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Amino Acid Incorporation by in Vitro Tumor and Liver Systems and Their Response to Exogenous Ribonucleic Acid M. A. O'NEAL* AND A. C. GRIFFINt (Deparimeni of Biochemistry, The Univertity of Texas M. D. Anderson Hoipital and Tumor Institute, Houston, Texa.@) SUMMARY A procedure is described for the isolation of amino acid-incorporating systems from Novikoff ascites tumor and from rat liver. Under the conditions employed the tumor system will incorporate approximately 80—70M@imolesof amino acid/mg ribosomal protein when C'4-labeled valine, phenylalanine, or lysine is added to the incubation medium. Addition of ribonucleic acid preparations from tumor and liver nuclei, tumor ribosomes, or tobacco mosaic virus to the tumor amino acid-incorporating system caused a 16-45 per cent increase in amino acid incorporation. Addition of polyuridylic acid to the tumor system with C'4-i@-phenylalanine caused a seven- to tenfold increase in the incorporation of this amino acid. These findings indicate that this mammalian system responds to the same nucleotide coding sequence for phenylalanine as E. coli and provide evidence that the tumor-incorporating system will respond to a limited extent to natural ribonucleic acids and to synthetic polynucleotides. Extensive progress has been made In all these systems, in establish ing the function of an informational or messenger ribonucleic acid (RNA) in protein formation, with the use of normal and phage-infected bacterial sys tems (6, 8, Q4) as well as mammalian tissues (9. 16, @O).The observation of Nirenberg and Matthaei (18) that addition of polyuridylic acid to activat ing components and ribosomes isolated from E. coli causes the formation of a polypeptide posed entirely stimulus for of phenylalanine identification com provided of a number the of the . although cate that discrepancies and code degeneracy it may be more complex first considered. A variety of mammalian than ity Division of Biology, California acid Insti November bearing minutes t American Cancer Society Professorof Biochemistry. for publication their capacity RNA's, to polyuridylic, respond and to various polycytidylic 7-day-old transplants. ‘Thefluid was immediately chilled, and all subsequent opera tions were carried out at O@—5@ C. The cells were centrifuged at 750 r.p.m. for 5 minutes and were washed 5 times with a solution containing .O@M glucose, .14 M NaCl, and .04 M Tris, pH 8.5 (11). After a final centrifugation at 1600 r.p.m. for 5 tute of Technology, Pasadena, California. Reeived and rats was at incorporating systems has been investigated, in eluding liver (7), reticulocytes (@1), Ehrlich ascites tumor cells (11), pancreas (@5), and cell nuclei (1). S Present address: the amino acid in MATERIALS AND METHODS Novikoff ascites fluid was aspirated from albino mdi in vitro amino however, may be attributed to the inherent RNA associated with ribosomes. Re cently, several groups have demonstrated a strik ing effect of polyuridylic acid on phenylalanine uptake by mammalian incorporating systems (@, 3, 5, 16, @8). In the present paper ribosomal fractions iso lated from Novikoff ascites cells and from rat liver are compared with respect to their inherent activ natural acids. specific polynueleotide coding units corresponding to most of the amino acids (14, @8).These findings, along with other observations (@, @7, @9),sup port the concept of a more or less universal code, . corporation messenger the ascites were stirred water. The 5, 1962. cells, now free of erythrocytes, with 6 volumes of cold, deionized cells were allowed to stand for 5 628 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1963 American Association for Cancer Research. ‘O'NEALAND GRIFFIN—Amino minutes before being disrupted in a Potter-Elveh jem homogenizer. The course of the homogeniza tion was followed under a phase-contrast micro scope to achieve the maximum cell rupture without damage to the nuclei. In all preparations more than 90 per cent of the cells were ruptured. After the homogenate was adjusted to .005 M MgCl2, .O2@5M KC1, and .25 M sucrose, it was centrifuged at 15,000X g for 20 minutes in the Spinco preparative ultracentrifuge, Model L, rotor #21. The resulting supernatant solution, free of cell debris, nuclei, and mitochondria, was care fully aspirated from under a lipide layer and was centrifuged for 2 hours at 105,000X g (rotor #40). The supernatant solution (5100) was aspirated, and the pellets were resuspended in a 2.5 per cent deoxycholate solution in 0.2 Mglycyiglycine buffer, pH 8.0 (11). After gentle homogenization in a Potter-Elvehjem homogenizer, the solution was diluted with 28 volumes of standard buffer (.005 M MgC12, .025 M KC1, .@5 M sucrose, and pH 7.6) and again centrifuged @ M Tris, pH 5. Both fractions were frozen and stored at —15°C. The yield of ribosomes from 185 ml. of the ascites fluid, averaging 50 X 1O@cells per ml., was approximately 100 mg. of protein. In a few cases, the 15,000 X g supernatant fraction was used in the incubation without separation of the ribo somes and the activating system. Both the ribo somes and the 5100, pH 5, fraction retained their activity for 1—2weeks when stored at —15°C., with only slight loss. Protein was determined by a modification of the method of Lowry et at. (1%). U@C'&.z,.Va1ine (25.9 mc/mmole) was obtained from Nuclear-Chicago Corp., U-C'4-i@-phenylal anine (30 mc/mmole), and U-C―-frlysine (8.3 mc/m.mole) were obtained from Yolk Radio chemical Company. The crystalline ribonuclease came from Worthington Biochemical Corporation. The synthetic polynucleotides were obtained from Miles Chemical Co., and the TMV-RNA was a gift of Dr. George Cochran, Department of Botany, Utah State University. The solutions of 629 in Vitro cold amino acids used in the incubations contained 1 @tmole/mlof each of the following framino acids : alanine, arginine, asparagine, aspartic acid, cysteine, cystine, glutamic acid, glutamine, gly cine, histidine, isoleucine, leucine, lysine, methio nine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. One or more TABLE LIVER AND ASCITES 1 TUMOR AMINO ACm INCORPORATING SYSTEM proteinComplete Tumor system@qsmoIes valine-C'4/mg . ribosomal system5 —S100, pH 5 3@ 0.6 26 4 —ATP,GTP —AlT, GTP, PEP, PEP kinase —Aminoacids 1@I@Cl2 +Ribonuclease 1.5 (10 pg.) +Liver S100, pH 5 for Tumor S100, pH5 @40t 21Liver at 105,000X g for 2 hours. The deoxycholate treatment was re peated, and the final ribosomal pellets were resus pended in a minimal amount of the standard buffer. Ribosomal ‘preparations from liver ‘ tissue were prepared by the same method but were treated only once with deoxycholate. The 5100 fraction was brought to pH 5.0 with 1 M acetic acid, allowed to stand for 15 minutes in the cold, and the flocculent precipitate was collected by centrifugation. This precipitate, redissolved in standard buffer to give a protein concentration of approximately 10—iS mg/ml, constituted the amino acid-activating system and was designated 5100, @ .05 Acid Incorporation system . 8@ 0.6 Complete system:5 @-‘--s$100,' p'H@5 —ATP, GTP —AlP, —MgCl2 1 1 1.4 PEP, PEP kinase 1 +Ribonuclease +Tumor S100, pH 5 for Liver S100, 16 pHS9 S The incubation medium consisted of the following: Tumor System S100, pH 5 = 0.3 mg. protein; ribosomes, —1.0 mg. protein; U-C'4-L-valine, 4.4 X 10@ pmole; 20 L-amino acids minus valine, .09-5 smole each; AlP, 0.25 zmole; GTP, 7.5 X 10—i .amole; phosphoenolpyruvate (Na), 2.5 @imole; phosphoenolpyruvate kinase, 5.0 pg., MgC12,S smole. Buffer was added to a total volume of 0.5 ml., and the reaction mix tures were incubated at 37°C. for 30 minutes. Samples of 0.05 ml. were removed, precipitated with cold TCA, extracted with hot TCA, and counted. Liver system was identical with the above incubation medium, S100, pH 5, and ribosomes substituting the same amounts were prepared from liver. of t Average value, obtained from fifteen separate ribosomal preparations (range, 27-60). Average value, obtainedjrom preparations fifteen separate ribosomal (range, 0.6—5). § Average value, obtained from four separate ribosomal preparations â€C̃'4-labeled (range, 4—19). amino acids were added to each incu bation tube, and the corresponding unlabeled amino acids were deleted from the mixture. The complete reaction mixtures and the incuba tion procedure are described in the legend to Table 1. After incubation the mixtures were chilled, and 0.05-ml. samples of each were pipetted in dupli cate onto 2.3-cm. paper discs, cut from Whatman #3 MM filter paper. The discs were immediately immersed in a large volume of cold 5 per cent TCA (at least 15 ml/disc) and swirled for 10 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1963 American Association for Cancer Research. @ @ @ T@1 630 . Cancer minutes. The discs were washed once more with cold TCA, then extracted with hot 5 per cent TCA at 90@C. for 7 minutes, followed by one cold TCA wash and two ethanol washes to remove the TCA. An additional ether wash did not affect the final count, and this step was eliminated from the routine procedure. The discs were dried with heat, and the radioactivity was determined in a Packard Tricarb liquid scintillation counter (Model EX). The paper-disc method outlined is a modification of the method described by Mans and Novelli (13). The counting efficiency of the system varied from 50 to 55 per cent. The ribonucleic acid of Novikoff ascites and liver cell fractions was prepared by phenol extrac tion from the ribosomes and Si®, or from nuclei isolated from the 15,000X g pellet. An equal vol TABLE 2 COMPARISON OF AMINO ACID INcoRPoRATION AUU@O ACID/MG [email protected] corporated approximately 40 s@moles of U-Ct4-r.@valine/mg of ribosomal protein. Omission of the 5100, pH 5, fraction from the incubation medium reduced the valine incorporation to 2—3 @smoles, indicating that the ribosomal preparations were essentially free of the amino acid-@activating corn ponents. The over-all amino acid activation, as de termined from the radioactivity of the cold TCA precipitable material, was in the range of 75—100 ,@moles/mg ribosomal protein for valine-C'4. Omission of the energy or energy-regenerating components greatly reduced the amino acid in corporation (Table 1). Ribonuclease also de stroyed the capacity of the system to incorporate valine-C'4. Deletion of the cold amino acids from the incubation medium caused a 40 per cent de crease in incorporation similar to that reported by Matthaei and Nirenberg for their E. coli system (15). Overnight.dialysis of the ribosomal and S100, pH 5, fractions did not reduce this value further, BY TUMORAND Lxvr@R in Vifro SYSTEMS psorzixtTumorLiver+U-C'4.L-valine 0 %O1. 2@, iviay 1963 Research RIDOSOMAL iNCUBATION SYSTEMIi,LM0LE$ +U-C―.L-phenylalanine 30 6 +U-C1'-L-lysine +Above three amino acids43 70 10 1467 26 S Incubation medium and proceduresas described in “Meth. ods―and in footnotes to Table 1. In above studies mixture of cold amino acids, exclusive of labeled amino acid(s) was add ed to system. t Average of three or more comparable assays. nine of redistilled phenol (80 per cent) was added to the tissue fraction suspended in dilute phos phate buffer, pH 7.0, and the mixture was stirred at 5°C. for 15 minutes. After centrifugation, the aqueous layer was washed twice with 80 per cent phenol. The final aqueous solution was made to 1 per cent with respect to sodium acetate, and the RNA was precipitated by the addition of 2 vol umes of chilled ethanol. The precipitated RNA was redissolved in standard buffer and extracted 5 times with at least 2 volumes of ether each time. The ether was removed by nitrogen, the RNA con centration was determined by absorption at 260 mis, and the solutions were frozen and stored at —15°C. RESULTS The characteristics of the Novikoff ascites tu mor and rat liver amino acid-incorporating sys tems are shown in Table 1. With optimal ratios of s100, pH 5, to ribosomes, the tumor system in and, owing to a slight loss of amino acid-incorpo rating activity, dialysis was generally omitted. Both the tumor and liver incorporating systems were dependent upon Mg+ + for activity. The liver system, utilizing valine-C'4, incorpo rated an average of 9.8 @pmoles/mg ribosomal pro tein. Omission of S100, pH 5, ATP-GTP, or the energy-regenerating system reduced this incorpo ration to low levels. Addition of ribonuclease to the incubation medium also inhibited incorporation (Table 1). Rate studies showed that, for both the tumor and liver systems, the over-all reaction proceeded in a linear fashion for 10—iSminutes and reached equilibrium at 30-40 minutes. Centrifugation of these 30-minute incubation mixtures at 105,000X 9 demonstrated that 90 per cent of the radioactiv ity remains associated • with the sedimentable, ribosomal fraction, This evidence suggests that, whatever the mechanism of polypeptide. release from the ribosomes may be, it is either not present or inactive in this system, and that the equilibrium which is@reached after 40 minutes may be due to this fact. Lamborg (10) has presented some new findings recently regarding the release of protein from ribosoines. â€ẫ€¢ The S100, pH 5, and ribosomal fractions of tu mor and liver are interchangeable to some extent (Table 1), although further studies will be re quired to establish whether the transfer factors of liver will operate with highly purified tumor ribo somes. The abilities of li@rer and tumor systems to incorporate specific amino acids are compared in Table 2. Tumor ribosomes incorporated 4—Stimes more valine and phenylalar,ine than did liver Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1963 American Association for Cancer Research. O'Na&L ribosomes, and an even AND GRIFFIN—Am@nO Acid Incorporathn higher proportion of lysine. The effects of exogenous natural RNA and synthetic polynucleotides on the amino acid incorporating systems are shown in Table 3. Ad dition to the tumor incubation systems of RNA preparations which may contain template infor mation has resulted in consistent increases in amino acid incorporation, ranging from @0to 100 per cent. Nuclear and ribosomal RNA prepared from ascites tumor cells Stimulated the incorpora 631 in Vitro The addition of polyuridylic acid to the tumor system markedly stimulated the incorporation of phenylalanine-C'4, and as little as 50 @ig.of the synthetic polynucleotide resulted in a seven-fold average increase in the radioactivity (Table 3). In some instances, the incorporation was raised from 27 to approximately 350 M@moles phenylalanine C'4/mg ribosomal protein. The addition of poly uridylic acid to the chilled systems after incuba tion was without effect. The specificity of this synthetic messenger as a template for the forma TABLE S EFFECT OP ADDITION 0@ RIB0NucLEIc ACID PREPARATIONS AND POLYNUCLEOTIDES TO TUMOR AND lAYER in Vitro AMINO ACID-INCORPORATING SYSTEMS PROWSC'4-i-ValineC'4-a-Phenylal&nineC'4-LysineC'4-L-Valine AMINOACIDmCOIPO*AflONVALUES,pi@iMOL5$/MV @iPOW@AL SYSTEM5Avxn.&ox C'4-Phenylal. C'4-n-LysineComplete +Aseites +Ascites @ tumor: nuclear RNA rib. RNA 47 (+85%) 48 (+23% +Liver nuclear RNA 22 (—86@' +Liver nuclear RNAt +Liver RNA +TMV.RNA (50 pg.) ±Polyuridylic acid 46 21 42 37 (100 pg.) acid (1004) (+30 (—38 (+90% (+ 6% 81 (+ 15%) +Polycytidylie 26 (— 4%) +Polyinosinic acid (100 @Lg.) 22 (— 18%) Completeliver: +Ascites 6 .5 7 (+ 8%) 7 (+ 8%)27 nuclear RNA +TMV-RNA +Polyuridylic acid 91 (+45%)129 174 (+34%) 190 (+710%) 6 26 (+430%)63 (100 pg.)85 S Incubation medium and procedure are described in “Methods― and in footnote to Table 1. In above studies a mixture of cold amino acids, exclusive of C'4.labeled amino acid(s), was added. Ap. proximately 600 pg. of RNA preparations were added to incubation mixtures unless otherwise noted. t Livernuclear RNwas added 15-20minutes afterthestartofincubation. @ tion ofeach of the three labeled amino acids, and collectively the effect was shown to be additive. It is possible, however, that some of the radio activity attributed to incorporation of lysine-C'4 may be caused by other combinations of this amino acid through its epsilon amino group (26). RNA prepared from total liver inhibited amino acid incorporation but stimulated amino acid activation. The proportion of messenger RNA in such a preparation is undoubtedly low, and con tamination by proteolytic enzymes or ribonuclease is a distinct possibility. To a variable extent RNA added to an incubation in progress caused a greater amino acid incorporation than was obtained when the RNA was added at the start of incubation. tion of a polypeptide composed solely of phenyl alanine was evidenced by its incapacity to stimu late the incorporation of either valine or lysine. Polycytidylic acid and polyinosinic acid were with out effect on the incorporation of phenylalanine. The response of the tumor-phenylalanine system to polyuridylic acid was dependent upon ATP and GTP, the energy regeneration system, Mg+ +, and was inactivated by ribonuclease. The liver system responded only slightly to the addition of either TMV-RNA or ascites nuclear RNA. Polyuridylic acid, however, did stimulate the incorporation of phenylalanine-C'4 to some extent. Further work will be necessary to assess the' effect of exogenous RNA on liver ribosomes. Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1963 American Association for Cancer Research. 632 Cancer Research The tumor nents resemble DISCUSSION amino acid-incorporating in many respects Studies on the nature of RNA attachment compo comparable sys tems obtained from E. coli (15, 17), although it has been impossible thus far to remove the inherent messenger RNA of the tumor ribosomes. In pre liminary studies, preincubation of tumor ribo somes reduced their amino acid-incorporating abil ity by 30 per cent, a reduction which might mdi cate the removal of ribosomal messenger RNA. These preincubated ribosomes would not respond to exogenous RNA, although the addition of polyuridylic acid and phenylalanine-C14 in a seven-fold tion (5). This did result increase in amino acid incorpora level of incorporation did not ap proach that obtained when polyuridylic acid and labeled phenylalanine were added to nonpreincu bated ribosomes. With this tumor cell system it has been possible to produce a small but consistent increase in amino acid incorporation by the addition of most natural RNA preparations. With an incorporation of 30—60 @molesof valine, phenylalanine, or ly sine per mg. of ribosomal protein, one could pre dict an over-all incorporation, based on twenty amino acids, of the order of 800 @mo1es.The natural RNA's which have been shown to increase the incorporation of individual amino acids by 16—45per cent would raise the theoretical total incorporation to as much as 1150 j@moles. The addition of polyuridylic acid produces a seven-fold increase in phenylalanine incorporation. However, if we assume that this polynucleotide synthesis of a protein composed directs entirely the of phenylalanine, a direct comparison with the natural RNA's must be made on the basis of the theoretical total incorporation. Calculated in this manner, the polyuridylic acid stimulates amino acid incorporation by 35 per cent, a figure which compares favorably with those for the natural RNA's. It appears, then, that under the conditions defined here all the exogenous messenger polyribo nucleotides stimulated amino acid incorporation to roughly the same extent. Arnstein et a]. (3) have observed that RNA isolated from rabbit reticu locyte ribosomes stimulated amino acid incorpora tion by 30—SOper cent, which is also in agreement with our findings. Maxwell (16) has recently re ported that the addition of liver microsomal RNA to a liver cell-free system stimulated the incorpo rationof all amino acids tested. The increased in corporation indicated for phenylalanine ranged from 30 to 90 per cent. Further extensive investi gation is thus required with mammalian systems to produce a messenger RNA stimulation of the magnitude reported for microbial Vol. 23, May ribosomes. ribosomes, amino acid polymerization, 1963 to the protein re lease mechanisms, or concentration of the 100 S ribosomes (19) from tumor cells may result in more active incorporating systems. It is apparent from the polyuridylic acid phenylalanine experiments that this mammalian system behaves like E. coli with respect to the cod ing unit (UT,IU) reported for this amino acid (18). Recently, we have noted that polycytidylic acid will stimulate proline incorporation by the tumor system. At least one of the coding units for proline in the tumor system is (CCC), and this same code is inherent in some microbial systems (4). There is further indication from Maxwell's investigations that the RNA coding units are the same for six amino acids in liver and E. coli systems (16). To what further extent tumor and other mammalian ribosomes will correspond to the codes and the degree of degeneracy in the codes still remains to be established. Although we will continue our efforts to produce tumor ribosomes with a lowered inherent mes senger RNA, we believe that the present system may be useful in the study of many factors that influence protein biosynthesis in mammalian sys tems. From the observations made thus far it ap pears that protein synthesis in the Novikoff ascites tumor cells closely resembles that seen in other mammalian and microbial systems. Further comparative studies with regard to chemical and physical characteristics of specific-transfer ribo nucleic acids, specificity of transfer factor(s), and mechanisms of release should reveal whether of peptides from ribosomes basic differences do exist between normal forming systems. malignant and tissue protein ACKNOWLEDGMENTS The authors wish to acknowledge the contribution of Mrs. Virginia Ward to this study. The research was supported in part by grants from the American Cancer Society and the Robert A. Welch Foundation. REFERENCES 1. ALLFREY, V. G., and MInsKY, A. E. In: R. J. C. HARRiS (ed.), Protein Biosynthesis, pp. 49-81. New York: Aca demic Press, 1961. 2. ARNSTEIN, H. R. V.; Cox, R. A.; and Hu@v, J. A. Function of Polyuridylic Acid and Ribonucleic Acid in Protein Bio synthesis by Ribosomes from Mammalian Reticulocytes. Nature, 194: 1042—44,1962. 3. . 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