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Nucleic acid metabolism of a estrogen dependent adrenal cortical tumor Redman, Lyle Wharton 1968

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NUCLEIC ACID METABOLISM OF A ESTROGEN DEPENDENT ADRENAL CORTICAL TUMOR by I.YLE WHARTON REDMAN B . S c , U n i v e r s i t y o f B r i t i s h C o l u m b i a , 196 A T h e s i s S u b m i t t e d i n P a r t i a l F u l f i l m e n t o The Requirements f o r t h e Degree o f MASTER OF SCIENCE i n t he Department o f B i o c h e m i s t r y We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d " The U n i v e r s i t y o f B r i t i s h C olumbia December, 196 8 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r a n a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l m a k e i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d S t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s m a y b e g r a n t e d b y t h e H e a d o f m y D e p a r t m e n t o r b y h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t b e a l l o w e d w i t h o u t m y w r i t t e n p e r m i s s i o n . D e p a r t m e n t T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8 , C a n a d a ABSTRACT The work i n t h i s thesis consisted of i n i t i a l experi-ments designed to elucidate the role of hormones i n a hormonal dependent tumor. Various aspects of nucleic acid synthesis i n a hormone dependent tumor i n the presence (growing) and absence (regressing) of the hormone were studied. The rates of nucleic acid synthesis were studied i n whole animals by i n j e c t i n g radioactive formate and allowing the animal to incorporate r a d i o a c t i v i t y for various periods of time. Nucleic acids were extracted by PAS, phenol pro-cedure and separated on a MAK column. Labelling of a l l species of nucleic acid was decreased i n regressing tumors. In order to determine whether estrogen i s acting d i r e c t -l y on c e l l s or at some i n d i r e c t p hysiological l e v e l ; the a b i l i t y of c e l l s from growing and regressing tumor to synthe-s i z e nucleic acids i n v i t r o was determined. Results of ex-periments with these c e l l suspensions demonstrate that c e l l s from the regressing tumor had a decreased a b i l i t y to synthe-size nucleic acids r e l a t i v e to growing tumor. The rate of DNA synthesis was decreased somewhat more than RNA. In preliminary experiments the a c t i v i t y of DNA dependent DNA polymerase and RNA polymerase from regressing tumor was compared with the same enzyme i n growing tumor. The s p e c i f i c a c t i v i t y of both RNA and DNA polymerase was decreased i n the regressing tumor. i i In target tissue l i k e uterus stimulation with e s t r a d i o l r e s u l t s i n an increased rate of synthesis of several species of RNA. In the tumor system used i n these preliminary experi-ments, stimulation with estrogens has a greater e f f e c t on the synthesis of DNA than RNA. i i i ACKNOWLEDGEMENTS The work i n t h i s thesis was performed under the super-v i s i o n of Dr. J.F. Richards, Cancer Research Center, Univer-s i t y of B r i t i s h Columbia; his assistance and advice were greatly appreciated. I would also l i k e to thank Dr. R.L. Noble for providing the necessary apparatus and equipment. The techni c a l assistance given by M. Garland and M. Thomson i s also g r a t e f u l l y acknowledged. F i n a l l y I would l i k e to thank the National Cancer I n s t i t u t e of Canada for providing the f i n a n c i a l support needed to perform th i s work.' i v TABLE OF CONTENTS Page ABSTRACT 1 ACKNOWLEDGEMENTS i ; L TABLE OF CONTENTS . j _ v LIST OF TABLES v i i LIST OF FIGURES vi±i LIST OF ABBREVIATIONS i x INTRODUCTION i A. I n d u c t i o n o f tumors by hormone im b a l a n c e 1 B. The e f f e c t o f e s t r o g e n t r e a t m e n t on normal t a r g e t 5 t i s s u e C. S t i m u l a t i o n o f normal a d r e n a l t i s s u e w i t h e s t r o - 10 gens EXPERIMENTAL 12 A. M a t e r i a l s and Methods 12 1) Treatment o f A n i m a l s 12 2) R a d i o a c t i v i t y 12 (a) Source o f c h e m i c a l s 12 (b) C h e m i c a l d e t e r m i n a t i o n s o f DNA and RNA 13 3) E x t r a c t i o n o f N u c l e i c A c i d s 13 (a) Source of c h e m i c a l s 13 (b) Method (paramino s a l i c y l a t e ) 13 (c) D i s u l p h o n a t e , paramino s a l i c y l a t e method 14 4) P r e p a r a t i o n o f a c i d s o l u b l e f r a c t i o n and s e p a r a - 14 t i o n o f RNA and DNA 5) P r e p a r a t i o n o f c e l l s u s p e n s i o n s 15 6) Chromatography o f n u c l e i c a c i d s on MAK columns 15 V Page (a) Source of chemicals 15 (b) Preparation of methylated albumin 16 (c) Preparation of MAK 16 (d) Preparation of MAK columns . 17 7) DNA and RNA polymerase assays 17 (a) Source of chemicals 17 (b) DNA polymerase preparation 18 (c) RNA polymerase preparation 18 (d) Post incubation procedures for RNA and 19 DNA polymerase assays B. Results 1) Comparison of extraction methods 2) Standardization of the MAK column (a) Chromatography of standards (b) Color determinations of nucleic acid f r a c -: tions (c) Digestion with degradative enzymes 31 3) In vivo experiments 35 (a) Choice of precursors 35 (b) C11* sodium formate incorporation into RNA 35 and DNA (c) The e f f e c t of hormone removal on the synthe- 40 s i s of i n d i v i d u a l -nucleic acids 4) Precursor supply i n growing and regressing tumors 42 5) Iii v i t r o experiments 43 6) Comparison of DNA polymerase a c t i v i t y i n growing 45 and regressing tumors v i Page 7) RNA polymerase a c t i v i t y i n growing and r e - 47 g r e s s i n g tumors C. D i s c u s s i o n BIBLIOGRAPHY 51 59 V I I LIST OF TABLES T a b l e Page No. 1. Comparison o f t e c h n i q u e s f o r n u c l e i c a c i d e x t r a c t i o n s . 22 2. C l l f sodium f o r m a t e i n c o r p o r a t i o n i n t o n u c l e i c a c i d s o f thymus, s p l e e n and l i v e r t i s s u e s . • E x t r a c t i o n o f n u c l e i c a c i d s from t h e s e t i s s u e s by s t a n d a r d s a l t t r e a t m e n t o r paramino s a l i c y -l a t e p h e n o l . 23 3. Comparison o f C 1 I f sodium f o r m a t e i n c o r p o r a t i o n i n t o n u c l e i c a c i d f r a c t i o n s o f v a r i o u s t i s s u e s of e s t r o n e p e l l e t e d a n i m a l s . 36 4. C l h sodium f o r m a t e l a b e l l i n g o f RNA and DNA from g r o w i n g and r e g r e s s i n g tumor t i s s u e i n v i v o . 39 5. The e f f e c t o f hormone remo v a l on t h e s y n t h e s i s o f i n d i v i d u a l n u c l e i c a c i d s p e c i e s o f tumor t i s s u e . 41 6. D i s t r i b u t i o n o f r a d i o a c t i v i t y i n t h e a c i d s o l u b l e f r a c t i o n o f g r o w i n g and r e g r e s s i n g tumor t i s s u e . 44 7. C 1" U r i d i n e and C1k TdR l a b e l l i n g o f RNA and DNA by c e l l s u s p e n s i o n s p r e p a r e d from g r o w i n g and r e g r e s s i n g tumors. 46 8- The DNA polymerase a c t i v i t y from the 1 0 5 , 0 0 0 g : s u p e r n a t a n t from c e l l homogenates o f gro w i n g and r e g r e s s i n g tumor t i s s u e . 50 9. lA c o m p a r i s o n o f the a c t i v i t y o f n u c l e a r RNA po l y m e r a s e s i n g r o w i n g and r e g r e s s i n g tumor t i s s u e . 52 v i i i LIST OF FIGURES Figure - Page 1 I d e n t i f i c a t i o n of contaminating polysaccharides found i n nucleic acid f r a c t i o n s , extracted from tissues with paraminosalicylate, phenol. 25 2 A t y p i c a l e l u t i o n pattern of u l t r a v i o l e t absorbing material i n a t o t a l nucleic acid extract. 26 3(a) E l u t i o n pattern of standard yeast sRNA. 28 3(b) E l u t i o n pattern of standard c a l f thymus DNA. 29 3(c) E l u t i o n pattern of r a t l i v e r R-RNA. 30 4(a) E l u t i o n pattern of u l t r a v i o l e t absorbing material i n a t o t a l nucleic acid extract a f t e r digestion with pancreatic R-RNase. 32 4(b) E l u t i o n pattern of u l t r a v i o l e t absorbing material i n a t o t a l nucleic acid extract a f t e r digestion with pancreatic DNase. 33 4(c) E l u t i o n pattern of u l t r a v i o l e t absorbing material i n a t o t a l nucleic acid extract a f t e r digestion with a amylase. 34 5(a) Incorporation of C 1 k dATP into DNA by c e l l free systems - e f f e c t of protein concentration. 48 5(b) Incorporation of C 1 "* dATP into DNA by c e l l free systems at d i f f e r e n t times. 49 i x LIST OF ABBREVIATIONS ACTH - Adreno c o r t i c a l trophic hormone . ATP - 5' triphosphate'of adenosine dATP - 5' triphosphate of deoxyadenosine AMP - 5' monophosphate of adenosine CTP . - 5' triphosphate of cytid i n e dCTP - 5' triphosphate of deoxycytidine DNA - Deoxyribonucleic acid DNase - Deoxyribonuclease GTP - 5' triphosphate of guanosine dGTP - 5' triphosphate of deoxyguanosine yg - 10 ^  gms MAK - Methylated albumin Kieselguhr mRNA - Messenger RNA PCA - Per c h l o r i c a c i d R-RNA - Ribosomal RNA S - Svedberg unit sRNA " - 'Soluble RNA* TTP - 5' triphosphate of thymidine TdR - Thymidine UV - U l t r a v i o l e t - UR - Uridine UTP - 5' triphosphate of uridine - 1 -INTRODUCTION A. Induction of.tumors, by hormone imbalance Hormonal imbalance has been implicated as a cause of tumorigenesis i n various- organs of the endocrine system. In some cases tumorigenesis seems to r e s u l t from abnormally high amounts of hormone, other tumors have resulted from removal of normal stimulus. In t h i s laboratory tumors of the adrenal cortex were developed i n rats subjected to prolonged exposure to high l e v e l s of estrogens. This i s an unusual tumor i n that most adrenal tumors a r i s e because of decrease i n estro-gen content. Endocrine carcinogenesis became a major aspect of cancer research a f t e r Lacassagne (1) f i r s t demonstrated that the n a t u r a l l y occurring s t e r o i d hormone estrone was implicated i n the cause of breast cancer i n mice. This led gradually to general recognition that hormones are s i g n i f i c a n t factors not only i n the aetiology, but also i n the prevention and t r e a t -ment of cancer. There are, broadly speaking, two d i f f e r e n t types of hormonal cancers which occur i n animals and man. To describe these tumor types the terms "hormone dependent" and "hormone-independent" were f i r s t used by Huggins and Scott (2) to denote those neoplasms which depend upon hormones fo r t h e i r maintenance or those which originate by hormonal stimulus and l a t e r become autonomous. Dependent growths are often composed of c e l l s which have been stimulated to p r o l i t e r a t e because of hormonal imbalance, but i n some instances they can be induced • - 2 -by e x t r i n s i c agents such as i r r a d i a t i o n s , carcinogenic hydro-carbons or hormones i n combination with i r r a d i a t i o n . Noble (3) has written a comprehensive review on the re-l a t i o n s h i p between endocrine tumors and hormones. Prolonged exposure of high lev e l s of estrogen have been found to produce a v a r i e t y of tumors of endocrine t i s s u e . Tumor induction i n the anterior p i t u i t a r y of mice and rats was produced by prolonged stimulation with estrogen (4,5,6). Adenomas of the intermediate lobe of the p i t u i t a r y have been observed r a r e l y i n r a t s , but more frequently.'in hamsters, a f t e r estrogen treatment. Tumors of the cervix and vagina have been reported i n many st r a i n s of mice follow-ing prolonged stimulus with estrogen (12,13,14,15,16). The e f f e c t s of treatment with estrogens on mammary tumor induc-t i o n i n mice have been studied extensively and reviewed by Shim Kim (17). The prolonged administration of estrogens to rats has been followed by a high incidence of mammary multiple carcinomas (18,19); some showed metastases. I n t e r s t i t i a l c e l l tumors have been produced i n mice and rats that have been exposed to estrogens for prolonged periods (20,21). Fibromas of the epididymis have been described i n the hamster following prolonged treatment with estrogen plus androgen. Regression of the tumors followed withdrawal of therapy (22). In contrast a number of tumors have arisen by decreased estrogen stimulus. Ovarian tumors have been r e a d i l y produced i n gonadecto-mized mice and rats by transplanting the ovary into tissues from which the venous return enters the p o r t a l system, and - 3 -the l i v e r inactivates the estrogenic hormones (23,241. Rats treated for a year or more with growth- hormone tend to. develop, tumors of the adrenal medulla. Tumors of the adrenal cortex have developed i n c e r t a i n s t r a i n s of mice, r a t s , guinea pigs and hamsters gonadectomized early i n l i f e . They were frequently associated with mammary tumor develop-ment. Wholley (25) i n extensive studies with tumor bearing mice, found that transplanted tumors, grew equally well i n normal and gonadectomized mice. Most adrenal tumors des-cribed i n the l i t e r a t u r e have arisen from decreased levels of estrogens or gonadectomy. These tumors showed evidence of abnormal hormone production. Estrogenic, androgenic and cor-t i c o i d e f f e c t s were produced by such, lesions (25,26).. The induction of adrenocortical tumors by gonadectomy could be prevented by the administration of s t i l b e s t r o l and other estrogens or androgens, or by hypophysectomy, but not by the administration of cortisone. Secretion of sex hormone re-sumed when hypophysectomized rats bearing such neoplasms were given the gonadotrope, whereas ACTH was i n e f f e c t i v e i n re-storing secretory a c t i v i t y of the adenomas. Franks and Chesterman (27) found hyperplasia and adeno-mata of the adrenal cortex i n hamsters treated with s t i l b o e s t -r o l . Gardner (28) has reported that some stra i n s of i n t a c t mice acquiring p i t u i t a r y adenomas a f t e r treatment with estro-gens frequently develop adrenal tumors. Noble (29) has re-cently reported an estrogen dependent u n i l a t e r a l adrenal c o r t i c a l carcinoma i n an inbred s t r a i n of hooded female rats that were stimulated continuously for 5-8 months with estrone p e l l e t s . This s t r a i n of rats normally shows about a 1% i n c i -dence of spontaneous adrenal tumors but a f t e r estrone t r e a t -ment the incidence r i s e s to 25%. The normal adrenals seem predisposed to become carcinomas; often there are adenomas present. The carcinomas appear to arise from the f a s i c u l a r -zone. They are highly anaplastic with m i t o t i c figures. The transplanted tumors a r i s e over a 4 month period with a 9 5% take, some are hemorrhagic and ne c r o t i c . This i s apparently the only reported instance of production of tumors of the adrenal cortex by prolonged treatment with excess estrogen, i n con-t r a s t with gonadectomy or decreased estrogen. In addition t h i s tumor i s hormone dependent, i n contrast with hormone inde-pendent tumors a r i s i n g from treatment with'low leve l s of. estro-gens . The mechanism by which estrogen or other s t e r o i d hormones act to induce the neoplastic state i s s t i l l unknown/ i n fac t i t i s not known even how steroids e f f e c t t h e i r p r o l i f e r a t i v e processes i n normal target t i s s u e s . Lazarev (30) postulated that only those hormones which possess the property of stimu-l a t i n g p r o l i f e r a t i v e processes can cause the development of tumors. Before these hormones can exert t h e i r carcinogenic action, several conditions are necessary, notably: 1) an i n -crease i n t h e i r l e v e l ; 2) t h e i r continuous action; and 3) t h e i r prolonged action. Lazarev (30,35) i n a hypothesis for the mechanism of the carcinogenic action of hormones proposes that hormones can stimulate p r o l i f e r a t i o n and cause de-dif-' f e r e n t i a t i o n of c e l l s . The carcinogenic e f f e c t arises only - 5 -a f t e r the prolonged and continuous action of hormones. The cel l s , under t h i s s t i m u l i become hyperplastic, then precancer-ous, and f i n a l l y a tumor develops. Because so l i t t l e i s known about hormone-induced tumor genesis, some investigators f e e l an understanding may develop through a study of the mechanisms by which s t e r o i d hormones produce t h e i r normal p h y s i o l o g i c a l e f f e c t s i n target organs. B. The e f f e c t of estrogen treatment on normal target t i s s u e Model systems l i k e mammary gland, ovaries, uterus, testes, and prostate, have been used to investigate the r o l e of hor-mones i n control of t h e i r target t i s s u e s . The e f f e c t of estrogen on the hormonal control of rat uterus i s the most investigated system to date. The p h y s i o l o g i c a l effects of estrogen i n the mammalian uterus are mediated by increases i n the synthesis of RNA and protein (31-34). It was clear that an acceleration of syn-thesis of ribosomal RNA and of ribosomes was an e s s e n t i a l feature i n the early action of estrogen i n i t s target organ (35-37). I t i s also known that the growth of the estrogen-d e f i c i e n t uterus was induced by treatment i n utero with RNA extracted from the organ stimulated by the hormone (33,34). The r o l e of messenger RNA i s not determined yet. Further-more i t was also uncertain whether, for early estrogen action, genetic t r a n s c r i p t i o n was t r u l y stimulated p r i o r to enhance-ment of cytoplasmic genetic t r a n s l a t i o n . The general.topics deal with the synthesis of RNA and protein i n the nucleus, the binding i n vivo of t r i t i a t e d 170-e s t r a d i o l to chromatin and the l a t t e r ' s template a c t i v i t y • - 6 -assayed i n v i t r o . Equally important i n the e f f e c t s of estra-d i o l on target tissue i s the transport of RNA from nucleus to cytoplasm, the formation of polyribosomes and the v a r i a t i o n i n the incorporation of amino acids by the polyribosomes as assayed i n the c e l l - f r e e system. The r a t i o s of t o t a l amounts of nuclear RNA to DNA and of protein to DNA ex h i b i t only minor decreases a f t e r ovariectomy, and only s l i g h t increases during the estrous phase but the amounts of RNA and protein i n the cytoplasm of an uterine c e l l from an ovariectomized' animal are much lower than i n normal uterine c e l l s (38). Thus the observation that i n the uterus the concentration of cytoplasmic RNA, but not of nu-clea r RNA, i s alt e r e d markedly by ovariectomy, estrous cyc l e , or hormone treatment constitutes one l i n e of evidence that estrogen i n some way regulates nuclear synthesis of RNA, i t s transport to, and i t s accumulation i n , the cytoplasm. Much of the r a p i d l y labeled RNA or nuclear RNA synthesized i n higher organisms under hormonal stimulus i s ribosomal pre-cursor RNA p a r t i c l e s . Hamilton (38) showed that there was a 500% increase i n the incorporation of t r i t i a t e d uridine into uterine nuclear RNA 20 minutes a f t e r hormone administration to an ovariectomized r a t . Sedimentation studies have shown that the l a b e l i s incorporated into 28s and 18s R-RNA or i t s precursor (39-42). Studies indicate that there i s no accumu-l a t i o n of RNA for any length of time a f t e r hormone administra-t i o n . This suggests that the a d d i t i o n a l nuclear RNA synthe-sized i n response to the hormone i s eithe r turned over into - 7 -t h e c y t o p l a s m (4 3) o r degraded i n t h e n u c l e u s ( 4 4 ) . I n some i s o t o p i c s t u d i e s t h e hormone s t i m u l a t e d s y n t h e s i s o f R N A was s t u d i e d s i m u l t a n e o u s l y i n t h e n u c l e u s and c y t o p l a s m (45-47). S h o r t p u l s e s r e v e a l e d a hormonal e f f e c t o n l y on n u c l e a r R N A , b u t l o n g e r e x p o s u r e t o t h e i s o t o p e i n d i c a t e d an a c c e l e r a t e d s y n t h e s i s o f a l l t y p e s of R N A . The enzyme r e s p o n s i b l e f o r R N A f o r m a t i o n i s a D N A dependent R N A p o l y m e r a s e . The a c t i v i t y o f t h i s enzyme can be a l t e r e d by v a r y i n g t h e i o n i c s t r e n g t h o f t h e medium. H a m i l t o n (38) has.shown t h a t t h e r e a r e two d i f f e r e n t R N A p o l y m e r a s e a c t i v i t i e s ; one s e n s i t i v e t o ( N H ^ ) 2 S O i * and t h e o t h e r n o t . I t has been s u g g e s t e d t h a t t h e s t i m u l a -t o r y e f f e c t o f h i g h s a l t c o n c e n t r a t i o n on R N A polymerase a c -t i v i t y i s due t o d i s s o c i a t i o n o f n u c l e o p r o t e i n complexes w i t h consequent enhancement o f t e m p l a t e a c t i v i t y . W i d r e l l has found t h a t ( N H i t ) 2 S O i t s h i f t s t h e t y p e o f R N A b e i n g s y n -t h e t i z e d f r om p r i m a r i l y r i b o s o m a l t o D N A - l i k e i n base compo-s i t i o n and t h a t hormonal e f f e c t s w h i c h o c c u r i n t h e absence o r p r e s e n c e o f ( N H i t ) 2 S O i t r e f l e c t t h e e f f e c t s o f t h e s e h o r -mones on t h e s y n t h e s i s o f R - R N A and D N A - l i k e R N A , r e s p e c t i v e l y . The f r a c t i o n o f R N A p o l y m e r a s e a c t i v i t y i n s e n s i t i v e t o ( N H i t ) 2 S 0 i + was s t i m u l a t e d 2-4 hours a f t e r a s i n g l e i n j e c t i o n o f e s t r a d i o l b u t t h e s e n s i t i v e f r a c t i o n was not s t i m u l a t e d u n t i l 24 h o u r s l a t e r ( 3 8 ) . Thus i t seems t h a t t h e hormonal s t i m u l a t i o n o f t a r g e t t i s s u e n u c l e a r R N A polymerase i s i n i -t i a l l y r e s t r i c t e d t o t h e s y n t h e s i s o f t h e r i b o s o m a l t y p e of R N A and o n l y a t l a t e r t i m e s , o r n o t a t a l l , w i l l t h e r e be an - 8 -e f f e c t t h a t c a n be i n t e r p r e t e d as t h e s y n t h e s i s o f D N A - l i k e RNA. H a m i l t o n (38) h a s shown t h a t e s t r o g e n a d m i n i s t r a t i o n t o an o v a r i e c t o m i z e d r a t i n c r e a s e s t h e t e m p l a t e a c t i v i t y o f . u t e r i n e c h r o m a t i n b y 26% a t 30 m i n u t e s a f t e r hormone t r e a t -ment and a t 8 h o u r s t h i s ' a c t i v i t y h a d i n c r e a s e d 300% o v e r t h e c o n t r o l . The r a t i o o f RNA t o DNA o f t h e c h r o m a t i n showed a s i g n i f i c a n t i n c r e a s e a f t e r o n l y 15 m i n u t e s o f e s t r o g e n a c t i o n i n v i v o . From 12 t o 24 h o u r s t h e r a t i o o f p r o t e i n t o DNA i n t h e ' C h r o m a t i n a l s o i n c r e a s e d . T h e r e was a p a r a l l e l d e c r e a s e i n t h e r a t i o o f h i s t o n e t o DNA. The p h y s i o l o g i c a l i m p l i c a t i o n o f t h e s e f i n d i n g s i s n o t c l e a r y e t , b u t t h e i n c r e a s e i n c h r o -m a t i n RNA may be a p r e r e q u i s i t e f o r h o r m o n a l s t i m u l a t i o n o f t h e t e m p l a t e a c t i v i t y o f t h e o r g a n ' s c h r o m a t i n . D i r e c t e v i d e n c e t h a t hormones s e l e c t i v e l y c o n t r o l t h e t r a n s c r i p t i o n o f mRNA i s l a c k i n g . B a r o n d e s (48) and o t h e r s (49) h a v e f o u n d t h a t t h e t e m p l a t e a c t i v i t y o f RNA e x t r a c t e d f r o m n u c l e i o r r i b o s o m e s c a n be e s t i m a t e d by i t s a b i l i t y t o s t i m u l a t e amino a c i d i n c o r p o r a t i o n i n t o p r o t e i n i n a c e l l f r e e s y s t e m . R e s u l t s h a v e i n d i c a t e d t h a t a t l e a s t p a r t o f t h e a d -d i t i o n a l RNA s y n t h e s i z e d u n d e r h o r m o n a l s t i m u l a t i o n i s mRNA. A l t h o u g h e v i d e n c e f o r t h e t r a n s c r i p t i o n o f mRNA m o l e c u l e s c o d -i n g f o r s p e c i f i c p r o t e i n s i n h i g h e r o r g a n i s m s h a s y e t t o come; some i n t e r e s t i n g s i t u a t i o n s do e x i s t . G o r s k i (50) h a s shown t h e i n d u c t i o n o f t h e s y n t h e s i s o f a s i n g l e p r o t e i n o c c u r r e d w i t h i n 30 m i n u t e s a f t e r e s t r o g e n t r e a t m e n t and p r i o r t o t h e o v e r a l l s t i m u l a t i o n o f p r o t e i n s y n t h e s i s . The s y n t h e s i s o f t h i s e s t r o g e n - i n d u c e d p r o t e i n was s t i l l d e m o n s t r a b l e b u t a t l o w e r e d l e v e l s i n t h e p r e s e n c e o f a c t i n o m y c i n D s u g g e s t i n g - 9 -that either i t s synthesis i s under t r a n s l a t i o n a l control or that RNA synthesis i n the s p e c i f i c gene locus for t h i s protein i s not e f f e c t i v e l y suppressed by actinomycin D. This may mean that there i s a stable mRNA present i n the cytoplasm that i s activated by estrogen. In an i n vivo pulse experiment estro-gen depressed the incorporation, 30-90 minutes a f t e r i n j e c t i o n , of radioactive amino acids into proteins of d i f f e r e n t c e l l organelles (38,51). This depression may be due to a drain on -ATP- supply. Observations on the a b i l i t y of polysomes to func-t i o n i n a c e l l free protein synthesizing system lead, to the conclusion that an e s s e n t i a l feature of the early action of estrogen i n the uterus i s the appearance i n the cytoplasm, of new polyribosomes, having amino acid incorporation properties d i f f e r e n t from the old ones. Jensen (52-54) Maurer and Chalkley (55) have described properties of e s t r a d i o l receptors found both i n the cyto-plasm and nucleus of target t i s s u e s . They suggest that the estrogen binds to a protein component of chromatin. In summary the early action of 17B-estradiol on uterine c e l l s appears to be (i) binding of the hormone to the chroma-t i n i n the nucleus, ( i i ) stimulation of synthesis of chromo-somal and ribosomal RNA i n conjunction with chromosomal and nucleolar a c t i v i t y , ( i i i ) an acceleration of the rate of f o r -mation of ribosomal precursor p a r t i c l e s ; (iv) an acceleration of transport of the ribosomal precursor p a r t i c l e s with attached messenger RNA to the cytoplasm, and (v) an accumula-tio n of new polyribosomes i n the cytoplasm having d i f f e r e n t - 10 -amino acid incorporating properties compared to those of' the old ones. C. Stimulation of normal adrenal tissue with estrogens. It i s d i f f i c u l t to assess the function of estrogen i n tumorigenesis o r l t s e f f e c t on normal adrenal t i s s u e . The .administration of estrogen stimulates growth of the adrenal glands, and ACTH production by the p i t u i t a r y , but causes a reduction i n the content of c h o l e s t e r o l , ascorbic acid, c o r t i coids and glucose metabolites i n mice and rats (56-60). A l l these changes, being s i m i l a r to those produced by ACTH, have been interpreted as a manifestation of increased a c t i v i t y , i n the adrenal cortex. These e f f e c t s do not occur i n the hypo-physectomized rat; subsequently the presence of the p i t u i t a r y gland i s indispensable for the response of the adrenal cor-tex to estrogen treatment. The stimulation of adrenal growth and protein synthesis by ACTH has been found to be largely dependent on changes i n RNA metabolism mediated by RNA poly-merase (61). I t would seem then that e s t r a d i o l stimulates the p i t u i t a r y to release ACTH which stimulates RNA synthesis. Estrogen may also have a d i r e c t e f f e c t on the adrenal. The work described i n t h i s thesis i s a comparison of the r o l e of estrogen i n maintaining growth of the adrenal tumor t i s s u e , with the r o l e of estrogen i n target t i s s u e . I n i t i a l studies were designed to compare nucleic .acid-metabolism i n growing tumors with tumors that have stopped growing, by evidence of a decreased s i z e , as a r e s u l t of removal of the source of estrogen, i n an attempt to assess - 11 -t h e f u n c t i o n o f t h e e s t r o g e n i n m e t a b o l i s m o f t h e a d r e n a l tumor. The work d e s c r i b e d i n t h i s t h e s i s i n v o l v e s t h r e e d i f -f e r e n t t y p e s of- e x p e r i m e n t s : . 1) P e l l e t e d and u n p e l l e t e d tumor b e a r i n g a n i m a l s were e x p o s e d i n v i v o t o r a d i o a c t i v e n u c l e i c a c i d p r e c u r s o r s , t o compare t h e a b i l i t i e s o f t u m o r s and o t h e r t i s s u e s t o s y n t h e -s i z e n u c l e i c a c i d s . The m c l e i c a c i d s were e x t r a c t e d w i t h p a r a m i n o s a l i c y l a t e , p h e n o l , m i x t u r e and f r a c t i o n a t e d on a m e t h y l a t e d a l b u m i n k i e s e l g u h r c o l u m n . 2) The p o s s i b i l i t y e x i s t s t h a t t h e hormone may be e x e r t i n g i t s a c t i o n b y a l t e r i n g some p a r a m e t e r i n t h e w h o l e a n i m a l and n o t d i r e c t l y e f f e c t i n g t h e tumor c e l l s . I n o r d e r t o e l i m i n a t e t h i s c o n s i d e r a t i o n , i n v i t r o e x p e r i m e n t s were d e s i g n e d u s i n g a v a r i e t y o f r a d i o a c t i v e p r e c u r s o r s t o examine t h e r a t e o f f o r m a t i o n o f RNA and DNA s p e c i e s i n t h e c e l l s f r o m t h e tumor. 3) I n p r e l i m i n a r y e x p e r i m e n t s t h e n u c l e i c a c i d p r e c u r s o r s u p p l y and t h e a c t i v i t i e s o f c e r t a i n enzymes ( i . e . RNA p o l y -m e r a s e and DNA p o l y m e r a s e ) w e r e compared i n tumor t i s s u e f r o m p e l l e t e d and u n p e l l e t e d a n i m a l s . E s t r o g e n a p p e a r s t o s t i m u -l a t e t h e r a t e o f RNA and DNA s y n t h e s i s i n tumor t i s s u e . - 12 -EXPERIMENTAL A.• Materials and Methods 1) Treatment of Animals An inbred s t r a i n of female hooded rats was used i n these experiments. The animals were fed purina laboratory p e l l e t s and water ad libitum. The tumor passage was affected by i n j e c -t i n g c e l l s subcutaneously i n the neck region of pe l l e t e d animals. Tumor regression was recorded by changes i n t o t a l body weight and tumor measurement. The depelleted animals used i n a l l experiments were allowed to regress for long periods of time (14 days) i n order that large changes i n meta-b o l i c rates may be seen. Radioactive precursors were injected i n t r a p e r i t o n e a l l y ; and the animals k i l l e d by stunning i n ether and then decapi-t a t i n g . The tissues were removed r a p i d l y , frozen i n ethanol-C0 2 and stored at -20°C. 2) Ra d i o a c t i v i t y (a) Source of chemicals - Brays l i q u i d s c i n t i l l a t o r (62). The reagents used i n preparing the l i q u i d s c i n t i l l a t o r described i n t h i s thesis were obtained from the following sources: p-dioxane ( p u r i f i e d ) , ethylene g l y c o l , absolute methanol, naph-thalene ( r e c r y s t a l l i z e d ) from Fisher S c i e n t i f i c Co., 2,5 d i -phenyl oxazole (PPO) and 14 di-2-(5-phenyl oxazolyl) benzene (POPOP) from -Pilot Chemicals Inc. The composition of the l i q u i d s c i n t i l l a t o r i s as follows: naphthalene 60 gm, (PPO). 4 gm, POPOP 0-2 gm, methanol (abso-lute) 100 ml, ethylene g l y c o l 20 ml and p-dioxane to make 1 l i t e r . - 13 -(b) C h e m i c a l d e t e r m i n a t i o n s ' o f DNA and RNA The amounts o f t h e s e m a t e r i a l s i n f r a c t i o n s o b t a i n e d by chromatography on MAK columns and the amounts i n t o t a l t i s s u e : e x t r a c t s were d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y by t h e d i -p h enylamine and o r c i n o T c o l o r r e a c t i o n s r e s p e c t i v e l y (63,64). 3) E x t r a c t i o n o f N u c l e i c A c i d s (a) S ource o f c h e m i c a l s The r e a g e n t s used i n t h e e x t r a c t i o n p r o c e d u r e were ob-t a i n e d f r om t h e f o l l o w i n g s o u r c e s : sodium paramino s a l i c y l a t e *K & K L a b o r a t o r i e s , C a l i f o r n i a , - 9 0 % l i q u i d p h e n o l , m - C r e s o l , 8-OH q u i n o l i n e , b e n t o n i t e , N a p h t h a l e n e 1,5 d i s u l p h o n a t e a l l were p u r c h a s e d from F i s h e r S c i e n t i f i c . (b) Method In t h e paramino s a l i c y l a t e p r o c e d u r e (65-68) d e s c r i b e d by K i r b y , weighed samples o f t i s s u e were homogenized i n 15 volumes o f 6% paramino s a l i c y l a t e (17 mg/ml B e n t o n i t e ) a t 21,000 r.p.m. i n an i c e c o o l e d S e r v a l homogenizer. F i f t e e n volumes o f 90% p h e n o l , 10% m - c r e s o l , 0.1% 8 OH q u i n o l i n e were added t o the m i x t u r e , w h i c h was shaken f o r 1 h r a t room t e m p e r a t u r e on a w a t e r b a t h s h a k e r . The two phase system was c e n t r i f u g e d a t 1000 x g , 0°C, f o r 1 h r i n a U n i v e r s a l r e -f r i g e r a t e d c e n t r i f u g e . The aqueous l a y e r was removed by p a s t e u r p i p e t t e , t h e i n t e r f a c e and p h e n o l l a y e r was r e -e x t r a c t e d w i t h 5 volumes o f 6% paramino s a l i c y l a t e as des-c r i b e d above. The p o o l e d aqueous l a y e r s were e x t r a c t e d t h r e e t i m e s w i t h e t h y l e t h e r t o remove r e s i d u a l p h e n o l and t h e - e t h e r was removed t h r o u g h e v a p o r a t i o n i n a s t r e a m o f n i t r o -g e n . The n u c l e i c a c i d s were p r e c i p i t a t e d f r o m s o l u t i o n by t h e a d d i t i o n o f two t o f o u r v o l u m e s o f i c e c o o l e d 9 5% e t h a n o l f o l l o w e d by s t o r a g e o v e r n i g h t a t 0 ° C . (c) D i s u l p h o n a t e , p a r a m i n o s a l i c y l a t e method (65-63) W e i g h e d s a m p l e s o f t i s s u e were h o m o g e n i z e d i n 15 v o l u m e s •of 0.5% n a p h t h a l e n e , 1-5 d i s u l p h o n a t e , s h a k e n w i t h 15 v o l u m e s 90% p h e n o l , 10% m - c r e s o l , 0.1% 8 - h y d r o x y q u i n o l i n e and t h e a q ueous l a y e r w i t h d r a w n . The i n t e r f a c e and p h e n o l l a y e r were r e - e x t r a c t e d w i t h 15 v o l u m e s e a c h o f 6% p a r a m i n o s a l i c y l a t e , 0.7 mg/ml b e n t o n i t e ) , and 90% p h e n o l , 10% m - c r e s o l , 0.1% 8-h y d r o x y q u i n o l i n e . 4) P r e p a r a t i o n o f a c i d s o l u b l e f r a c t i o n and s e p a r a t i o n  o f RNA and DNA (6 9) W e i g h e d amounts o f t i s s u e were h o m o g e n i z e d i n c o l d 0.7 N p e r c h l o r i c a c i d , i n a S e r v a l h o m o g e n i z e r , and c e n t r i f u g e d f o r 10 m i n u t e s a t 1000 x g i n a r e f r i g e r a t e d c e n t r i f u g e d s e t a t 0 ° C . The s u p e r n a t a n t was s a v e d and enough 5 N KOH a d d e d t o remove t h e p e r c h l o r a t e i o n ; t h e pH was a d j u s t e d t o 6.0. T h i s c o n s t i t u t e s t h e a c i d s o l u b l e f r a c t i o n . The a c i d i n s o l u b l e p r e c i p i t a t e was e x t r a c t e d o n c e w i t h e a c h o f a c e t o n e , e . t h a n o l , e t h a n o l : c h l o r o f o r m ( 1 : 1 ) , e t h a n o l : e t h e r (3:1) and e t h e r ; e a c h s u p e r n a t a n t was d i s c a r d e d and t h e f i n a l p r e c i p i t a t e was d i s t r i b u t e d o v e r t h e t u b e s t o d r y . N u c l e i c a c i d s were o b t a i n e d f r o m t h e d r y m a t e r i a l by e x t r a c t i n g w i t h h o t 10% N a C l a t pH 7.4, and p r e c i p i t a t i n g o v e r n i g h t w i t h - 15 -2-4 volumes of i c e c o o l e d 95% e t h a n o l . The n u c l e i c a c i d p r e c i p i t a t e was c o l l e c t e d by c e n t r i -f u g a t i o n , d i s s o l v e d i n 1 volume of 0.3 N KOH/gm t i s s u e arid i n c u b a t e d a t 37°C f o r 18-22 h r s . The DNA was p r e c i p i t a t e d by a c i d i f i c a t i o n w i t h 0.2 volumes of c o l d 3 N HC1. A l i q u o t s of the supernatant and the p r e c i p i t a t e were used f o r determina-t i o n s of r a d i o a c t i v i t y , r i b o s e and DNA. 5) P r e p a r a t i o n o f c e l l suspensions Tumors were removed from animals, minced w i t h s c i s s o r s and homogenized s e v e r a l times i n Robinson's Media (70) w i t h a l o o s e - f i t t i n g g l a s s hand homogenizer. Connective t i s s u e was removed by f i l t e r i n g , through 2 l a y e r s of gauze. A l i q u o t s of the c e l l s u s p e n s i o n were then added to the i n c u b a t i o n f l a s k s , which c o n t a i n e d Robinson's Medium (70); a b u f f e r e d s a l t s p l u s g l u c o s e media, made 10% w i t h r e s p e c t to hbrse serum; and r a d i o a c t i v e p r e c u r s o r s . The i n c u b a t i o n s were stopped by p l a c i n g the f l a s k s on i c e . The n u c l e i c a c i d s were e x t r a c t e d by homogenizing t h e c e l l suspension i n an e q u a l volume of 6% paramino s a l i c y l a t e plus b e n t o n i t e , w i t h a s m a l l p o t t e r homogenizer. The mixture was shaken f o r 1 hr a t room temperature w i t h an equal volume of 90% phenol, -10% m - c r e s o l . The aqueous l a y e r was s e p a r a t e d from the phenol l a y e r by c e n t r i f u g a t i o n and t r e a t e d as p r e v i o u s l y des-c r i b e d t o o b t a i n RNA and DNA f r a c t i o n s . 6) Chromatography of n u c l e i c a c i d s on MAK columns (a) Source of chemicals K i e s e l g u h r was purchased i n the grade s o l d as " H y f l o S u p e r c e l " by the J o h n s - M a n v i l l e Products C o r p o r a t i o n , New York - 16 -C i t y . "Fraction V" bovine serum albumin powder, "for micro-b i o l o g i c a l use" was purchased from the Armour Laboratories, Chicago. Methylated albumin was purchased from the Worthing-ton Biochemical Corporation. (b) Preparation of methylated albumin (71) Five gm of albumin were suspended i n 500 mis of absolute methyl alcohol and 4.2 mis of 12 N hydrochloric acid were .added. The mixture was allowed to stand i n the dark f o r 3 days or more with occasional shaking. The p r e c i p i t a t e was c o l l e c t e d and washed twice with both methyl alcohol and anhydrous ether. Most of the ether was evaporated i n the a i r and then i n vacuo over KOH p e l l e t s . The material was reduced to a powder and stored over KOH. The methylated pro-t e i n was used i n the form of a 1% solution i n water. (c) Preparation of MAK The fines were removed from the Kieselguhr by making a s l u r r y with 0.2 M buffered saline and repeatedly decanting the l i g h t e r material. A suspension of 20 gm of Kieselguhr i n 100 mis of 0.1 M buffered s a l i n e was b o i l e d (to expel air) and cooled. Five ml of 1% e s t e r i f i e d albumin were added. The mixture was s t i r r e d and an addi t i o n a l 20 mis of s a l i n e added. This suspension i s methylated albumin Kieselguhr or MAK. The MAK must be pre-pared fresh each day or the column w i l l lose i t s capacity. Buffered s a l t solutions were prepared to contain 0.0.5 M phosphate buffer (NaaHPOit and KH 2P0i t), appropriate to give a f i n a l pH> of 6.7. The solutions were preserved by addition of a few drops of chloroform to each b o t t l e . - 17 -(d) Preparation of MAK columns The chromatographic columns have an inner j o i n t with a sealed-in f r i t t e d glass d i s c . Both 1 cm and 2 cm diameter columns were used with varying amounts of MAK. - The MAK was poured d i r e c t l y into the column, the excess buffered saline was driven down to the l e v e l of the packed material by apply-ing 4 lbs pressure with nitrogen. The columns were washed with 10 volumes of buffered s a l i n e to 1 volume of MAK. Samples were added to the column i n 0.2 M NaCl .05 M phosphate buf f e r , pH 6.7 and allowed to soak into column u n t i l the f i r s t f r a c t i o n of u l t r a v i o l e t absorbing material at 265 my was eluted with the s t a r t i n g buffer. Material was then eluted from the column with a l i n e a r s a l t gradient pumped onto the column with a Beckman solvent pump (Model 746), at 2 mis/minute. Fractions were c o l l e c t e d on a Gilson automatic f r a c t i o n c o l l e c t o r , scanned for u l t r a v i o l e t l i g h t absorbance at 265 my and r a d i o a c t i v i t y . A f t e r the gradient was fin i s h e d more u l t r a v i o l e t absorbing material at 265 my was eluted with 1.5 M NR\OH. In order to prevent degradation, the 1.5 N N H 4 O H was neutralized with concentrated HC1 as soon as i t came of the column. This technique was l a t e r replaced by e l u t i n g with 0.2% sodium dodecyl sulphate i n 0.4 M buffered NaCl, some-times t h i s was followed by 1.5 N NH40H treatment. 7) DNA and RNA polymerase assays (a) Source of chemicals The reagents used i n these incubations were obtained from the following sources: C 1 k UTP (u) New England Nuclear UTP, CTP, GTP, ATP, Schwarz Bioresearch Inc., New York, d i -t h i o t h r e i t o l , Sigma Chemical Co., St. Louis, 8-C 1 4 dATP, dATP, dCTP, TTP, dGTP, Schwarz Bioresearch Inc., New York. (b) DNA polymerase preparation (72) Weighed amounts of minced tissue were suspended i n 3 volumes of 50 M T r i s buffer containing 5 mM MgCl 2 and 1 mM d i t h i o t h r e i t o l at pH 7.5. The mixture was then homogenized i n a Serval homogenizer at 16,000 r.p.m. for.60-90 seconds; a l l operations were c a r r i e d out on i c e . The homogenate was spun i n a c e l l u l o s e n i t r a t e centrifuge tube for 20 minutes at 30,000 g, 0°C i n a Spinco centrifuge Model L-40. The supernatant was f i l t e r e d through several layers of gauze i n t o fresh c e l l u l o s e n i t r a t e tubes and spun at 105,000 g, 0°C for 1 hour, the supernatant was again f i l t e r e d through gauze and an a l i q u o t set aside for protein determination. Aliquots of the supernatant were now added to the incuba-t i o n tubes. Each tube contained 0.1 yc S-^C-dATP (2 8 my moles), 12.2 mymoles dATP, 80 mymoles of each of dGTP, dCTP and TTP, 1 ymole d i t h i o t h r e i t o l , 1 ymole MgCl 2, T r i s buffer pH 7.8 to f i n a l concentration of 50 mM i n a t o t a l volume of 0.5 mis, 25 yg denatured c a l f thymus DNA and 1 ymole ATP. The incubation was c a r r i e d on at 37° C i n a water bath. (c) RNA polymerase preparation (73-76) Weighed tissue was minced and then suspended i n 10 volumes 0.25 M sucrose containing 1 mM MgCl 2 and 3 mM CaCl 2 - 19 -The s u s p e n s i o n was homogenized f o r 3 seconds i n an i c e c o o l e d S e r v a l homogenizer, f o l l o w e d by t w e l v e s t r o k e s on a l a r g e P o t t e r - E l v e h j e m homogenizer. The homogenate was f i l t e r e d t h r o u g h n y l o n and spun f o r 10 m i n u t e s a t 1000 g, 0°C, i n an I n t e r n a t i o n a l r e f r i g e r a t e d c e n t r i f u g e . The s u p e r n a t a n t was d i s c a r d e d , p e l l e t s were t a k e n up i n a s m a l l amount o f 0.25 M s u c r o s e (1 mM M g C l 2 a n d 3 M C a C l 2 ) and l a y e r e d o v e r 10 volumes o f 0.32 M s u c r o s e (1 mM M g C l 2 a n d 3 mM C a C l 2 ) i n 12 ml c e n t r i -f uge t ubes and spun a t 1000 g, 0°C f o r 10 m i n u t e s . The s u p e r -n a t a n t was d i s c a r d e d , t h e p e l l e t s t a k e n up i n a s m a l l volume o f 0.25 M s u c r o s e (1 mM M g C l 2 and 3 mM C a C l 2 ) and l a y e r e d o v e r 9.0 mis o f ' 1.6 M s u c r o s e w i t h 1 mM M g C l 2 and 3 mM C a C l 2 i n c e l l u l o s e n i t r a t e t u b e s and spun a t 100,000 g, 0°C f o r •,40 m i n u t e s . The s u p e r n a t a n t was d i s c a r d e d and t h e - n u c l e a r p e l l e t s r e s u s p e n d e d i n 0.25 M s u c r o s e i n 12 ml c e n t r i f u g e t u b e s , spun a t 1000 g, 0°C f o r 10 m i n u t e s . T h i s l a s t s t e p was r e p e a t e d and the f i n a l n u c l e a r p e l l e t was t a k e n up i n 1/10 o f t h e o r i g i n a l d i l u t i n g volume. A l i q u o t s o f t h e s u s p e n s i o n o f n u c l e i were now added t o t h e s t a n d a r d i n c u b a t i o n mix. Each tube c o n t a i n e d 0.1 yc 1 "*C UTP (0.2857 mymoles), 20 mymoles UTP, 160 mymoles each o f CTP, GTP, ATP, 1 umole M n C l 2 , 4 mymoles M g C l 2 , 1 ymole d i -t h i o t h r e i t o l and T r i s - m a l e a t e b u f f e r pH 7.8 made up t o a f i n a l c o n c e n t r a t i o n o f 50 mM i n 0.5 ml t o t a l volume. (d) P o s t i n c u b a t i o n p r o c e d u r e s f o r RNA and DNA p o l y m e r a s e a s s a y s The r e a c t i o n s were t e r m i n a t e d by p l a c i n g t u b e s on i c e . - 20 -The m i x t u r e , was, t r a n s f e r r e d , by p a s t e u r p i p e t t e t o c h i l l e d 12 ml c e n t r i f u g e t u b e s , e a c h . c o n t a i n i n g 0.4 ml o f c o l d 7% HCIO^. A s o l u t i o n c o n t a i n i n g 500 ug o f c a l f thymus DNA was added t o each t u b e as a r i n s e and t h e n t r a n s f e r r e d t o t h e HC1CK a c i d tubes and mixed. The tubes were spun a t 1000 g, 0°C, f o r 10 m i n u t e s ; the s u p e r n a t a n t s were s e t a s i d e f o r c o u n t i n g and t h e p r e c i p i t a t e s r e s uspended i n 6 mis o f 1% HC1CH. The m i x t u r e was spun a t 1000 g , 0°C, f o r 10 minu t e s and re s u s p e n d e d i n 3 mis o f 1% HClOi*, spun and S u p e r n a t a n t s p o o l e d . The f i n a l p r e c i p i t a t e was d i s s o l v e d i n 1.5 ml o f 0.2 N NH^OH and c o u n t e d . B. R e s u l t s 1) Comparison o f e x t r a c t i o n methods D i f f e r e n t methods f o r e x t r a c t i n g n u c l e i c a c i d s p e c i e s were i n v e s t i g a t e d t o e s t a b l i s h a c o n v e n i e n t , q u a n i t a t i v e t e c h n i q u e s u i t a b l e f o r r o u t i n e u s e . The n a p h t h a l e n e d i s u l p h o n a t e - p h e n o l t r e a t m e n t l i b e r a t e s sRNA a n d r - R N A i n t o t h e aqueous phase. When t h e r e m a i n i n g i n t e r f a c e and p h e n o l l a y e r a r e r e - e x t r a c t e d w i t h paramino s a l i c y l a t e and p h e n o l . t h e aqueous l a y e r c o n t a i n s mRNA and DNA. D i r e c t e x t r a c t i o n w i t h paramino s a l i c y l a t e and p h e n o l s o l u b i l i z e s t h e t o t a l n u c l e i c a c i d f r a c t i o n i n t o t h e aqueous-phase. S e v e r a l e x p e r i m e n t s were done t o compare t h e e f f i -c i e n c y o f t h e s e methods t o t h a t o f a s t a n d a r d s a l t e x t r a c t i o n . I n t h i s c o m p a r a t i v e s t u d y weighed samples o f r a t l i v e r were e x t r a c t e d w i t h Na d i s u l p h o n a t e , paramino s a l i c y l a t e , - 21 -phenol, or with, paramino, s a l i c y l a t e phenol, mixture as described i n the methods. The r e s u l t s of these experiments': are shown i n Table I. I t i s necessary to re-extract the in t e r f a c e and phenol layer several times to e f f e c t complete s o l u b i l i z a t i o n of a l l nucleic acid f r a c t i o n s . The extracts were shaken on a water bath for 1 hr at room temperature; one paramino s a l i c y l a t e extract was s t i r r e d for 1 hr with a magnetic s t i r r e r . Extraction of tissues, with paramino s a l i c y l a t e i s more e f f i c i e n t and convenient than with Na disulphonate; e s p e c i a l l y i n regard to s o l u b i l i z a t i o n of DNA. S t i r r i n g appears c r u c i a l i n these experiments. In the experiment whose re s u l t s are shown i n Table II 4 male hooded rats were, injected with 25 ^curies of C11* Na formate (3.76 mc/min). The animals were k i l l e d 4 1/2 hours l a t e r by stunning with ether and then decapitated. The tissues were removed, frozen i n dry i c e and ethanol. Weighed samples were extracted with paramino s a l i c y l a t e , phenol or by a standard s a l t extraction, acid p r e c i p i t a t i o n method (69). The t o t a l amounts of nucleic acids extracted with the standard s a l t extraction method were less than that extrac-ted by paramino s a l i c y l a t e ; but the s p e c i f i c a c t i v i t i e s were higher. The s a l t extraction method may be l i b e r a t i n g nucleic acid species whose rate of synthesis i s d i f f e r e n t . The data indicates that the paramino s a l i c y l a t e method i s a more con-venient guanitat.ive and routine procedure for extracting - 22 -TABLE I. Comparison of techniques f o r nucleic acid extractions Tissue No. of Disulphonate PAS (H 20 bath) PAS & Magnetic Exts. (H 20 b-ath) S t i r r e r yg RNA yg DNA yg RNA yg DNA yg RNA yg DNA /qm • /qm /gm /qm /gm /gm Liver I 750 4.8 25.0 7835.2 1182.5 9015.2 250.0 II 1359.36 12.5 1543.44 425.0 2119.28 257.0 III 118.0 35.0 410.17 375.0 390.82 160.0 IV 15.0 0.0 87.23 34.5 89.96- 160.0 Total 8983.66 72.5 9876.04 2017.0 11615.26 827.0 - 23 -.TABLE I I . C 1 ** sodium formate incorporation into nucleic acids  of thymus, spleen and l i v e r t i s s u e s . Extraction of nucleic  acids from these tissues by standard s a l t treatment or paramino  s a l i c y l a t e phenol. Standard Salt Extraction PAS Extraction Tissue yg RNA /am yg DNA /gm S.A. RNA S.A. DNA yg RNA /gm yg DNA /crm S.A. RNA S.A. DNA Thymus - 28,305.7 - 1.56 4250 25,992 .807 1.205 Spleen 2000 .9,940 4.24 9.64 6365 19,580 2.68 6.83 Liver 16m 6020 380 .27 .684 8600 2,140 .44 1.178 Liv e r 56m 6810 318. 8 . 199 . 898 8530 2,934 .343 .516 - 24 -nucleic acids from a l l tissues; then the standard s a l t extrac-t i o n method or disulphonate plus paramino s a l i c y l a t e one. One disadvantage with the paramino s a l i c y l a t e ..phenol extrac-t i o n procedure i s the presence of contaminating polysaccharides When the t o t a l nucleic acid f r a c t i o n was extracted with para-.mino s a l i c y l a t e and. d i v i d e d i n t o RNA and DNA with 0.3 N KOH an insoluble residue remained behind. This residue was hydrolyzed with 0.6 N HCl i n a b o i l i n g water bath for 2 1/2 hours. The hydrolysate was spotted on a' chromatogram (Fig. 1) which was developed i n an ascending solvent (isopropanol water 4:1) for 30 hours. Material i n the hydrolysate ran with the same Rf value as glucose standard; t h i s was interpreted as the contaminating material being gly-cogen. In subsequent experiments polysaccharides were re-moved from the t o t a l nucleic f r a c t i o n i n 0.2 M NaCl .05 M phosphate buffer by centrifugation. An a l t e r n a t i v e to t h i s method was the removal of polysaccharides by digestion with . a amylase., t h i s treatment did not a l t e r the spectra of a t o t a l nucleic acid f r a c t i o n , as w i l l be seen l a t e r . 2) Standardization of the MAK column (a) Chromatography of standards Figure 2 depicts a t y p i c a l e l u t i o n pattern of u l t r a v i o -l e t absorbing material at 265 my obtained, when a t o t a l nu-c l e i c acid f r a c t i o n which was extracted by the paramino s a l i -cylate phenol c r e s o i method., was chromatographed on a MAK column. The peaks were i d e n t i f i e d by comparison with standards UNKNOWN GLUCOSE RIBOSE Figure 1. I d e n t i f i c a t i o n of contaminating material found i n n u c l e i c acid f r a c t i o n s extracted from tissues with equal volumes of 6% p-aminosalicylate and 90% phenol 10% m-cresol. The contaminating material "as hydroly?ed.in 0.6 N HC1 i n a b o i l i n g "ater bath for 2^ hours. The hydrolysate. was spotted on a chromatogram, developed i n an ascending solvent ( i s o -propanol T'ater 4:1 ) for 30 hours, and detected by dipping the chromatogram f i r s t i nto a saturated AgN05 Acetone solution and then into a 0.5% NaOH 100% ethanol solution. - 26 -D. P. M. 0 10 20 30 40 50 70 80 90 F i g u r e 2. A t y p i c a l e l u t i o n p r o f i l e on a 10 ml MAK column of u l t r a v i o l e t a b s o r b i n g m a t e r i a l , e x t r a c t e d from growing tumor t i s s u e w i t h equal volumes of 6% p - a m i n o s a l i c y l a t e and 90% phenol - 10% m - c r e s o l . 1.0 mg of n u c l e i c a c i d s put on column i n 0.2 m NaCl (.05 m phosphate b u f f e r , pH 6.7)- flow r a t e 2 mis per minute- 5.0 ml f r a c t i o n s c o l l e c t e d . 1. G r a d i e n t s t a r t e d 0.4 m NaCl -- 1.5 NaCl (0.05 m phosphate b u f f e r , pH 6.7 500 mis) 2. 1.5 m NH^OH - 27 -sugar'determinations and degradation with enzymes. Figures 3 ( a ) , (b) and (c) are e l u t i o n patterns of u l t r a v i o l e t absorb-ing material of standard s RNA, c a l f thymus DNA and r a t l i v e r R-RNA (77). sRNA was eluted from the column in a stepwise "manner with 0.4 M NaCl .05 M. phosphate buffer pH 6.7, DNA at 0.6 M and R-RNA with 0.8 M. The same res u l t s were obtained when e l u t i o n was done with a l i n e a r s a l t gradient. R-RNA was prepared by extracting a t o t a l nucleic acid f r a c t i o n twice with cold 3 M^sodium acetate pK 6.0. This removed DNA, sRNA and glycogen into the supernatant. (b) Color determinations of. nucleic acid f r a c t i o n s After a t o t a l nucleic acid sample was fractionated on a MAK column by el u t i n g with a l i n e a r s a l t gradient, a l i -quots from each tube were assayed for r a d i o a c t i v i t y on a s c i n t i l l a t i o n spectrometer. Contents of the tubes within an i n d i v i d u a l nucleic acid f r a c t i o n were pooled and dialyzed against d i s t i l l e d H 20 at 4°C. The eluate was l y o p h i l i z e d on a thermovac freeze dryer (Model FD2). The residue containing DNA v/as dissolved i n a small amount of 0.1 N NHi+OH and the diphenyl amine reaction (6 3) performed on an ali q u o t . Simi-l a r l y the residue containing RNA was dissolved i n water and the o r c i n o l color determination performed (64). Color deter-minations indicated that peaks No. (a) degradation peak, (b), (c), (d), (e) eluated with 1.5 M NH^OH or .2% SDS i n .4 M buffered s a l i n e a l l were ribose containing peaks; the diphenyl amine color determination of these peaks showed very l i t t l e contamination with DNA when compared with color development 28 -0.7 0 o6 0.5 o 0 10 20 30 40 Fraction Noc ,Figure 3 (a). U l t r a v i o l e t absorption pattern of Standard yeast sRNA, eluted from a 4.0 ml MAK column. 0.400 mg of sRNA put on column i n 0.2 m NaCl (.05 m phosphate buffer, pH 6 . 7 ) - flow rate 2 mis per minute - 5.0 ml f r a c t i o n s c o l l e c t e d , l r 0.4 m NaCl (0.05 m phosphate buffer, pH 6 . 7 ) 0.9 0.8 0.7 o 0.6 -o o n 5 0 10 20 30 40 50 60 Fraction No0 F i g u r e 3 ( b ) . U l t r a v i o l e t a b s o r p t i o n p a t t e r n of Standard c a l f thymus- DNA, e l u t e d from a 4.0 ml MAK column. 0,400 mg of DMA put on column i n 0.2 m NaCl (.05 m phosphate b u f f e r , pH 6.7) - f l o w r a t e 2 mis per minute - 5.0 ml f r a c t i o n s c o l l e c t e d . 1. - 0.6 m NaCl (0.05 m phosphate b u f f e r , pH 6.7) - 30 -1.2 1.1 0.9 0.8 0.8 0 10 20 30 40 50 60 70 Fraction No. Figure 3 ( c ) . U l t r a v i o l e t absorption pattern of r a t l i v e r r-RNA, eluted from a 4.0 ml MAK column. The r-RNA ^as pre-pared by .extracting a t o t a l n u c l e i c acid: preparation twice w i t h , c o l d 3 m sodium acetate, pH 6.0 0.400 mg of r-RNA put on column i n 0.2 m NaCl (.05 m phosphate buffer, pH 6.7) - flow rate 2 mis per minute - 5.0 ml f r a c t i o n s c o l l e c t e d . -1. - 0.8 m NaCl (0.05 phosphate buffer, pH 6.7) 2. - 1.2 m NaCl (0.05 phosphate buffer, pH6.7) 3. - 1.5 m NH<jOH - 31 -i n . t h e DNA. p e a k . (c) D i g e s t i o n w i t h d e g r a d a t i v e enzymes F i g u r e 4(a) shows an e l u t i o h p a t t e r n o f u l t r a v i o l e t a b s o r b i n g m a t e r i a l a t 265 my, i n a t o t a l n u c l e i c a c i d ex-t r a c t f r o m p e l l e t e d tumor b e a r i n g a n i m a l s a f t e r d i g e s t i o n w i t h p a n c r e a t i c RNase ( W o r t h i n g t o n . B i o c h e m i c a l Corp) R-RNA was t h e o n l y f r a c t i o n removed by. t h i s t r e a t m e n t . T h i s d i -g e s t i o n was p e r f o r m e d i n 0.2 M N a C l .05 M p h o s p h a t e pH 6.7, t h e s e c o n d i t i o n s may h a v e b e e n t o o m i l d f o r e n z y m a t i c de-g r a d a t i o n o f sRNA. F i g u r e 4b shows an e l u t i o n p a t t e r n o f u l t r a v i o l e t a b s o r b i n g m a t e r i a l a t 265 my f r o m a t o t a l nu-c l e i c a c i d e x t r a c t o f tumor t i s s u e f r o m a p e l l e t e d a n i m a l a f t e r d i g e s t i o n , w i t h p a n c r e a t i c DNase ( W o r t h i n g t o n B i o c h e m i -c a l C o r p . ) . The enzyme d e g r a d e d o n l y t h e DNA f r a c t i o n . F i g u r e 4 (c) shows an e l u t i o n p a t t e r n o f u l t r a v i o l e t a b s o r b -i n g m a t e r i a l a t 265 my f r o m a t o t a l n u c l e i c e x t r a c t o f tumor t i s s u e f r o m a p e l l e t e d a n i m a l t h a t w a s d i g e s t e d w i t h a amylase; t h e n u c l e i c a c i d s p e c i e s a r e n o t a f f e c t e d b y t h i s enzyme. I t was c o n c l u d e d t h a t t h e f r a c t i o n s e l u t e d f r o m t h e MAK c o l u m n w i t h i n c r e a s i n g s a l t c o n c e n t r a t i o n s were t h e d e g r a -d a t i o n p e a k , sRNA, xRNA, DNA, R-RNA and yRNA. xRNA may be s i m i l a r t o 5 sRNA, r e p o r t e d i n t h e l i t e r a t u r e (84) . Some a u t h o r s h a v e c o n s i d e r e d yRNA r a p i d l y l a b e l l e d RNA. S e p a r a t i o n o f t o t a l n u c l e i c a c i d s p e c i e s by c h r o m a t o -g r a p h y on MAK c o l u m n s i s a f a s t , e f f i c i e n t , r o u t i n e p r o c e d u r e . The c o l u m n s t a k e f i v e m i n u t e s t o p r e p a r e and a p p r o x i m a t e l y one - i 32 -as 0.7 0.6 0.5 •c . 0 10 20 30 40 50 60 70 80 90 Fraction No. Figure 4 (a). E l u t i o n pattern from a 4 ml MAK column, of u l t r a v i o l e t absorbing material i n a t o t a l n u c l e i c acid extract a f t e r digestion with pancreatic Rnase. 0.-400 mg of n u c l e i c acids put on column i n 0.-2 m NaCl (.05 m phosphate buffer, pH 6.7) - flow rate 2 mis per minute - 5.0 ml f r a c t i o n s c o l l e c t e d . 1. - 0.2 m NaCl (0.05 m phosphate, buffer, pH 6.7) ' .2. - Gradient started 0.4 m — 1.5 m NaCl (0.05 m phosphate buffer, pH 6.7 500 mis) 3. - 1.5 m NH.OH 33 -0,8 0.7 0.6 J 0 10 20 30 40 50 Fraction No. Figure 4 (b). E l u t i o n pattern from a 4 ml MAK column of u l t r a v i o l e t absorbing material i n a t o t a l n u c l e i c acid extract a f t e r digestion with pancreatic Dnase. 0.400 mg of nucleic acids put on column i n 0.2.m NaCl (.05 m phosphate buffer, pH 6.7)- flow rate 2 mis per minute - 5.0 ml f r a c t i o n s c o l l e c t e d . J^. Gradient started 0.4 m --.1.5 m. NaCl (0.05 m phosphate buffer, pH 6.7 500 mis) 2. - 0.2% SDS (Sodium dodecyl sulphate) i n 0.4 m NaCl (0.05 m phosphate buffer, pH 6.7) 3. - 1.5 m NH^OH - 34, 1.4 1.3 U 1.1 1.0 0 10 20 30 40 50 60 70 80 90 100 110 Fraction No. Figure 4 ( c ) . E l u t i o n pattern from a 4 ml MAK column, of u l t r a v i o l e t absorbing material i n a t o t a l n u c l e i c acid extract a f t e r digestion with c£ amylase. 0.400 mg of nu c l e i c acids put on column i n 0.2 m NaCl (...05 m phosphate buffer, pH 6.7) - flow'rate 2 mis per minute 5.0 ml f r a c t i o n s ^ c o l l e c t e d . Vj - 0.2 m NaCl (0.05 m phosphate buffer, pH 6.7) 2. - Gradient started 0.4 m-- 1,5 m NaCl (0.05 phosphate buffer, pH 6.7 500 mis) - 35 -hour to. equilibrate,, depending on the si z e of the column. The nucleic acid f r a c t i o n s can be eluted from the larger columns (10 ml MAK) i n 3 hours, whereas the smaller columns (4 ml MAK) take only one and h a l f hours. The development of routine extraction and separation methods has been discussed. These methods were used to analyze d i f f e r e n t nucleic acid species under a variety of b i o l o g i c a l conditions. 3) In vivo experiments (a) Choice of precursors 2-C11* glycine, 6-C1 "*-orotate and C ^ - u r i d i n e (U) were used as radioactive precursors to study rates of nucleic acid synthesis i n vivo. These precursors l a b e l l e d the RNA and DNA fr a c t i o n s of normal tissues; but did not la b e l those of the tumor s u f f i c i e n t l y to produce s i g n i f i c a n t counts i n each f r a c t i o n a f t e r spreading of the sample by el u t i o n from a MAK column. The nucleic acid species of pe l l e t e d and un-pe l l e t e d tumor bearing animals were l a b e l l e d by incorporation for various periods of time of 100 usuries c l l f Na formate (3.76 mc/mmole) injected i n t r a p e r i t o n e a l l y . The data presented i n Table III i l l u s t r a t e s the d i f -ferences i n l a b e l l i n g of nucleic acids i n tissues of pel l e t e d tumor bearing animals that have been injected with 100 [icuries C1k Na formate 4 and 10 hrs previously. (b) C 1 sodium formate incorporation into RNA and DNA Aft e r p e l l e t e d tumor bearing animals were exposed to 100 (icuries C11* Na formate (3.76 mc/mmole) for 4 hrs. The synthe-t i c a c t i v i t y of tumor DNA was only one ha l f of that seen for TABLE I I I . Comparison of C1** formate incorporation into nucleic acid f r a c t i o n s of various t i s s u e s o f e s t r o n e p e l l e t e d a n i m a l s . T i s s u e yg RNA 1 gm t i s s . y g DNA 1 gm tiss Deg sRNA xRNA DNA R-RNA yRNA L i v e r . 4 h r p u l s e p e l l e t e d 12,692.4 605.0 CPM yg SA 280 327.5 . 855 1,660 139.87 11. 87 310 96.77 3.2 240 28.0 8.57 110 207.5 0.53 210 497.6 0.42 L i v e r 10 h r p e l l e t e d 12,160.1 2,655.0 CPM yg SA 210 124.4 1.69 220 332.8 0.66 170 82.65 2.06 416 12 8.0 3.225 70 60 0.7 0.117 110 75.56 1. 46 Tumor 4 h r p e l l e t e d 5,117.7 2,520.0 CPM yg SA 50 44.1 1.13 420 105.46 3.98 540 99.16 5.45 696 212 3.28 70 212.65 0.33 250 50.4 4.96 Tumor 10 h r p e l l e t e d 11,574.4 680.0 CPM yg SA 1,490 148.0 10.06 370 121. 2 3.05 1,000 91.3 17.52 4,752 30 158.4 4,860 653.82 7.43 200 143.12 1.39 S p l e e n 4 h r p e l l e t e d 10,205.58 4,560.0 CPM yg SA 230 96.01 2.4 650 120.01 5. 42 210 155.81 1.35 2,384 302.0 7. 89 4,600 394.1 11.67 260 111.75 2. 33 S p l e e n 10 h r p e l l e t e d 12,894.4 3,193.4 CPM yg SA 20,460 177.83 115.05 11,9 50 179.4 66.61 3,950 169.96 23.24 232 13 2 1.76 12,480 671.22 18.59 1,760 121.98 14.42 - 37 -l i v e r a n d s p l e e n DNA. U n d e r t h e s e l a b e l l i n g c o n d i t i o n s t h e d e g r e e o f l a b e l l i n g o f RNA s p e c i e s f r o m tumor, l i v e r a nd s p l e e n t i s s u e s we're a ' l l s i m i l a r e x c e p t , t h e s p l e e n R—RNA" f r a c -t i o n was l a b e l l e d t h i r t y t i m e s f a s t e r t h a n t h e R-RNA s p e c i e s o f tumor and l i v e r . A f t e r e x p o s u r e o f t h e p e l l e t e d tumor b e a r i n g a n i m a l t o 100 |j,curies C 1" 1 Na f o r m a t e f o r 10 h r s . The s y n t h e t i c a c -t i v i t y o f tumor DNA f r a c t i o n was f i f t y t o e i g h t y t i m e s t h a t f o u n d f o r l i v e r a n d s p l e e n DNA. The r a t e s o f l a b e l l i n g l i v e r a n d s p l e e n DNA a c t u a l l y d e c r e a s e d f r o m t h e i r 4 h r l e v e l s . T h i s l a r g e i n c r e a s e i n t h e s p e c i f i c a c t i v i t y o f tumor DNA may be an e x p r e s s i o n o f t h e l e n g t h o f t i m e t h a t i s n e c e s s a r y f o r t h e r a d i o a c t i v e i s o t o p e , t o r e a c h maximum c o n -c e n t r a t i o n i n t h e tumor c e l l s . I t may t a k e tumor t i s s u e l o n g e r t o a c c u m u l a t e a c r i t i c a l c o n c e n t r a t i o n o f t h e i s o t o p e b e c a u s e i t i s f a r t h e r f r o m t h e s i t e o f i n j e c t i o n t h a n t h e o t h e r t i s s u e s . I f t h i s d i f f e r e n c e i n t h e r a t e o f s y n t h e s i z -i n g tumor DNA i s due t o s u p p l y , t h e n tumor t i s s u e f r o m p e l -l e t e d a n i m a l s i s d i v i d i n g v e r y r a p i d l y . A f t e r e x p o s u r e t o 100 p c u r i e s o f C 1 4 Na f o r m a t e (3.76 mc/mmole) f o r 10 h o u r s t h e r a t e o f l a b e l l i n g RNA s p e c i e s o f l i v e r t i s s u e h a d d e c l i n e d f r o m t h e 4 h r l e v e l , b u t t h e s p e c i -f i c a c t i v i t i e s o f theRNA f r a c t i o n s o f tumor and s p l e e n t i s -s u e s were i n c r e a s e d g r e a t l y o v e r t h e i r 4 h r l e v e l s , e s p e c i a l l y t h e d e g r a d a t i o n , sRNA and xRNA f r a c t i o n s . T h e r e i s one m a j o r d i f f e r e n c e b e t w e e n tumor and s p l e e n t i s s u e and t h i s i s i l l u s t r a t e d a t t h e 10 h r l a b e l l i n g p e r i o d . The r a t e o f l a b e l l i n g t h e RNA f r a c t i o n s o f b o t h t i s s u e s and - 38 -tumor DNA i n c r e a s e s w i t h t h e l e n g t h o f e x p o s u r e t o t h e i s o -t o p e ; b u t t h e r a t e o f l a b e l l i n g s p l e e n DNA d e c r e a s e s a f t e r e x-'p'o'sure' to- r a d i o a c t i v e f o r m a t e f o r 4 h r s . T h i s p r e l i m i n a r y e v i d e n c e i n d i c a t e s t h a t t h e c e l l s o f tumor t i s s u e a r e s m a l l and r a p i d l y d i v i d i n g when u n d e r h o r m o n a l c o n t r o l . The d a t a s u m m a r i z e d i n T a b l e IV i s a c o m p a r i s o n o f t h e a b i l i t y o f g r o w i n g and r e g r e s s i n g tumor t i s s u e t o i n c o r p o r a t e r a d i o a c -t i v e p r e c u r s o r s i n t o t o t a l RNA and DNA. The n u c l e i c a c i d f r a c t i o n s were e x t r a c t e d by t h e s t a n d a r d s a l t e x t r a c t i o n method f r o m tumor t i s s u e o f p e l l e t e d and u n p e l l e t e d f e m a l e h o o d e d r a t s t h a t h a d b e e n s u b j e c t e d t o 100 u s u r i e s C 1 1* Na f o r m a t e (3.76 mc/mmole) f o r 4 and 10 h o u r s . The r a t e o f l a b e l l i n g o f t o t a l RNA a n d DNA f r a c t i o n s were r e d u c e d more t h a n 50% i n d e p e l l e t e d a n i m a l s . T h i s d e c r e a s e was observed a f t e r e x p o s u r e t o t h e i s o t o p e f o r b o t h i n t e r v a l s . The d a t a f r o m t h e s e p r e l i m i n a r y e x p e r i m e n t s i n d i c a t e t h a t t h e s y n t h e -s i s o f b o t h RNA and DNA i n tumor t i s s u e i s u n d e r h o r m o n a l c o n t r o l . I n o r d e r t o d e t e r m i n e i f t h e s y n t h e s i s o f any p a r -t i c u l a r s p e c i e s o f RNA o r DNA was p r e f e r e n t i a l l y a f f e c t e d b y t h e p r e s e n c e o f t h e hormone t h e n u c l e i c a c i d s o f g r o w i n g and r e g r e s s i n g tumor t i s s u e were e x t r a c t e d and s e p a r a t e d . The d a t a s u m m a r i z e d i n T a b l e V i s a c o m p a r i s o n o f t h e a b i l i t y o f g r o w i n g and r e g r e s s i n g tumor t i s s u e t o s y n t h e s i z e d i f f e r -e n t s p e c i e s o f RNA and DNA. The n u c l e i c a c i d f r a c t i o n s were e x t r a c t e d by t h e p - a m i n o s a l i c y l a t e method f r o m tumor t i s s u e o f p e l l e t e d and u n p e l l e t e d - 39 -TABLE IV. C11* sodium formate l a b e l l i n g of RNA and DNA  from growing and regressing tumor tissue i n vivo. Conditions of Incubation (hr) Precursor DNA CPM/yDNA RNA CPM/yribose Growing 4 formate 4.719 47.996 Growing 10 I I 5.725 49.507 Regressing 4 1.472 27.483 Regressing 10 I I 2.047 22.119 - 40 -female hooded rats that had been subjected to 100 usuries of C l k Na formate for 4, 10, and 16 hrs. The nu c l e i c acid species were separated' by chromatography on a 10 ml MAK column, and the f r a c t i o n s eluted with a l i n e a r s a l t gradient (0.4 M NaCl with .05 M PO^, pH 6.7 > 1.5 M NaCl). A l l RNA and DNA containing f r a c t i o n s extracted from tumor t i s s u e of depelleted female hooded rats were l a b e l l e d at a slower rate than those extracted from tumor tissue of p e l l e t e d animals. Some species are more highly l a b e l l e d than others regardless of the length of time the animal i s ex-posed to the isotope ( i . e . degradation f r a c t i o n , sRNA, xRNA, DNA, R-RNA and yRNA) S.A.'s 19.8, 10.4, 5.34, 6.0, 2.17 and 20.2 r e s p e c t i v e l y f o r 4 hrs exposure - p e l l e t e d animal. The data summarized i n Table III i l l u s t r a t e d that exposure of the tumor bearing animal for 10 hrs to radioactive Na formate l a b e l l e d a l l n u c l e i c acid species optimally; t h i s observation i s again borne out i n the experiments summarized ;in Table V. (c) The e f f e c t of hormone removal on the synthesis of  i n d i v i d u a l n u c l e i c acids If a comparison i s made of the e f f e c t of hormone removal on the i n d i v i d u a l n u c l e i c a c i d species l a b e l l e d a f t e r ex-posure of the animal to isotope for 10 hrs the s p e c i f i c ac-t i v i t y o f : t h e DNA containing f r a c t i o n was reduced 15 f o l d . The s p e c i f i c a c t i v i t i e s of a l l other f r a c t i o n s containing nuc l e i c acids was reduced 4-6 f o l d . If other exposure times are considered the pattern of reduction i n l a b e l l i n g of a TABLE V. -The e f f e c t o f hormone rem o v a l on t h e s y n t h e s i s o f i n d i v i d u a l n u c l e i c a c i d s p e c i e s o f tumor t i s s u e . D e s c r i p t i o n P u l s e Time 100 yc Clh f o r m a t e D e g r a d a t i o n 4S xRNA DNA R-RNA yRNA Growing tumor 4 h r s 19. 8 10. 4 5.34 6.0 2.17 20.2 D e p e l l e t e d A n i m a l 4 h r s 11. 38 8.38 7.76 1.06 9.02 5.66 Growing.tumor 10 h r s 32.15 35.9 12.84 31.2 76 .0 42.8 D e p e l l e t e d A n i m a l 10 h r s 6.82 9. 85 - 2.54 12.75 9.63 Growing tumor 16 h r s 19.4 18.2 - 10.26 20.73 8.7 D e p e l l e t e d A n i m a l 16 h r s 12.68 1.8 4.47 3.45 3.28 2.86 - 42 -nucleic acid species i s not altered d r a s t i c a l l y . When a v i s u a l comparison was ^ made of the el u t i o n pat-terns" Of u l t r a v i o l e t absorbing material a't 2 65 my from grow-ing and regressing tumor tissu e the R-RNA f r a c t i o n appeared to be q u a n t i t a t i v e l y reduced i n size i n the regressing tissue more than the other f r a c t i o n s . This disproportionate reduc-t i o n of the R-RNA f r a c t i o n was further seen when the t o t a l amounts of RNA for each f r a c t i o n were compared. Despite the implication that R-RNA synthesis i n tumor tissu e i s prefer-e n t i a l l y c o n t r o l l e d by the presence of estrogens the rate of l a b e l l i n g of the DNA containing f r a c t i o n i s reduced to a greater degree i n the depelleted animal exposed to C1k Na formate for 10 hrs. than the R-RNA f r a c t i o n . The preliminary evidence thus f a r presented seems to indicate that hormone removal affects the rate of synthesis of DNA to a greater degree than the rate of synthesis of RNA i n tumor t i s s u e . Growth then of the adrenal tumor, as seen by an increase i n size and weight of the tumor i n pe l l e t e d a n i -mals, may be brought about by c o n t r o l l i n g the size a tumor c e l l obtains and the rate i t d i v i d e s . 4) Precursor supply i n growing and regressing tumors The precursor supply i n growing and regressing adrenal tumors was examined i n preliminary experiments to determine i f the supply of nucleotides was 3Mt±ng, in the regressing tumor compared to the growing tumor; and might cause a decreased rate of synthesis of RNA and DNA. Weighed amounts of tumor - 43 -tissue from p e l l e t e d and unpelleted animals that were exposed for 4 and 10 hrs to 100 ucuries c1!*Na formate were extracted with cold 6.7 N HClOij acid to obtain an acid soluble f r a c t i o n . This f r a c t i o n was chromatographed on a 1 x 3 cm DEAE c e l l u -lose column i n the bicarbonate form. The nucleosides, bases and unused l a b e l were eluted q u a l i t a t i v e l y from the column . with 0.005 M N H 4 H C O 3 pH 8.6; the nucleotide f r a c t i o n was eluted from the column with 1 M N H 4 H C C 3 pH 8.6. The data sum-marized i n Table VI are the r e s u l t s of many experiments i n which the t o t a l amount of r a d i o a c t i v i t y i n the nucleotide f r a c t i o n was examined. The amount of radioactivity/gm of tissue i n the nucleotide f r a c t i o n of depelleted tumor bearing animals was less than i n animals with growing tumors; a f t e r exposure to 100 |j,curies of C l l f Na formate (4.01 mc/mmole) for 4 and 10 hours. The microscopic and macroscopic morpho-logy of growing and regressing adrenal tumors shows a higher preponderance of connective tissue i n the regressing than i n the growing tumors. Preliminary evidence suggests then that hormone removal causes a reduction i n c e l l d i v i s i o n and growth, with the net e f f e c t of fewer cells/gm regressing tumor. If there were fewer cells/gm tissue then there would be less r a d i o a c t i v i t y i n the nucleotide f r a c t i o n and t o t a l amounts of RNA and DNA would be reduced. In order to eliminate t h i s and eliminate the p o s s i b i l i t y that estrogen i s acting at a s i t e other than within the c e l l i n v i t r o experiments were performed. 5) In v i t r o experiments In order to e s t a b l i s h that the hormone was not exerting TABLE VI. D i s t r i b u t i o n of r a d i o a c t i v i t y i n the acid soluble f r a c t i o n of  growing and regressing tumor t i s s u e . Precursor Description and L a b e l l i n g Time Radioactivity i n Nucleotide Fra c t i o n C.P.M./gm of Tissue Na formate Growing tumor 4 hrs 19,105 Na formate Growing tumor 4 hrs 20,129 Na formate Tumor from depelleted animal 4 hrs 12,701 Na formate Tumor from depelleted animal 4 hrs 15,228 Na formate Growing tumor 10 hrs 18,354 Na formate Growing tumor 10 hrs 17,632 Na formate Tumor from depelleted 9,720 - 45 -i t s stimulatory action i n d i r e c t l y by a l t e r i n g other parameters i n the whole animal instead of acting d i r e c t l y at the c e l l l e v e l single c e l l suspensions were prepared from growing and regressing tumors, and aliquots were incubated i n Robinsons media (10% with respect to horse serum) with radioactive pre-cursors for periods up to 2 hrs. Under.these conditions 2-rC 1 1* TdR and C1"* Na formate were incorporated l i n e a r l y into the DNA f r a c t i o n over a period of 2 hours. C l l f Na formate, 8-C11* hypoxanthine, C 1''-uridine (U) and 6-C1 ^-oratate incor-poration into RNA was completed within 30 minutes. The data summarized i n Table VII i s a comparison of the a b i l i t y of cells, from growing and regressing tumor tissue to synthesize RNA and DNA, l a b e l l e d with C 1 l*-2-uridine (495 mc/ mmole) and 2-C1'*-TdR (54.5 mc/mmole) respectively. The tumor was allowed to regress for 14 days, at t h i s time the rate of formation of DNA was reduced but the rate of RNA synthesis was not i n Experiment II and I I I . Tumor l i n e s may vary i n t h e i r synthetic a c t i v i t i e s . Table VII, Experiment I shows a com-parison of RNA l a b e l l e d with C 1''-uridine (U) 6-C 1 1*-orotate, and DNA l a b e l l e d with 2-Cllf-TdR, by tumor c e l l s from p e l l e t e d and unpelleted animals. The rate of synthesis of RNA and DNA i n t h i s tumor l i n e depends on the presence of the hormone. Any influence by c e l l numbers on the a b i l i t y of c e l l s to synthesize i n v i t r o RNA and DNA has been eliminated by examin-ing the rate of synthesis of these*molecules. 6) Comparison of DNA polymerase a c t i v i t y i n growing and  regressing tumors TABLE VII. C l l f Uridine and C l l 4TdR l a b e l l i n g of RNA and DNA by c e l l suspensions  prepared from growing and regressing tumors. Type of Tumor Precursor S.A. of DNA S.A. of RNA Experiment I 2-C1'tTdR C l l fUr (U) Growing tumor male (6A-8) unpelleted male 14 days 11.84 5.88 Average of 6 determinations 697.1 569.1 Average of 8 determinations Experiment I 6-C 1"oratate 6-C 1 Horatate Growing tumor male (6A-8) tinpelleted male 14 days no incorporation 67.0 61.8 Average of 8 determinations Experiment II 2-C1"TdR C i 4Ur (U) Growing tumor female (ovarectomized) (3A-8) depelleted female 14 days Growing tumor male depelleted male 14 days 3.755 1.530 2.035 0.3542 49.606 62.876 47.947 50.967 Experiment III 2-C i HTdR C 1 4 U r (U) Growing tumor female (6A-9) depelleted female 14 days 8.03 2.308 Average of 4 determinations 392.453 472.611 Average of 4 determinations - 47 -Although the method used (72) to s o l u b i l i z e DNA polymerase produces a crude homogenate, the enzymatic a c t i v i t y of t h i s f r a c t i o n has requirements s i m i l a r to various p u r i f i e d mamma-l i a n enzymes (72-76). Figure 5(a) i s an enzyme concentration curve for DNA polymerase. The incorporation of 8-C 1 4 dATP into acid insoluble counts by DNA polymerases from both grow-ing and regressing tumor tissue was dependent on protein concentration. Figure 5(b) shows a time curve for DNA poly-merase. The incorporation of 8-C11* dATP into acid insoluble counts hy DNA polymerases from regressing and growing tumor tissue was l i n e a r for 8 and 15 minutes respectively, with optimum amounts of enzyme. The a c t i v i t y of the enzyme from regressing tumor i s lower under a l l conditions. The res-ponse to increasing amounts of primer DNA i s s i m i l a r i n re-gressing tumor to growing tumor. The r e s u l t s i n Table VIII indicate that on the basis of s p e c i f i c a c t i v i t y measurements there i s proportionately 50% less a c t i v i t y i n the f r a c t i o n from the regressing tumor. These re s u l t s agree with e a r l i e r findings; the a f f e c t of removing the hormone from tumor bearing animals appears to be directed more at the l e v e l of DNA synthesis than RNA syn-thesis . 7) RNA polymerase a c t i v i t y i n growing and regressing  tumors The experiments reported here are of a very preliminary nature. - 48 -•>'• Growing Regressing 0.1300 . • 0.1200 'l 0.1100 moles 1.0 2.0 3.0 Mg. Protein Figure 5 (a). A c t i v i t y of "DNA polymerase i n homogenates of growing and regressing tumor tissue. Incorporation of C'^cL ATP into DNA - E f f e c t of protein concentration. Each tube contained 0.1 p,c 8 - C'^d ATP (28 m p, moles), 12.2 m \± moles d ATP, 80 m p, moles of each of d GTP, d CTP and TTP, 1 \i mole d i t h i o t h r e i t o l , 1 p, mole MgCl8 , T r i s buffer pH 7.8 to f i n a l concentration of 50 m Molar i n a t o t a l voltime of 0.5 mis, 25 (j,g denatured c a l f thymus DNA and 1 p, mole ATP. The incubation was c a r r i e d on at 37 C i n a water bath. m p, moles C* 4 d ATP incorporated 0.1500 0.1400 0.1300 0.1200 0.1100 0.1000 0.0900 0.0800 0.0700 0.0600 0.0500 0.0400 0.0300 0.0200 0.0100 ^ 0.0000 - 49 -Growing Regressing 10 2*0 30 40 Time (Mins) Figure 5 (b). Incorporation of d ATP int o DNA by c e l l free systems at d i f f e r e n t times. Each tube contained 0.1 \±c 8 - C H d ATP (28 m n moles) , 12.2 m u, moles d ATP, 80 m p. moles of each of d GTP, d CTP and TTP, 1 p mole d i t h i o t h r e i t o l , 1 p. mole MgCl & , T r i s buffer pH 7.8 to f i n a l concentration of 50 m Molar i n a t o t a l volume of 0.5 mis, 25 p,g denatured c a l f thymus DNA and 1 p, mole ATP. The incubation was ca r r i e d on at 37*C i n a water bath. - 50 -TABLE V I I I The DNA p o l y m e r a s e a c t i v i t y front t h e 105,000  g s u p e r n a t a n t f r o m c e l l h o mogenates o f g r o w i n g and  r e g r e s s i n g tumor t i s s u e . I n c u b a t i o n C o n d i t i o n s m y m o l e s o f C 1 "*dAMP i n c o r p o r a t e d /mg P r o t e i n / 3 0 ' i n c u b . G r o w i n g Reg. Med. ENZ 15' i n c u b . a t 37°C 0.07292 0.02186 •^denatured DNA 0.02358 0.037 4 + u n d e n a t u r e d DNA (25 yg) 0.09126 0.03764 +low amt. d e n . DNA (10 yg) 0.05828 0.2784 + h i g h amt. d e n . DNA (75 yg) 0.1672 0.06612 - 51 -The r a t e o f i n c o r p o r a t i o n o f C 1 l fUTP (U) i n t o a c i d i n -s o l u b l e c o u n t s was n o t l i n e a r a f t e r 3 m i n u t e s i n c u b a t i o n w i t h RNA p o l y m e r a s e s f r o m b o t h g r o w i n g and r e g r e s s i n g tumor t i s s u e . D e g r a d a t i v e enzymes may be r e s p o n s i b l e f o r some o f t h i s d e -c r e a s e i n s y n t h e t i c a c t i v i t y . The a c t i v i t y seems t o r e a c h a p l a t e a u a f t e r a v e r y s h o r t ± i m e i n t e r v a l , f o r t h i s r e a s o n we were n o t a b l e t o e s t a b l i s h an a d e q u a t e t i m e c u r v e . I f a c o m p a r i s o n i s made a t any one t i m e i n t e r v a l b e t w e e n RNA p o l y -m e r a s e s f r o m g r o w i n g and r e g r e s s i n g tumor t i s s u e a d i f f e r -e n c e c a n be s e e n . RNA p o l y m e r a s e s f r o m g r o w i n g and r e g r e s -s i n g tumor t i s s u e showed a g r e a t e r a f f i n i t y f o r M n + + t h e n M g + + , b o t h enzymes a r e s t i m u l a t e d b y ( N H 4 ) 2 S O i f , t h e i r r e s u l t s a r e s e e n i n T a b l e IX. A f t e r 7 m i n u t e s i n c u b a t i o n t i m e when t h e a c t i v i t i e s h a d p l a t e a u e d a d i f f e r e n c e i n a c t i v i t y c a n be s e e n b e t w e e n RNA p o l y m e r a s e s o f g r o w i n g and r e g r e s s i n g tumor t i s s u e . T a b l e IX i l l u s t r a t e s t h a t i n most c a s e s t h e enzyme 'from r e g r e s s i n g t i s s u e i n c o r p o r a t e s C1** UTP (U) i n t o a c i d i n -s o l u b l e c o u n t s a t a l o w e r r a t e t h a n t h e enzyme f r o m g r o w i n g tumor t i s s u e . I n summary, t h e hormone a p p e a r s t o be s t i m u l a t i n g t h e s y n t h e s i s o f DNA and t o a l e s s e r d e g r e e t h e s y n t h e s i s o f RNA b y a l t e r i n g t h e s y n t h e t i c a b i l i t i e s o f DNA d e p e n d e n t , DNA and RNA p o l y m e r a s e s . T h i s l e v e l o f h o r m o n a l s t i m u l a t i o n o f a d r e -n a l tumor t i s s u e i s o b s e r v e d b o t h in v i v o and i_n v i t r o . C. D i s c u s s i o n The p a r a m i n o s a l i c y l a t e method o f e x t r a c t i n g n u c l e i c a c i d s p r o v e d t o be c o n v e n i e n t , and t h e r e c o v e r y o f DNA and - 52 -TABLE IX A c o m p a r i s o n .of. the . a c t i v i t y o f  n u c l e a r RNA.polymerases i n growing- and r e g r e s - s i n g tumor t i s s u e . I n c u b a t i o n C o n d i t i o n s Mymoles o f C u UMP i n c o r p o r a t e d /mg DNA/30' Growing Reg. Med. amt. o f N u c l e i ; 7' i n c u b . a t 37° C 15.5 12.9 5 -Mg + Mn .5 ym 16.85 12.48 + ( N H O 2 S O 4 35.95 30.8 + Mn 1.0 ym 16.45 12.85 + Mn 2.0 ym 14.95 11.17 + Mn 4.0 ym 14.29 10.92 -MN + Mg 1.0 ym 12.25 7.95 . + Mg 8.0 ym 18. 86 13.64 - 53 -RNA i s more e f f i c i e n t t h a n w i t h t h e d i s u l p h o n a t e paramino s a l i c y l a t e t r e a t m e n t o r s t a n d a r d s a l t e x t r a c t i o n . The m i l d e r t r e a t m e n t o f e x t r a c t i n g n u c l e i c a c i d s w i t h paramino s a l i c y l a t e and d e n a t u r i n g t h e p r o t e i n s ,with a p h e n o l -c r e s o l m i x t u r e has the d i s a d v a n t a g e o f e x t r a c t i n g g l y c o g e n as w e l l as t h e n u c l e i c a c i d s . A l t h o u g h t h e r e i s no c o n c l u -s i v e e v i d e n c e o f complete r e m o v a l o f p o l y s a c c h a r i d e s by t h e i r r e l a t i v e i n s o l u b i l i t y i n b u f f e r e d s a l i n e s o l u t i o n s o r by e n z y m a t i c d i g e s t i o n , t h a t w h i c h may remain does n o t appear t o i n t e r f e r e w i t h s e p a r a t i o n o f n u c l e i c a c i d s by column chromatography. The e l u t i o n p a t t e r n s o b s e r v e d i n t h e p r e s e n t s t u d y w i t h m e t h y l a t e d a l b u m i n K i e s e l g u h r columns a re c o n s i s t e n t w i t h t h a t r e p o r t e d i n t h e l i t e r a t u r e . Chromatography on columns o f m e t h y l a t e d b o v i n e serum a l b u m i n a b s o r b e d on K i e s e l g u h r has p r o v e d a v e r y u s e f u l t o o l t o f r a c t i o n a t e n u c l e i c a c i d s . The s e p a r a t i o n of t h e b r o a d c l a s s e s o f RNA and DNA i s c l e a r , and r e c o v e r y o f undenatured n u c l e i c a c i d s i s v i r t u a l l y q u a n i t a t i v e . P h i l i p s o n (78) ex-t r a c t e d n u c l e i c a c i d s from H e l a c e l l s and chromatographed them on a MAK column; he r e p o r t e d a d e g r a d a t i o n , sRNA, DNA and R^RNA peaks. E l l e m (74,80) and Yoshikawa (81) have found t h a t a l a r g e p a r t o f r a p i d l y s y n t h e s i z e d RNA from L c e l l s i s bound t o o t e n a c i o u s l y t o t h e MAK column t o be e l u t e d by a s a l t g r a d i e n t ; i t c o u l d be e l u t e d e i t h e r w i t h 1.5 M' N H 1 4 O H o r L 5 M N a C l a t an e l e v a t e d t e m p e r a t u r e (90°C) . - 54 -Many other authors (82-94) have extracted nucleic acids from plant and animal tissues; and chromatographed them on Mandel and Hershey's 3 layered columns (82) or Sueoka's (83) one layered column and obtained r e s u l t s s i m i l a r to those re-ported here. Fraser (84.) recently has reported a 5S micro-somal RNA f r a c t i o n that i s eluted from the MAK column close to. our x-RNA f r a c t i o n . The n u c l e i c acid species eluted from the MAK columns i n t h i s project were i d e n t i f i e d i n the. following order and manner. sRNA f r a c t i o n by sugar determination and column •chromatography of standard sRNA. The DNA component was iden-t i f i e d by these .two methods and degradation with DNase I which was (RNase f r e e ) . The R-RNA f r a c t i o n was examined by , a l l three methods. Some authors omit the degradation f r a c t i o n when reporting t h e i r r e s u l t s , t h i s r e l a t i v e l y r a p i d l y l a b e l l e d species may contain oligoribonucleotides that are i n s e n s i t i v e t o RNase digestion under the present incubation conditions. The sRNA species was RNase i n s e n s i t i v e while R-RNA was RNase s e n s i t i v e . I t was f e l t that the incubation conditions em-ployed were not d r a s t i c enough to e f f e c t hydrolysis of the h e l i c a l areas i n the sRNA molecule. Many authors r e f e r to the material eluted with 1.5 M NR\OH or 0.2% sodium dodecyl sulphate i n buffered 0.4 M NaCl as r a p i d l y l a b e l l e d DNA-like RNA, or mRNA; (95,96) others r e f e r to t h i s peak as. single ^stranded DNA. (9 7). RNase digestion and color determinations of material i n t h i s f r a c t i o n do not tend to substantiate these t h e o r i e s . mRNA i s degraded i n 5 m i n u t e s w i t h RNase a t 0°C, ( 9 8) w h i l e t h e yRNA was RNase i n s e n s i t i v e and d i d n o t c o n t a i n •DNA, as- shown b y - c o l o r r e a c t i o n and eri'zymatlc d e g r a d a t i o n ; th*e s p e c i f i c a c t i v i t y o f yRNA was h i g h e r t h a n R-RNA b u t s i m i l a r t o sRNA and t h e d e g r a d a t i o n f r a c t i o n . F u r t h e r i n v e s t i g a t i o n s w i t h d i f f e r e n t i s o l a t i o n methods may h e l p t o i d e n t i f y t h e de-g r a d a t i o n , xRNA and yRNA pe a k s . F o r t h e p r e s e n t p u r p o s e s t h e MAK column appears s u i t a b l e f o r d i f f e r e n t i a t i n g t h e g e n e r a l s p e c i e s o f n u c l e i c a c i d s . On .comparing tumor, l i v e r and s p l e e n t i s s u e s from p e l -l e t e d a n i m a l s , exposed f o r 4 h r s t o r a d i o a c t i v e Na f o r m a t e , i t a p pears t h a t tumor t i s s u e s a r e s y n t h e s i z i n g n u c l e i c a c i d s q u i t e s l o w l y ; b u t on comparing s y n t h e s i s a f t e r 10 h r s expos-u r e t o t h e i s o t o p e , n u c l e i c a c i d s y n t h e s i s compared t o o t h e r t i s s u e s a ppears t o have r e a c h e d an optimum. T h i s may be due t o t h e remoteness o f t h e tumor from t h e s i t e o f i n j e c t i o n o r t h i s t i m e i s t a k e n t o l a b e l t h e p r e c u r s o r p o o l o f tumor t i s s u e and he nee l a b e l RNA and DNA m a x i m a l l y . When o p t i c a l d e n s i t y s c ans o f u l t r a v i o l e t a b s o r b i n g m a t e r i a l a t 265 my from g r o w i n g and r e g r e s s i n g tumor t i s s u e were examined t h e R-RNA f r a c t i o n f r om r e g r e s s i n g tumor appeared d i m i n i s h e d v i s u a l l y and s p e c t r o p h o t o m e t r i c a l l y more t h a n o t h e r f r a c t i o n s . One e x p l a n a t i o n f o r t h e s e o b s e r v a t i o n s may be t h a t t h e numbers, o f c e l l s / g m o f r e g r e s s i n g tumor t i s s u e a r e l e s s t h a n i n t h e g r o w i n g tumor; b o t h m i c r o s c o p i c and m a c r o s c o p i c o b s e r -v a t i o n s t e n d t o s u p p o r t t h i s . - 56 -P r e c u r s o r supply, may be l i m i t e d . N u c l e o t i d e s u p p l y i s ATP dependent, t h e f o r m a t i o n , t r a n s p o r t a t i o n , o r u t i l i z a t i o n o f t h i s e n e r g y s o u r c e may be a f f e c t e d when the e s t r o n e p e l l e t i s removed. These o b s e r v a t i o n s can be e x p l a i n e d v e r y e a s i l y -i f t h e r e a r e fewer c e l l s / g m o f t i s s u e i n t h e r e g r e s s i n g tumor and hence l e s s RNA and DNA. However s p e c i f i c a c t i v i t i e s w h i c h a r e a b e t t e r r e f l e c t i o n o f n u c l e i c a c i d s y n t h e s i s show t h a t a l l f r a c t i o n s i n r e g r e s s i n g tumors a r e b e i n g l a b e l l e d a t a s l o w e r r a t e t h a n i n g r o w i n g tumor. The f a t e o f l a b e l l i n g o f the DNA f r a c t i o n i s r e d u c e d t o a g r e a t e r degree t h a n t h e o t h e r s p e c i e s . I n o r d e r t o e l i -m i n a t e any e f f e c t t h a t fewer c e l l s / g m o f t i s s u e may have and t h e p o s s i b i l i t y t h a t t h e hormone may be a f f e c t i n g t h e a n i m a l a t an o r g a n l e v e l and n o t i n t r a c e l l u l a r l y , i n v i t r o e x p e r i -ments u t i l i z i n g a v a r i e t y o f p r e c u r s o r s were p e r f o r m e d . The a b i l i t y o f g r o w i n g v.nd r e g r e s s i n g tumor t i s s u e t o s y n t h e s i z e n u c l e i c a c i d s was compared. I n some tumor l i n e s b o t h RNA and DNA s y n t h e s i s was decreased' a f t e r tumor b e a r i n g r a t s were d e p e l l e t e d f o r 14 days whereas- i n o t h e r l i n e s j u s t DNA s y n t h e -s i s was d e c r e a s e d . These o b s e r v a t i o n s i n v i t r o supplement t h e d a t a o b t a i n e d i n whole a n i m a l e x p e r i m e n t s , hormone r e -moval a f f e c t s t h e s y n t h e s i s o f RNA and DNA ( i . e . RNA and DNA p o l y m e r a s e ) . The a c t i v i t y o f key enzymes i n t h e s y n t h e s i s o r d e g r a d a -t i o n o f n u c l e i c a c i d s may be a f f e c t e d by t h e r e m o v a l o f the hormonal s t i m u l u s . Some v e r y p r e l i m i n a r y e x p e r i m e n t s were done t o compare a c t i v i t i e s o f RNA, DNA p o l y m e r a s e s i n g r o w i n g - 57 -and r e g r e s s i n g tumor t i s s u e . T h e s e enzymes were n o t p u r i f i e d b e c a u s e a c o m p a r i s o n b e t w e e n t h e enzyme a c t i v i t y i n g r o w i n g and r e g r e s s i n g tumor t i s s u e was a l l t h a t : w a s d e s i r e d . The a c t i v i t y o f D N A p o l y m e r a s e f r o m g r o w i n g and r e g r e s -s i n g tumor t i s s u e i s d e p e n d e n t on p r o t e i n c o n c e n t r a t i o n . The enzyme o f r e g r e s s i n g tumor t i s s u e i s n o t s o d e p e n d e n t on p r o t e i n c o n c e n t r a t i o n f o r i t s a c t i v i t y as t h e enzyme i n grow-i n g tumor t i s s u e ; t h i s may r e f l e c t t h e pr.esence o f d e g r a d a -t i v e enzymes. B o t h enzymes p r e f e r d e n a t u r e d D N A as a p r i m e r i n p r e f e r e n c e t o n a t i v e D N A . V7hen t h e e s t r o n e p e l l e t was removed f r o m tumor b e a r i n g a n i m a l s t h e a c t i v i t y o f D N A d e p e n -d e n t D N A p o l y m e r a s e i n tumor t i s s u e was r e d u c e d 5 0 % . The n u c l e a r R N A p o l y m e r a s e s f r o m g r o w i n g and r e g r e s s i n g -'•••feumor t i s s u e b o t h show p r e f e r e n c e f o r M n + + o v e r M g + + , and a r e s t i m u l a t e d b y ( N H i t ) 2 S 0 i t . One o f t h e m a i n d i f f i c u l t i e s i n t h i s p r e l i m i n a r y e x p e r i m e n t was t h a t t h e a c t i v i t y o f R N A p o l y m e r a s e was n o t l i n e a r a f t e r 3 m i n u t e s i n c u b a t i o n t i m e . I f one compares t h e s p e c i f i c a c t i v i t y o f R N A p o l y m e r a s e f r o m g r o w i n g tumor w i t h t h a t o f r e g r e s s i n g tumor a f t e r a p l a t e a u i n a c t i v i t y i s r e a c h e d , t h e enzyme f r o m g r o w i n g tumor has a h i g h e r s p e c i f i c a c t i v i t y . T h e s e r e s u l t s complement t h o s e o b t a i n e d i n w h o l e a n i m a l and in v i t r o e x p e r i m e n t s . The t o t a l amount o f r a d i o a c t i v i t y p e r gram o f t i s s u e i n t h e n u c l e o t i d e f r a c t i o n o f r e g r e s s i n g tumor i s l e s s t h a n i n t h e n u c l e o t i d e f r a c t i o n o f g r o w i n g tumor. I n summary, we h a v e s t u d i e d t h e g e n e r a l n u c l e i c a c i d m e t a b o l i s m o f an e s t r o g e n d e p e n d e n t a d r e n a l c o r t i c a l tumor. The tumors r e g r e s s i n s i z e and s y n t h e t i c a c t i v i t y a f t e r t h e - 58 -estrone p e l l e t has been removed. I f the p e l l e t i s not r e -placed the tumor w i l l regress t o a s m a l l nodule and stay i n t h i s i n a c t i v e form u n t i l again s t i m u l a t e d by estrogens. Ex-periments i n which the RNA and DNA s y n t h e s i s were measured under a v a r i e t y of c o n d i t i o n s ( i n v i v o , acid, s o l u b l e , i n v i t r o DNA and RNA polymerase experiments) i n d i c a t e t h a t the s y n t h e s i s of a l l species of n u c l e i c a c i d are reduced upon hormone removal. The syn t h e s i s of DNA i s reduced more than the s y n t h e s i s of RNA, more work i s needed before conclusions can be drawn as t o the a c t i o n of estrogens i n t h i s tumor system. Some i n t e r e s t i n g experiments t h a t should be performed might i n c l u d e l o o k i n g at the e f f e c t of hormone removal on RNA metabolism a f t e r short time i n t e r v a l s , i . e . 4-5 hours, the e f f e c t of i n j e c t i n g 173 e s t r a d i o l i n t o animals or the a d d i t i o n of 173 e s t r a d i o l t o in_ v i t r o c e l l i n c u b a t i o n s , t o see i f a r e v e r s a l of the decrease i n RNA and DNA metabo-l i s m of r e g r e s s i n g tumors i s p o s s i b l e . 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