d that the tumourigenic phenotype i s r e c e s s i v e , although i t i s p o s s i b l e to i s o l a t e a c t i v a t e d ras genes from the tumours. The appearance of the two tumours, r e t i n o b l a s t o m a and Wilm's tumour, i s a s s o c i a t e d with the l o s s of s p e c i f i c p a r t s of the genome, which are presumably i n v o l v e d i n l o s s of a suppressor f u n c t i o n and e x p r e s s i o n of o n c o g e n i c i t y (Cavenee et a l . , 1 9 8 3 ; Godbout et a_l. , 1 9 8 3 ; Hansen et a l . , 1 9 8 5 ; Koufos et al_. , 1 9 8 5 ) . The r e c e s s i v e nature of oncogenesis i s f u r t h e r supported by the suppression of the transformed phenotype i n somatic c e l l h y b rids i n v o l v i n g normal and transformed c e l l s (Guerts van K e s s e l et a_l. , 1 9 8 1 ; K l i n g e r , 1 9 8 0 , 1 9 8 2 , Stanbridge et a l . , 1 9 8 2 ) . The a b i l i t y of the normal c e l l s to suppress the e f f e c t s of a c t i v a t e d oncogenes i n c e l l s that are otherwise capable of being transformed by the oncogene i m p l i e s that at l e a s t part of the a b i l i t y of normal c e l l s to r e s i s t t r a n s f o r m a t i o n may r e s t i n an a b i l i t y to suppress the a c t i v i t y of an oncogene (Knudson, 1985). The i n d u c t i o n of an oncogenic phenotype by ras and myc i n SHE c e l l s i s c o r r e l a t e d with monosomy f o r chromosome 15 (Oshimura et a l . , 1985). This l o s s of g e n e t i c m a t e r i a l i s again a s s o c i a t e d with the e x p r e s s i o n of the transformed phenotype, although the e f f e c t s of the l o s s of suppression are somewhat v a r i a b l e (Thomassen et al_. , 1 985 ). The a b i l i t y of c e r t a i n oncogenes to produce autonomous growth i n many immortalized c e l l l i n e s , but not i n the primary c u l t u r e s from which the permanent l i n e s are d e r i v e d , would a l s o appear to r e f l e c t t h i s suppression of oncogene a c t i v i t y (Auersperg et a l . , 1981; Auersperg and Calderwood, 1984). Tumour t i s s u e s have been e x t e n s i v e l y examined f o r the appearance of a l t e r e d or a c t i v a t e d forms of c e l l u l a r oncogenes (see Slamon et^ al.> 1984 f o r a r e v i e w ) . Tumours de r i v e d from a v a r i e t y of d i f f e r e n t t i s s u e s can c o n t a i n the same a c t i v a t e d oncogene. Oncogenes have been placed under the c o n t r o l of i n d u c i b l e or t i s s u e s p e c i f i c promoters and used to c r e a t e t r a n s g e n i c mice ( B r i n s t e r e_t al_. , 1984; Stewart et a^L. , 1984; Adams et a l . , 1985). In the t r a n s g e n i c mice, tumours form only i n the t i s s u e s i n which the oncogene might be expected to be expressed, demonstrating that some oncogenes are able to cause tumours i n a v a r i e t y of t i s s u e s . The tumours that a r i s e a l s o appear to be c l o n a l , suggesting that complete t r a n s f o r m a t i o n i s 35 i n f r e q u e n t , and s u p p o r t i n g the m u l t i s t e p nature of c a r c i n o g e n e s i s i n v i v o . The b a s i s f o r the c o o p e r a t i o n of two oncogenes i n the t r a n s f o r m a t i o n of primary c e l l s i s s t i l l r a t h e r obscure. The i n t r o d u c t i o n of an overexpressed c-myc i n t o v a r i o u s c e l l l i n e s renders the c e l l s more respo n s i v e to EGF (Stern et a l . , 1 9 8 6 ; Balk et a l . , 1 9 8 5 ) , and a s i m i l a r i n c r e a s e i n s e n s i t i v i t y to TGF-06might a l s o be expected. Since e x p r e s s i o n of ras induces the p r o d u c t i o n of TGF-cC and TGF-y3, the c e l l s e x p r e s s i n g both oncogenes might be expected to show an enhanced response to a u t o c r i n e s t i m u l a t i o n than those e x p r e s s i n g e i t h e r oncogene alone. I t i s not c l e a r that t h i s mechanism could apply to other combinations of c o o p e r a t i n g oncogenes as some of the oncogenes are not a s s o c i a t e d with TGF p r o d u c t i o n . The model d e r i v e d from these data does not account f o r the s u p p r e s s i o n of t r a n s f o r m a t i o n by c o o p e r a t i n g oncogenes a s s o c i a t e d with primary c e l l s . F i n a l l y , the p r o d u c t i o n of TGF-oc appears to be d i s p e n s a b l e i n ras induced t r a n s f o r m a t i o n (Pruss et a l . , 1 9 7 8 ; McKay e_t a l . , 1 986 ), suggesting that t r a n s f o r m a t i o n by ras can occur i n the absence of T G F - o c s t i m u l a t i o n of growth. 1 . 5 T i s s u e C u l t u r e i n the Examination of Transformation  1 . 5 . 1 Establishment of C e l l Lines Most of the work examining t r a n s f o r m a t i o n by oncogenes has been performed i n v i t r o using c e l l s i n t i s s u e c u l t u r e , as t h i s f a c i l i t a t e s o b s e r v a t i o n and manipulation of the t r a n s f o r m i n g c e l l s . Many of the experiments use f i b r o b l a s t i c c e l l s , although c e l l s with d i f f e r e n t o r i g i n s and f u n c t i o n s have a l s o been used. F i b r o b l a s t s can be e s t a b l i s h e d i n c u l t u r e from t i s s u e s explants as the c e l l s r e a d i l y migrate out of t i s s u e and w i l l grow r a p i d l y under c u l t u r e c o n d i t i o n s . Other c e l l types can be e s t a b l i s h e d i n c u l t u r e by a v a r i e t y of techniques. S t e r o i d o g e n i c c e l l s from the adrenal cortex can be e s t a b l i s h e d i n e s s e n t i a l l y the same f a s h i o n as f i b r o b l a s t i c c e l l s ( S l a v i n s k i et a_l. , 1974). The c e l l s e s t a b l i s h e d i n primary c u l t u r e are presumed to most a c c u r a t e l y r e f l e c t normal c e l l s in v i v o , although most primary c u l t u r e s show responses s i m i l a r to those evoked by t r a u m a t i z i n g the t i s s u e from which they are d e r i v e d ( B a r r e t t et al_. , 1984; C o l l i n s e_t al_. , 1985; Jaye et a l . , 1985). The c e l l s grow to a s p e c i f i c d e n s i t y , at l e a s t p a r t i a l l y determined by the c u l t u r e c o n d i t i o n s , and then a r r e s t growth and remain quiescent u n t i l the c e l l s are d i l u t e d by passaging. The m a j o r i t y of the c e l l s that are e s t a b l i s h e d i n c u l t u r e have r e l a t i v e l y l i m i t e d l i f e spans, and senesce and die a f t e r a c h a r a c t e r i s t i c l e n g t h of time i n c u l t u r e . In many s p e c i e s a small percentage of the c e l l s w i l l s u r v i v e t h i s p e r i o d of c r i s i s and become immortalized or permanent c e l l l i n e s and do not appear to s u f f e r c r i s i s a g ain. C r i s i s appears to r e s u l t from an i n s u f f i c i e n t number of c e l l s being able to r e p l i c a t e and grow to confluence (Merz and Ross, 1969). Immortalized c e l l s , although u s e f u l i n that they r e p r e s e n t an homogeneous pool of c e l l s that are adapted to growth i n t i s s u e c u l t u r e , express a number of phenotypes that d i s t i n g u i s h them from l i n e s that are not immortalized and c e l l s i n v i v o . The most important of these d i f f e r e n c e s i n the c u r r e n t context i s the r e l a t i v e ease of t r a n s f o r m a t i o n of e s t a b l i s h e d c e l l l i n e s . Transformation of immortalized c e l l l i n e s o f t e n appears to r e q u i r e only a s i n g l e a c t i v a t i n g change. Some c e l l l i n e s appear to be r e s i s t a n t to the t r a n s f o r m i n g e f f e c t s of a s i n g l e oncogene and show s i m i l a r requirements f o r c o o p e r a t i n g oncogenes as have been d e s c r i b e d i n primary c e l l s (Franza et al_. , 1 986 ). I t i s of i n t e r e s t to note that c e l l u l a r i m m o r t a l i t y can a p p a r e n t l y be suppressed i n somatic c e l l h y b rids ( P e r e i r a -Smith and Smith, 1983), but i t i s not known i f t h i s can be c o r r e l a t e d with the suppression of t r a n s f o r m a t i o n seen i n h y b r i d s of normal and transformed c e l l s . 1.5.2 R e g u l a t i o n of C e l l Growth by Growth F a c t o r s The growth of normal c e l l s i n t i s s u e c u l t u r e i s dependent on serum growth f a c t o r s . Normal c e l l l i n e s r e q u i r e a serum supplement i n the medium to support growth and r e p l i c a t i o n , but many transformed c e l l s do not. The c e l l s grown from many spontaneous tumours a l s o r e q u i r e serum f a c t o r s f o r growth i n v i t r o (McCulloch et_ a_l. , 1974) and show approximately the same low r a t e of s u c c e s s f u l establishment of immortalized l i n e s i n c u l t u r e as normal c e l l s d e r i v e d from the same t i s s u e . The serum growth f a c t o r s are p o l y p e p t i d e chains ranging from 5,000 to 28,000 d a l t o n s . They appear to act by b i n d i n g to c e l l s u r f a c e r e c e p t o r s s p e c i f i c f o r the growth f a c t o r and a c t i v a t i n g a r e g u l a t o r y pathway ( H e l d i n and Westermark, 1984). The growth f a c t o r s are de r i v e d both from the rupture of p l a t e l e t s i n the p r e p a r a t i o n of serum and from plasma and f i v e s p e c i f i c f a c t o r s appear to be l a r g e l y r e s p o n s i b l e f o r the mitogenic e f f e c t s of serum: EGF, PDGF, IGF-II, TGF-^3 and FGF (Pledger et a l . , 1982). These growth f a c t o r s can be d i v i d e d i n t o two groups based on t h e i r r o l e i n the s t i m u l a t i o n of c e l l d i v i s i o n . PDGF and FGF appear to act as i n i t i a t i n g f a c t o r s and s t i m u l a t e serum s t a r v e d , growth a r r e s t e d c e l l s to pass the f i r s t r e s t r i c t i o n p o i n t 10 to 12 hours before DNA r e p l i c a t i o n . C e l l s that have been t r e a t e d with PDGF or FGF only do not u s u a l l y progress to DNA s y n t h e s i s or c e l l d i v i s i o n but a r r e s t at a second r e s t r i c t i o n p o i n t 4 to 8 hours before DNA s y n t h e s i s . Treatment of the c e l l s with e i t h e r EGF or IGF-II, p r o g r e s s i o n or competence f a c t o r s , w i l l then r e s u l t i n the s t i m u l a t i o n of DNA s y n t h e s i s . The mitogenic e f f e c t s of serum appear to r e s u l t from the co o p e r a t i o n of these two groups of growth f a c t o r s . 1.5.3 Transformation and Growth F a c t o r Independence The t r a n s f o r m a t i o n of c e l l s by many of the oncogenes r e s u l t s i n e i t h e r a p a r t i a l or complete l o s s of the normal serum requirements f o r growth. Some of the tumour d e r i v e d c e l l l i n e s produce growth f a c t o r s that appear to be i n v o l v e d 39 i n an a u t o c r i n e s t i m u l a t i o n of c e l l growth (Reeve e_t a l . , 1985; Scott et a l . , 1985; Todaro et a l . , 1980; van Ke s s e l et <|1. , 1985; van Zoelen et a l . , 1 985 ). Expression of growth f a c t o r s can r e s u l t i n the tumourigenic con v e r s i o n of normal c e l l s . The i n t r o d u c t i o n of the gene f o r TGF-oc i n t o a r a t c e l l l i n e (Rosenthal et _ a l . , 1986), or expre s s i o n of the s i s oncogene, which i s c l o s e l y r e l a t e d to PDGF (Besmer e_t a l . , 1983) can transform c e l l s . These data i n d i c a t e that e x p r e s s i o n of growth f a c t o r s can be a s s o c i a t e d with oncogenesis i n viv o as w e l l as i n v i t r o . I t has been p o s s i b l e to demonstrate the exp r e s s i o n of both the c - s i s oncogene and c-myc i n human p l a c e n t a l c e l l s suggesting a normal a u t o c r i n e c o n t r o l of growth i n these c e l l s (Goustin et a l . , 1985). I t has a l s o been p o s s i b l e to demonstrate the expres s i o n of TGF-oC i n embryos l i n k i n g the exp r e s s i o n of t h i s growth f a c t o r to embryonic growth (Lee et a_l_. , 1 985 ). I t i s becoming c l e a r that the requirements f o r growth r e g u l a t i o n i n viv o are s i g n i f i c a n t l y d i f f e r e n t from those i n v i t r o . The i n t e r a c t i o n between c e l l s of d i f f e r e n t types can s i g n i f i c a n t l y a l t e r the response of c e l l s to growth f a c t o r s ( J e f f e r s o n et a_l. , 1984a, 1984b). To study t r a n s f o r m a t i o n i n t i s s u e c u l t u r e i t has been necessary to i d e n t i f y the changes seen i n v i t r o that c o r r e l a t e with the a c q u i s i t i o n of t u m o u r i g e n i c i t y . The i n t r o d u c t i o n of a c t i v a t e d oncogenes i n t o most c e l l s r e s u l t s i n changes i n morphology, i n c r e a s e d s a t u r a t i o n d e n s i t y , reduced serum requirements and anchorage independent growth. I t has not been p o s s i b l e to show a c o m p l e t e c o r r e l a t i o n o f a n y o f t h e s e p h e n o t y p e s w i t h t u m o u r i g e n i c i t y , b u t a n c h o r a g e i n d e p e n d e n c e s h o w s t h e s t r o n g e s t c o r r e l a t i o n ( K a h n a n d S h i n , 1 9 7 9 ; C i f o n e a n d F i d l e r , 1 9 8 0 ) . I t i s o f i n t e r e s t t o n o t e t h a t t h e a c q u i s i t i o n o f r e d u c e d s e r u m r e q u i r e m e n t s f o r g r o w t h a n d a n c h o r a g e i n d e p e n d e n c e a p p e a r s t o b e c o i n c i d e n t a n d c o r r e l a t e d w i t h a h i g h l y t u m o u r i g e n i c p h e n o t y p e i n t h e 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 u e r s p e r g e_t a l . , 1 9 8 6 ) . I t i s n o t c l e a r why a n c h o r a g e i n d e p e n d e n c e s h o u l d b e c o r r e l a t e d w i t h t u m o u r i g e n i c i t y , a l t h o u g h i t i s t h o u g h t t h a t t h e a b i l i t y t o g r o w d e s p i t e t h e l a c k o f a n a p p r o p r i a t e s u b s t r a t e w o u l d a l l o w t u m o u r c e l l s t o g r o w i n a p p r o p r i a t e l y i n t h e a n i m a l . A n c h o r a g e i n d e p e n d e n c e i s u s e d a s a p a r a m e t e r t o r e f l e c t t r a n s f o r m a t i o n i n v i t r o . 41 CHAPTER 2 MATERIALS AND METHODS 2.1 C e l l s Establishment of primary a d r e n o c o r t i c a l c e l l c u l t u r e s . Two to three month o l d F i s c h e r r a t s were k i l l e d by c e r v i c a l d i s l o c a t i o n and the adre n a l glands were removed a s e p t i c a l l y and minced. The adre n a l c e l l s from each r a t were maintained and grown as separate c e l l l i n e s throughout a l l the experiments. The minced adrenals were allowed to a t t a c h to the t i s s u e c u l t u r e d i s h f o r 30 to 60 minutes before the d i s h was f i l l e d with Dulbecco's modified Eagles medium with 25% f e t a l bovine serum and p e n i c i l l i n (1OOU/ml)/streptomycin (50ug/ml) (DMEM + 25% FBS). The t i s s u e explants were then incubated at 37°C, 5% C0 2 i n a h u m i d i f i e d atmosphere. A f t e r 10 to 14 days f i b r o b l a s t i c c e l l s from the adre n a l glands had grown to confluence i n the 60 mm d i s h e s . The c e l l s were then t r y p s i n i z e d and f r o z e n i n DMEM + 2 0% FBS + 10? dimethyl s u l p h o x i d e . The r a t lung f i b r o b l a s t s and r a t embryo f i b r o b l a s t s were e s t a b l i s h e d by e s s e n t i a l l y the same process as that d e s c r i b e d f o r the adre n a l cortex c e l l s . The r a t embryo f i b r o b l a s t s were from F i s c h e r r a t embryos. E s t a b l i s h e d r a t and mouse c e l l l i n e s . The r a t 2 c e l l l i n e (Topp, 1981) was grown i n DMEM + "\0% FBS. The c e l l s were s t o r e d f r o z e n i n l i q u i d n i t r o g e n (1N 2) and thawed and expanded when the c e l l s were r e q u i r e d f o r assays or experiments. The FSV-transformed r a t 2 c e l l s r e s u l t e d from t r a n s f e c t i o n of the r a t 2 c e l l l i n e with a p r o v i r a l clone of the a v i a n , a c u t e l y oncogenic r e t r o v i r u s F u j i n a m i sarcoma v i r u s (FSV) (courtesy of G. Weinmaster). The T-ROG c e l l l i n e was d e r i v e d from a Ki-MSV-infected F i s c h e r r a t o v a r i a n g r a n u l o s a . c e l l c u l t u r e as the f u l l y transformed l i n e that grew out a f t e r passaging the i n f e c t e d c u l t u r e (from N. Auersperg) ( H a r r i s o n and Auersperg, 1981 ) . The s t r a i n A c e l l s were d e r i v e d (by N. Auersperg) from c u l t u r e s grown out from r a t adrenals u n t i l the c e l l s had passed through senescence and the l i n e was permanently e s t a b l i s h e d i n c u l t u r e . The c e l l s were contact i n h i b i t e d and showed a normal f i b r o b l a s t i c morphology. S t r a i n A2 was d e r i v e d from s t r a i n A c e l l s by Ki-MSV i n f e c t i o n and showed serum independent and anchorage independent growth and was tumourigenic. The NIH 3T3 c e l l l i n e i s an e s t a b l i s h e d l i n e d e r i v e d from mouse embryo f i b r o b l a s t s (Todaro and Green, 1 9 6 3 ) and was obtained from ATCC. The Ki-MSV nonproducer NIH 3T3 l i n e was obtained by i n f e c t i o n of NIH 3T3 c e l l s with a low t i t r e of Ki-MSV and i s o l a t i o n of a focus of nonproducing c e l l s (from D. Lowy). The c a l u - 1 l i n e , d e r i v e d from a human lung adenocarcinoma, c o n t a i n i n g an a c t i v a t e d c-ras oncogene, was obtained from ATCC and maintained as recommended by the ATCC . 43 2.2 V i r u s e s The four a c u t e l y oncogenic r e t r o v i r u s e s used i n these experiments were Ki-MSV, 3611, MMCV and 2-1. Ki-MSV and 3611 are n a t u r a l i s o l a t e s d e r i v e d from tumours ( K i r s t e n and Mayer, 1 967 ; Rapp e_t a l . , 1983a). Stocks of Ki-MSV(Mo-MLV) were obtained by t r a n s f e c t i n g the Ki-MSV transformed nonproducing NIH 3T3 c e l l l i n e with a genomic clo n e , pMLV-48, of the r e p l i c a t i o n competent helper v i r u s Moloney murine leukemia v i r u s (Mo-MLV) (from D. M i l l e r ( M i l l e r and Verma, 1984)). The c e l l s were expanded through 2 passages a f t e r t r a n s f e c t i o n and v i r u s was harvested at 4 hour i n t e r v a l s i n t o DMEM + 1$ c a l f serum. The supernatant was concentrated i n an Amicon c o n c e n t r a t o r to 40 to 50 f o l d and then s t o r e d i n a l i q u o t s at -80°C. Both the 3611 and MMCV were harvested i n the same way. The 3611(Mo-MLV) v i r u s was obtained from a producing NIH 3T3 c e l l l i n e ( courtesy of U.R. Rapp). The t i t r e s of the Ki-MSV and 3611 were determined by focus formation on r a t 2 c e l l s . The v i r u s stock was brought to 4 ug/ml polybrene and then s e r i a l l y d i l u t e d i n DMEM + 4 ug/ml polybrene. Rat 2 c e l l s , approximately 40 to 60% c o n f l u e n t , were t r e a t e d with 0.5 ml of the v i r u s d i l u t i o n s f o r 1 hour at 37^C. The p l a t e s were f i l l e d with medium and incubated as f o r the u n i n f e c t e d c e l l s f o r 5 to 7 days when the f o c i were counted. MMCV i s a r e p l i c a t i o n d e f e c t i v e , t r a n s f o r m i n g r e t r o v i r u s c o n s t r u c t e d i n v i t r o (Vennstrom et a l . , 1984) and 44 provided i n an NIH 3T3 producer l i n e . The v i r u s was d e r i v e d from Ha-MSV LTR's and a Mo-MLV 5* noncoding r e g i o n and s p l i c e s i t e . The avian v-myc oncogene was cloned from the OK 10 a c u t e l y t r a n s f o r m i n g r e t r o v i r u s . T h i s v e r s i o n of the oncogene was chosen as i t con t a i n s a 3 ' s p l i c e acceptor s i t e and i s f r e e of r e t r o v i r a l gag sequences. The t i t r e was determined by the i n d u c t i o n of anchorage independent growth i n r a t 2 c e l l s . The r a t 2 c e l l s were i n f e c t e d with MMCV and a f t e r 48 hours the c e l l s were t r y p s i n i z e d and s o f t agar assays were set up at 10^ c e l l s from each v i r u s c o n c e n t r a t i o n i n a 60 mm d i s h . A f t e r 12 to 14 days the v i r u s d i l u t i o n which gave between 15 and 30$ colony formation was determined and the v i r u s c o n c e n t r a t i o n c a l c u l a t e d . The 2-1 v i r u s i s a c o n s t r u c t e d oncogenic murine r e t r o v i r u s that c o n t a i n s the avian v - s r c gene from Rous sarcoma v i r u s . The v i r u s was c o n s t r u c t e d by S. Anderson (Anderson and S c o l n i c k , 1983) and very k i n d l y provided by K. Humphries as high t i t r e v i r u s stock using the amphotropic helper 4070. I n f e c t i o n was c a r r i e d out as d e s c r i b e d f o r K i -MSV and the i n f e c t e d c e l l s were maintained i n a l e v e l C f a c i l i t y . 2.3 I n f e c t i o u s Centre Assays The number of adrenal cortex c e l l s i n f e c t e d by e i t h e r Ki-MSV or 2-1 was estimated by an i n f e c t i o u s centre assay. The a d r e n a l cortex c e l l s were t r y p s i n i z e d at the end of the passage i n which they were i n f e c t e d , counted and p l a t e d at 10 , 10-3 and 10 c e l l s on subconfluent monolayers of u n i n f e c t e d r a t 2 c e l l s . Ki-MSV-transformed r a t 2 c e l l s were used as a p o s i t i v e c o n t r o l f o r focus formation e f f i c i e n c y . The c e l l s were allowed to a t t a c h f o r 4 to 6 hours and then covered with an o v e r l a y of 0.6$ agarose i n DMEM plus 10$ FBS. The c e l l s were then incubated f o r 10 to 12 days and the f o c i were then counted. The number of f o c i formed by the i n f e c t e d adrenal cortex c e l l s was c o r r e c t e d f o r the e f f i c i e n c y of focus formation, determined from the Ki-MSV (Mo-MLV)-infected r a t 2 c e l l s and the percentage of 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 estimated. I t was assumed that the e f f i c i e n c y of focus formation i n the i n f e c t i o u s centre assay by the Ki-MSV-infected r a t 2 c e l l s was g r e a t e r than or equal to the e f f i c i e n c y of the Ki-MSV-infected adrenal cortex c e l l s . The MMCV-infected adrenal cortex c e l l s d i d not produce a d e t e c t a b l e response i n t h i s assay. The i n f e c t i o u s centre assay has a l s o been performed with Ki-MSV-infected r a t 2 and adrenal cortex c e l l s t r e a t e d with mitomycin C to i n h i b i t c e l l r e p l i c a t i o n and no s i g n i f i c a n t d i f f e r e n c e was seen between the mitomycin C t r e a t e d and untreated c e l l s . 2.4 Anchorage Independent Growth The bottom agar l a y e r was composed of 1X DMEM plus p e n i c i l l i n and streptomycin, 5X MEM vi t a m i n s , 0.6$ agarose, s t e r i l e d i s t i l l e d water to volume and e i t h e r 25% FBS or 5% horse or c a l f serum as was r e q u i r e d f o r the assay. The top agar contained 1X DMEM plus a n t i b i o t i c s , 5X MEM vi t a m i n s , 0.35$ agarose, s t e r i l e d i s t i l l e d water to volume, 25$ FBS, 5% horse or c a l f serum, and exogenous p u r i f i e d growth f a c t o r s as necessary. 1 u \ 3 x 10^ or 10 5 c e l l s i n 0.1 to 0.2 ml were suspended i n 2.5 to 3.0 ml of the top agar mix f o r each 60 mm d i s h . The s o f t agars were covered with DMEM plus a n t i b i o t i c s and serum with or without exogenous growth f a c t o r s as i n d i c a t e d and incubated f o r 2 to 3 weeks. 2.5 R a d i o l a b e l l i n g of C e l l s C e l l s were seeded and grown i n DMEM + serum as r e q u i r e d u n t i l the c e l l s were 50 to 80% c o n f l u e n t . The c e l l s were washed once with phosphate f r e e s a l i n e and then incubated 8 to 12 hours with 300-500 uCi of ^ 2P-orthophosphate ( c a r r i e r f r e e , ICN Pharmaceuticals) i n phosphate f r e e DMEM with 3% FBS f o r c e l l s incubated i n 10 to 25% FBS and 3% HS f o r c e l l s grown i n 3% HS. A f t e r l a b e l l i n g the c e l l s were washed once i n i c e c o l d T r i s - s a l i n e (25 mM T r i s , 140 mM NaCl, 5 mM KC1, 0.7 mM Na 2HP0 4, 5.5 mM D-glucose, [pH 7.4]). The c e l l s were then scraped i n t o 1 ml of l y s i s b u f f e r ( f i n a l volume). For immunoprecipitations with the a n t i p 2 1 r a s monoclonal antibody Y13-259 the l y s i s b u f f e r used was 100 mM NaCl, 5 mM MgCl 2, 5mM T r i s - H C l (pH 7.5), 1$ v/v T r i t o n X-100, 0.5% w/v SDS, 1mM l e u p e p t i n . The l y s i s b u f f e r used f o r immunoprecipitations with other a n t i b o d i e s was 10 mM T r i s -HCl pH 7.5, 100 mM NaCl, 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 . The l y s a t e was c e n t r i f u g e d at 4°C, 35,000 x g f o r 30 minutes and the supernatant was recovered and incubated with the ap p r o p r i a t e antiserum or f r o z e n o v e r n i g h t . C e l l s were l a b e l l e d with 47 [ ] m e t h i o n i n e i n a s i m i l a r manner except that the c e l l s were washed with T r i s - s a l i n e and then incubated with 150 uCi [ ^ S ] m e t h i o n i n e (800-1,000 Ci/mmol; Amersham Corp.) i n methionine f r e e DMEM with 3$ FBS or 3 $ HS. The c e l l s were ly s e d and t r e a t e d as above. 2.6 Immunoprecipitation The c l a r i f i e d c e l l l y s a t e s were incubated on i c e with 1 to 5 u l of the a p p r o p r i a t e antibody e s s e n t i a l l y as d i r e c t e d by the s u p p l i e r f o r 60 minutes and then f o r a f u r t h e r 60 minutes with 50 u l of a 10$ suspension of Staphyloccus  aureus s t r a i n Cowan I (IgG sorb, The Enzyme Center) or f o r the r a t monoclonals 50 u l of a 10$ suspension of S_. aureus coated with r a b b i t a n t i - r a t IgG (Roc R i g ) . The S_. aureus and S. aureus + Woe Rig were prepared by c e n t r i f u g a t i o n and resuspension i n the l y s i s b u f f e r 3 times before adding to the c e l l l y s a t e . The immune complex was then p e l l e t e d i n a microfuge f o r 15 to 20 seconds and washed 5 to 6 times with the l y s i s b u f f e r . The immunoprecipitations of l a b e l l e d c e l l l y s a t e s were then washed i n 1M NaCl, 10 mM T r i s - H C l (pH 8.0), 0.1$ v/v NP40. The immunoprecipitated m a t e r i a l was r e l e a s e d by i n c u b a t i o n i n SDS g e l sample b u f f e r at 37^C f o r 10 minutes and the supernatant c o l l e c t e d and f r o z e n at -80°C u n t i l use. 2.7 Immune Complex Kinase Reaction For the immune complex kinase r e a c t i o n 100 mm d i s h c u l t u r e s were l y s e d i n 1 ml of modified RIPA b u f f e r (0.15 M NaCl, 1$ NP40, 1$ NaDOC, 0.1$ SDS, 10 mM NaPhosphate pH 7.0, 2 mM EDTA, 14 mM 2-mercaptoethanol, 1 mM l e u p e p t i n and 50 mM NaF) at 4°C. The l y s a t e was c l a r i f i e d as above. 0.1 mis of the c l a r i f i e d supernatant was immunoprecipitated with the a n t i - p 6 0 s r c monoclonal antibody 327 ( L i p s i c h et a l . , 1983) f o r a f u r t h e r 30 minutes and incubated with RotMIg coated S.  aureus f o r a f u r t h e r 30 minutes. The immunoprecipitates were c o l l e c t e d by c e n t r i f u g a t i o n through a 10$ sucrose pad and then washed three times i n the modified RIPA and once i n 0.1 M NaCl, 10 mM PIPES pH 7.0. For the kinase r e a c t i o n s the immunoprecipitates were resuspended i n 10 u l of 20 mM PIPES pH 7.0, 10 mM M n C l 2 > 5 t o 1 0 u C i [ y - 3 2 P ] ATP (-3000 Ci/mmol, Amersham) and 10 ug a c i d t r e a t e d r a b b i t muscle enolase (Sigma) (Cooper et a l . , 1984), and incubated at 30 UC f o r 10 minutes. The r e a c t i o n s were stopped by adding an equal volume of two f o l d concentrated SDS g e l sample b u f f e r . 2.8 SDS-polyacrylamide Gel E l e c t r o p h o r e s i s Samples were prepared f o r e l e c t r o p h o r e s i s by heating at 100°C f o r 3 minutes i n SDS g e l sample b u f f e r (2$ SDS, 5$ 2-mercaptoethanol, 10 mM T r i s - H C l pH 6.8, 10$ v/v g l y c e r o l , 0.001$ bromphenol b l u e ) . E l e c t r o p h o r e s i s was performed using an SDS-polyacrylamide d i s c o n t i n u o u s b u f f e r system as d e s c r i b e d by Laemmli (1970). The g e l s used a 4.5$ p o l y a c r y l a m i d e s t a c k i n g g e l and e i t h e r a 12.5$ ( f o r p 2 1 r a s and p 6 0 s r c ) or 10.5$ ( f o r p 5 7 v _ m y c ) pol y a c r y l a m i d e s e p a r a t i n g g e l . The acrylamide stock s o l u t i o n was 29.2$ w/v acrylamide and 0.8$ (w/v) N,N 1-bis-methylene acrylamide. The running b u f f e r was 0.025 M T r i s , 0.192 M g l y c i n e and 4 9 0.1$ SDS (pH 8.3). The denatured p r o t e i n s were e l e c t r o p h o r e s e d at constant power of 3 to 4 watts per g e l . The g e l s were then f i x e d and s t a i n e d o v e r n i g h t i n Coomassie b r i l l i a n t b l ue, 7 . 5 $ a c e t i c a c i d and 4 5 $ methanol i n d i s t i l l e d water, to l o c a t e molecular weight markers (Sigma). The g e l s were d e s t a i n e d i n 7 . 5 $ a c e t i c a c i d and 4 5 $ methanol i n d i s t i l l e d water. The g e l s of m a t e r i a l l a b e l l e d with r 3 5 3 [ J S]methionine were t r e a t e d with En-'Hance (New England Nuclear Corp.) f o l l o w i n g the manufacturer's i n s t r u c t i o n s . The g e l s were then d r i e d onto 3MM f i l t e r paper on a Hoefer s l a b g e l d r i e r , and exposed to XAR -5 f i l m (Kodak) at -80°C. An i n t e n s i f y i n g screen (Dupont, L i g h t n i n g Plus) was used with g e l s c o n t a i n i n g - l a b e l l e d m a t e r i a l . 2 . 9 Pulse L a b e l l i n g of P r o t e i n s For pulse-chase and time course i n c o r p o r a t i o n experiments the c e l l s were l a b e l l e d e s s e n t i a l l y as d e s c r i b e d above except the c e l l s were incubated i n DMEM-methionine plus 5$ FBS f o r 4 hours p r i o r to l a b e l l i n g . The [ 3 5 s ] -methionine was added d i r e c t l y to the methionine f r e e medium i n which the c e l l s had been i n c u b a t e d . For the pulse-chase experiments the c e l l s that had been st a r v e d as d e s c r i b e d were pulsed with methionine f o r 1 hour. The medium was then removed and r e p l a c e d by prewarmed DMEM plus 10$ FBS and 10 mM u n l a b e l l e d methionine. The c e l l s were maintained i n t h i s medium u n t i l h a r v e s t . 10 mM methionine was chosen as t h i s had been shown to i n h i b i t g r e a t e r than 9 5 $ of the 50 i n c o r p o r a t i o n of [35 S] methionine when added to the c e l l s at the same time. 2.10 T r y p t i c Peptide A n a l y s i s The t r y p t i c peptide mapping a n a l y s i s was e s s e n t i a l l y as p r e v i o u s l y d e s c r i b e d (Weinmaster et a l . , 1984). The g e l s l i c e of the a p p r o p r i a t e band was swollen i n e l u t i o n b u f f e r , the backing paper removed and crushed. 5 ml of e l u t i o n b u f f e r (50 mM NH 4HC0 3, 0.1$ SDS) was added, brought to 5$ 2-mercaptoethanol and b o i l e d f o r 5 minutes, then shaken at 3 7°C o v e r n i g h t . The supernatant was c o l l e c t e d and the g e l fragments washed once i n 2 ml of e l u t i o n b u f f e r . 50 ug of bovine gamma g l o b u l i n was added to the pooled supernatants which were then made 20$ i n t r i c h l o r o a c e t i c a c i d , and s t o r e d at 4^c o v e r n i g h t . The p r o t e i n was recovered by c e n t r i f u g a t i o n at 15000xg f o r 30 minutes and the p e l l e t washed twice i n e t h a n o l . The d r i e d p e l l e t was o x i d i z e d i n performic a c i d (100 u l formic a c i d , 25 u l methanol and 40 u l performic a c i d (30$ H 2 o 2 and 98$ formic a c i d [1:9])) f o r 2 hours i n an i c e s l u r r y . The r e a c t i o n was terminated by d i l u t i o n i n 3 ml d i s t i l l e d water and the sample l y o p h i l i z e d . The o x i d i z e d p r o t e i n was d i g e s t e d with 5 ug of L-(1-t o s y l a m i d o - 2 - p h e n y l ) e t h y l chloromethyl k e t o n e - t r e a t e d t r y p s i n (Worthington) i n 0.5 ml of 50 mM NH^HCOg f o r 6 to 8 hours at 37°C. The d i g e s t was d i l u t e d to 3 ml with d i s t i l l e d water and l y o p h i l i z e d . The sample was l y o p h i l i z e d two more times from d i s t i l l e d water and the d i g e s t e d p r o t e i n sample was resuspended i n 10 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 spotted onto a 20 x 20 cm t h i n - l a y e r c e l l u l o s e p l a t e (E. Merck Lab). The sample was e l e c t r o p h o r e s e d i n pH 2.1 b u f f e r (water:88$ formic a c i d : a c e t i c a c i d = 90:2:8 by volume) at 1,000 v o l t s f o r 45 minutes. A f t e r e l e c t r o p h o r e s i s the p l a t e was a i r d r i e d and chromatographed p e r p e n d i c u l a r 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 i n N-b u t a n o l : a c e t i c a c i d : w a t e r : p y r i d i n e (75:15:60:50, by volume). The p l a t e was a i r d r i e d and sprayed with E n h a n c e and exposed to XAR-5 f i l m at -80°C. 2.11 Southern H y b r i d i z a t i o n The genomic DNA was prepared from c e l l l i n e s e s s e n t i a l l y by the procedure d e s c r i b e d i n M a n i a t i s e_t a l . (1982). The p u r i f i e d DNA was d i g e s t e d with e i t h e r BamHI or H i n d l l l (BRL-Gibco), phenol e x t r a c t e d 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 was r e d i s s o l v e d i n water and an equal volume of two-fold concentrated Tris-borate-EDTA l o a d i n g b u f f e r was added and the samples were el e c t r o p h o r e s e d i n a 0.75$ (w/v) agarose submarine g e l . A f t e r e l e c t r o p h o r e s i s the g e l was t r e a t e d with 0.25 N HC1 f o r 15 minutes and denatured i n 0.5 M NaOH, 1.5 M NaCl f o r 30 minutes. The g e l was then covered with n e u t r a l i z i n g s o l u t i o n (1.5 M NaCl, 0.5 M T r i s - H C l pH 7.2, 0.001 M Na 2EDTA) f o r 30 minutes. The DNA was then t r a n s f e r r e d by c a p i l l a r y b l o t t i n g to Hybond-N (Amersham) nylon membranes e x a c t l y as d e s c r i b e d by the manufacturer. The DNA was f i x e d to the membrane by U.V. i r r a d i a t i o n . The membrane was washed i n 0.1X SSC (1X SSC: 0.15 M NaCl, 0.015 M N a ^ i t r a t e ) , 0.5$ SDS, at 65 C f o r 60 minutes and then p r e h y b r i d i z e d i n 6X SSC, 5X Denhardt's s o l u t i o n (100X Denhardt's s o l u t i o n : 2$ (w/v) bovine serum albumin, 2% (w/v) F i c o l l , 2% p o l y v i n y l p y r o l l i d o n e ) , 0.5$ SDS, and 20 ug/ml s o n i c a t e d , denatured salmon sperm DNA at 65°C f o r 4 to 8 hours. The avian v-myc probe was prepared from a 1.5 kb fragment from pMC38 (a g i f t of J.M. Bishop, (Vennstrom et a l . , 1981)) c o n t a i n i n g the MC29 v-mvc gene. The fragment was l a b e l l e d by nick t r a n s l a t i o n ( M a n i a t i s et a l . , 1982) with [c*' 3 2P] dCTP (3,000 Ci/mmol, New England Nuclear Corp.) and then separated from 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 chromatography on Sephadex G-50. The probe was denatured with the s o n i c a t e d salmon sperm DNA and the h y b r i d i z a t i o n was performed i n the same b u f f e r as the p r e h y b r i d i z a t i o n f o r 24 hours at 65°C. The membrane was then washed once i n 2X SSC, 0.1$ SDS at 65°C f o r t h i r t y minutes, twice i n 2X SSC, 0.5$ SDS at 65°C f o r t h i r t y minutes and once i n 0.1X SSC at 65^C f o r 10 minutes. The membrane was then a i r d r i e d , and exposed to XAR-5 f i l m with an i n t e n s i f y i n g screen at -80°C. 2.12 HPLC A n a l y s i s of S t e r o i d Products from the Y-1  A d r e n o c o r t i c a l C e l l Line The s t e r o i d products were p u r i f i e d on a SepPak (Waters-M i l l i p o r e ) from the medium of the p a r e n t a l and v i r u s i n f e c t e d Y-1 c e l l l i n e s (Ramirez et a l . , 1982).The column was prepared by washing with dichloromethane and then PBS. The medium was then passed over the column dropwise and washed again with PBS. The s t e r o i d s were then e l u t e d wth dichloromethane and d r i e d under a stream of n i t r o g e n . A f t e r d r y i n g the s t e r o i d s were resuspended i n e i t h e r methanol or a c e t o n i t r i l e . The HPLC was performed on a Waters C^g reve r s e phase, 10 um bead s i z e column at room temperature at a flow r a t e of 1 ml per minute. The sample was loaded i n 15 to 30 u l volume at 30$ a c e t o n i t r i l e i n water. The column was run at 30$ a c e t o n i t r i l e i n water f o r 15 minutes and the s t e r o i d s e l u t e d i n a 30 to 70$ l i n e a r g r a d i e n t of a c e t o n i t r i l e i n water over 25 minutes. The e l u t i o n of s t e r o i d s was monitored at 254 nm and recorded and i n t e g r a t e d on a Hewlett-Packard 3 3 9 0 A I n t e g r a t o r . A l l s o l v e n t s were of spectrophotometric grade and f i l t e r e d j u s t p r i o r to use. 2.13 Radioimmunoassay of C u l t u r e Supernatant of Ki-MSV/MMCV  Transformed Rat Adrenal Cortex C e l l s f o r S t e r o i d P r o d u c t i o n The antibody used was a r a b b i t anti-progesterone-11 (X -BSA ( M i l e s - Y e d a ) . The t r a c e r was [1,2,6,7-^] progesterone (100 uCi/mmol, New England Nuclear Corp) and the c o l d progesterone (4-pregnen - 3,20-dione) was obtained from Sigma and d i l u t e d i n e t h a n o l . The standards were made from the stock progesterone by s e r i a l d i l u t i o n s i n PBSG (7.94 g / 1 NaCl, 0.46 g / 1 NaH 2P0 4, 1.01 g / 1 Na 2HP0 1 |, 1g/l g e l a t i n ) . Dextran coated c h a r c o a l was prepared by mixing 0.625 g Norit - A c h a r c o a l ( F i s h e r Chemicals) and 0.0625 g Dextran T-70 i n 100 mis f i n a l volume PBSG. The Ki-MSV/MMCV transformed c e l l l i n e s were seeded i n t o a 35 mm d i s h i n DMEM with 10$ FBS and allowed to a t t a c h o v e r n i g h t . The c e l l s were incubated f o r 1 hour i n DMEM without serum. The c e l l s were then incubated i n DMEM with 5 x 10~9 g pregnenolone (3 mis f i n a l volume) f o r 4 hours at 3 7^C. The medium was c e n t r i f u g e d f o r 5 minutes i n a microfuge at 4°C and the supernatant stor e d at -20°C. The a n t i - p r o g e s t e r o n e antibody, progesterone t r a c e r and sample or standard were mixed i n a 400 u l f i n a l volume with approximately 2500 cpm of the progesterone t r a c e r , and incubated at room temperature f o r 1 hour. The samples were then c h i l l e d on i c e and 200 u l of dextran coated c h a r c o a l was added. A f t e r 10 minutes the samples were c e n t r i f u g e d at 2500 x g f o r 15 minutes and the supernatant decanted. The supernatants were mixed with 3 mis of ACS s c i n t i l l a t i o n f l u i d (Amersham) and the counts were determined by s c i n t i l l a t i o n counting CHAPTER 3 3.0 Expression of V i r a l p 2 1 r a s during A c q u i s i t i o n of a  Transformed Phenotype by Rat Adrenal Cortex C e l l s I n f e c t e d  with K i r s t e n Murine Sarcoma V i r u s  3.1 INTRODUCTION The a n a l y s i s of oncogenic t r a n s f o r m a t i o n i n v i t r o has sought to d e f i n e the molecular mechanisms i n v o l v e d i n c a r c i n o g e n e s i s . S i n c e oncogenesis in v i v o appears to be a m u l t i s t e p process there has been c o n s i d e r a b l e e f f o r t (see 1.4 of the I n t r o d u c t i o n ) to e l u c i d a t e the reasons behind the requirement f o r m u l t i p l e changes and mimic the process i n  v i t r o . Transformation of primary c e l l s by the t r a n s f o r m i n g r e t r o v i r u s e s can appear to i n v o l v e complementing g e n e t i c or e p i g e n e t i c changes. I n f e c t i o n of murine bone marrow c e l l s by Abelson murine leukemia v i r u s (Whitlock and Witte, 1981) or an s r c - c o n t a i n i n g murine r e t r o v i r u s (Spooncer et  a l . , 1984) produces c u l t u r e s that have a reduced dependence on exogenous serum growth f a c t o r s . The c u l t u r e s i n f e c t e d with Abelson murine leukemia v i r u s become serum independent f o r growth and a l s o a c q u i r e a h i g h l y tumourigenic phenotype i n a stepwise f a s h i o n that a p p a r e n t l y i n v o l v e s a c t i v a t i o n of c e l l u l a r genes (Whitlock et a_l. , 1 9 8 3 ) - Transformation of avian c e l l s from d i f f e r e n t i a t e d t i s s u e s a l s o shows c o o p e r a t i v i t y between r e t r o v i r a l l y borne oncogenes (Adkins et a l . , 1984: Alema et a l . , 1985b; Kahn et a l . , 1984). Most s t u d i e s on the a c t i o n of v i r a l or c e l l u l a r oncogenes have employed c e l l l i n e s or embryonic c e l l s , which resemble malignant c e l l s i n some of t h e i r c h a r a c t e r i s t i c s (Sherbert, 1982). In c o n t r a s t most spontaneous tumours a r i s e i n a d u l t d i f f e r e n t i a t e d e p i t h e l i a l c e l l s . . Some oncogenes, the ras genes, have been a s s o c i a t e d with tumours from a v a r i e t y of t i s s u e s (reviewed i n Barbacid, 1986), but i t appears that the molecular mechanisms of t r a n s f o r m a t i o n are not i d e n t i c a l f o r a l l c e l l s (Jenuwien et_ a_l. , 1985 ). C u l t u r e s that appear to resemble stem c e l l s of the r a t adrenal c o r t e x , a s t e r o i d - s e c r e t i n g g l a n d u l a r e p i t h e l i u m , have been used to examine the r o l e of oncogenes i n the t r a n s f o r m a t i o n and c e l l development of e p i t h e l i a l c e l l types. I n f e c t i o n of r a t adrenal cortex c e l l s by Ki-MSV presents a model system i n which to i n v e s t i g a t e the e f f e c t of a ras oncogene on c e l l growth and d i f f e r e n t i a t i o n . Normal r a t adrenal cortex c e l l s can be grown from t i s s u e explants i n 25% f e t a l bovine serum (FBS) as s t e r o i d o g e n i c , a d r e n o c o r t i c o t r o p h i c hormone r e s p o n s i v e c e l l s ( S l a v i n s k i et a_l. , 1974). These c e l l s grow a c t i v e l y , adopt a f i b r o b l a s t i c morphology and can metabolize pregnenolone to in t e r m e d i a t e s of the s t e r o i d o g e n i c pathway. In the presence of 3% horse serum (HS) these c e l l s grow sl o w l y , appear e p i t h e l i a l and express a more complex s t e r o i d o g e n i c c a p a c i t y . The c e l l s c u l t u r e d i n FBS resemble f i b r o b l a s t i c stem c e l l s of the adrenal cortex and those i n HS appear to be more h i g h l y d i f f e r e n t i a t e d . The i n t e r c o n v e r s i o n of the two c e l l types i n c u l t u r e suggests that they represent i n t e r m e d i a t e s of the same d i f f e r e n t i a t i o n pathway i n v i v o . When adrenal cortex c u l t u r e s are i n f e c t e d with Ki-MSV and c u l t u r e d i n 25% FBS f o c i appear (Auersperg et al_. , 1977; Auersperg et a l . , 1981). When these c u l t u r e s are passaged i n t o 3% HS, u s u a l l y a l l of the c e l l s r e v e r t to the slow-growing, e p i t h e l i a l morphology, while s i b l i n g c u l t u r e s i n 25% FBS con t i n u e to express f o c i . With f u r t h e r passaging i n 25% FBS the f o c i become serum independent and the c u l t u r e s become o v e r t l y transformed. C u l t u r i n g of the Ki-MSV-i n f e c t e d adrenal c e l l s i n 3% HS delays and can, i n some cases, prevent appearance of the transformed phenotype (Auersperg and Calderwood, 1984; Auersperg et a l . , 1981). These r e s u l t s are of i n t e r e s t f o r two reasons: f i r s t , they i n d i c a t e a m u l t i s t e p t r a n s f o r m a t i o n process f o r adrenal cortex c e l l s i n f e c t e d with Ki-MSV; and second, t r a n s f o r m a t i o n of the Ki-MSV-infected adrenal cortex c e l l s i s s u b j e c t to environmental r e g u l a t i o n by serum f a c t o r s which a l s o appear to modulate the s t a t e of d i f f e r e n t i a t i o n of u n i n f e c t e d c e l l s . There has been some evidence that the degree 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 can be c o r r e l a t e d with the l e v e l of expression of the ras oncogene (Winter and Perucho, 1986), and so i t was of i n t e r e s t to determine whether the l e v e l of v i r a l p 2 1 r a s i n the Ki-MSV-inf ect ed adrenal c e l l s could be c o r r e l a t e d with t h e i r degree of tr a n s f o r m a t i o n or was constant. The amount of p 2 1 r a s expressed during t r a n s f o r m a t i o n was unchanged, suggesting 58 that a d d i t i o n a l changes must occur i n the i n f e c t e d c e l l s to allow expression of a transformed phenotype. R ESULTS 3.2 Expression of V i r a l p 2 1 r a s i n Ki-MSV-infected Rat  Adrenal Cortex C e l l s i n the Passages Immediately F o l l o w i n g  I n f e c t i o n . Rat adrenal cortex c e l l s were i n f e c t e d at the end of the f i r s t passage with Ki-MSV(Ki-MLV) and c u l t u r e d as d e s c r i b e d i n chapter 2. To examine p 2 1 r a s expression the Ki-MSV-inf ected adrenal cortex c e l l s were m e t a b o l i c a l l y l a b e l l e d with [ 3 5 S ] methionine, which l a b e l s both v i r a l and c e l l u l a r p 2 1 r a s . The r e l a t i v e amount of l a b e l i n c o r p o r a t e d i n t o the p 2 1 r a s from the c u l t u r e s incubated with [ 3 5 S ] -methionine should r e f l e c t the r e l a t i v e amount of p 2 1 r a s present. C u l t u r e s were a l s o t r e a t e d with 3 2 P i , which l a b e l s only the v i r a l p 2 1 r a s to d e t e c t a b l e l e v e l s under the c o n d i t i o n s used (Papageorge _et . a l . , 1982). The s p e c i f i c i n c o r p o r a t i o n of 3 2 P i i n t o the v i r a l p 2 1 r a s occurs because the v i r a l ras gene has s u s t a i n e d a mutation that a l t e r s codon 59 to code f o r a t h r e o n i n e which can then act as a phosphoacc eptor (Shih e_t a l . , 1982a; Tsuchida e_t a_l. , 1982 ). C e l l s from passages 2, 3 and 5 a f t e r v i r a l i n f e c t i o n were c u l t u r e d i n 25% FBS or 3% HS. The m a j o r i t y of the i n f e c t e d c e l l s i n 25% FBS had a normal f i b r o b l a s t i c morphology ( F i g . 3.1), but some transformed c e l l s appeared at a l l passages assayed, being p a r t i c u l a r l y apparent i n 25% FBS, f i v e passages a f t e r i n f e c t i o n (panel E) where between 1/4 and 1/2 of the c e l l s appeared m o r p h o l o g i c a l l y transformed. The c u l t u r e s i n 3% HS showed a more f l a t t e n e d F i g u r e 3.1. E a r l y passages of KiMSV-infected r a t adrenal cortex c e l l s grown with 25% f e t a l bovine or 3% horse serum supplements. A r a t adr e n a l cortex primary c u l t u r e was prepared and i n f e c t e d with KiMSV(KiMLV) at passage one at m.o.i.'s of 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 passaged i n t o 25% FBS or 3% HS at the beginning of each of the passages noted below. The KiMSV-i n f e c t e d adrenal cortex c e l l s were p l a t e d i n 10 mm w e l l s atpassage 2 (A and B), passage 3 (C and D) and passage 5 (E and F ) , and c u l t u r e d i n 25% FBS (A,C,E) or 3% HS (B,D,F). C e l l s were then c u l t u r e d f o r 4-17 days p r i o r to photomicroscopic examination. Photomicrographs are at 100X m a g n i f i c a t i o n . e p i t h e l i a l morphology, grew more slowly and contained few or no m o r p h o l o g i c a l l y transformed c e l l s . These c u l t u r e s were examined f o r v i r a l p 21 r a s, expression by l a b e l l i n g with [ 3 ^ S ] m e t h i o n i n e or 3 2 P i and immunoprecipitating c e l l l y s a t e s , normalized f o r T C A - 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 , with the a n t i - p 2 1 r a s monoclonal antibody Y13-259 (Furth et a l . , 1982), or with normal r a t serum. Immunoprecipitation of u n i n f e c t e d adrenal cortex c e l l s l a b e l l e d with [ 3 5 S ]methionin e r e v e a l e d a c e l l u l a r p 2 1 r a s doublet ( F i g . 3.2, lane 9) (Shih et a l . , 1982b). The [ 3 5 S ] -methionine l a b e l l e d Ki-MSV-inf ect ed c e l l s from passages 2, 3 and 5 expressed a higher l e v e l of p 2 1 r a s than the u n i n f e c t e d c e l l s as i n d i c a t e d by the more i n t e n s e l y l a b e l l e d band ( F i g . 3.2, lanes 1 and 5). Immunoprecipitation of the 3 2 P i l a b e l l e d i n f e c t e d c e l l s showed the presence of a s p e c i f i c band that comigrated with the more slowly m i g r a t i n g r 3 5 L S]methionine l a b e l l e d band ( F i g . 3.2, lanes 3 and 7) as i s c h a r a c t e r i s t i c of the phosphorylated v i r a l ras product (Papageorge et al., 1982). Ki-MSV p 2 1 v " r a s , s p e c i f i c a l l y i d e n t i f i e d by i t s a b i l i t y to be l a b e l l e d with 3 2 P i , was produced i n a l l three e a r l y passage c u l t u r e s of the i n f e c t e d adrenal c e l l s under both serum c o n d i t i o n s , r e g a r d l e s s of the r e l a t i v e degree of t r a n s f o r m a t i o n or the phenotype of the adrenal cortex c e l l s . The [ 3 5 S ] m e t h i o n i n e l a b e l l e d bands of p 2 1 r a s were excised from the g e l and subjected to s c i n t i l l a t i o n counting (Table 3.1). The amount of v i r a l F i g u r e 3.2 A n a l y s i s of p 2 1 r a s expression i n KiMSV-infected and u n i n f e c t e d r a t adrenal cortex c e l l s . Immediately f o l l o w i n g photography the 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 with [ 3 5 s ] m e t h i o n i n e (lanes 1, 2, 5, 6, 9 , 10) or with 3 2 P i (lanes 3 , 4, 7, 8) i n 0 . 3 ml Dulbecco's modified Eagle's medium l a c k i n g methionine or phosphate r e s p e c t i v e l y and supplemented with 5$ FBS f o r the c e l l s p r e v i o u s l y grown i n 25$ FBS (lanes 1-4 ,9,10) or 3$ HS (lanes 5-8) f o r the 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 , normalized 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 -p 2 1 r a s monoclonal antibody Y13-259 (lanes 1, 3 , 5, 7, 9) or normal r a t serum (lanes 2, 4, 6, 8, 10). The immunoprecipitates were analyzed 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 Methods. 64 65 TABLE 3. 1 [ 3 5 S ] I n c o r p o r a t i o n i n t o p 2 1 r a s i n Ki-MSV-infected and Uninfected Adrenal Cortex C e l l s Passage Serum Ki-MSV [ 3 5 S ] c o u n t s counts/minute number supplements i n f e c t i o n per minute c o r r e c t e d f o r i n band t o t a l i n c o r p o r a 2 a FBS + 675 -HS + 681 -FBS - 295 -3 a FBS + 936 -HS + 474 573 FBS - 280 -30 b FBS + 1 201 1201 HS + 238 954 3 b FBS + 81 1 1286 2b FBS - 403 403 Blank _ _ 58 58 Table 3. 1 The areas of the g e l s shown i n F i g u r e s 3.2 and 3-5 corresponding to p 2 1 r a s were excised and placed 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 counted f o r t 3 ^ S ] m e t h i o n i n e . The l y s a t e f o r the immunoprecipitations shown i n F i g u r e 3.5 f o r the c e l l s i n HS at passage 30 or FBS at passage 3 contained fewer counts than the c e l l s i n FBS at passage 30 and so the counts 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 ] counts i n p21 on the g e l shown i n F i g u r e 3.2. [ 3 5 S ] counts i n p 2 1 r a s on the g e l shown i n F i g u r e 3.4. p21 was approximately the same i n both c u l t u r e c o n d i t i o n s at each passage. There were some d i f f e r e n c e s w i t h i n experiments (6 experiments on 3 c u l t u r e s ) , but no c o n s i s t e n t b i a s i n the l e v e l of p 2 1 r a s production between the c u l t u r e c o n d i t i o n s was noted. Another band (M r " 27,000) was a l s o s p e c i f i c a l l y immunoprecipitated by the Y13-259 a n t i - p 2 1 r a s monoclonal antibody i n the [ 3 S]methionine l a b e l l e d c u l t u r e s ( F i g u r e 3.2) and i s d i s c u s s e d f u r t h e r i n chapter 7. 3.3 V i r a l p 2 1 r a 3 Expression i n P a r t i a l l y and F u l l y  Transformed Ki-MSV-infected Rat Adrenal Cortex C e l l s . As the Ki-MSV-infected adrenal cortex c e l l s are passaged i n the presence of 25% FBS the c u l t u r e g r a d u a l l y a c q u i r e s a transformed phenotype (Auersperg and Calderwood, 1984). To determine i f the p r o g r e s s i o n to a f u l l y transformed phenotype could be c o r r e l a t e d with any change i n the l e v e l of v i r a l p 2 1 r a s expression, an adrenal cortex c u l t u r e was examined at passage t h r e e , two passages a f t e r i n f e c t i o n with Ki-MSV, at which stage few c e l l s appeared m o r p h o l o g i c a l l y transformed ( F i g . 3-3 C), and again at passage 30, when the c u l t u r e had become f u l l y transformed and serum independent f o r growth ( F i g . 3.3 A and B). The c u l t u r e s shown i n F i g u r e 3.3 were r a d i o l a b e l l e d with [ 3 5 S ] m e t h i o n i n e or 3 2 P i and the l a b e l l e d c e l l l y s a t e s were subjected to immunoprecipitation with anti-p21 monoclonal antibody as d e s c r i b e d b e f o r e . The l e v e l of v i r a l p 2 1 r a s expression as r e v e a l e d by 3 2 P i l a b e l l i n g was e s s e n t i a l l y the same i n a l l three c u l t u r e s F i g u r e 3-3. Morphology of KiMSV-infected r a t adrenal cortex c e l l s at e a r l y and l a t e passages (100X). Rat adrenal cortex c e l l s i n f e c t e d with KiMSV at the f i r s t passage were c a r r i e d u n t i l passage 30, by which time the c e l l s had acquired a f u l l y transformed phenotype and were serum-independent f o r growth. The KiMSV-infected c e l l s at passage 30 (29 passages 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 the presence of 25% FBS (A) or 3% HS (B). The c e l l s used i n t h i s experiment were from the same o r i g i n a l c u l t u r e shown i n Fig u r e 1. An a l i q u o t of the same KiMSV-infected adrenal cortex c e l l s f r o z e n i n the f i r s t passage a f t e r i n f e c t i o n was thawed, grown i n 25% FBS and examined at passage 3 (C) at the same time as the c u l t u r e s i n passage 30. 68 F i g u r e 3 . 4 . E x p r e s s i o n of v i r a l p 2 1 r a s i n e a r l y and l a t e passage c u l t u r e s of KiMSV-infected adrenal cortex 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 r a s : KiMSV-infected r a t adrenal cortex c e l l s (TrA) at passage 30 (lanes 1 - 6 ) or passage 3 (lanes 7 - 9 ) c u l t u r e d i n 25$ FBS (lanes 1 - 4 , 7-9)or 3$ HS (lanes 5 and 6 ) . Uninfected adrenal cortex c e l l s (AC) were a l s o analyzed (lanes 10 and 11). 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 with ^2Pi (lanes 1, 2, 5, 7 ) or [ 3 5 S ] -methionine (lanes 3, 4 , 6 , 8-11). 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 equal 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 r a s Y13-259 antibody (lanes 1> 3 » 5-8, 10) or with normal r a t serum (lanes 2, 4 , 9, 11); the l y s a t e f o r lane 6 contained only 20$ of the TCA-i n s o l u b l e [-"Si methionine counts per minute compared to other [ 3 5 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 analyzed by e l e c t r o p h o r e s i s on a 12.5$ SDS-polyacrylamide 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. TrA AC passage •< P-30 p-3 • serum FBS HS FBS 1 -2 3 4 5 6 7 8 9 1011 - 205 - 97 - 68 - 45 •30 - 24 ( F i g . 3.4 lanes 1, 5 and 7). The t o t a l s y n t h e s i s of p 2 1 r a s was determined by [ 3 5 s ] methionine l a b e l l i n g and appeared to be the same i n both the e a r l y and l a t e passage Ki-MSV-i n f e c t e d adrenal cortex c e l l s when normalized f o r the r e l a t i v e r a t e s of t 3 5 S ] methionine i n c o r p o r a t i o n ( F i g . 3-4 lanes 3, 6 and 8 and t a b l e 3-1). The t o t a l l e v e l of [ 3 5 s ] -methionine l a b e l l e d p 2 1 r a s i n Ki-MSV-inf ected adrenal cortex c e l l s was approximately f i v e - f o l d g r e a t e r than the l e v e l of endogenous p 2 1 r a s i n corresponding u n i n f e c t e d adrenal c e l l s ( F i g . 3-4 lane 10) as q u a n t i t a t e d by the counting of excised bands. The in c r e a s e d l e v e l of p 2 1 r a s i n v i r u s i n f e c t e d c e l l s was presumably due to the expression of the v i r a l ras gene. These r e s u l t s suggested that p r o g r e s s i o n of Ki-MSV-i n f e c t e d adrenal cortex c e l l s to the f u l l y transformed phenotype was not accompanied by enhanced production of v i r a l p 2 1 r a s . 72 3.4 DISCUSSION The data presented here suggest that the expression of Ki-MSV-encoded p 2 1 r a s i s not of i t s e l f s u f f i c i e n t to induce the acute t r a n s f o r m a t i o n of f r e s h l y c u l t u r e d r a t adrenal cortex c e l l s . E a r l y passage c e l l s i n f e c t e d with Ki-MSV and expressing l e v e l s of v i r a l p 2 1 r a s t y p i c a l of Ki-MSV-transformed c e l l l i n e s can r e t a i n t h e i r serum s e n s i t i v i t y and r e v e r t to a slow-growing, normal e p i t h e l i a l phenotype i n the presence of HS even a f t e r the i n i t i a l appearance of transformed f o c i . The r e v e r t e d c e l l s c o n t i n u e to express high l e v e l s of v i r a l p 2 1 r a s d e s p i t e t h e i r a pparently normal e p i t h e l i a l , s t a t i o n a r y phenotype. I t would appear that the medium serum supplement does not a f f e c t the l e v e l of expression of v i r a l p 2 1 r a s , but r a t h e r that the l i m i t e d t r a n s f o r m i n g a c t i v i t y of Ki-MSV p 2 1 r a s i n e a r l y passage adrenal cortex c e l l s r e q u i r e s the presence of a f a c t o r ( s ) i n FBS. As suggested by previous experiments (Auersperg and Calderwood, 1984 ; Auersperg e_t a l . , 1981; H a r r i s o n and Auersperg, 1981) i t seems l i k e l y that a growth f a c t o r ( s ) i n FBS which i s absent from HS f u n c t i o n a l l y cooperates with p 2 1 r a s i n focus formation. The f u l l t r a n s f o r m a t i o n of adrenal cortex c u l t u r e s a f t e r many passages i n FBS was not a p p a r e n t l y accompanied by i n c r e a s e d expression of p21 . Both e a r l y and l a t e passage Y* 3. S c e l l s s y n t h e s i z e s i m i l a r amounts of Ki-MSV p21 , suggesting that the development of serum i n s e n s i t i v i t y i n l a t e passage c e l l s r e q u i r e s a step or steps i n a d d i t i o n to expression of the v i r a l ras product. T h i s p r o g r e s s i o n to an o v e r t l y transformed phenotype may i n v o l v e c e l l u l a r g e n e t i c or e p i g e n e t i c changes r e s u l t i n g i n a c t i v a t i o n of a c o o p e r a t i n g c e l l u l a r gene, the expression of tumour growth f a c t o r s or l o s s of the a b i l i t y to suppress the transformed phenotype. Klempnauer et _al . (1984) have shown that the d i f f e r e n t i a t i o n of avian m y e l o b l a s t o s i s v i r u s transformed myeloblasts to macrophages induced by phorbol ester i s accompanied by a r e d i s t r i b u t i o n of the p 4 5 v - m v ^ transfo r m i n g p r o t e i n from the nucleus to the cytoplasm of the c e l l . A s u b c e l l u l a r r e d i s t r i b u t i o n of p 2 1 r a s a p p a r e n t l y accompanies the p r o g r e s s i o n of t r a n s f o r m a t i o n i n r a t kidney c e l l s (Myrdal and Auersperg, 1985). Thus, a change i n v i r a l p 2 1 r a s a c t i v i t y more s u b t l e than simple v a r i a t i o n i n expression may be important i n the a l t e r e d phenotypes of the i n f e c t e d adrenal cortex c e l l s . The o b s e r v a t i o n s reported here suggest that the a c t i o n of an oncogenic p 2 1 r a s can be dependent on environmental f a c t o r s which i n f l u e n c e the d i f f e r e n t i a t e d phenotype of the c e l l . T h i s dependence can be r e l i e v e d by as yet unknown events, presumably i n v o l v i n g the a l t e r a t i o n of a c e l l u l a r gene, which can r e s u l t i n a f u n c t i o n a l complementation of the t r a n s f o r m i n g a c t i v i t y of v i r a l p 2 1 r a s . The e f f e c t s of v i r a l ras oncogenes i n a v a r i e t y of primary c e l l types from d i f f e r e n t t i s s u e s have been i n v e s t i g a t e d and some Ki-MSV-i n f e c t e d c e l l s can s t i l l respond to exogenous s i g n a l s by d i f f e r e n t i a t i n g ( P i e r c e and Aaronson, 1985), while others cannot (Yuspa et _al. , 1985 ). I t would appear that some types of c e l l s may r e q u i r e a coope r a t i n g oncogene i n a d d i t i o n to v-r as f o r t r a n s f o r m a t i o n (Rhim et aJL. , 1985 ; Yoakum et _a l . , 1985), but others may be transformed by ras alone under s p e c i f i c c o n d i t i o n s (Land et al.. , 1986; Spandidos and W i l k i e , 19 84). I t has been demonstrated that h i g h l y ov er expr ess ed , a c t i v a t e d ras genes can transform primary c e l l s , and that tumourigenic v a r i a n t s of some l i n e s c o n t a i n over expr ess ed ras genes (Winter and Perucho, 1986), but a c q u i s i t i o n of a h i g h l y transformed phenotype by the a d r e n o c o r t i c a l c e l l s does not appear to c o r r e l a t e with a f u r t h e r i n c r e a s e i n expression of the v-ras gene. These r e s u l t s f u r t h e r demonstrate the requirement f o r s e v e r a l a c t i v a t i n g steps i n normal primary c e l l s to g i v e f u l l t r a n s f o r m a t i o n . The p o s s i b i l i t y that the c e l l u l a r K i - r a s gene may be a c t i v a t e d i n some adrenal cortex tumours and c o n t r i b u t e to malignant p r o g r e s s i o n i i i v i v o i s supported by both the a m p l i f i c a t i o n of the K i - r a s 2 gene and i t s g r e a t l y i n c r e a s e d expression i n the Y-1 a d r e n o c o r t i c a l tumour de r i v e d c e l l l i n e (Schwab et a l . , 1983). CHAPTER 4 4.0 Transformation of Rat Adrenal Cortex C e l l s by r„as. and  mvc: Evidence f o r the Requirement f o r a Fu r t h e r C e l l u l a r Chang e 4.1 INTRODUCTION The r e s u l t s d e s c r i b e d i n the previous chapter had i n d i c a t e d a requirement f o r c e l l u l a r changes to cooperate with the expression of the v i r a l ras oncogene i n the t r a n s f o r m a t i o n of the adrenal cortex c e l l s . I t has been demonstrated i n other systems examining e a r l y passage, nonimmor t a l i z ed c e l l s that some v i r a l and c e l l u l a r oncogenes can complement the a c t i v i t y of an oncogenic ras to r e s u l t i n t r a n s f o r m a t i o n when e i t h e r oncogene alone could not (Land et a l . , 1 983a; Ruley, 1 983 ). The myc oncogene appears to be ab l e to cooperate with ras i n transfo r m i n g a v a r i e t y of c e l l s (Murray et E Q . , 1 983 ; Taya et a l . , 1984 ), although there can be exceptions (Jenuwein et a l . , 1985) and these oncogenes have come to be considered r e p r e s e n t a t i v e of two coo p e r a t i n g c l a s s e s of oncogenes. I t would appear that t r a n s f o r m a t i o n of c e l l s by ras alone or ras and myc together i n v i v o (Oshimura _et a_l. , 1985 ; Thomassen et al_. , 1 985 ) may r e q u i r e a f u r t h e r c e l l u l a r change. There a l s o appears to be a requirement f o r a f u r t h e r step i n the t r a n s f o r m a t i o n of hematopoietic c e l l s _in v i t r o (Stevenson and Volsky, 1986 ; Vogt et j a l . , 1 986), but th e r e has not been a s i m i l a r requirement d e s c r i b e d f o r f i b r o b l a s t s . The adrenal cortex c e l l s , because of t h e i r d e r i v a t i o n from an a d u l t , hormone producing e p i t h e l i u m , represent a u s e f u l system f o r the examination of t r a n s f o r m a t i o n . I t i s s t i l l of i n t e r e s t that a change i n the c o n c e n t r a t i o n of the medium serum supplement can lead to both morphological as w e l l as f u n c t i o n a l and growth r e l a t e d changes. The requirement f o r a high serum supplement to allow expression of the Ki-MSV-induced morphological a l t e r a t i o n s i n the adrenal cortex c e l l s has been used to d e f i n e a f u r t h e r c e l l u l a r change r e q u i r e d f o r t r a n s f o r m a t i o n . The a c q u i s i t i o n of a serum independent transformed phenotype by the Ki-MSV- i n f e c t e d a d r e n o c o r t i c a l c e l l s has been c l o s e l y a s s o c i a t e d with the a b i l i t y to grow i n s o f t agar and a h i g h l y tumourigenic phenotype (Auersperg et a_l. , 1 986 ). The oncogenes used i n these experiments were introduced by r e t r o v i r u s e s as t h i s allows the i n t r o d u c t i o n of a f o r e i g n gene i n a r e l a t i v e l y g e n t l e and e f f i c i e n t manner. The v i r u s e s used were: Ki-MSV(Mo-MLV) ( K i r s t e n and Mayer, 1967), c o n t a i n i n g an a c t i v a t e d v-ras gene; and MMCV(Mo-MLV), c o n t a i n i n g an avian v-myc gene cloned i n t o a murine r e t r o v i r u s c o n s t r u c t (from B. Vennstrom) (Vennstrom et a l . , 1984) (see F i g u r e 4.1). Ki-MSV and MMCV were pseudotyped with the same helper v i r u s , 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 the two v i r u s e s to i n t r o d u c e the two oncogenes i n t o the a d r e n o c o r t i c a l c e l l s . P r o d u c t i v e i n f e c t i o n by r e t r o v i r u s e s r e s u l t s i n a block to r e i n f e c t i o n by the same helper v i r u s . Each l i n e of F i g u r e 4.1. Diagram of the p r o v i r a l form of MMCV (taken 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 of MMCV i s diagrammed showing the r e s t r i c t i o n s i t e s used. The 2.5 Kb BamHI fragment d e r i v e d from the OK-10 v i r u s i s i n d i c a t e d . The remainder of the v i r a l genome i s de 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 le n g t h and i s shown approximately to s c a l e . Hind III BamM LTR lll E figure 4.1 OK-TO fragment v-myc BamM I I LTR a d r e n o c o r t i c a l c e l l s was e s t a b l i s h e d as a t i s s u e explant from the adrenal glands of a s i n g l e r a t by p r e v i o u s l y d e s c r i b e d methods ( S l a v i n s k i et a_l. , 1974). Each l i n e was then maintained and s t u d i e d s e p a r a t e l y so that any v a r i a b i l i t y between the l i n e s would be apparent. In examining t r a n s f o r m a t i o n of the adrenal cortex c e l l s by ras and myc the a b i l i t y to form c o l o n i e s i n s o f t agar was chosen as the end p o i n t f o r t r a n s f o r m a t i o n i n v i t r o as t h i s i s thought to represent the expression of a h i g h l y tumourigenic phenotype. There i s c o n s i d e r a b l e precedent f o r the a s s o c i a t i o n of s o f t agar growth and t r a n s f o r m a t i o n and i t appears to be the phenotype i n v i t r o that i s most s t r o n g l y a s s o c i a t e d with t u m o u r i g e n i c i t y (Kahn and Shin, 1979; C i f o n e and F i d l e r , 1980). R ESULTS 4.2 In d u c t i o n of Focus Formation and Mo r p h o l o g i c a l  Transformation by Ki-MSV and MMCV. The r a t adrenal cortex c e l l l i n e s were i n f e c t e d i n second passage with Ki-MSV alone, MMCV alone or Ki-MSV and MMCV together. The morphological responses of the c e l l s to the v a r i o u s i n f e c t i o n s are shown i n F i g u r e 4.2. As can be seen i n the photographs both Ki-MSV and MMCV were ab l e to produce f o c i of a l t e r e d c e l l s by themselves. MMCV-infection produced c e l l s that had a more e p i t h e l i o i d morphology than the normal c e l l s , being more polygonal i n shape. This i s s i m i l a r to changes that have been d e s c r i b e d i n a NRK c e l l l i n e expressing elevated l e v e l s of c-myc (Stern _et a l . , 1 986 ). Ki-MSV i n f e c t i o n r e s u l t e d i n the appearance of c e l l s that showed an elongated, r e f r a c t i l e morphology c h a r a c t e r i s t i c of v-ras induced t r a n s f o r m a t i o n , but the number of f o c i induced was much s m a l l e r than the number of c e l l s i n f e c t e d (Table 4.1 and 4.2). The f o c i that r e s u l t e d from Ki-MSV i n f e c t i o n were smal l and the morphological a l t e r a t i o n s r e l a t i v e l y s u b t l e . The Ki-MSV/MMCV-infected c e l l s showed s i m i l a r but more pronounced morphological a l t e r a t i o n s when compared to the c e l l s i n f e c t e d with Ki-MSV alone. The c o - i n f e c t i o n of the c e l l s with Ki-MSV and MMCV r e s u l t e d i n the formation of a g r e a t e r number of f o c i (Table 4.1) and c e l l s that were more rounded and r e f r a c t i l e than e i t h e r v i r u s alone. There was some v a r i a b i l i t y between l i n e s i n the number of f o c i t h a t were formed i n the Ki-MSV/MMCV-infected F i g u r e 4.2. Focus formation i n e a r l y passage r a t adrenal cortex c e l l s i n f e c t e d with Ki-MSV, MMCV or both. Rat adrenal c o r t e x c e l l s were thawed and passaged twice 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 at 1X10^ and i n f e c t e d when 50$ c o n f l u e n t with Ki-MSV (A), MMCV (B) or K i -MSV/MMCV (C) at m.o.i.'s of 2-4 i n a 60mm d i s h . The c e l l s were then maintained i n 25$ FBS u n t i l examination. The photomicrographs (100X) were taken 7 days a f t e r i n f e c t i o n . TABLE 4.1 Focus Formation i n Rat Adrenal Cortex 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 C e l l L i n e Number of Foci/60 mm Dish Ki-MSV/MMCV Ki-MSV MMCV. A B C E F 25 .5 5.5 1 2 1.01 15.5 0 0 0 6 1 - (10) - (2) - (3) - (5) - (0) Rat adrenal cortex c e l l s were i n f e c t e d (as d e s c r i b e d i n the legend to F i g u r e 4.2) i n second passage with e i t h e r Ki-MSV, MMCV or both. The 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 while each of the 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 appeared 3 to 5 days a f t e r i n f e c t i o n and were counted 5 to 7 days a f t e r i n f e c t i o n . F o c i of 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 the c u l t u r e s i n f e c t e d with MMCV alone, but the morphology was d i s t i n c t from that seen i n the c u l t u r e s i n f e c t e d with K i -MSV/MMCV or Ki-MSV alone. The number of f o c i formed i n the MMCV-infected c u l t u r e i s shown i n brackets to i n d i c a t e the a b i l i t y of the c e l l s to respond to t h i s v i r u s . c u l t u r e s ( T a b l e 4.1), and the t r a n s f o r m a t i o n by Ki-MSV/MMCV of the R EF 52 c e l l l i n e , which a l s o r e q u i r e s c o o p e r a t i n g oncogenes, was c o n s i d e r a b l y more e f f i c i e n t . To analyze the ba s i s of these r e s u l t s the number of a d r e n o c o r t i c a l c e l l s l i k e l y to be c o i n f e c t e d by Ki-MSV and MMCV was estimated. The number of c e l l s i n f e c t e d by Ki-MSV i n each l i n e was determined by an i n f e c t i o u s c e n t r e assay and the probable number of c e l l s i n f e c t e d with MMCV was estimated by using the r e l a t i v e t i t r e s of the two v i r u s stocks determined on the rat-2 c e l l l i n e . The number of c e l l s l i k e l y to be c o i n f e c t e d was then c a l c u l a t e d . As can be seen (Table 4.1 and 4.2) the e f f i c i e n c y of i n f e c t i o n was lowest i n the c u l t u r e s i n which focus formation was lowest. The a c t u a l number of f o c i was 5-10X higher than the number p r e d i c t e d from the estimated number of c o - i n f e c t e d c e l l s (Table 4.1), which suggested that the coexpression of myc and ras was s u f f i c i e n t to r e s u l t i n morphological t r a n s f o r m a t i o n . The di s c r e p a n c y between the p r e d i c t e d and a c t u a l number of f o c i presumably a r i s e s from experimental e r r o r s and i n a c c u r a c i e s i n the assumptions r e q u i r e d f o r c a l c u l a t i o n s , but th e r e does not appear to be a l a r g e number of c o - i n f e c t e d c e l l s that are unable to form f o c i i n t h i s system, although t h e i r presence can not be s t r i c t l y r u l e d out. I t can be seen i n F i g u r e 4.3 that the v i r a l l y i n f e c t e d a d r e n o c o r t i c a l c e l l s had a s e l e c t i v e advantage over the un i n f e c t e d c e l l s , as the c u l t u r e s showed an i n c r e a s i n g 85 TABLE 4 .2 I n f e c t i o u s Centre Assay of Ki-MSV/MMCV-infected Adrenal Cortex C e l l s Adrenal C u l t u r e V i r u s I n f e c t i o n % C e l l s Estimated Number In f e c t e d of F o c i / 1 0 6 with Ki-MSV C e l l s A K/M 0.04 2 K 0.02 B K/M 0.005 1 K 0.005 K/M K 0.005 0.03 K/M K 0.12 0.11 20 Rat 2 K 25.0 K= Ki-MSV M= MMCV C e l l s from the experiment shown i n Table 4.1 were taken at the end of the passage i n which they 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, at 10 2, 10-* and 10 c e l l s . The c e l l s were allowed to a t t a c h f o r 8 to 10 hours and then the medium was removed and re p l a c e d with a 0.6% agar o v e r l a y . A p o s i t i v e c o n t r o l of Rat 2 c e l l s i n f e c t e d with Ki-MSV as de s c r i b e d f o r i n f e c t i o n of the adrenal cortex c e l l s (Table 4.1) was i n c l u d e d to monitor the e f f i c i e n c y of the assay. The f o c i were counted 13 to 14 days a f t e r p l a t i n g the c e l l s . Adrenal cortex c e l l s i n f e c t e d with MMCV alone d i d not form any d e t e c t a b l e f o c i i n t h i s assay. The estimated number of 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 the p r o b a b i l i t y of a c e l l being 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 then m u l t i p l y i n g by 10 6 as the number of c e l l s i n a d i s h at the time of i n f e c t i o n . This assumes that the presence of one v i r u s does not i n t e r f e r e with the i n f e c t i o n of the other and that the t i t r e s of the two v i r u s e s are the same. F i g u r e 4.3. Overgrowth of i n f e c t e d adrenal 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 transformed 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 passaged and maintained i n 25% FBS. The photomicrographs (100X) shown here were taken i n the second passage a f t e r i n f e c t i o n . A) Ki-MSV-infected adrenal cortex c e l l s ; B) MMCV-inf ect ed adrenal cortex c e l l s ; C) K i -MSV/MMCV-infected adrenal cortex c e l l s . The morphological t r a n s f o r m a t i o n seen i n the 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 not r e f l e c t e d i n the a b i l i t y of the c e l l s to grow i n s o f t agar i n the course of t h i s experoment which was the parameter used to d e f i n e a f u l l y transformed phenotype. 87 number of 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 with f u r t h e r passaging. The i n c r e a s e i n v i r a l l y i n f e c t e d c e l l s i s u n l i k e l y to occur by v i r u s spread as expression of the helper v i r u s r e s u l t s i n a block to s u p e r i n f e c t i o n . 4.3 The Expression of a Transformed Morphology and Growth by  Ki-MSV/MMCV-infected A d r e n o c o r t i c a l C u l t u r e s Requires a High  Con c e n t r a t i o n of Serum. Previous work on the t r a n s f o r m a t i o n of r a t adre n a l cortex c e l l s by Ki-MSV had shown that the expression of the transformed morphology and growth e a r l y a f t e r i n f e c t i o n r e q u i r e d the presence of a high c o n c e n t r a t i o n of serum (10 to 25% FBS) i n the medium (Auersperg and Calderwood, 1984). I f the Ki-MSV-inf ect ed c e l l s were grown i n the presence of low serum (1 to 5% HS) they r e v e r t e d to a normal, e p i t h e l i a l - l i k e morphology. S i n c e the i n t r o d u c t i o n of myc i n t o c e l l s has been a s s o c i a t e d with a r e d u c t i o n i n the serum requirement f o r growth (Armelin e i a l . , 1 984 ; Keath et al.. , 1985), the c o i n f e c t e d l i n e s were examined f o r the a b i l i t y to grow and express a transformed morphology i n the presence of a low serum supplement (5% HS). As i s c l e a r l y demonstrated i n F i g u r e 4.4 the K i -MSV/MMCV-inf ect ed l i n e s s t i l l r e q u i r e d the presence of a high serum supplement to express the a l t e r a t i o n s i n morphology and growth a s s o c i a t e d with c o - i n f e c t i o n . A s i m i l a r requirement f o r serum was a l s o seen i n both s i n g l y i n f e c t e d l i n e s . A d d i t i o n of FBS to 25% r e s u l t e d i n the appearance of the morphology c h a r a c t e r i s t i c 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 of the transformed morphology induced by ras and myc i n r a t adre n a l cortex c e l l s . Three passages a f t e r i n f e c t i o n the 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 with Ki-MSV and MMCV were p l a t e d i n DMEM with 25% FBS and a f t e r 24 hours were s h i f t e d i n t o DMEM with 3% HS on the a p p r o p r i a t e p l a t e s and the photomicrographs (40X) were taken 7 days l a t e r a f t e r being maintained i n e i t h e r 25% FBS (A) or 3% HS (B). The c e l l s i n the 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 transformed morphology that was d i s t i n g u i s h a b l e from the morphologies of the s i n g l y i n f e c t e d c u l t u r es. 90 Figure 4.5 Serum dependence of the exp r e s s i o n of the a l t e r e d morphology i n Ki-MSV/MMCV-infected r a t adrenal cortex c e l l s . Two passages a f t e r i n f e c t i o n the Ki-MSV/MMCV-i n f e c t e d a d r e n a l cortex c e l l s were p l a t e d i n DMEM with 25% FBS and a f t e r 24 hours a l l c u l t u r e s were s h i f t e d i n t o DMEM with 3% HS. Photomicrographs (100X) were taken a f t e r one week (1, 3) and the areas photographed were marked. The medium was then made up to 25% FBS and a f t e r a f u r t h e r seven days the same areas were again photographed (2,4 r e s p e c t i v e l y ) . were grown i n 25% FBS, d e s p i t e the continued presence of 5% HS ( F i g u r e 4.5), i n d i c a t i n g that the absence of FBS r a t h e r than the presence of any f a c t o r s i n HS led to a mo r p h o l o g i c a l l y normal phenotype, as i s a l s o the case f o r the Ki-MSV-inf ect ed a d r e n o c o r t i c a l c e l l s (Auersperg et a l . , 1981). 4.4 A d d i t i o n of a P u r i f i e d Growth Fa c t o r can Replace the Requirement f o r a High Serum Supplement C o n c e n t r a t i o n i n K i - MSV/MMCV-infected C e l l s . C u l t u r i n g the Ki-MSV/MMCV-infected a d r e n o c o r t i c a l c e l l s i n low c o n c e n t r a t i o n s of FBS (1 to 5%) or c a l f serum (_< 10$) gave a s i m i l a r m o r p h o l o g i c a l r e v e r s i o n as s u b - c u l t u r i n g of the c e l l s i n t o 5% HS. This r e s u l t suggested that the a c t i v e component of serum might be the growth f a c t o r s . In an attempt to address t h i s question d i r e c t l y c u l t u r e s i n f e c t e d with Ki-MSV, MMCV or Ki-MSV/MMCV were p l a t e d and maintained i n 5% HS u n t i l they had reached c o n f l u e n c e . P u r i f i e d growth f a c t o r s (EGF, PDGF, FGF and IFG-II) were then added i n the presence of 5% HS e i t h e r alone or i n v a r i o u s combinations (see legend to F i g u r e 4.6). Of these growth f a c t o r s only EGF appeared to be abl e to induce a morphological response i n the Ki-MS V/MMC V - i n f ect ed c u l t u r e s s i m i l a r to that seen i n response to the a d d i t i o n of FBS. I n t e r e s t i n g l y , none of the growth f a c t o r s alone or i n combination i n c l u d i n g EGF appeared to be ab l e to mimic the e f f e c t s of FBS on the c e l l s i n f e c t e d with e i t h e r Ki-MSV or MMCV alone. These r e s u l t s suggested that EGF or an EGF-like a c t i v i t y might be the a c t i v e component of FBS that was F i g u r e 4.6. Mo r p h o l o g i c a l response of Ki-MSV/MMCV-infected adrenal cortex c e l l s to p u r i f i e d growth f a c t o r s . K i -MSV/MMCV-infected adrenal cortex c e l l s three passages a f t e r i n f e c t i o n , before c e l l s were cloned, were p l a t e d i n DMEM with 25$ FBS and a f t e r 24 hours were s h i f t e d i n t o DMEM + 5% HS and maintained i n low serum f o r two weeks. At the end of two weeks PDGF (10 ng/ml f i n a l ) , EGF (0.5 nM f i n a l ) and IGF-II (10 nM f i n a l ) were added to the c e l l s i n DMEM + 5% HS. One d i s h had f r e s h DMEM with 5% HS added and another was s h i f t e d to DMEM with 25$ FBS and f r e s h medium was added at the same time as f u r t h e r growth 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 growth f a c t o r s at 1/2 the i n i t i a l c o n c e n t r a t i o n was added at 24 and 48 hours a f t e r the i n i t i a l treatment and again at 1/10 the 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 the f i r s t growth f a c t o r a d d i t i o n . The photomicrographs were taken two weeks a f t e r the f i n a l a d d i t i o n of growth f a c t o r s . A l l p o s s i b l e combinations of the three growth f a c t o r s have been t r i e d and a r e p r e s e n t a t i v e s e l e c t i o n of photographs are shown EGF (A), 25$ FBS (B), PDGF + IGF-II (C), IGF-II (D), 5$ HS (E) and PDGF ( F ) . FGF has a l s o been t r i e d but f a i l e d to e l i c i t a s i g n i f i c a n t response e i t h e r alone and with PDGF or IGF-II. The r e s u l t s are not shown. 95 r e q u i r e d f o r ras and myc induced morphological t r a n s f o r m a t i o n of the a d r e n o c o r t i c a l c e l l s . 4. 5 A c q u i s i t i o n of Serum Independent Growth and Anchorage  Independent Growth. Anchorage independent growth was the i n  v i t r o parameter used to monitor the expression of a f u l l y transformed phenotype. The Ki-MSV/MMCV-infected c e l l s d i d not show any s i g n i f i c a n t a b i l i t y to form c o l o n i e s i n s o f t agar u n t i l the c u l t u r e s were ab l e to grow and maintain a transformed morphology i n low serum. In t h r e e of the four l i n e s i t was p o s s i b l e to demonstrate that expression of reduced serum requirements and anchorage independent growth, was c o i n c i d e n t (Table 4.3). The f o u r t h l i n e was not t e s t e d f o r colony formation i n the passage i n which i t appeared to be serum independent, but was p o s i t i v e f o r s o f t agar growth i n the next passage. The c e l l s were a b l e to form c o l o n i e s i n s o f t agar i n both high and low serum as soon as anchorage independent growth was expressed. These r e s u l t s i n d i c a t e d that ras and myc were not s u f f i c i e n t to r e s u l t i n the immediate t r a n s f o r m a t i o n of the e a r l y passage a d r e n o c o r t i c a l c e l l s as r e f l e c t e d i n anchorage independent growth. The delay i n the appearance of anchorage independent growth suggested some f u r t h e r c e l l u l a r change was r e q u i r e d b e f o r e the c e l l s were ab l e to express a l l of the c h a r a c t e r i s t i c s a s s o c i a t e d with a transformed phenotype. The a d d i t i o n of exogenous growth f a c t o r s i n the presence of high or low serum d i d not s t i m u l a t e colony formation i n s o f t agar before the c e l l s became serum independent, even though EGF was 97 TABLE 4.3 Number of Passages Between I n f e c t i o n and Expression of Serum Independence and Anchorage Independent Growth Lin e Serum Independence Anchorage Independence A 5 6« C 4 4 E 4 4 F 6 6 Serum independence was assayed at each passage a f t e r i n f e c t i o n by s w i t c h i n g the medium from 25% FBS to 5% HS a f t e r the c e l l s had attached. Serum independence of the K i -MSV/MMC V - i n f ect ed adrenal cortex c e l l s was assessed by growing the c e l l s i n 3-5% HS and examining the c e l l u l a r morphology and growth p a t t e r n . At the end of each passage 1fj5 c e l l s were p l a t e d i n s o f t agar i n a 60 mm d i s h and c u l t u r e d f o r 2 to 3 weeks. The expression of anchorage independent growth was taken as the f i r s t passage i n which any c o l o n i e s were d e t e c t a b l e by mic r o s c o p i c examination. In each c u l t u r e t h e r e were no countable c o l o n i e s un,til the passage i n d i c a t e d i n the t a b l e , at which time g r e a t e r than 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 under the colony counter. When the c o l o n i e s were r e a d i l y v i s i b l e they were counted using a colony counter. The adrenal cortex c u l t u r e s i n f e c t e d with e i t h e r Ki-MSV or MMCV did not form c o l o n i e s i n s o f t agar during the course of the experiment. "growth i n s o f t agar not checked at passage 5. s u f f i c i e n t to e l i c i t a transformed morphology. Neither of the s i n g l y i n f e c t e d l i n e s formed any c o l o n i e s i n s o f t agar during the course of these experiments ( F i g u r e 4 . 7 ) . 4.6 I s o l a t i o n of Transformed Lines from Ki-MSV/MMCV  In f e c t e d C u l t u r e s . To determine the o r i g i n of the s u s c e p t i b l e c e l l s and requirements f o r t r a n s f o r m a t i o n , c l o n a l l i n e s were i s o l a t e d from the transformed, c o - i n f e c t e d c u l t u r e s . These l i n e s were examined f o r the presence of the v i r a l oncogenes, or t h e i r products, which should r e f l e c t the requirements f o r i n i t i a t i o n and maintenance of the transformed s t a t e . The l i n e s were assayed f o r the a b i l i t y to m e t abolize a p r e c u r s o r of the s t e r o i d o g e n i c pathway to i n t e r m e d i a t e s , which i n d i c a t e d whether the c e l l s were d e r i v e d from the adre n a l cortex (Auersperg et a l . , 1 9 7 7 ; Wiebe et a l . , i n p r e s s ) . The f i r s t method used to i s o l a t e cloned l i n e s from the po p u l a t i o n of transformed c e l l s was to i s o l a t e c o l o n i e s from the s o f t agar assays and grow the l i n e s from the c o l o n i e s . The second method was based on the appearance of f o c i i n monolayers of Ki-MSV/MMCV-infected c e l l s maintained i n low serum. These f o c i arose a p p a r e n t l y spontaneously a f t e r the monolayers had been maintained i n 5% HS f o r 2 to 4 weeks ( F i g u r e 4 . 8 ) . Cloning c y l i n d e r s were used to i s o l a t e the c e l l s i n the f o c i and l i n e s were grown d i r e c t l y from the c e l l s i s o l a t e d . Spontaneous f o c i a l s o arose i n the c u l t u r e s F i g u r e 4.7. Anchorage independent growth of Ki-MSV/MMCV-i n f e c t e d a d r e n a l cortex c e l l s . The i n f e c t e d adrenal cortex c e l l s were assayed f o r anchorage independent growth by suspending 10^ c e l l s i n 3 mis of 0.35$ agarose i n DMEM with e i t h e r 25$ FBS or 5$ HS. The c e l l s were incubated f o r 2 to 3 weeks and the c o l o n i e s counted and photographed (40X). A) Ki-MSV/MMCV-infected c e l l s ; B) Ki-MSV-infected c e l l s ; and C) MMCV-infected c e l l s . A l l photomicrographs are of s o f t agar assays done i n the presence of 5$ HS, but the s u b s t i t u t i o n of 25$ FBS d i d not a l t e r the r e s u l t s . The s o f t agars from the 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 or smal l aggregates (approximately 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 areas on the dark background. The Ki-MSV/MMCV-infected 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. Formation of a spontaneous focus i n the presence of 5% HS. The Ki-MSV/MMCV-infected r a t adrenal cortex c e l l s were p l a t e d i n DMEM with 25% FBS and 24 hours l a t e r the medium was changed to DMEM with 5% HS. A f t e r three to fou r weeks f o c i arose 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 photomicrograph (100X) i n the f i g u r e was taken four weeks a f t e r the c u l t u r e was p l a t e d . 102 i n f e c t e d with Ki-MSV alone, but no transformed l i n e s could be i s o l a t e d from these spontaneous f o c i . 4.7 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  Lines f o r the Presence of the V i r a l Oncogenes and t h e i r  Products and S t e r o i d o g e n i c A b i l i t y . Rapid t r a n s f o r m a t i o n of the a drenal cortex c e l l s appeared to r e q u i r e the presence of both ras and myc. The i s o l a t e d c e l l l i n e s were examined f o r the presence of the two v i r a l oncogenes. The c e l l l i n e s were examined f o r the presence of v i r a l p 2 1 r a s by immunopr e c i p i t a t i o n of c e l l l y s a t e s a f t e r l a b e l l i n g in_ v i v o with J P i (Papageorge et a l . , 1982). This p r o v i d e s a simple method f o r demonstrating the presence of the v-ras product as a s p e c i f i c amino a c i d change allows the p 2 1 v - r a s to act as a phosphate acceptor, where the p 2 1 c - r a s does not (Shih et al., 1982a; Tsuchida et a l . , 1982). Nineteen of the twenty l i n e s examined contained the v i r a l p 2 1 r a s ( i l l u s t r a t i v e r e s u l t s shown i n F i g u r e 4.9). S i x t e e n of the l i n e s examined f o r the expression of v-ras have a l s o been probed f o r the presence of v-myc by Southern b l o t t i n g . The genomic DNA from both the cloned and uncloned, transformed a d r e n o c o r t i c a l l i n e s was d i g e s t e d with BamHI, which l i b e r a t e s a 2.5 k i l o b a s e fragment c o n t a i n i n g the v-myc gene from the i n t e g r a t e d p r o v i r u s , and probed with the a vian v-myc gene from the avian myelocytomatosis v i r u s , MC29 (Vennstrom et a_l_. , 1981). Of the s i x t e e n l i n e s examined a l l contained the avian v-myc gene seen as a 2.5Kb. Figure 4.9. A n a l y s i s of p 2 1 r a s e x p r e s s i o n i n Ki-MSV/MMCV-i n f e c t e d r a t adrenal cortex c e l l s . The transformed c e l l l i n e s from the Ki-MSV/MMCV-infected r a t adrenal cortex 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 with 10$ FBS and allowed to a t t a c h f o r 24 hours. The c e l l s were then l a b e l l e d with 300 uCi of 3 2 P i i n DMEM l a c k i n g phosphate and supplemented with 2$ FBS f o r twelve hours. The l a b e l l e d c e l l s were then 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 counts determined. As a l l the l y s a t e s f e l l w i t h i n a two-fold range they were immunoprecipitated without f u r t h e r adjustment. The l y s a t e s were immunoprecipitated with the K i - r a s s p e c i f i c a n t i - p 2 1 r a s monoclonal antibody Y13-259 (lanes d-2 and d-5) or without an a n t i - r a s antibody (lane-Ab) which acts as a negative c o n t r o l . The immunoprecipitates were washed and analyzed by e l e c t r o p h o r e s i s through a 12.5$ SDS-polyacrylamide g e l . Gels were s t a i n e d f o r molecular weight markers, d r i e d and exposed to XAR-5 f i l m f o r 5 days at room temperature. F i g u r e 4.10. Demonstration of the presence of v-myc i n transformed c e l l l i n e s d e r i v e d from the Ki-MSV/MMCV-infected r a t a d r e n a l cortex c u l t u r e . Genomic DNA was i s o l a t e d from 10, 100 mm dishes 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 manufacturer using 10 ug of DNA from each l i n e , a three f o l d excess of enzyme and incubated f o r 2 hours. The enzyme r e a c t i o n was then e x t r a c t e d once with phenol-chloroform 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 resuspended i n 1X TBE and e l e c t r o p h o r e s e d on a 0.75$ agarose 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 to a nylon membrane, Hybond-N (Amersham) and probed as d i r e c t e d by the manufacturer. The DNA was probed with a v-myc c o n t a i n i n g fragment d e r i v e d from the molecular clone of the MC29 v i r u s , pMC38, which i s s p e c i f i c f o r the avian myc gene contained i n the MMCV under the c o n d i t i o n s used (see F i g u r e 5.5, Vennstrom et a l . , 1984). The DNA's i n the lanes marked B were d i g e s t e d with BamHI which should r e l e a s e a 2.5 Kb fragment from the i n t e g r a t e d p r o v i r u s that c o n t a i n s the v-myc gene. The DNA's i n the lanes marked H were cleaved with H i n d l l l which cuts once i n the p r o v i r u s o u t s i d e the v-myc gene to g i v e a fragment g r e a t e r than 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 are i n d i c a t e d by arrowheads beside each l a n e . AC e k m_DNA d e r i v e d from Ki-MSV/MMCV-infected, uncloned l i n e E a f t e r the c u l t u r e had become serum independent f o r growth. A l l other lanes are of 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-1b/L), or c o l o n i e s i n s o f t agar ( e - d 1 ,1 2 ,1 4 ) . 107 108 band marked i n t h e f i g u r e as v-myc ( r e p r e s e n t a t i v e r e s u l t s shown i n F i g u r e 4 . 1 0 ) . F i v e l i n e s d e r i v e d f r o m t h e K i -MSV/MMCV-inf e c t ed a d r e n o c o r t i c a l c e l l s w e r e a l s o e x a m i n e d f o r e x p r e s s i o n o f t h e p r o d u c t o f t h e v-myc g e n e by immunopr e c i p i t a t i o n . A l l f i v e l i n e s c h o s e n e x p r e s s e d t h e a v i a n v-myc p r o d u c t ( F i g u r e 4 . 1 1 ) , d i s t i n g u i s h a b l e f r o m t h e mammalian c-myc p r o d u c t as t h e y h a v e d i f f e r e n t Mr's by SDS-PAGF: (Hann _et a _ l . , 1983 ). The p r e s e n c e o f v-myc i n a l l 21 l i n e s p r o b e d and t h e e x p r e s s i o n o f v - r a s i n 19 o f 20 l i n e s e xamined s t r o n g l y i n d i c a t e s t h a t t h e p r e s e n c e o f b o t h o n c o g e n e s i n a c e l l r e s u l t s i n t r a n s f o r m a t i o n more e f f i c i e n t l y t h a n e i t h e r o n c o g e n e a l o n e . The r e s u l t s f r o m t h e a p p e a r a n c e o f serum and 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 i n t h e 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 e l l s s u g g e s t e d t h a t some f u r t h e r g e n e t i c or e p i g e n e t i c c h a n g e i n t h e c e l l s was n e c e s s a r y t o a l l o w t r a n s f o r m a t i o n . I f t h i s were t h e c a s e t h e n t h e s e r u m i n d e p e n d e n t c u l t u r e s h o u l d h a v e a r i s e n f r o m t h e c l o n a l e x p a n s i o n o f a s m a l l number o f c e l l s t h a t had a c q u i r e d t h e 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 . The i n t e g r a t i o n s i t e o f MMCV can be mapped u s i n g H i n d l l J w h i c h o n l y c u t s t h e i n t e g r a t e d p r o v i r u s o n c e ( F i g u r e 4 . 1 ) . R e t r o v i r a l i n t e g r a t i o n i s an e s s e n t i a l l y random p r o c e s s and can a c t as a c l o n a l m a r k e r . The r e s u l t s a r e most c l e a r l y i l l u s t r a t e d by t h e p a t t e r n i n t h e DMA o f t h e u n c l o n e d Ki-MSV/MMCV-inf e c t e d l i n e s d e r i v e d f r o m t h e i n i t i a l i n f e c t i o n by p a s s a g i n g u n t i l t h e c u l t u r e a p p e a r e d f u l l y t r a n s f o r m e d . I n each o f t h e t h r e e u n c l o n e d 109 F i g u r e 4.11. E x p r e s s i o n o f P 5 7 V - m y e 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 f r o m 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 . 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 and l a b e l l e d w i t h 250 u C i o f [ 3 5 S ] _ m e t h i o n i n e f o r 2 h o u r s 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 were l y s e d i n a RIPA b u f f e r (1$ T r i t o n X-100, 0.5$ NaDOC, 0.1$ SDS, 100 mM N a C l , 20 mM T r i s -H C l (pH 7 . 5 ) , 1 mM EDTA), c l e a r 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 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 serum ( l a n e s 1 ) . The i m m u n o p r e c i p i t a t e s were washed as recommended by 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 10.5$ 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 t r e a t e d w i t h 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 . ) as s u g g e s t e d by 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 e n d o g e n o u s r a t c-myc p r o d u c t . 110 d - 3 d -5 a - 1 a - 7 1 2 1 2 1 2 1 2 . 205 '116 "97 .68 <p64 c-myc cp57 v-myc •45 •30 l i n e s examined (A, C and E, c e l l l i n e E i s shown as an example) a s i n g l e band was present i n the H i n d l l l d i g e s t of the genomic DNA ( F i g u r e 4.10, A C e R M ) . When the cloned l i n e s d e r i v e d from these t h r e e c u l t u r e s were examined i t was apparent that the p a r e n t a l c u l t u r e was not c l o n a l ( F i g u r e 4.10 lanes e-c11,12 , l 4 , e - 4/L,1b/L). The v a r i a t i o n i n s i z e of the H i n d l l l , v-myc c o n t a i n i n g fragment, between the genomic DNA's of the cloned l i n e s c l e a r l y i n d i c a t e d that the uncloned c u l t u r e contained a mixture of c e l l s from d i f f e r e n t i n f e c t i o n / i n t e g r a t i o n events. This suggested that the p a r e n t a l l i n e was l a r g e l y composed of a s i n g l e c l o n e that had overgrown the c u l t u r e . The overgrowth was ap p a r e n t l y not complete, but the d i f f e r e n t c e l l s presumably represented a m i n o r i t y i n the c u l t u r e as t h e i r c h a r a c t e r i s t i c fragments were not d e t e c t a b l e i n the DNA from the uncloned l i n e s . These data s t r o n g l y support the hypothesis that the serum independent c e l l s i n the Ki-MSV/MMCV-inf ect ed c u l t u r e s arose as the r e s u l t of a low frequency, g e n e t i c or e p i g e n e t i c event. The experiments thus f a r have used c e l l s d e r i v e d from the a d u l t r a t adrenal cortex as a model f o r t r a n s f o r m a t i o n . S i n c e i t was p o s s i b l e that c e l l s not d e r i v e d from the adrena l cortex were present and s u s c e p t i b l e to tr a n s f o r m a t i o n i n the o r i g i n a l c u l t u r e s , eight of the cloned l i n e s were assayed f o r t h e i r a b i l i t y to produce s t e r o i d s , a s p e c i f i c marker f o r a d r e n o c o r t i c a l c e l l s . The c e l l s were given pregnenolone as a prec u r s o r of the s t e r o i d b i o s y n t h e t i c pathway and assayed f o r the production of progesterone by a RIA. At l e a s t s i x of the eight l i n e s appeared to be producing some s t e r o i d i n t e r m e d i a t e s (the r e s u l t s of s i x l i n e s are shown i n Table 4.4) and confirmed the a d r e n o c o r t i c a l o r i g i n of some of the transformed c e l l s i n d i c a t i n g t hat the pathway of t r a n s f o r m a t i o n d e f i n e d i n  v i t r o f o r the a d r e n o c o r t i c a l c e l l s should be an a p p l i c a b l e model f o r c e l l s capable of expressing a h i g h l y d i f f e r e n t i a t e d phenotype. 113 TABLE 4.4 Conversion of Pregnenolone to an Intermediate of the St e r o i d o g e n i c Pathway, Progesterone, by Ki-MSV/MMCV-transformed Rat Adrenal Cortex C e l l Lines Standard Curve Counts-bound Sample Counts-bound (ng u n l a b e l l e d prog est eron e/ml) 2.56 387 + 1 2 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 + 41 n o n s p e c i f i c b i n d i n g 235 + 105 medium blank 1836 + 32 The transformed l i n e s d e r i v e d from the Ki-MSV/MMCV-inf ected adrenal cortex c u l t u r e s were p l a t e d i n 35mm dishes 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 then incubated i n DMEM f o r 45 minutes. The c e l l s were incubated i n 3 ml. DMEM with 5x10~^M pregnenolone f o r four hours and the medium was spun i n a microfuge f o r 5 minutes at 4°C. A sample of the 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 with the other samples to act as a blank c o n t r o l . The samples were processed as d e s c r i b e d i n M a t e r i a l s and Methods. The standard curve with c o l d progesterone i s shown to i n d i c a t e the s e n s i t i v i t y of the assay and the r e s u l t s f o r s i x of the eight samples processed are presented. The l i n e d e s i g n a t i o n s are the same as i n other f i g u r e s i n t h i s chapter. Each l i n e was assayed i n t r i p l i c a t e and the standard e r r o r was estimated. 4.8 DISCUSSION The data presented here i n d i c a t e that i n v i t r o 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 c e l l s by the ras and myc oncogenes can appear to r e q u i r e at l e a s t t h r e e s t e p s . The i n i t i a l i n a b i l i t y of Ki-MSV/MMCV-infected c u l t u r e s to form c o l o n i e s i n s o f t agar, even i n 25% FBS, d e s p i t e the presence of c e l l s that express a h i g h l y transformed morphology, appears to r e f l e c t the need f o r a fundamental change i n the i n f e c t e d c e l l s to allow ras and myc to induce anchorage independent growth. The a c q u i s i t i o n of anchorage independent growth i s c o r r e l a t e d with the l o s s of the serum dependency of the transformed morphology induced by K i -MSV/MMCV i n f e c t i o n . I t i s of p a r t i c u l a r i n t e r e s t that although the a c q u i s i t i o n of anchorage independent and serum independent growth were a p p a r e n t l y c l o s e l y c o r r e l a t e d , the presence of 25% FBS or exogenous growth f a c t o r s was i n s u f f i c i e n t to s t i m u l a t e anchorage independent growth. The emergence of a s i n g l e dominant c l o n e i n the Ki-MSV/MMCV-i n f e c t e d c u l t u r e s , as r e f l e c t e d i n the Southern b l o t s , supports the idea that a f u r t h e r event that occurs at a low frequency i s r e q u i r e d f o r the p r o g r e s s i o n of t r a n s f o r m a t i o n i n the a d r e n o c o r t i c a l c e l l s r a t h e r than simple overgrowth of the c u l t u r e by the Ki-MSV/MMCV-infected c e l l s (Land et a l . , 1986) . The e f f i c i e n t formation of f o c i i n response to co-i n f e c t i o n by Ki-MSV and MMCV c l e a r l y i n d i c a t e s that these 115 two oncogenes can cooperate and are s u f f i c i e n t to induce the morphological a l t e r a t i o n s a s s o c i a t e d with t r a n s f o r m a t i o n when the c e l l s are grown i n high serum or with an exogenous growth f a c t o r , EGF. The serum dependence of the transformed phenotype induced by Ki-MSV/MMCV-infection i s r e m i n i s c e n t of the dependence seen i n Ki-MSV-inf ect ed c e l l s (Auersperg and Calderwood, 1984). The e f f i c i e n t i n d u c t i o n of morphological a l t e r a t i o n s by c o - i n f e c t i o n stands i n sharp c o n t r a s t to the low percentage of c e l l s i n f e c t e d by Ki-MSV that d i s p l a y a transformed phenotype. The i n c r e a s e d s i z e and more pronounced morphological a l t e r a t i o n s i n the f o c i formed i n the Ki-MSV/MMCV-infected c u l t u r e s r e l a t i v e to the c u l t u r e s i n f e c t e d with Ki-MSV alone i n d i c a t e s the a b i l i t y of the myc oncogene to enhance the transforming a c t i v i t y of v - r a s . I t appears that myc f u r t h e r reduced the serum requirements f o r expression of a l t e r e d morphology and growth, as EGF was s u f f i c i e n t to produce an e f f e c t s i m i l a r to the a d d i t i o n of FBS i n the Ki-MSV/MMCV-infected c e l l s but not i n Ki-MSV-i n f e c t e d c u l t u r e s . I t i s not c l e a r what f a c t o r ( s ) i n FBS v-myc was a b l e to r e p l a c e , but the four growth f a c t o r s used i n these experiments (EGF, FGF, PDGF and IGF-II) were unable to s u b s t i t u t e f o r FBS i n e i t h e r of the s i n g l y i n f e c t e d c u l t u r es. There have been previous r e p o r t s that a f u r t h e r c e l l u l a r change i n a d d i t i o n to the i n t r o d u c t i o n of ras and myc oncogenes may be r e q u i r e d to produce t u m o u r i g e n i c i t y i n S y r i a n hamster embryo f i b r o b l a s t s (Oshimura e_t a l . , 1 985 ; Thomassen et _a_l. , 1 985 ). This c e l l u l a r change i s a s s o c i a t e d with monosomy f o r chromosome 15 i n the tumours formed. The l o s s of su p p r e s s i o n of the transformed phenotype i n c e l l h y brids between normal and transformed c e l l s i s a l s o a s s o c i a t e d with the l o s s of s p e c i f i c chromosomes ( K l i n g e r 1980,1982; S t o l e r and Bouck, 1985; Stanbridge et a l . , 1982). There i s as yet no c l e a r demonstration that the steps i n v o l v e d i n the expression of a h i g h l y transformed phenotype i n the two systems i s the same. The advantage of the system d e s c r i b e d here i s that i t provides a t i s s u e c u l t u r e model system of t r a n s f o r m a t i o n by ras and myc that appears to r e q u i r e an a d d i t i o n a l c e l l u l a r change, which i s a s s o c i a t e d with the expression of phenotypes that have been l i n k e d with t u m o u r i g e n i c i t y , serum independent growth and anchorage independence. The expression of serum independent growth and anchorage independence i n Ki-MSV-inf ect ed r a t adrenal cortex c e l l s has been a s s o c i a t e d with a h i g h l y tumourigenic phenotype (Auersperg jet a l . , 1 986 ). Colony formation i n s o f t agar i s the i n v i t r o phenotype that i s most c l o s e l y c o r r e l a t e d with t u m o u r i g e n i c i t y (Kahn and Shin, 1979; C i f o n e and F i d l e r , 1980). I t might be expected that the a c q u i s i t i o n of anchorage independent • growth i n the Ki-MSV/MMCV-infected a d r e n o c o r t i c a l c e l l s would r e f l e c t a s i m i l a r i n c r e a s e i n t u m o u r i g e n i c i t y . The c e l l u l a r change necessary i n the a d r e n o c o r t i c a l c e l l s to g i v e r i s e to anchorage independence may be s i m i l a r to that r e q u i r e d to g i v e r i s e to t u m o u r i g e n i c i t y i n the S y r i a n hamster embryonic f i b r o b l a s t s . The serum dependence f o r morphological t r a n s f o r m a t i o n of the Ki-MSV/MMCV-infected c e l l s i n d i c a t e s that t r a n s f o r m a t i o n of the c e l l s can be modulated by e x t e r n a l c o n d i t i o n s . I n f e c t i o n of c e l l l i n e s by Ha-MSV or Ki-MSV r e s u l t s i n the pro d u c t i o n of the transforming growth f a c t o r TGF-ocand TGF-y3 (Anzano et a l . , 1983 ). These TGF's are able to induce a transformed phenotype i n c e l l l i n e s i n the absence of the v-ras oncogene (Ozanne et a l . , 1 9 8 0 ; Kaplan et a l . , 1 9 8 2 ; Kaplan and Ozanne, 1 9 8 3 ) . The TGF's cooperate i n producing a transformed phenotype with TGF-/3 enhancing the TGF-oc t r a n s f o r m i n g a c t i v i t y (Assoian et a_l. , 1 983 , 1 984 ; Roberts e_t ^ 1 . , 1 9 8 4 ; Massague, 1 9 8 5 ) . TGF-©c i s r e l a t e d to EGF (Marquardt et a l . , 1 9 8 4 ; Derynck et a l . , 1984) and appears to exert i t s e f f e c t through the EGF r e c e p t o r (Carpenter, 1 9 8 3 ; Todaro et a_l. , 1 9 8 0 ; DeLarco and Todaro, 1 9 8 2 ) . The a b i l i t y of EGF to s u b s t i t u t e f o r FBS i n the i n d u c t i o n of morphological a l t e r a t i o n s i n the Ki-MSV/MMCV-i n f e c t e d c e l l s i m p l i e s that d e s p i t e the presence of the v-ras oncogene the transformed c e l l s are e i t h e r not producing TGF-oc or are not responding to the l e v e l s produced. I t has been shown that EGF treatment of myc over expressing NRK c e l l s can r e s u l t i n anchorage independent growth (Stern et a l . , 1 9 8 6 ) . EGF d i d not induce anchorage independent growth or morphological a l t e r a t i o n s i n the c u l t u r e s i n f e c t e d with MMCV alone or with Ki-MSV. These r e s u l t s imply that t h e r e i s a r e s t r i c t i o n to myc a c t i v i t y i n n o n e s t a b l i s h e d c e l l s which i s not present i n the NRK c e l l l i n e . The a c q u i s i t i o n of anchorage independent growth i n the Ki-MSV/MMCV-infected a d r e n o c o r t i c a l c e l l s would appear to r e s u l t from the l o s s of suppression of the a c t i v i t i e s of one or both oncogenes. I t should be p o s s i b l e to use t h i s i n  v i t r o model system to e l u c i d a t e the nature of the c e l l u l a r change r e q u i r e d to allow anchorage independent growth i n the r a t a d r e n a l cortex c e l l s expressing v-ras and v-myc. CHAPTER 5 5.0 myc and s r c Cooperate i n the i n v i t r o Transformation of E a r l y Passage Rat A d r e n o c o r t i c a l C e l l s 5.1 INTRODUCTION The oncogenes that have been shown to be a b l e to cooperate i n t r a n s f o r m a t i o n of primary c e l l s can be d i v i d e d i n t o two groups that are c o r r e l a t e d with the l o c a t i o n of t h e i r products. One group i n c l u d e s those l o c a t e d i n the nucleus, p62/64 myc, polyoma l a r g e T (PyLT), p53, the other group i n c l u d e s those l o c a t e d i n the cytoplasm, p 2 1 r a s , and polyoma middle T (Py MT)(Land et a l . , 1983a; Ruley, 1983). The r o l e of polyoma middle T as a c o o p e r a t i n g oncogene i s p a r t i c u l a r l y i n t e r e s t i n g as i t i s thought to exert i t s oncogenic e f f e c t by a c t i v a t i n g p 6 0 c - s r c (Courtneidge, 1985; Bolen et a l . , 1984). The a b i l i t y of Py MT to cooperate with myc and Py LT i n primary c u l t u r e s has been w e l l e s t a b l i s h e d , implying that an a c t i v a t e d s r c gene could play a s i m i l a r r o l e , but t h i s has not been d i r e c t l y examined i n mammalian c e l l s . Cooperation between s r c and myc has been shown i n the t r a n s f o r m a t i o n of avian chondroblasts (Alema et a l . , 1985 ) and between s r c and other oncogenes i n avian hematopoietic c e l l s (Adkins et a_l. , 1984 ). Using the avian v - s r c c o n t a i n i n g mammalian r e t r o v i r u s , 2-1 (Anderson and S c o l n i c k , 1983) the a b i l i t y of s r c to cooperate with myc or ras was examined i n r a t a d r e n o c o r t i c a l c e l l s . The murine r e t r o v i r u s 2-1 was c o n s t r u c t e d from s t r u c t u r a l elements of the murine r e t r o v i r u s e s and the v - s r c gene from RSV. The c o n s t r u c t can express a genomic RNA that can be packaged and r e p l i c a t e d by one of the r e p l i c a t i o n competent murine r e t r o v i r u s e s . The helper v i r u s used was the amphotropic v i r u s 4070, which i s capable of i n f e c t i n g a broad range of host s p e c i e s i n c l u d i n g but not l i m i t e d to mice and r a t s ( f o r a reveiw see Weiss e£ a l . , 1985). The amphotropic v i r u s uses a rec e p t o r that i s d i s t i n c t from the one used by the e c o t r o p i c v i r u s , so that previous i n f e c t i o n by Mo-MLV, the e c o t r o p i c helper used with Ki-MSV and MMCV, would not prevent s u p e r i n f e c t i o n by 2-1 packaged by the amphotrope 4070. This process was used to i n t r o d u c e the v-src gene i n t o c u l t u r e s i n f e c t e d by e i t h e r Ki-MSV or MMCV, to examine the a b i l i t y of the v - s r c gene to cooperate with e i t h e r ras or myc i n the tr a n s f o r m a t i o n of the non e s t a b l i s h e d r a t a d r e n o c o r t i c a l c e l l s . Previous work had demonstrated that the v- s r c gene alone could induce t r a n s f o r m a t i o n of e a r l y passage SHE c e l l s (Gilmer et al_. , 1985), u n l i k e ras which showed co o p e r a t i o n with myc (Oshimura _et a_l. , 1 985 ). The f o l l o w i n g experiments d e s c r i b e an i n v i t r o a n a l y s i s of v - s r c a c t i o n i n e a r l y passage c e l l s . The v - s r c oncogene induced a transformed phenotype i n the a d r e n o c o r t i c a l c e l l s that i s more independent of environmental c o n d i t i o n s than that induced by v-r a s , but s t i l l shows an a b i l i t y 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 to  S u p e r i n f e c t i o n by the s r c Co n t a i n i n g R e t r o v i r u s , 2-1. The adrenal cortex c u l t u r e s A,C,E and F, d e s c r i b e d i n chapter f o u r , were passaged twice a f t e r i n f e c t i o n by e i t h e r Ki-MSV or MMCV. These c u l t u r e s were then s u p e r - i n f e c t e d with a high t i t r e stock of 2-1. Uninfected adrenal c e l l s have a l s o been i n f e c t e d with 2-1. The i n f e c t i o n of the a d r e n o c o r t i c a l c e l l s by the 2-1 v i r u s r e s u l t e d i n the appearance of f o c i of r e f r a c t i l e , m o r p h o l o g i c a l l y transformed c e l l s that could be r e a d i l y d i s t i n g u i s h e d even a g a i n s t the background of transformed c e l l s i n e i t h e r the Ki-MSV- or MMCV-infected c u l t u r e s ( F i g . 5.1). The number of f o c i formed i n the a d r e n o c o r t i c a l c e l l s p r e v i o u s l y i n f e c t e d with MMCV was gr e a t e r than that formed i n e i t h e r the Ki-MSV-infected or un i n f e c t e d c e l l s (Table 5.1). The c e l l s i n f e c t e d with 2-1 alone were subjected to an i n f e c t i o u s c e n t r e assay which i n d i c a t e d that approximately 1% of the 0.5-1X10 6 t r e a t e d c e l l s were p r o d u c t i v e l y i n f e c t e d . The number of c e l l s that should have been i n f e c t e d per d i s h was aproximately 1 to 2 x 101* while the number of f o c i formed was c o n s i d e r a b l y l e s s (10-30 foci/60mm d i s h ) , i n d i c a t i n g that i n f e c t i o n by the v-src c o n t a i n i n g v i r u s , 2-1 was i n s u f f i c i e n t to r e s u l t i n tr a n s f o r m a t i o n of most n o n e s t a b l i s h e d c e l l s . The 2-1 sup er i n f ec t ed adrenal cortex c e l l s were fol l o w e d over two passages. As can be seen i n F i g . 5.1, i n the c u l t u r e s that had been p r e v i o u s l y i n f e c t e d with MMCV the Figure. 5.1. C e l l morphology i n MMCV- or Ki-MSV-infected 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 sr c 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 with e i t h e r Ki-MSV or MMCV were passaged twice and then s u p e r i n f e c t e d with a high t i t r e stock of 2-1 (10 6 focus forming u n i t s / m l ) . The c u l t u r e s were maintained i n DMEM with 25% FBS and passaged twice before photomicrographs were taken (200X, phase c o n t r a s t ) . The photograph of the 2-1/MMCV-infected c e l l s (A) i s r e p r e s e n t a t i v e of the c u l t u r e , while the photograph of the 2-1/Ki-MSV-infected c e l l s (B) was chosen to i n c l u d e one of the f o c i i n the c u l t u r e as w e l l as the c e l l s e x p r e s s i n g a normal morphology. 123 TABLE 5.1 C e l l L i n e V i r u s I n f e c t i o n s Number of F o c i A 2-1/Ki-MSV 23 2-1/MMCV 111 B 2-1/Ki-MSV 6 2-1/MMCV 28 Focus formation induced 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 MMCV-infected adrenal cortex c e l l s were passaged twice and then sup e r i n f ect ed with high t i t r e stocks of 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 then incubated f o r a f u r t h e r f i v e days and the f o c i counted. The morphology of the f o c i induced 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 that induced by Ki-MSV or MMCV. 125 transformed c e l l s overgrew the c u l t u r e much more r a p i d l y than i n the c u l t u r e p r e v i o u s l y i n f e c t e d with Ki-MSV. These r e s u l t s were seen i n a l l four c u l t u r e s assayed, and presumably r e f l e c t e d the a b i l i t i e s of the v i r a l oncogenes to cooperate with each other. There was no corresponding morphological change i n the c u l t u r e s i n f e c t e d with only K i -MSV or MMCV that would have accounted f o r the appearance of the h i g h l y transformed c e l l s . The r e s u l t s d e s c r i b e d f o r t r a n s f o r m a t i o n by ras and myc had shown that the m o r p h o l o g i c a l l y transformed c e l l s that r e s u l t e d from c o i n f e c t i o n by Ki-MSV and MMCV i n i t i a l l y r e q u i r e d a high serum supplement f o r the expression of a transformed morphology. The c e l l s s u p e r i n f e c t e d with 2-1 or i n f e c t e d with 2-1 alone were a l s o t e s t e d f o r serum s e n s i t i v i t y of the transformed phenotype at each passage a f t e r s u p e r i n f e c t i o n . The c u l t u r e s demonstrated the a b i l i t y to express a transformed morphology and growth i n medium with a low serum supplement i n the passage immediately f o l l o w i n g i n f e c t i o n . These r e s u l t s i n d i c a t e d that the transformed phenotype induced by v- s r c was l a r g e l y serum independent at the time of i t s appearance, u n l i k e the e f f e c t of ras induced t r a n s f o r m a t i o n . 5.3 Anchorage Independent Growth. The appearance of serum independent growth i n c u l t u r e s transformed by Ki-MSV or K i -MSV/MMCV has been c o r r e l a t e d with anchorage independent growth. The c u l t u r e s s u p e r i n f e c t e d with 2-1 were t e s t e d f o r t h e i r a b i l i t y to form c o l o n i e s i n s o f t agar two passages a f t e r i n f e c t i o n . As can be seen the 2-1/MMC V - i n f ec t ed c u l t u r e s formed c o l o n i e s at high e f f i c i e n c y ( F i g u r e 5.2a). The 2-1/Ki-MSV-infected c e l l s formed s m a l l e r c o l o n i e s at a lower e f f i c i e n c y than the 2-1/MMCV-infected c e l l s . As with the appearance of anchorage independent growth i n the K i -MSV/MMCV-inf ect ed c u l t u r e s , the 2-1 sup e r i n f ect ed c u l t u r e s were a l s o a b l e to form c o l o n i e s i n s o f t agar i n low serum. I f the 2-1/MMC V - i n f ect ed c e l l s were p l a t e d at a lower d e n s i t y the percentage of c e l l s that formed c o l o n i e s and the s i z e of the c o l o n i e s that r e s u l t e d i n the s o f t agars with low serum was much lower than that at a higher c e l l d e n s i t y i n low serum ( F i g u r e 5.2b and Table 5.2), showing a n o n l i n e a r decrease r e l a t i v e to the number of c e l l s assayed. These r e s u l t s i m p l i e d that the c e l l s transformed by 2-1/MMCV s t i l l needed to c o n d i t i o n the medium i n which they were c u l t u r e d f o r optimal growth to occur. The growth r a t e of 2-1/MMCV-infected c u l t u r e s i n high and low serum was examined and as can be seen i n F i g u r e 5.3 and Table 5.3 ther e was a d i s t i n c t slowing of the growth r a t e i n the transformed c u l t u r e s grown i n low serum, although the c e l l s s t i l l grew a c t i v e l y . 5.4 C h a r a c t e r i z a t i o n of Three Transformed Lines f o r the  Presence of the V i r a l Oncogenes. As was di s c u s s e d i n the previous chapter, to d i r e c t l y i m p l i c a t e the need f o r co-expression of the two oncogenes f o r t r a n s f o r m a t i o n of a c e l l , i t was necessary to demonstrate the presence of the oncogenes or t h e i r products i n c l o n a l c e l l l i n e s . T h i s was Figure 5.2. Colony formation i n s o f t agar by the 2-1 s u p e r i n f e c t e d adrenal cortex c u l t u r e s . The Ki-MSV- and MMCV-infected c u l t u r e s s u p e r i n f e c t e d with 2-1 were passaged twice and then assayed f o r anchorage independent growth. The c e l l s were suspended at 3 x 10^ or 10 c e l l s i n 2 mis of 0.35$ agarose, DMEM and 5% c a l f serum e s s e n t i a l l y as des c r i b e d i n the 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 incubated f o r s i x t e e n days and photographs were taken on an i n v e r t e d microscope at low power. A) 2-1/MMCV-i n f e c t e d adrenal cortex c e l l s seeded at 3 x 10^ c e l l s / w e l l . B) 2-1/MMCV-infected adrenal cortex c e l l s seeded at 1 x 10 c e l l s / w e l l . C) 2j-1/Ki-MSV-infected ad r e n a l cortex c e l l s seeded at 3 x 10^ c e l l s / w e l l . The photomicrographs (25X) are from r e g u l a r o p t i c s and the c e l l s and c o l o n i e s appear as dark areas on the l i g h t background ( c o n t r a s t F i g u r e 4.7). TABLE 5.2 Anchorage Independent Growth of 2-1/MMCV-inf ect ed Adrenal Cortex C e l l s C e l l L i n e Number of C e l l s Number of C o l o n i e s A 3 x 10* 1 83 10* 2 C 3 x 10* 261 10* 21 E 3 x 10* 77 10* 5 transformed c u l t u r e s d e r i v e d from the 2 s u p e r i n f e c t i o n of the MMCV-inf ect ed adrenal cortex c e l l s were p l a t e d i n 0.35$ agarose with 5% c a l f serum. The s o f t agars were t h r e e mis f i n a l volume i n a 60 mm d i s h with e i t h e r 3 x 10* or 10* c e l l s i n each d i s h , as noted. The number of macroscopic c o l o n i e s were counted at the end of thr e e weeks as being r e p r e s e n t a t i v e of the e f f i c i e n c y of colony formation and growth as seen by mi c r o s c o p i c examination. Figure 5.3- Growth r a t e of 2-1/MMCV-infected adrenal cortex c e l l s i n the presence of high or low serum supplements. A l i n e of 2-1/MMCV-infected adrenal cortex c e l l s was p l a t e d i n 60 mm dishes i n DMEM with 10$ FBS and allowed to a t t a c h o v e r n i g h t . A f t e r 12 hours h a l f the p l a t e s were s h i f t e d i n t o DMEM with 5$ c a l f serum. P l a t e s were harvested i n t r i p l i c a t e at approximately twelve hour i n t e r v a l s and the c e l l s counted. ( - X - ) 2-1/MMCV-infected c e l l s grown i n DMEM + 10$ FBS, (-#-) 2-1/MMCV-infected c e l l s grown i n DMEM + 5$ c a l f serum. TABLE 5.3 C e l l L i n e Serum C o n c e n t r a t i o n Number of C e l l s (x10 b) A 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 E 1 0$ FBS 3 .59 + 0 .40 10$ CS 2 .35 + 0.26 5$ CS 1 .86 + 0.21 2% CS 1.17 + 0.13 Growth of 2-1/MMCV-infected adrenal cortex c e l l s i n the presence of d i f f e r e n t serum supplements. The 2-1/MMCV-i n f e c t e d a d r e n a l cortex c e l l s were p l a t e d i n 60 mm dishes i n DMEM with 10$ FBS and allowed to a t t a c h f o r eight hours. A f t e r eight hours the c e l l s were s h i f t e d to f r e s h medium with the serum supplement i n d i c a t e d i n the t a b l e , each serum c o n c e n t r a t i o n being assayed i n d u p l i c a t e on each l i n e . The c e l l s were incubated f o r 48 hours, harvested and counted. The e r r o r given i s the standard e r r o r estimated from the d u p l i c a t e samples. p a r t i c u l a r l y important with the 2-1 v i r u s , as i t a p p a r e n t l y could r a p i d l y transform the a d r e n o c o r t i c a l c e l l s i n the absence of another v i r a l oncogene. Three l i n e s were cloned out of s o f t agar from two d i f f e r e n t 2-1/MMCV-infected c u l t u r e s and were analyzed f o r the enhanced expression of poO , as an i n d i c a t i o n of v - s r c expression, and the presence of the v-myc oncogene. As can be seen i n F i g u r e 5.4 the thr e e 2-1/MMC V - i n f ect ed l i n e s a l l expressed elevated l e v e l s of p 6 0 s r c r e l a t i v e to the Ki-MSV/MMCV-infected l i n e , c l e a r l y i n d i c a t i n g the presence of v- s r c i n the transformed c e l l s . The presence of the v-myc oncogene was probed using a Southern b l o t as d e s c r i b e d i n chapter 4. The DNA's of the thr e e transformed l i n e s were d i g e s t e d with BamHI which w i l l e x c i s e the v i r a l oncogene fragment from MMCV (see F i g u r e 4.1) and compared to a l i n e transformed by ras and myc, and a F i s c h e r r a t d e r i v e d untransformed c e l l l i n e , r a t - 2 . Two of the t h r e e 2-1/MMCV-infected l i n e s ( F i g u r e 5.5 a-1, e-3) contained a fragment that h y b r i d i z e d to the avian v-myc probe of the same s i z e as that i n the l i n e transformed by ras and myc ( F i g . 5.5 c-16). There was no corresponding fragment i n the normal rat-2 c e l l l i n e i n d i c a t i n g that the h y b r i d i z a t i o n i s s p e c i f i c f o r the avian myc gene. Figure 5.4. Examination of cloned c e l l l i n e s d e r i v e d from the 2-1/MMCV-infected adrenal cortex c u l t u r e s f o r e l e v a t e d p 6 0 s r c kinase a c t i v i t y . The three 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 d i s h e s . When the c e l l s were approximately 50$ c o n f l u e n t the c e l l s were l y s e d , c l a r i f i e d and store d at -80°C u n t i l use. One of the Ki-MSV/MMCV-infected c e l l l i n e s grown out from a s o f t agar colony 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 immuno-p r e c i p i t a t e d with the a n t i - s r c antibody 327. The immunoprecipitates were l a b e l l e d i n an in_ v i t r o kinase r e a c t i o n i n the presence of the exogenous s u b s t r a t e enolase and then analyzed on a 12.5$ SDS-polyacrylamide g e l . The g e l was s t a i n e d with Coomassie B r i l l i a n t Blue to show the enolase, d r i e d and exposed to f i l m with a screen at -80°C. 135 Ki-MSV F i g u r e 5.5. Southern a n a l y s i s of the 2-1/MMCV-infected adrenal cortex c e l l l i n e s f o r the presence of v-myc. Genomic DNA was prepared from the three cloned 2-1/MMCV-i n f e c t e d a d r e n a l cortex c e l l l i n e s . One of the DNA samples from a Ki-MSV/MMCV-infected l i n e , C - C16 (Figure 4.10), p r e v i o u s l y examined was i n c l u d e d f o r comparison. 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 negative c o n t r o l . Ten micrograms of each of the DNA samples were d i g e s t e d with BamHI and the fragments were separated by e l e c t r o p h o r e s i s on a 0.75$ agarose g e l . T r a n s f e r and probing of the samples was done as d e s c r i b e d above, and the b l o t was exposed to f i l m with a screen at -80°C f o r s i x days. Ki-MSV 2 1 / MMCV M M + C V Rat a-l e-l e-3 c-16 -2 v - m y c 5.5 DISCUSSION The r e s u l t s presented here demonstrate that while the v - s r c oncogene can r a p i d l y transform n o n e s t a b l i s h e d c e l l s to serum independent, anchorage independent growth i t does so at a low e f f i c i e n c y . v - s r c i s app a r e n t l y a b l e to cooperate with v-myc but not v-ras to induce t r a n s f o r m a t i o n more e f f i c i e n t l y than e i t h e r oncogene alone i n the e a r l y passage adrenal cortex c e l l s . The presence of elevated l e v e l s of P°0 i n the th r e e l i n e s c h a r a c t e r i z e d c l e a r l y i n d i c a t e s the involvement of the v - s r c gene i n the t r a n s f o r m a t i o n of the adrenal cortex c e l l s . The v i r a l myc oncogene i s present i n two of the th r e e l i n e s assayed by Southern b l o t t i n g i n d i c a t i n g that although v-myc .need not be present i t occurs with the v - s r c at a r e a d i l y d e t e c t a b l e frequency. The g r e a t e r number of f o c i and s i g n i f i c a n t l y more r a p i d overgrowth of the 2-1/MMCV-infected c u l t u r e by transformed c e l l s than occurs i n the Ki-MSV/2-1- or 2 - 1 - i n f e c t e d c u l t u r e s supports the idea that v - s r c i s ab l e to cooperate with v-myc but not v-ras to transform the a d r e n o c o r t i c a l c e l l s more e f f i c i e n t l y . I t has been p r e v i o u s l y demonstrated that polyoma middle T, which appears to transform c e l l s by a c t i v a t i n g p 6 0 c ~ s r c , can cooperate with myc (Courtneidge, 1985; Bolen et a l . , 1984, Land et a_l. , 1983). The r e s u l t s with polyoma MT provided i n d i r e c t evidence that myc and s r c might cooperate i n v i t r o i n the 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 f i b r o b l a s t s , but the r e s u l t s d e s c r i b e d i n t h i s chapter are the f i r s t d i r e c t demonstration. Cooperation i n tr a n s f o r m a t i o n by v - s r c and v-myc has a l s o been shown i n avian c e l l s (Alema et a_l., 1985b; Adkins et a_l. , 1984). The r e s u l t s presented here suggest that v - s r c and v-myc can r a p i d l y induce a h i g h l y transformed phenotype i n the adrenal c o r t e x c e l l s without the apparent need f o r a f u r t h e r , independent c e l l u l a r change. The r a p i d t r a n s f o r m a t i o n i s r e f l e c t e d i n the s u p e r i n f e c t e d c e l l s i n the expression of a reduced serum dependency f o r growth i n the passage a f t e r s u p e r i n f e c t i o n and anchorage independent growth when f i r s t assayed two passages a f t e r s u p e r i n f e c t i o n . T h i s r a p i d a c q u i s i t i o n of anchorage independent growth by the 2-1/MMCV-infected c u l t u r e s c o n t r a s t s with the i n a b i l i t y of the Ki-MSV/MMCV-infected c u l t u r e s to form c o l o n i e s i n s o f t agar i n the passages immediately f o l l o w i n g i n f e c t i o n . The transformed phenotype induced by 2-1 by i t s e l f , u n l i k e Ki-MSV, a l s o appeared to be r e l a t i v e l y serum independent. The t r a n s f o r m a t i o n of the a d r e n o c o r t i c a l c e l l s by v - s r c and v-myc appears to f i t a two step pathway. This stands i n c o n t r a s t to t r a n s f o r m a t i o n by v-ras and v-myc which appears to f o l l o w a t h r e e step pathway. This i m p l i e s that t r a n s f o r m a t i o n of e a r l y passage c e l l s by v - s r c and v-myc i s presumably not s u b j e c t to the c e l l u l a r s u ppression that appears to i n h i b i t t r a n s f o r m a t i o n by v-ras and v-myc ( t h i s t h e s i s and Oshimura e_t a l . , 1985; Thomassen g_fc a l . , 1985; Stevenson and Volsky, 1986; Vogt et a l . , 1986). The d i f f e r e n c e i n the pathways of t r a n s f o r m a t i o n in. v i t r o by v-src and myc r e l a t i v e to ras and myc c o r r e l a t e with the pathways of development of t u m o u r i g e n i c i t y (Oshimura et a l . , 1985; Gilmer et a l . , 1985). I n d u c t i o n of t u m o u r i g e n i c i t y by v - s r c and myc does not appear to r e q u i r e any f u r t h e r c e l l u l a r change, but t r a n s f o r m a t i o n by ras and myc does. The 2-1/MMCV-infected c u l t u r e s were not e n t i r e l y serum independent f o r growth. Although t h e r e was no apparent change i n the c e l l u l a r morphology of the 2-1/MMCV-infected c e l l s when c u l t u r e d with a low serum supplement, t h e r e was a drop i n the growth r a t e of the c e l l s maintained i n low serum. The e f f i c i e n c y of colony formation i n s o f t agar was a l s o c o n s i d e r a b l y reduced when the c e l l s were assayed at low d e n s i t y i n low serum. These r e s u l t s imply that the c e l l s transformed by s r c and myc can s t i l l show some serum dependence, although the requirements are c o n s i d e r a b l y reduced. The b a s i s of the r e s i d u a l serum dependence i s not c l e a r , but the e f f e c t of c e l l d e n s i t y on colony formation i n s o f t agar pro v i d e s some i n d i c a t i o n that an a u t o c r i n e mechanism may be i n v o l v e d . ^ * v ~ s r c poO appears to be a b l e to i n i t i a t e a f u l l round of c e l l d i v i s i o n i n c e l l l i n e s i n the apparent absence of any e x t e r n a l f a c t o r s , w h ile l i n e s expressing v-ras r e q u i r e some serum f a c t o r s to e f f i c i e n t l y t r a v e r s e the c e l l c y c l e (Durkin and W h i t f i e l d , 19 84, 1986). The r e l a t i v e independence from serum f a c t o r s of the v - s r c transformed c e l l l i n e r e l a t i v e to the v-ras expressing l i n e i n the i n d u c t i o n of c e l l d i v i s i o n does seem to p a r a l l e l the d i f f e r e n c e s i n the a c t i o n of the v-ras and v - s r c oncogenes i n transf o r m i n g the adrenal cortex c e l l s , although i t i s not known what common r e g u l a t o r y mechanisms may u n d e r l i e the two phenotypes, anchorage independent growth and c e l l d i v i s i o n i n a serum deprived monolayer. CHAPTER 6 6 . 0 E f f e c t s of V i r a l Oncogenes on the S t e r o i d o g e n i c A b i l i t y  of 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 INTRODUCTION The formation of tumours i n the human adrenal cortex i s f r e q u e n t l y a s s o c i a t e d with o v e r p r o d u c t i o n of some of the g l u c o c o r t i c o i d s t e r o i d s , with attendant metabolic problems (Mulrow 1 9 8 6 ) . The expression of a t e r m i n a l l y d i f f e r e n t i a t e d phenotype i s o f t e n a s s o c i a t e d with the l o s s of the a b i l i t y of a c e l l to d i v i d e (Weir and S c o t t , 1986 and r e f e r e n c e s t h e r e i n ) . The expression of v i r a l oncogenes i n d i f f e r e n t i a t e d c e l l s appears to r e s u l t i n an i n c r e a s e i n p r o l i f e r a t i o n of the c e l l s and a l o s s of some aspects of normal d i f f e r e n t i a t e d f u n c t i o n (Bishop, 1984; K l e i n and K l e i n , 1985, 1 9 8 6 ) . The a b i l i t y of v-ras and v - s r c to i n h i b i t growth and s t i m u l a t e d i f f e r e n t i a t i o n of the pheochromocytoma c e l l s would appear to be an exception but u n d e r l i n e s a r o l e f o r the oncogenes i n modulating the d i f f e r e n t i a t e d phenotype of these c e l l s (Bar-Sagi and Feramisco, 1985; Noda et a l . , 1985; Alema et a l . , 1985a). The mouse a d r e n o c o r t i c a l tumour c e l l l i n e , Y-1, produces some s t e r o i d end products ( F i g u r e 6.1) and can respond to a d r e n o c o r t i c o t r o p h i c hormone (ACTH), a normal inducer of s t e r o i d o g e n e s i s , by i n c r e a s i n g s t e r o i d production (Kowal and F i e d l e r , 1968). The Y-1 s t e r o i d o g e n i c pathway rep r e s e n t s a truncated v e r s i o n of the normal, i n v i v o pathway. , The Y-1 l i n e c o n t a i n s an a m p l i f i e d and Figure 6.1. S t e r o i d o g e n i c pathway of Y-1 a d r e n o c o r t i c a l tumour c e l l s (taken from Kowal and F i e d l e r , 1968). The pathway i s shown from the f i r s t i n t e r m e d i a t e of the s t e r o i d o g e n i c pathway, pregnenolone. Pregnenolone i s d e r i v e d from c h o l e s t e r o l by a s i n g l e step mediated by cytochrome P - 4 5 0 s i d e chain cleavage. The major s e c r e t e d products i n d e c r e a s i n g order are 11/3 -hydroxyprogesterone, 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 £ -dihydroprogesterone . Progesterone and 20<X -dihydroprogesterone are a l s o d e t e c t a b l e as minor products. PREGNENOLONE » 20cf-DIHYDROPREGNENOLONE PROGESTERONE -> 20^-DIHYDROPROGESTERONE PROGESTERONE -> 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 2 gene that i s presumably i n v o l v e d i n the t r a n s f o r m a t i o n of the c e l l s (Schwab et a l . , 1983). The r e g u l a t i o n of s t e r o i d o g e n e s i s has been e x t e n s i v e l y s t u d i e d (Schimmer, 1980) and the metabolic pathway and i n t e r m e d i a t e s c h a r a c t e r i z e d . The Y-1 c e l l l i n e seemed to rep r e s e n t a u s e f u l t a r g e t to study the e f f e c t of s p e c i f i c oncogenes on the expression of the d i f f e r e n t i a t e d phenotype. RESULTS 6 .2 The E f f e c t s of R e t r o v i r a l l y Borne Oncogenes on the  Morphology and Growth of the Y-1 C e l l L i n e . The p a r e n t a l Y-1 c e l l s were i n f e c t e d by MMCV, c o n t a i n i n g the avian v-myc, Ki-MSV, c o n t a i n i n g the v-ras oncogene, or 3611 which expresses the v - r a f oncogene (Rapp et a l . , 1983a, 1983b). A l l the oncogenic v i r u s e s used were packaged by a Mo-MLV he l p e r . I n f e c t i o n of the Y-1 c e l l s by the oncogenic r e t r o v i r u s e s d i d not r e s u l t i n any overt change i n the c e l l u l a r morphology of the Y-1 c e l l s ( F i g u r e 6.2). The i n f e c t e d Y-1 c e l l s a l s o r e t a i n e d the a b i l i t y to respond m o r p h o l o g i c a l l y to inducers of s t e r o i d o g e n e s i s i n the same f a s h i o n as the u n i n f e c t e d Y-1 c e l l s (Kowal and F i e d l e r , 1968) . The Y _ - )M M C V C E L L i ± n e w a s compared to the p a r e n t a l Y-1 c e l l s f o r r a t e of growth under normal c o n d i t i o n s . The growth r a t e was determined by f o l l o w i n g p r o t e i n accumulation i n the two c u l t u r e s . There was no s i g n i f i c a n t d i f f e r e n c e i n the growth r a t e of the two c e l l l i n e s . Treatment of the Y-1 c e l l s with ind u c e r s of s t e r o i d o g e n e s i s , such as ACTH or f o r s k o l i n , i n h i b i t s t h e i r growth (Weidman and G i l l , 1976; G i l l and Weidman, 1977; Moriwaki et a l . , 1982). The Y-1 c e l l l i n e and the MMCV-infected d e r i v a t i v e were t r e a t e d with f o r s k o l i n o v e r n i g h t , and the i n c o r p o r a t i o n of [ 3H] -thymidin e was determined to assess the a b i l i t y of the v i r a l oncogene to r e l i e v e the f o r s k o l i n induced growth i n h i b i t i o n (Table 6.1). The Y - 1 ^ ^ l i n e appeared to be l e s s s u s c e p t i b l e to Figure 6.2. Morphology of Y-1 and y - 1 - c e l l s . Y-1 c e l l s were i n f e c t e d with a high t i t r e stock of MMCV (2X10 6 colony forming u n i t s / m l ) as d e s c r i b e d i n Methods. A f t e r two passages the c e l l s were photographed on a L e i t z i n v e r t e d microscope with phase c o n t r a s t . Both c u l t u r e s had been maintained and passaged 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 areas were chosen. 148 TABL E 6. 1 I n h i b i t i o n of DNA Syn t h e s i s by F o r s k o l i n i n the Y-1 and Y - 1 M M C V L i n es C e l l L i n e [ 3 H ] t h y m i d i n e Uptake [ 3 H ] t h y m i d i n e Uptake ^ I n h i b i t i o n i n Untreated C e l l s i n C e l l s Treated with F o r s k o l i n Y-1 Y-1 MMCV 24,733 + 2458 43 ,225 + 316 7,300 + 866 21 ,300 + 3 ,593 70? 51? Y-1 and Y - 1 H n u v c e l l s were seeded i n t o a 96 w e l l t r a y at 1 to 2 x 10 c e l l s per w e l l and allowed to a t t a c h and grow f o r 2 days. F o r s k o l i n ,was then added to the medium to 5 x 10~ 5 M and the c e l l s were incubated f o r 14 hours. One uCi of [^H]thymidine was then added to each w e l l and the c e l l s were then incubated f o r a f u r t h e r s i x hours. The i n c o r p o r a t i o n of [ 3 H ] - thymidine 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 determined. Each datum i s an average of three d eterminations and the e r r o r s are given as standard d e v i a t i o n s . the f o r s k o l i n mediated i n h i b i t i o n of -thymidine uptake than the Y-1 c e l l s . The d i f f e r e n c e between Y-1 and Y - 1 m m c v i n thymidine uptake, although s m a l l , was appa r e n t l y s i g n i f i c a n t . 6.3 A n a l y s i s of S t e r o i d Production from the R e t r o v i r a l l y I n f e c t e d and Uni n f e c t e d Y-1 l i n e s . The major s t e r o i d products of the Y-1 c e l l l i n e are f l u o r e s c e n t at low pH (Kowal and F i e d l e r , 1968) a l l o w i n g a q u a n t i t a t i v e measurement of the accumulation of the s t e r o i d products i n the medium. The Y-1 l i n e s i n f e c t e d with the oncogenic r e t r o v i r u s e s were compared to the u n i n f e c t e d , p a r e n t a l Y-1 c e l l l i n e and Y-1 c e l l s i n f e c t e d with Mo-MLV alone. The secr e t e d s t e r o i d s were extracted from the medium, d r i e d and resuspended i n 100$ ethanol. The f l u o r e s c e n c e was then measured and normalized f o r the r e l a t i v e amounts of p r o t e i n i n each c u l t u r e . There was no d e t e c t a b l e d i f f e r e n c e i n the prod u c t i o n of f l u o r o g e n i c m a t e r i a l between the p a r e n t a l c e l l l i n e and the Mo-MLV-inf ect ed Y-1 l i n e . I n f e c t i o n of the Y-1 c e l l l i n e with any of the oncogenic r e t r o v i r u s e s r e s u l t e d i n a 2 to 3 f o l d i n c r e a s e i n the pro d u c t i o n of f l u o r o g e n i c m a t e r i a l (Table 6.2) r e l a t i v e to the u n i n f e c t e d c e l l l i n e . To f u r t h e r c h a r a c t e r i z e the e f f e c t of the int r o d u c e d oncogenes on the phenotype of the Y-1 c e l l s , the response of the i n f e c t e d c e l l s to an inducer of s t e r o i d o g e n e s i s was analyzed. For these experiments f o r s k o l i n , a potent inducer of a denylate c y c l a s e a c t i v i t y , was used to induce s t e r o i d Table 6 .2 C e l l s to be assayed f o r s t e r o i d s were washed twice i n c u l t u r e medium and incubated i n f r e s h medium with f o r s k o l i n added i f necessary f o r two hours. The medium was harvested and c e n t r i f u g e d at 1600 x g f o r 10 minutes. The c l e a r e d medium was then e x t r a c t e d with one volume of dichloromethane. The d i c h l o r o - methane was then extracted with s u l p h u r i c a c i d : e t h a n o l (65:35) and a f t e r 30 minutes the f l u o r e s c e n c e was measured on a Perkin-Elmer 650-10S f l u o r e s c e n c e spectrophotometer with e x c i t a t i o n at 470 nm and d e t e c t i o n at 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 the amount of f l u o r o g e n i c m a t e r i a l was normalized to the amount of c e l l u l a r p r o t e i n i n the dishes as determined using a BioRad p r o t e i n assay. The c u l t u r e medium was a l s o assayed and t h i s value has been s u b t r a c t e d from the f l u o r e s c e n c e . A) Comparison of the pro d u c t i o n of f l u o r o g e n i c s t e r o i d s by each of 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 . B) Co n - c e n t r a t i o n curve of the e f f e c t of f o r s k o l i n on the production of f l u o r o g e n i c s t e r o i d s by the Y-1 and y - 1 M M c v c e l l l i n e s . Measurement of S t e r o i d Production from Y-1 C e l l s and Viru s Infected D e r i v a t i v e s by Acid Induced F l u o r e s c e n c e A) C e l l R e l a t i v e Fold Increase R e l a t i v e L i n e Fluorescence to Untreated Y-1 C e l l s Y-1 1.1 Y - 1 M M C V 3.1 2.2 ^ Y - 1 3 6 1 1 2.8 2.0 | Y - 1 K i 3.6 2.6 - j * B) F o r s k o l i n c o n c e n t r a t i o n (M) 10" 8 10-7 ,-6 R e l a t i v e F l u o r e s c e n c e 10" 10 -5 Y-1 5.5 6.4 6.6 10.6 14.4 y_-|MMCV 12.0 14.3 20.0 29.4 43.2 Fold Increase 2.2 2.2 3.0 2.8 3.0 p r o d u c t i o n (Moriwaki et a l . , 1982), but s i m i l a r r e s u l t s were obtained using ACTH. Each of the i n f e c t e d Y-1 l i n e s responded to f o r s k o l i n by an in c r e a s e d output of f l u o r o g e n i c s t e r o i d s s i m i l a r to that seen i n the u n i n f e c t e d Y-1 c e l l l i n e . The pr o d u c t i o n of f l u o r o g e n i c s t e r o i d s i n the Y-I^MCV l i n e remained approximately 3 f o l d higher than i n the un i n f e c t e d Y-1 c e l l l i n e at a l l f o r s k o l i n c o n c e n t r a t i o n s examined (T a b l e 6.2B). The s t i m u l a t i o n of s t e r o i d p r o d u c t i o n by f o r s k o l i n was approximately the same i n a l l the i n f e c t e d c e l l l i n e s , r e s u l t i n g i n a maintenance of the enhanced s t e r o i d o g e n e s i s r e l a t i v e to the u n i n f e c t e d Y-1 c e l l l i n e . The net r e s u l t of the i n f e c t i o n and f o r s k o l i n treatment was a 7 to 8 f o l d i n c r e a s e i n the prod u c t i o n of f l u o r o g e n i c s t e r o i d s r e l a t i v e to the u n i n f e c t e d , untreated Y-1 c e l l l i n e . Because the f l u o r e s c e n c e assay f o r s t e r o i d s d e t e c t s a l l s t e r o i d s with a 3ket o , 4 A - s t r u c t u r e , the assay r e f l e c t s a pool of products. To examine the products s e p a r a t e l y the s t e r o i d s were i s o l a t e d and examined by r e v e r s e phase HPLC. The s t e r o i d s produced by the Y-1 c e l l l i n e are r e a d i l y s e p a r a b l e by HPLC on a C^g r e v e r s e phase column (Ramirez et a l . , 1982; D'Agostino e£ a l . , 1984) . The thr e e major s t e r o i d products of the Y-1 c e l l l i n e were w e l l separated and t h e r e was l i t t l e i n t e r f e r i n g background ( F i g . 6.3). The pa t t e r n d e r i v e d from the Mo-MLV-inf ect ed Y-1 l i n e was e s s e n t i a l l y i d e n t i c a l to the p a t t e r n from the u n i n f e c t e d Y-1 l i n e . The s t e r o i d s produced by the y - 1 M M C V l i n e showed a F i g u r e 6.3. Demonstration of comigration of s t e r o i d products d e r i v e d from the Y-1 and y_iMMCV c e l l i i n e s . The s t e r o i d products s e c r e t e d by the Y-1 and Y-1^ M (^ c e l l l i n e s were p u r i f i e d from the 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 products were resuspended 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 the HPLC. The column was a C^g reverse phase column of 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 loaded at 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 of 30 to 70? a c e t o n i t r i l e i n water. The e l u t i o n of the s t e r o i d s was monitored at 254 nm and recorded by a Hewlett-Packard 3390A I n t e g r a t o r . Each of the samples from the Y-1 and y_1MMCV c e l l l i n e s w a g r u n s e p a r a t e l y (Y-1 and Y - 1 M M C V ) and then mixed.,and (Y-1 + Y-I^CV) rerun under the same c o n d i t i o n s p a t t e r n that was very s i m i l a r to that from the Y-1 l i n e ( F i g u r e 6 . 3 ) . The i n t e g r a t i o n of the peaks showed that the r e l a t i v e amounts i n each peak were e s s e n t i a l l y the same f o r both the Y - 1 M M C V c e l l s and the u n i n f e c t e d Y-1 c e l l s (Table 6 . 3 ) . Mixing of the two samples confirmed that the s t e r o i d products d i d comigrate, c l e a r l y i n d i c a t i n g that the s e c r e t e d end products were the same f o r both the Y-1^MCV c e n s a n c j the u n i n f e c t e d c e l l s . Peak 1 has been i d e n t i f i e d as 20c6-dihydroprogesterone by comigration with a p u r i f i e d standard. Peaks 2, 3 and 4 are thought to be 1 1^-dihydroprog est eron e, 1 y}-2 00C-dihydroprog est eron e and 11 keto-20tf-d i h y d r opr og est eron e r e s p e c t i v e l y , based on t h e i r expected order of e l u t i o n from the column. Examination of the s t e r o i d s produced by the Ki-MSV- and 361 1-infect ed Y-1 c e l l s r e v e a l e d some s i g n i f i c a n t d i f f e r e n c e s when compared to the u n i n f e c t e d Y-1 c e l l s . The s t e r o i d products s e c r e t e d by the Y - 1 K i " M S V and Y-13611 c e i i s were the same as those s e c r e t e d by the u n i n f e c t e d Y-1 c e l l s , as can be seen i n the t r a c e s of the samples ( F i g u r e 6 . 4 ) . The i n t e g r a t i o n of the peaks shows that the r e l a t i v e amounts of the s t e r o i d s s e c r e t e d v a r i e d s i g n i f i c a n t l y between the two i n f e c t e d l i n e s and the u n i n f e c t e d Y-1 l i n e . Both Y-1361 1 a n d Y^Ki-MSV l i n e s s h o w e d an enhanced p r o d u c t i o n of the more hydrophobic s t e r o i d s , progesterone and 20 OC -dihydroprog est erone (peak 1) and 11yC3-dihydroprog est erone (peak 2), when compared to the u n i n f e c t e d Y-1 l i n e (Table 6 . 3 ) . T h i s s h i f t i n the s t e r o i d s produced was p a r t i c u l a r l y F i g u r e 6.4. Comparison of the s t e r o i d products produced by the Y-1, y_<|MMCV a n d Y_ 1Ki-MSV c e l l l i n e s , . The s t e r o i d products of the Y-1, y - 1 M M C V and Y - 1 K l _ M b V c e l l l i n e s were prepared and analyzed as d e s c r i b e d i n the legend of f i g u r e 6.3. The t r a c e s shown are of each sample run i n d i v i d u a l l y and the comigrating peaks are i n d i c a t e d (1-4). A c e t o n i t r i l e 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 . 159 TABLE 6.3 A n a l y s i s of the R e l a t i v e P roduction of the S t e r o i d Products of the V i r a l l y i n f e c t e d Y-1 C e l l Lines C e l l L i n e Peak (p ere ent of the sum of p eaks 1 , 2, 3 and 1 2 3 4 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 Y_iKi-MSV 17.5 71.6 5.3 5.5 89 • 1 10.9 Y-13611 11.9 69.0 9.6 9.4 80 .9 19.1 The e l u t i o n of s t e r o i d s was monitored by a Hewlett Packard 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 a u t o m a t i c a l l y i n t e g r a t e d . Traces i n which a l l the peaks were shown without t r u n c a t i o n were used and the areas of the peaks 1 to 4 were summed. The percentage of each peak of the t o t a l was d et ermin ed. n o t i c e a b l e i n the Y - 1 K l _ M S V l i n e where i t would appear that a l l of the 2 to 3 f o l d i n c r e a s e i n the p r o d u c t i o n of f l u o r o g e n i c s t e r o i d s could be accounted f o r by the i n c r e a s e d accumulation of peaks 1 and 2 (20oC -dihydroprogest eron e and 11y3-dihydroprogesterone) . The changes i n response to i n f e c t i o n by 3611 and Ki-MSV i n d i c a t e d that these oncogenes r e s u l t e d i n a f u r t h e r t r u n c a t i o n of the s t e r o i d o g e n i c pathway than had a l r e a d y occurred i n the Y-1 c e l l s . 6.4 DISCUSSION The e f f e c t s of the simian adenovirus SA-7 v i r a l oncogenes on the Y-1 c e l l l i n e were p r e v i o u s l y examined by L e f e v r e et a l . (1981). I n f e c t i o n by SA-7 r e s u l t e d i n an almost complete l o s s of normal s t e r o i d o g e n i c a b i l i t y , although the c e l l s were ab l e to convert c h o l e s t e r o l to pregnenolone and respond to ACTH. The l i n e s i n f e c t e d by the a c u t e l y oncogenic r e t r o v i r u s e s continued to produce in t e r m e d i a t e s of the s t e r o i d o g e n i c pathway, but the r a t e of s y n t h e s i s was elevated to approximately the same extent as that seen when s t e r o i d p r o d u c t i o n of the Y-1 c e l l s was induced by f o r s k o l i n . The responsiveness of the i n f e c t e d Y-1 c e l l s to inducers of s t e r o i d o g e n e s i s i n d i c a t e s that the f l u o r o g e n i c m a t e r i a l i s composed of s t e r o i d s and not contaminants that are induced by r e t r o v i r a l i n f e c t i o n , and that the r e g u l a t o r y pathways f o r s t e r o i d o g e n e s i s remain i n t a c t . The i n t r o d u c t i o n of the v-myc oncogene appears to enhance the expression of the d i f f e r e n t i a t e d phenotype of the Y-1 c e l l s as r e f l e c t e d i n the i n c r e a s e d output of f l u o r o g e n i c s t e r o i d s . The elevated p r o d u c t i o n of a l l the s t e r o i d m e t a b o l i t e s i n the y - 1 M M C V c e l l s i m p l i e s that t h e r e i s no t r u n c a t i o n of the Y-1 s t e r o i d o g e n i c pathway. Although the Y-1 c e l l s c o n t a i n an a m p l i f i e d and overexpressed c - K i -ras 2 gene (Schwab et a l . , 1 983 ) the e f f e c t of v-myc was to enhance s t e r o i d p r o d u c t i o n , suggesting that the i n t e r a c t i o n 162 of the myc oncogene with the over expr ess ed ras proto-oncogene can r e s u l t i n the i n c r e a s e d expression of a d i f f e r e n t i a t e d phenotype i n the Y-1 c e l l l i n e , r a t h e r than the l o s s of d i f f e r e n t i a t i o n a s s o c i a t e d with t r a n s f o r m a t i o n . The o v e r e x p r e s s i o n of the myc oncogene i n NRK c e l l s can i n c r e a s e the s u s c e p t i b i l i t y of the c e l l s to the i n d u c t i o n of anchorage independent growth by growth f a c t o r s (Land et a l . , 1986). I t would appear that myc i s a b l e to enhance the expression of other aspects of growth and d i f f e r e n t i a t i o n . In a d r e n o c o r t i c a l c e l l s the i n d u c t i o n of i n c r e a s e d s t e r o i d o g e n e s i s i s a s s o c i a t e d with a r e d u c t i o n i n growth r a t e (Weidman and G i l l , 1976; G i l l and Weidman, 1977). In the y_i MMCV i i n e the i n c r e a s e i n s t e r o i d o g e n e s i s occurs i n the absence of a d e t e c t a b l e change i n growth r a t e and a s l i g h t r e l i e f of the i n h i b i t i o n of DNA r e p l i c a t i o n caused by f o r s k o l i n treatment. The i n f e c t i o n of avian hematopoietic c e l l s by v-myc expressing v i r u s e s r e s u l t s i n d i s t i n c t and r e p r o d u c i b l e changes i n c e l l phenotype (Symonds et a l . , 1986). Many aspects of the normal d i f f e r e n t i a t e d phenotype are s t i l l expressed by the myc transformed c e l l s as i s the case i n the y - 1 M M C V c e l l s . I n f e c t i o n of the Y-1 c e l l s with Ki-MSV or 3611 r e s u l t e d i n a l t e r a t i o n s i n the r e l a t i v e p r o d u c t i o n of the s e c r e t e d s t e r o i d s . I t i s not understood why a l l the oncogenic v i r u s e s produce a s i m i l a r i n c r e a s e i n the production of f l u o r o g e n i c s t e r o i d s , but i t would appear that the mechanisms i n v o l v e d are d i s t i n c t . Both Ki-MSV and 3611 appear to r e s u l t i n a f u r t h e r t r u n c a t i o n of the s t e r o i d o g e n i c pathway, so that both the v-ras and v - r a f oncogenes seem to f u r t h e r c u r t a i l the d i f f e r e n t i a t e d phenotype of the Y-1 c e l l s . These r e s u l t s i n d i c a t e that the means by which the v-ras and v - r a f products i n t e r a c t with the d i f f e r e n t i a t e d phenotype of the Y-1 c e l l s i s d i s t i n c t from that of the v-myc product, and that even the f i r s t few steps of the s t e r o i d o g e n i c pathway can respond i n d i s t i n c t ways to changes i n c e l l phenotype. I t has been p r e v i o u s l y demonstrated that v-ras i s more e f f i c i e n t than c-ras i n t r a n s f o r m i n g c e l l s (Spandidos and W i l k i e , 1984). The Y-1 c e l l s overexpress c - K i - r a s 2 so that the a l t e r a t i o n s i n s t e r o i d p r o d u c t i o n induced by the v i r a l p 2 1 r a s provides f u r t h e r evidence that the a c t i o n s of a c t i v a t e d and normal ras genes can be d i s t i n c t . Work with the ras oncogenes has i n d i c a t e d that they can modulate expression of the d i f f e r e n t i a t e d phenotype i n many types of c e l l s (Noda et a_l. , 1985; Yuspa e_t a l . , 1 985 ; Wiebe et a l . , i n p r e s s ) . The Y-1 c e l l s appear to respond to the ras and r a f oncogenes by d e d i f f er e n t i a t i o n . I t would appear from the r e s u l t s presented here that v-ras can r e s u l t i n changes i n the Y-1 s t e r o i d p r o d u c t i o n that are d i s t i n g u i s h a b l e from those induced by v - r a f : however, as the i n f e c t e d l i n e s were not cloned i t i s p o s s i b l e that t h i s r e f l e c t s the presence of u n i n f e c t e d c e l l s i n the Y-13611 c u l t u r e . The t i t r e s of the two v i r u s s t o c k s were s i m i l a r (3611=6X10 5; Ki-MSV=10^) and both used the same helper v i r u s (Mo-MLV). Assuming that the i n t r o d u c t i o n of the two oncogenes s t i m u l a t e d the pro d u c t i o n of f l u o r o g e n i c s t e r o i d s to the same extent and that the two v i r u s e s i n f e c t e d with the same e f f i c i e n c y i t would appear that the c u l t u r e s examined were probably i n f e c t e d to the same extent. The e f f i c i e n c y of i n f e c t i o n was not measured d i r e c t l y and the percent of c e l l s i n f e c t e d i s not known, but the d i s t i n c t i v e e f f e c t s i n each of the i n f e c t e d c u l t u r e s i n d i c a t e s the e f f e c t s that would be seen i n a pure p o p u l a t i o n of i n f e c t e d c e l l s . 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 Related to p 2 1 r a s i s  Expressed i n Human and Rat Primary T i s s u e C u l t u r e C e l l s 7.1 INTRODUCTION In the experiments examining the t r a n s f o r m a t i o n of e a r l y passage r a t c e l l s by Ki-MSV a novel band of 27,000 Mr was s p e c i f i c a l l y immunoprecipitated i n a d d i t i o n to p 2 1 r a s i n the e a r l y passages a f t e r i n f e c t i o n . T h i s band had not been repor t e d a s s o c i a t e d with K i - M S V - i n f e c t i o n of c e l l s p r e v i o u s l y (Shih et a l . , 1979 ; Young et al. , 1 979 ). The ras f a m i l y of genes has a l r e a d y been demonstrated to c o n t a i n at l e a s t t h r e e f u n c t i o n a l genes i n mammals, Ha-ras, K i - r a s and N-ras , and to have r e p r e s e n t a t i v e genes i n other s p e c i e s i n c l u d i n g i n v e r t e b r a t e s such as D r o s o p h i l a melanogast er ( S h i l o and Weinberg, 1981) and s i n g l e c e l l organisms such as yeast (Powers et a l . , 1984; DeFeo-Jones et a l . , 1983) and D i c t y o s t elium d i s c o i d eum (Reymond et a_l. , 1984; Pawson et a l . , 1985). The d i v e r s i t y of molecular weights of these p r o t e i n s i n d i c a t e d that products of the ras f a m i l y could cover a wide range of s i z e s . The occurence and expression of members of the ras gene f a m i l y i n such d i v e r s e organisms suggests a fundamental r o l e f o r ras gene products i n c e l l u l a r r e g u l a t i o n . I n t e r e s t i n the ras and r a s - r e l a t e d genes stems from the frequent 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 ras genes with human and animal tumours from a v a r i e t y of t i s s u e s and t h e i r widespread expression i n normal c e l l s (see Barbacid, 1986). Although the r o l e of the ras gene products are not understood, t h e r e i s c o n s i d e r a b l e c i r c u m s t a n t i a l evidence to suggest that they are a b l e to modify the r e g u l a t i o n of c e l l d i f f e r e n t i a t i o n and p r o l i f e r a t i o n . M i c r o i n j e c t i o n of p u r i f i e d p 2 1 r a s i n t o NIH 3T3 c e l l s a r r e s t e d i n low serum can s t i m u l a t e DNA s y n t h e s i s and d i v i s i o n (Feramisco et a l . , 1984), while m i c r o i n j e c t i o n of an a n t i - p 2 1 r a s antibody can i n h i b i t c e l l d i v i s i o n (Mulcahy et a l . , 1985). In yeast the RAS gene products appear to r e g u l a t e c e l l growth i n response to environmental s t i m u l i and to be r e q u i r e d f o r the i n i t i a t i o n of s p o r u l a t i o n (Kataoka e_t a_l. , 1984). The expression of the ras gene products i n D i c t y o s t elium appears to be h i g h e s t i n d i v i d i n g c e l l s (Pawson et a l . , 1985). The ras gene products are r e l a t e d to the GTP b i n d i n g r e g u l a t o r y p r o t e i n s Gs, t r a n s d u c i n , and EF-Tu (Jurnak, 1985; Leberman and Egner, 1 984 ; L o c h r i e et a l . , 1985 ; Tanabe et a l . , 1 985 ), and r e p r e s e n t a gene f a m i l y w i t h i n the s u p e r f a m i l y of genes encoding GTP b i n d i n g r e g u l a t o r y p r o t e i n s . E l u c i d a t i o n of ras gene f u n c t i o n i n mammalian c e l l s r e q u i r e s that the complexity of genes encoding the ras p r o t e i n s and any r e l a t e d p o l y p e p t i d e s be determined. The novel 27,000 Mr p r o t e i n appears to be a r a s - r e l a t e d p o l y p e p t i d e that i s widely expressed i n normal c e l l s . R ESULTS 7.2 Survey of p27 Expression. The expression of ras p r o t e i n s i n primary c u l t u r e s of r a t and human c e l l s was examined by immunopr e c i p i t a t i o n of [ 3 5 s ] m e t h i o n i n e l a b e l l e d c e l l l y s a t e s with the a n t i - p 2 1 r a s monoclonal antibody Y13-259 (Furth et a l . , 1982). This antibody r e c o g n i z e s a h i g h l y conserved a n t i g e n i c determinant w i t h i n the ras p r o t e i n s and i s a b l e to p r e c i p i t a t e a l l known forms of mammalian p 2 1 r a s as w e l l as the p r o t e i n s encoded by _S_. c er e v i s i a e , D.  d i s c o i d eum and D_. melanogaster ras genes (DeFeo-Jones et  a l . , 1983; Pawson et a_l. , 1 985 ). The c u l t u r e s examined i n c l u d e d c e l l s of e p i t h e l i a l o r i g i n , r a t ovarian s u r f a c e e p i t h e l i u m , and other hormone s e c r e t i n g c e l l s , the ovarian g r a n u l o s a c e l l s and adrenal cortex c e l l s , as w e l l as f i b r o b l a s t i c c e l l s from the lung and muscle f a s c i a and complex mixtures of d i f f e r e n t i a t e d c e l l s from other organs. Rat or human primary c u l t u r e s were grown u n t i l n e a r l y c o n f l u e n t and then m e t a b o l i c a l l y l a b e l l e d f o r 16 hours with C35s] methionine or passaged once b e f o r e l a b e l l i n g . A l i q u o t s of c e l l l y s a t e s c o n t a i n i n g e q u i v a l e n t TCA p r e c i p i t a b l e - [ 3 5 s ] cpm were immunoprecipitated with Y13-259 or an i r r e l e v a n t r a t monoclonal antibody. A l i n e of r a t ovarian g r a n u l o s a c e l l s transformed by Ki-MSV ( H a r r i s o n and Auersperg, 1981) was l a b e l l e d and immunoprecipitated i n p a r a l l e l as a p o s i t i v e c o n t r o l f o r the immunoprecipitation. C e l l u l a r p 2 1 r a s was s p e c i f i c a l l y immunoprecipitated from a l l primary c u l t u r e c e l l s , and c e l l l i n e s examined ( f i g u r e 7.1). This r e s u l t extends the number of types of c e l l s examined f o r the expression of p 2 1 r a s . The a n t i - p 2 1 r a s immunoprecipitation of the Ki-MSV transformed r a t ovarian granulosa c e l l s r e a d i l y demonstrated expression of v i r a l p 2 1 r a s (lane 1, f i g u r e 7.1). In a d d i t i o n another p r o t e i n with an approximate Mr of 27,000 was immunoprecipitated from a l l the primary c u l t u r e s , but not from the Ki-MSV-transformed c e l l l i n e. This newly i d e n t i f i e d p r o t e i n appeared to be as widely expressed as p 2 1 r a s i n primary r a t and human t i s s u e c u l t u r e ( F i g u r e 7-1 and Table 7.1) and was expressed i n some c e l l l i n e s such as rat-1 and a r a t adrenal cortex c e l l l i n e , but was not d e t e c t a b l e i n other l i n e s such as mouse NIH 3T3 f i b r o b l a s t s and the human calu-1 lung adenocarcinoma l i n e (see t a b l e 7.1 f o r a complete l i s t ) . The reason f o r the d i f f e r e n c e i n the degree of l a b e l l i n g of p 2 1 r a s and p27 between the d i f f e r e n t c u l t u r e s i s not e n t i r e l y c l e a r . Although the same number of counts was used i n each immunoprecipitation the i n t e n s i t y of the l a b e l p r e c i p i t a t e d appeared to drop with the decrease i n the a b i l i t y of the c u l t u r e to i n c o r p o r a t e [ 3 5 s ] methionine. 7.3 F r a c t i o n a t i o n of the L a b e l l e d Lysate of a p27 Expressing  C e l l L i n e . S e v e r a l p o s s i b l e explanations f o r the co-immunoprecipitation of 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 the expression of ras 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 Methods. The c e l l s from one 60mm d i s h were l a b e l l e d overnight at the end of f i r s t passage with 100 uCi 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 1.5x10 7 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 r a s monoclonal Y13-259. The p u r i f i e d products were separated on a 12.5? SDS-po l y a c r y l a m i d e g e l , En 3Hanced and exposed to XAR-5 f i l m at -80°C. Panel A: A Ki-MSV-transformed r a t ovarian granulosa 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 primary c u l t u r e s examined were: lane 2-ovarian granulosa c e l l s ; lane 3-lung f i b r o b l a s t s ; l a n e 4-adrenal c o r t e x parenchyma; l a n e 5-muscle f a s c i a ; lane 6-kidney stroma; Panel B: lane 1-adult lung f i b r o b l a s t s ; l a n e 2-preadult (prepubescent) lung f i b r o b l a s t s ; l a n e 3-adult adrenal cortex parenchyma; and lane 4-preadult adrenal c o r t e x . a = a n t i - p 2 1 r a s 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. 1 2 3 4 a b a b a b a b P 27 p21 c-ras tp27 ip21 eras Table 7.1 Expression of p27 in some Rodent and Human Cell Cultures. Cel ls Examined Origin of Cultures p27 Expression Rat-1 FSV-transformed Rat-1 Strain A Ki-MSV-trans formed Strain A NIH3T3 Ki-MSV-transformed NIH3T3 Calu-1 Rat embryo f ib rob las ts Rat hepatocytes Human foreskin f ib rob las ts NT, Immortal fps-transformed NT, Immortal ras-transformed NT, Immortal ras-transformed ras-transformed human carcinoma NT, primary culture •NT, primary culture NT, primary culture + + + +/-+ + Cel ls were labe l led and immunoprecipitated with Y13-259 ant i -p21 as described in the legend to Figure 7.1 and Materials and Methods and analyzed by SDS-PAGE. NT- nontransformed cu l tu res . Calu-1 is a c e l l l i n e iso lated from a human lung adenocarcinoma that contains as act ivated ras gene that i s thought to be involved in transformation of the c e l l s . The other l i nes are described in Materials and Methods. p27 might share an a n t i g e n i c determinant with p 2 1 r a s which i s recognized by the Y13-259 antibody; i f so, p27 might be a d i s t i n c t but r e l a t e d gene product or a h i g h l y modified form of p 2 1 r a s . Another p o s s i b i l i t y i s that p27 might be a s s o c i a t e d with p 2 1 r a s i n an immunoprecipitab1e complex. I n c r e a s i n g the c o n c e n t r a t i o n of SDS i n the c e l l l y s i s and immunoprecipitation b u f f e r s from 0? to 0.1? or 0.5? w/v d i d not appear to a f f e c t the r e l a t i v e y i e l d of p27 and p 2 1 r a s , making i t l e s s l i k e l y that p27 and p 2 1 r a s are i n a complex. To explore the p o s s i b i l i t y of formation of a p21 r a s/p27 complex, r a t lung f i b r o b l a s t s i n e a r l y passage were l a b e l l e d as above with [35s]methionine and ly s e d i n a b u f f e r c o n t a i n i n g 0.1? w/v sodium deoxycholate, and the c l a r i f i e d l y s a t e was then f r a c t i o n a t e d by s i z e using c e n t r i f u g a t i o n on a l i n e a r 5 to 20? w/v sucrose g r a d i e n t . The g r a d i e n t was f r a c t i o n a t e d and the f r a c t i o n s were then made up to 0.5? w/v SDS and immunoprecipitated with Y13-259 or c o n t r o l a n t i b o d i e s ( f i g u r e 7.2). I t was p o s s i b l e to demonstrate the immunoprecipitation of p27 i n the presence of 0.1? DOC suggesting that DOC would not i n t e r f e r e with the a n a l y s i s (data not shown). Both the p27 and p 2 1 r a s p r o t e i n s sedimented r e l a t i v e to s i z e markers as though they were monomolecular 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 t h a t p27 i s p r e c i p i t a t e d by the a n t i - p 2 1 r a s antibody by v i r t u e of a noncovalent i n t e r a c t i o n with p 2 1 r a s . F i g u r e 7.2 Rate-zonal f r a c t i o n a t i o n of [ 3 5 S ] methionine 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 lung f i b r o b l a s t s i n t h i r d passage were l a b e l l e d overnight with [ 3 5 S ] methionine and ly 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 the only detergent. 10° c.p.m. of the 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 sucrose g r a d i e n t at 50000rpm f o r 18 hours 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 to 0.5$ SDS and immunoprecipitated 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 molecular weight markers was run 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 the f r a c t i o n s analyzed on a 12.5$ SDS-polyacrylamide g e l and the g e l of the g r a d i e n t was marked to i n d i c a t e the f r a c t i o n that corresponded to the most i n t e n s e s t a i n i n g of the marker. a=anti p 2 1 r a s monoclonal Y13-259; b=negative c o n t r o l . 175 7.4 T r y p t i c P e p t i d e Mapping of p27. Si n c e t h e r e d i d not appear to be any complex formed between p27 and p 2 1 r a s , i t seemed probable that the co-immunoprecipitation of the two p r o t e i n s was due to the presence of a common epitope. To examine p27 f o r s t r u c t u r a l homology with p 2 1 r a s the two p r o t e i n s were compared by t r y p t i c p e p t i d e mapping. Both p r o t e i n s were i s o l a t e d from [ 3 5 S ] 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 s , e luted from a g e l , d i g e s t e d with t r y p s i n and subjected to two dimensional a n a l y s i s of the r e s u l t i n g t r y p t i c p e ptides using e l e c t r o p h o r e s i s at pH 2.1 i n the f i r s t dimension followed by chromatography i n the second dimension (see M a t e r i a l s and Methods). This process compares pe p t i d e s on the b a s i s of t h e i r primary sequence depending on number of charges and a f r a c t i o n a l power of the molecular weight f o r s e p a r a t i o n i n the f i r s t dimension and h y d r o p h o b i c i t y f o r s e p a r a t i o n i n the second dimension, and i s very s e n s i t i v e to changes i n amino a c i d sequence. The t r y p t i c p e p t i d e maps of p27 and p 2 1 r a s showed th r e e comigrating peptides (arrowed i n F i g u r e 7.3). Apart from these t h r e e spots the maps of p27 and c e l l u l a r p 2 1 r a s were d i f f e r e n t , i n d i c a t i n g that they a re d i s t i n c t p r o t e i n s and making i t u n l i k e l y that p27 i s a h i g h l y modified form of p 2 1 r a s . The t h r e e comigrating peptides i d e n t i f i e d i n F i g u r e 7.3 were observed i n t r y p t i c d i g e s t s of Ki-MSV and Ha-MSV v i r a l p 2 1 r a s , which are s i m i l a r to the c e l l u l a r p 2 1 r a s and d i s t i n c t from r a t p27. The c o - o r d i n a t e l y poor 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 of p27 and p 2 1 r d . Immunoprecipitated p27 and p 2 1 r a s were eluted from a g e l fragment, d i g e s t e d with t r y p s i n and analyzed by two dimensional t r y p t i c p e p t i d e mapping. The di g e s t e d p r o t e i n s were spotted at the o r i g i n (Or) and e l ectr ophor es ed (anode on the l e f t ) at 1000V f o r 45 minutes and then chromatographed (bottom to t o p ) . The p l a t e s were sprayed with En 3Hance (NEN) and exposed to XAR-5 f i l m at -80°C. The t r y p t i c peptides were analyzed both s e p a r a t e l y and mixed as noted i n the f i g u r e , and a cartoon of the mixed peptides i s drawn i n the lower r i g h t frame. The empty c i r c l e s i n d i c a t e the unique p27 p e p t i d e s , the c i r c l e s with v e r t i c a l hatching i n d i c a t e the unique p 2 1 r a s p e p t i d e s , and the s o l i d c i r c l e s r e p r e s e n t the peptides i n common between p27 and p 2 1 r a s . These shared peptides are a l s o arrowed i n the autoradiograms of the pe p t i d e maps. 177 conserved spots a and b suggests that they might be d i f f e r e n t l y m o d ified forms of a s i n g l e p e p t i d e . S i m i l a r r e s u l t s were obtained when the t r y p t i c p e p t i d e maps of p27 and p 2 1 r a s were done u s i n g e l e c t r o p h o r e s i s at pH 8.9 i n the f i r s t dimension (data not shown) suggesting that the c o m i g r a t i o n shown i n f i g u r e 7 .3 was not f o r t u i t o u s . T r y p t i c p e p t i d e a n a l y s i s of [ 3 5 S ] cys t ein e-lab e l l ed p r o t e i n s i s o l a t e d from the r a t lung f i b r o b l a s t s has r e v e a l e d a f u r t h e r 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 comigrates between maps of p27 and p 2 1 r a s . The peptides marked 1a and 1b i n F i g . 7 . 3d have been shown to comigrate with peptides from the ( c y s t e i n e - l a b e l l e d p 2 1 r a s . 7.5 Pulse-Chase L a b e l l i n g of p27. The maps of p27 and p 2 1 r a s are s u f f i c i e n t l y d i s t i n c t to suggest that these p r o t e i n s are not d i f f e r e n t l y modified forms of the same primary gene product. To i n v e s t i g a t e the p o s s i b i l i t y of a p r e c u r s o r - p r o d u c t r e l a t i o n s h i p between p 2 1 r a s and p27 f u r t h e r , r a t lung f i b r o b l a s t s were l a b e l l e d i n time-course and pulse-chase experiments to c h a r a c t e r i z e the r e l a t i v e r a t e s of s y n t h e s i s and degradation of p27 and p 2 1 r a s . When c e l l s were p u l s e - l a b e l l e d both p27 and p 2 1 r a s became l a b e l l e d w i t h i n f i f t e e n minutes and continued to accumulate l a b e l during longer i n c u b a t i o n times which suggested that the p r o t e i n s were independently s y n t h e s i z e d ( F i g u r e 7.4). When c e l l s were pulsed with [ 3 5 S ] methionine and then chased with an excess of n o n - r a d i o a c t i v e methionine the p r o t e i n s were degraded at d i f f e r e n t r a t e s . The p27 appeared to have F i g u r e 7.4 [ 3 5 S ] m e t h i o n i n e pulse-chase of p27 and p 2 1 r a s . A 60mm d i s h of r a t lung f i b r o b l a s t s were pulsed with 100 uCi of [ 3 5 S ] methionine f o r 0.25 hours ( l a n e 1), 0.5 hours ( l a n e 2), 1 hour ( l a n e 3 ) , 4.5 hours ( l a n e 4), 10 hours ( l a n e 5), and 24 hours ( l a n e 6 ). In lane 1 a l l of the l y s a t e was immunoprecipitated, i n lanes 2 and 3 a h a l f of the l y s a t e was used i n each immunoprecipitation and i n lanes 4-6 one tenth of the l y s a t e was used. a=anti-ras monoclonal Y13-259, b=negative c o n t r o l . B-Rat lung f i b r o b l a s t s were pulsed f o r one hour with 100 uCi [ 3 ^ S ] m e t h i o n i n e and chased with 10mM u n l a b e l l e d methionine f o r 3 hours ( l a n e 1), 7 hours ( l a n e 2), 11 hours ( l a n e 3 ) and 23 hours ( l a n e 4 ). Lane 5 i s an immunoprecipitation of r a t lung f i b r o b l a s t s l a b e l l e d f o r 24 hours as d e s c r i b e d above. a=anti-ras monoclonal Y13-259, b=negative c o n t r o l . 205 TI6 97 B 1 U 2 3 4 5^ a b a a a a b *•- II - 205 - 116 - 97 - 66 66 - 45 p27- -p21*f f . f 2 9 - 29 - 24 - 18 18 a h a l f - l i f e of 6 to 8 hours, while p 2 1 r a s had a h a l f - l i f e of approximately 14 to 16 hours as estimated by the disappearance of immunoprecipitable m a t e r i a l . Thus the turnover r a t e of p27 appeared to be approximately twice as r a p i d as that of p 2 1 r a s and t h e r e d i d not appear to be any c l e a r p r o d u c t - p r e c u r s o r r e l a t i o n s h i p between the two prot e i n s . In f i g u r e 7.1 normal r a t ovarian granulosa c e l l s and a Ki-MSV transformed d e r i v a t i v e c e l l l i n e were l a b e l l e d and immunoprecipitated under s i m i l a r c o n d i t i o n s and while p27 i s r e a d i l y d e t e c t a b l e i n the normal c e l l s i t i s not apparent i n the transformed c e l l s . Experiments comparing m o r p h o l o g i c a l l y normal and Ki-MSV transformed r a t adrenal cortex c e l l s (see f i g u r e 3-5) gave s i m i l a r r e s u l t s i n which p27 was expressed i n the normal c e l l s , but a p p a r e n t l y not i n the transformed c e l l s . In a r a t f i b r o b l a s t i c l i n e the Ki-MSV-transformed d e r i v a t i v e expressed a reduced but s t i l l d e t e c t a b l e amount of p27 ( f i g u r e 7.5) when compared to the p a r e n t a l l i n e . The i n c r e a s e i n p 2 1 r a s expression due to the presence of the v-ras gene i s apparent. S i m i l a r r e s u l t s have been obtained i n an NRK l i n e and i t s Ki-MSV-transformed d e r i v a t i v e . F i g u r e 7.5 Examination of p27 and p 2 1 r e l s expression i n a Ki-MSV transformed c e l l l i n e . An adrenal cortex c e l l l i n e , s t r a i n A ( l a n e b ) , and a Ki-MSV transformed 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 hours with 150uCi of [ 3^S ]methionin e. The c e l l s were lysed and immunopr e c i p i t a t ed as d e s c r i b e d above with 1.5x10^ TCA p r e c i p i t a b l e c.p.m. being used i n each immunoprecipitation. Lanes b and c were from immunoprecipitates using the a n t i - p 2 1 r a s monoclonal antibody Y13-259. Lane a i s a p a r a l l e l immunoprecipitate using s t r a i n A l y s a t e without antibody. The immunoprecipitates were analyzed on a 12.5$ SDS-polyacrylamid e g e l , Enhanced, d r i e d and autoradiographed. 7 . 6 DISCUSSION The 27,000 d a l t o n p r o t e i n d e s c r i b e d here appears to be a n t i g e n i c a l l y and s t r u c t u r a l l y r e l a t e d to p 2 1 r a s , but the product of a d i s t i n c t gene. This new p r o t e i n , p27, i s expressed i n a wide v a r i e t y of r a t and human primary t i s s u e c u l t u r e s , as i s p 2 1 r a s , and some c e l l l i n e s . The c e l l s examined were d e r i v e d from embryonic, immature and a d u l t animals and two mammalian s p e c i e s . The c e l l s r e p r e s e n t d i f f e r e n t embryonic l i n e a g e s , mesodermal and endodermal, and many types of d i f f e r e n t i a t e d c e l l s and should r e f l e c t most types of mammalian c e l l s . The expression of p27 i n a l l these primary c u l t u r e s i n d i c a t e s that i t may be s y n t h e s i z e d i n most, i f not a l l c e l l s , i n the animal. The expression of p27 may be induced when c e l l s are c u l t u r e d , as has been d e s c r i b e d f o r other p r o t e i n s ( B a r r e t t e_t a l . , 1984), but the presence of p27 i n r a t hepatocytes, which e s s e n t i a l l y do not grow i n c u l t u r e , s u r v i v i n g only 24 to 36 hours a f t e r i s o l a t i o n from the animal, strengthens the p r o b a b i l i t y that p27 i s expressed i n v i v o . There does not appear to be any d i r e c t c o r r e l a t i o n between c e l l growth and the expression of p27 or p 2 1 r a s f as they are both expressed i n t e r m i n a l l y d i f f e r e n t i a t e d , n o n d i v i d i n g c e l l s at l e v e l s comparable to those i n d i v i d i n g c e l l s . The expression of ras gene products i n n o n d i v i d i n g c e l l s has been d e s c r i b e d i n mammalian as w e l l as i n v e r t e b r a t e c e l l s , and i t appears that ras p r o t e i n s have r e g u l a t o r y f u n c t i o n s i n both growing and a r r e s t e d c e l l s (Segal and S h i l o , 1 9 8 6 ) . I t does not appear that p27 expression i s r e q u i r e d f o r c e l l v i a b i l i t y as i t i s a p p a r e n t l y absent from some c e l l l i n e s . I t i s p o s s i b l e that p27 i s present i n these c e l l s but i s turned over more r a p i d l y and so i s not detected under the l a b e l l i n g c o n d i t i o n s used here. T h i s apparent independence of expression of p 2 1 r a s and p27 f u r t h e r i n d i c a t e s that they are d i s t i n c t p r o t e i n s . The p27 p r o t e i n i s a p p a r e n t l y c l o s e l y r e l a t e d to c e l l u l a r p 2 1 r a s over a l i m i t e d r e g i o n minimally d e f i n e d by the c o - m i g r a t i n g t r y p t i c p e p t i d e s , but otherwise appears to be d i s t i n c t from p 2 1 r a s both i n sequence and s t a b i l i t y . The Y13-259 antibody presumably r e c o g n i z e s an epitope formed w i t h i n the r e g i o n of amino a c i d sequence conserved between p27 and p 2 1 r a s . This monoclonal antibody i s broadly c r o s s r e a c t i v e a g a i n s t e u k a r y o t i c ras p r o t e i n s , and i n a d d i t i o n to mammalian p 2 1 r a s r e c o g n i z e s the 34 and 39 kd p r o t e i n s encoded by S^ c e r e v i s i a e ras genes (DeFeo-Jones ^st a l . , 1983), a 23 kd .D . discoideum ras p r o t e i n (Pawson et, a l . , 1985), and 21 and 27 kd p r o t e i n s in_D. melanogast er. The major comigrating t r y p t i c p e p t i d e of mammalian p27 and p 2 1 r a s i s a l s o present i n t r y p t i c p e p t i d e maps of [35 S ]methionin e-lab e l l ed D. d i s c o i d eum and S. c e r e v i s i a e ras p r o t e i n s . A comparison of the r e l e v a n t d e r i v e d amino a c i d sequences showed that only a s i n g l e m e t h i o n i n e - c o n t a i n i n g t r y p t i c p e p t i d e i s s u f f i c i e n t l y h i g h l y conserved i n a l l t h r e e ras p r o t e i n s to account f o r the observed comigration (Powers et al.. , 1984; DeFeo-Jones et a l . , 1 9 8 3 ; Reymond et a l . , 1984; Tsuchida et a_l. , 1982). This f i v e amino a c i d p e p t i d e , comprising r e s i d u e s 6 9 to 73 of mammalian p 2 1 r a s with the sequence NH 2_asp-gln-tyr-met-arg-COOH i s conserved between a l l i d e n t i f i e d ras p r o t e i n s with the exception of the yeast R A S S C 1 and R A S S C 2 products which show a s i n g l e , c o n s e r v a t i v e amino a c i d change of a s p a r t a t e to glutamate, which should have a n e g l i g i b l e e f f e c t on the m o b i l i t y of the p e p t i d e i n the mapping procedure. This pentapeptide seems to be a l i k e l y c a n d i d a t e f o r the major comigrating spot of p 2 1 r a s and p27. This p e p t i d e i s l o c a t e d w i t h i n a h i g h l y conserved r e g i o n of the ras p r o t e i n s and i s c l o s e to amino a c i d s (59-6 3 ) that are i m p l i c a t e d i n the oncogenic a c t i v a t i o n of some transforming p 2 1 r a s p r o t e i n (Fasano et a l . , 1984) and i s p a r t of the epitope recognized by the monoclonal Y13-259 ( L a c a l and Aaronson, 1986). I t i s a l s o noteworthy that the t h r e o n i n e at r e s i d u e 59 of v i r a l p 2 1 r a s transforming p r o t e i n s i s phosphorylated, suggesting that t h i s r e g i o n of the p r o t e i n might p a r t i c i p a t e i n GTP h y d r o l y s i s (McCormick et a l . . 1985). The p e p t i d e that i s l a b e l l e d with both [ 3 ^ s ] c y s t e i n e and [ ^ ^ S]methionine (43-68, i d e n t i f i e d as 1a and b i n f i g u r e 7.4) that does not comigrate with any peptides i n p27 appears to be the p e p t i d e i n p 2 1 r a s immediately amino-terminal to the pentapeptide that i s thought to comigrate between p27 and p21 . This p e p t i d e i s the only t r y p t i c p e p t i d e i n p 2 1 r a s to c o n t a i n both c y s t e i n e and methionine (Tsuchida et a_l., 1982). The amino t e r m i n a l g i n can be modified i n the mapping procedure to g i v e more than one spot from a s i n g l e p e p t i d e . The two peptides are both encoded w i t h i n the second exon of mammalian p 2 1 r a s which would i n d i c a t e that p27 i s probably not d e r i v e d from a known ras gene by a l t e r n a t e s p l i c i n g . Recent f i n d i n g s suggest that the mammalian ras gene f a m i l y may be c o n s i d e r a b l y more ex t e n s i v e than p r e v i o u s l y d e s c r i b e d (Madaule and Axel, 1985). With the d i s c o v e r y of other r e l a t e d p r o t e i n s that comprise other f a m i l i e s of GTP-bi n d i n g r e g u l a t o r y p r o t e i n s i t would appear that the su p e r f a m i l y to which the ras genes belong w i l l prove to be q u i t e e x t e n s i v e . I t i s p o s s i b l e that p27 i s r e l a t e d to p 2 1 r a s over much of i t s sequence or i t may simply share a f u n c t i o n a l s i t e presumably i n v o l v e d i n n u c l e o t i d e b i n d i n g or h y d r o l y s i s which c o n t a i n s the epitope recognized by the Y13-259 antibody. The p 2 1 i a o p r o t e i n s appear to be f u n c t i o n a l l y analogous to the G p r o t e i n s that mediate r e c e p t o r c o n t r o l l e d modulation of adenyl c y c l a s e a c t i v i t y (Gilman, 1984). Thus p27 might r e p r e s e n t another GTP b i n d i n g r e g u l a t o r y p r o t e i n that i s d i s t i n c t from p 2 1 r a s but r e t a i n s s u f f i c i e n t homology to bind the a n t i - p 2 1 r a s monoclonal antibody. A number of c e l l u l a r genes such as N-myc and R-fos which have l i m i t e d homology to known proto-oncogenes have been i s o l a t e d (Schwab et a l . , 1 9 8 3 ; Lee et a l . , 1984; ) and i t seems l i k e l y that p27 i s the product of a gene that i s s i m i l a r l y r e l a t e d to the normal c e l l u l a r ras genes. I t appears that t r a n s f o r m a t i o n of c e l l s expressing p27 by Ki-MSV r e s u l t s i n a decrease or l o s s of p27 expression. The l o s s or r e d u c t i o n of p27 expression was observed i n four separate experiments. The mechanism that u n d e r l i e s t h i s change i n p27 pro d u c t i o n i s not known, but may be e i t h e r i n c r e a s e d p r o t e i n turnover or decreased s y n t h e s i s . The decrease of p27 expression i n c e l l s transformed by an oncogenic ras gene i m p l i e s a s p e c i f i c i t y i n the i n t e r a c t i o n that leads to the r e d u c t i o n of p27, but what r o l e t h i s decrease may p l a y i n t r a n s f o r m a t i o n i s obscure. Transformation of the R EF 52 c e l l l i n e by an a c t i v a t e d Ha-ras oncogene r e s u l t s i n a decrease i n expression of the normal p 2 1 r a s (Franza et a l . , 1986). I f p27 i n t e r a c t s with the same r e g u l a t o r y pathway as p 2 1 r a s t h i s may repr e s e n t an analogous s i t u a t i o n i n which expression of a normal c e l l u l a r gene product i s down r e g u l a t e d by a r e l a t e d , one og e n i c a l l y a c t i v a t e d one. CHAPTER 8 SUMMARY The r e s u l t s on the t r a n s f o r m a t i o n of the e a r l y passage adre n a l cortex c e l l s d e s c r i b e d here complement the models d e r i v e d from work on immortalized c e l l l i n e s and much of the previous work on primary c e l l s i n v i t r o . Transformation of many immortalized c e l l l i n e s by a c t i v a t e d oncogenes has been shown to be a s i n g l e h i t phenomenon. In permanent c e l l l i n e s that r e q u i r e m u l t i p l e s t e p s , the ras and myc oncogenes cooperate to produce complete t r a n s f o r m a t i o n . The e a r l y passage c u l t u r e s of the adrenal cortex c e l l s c o i n f e c t e d with Ki-MSV and MMCV did not immediately demonstrate anchorage independent growth (Table 4.3) i n d i c a t i n g t h a t a f u r t h e r change was necessary to allow expression of a f u l l y transformed phenotype. The low e f f i c i e n c y of i n d u c t i o n of focus formation by K i - M S V - i n f e c t i o n of the r a t adrenal cortex c e l l s (Table 4.1 and 4.2) i m p l i e s that the expression of a m o p h o l o g i c a l l y transformed phenotype may r e q u i r e some p r e d i s p o s i t i o n of the c e l l . The i n t r o d u c t i o n of myc seems to s u b s t i t u t e f o r the c e l l u l a r p r e d i s p o s i t i o n . E x t r a p o l a t i o n of e a r l i e r r e s u l t s suggested that the r o l e of myc i n t r a n s f o r m a t i o n by the two oncogenes was to enhance the a c t i v i t y of the a c t i v a t e d ras gene by reducing the t h r e s h o l d f o r t r a n s f o r m a t i o n by TGF's of the c e l l s (Stern et al., 1986). The d i f f e r e n t growth f a c t o r requirements f o r expression of morphological t r a n s f o r m a t i o n of the Ki-MSV-infected r e l a t i v e to the K i -MSV/MMCV-inf ect ed a d r e n a l c e l l s ( F i g u r e 4 . 6 ) i n d i c a t e s that the i n t r o d u c t i o n of the myc gene r e s u l t s i n complex phenotypic changes that i n c l u d e a l o s s or r e d u c t i o n i n the requirements f o r some serum f a c t o r s , but not serum ind ep end enc e. The requirement f o r a t h i r d step to complement ras and myc i n t r a n s f o r m a t i o n of nonimmortalized c e l l s i n v i t r o has a l s o been d e s c r i b e d f o r hematopoietic c e l l s (Stevenson and Volsky, 1 9 8 6 ; Vogt et a l . , 1 9 8 6 ; Schwartz et a l . , 1 9 8 6 ) . The c u l t u r e s d i d not show autonomous growth immediately f o l l o w i n g c o i n f e c t i o n . The appearance of h i g h l y transformed c u l t u r e s was c o r r e l a t e d with the emergence of a s p e c i f i c c l o n e of c e l l s (Schwartz et a l . , 1 9 8 6 ) i n the transformed c u l t u r e , suggesting that a f u r t h e r change was r e q u i r e d b e f o r e the c e l l s could express a h i g h l y transformed phenotype. The c u l t u r e s d i d show some phenotypic a l t e r a t i o n s s h o r t l y a f t e r i n f e c t i o n , but i t was not p o s s i b l e to demonstrate any changes i n the growth f a c t o r r equir ements. The morphological t r a n s f o r m a t i o n of the Ki-MSV/MMCV-i n f e c t e d f i b r o b l a s t s i n response to high c o n c e n t r a t i o n s of serum or EGF stands i n c o n t r a s t to the i n a b i l i t y of e i t h e r serum or EGF to s t i m u l a t e anchorage independent growth. The serum independence of the h i g h l y transformed c u l t u r e s i s presumably not s u f f i c i e n t i n i t s e l f to r e s u l t i n anchorage independence, although the expression of the phenotypes i s l i n k ed. The incomplete t r a n s f o r m a t i o n of e a r l y passage K i -MSV/MMCV-inf ect ed a d r e n a l c e l l s may r e s u l t from an i n h i b i t i o n of the a c t i v i t i e s of the oncogenes that seems to be l o s t when the c e l l s become anchorage independent f o r growth. The requirement f o r EGF to allow the expression of the m o r p h o l o g i c a l l y a l t e r e d phenotype i n the e a r l y passage Ki-MSV/MMCV-infected c e l l s , but the i n a b i l i t y of these c e l l s to form c o l o n i e s i n s o f t agar even i n the presence of high serum c o n c e n t r a t i o n s and exogenous EGF, i n d i c a t e s that the f u n c t i o n s of the ras and myc oncogenes i n e a r l y passage c e l l s are suppressed r e l a t i v e to immortalized c e l l l i n e s . The a s s o c i a t i o n of the i n d u c t i o n of t u m o u r i g e n i c i t y i n S y r i a n hamster embryo c e l l s by ras and myc with monosomy f o r chromosome 15 suggests that the i n d u c t i o n of a transformed phenotype by these two oncogenes r e q u i r e s a l o s s or r e d u c t i o n of s u p p r e s s i o n (Oshimura et al., 1985). I t has been demonstrated that normal c e l l s can suppress the a c t i o n of some oncogene products ( C r a i g and Sager, 1 985 ) and i t seems l i k e l y t h a t a s i m i l a r i n h i b i t i o n prevents ras and myc from r e a d i l y t r a n s f o r m i n g the adrenal cortex c e l l s . The combination of s r c and myc appears to be s u f f i c i e n t f o r t r a n s f o r m a t i o n , and i s exempt from the need f o r a f u r t h e r c e l l u l a r change, as the presence of the two oncogenes r e s u l t e d i n the r a p i d appearance of anchorage independent and serum independent growth. The transforming a b i l i t y of v - s r c alone i n the e a r l y passage adrenal cortex c e l l s appears to be more independent of environmental c o n d i t i o n s than that of v-ras alone, i n d i c a t i n g that the t r a n s f o r m a t i o n by v - s r c i s l e s s s u s c e p t i b l e to suppression than t r a n s f o r m a t i o n by v - r a s . The s u s c e p t i b i l i t y of the t r a n s f o r m a t i o n pathway to suppression seems to be determined by the i n t e r a c t i o n of v-ras or v - s r c with the c e l l i ndependently of myc. The work with the Y-1 a d r e n o c o r t i c a l c e l l s was done i n the hope that an a n a l y s i s of the changes i n the d i f f e r e n t i a t e d phenotype would p r o v i d e some c l u e s as to the mechanism of a c t i o n of the oncogene products. The myc oncogene appears to enhance the s t e r o i d p r o d u c t i o n of the Y-1 c e l l s , w h i le both v-ras and v - r a f seem to a l t e r the expression of the d i f f e r e n t i a t e d phenotype, as r e f l e c t e d i n s t e r o i d p r o d u c t i o n . Although the d i v i s i o n of the oncogenes based on t h e i r e f f e c t on Y-1 s t e r o i d o g e n e s i s separates these t h r e e oncogenes i n t o two groups that are s i m i l a r to those based on the a b i l i t y of the oncogenes to cooperate i n primary c e l l t r a n s f o r m a t i o n , t h i s work does not represent an exhaustive survey and i t i s not c l e a r that a l l the oncogenes from the two groups would show such a c l e a r cut d i s t i n c t i o n . Overexpression of c-myc can a l s o enhance c e l l u l a r response to EGF (Stern et a l . , 1986), and i t might be suggested that the f u n c t i o n of myc i s to enhance expression of s e v e r a l aspects of the phenotype of c e l l l i n e s . The K i -MSV/MMCV-inf ect ed c e l l s appear to have reduced serum requirements f o r expression of an a l t e r e d morphology r e l a t i v e to e i t h e r of the s i n g l y i n f e c t e d c u l t u r e s , implying that i n these c e l l s the myc may act to a l t e r the growth f a c t o r requirements of the c e l l i n a more complex f a s h i o n than simple enhancement of the transfo r m i n g e f f e c t of the v-ras oncogene. The suggestion that TGF-oc p r o d u c t i o n i s d i s p e n s a b l e f o r ras t r a n s f o r m a t i o n (McKay ^ t a l . , 1986) supports the idea that the r o l e of myc i n c o o p e r a t i v e t r a n s f o r m a t i o n may not be as simple as enhancement of TGF -C6 a c t i v i t y but may i n v o l v e enhancement of other p 2 1 r a s f u n c t i o n s , as w e l l as myc induced phenotypic a l t e r a t i o n s independent of r a s . The r o l e of myc i n co o p e r a t i o n with v-src i s not c l e a r as i t was not p o s s i b l e to d i s s e c t the phenotype of the transfo r m i n g c e l l s i n s u f f i c i e n t d e t a i l to det e c t any d i f f e r e n c e s between the 2-1/MMC V - i n f ect 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 of v-ras induced t r a n s f o r m a t i o n of e a r l y passage c e l l s with the l o s s of suppression of the a c t i v i t y of the oncogenes and the reduced expression of the p27 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 . Without any knowledge of the f u n c t i o n of p27, i t i s not p o s s i b l e to know i f the reduced expression of p27 i s d i r e c t l y i n v o l v e d or c o i n c i d e n t a l to expression of t r a n s f o r m a t i o n . The model of the t r a n s f o r m a t i o n pathway i s diagrammed i n F i g u r e 8.1. The most important f e a t u r e s are the requirements f o r at l e a s t one f u r t h e r change to r e s u l t i n a h i g h l y transformed phenotype a f t e r the c o i n t r o d u c t i o n of ras and myc, as compared to the two step pathway seen with t r a n s f o r m a t i o n by s r c or s r c and myc. The a b i l i t y to d e t e c t d i f f e r e n c e s i n the process of t r a n s f o r m a t i o n by the d i f f er ent. one og en es suggests that each oncogene may f o l l o w a somewhat d i f f e r e n t pathway. The s e n s i t i v i t y of the a d r e n o c o r t i c a l c e l l s to these d i f f e r e n c e s should p r o v i d e a u s e f u l system i n which to examine the changes a s s o c i a t e d with t r a n s f o r m a t i o n by the v a r i o u s oncogenes. F i g u r e 8.1 Diagramatic r e p r e s e n t a t i o n of the 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 a d r e n o c o r t i c a l c e l l s . The oncogenes used i n the experiments are i n d i c a t e d . The boxes i n the diagram r e p r e s e n t u n c h a r a c t e r i z e d steps i n the t r a n s f o r m a t i o n pathway that have been d e f i n e d by a v a r i e t y of experiments i n c l u d i n g those d e s c r i b e d i n t h i s t h e s i s . The p a r t i a l l y transformed phenotype i s d e f i n e d as the stage at which the c e l l s are m o r p h o l o g i c a l l y transformed, but serum and anchorage dependent f o r growth. The c e l l s are considered to be f u l l y transformed when they become anchorage independent. ras ras + myc src H src +- myc partially — transformed -fully transformed 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., Alexander, W.S., Cory, S., P a l m i t e r , R.D. and B r i n s t e r , R.L. (1985). The c-myc oncogene d r i v e n by immunoglobulin enhancers induces lymphoid malignancy i n t r a n s g e n i c mice. Nature 518:533-538. 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