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[CANCER RESEARCH 37, 1876-1882, June 1977] Effects of 6-Thioguanine on RNA Biosynthesis in Regenerating Rat Liver1 Christine K. Carrlco2 and Alan C. Sartorelli Departmentof Pharmacologyand Sectionof DevelopmentalTherapeutics,ComprehensiveCancerCenter,YaleUniversitySchoolof Medicine,NewHaven, Connecticut06510 SUMMARY 6-Thioguanine, at a dose of 40 mg/kg body weight, was administered to rats at 12 hr after partial hepatectomy; 6 hr later, liver polysomes and cell sap were isolated and utilized to measure the effects of this antimetabolite on protein synthesis in vitro. When radioactive leucine was used to label peptides synthesized in vitro, no difference was ob served between polyacrylamide gradient gel scans of sys tems derived from control regenerating liver and those from 6-thioguanine-treated regenerating liver. However, when radioactive tyrosine was used as the tracer to monitor syn thesized peptides, a depression in the 30,000-molecular weight region of scans of products synthesized in systems derived from 6-thioguanine-treated regenerating liver was observed. Recombination experiments showed this effect to be due to the pobysome component of the system. When equal amounts of polyadenybic acid-containing RNA from 6thioguanine-treated or control regenerating liver were added to a wheat germ in vitro protein-synthesizing system, pobyacrybamidegel scans of the products synthesized in the presence of radioactive tynosine showed that more peptides were synthesized from polyadenylic acid-containing RNA from 6-thioguanine-treated rats than from control polyade nylic acid-containing RNA. That this phenomenon might be the result of incorporation of the analog into RNA was shown by the finding that all types of RNA contained 6thioguanine, with the greatest concentration occurring in polyadenylic acid-containing RNA. the synthesis of other kinds of RNA was apparently unaf fected by the punine antimetabobite. These effects were initiated in the G1 phase of the cell cycle and appeared to constitute a new metabolic lesion(s) created by 6-TG; thus, it was of importance to attempt to localize more precisely the site of action of the pumine antimetabolite on these metabolic events. Earlier work by Wheeler et a!. (20) has shown that 6-TG acts in the G, phase of the cell cycle of H.Ep.2 cells to delay the G1- to S-phase transition; the biochemical alteration responsible for this action is un known but would appear to be unrelated to incorporation of the analog into DNA. GTP is required at several stages of the translational process; therefore, protein synthesis would appear to be a particularly vulnerable site for the action of a guanine ana bog. Roy et a!. (14), however, demonstrated that when 6-TG tniphosphate was substituted for GTP in an in vitro amino acid-incorporating system, not only did the thiopurine nu cleotide not inhibit the reaction, but in contrast, it was found to partially substitute for the natural substrate. Gray and Rachmeler (6) reported that 6-TG was incorporated into Escherichia coli tRNA and that this incorporation appar ently affected the amino acid acceptor activities of some of the tRNA's. 6-TG could also be envisioned to interfere with the syn thesis of certain proteins either by being incorporated into the specific mRNA molecules for these proteins or by affect ing the synthesis of the specific mRNA's themselves. The punine antimetabolite has been shown to be incorporated into both RNA and DNA (8, 9, 15, 17), but it is the incorpora tion into DNA to which cytotoxicity has been attributed (see, INTRODUCTION e.g., Ref. 10). The experiments described in this paper show that in vitro The previous paper of this series (4) has showrtthat 6-TG3 translation of certain groups of peptides is altered in cell has a major inhibitory effect on the partial hepatectomy free systems derived from 6-TG-treated regenerating liver induced synthesis of enzymes required to support DNA and that the basis of this effect resides in the polysomes. replication in regenerating rat liver without altering the mate Furthermore, 6-TG is preferentially incorporated into of total protein synthesis in vivo. These findings were ac poly(A)@ RNA, and this 6-TG-containing RNA is more effi companied by a depression of the synthesis of poly(A) cient in directing in vitro translation than is poIy(A)@RNA containing RNA in 6-TG-tneated regenerating liver, whereas without 6-TG. These effects are observed at a time when DNA synthesis I Support was provided by Grants CA-02817 and CA-16354 from the Na and 6-TG incorporation into these macro molecules in regenerating liver are minimal. tional Cancer Institute, USPHS. 2 Recipient of Training Grant GM-0059 from the General Medical Sciences, USPHS. The work described in this report is a portion of the work presented in partial fulfillment of the requirements for the Ph.D. degree. 3 The abbreviations used are: 6-TG, 6-thioguanine; poly(A), polyadenylic acid; poIy(A)@RNA, polyadenylic acid-containing RNA; SDS, sodium dodecyl sulfate; poly(A) RNA, polyadenylic acid-lacking RNA; PCA, perchloric acid. Received January 14, 1977; accepted March 21, 1977. 1876 MATERIALS AND METHODS Wheat germ, pynuvate kinase, phosphoenolpyruvate, RNase A, dithiothreitob, creatine phosphate, creatine phos phokinase, N-2-hydnoxyethybpipenazine-N'-2-ethanesulfonic CANCER RESEARCH VOL. 37 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1977 American Association for Cancer Research. 6-TG Action on RNA Biosynthesis acid, and carbonic anhydrase were obtained from Sigma of Roberts and Paterson (13). The peak turbid fractions Chemical Co., St. Louis, Mo.; acrylamide and N,N'-methyl were pooled, rapidly frozen in 0.5-mb batches, and stored at enebisacrybamide were purchased from Eastman Kodak —70°. Wheat germ in vitro protein synthesis assays were Co., Rochester, N. Y. CelIex-D anion-exchange resin was performed in a total volume of 0.5 ml, containing 0.2 ml obtained from Bio-Rad Laboratories, Richmond, Calif.; wheat germ S-30 fraction, 20 mM N-2-hydroxyethylpipera [4,5-3H (N)]Ieucine and elemental 355 were obtained from zine-N'-2-ethanesulfonic acid, pH 7.6 (adjusted with KOH), New England Nuclear, Boston, Mass.; [2,6-3H (N)Jtyrosmne 2 mi@idithiothreitob, 1 mM ATP, 20 @M GTP, 8 mM creatine was purchased from ICN Chemical Radioisotope Division, phosphate, 3 mM magnesium acetate, 100 mM KCI, 30 @M Irvine, Calif. fledisobv HP was obtained from Beckman In cold amino acid mix without tynosine, 50 @Ci[3,5-3H (N)]tyrosine, 20 /hg creatine phosphokinase, and 50 @g struments, Inc., Fullerton, Calif. 6-TG was the generous gift of Dr. George H. Hitchings of Bummoughs-Wellcome Re poIy(A)@RNA isolated as described previously (4). The meac search Laboratories, Research Triangle Park, N. C. tion was started by the addition of the 5-30 fraction and was Male Sprague-Dawley rats (Charles Riven Breeding Labo allowed to proceed for 30 mm at 25°,after which time 10 j.@g ratonies, Wilmington, Mass.), weighing between 170 and 200 RNase and EDTA to a final concentration of 10 mM were g, were housed routinely over corn cob bedding, and alter added. After incubation for 15 mm at 37°,the peptides were nating periods of 12 hr dark and 12 hr light were main precipitated by the addition of 2 volumes of 80% acetone at tamed. Before surgery, Purina rat chow and water were 00. The precipitate was collected by centnifugation and washed 3 times with 80% acetone, after which it was sus available ad libitum. Partial hepatectomies were performed pended in 0.0625 M Tmis-HCI,pH 6.8-2% SDS-10% glycerol under light ether anesthesia between 8:30 and 10:30 a.m. according to the method of Higgins and Anderson (7). 6-TG 0.1 M dithiothneitol-0.01% bromphenol blue for poly was dissolved in 0.9% NaCI solution with the aid of dilute acrylamide gel electrophonesis on gradient pobyacrylamide NaOH as described previously (4) and was injected i.p. at 12 gels. Linear (4 to 30%) gradient acrybamide gels were prepared hr after partial hepatectomy, at a dose of 40 mg/kg body weight; control animals received an equal volume of 0.9% by using an apparatus described by Caton and Goldstein (5). Chamber 1 contained 51.45 g acrylamide and 1.05 g N,N' NaCI solution. methylenebisacrybamide in 150 ml of 0.375 M Tnis-HCI, pH The rat liver in vitro protein-synthesizing system utilized was a composite of those described by Ragnotti et a!. (12) 8.8-0.1% (w/w) SDS, and Chambem2 contained 5.72 g acryl amide and 0.28 g N,N'-methylenebisacrybamide in 150 ml of and Atkins et a!. (2). Partially hepatectomized and normal animals were sacrificed by decapitation, and their livers the above Tnis buffer. Immediately before starting the gra were excised, minced, and homogenized in 2 volumes of dient former, 0.15 ml N ,N ,N' ,N―-tetnamethylethylenedia mine and 0.075 g of ammonium pemsulfate were added to 0.25 M sucrose-50 mM Tnis-HCI, pH 7.8-25 mM KCI-6 mM MgSO4. This homogenate was centrifuged at 12,000 x g ton each chamber and mixed well. The gels were allowed to 10 mm, and the supemnatant solution was mecentnifuged at polymerize overnight at room temperature, after which time 145,000 x g for 2 hr. The resulting pellet, resuspended in they were cut free from the excess gel encasing the glass incubation buffer (0.15 M sucnose-35 mM Tnis-HCI, pH 7.8-25 tubes. Gels prepared in this fashion could be stored under buffer in the cold for several weeks. mM KCI-10 mM MgSO4), was used as the source of poly somes in the cell-free system. The 145,000 x g supernatant Before electrophoresis of peptides, gels were subjected solution was passed through a Sephadex G-25 coarse cob to preelectrophoresis for 45 mm at 2 ma/gel. Samples were umn equilibrated with incubation buffer. The initial frac applied in a volume of 50 @b and subjected to ebectrophore tions of this column were used as the source of amino acid sis in a Buchlen Polyanalyst water-jacketed electrophomesis free cell sap for the cell-free incubations. cell at 2 ma/gel, until the bromphenol blue marker reached The cell-free incubation system consisted of 2 to 4 ml cell the bottom of the tube (approximately 4.5 to 5 hr). Electro sap; 0.5 to 2.0 ml polysomes (containing 10 to 20 mg RNA) phonesis buffer was 0.025 M Tnis-0.193 M glycmne, pH 8.3, in incubation buffer; 175 @g pyruvate kinase pen ml, 4 ml of containing 0.1% SDS. Gels were chopped into 2-mm seg a stock solution consisting of 2 mM ATP, 0.25 mM GTP, and ments directly from the electrophoresis tubes with a Gibson Aliquogel fractionator, and radioactivity in the gel sections 10 mM phosphoenolpyruvate, and 35 @Ci [3,5-3H (N)]tyrosine was determined in Beckman Redisolv HP using a Packard (24.6 Ci/mmole) or 100 @Ci[3,5-3H (N)Jleucmne (40 Ci! mmole) in a total volume of 10 ml. One ml of a 300 mM cold Tni-Cambliquid scintillation spectrometer. amino acid mix lacking only the radioactive amino acid Protein markers were run each time a series of gels was utilized was also added to the incubation flask. The reaction subjected to electrophoresis. The marker proteins used was started by the addition of polysomes and allowed to were cytochnome c, RNase A, and carbonic anhydmase, proceed at 37°for 1 hm,after which time 200 @g RNase A in possessing molecularweightsofl.17 x 10@,1.37 x 10@,and 10 mM EDTA were added. After 30 mm at 37°,the proteins 3.40 x 10@,respectively. Marker gels were stained with 0.1% Coomassie blue in 7.5% acetic acid-50% methanol and de were precipitated at 0°with 80% acetone. The precipitate was dissolved in sample buffer for linear (4 to 30%) gradient stained in 7.5% acetic acid-50% methanol. Following de polyacrylamide gel ebectrophonesis (0.0625 M Tmis-HCI, pH staining, the marker gels were scanned at 600 nm in a Beckman DU spectrophotometer equipped with a Gilford 6.8-2% SDS-10% glycerol-0.1 M dithiothreitol-0.01% bromo Model 2140 linear transport accessory utilizing a scanning phenol blue). aperture of 2.4 x 0.05 mm and a scanning mateof 1 cm/mm. The 5-30 fraction for the wheat germ in vitro protein 6-[35S]TG was synthesized according to the method of synthesizing system was prepared according to the method JUNE 1977 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1977 American Association for Cancer Research. 1877 C. K. Carrico and A. C. Sartorelli Morávek and Nejedb@(11). 6-TG was dried at 110°in a vacuum overP205forseveralhr.To a suspensionof500 mg (2.99 moles) of 6-TG in 200 ml of dry pymidine, which had been refluxing for3 hn, were added 10 mCi (1 to 20 mCi/mg) of elemental 355in benzene solution. The vial containing the @ RESULTS Typical gradient polyacrylamide gel profiles of peptides synthesized in the rat liver in vitro protein-synthesizing sys tem as measured by the incorporation of [3H]leucine are shown in Chart 1. No difference was detected in the pep 355 was rinsed 5 to 7 times with pynidine, and these tides synthesized in systems derived from either 18-hr par washes were added to the refluxing solution. The suspen sion was refluxed (protected by a calcium chloride tube) for tially hepatectomized control livers or from partially hepa 3.5 hr. The pynidine was removed in a vacuum. The residue tectomized , 6-TG-treated livers. However, when tyrosine was substituted for leucine as the was resuspended in tobuene, and this was removed in a vacuum to eliminate all pynidine from the product. The dry radioactive amino acid used in the rat liver in vitro protein synthesizing system, the peptide profiles on polyacrylamide residue was recrystallized from 480 ml of water after deco Ionization with charcoal and concentration of the filtrate to gels shown in Chart 2 were obtained; in these studies as 150 ml. The crystals were collected after 48 hr of mefrigera well as for those described in Chart 1 using leucine as the tion and dried with air and acetone. The yield was 115 mg measure of proteins synthesized, both the polysomes and the cell sap were derived from the same liver. The data show (23%) of 6-[35S]TG (3 mCi/mmole). For measurement of the incorporation of 6-TG into RNA, that in one particular region (3 to 5 cm), corresponding to matswere given 6-[35S]TG at 40 mg/kg body weight 12 hr approximately M.W. 30,000, the synthesis of peptides was significantly depressed in the in vitro protein-synthesizing after partial hepatectomy. Six hr later the matswere sacni ficed by decapitation, and their livers were excised and system derived from 6-TG-tmeated regenerating liver, rela homogenized in 3 volumes of 0.32 M sucmose-3mM MgCI2. tive to that from control regenerating liver. The average area Nuclear poly(A)@ RNA and poby(A) RNA were isolated as under the affected peaks from nine 6-TG-denived systems described previously (4). Cytoplasmic RNA was isolated was 52% of the average control area from 8 scans (p < 0.005). The reason for the amino acid specificity toward from the 700 x g supernatant solution after centnifugation to remove the mitochondnia and precipitation with 95% leucine and tyrosine in the action of 6-TG is unknown; presumably, it reflects the amino acid composition of the ethanob-2% potassium acetate at —20° overnight. The pre cipitate was dissolved in10 mM sodium acetate, pH 5.1-0.14 newly synthesized peptides, since the profiles of peptides synthesized in the presence of tyrosine and leucine differ M NaCI-0.01% sodium dextran sulfate-0.3% SDS, and the RNA was extracted and separated into its poly(A)@ and markedly with respect to peak position and height. poly(A) components as described (4). The absorbance at II' 260 and 280 nm was determined for each fraction. RNA CONTROL fractions were then hydrolyzed at 37°for 1 hr in 0.3 N KOH. 300 After neutralization with PCA, the hydrolysates were applied to Celbex-D anion-exchange columns (30 x 5 mm), equili E 200 brated with 0.02 M Tnis-HCI, pH 8.0. Following elution with z 11 ml of 0.02 M Tmis-HCI,pH 8.0, to remove 6-TG present as 0 the free base, nucleotides were eluted with 2 ml of 1 N HCI, i: 100 ‘4 and radioactivity therein was determined using Beckman 0 Redisolv HP in a Packard Tni-Carb liquid scintillation spec trometer. 0 C., 6-TG z The presence of 6-TG nucleotide in DNA was determined after an i.p. injection of 6-[35S]TGat 40 mg/kg body weight. w z Purified nuclei were isolated from 18- and 24-hr regenerat 200 ing liver as described previously (4). The 50,000 x g pellet w was resuspended in 1 ml of 0.32 M sucmose-3mM MgCI2 and 100 precipitated with 4.5 volumes of 0.5 N PCA. Following cen tnifugation at 270 x g for 10 mm, the pellet was dissolved in 0 4 ml of 0.3 N KOH and heated at 37°for 1 hr to hydrolyze the 0 1 2 3 4 5 6 7 8 RNA. Following the addition of 2.5 ml of 1.2 N PCA and DISTANCE MIGRATED (Cm) centnifugation at 270 x g for 10 mm, the pellet, which Chart 1. Gradient polyacrylamide gel electrophoretic scans of products consisted of DNA and protein, was dispersed in 4 ml of 0.5 N derived from the nat liven in vitro protein-synthesizing system using (‘I Q. — @ 300 -J @ I PCA and heated to 70°for 20 mm. The DNA hydrolysate was chilled and centrifuged at 270 x g for 10 mm. Abiquots of the supemnatant solution were taken for measurement of DNA concentration by the method of Burton (3) using calf thy mus DNA as a standard. The remainder of the supernatant solutions were neutralized with KOH and chromatographed on Ceblex-D anion-exchange resin; then, deoxynucleotide fractions collected, and radioactivity therein was deter mined as described above for RNA samples. 1878 I I I I I rH]Ieucine as the tracer. 6-TG ( 0) was injected i.p. into rats 12 hr after partial hepatectomy at a dose of 40 mg/kg body weight. The control group (•) received an equivalent volume of 0.9% NaCI solution. At 18 hr after partial hepatectomy the rats were sacrificed, and the liver polysomes and cell sap were isolated. The polysomes and cell sap were added to an in vitro system containing pyruvate kinase, [3H]leucine, ATP, GTP, phosphoenolpyruvate, and an amino acid mixture lacking leucine as described under “Materials and Methods.―Following incubation, peptides were subjected to electrophoresis on 4 to 30% linear gradient polyacrylamide gels. The gels were divided into 2mm sections, and the radioactivity in each segment was determined. Car bonic anhydrase (molecular weight of 34,000) used as a standard migrated 4.1 cm. CANCER RESEARCH VOL. 37 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1977 American Association for Cancer Research. 6-TG Action on RNA Biosynthesis 300 @ CONTROL 800 200 z 600 0 i: 100 E ‘4 400 U 0 Q. z 8300 z I‘4 0 6- TG “a @ 0 5. 200 U) 200 0 0 C., z @- ‘ii z 100 800 U, 0 0 0 1 2 3 4 5 6 DISTANCE MIGRATED (Cm) 7 8 Chant 2. Gradient polyacrylamide gel electrophoretic scans of products derived from the rat liver in vitro protein-synthesizing system using @H]tyrosineas the tracer. The data were obtained by procedures identical to those described in Chart 1 except that (3Hjtyrosine was used instead of leucine as the radioactive amino acid, and the cold amino acid mix lacked tyrosine instead of leucine. In an effort to determine whether the polysome compo nent or the cell sap component was responsible for the observed decrease by 6-TG in the synthesis of a peptide or peptides containing tyrosine, an experiment was performed in which polysomes from 18-hr control regenerating liver were added to an in vitro protein-synthesizing system con taming cell sap from 18-hr 6-TG-treated regenerating liver. A polyacrylamide gel profile from this experiment is shown in Chart 3 (bottom). Profiles of the product(s) synthesized in the presence of polysomes from control regenerating liver and cell sap from drug-treated regenerating liver indicate that the 30,000-M.W. region was present apparently in unab tered form (compare to control profile; Chart 3, top). The converse experiment, that is, the use of polysomes from 18-hr 6-TG-treated regenerating liver with cell sap from 18-hr control regenerating liven, was performed (Chart 4, top); the 6-TG gel profiles clearly demonstrated the induced inhibitory effect (Chart 4). From these experiments, it was concluded that the decrease in tyrosine-containing pep tides synthesized in the in vitro system derived from 6-TG treated regenerating liver was due to the polysome compo nent rather than to the cell sap component. To determine whether the observed effect might be due to a decrease in the quantity of specific mRNA's in polysomes from the livers of 6-TG-treated animals, a wheat germ in vitro protein-synthesizing system was used which allowed the addition of an equal quantity of poIy(A)@RNA from the livers of 6-TG-treated or control rats to the incubation mix. The peptides synthesized in vitro were separated on gra dient polyacrybamide gels, and representative duplicate ra dioactivity profiles of gels from preparations nun with con trol and 6-TG poly(A)@RNA are shown in Chart 5. The areas under the affected peaks from 6-TG-denived systems were 30% greater than the areas under the peaks from control systems (p < 0.005), indicating that more total peptides were being synthesized from poIy(A)@RNA from 6-TG I- 600 400 200 2 3 DISTANCE 4 5 MIGRATED 6 7 8 (cm) Chart 3. Gradient polyacrylamide gel electrophoretic scans of products from a recombined rat liver in vitro protein-synthesizing system in which polysomes from untreated partially hepatectomized animals and cell sap from 6-TG-treated rats were used. The data were obtained by procedures identical to those described in Chart 2, except that in both cases polysomes were obtained from control partially hepatectomized rat liver and cell sap from either 6-TG-treated or untreated regenerating liven. treated rats than from control poby(A)@RNA. That this effect might be due to the presence of 6-TG in mRNA was shown by the finding that 6-TG was incorporated into all species of RNA as a nucleotide (Table 1). Much more 6-TG was incorporated into poIy(A)@ RNA than was incorpo rated into poly(A) RNA under these conditions. This effect was particularly striking in the nucleus where poby(A)@ RNA incorporated 50 times more 6-TG than did poly(A) RNA. 6TG was also incorporated into the DNA of regenerating rat liver. As expected, at 18 hr after partial hepatectomy, the liver DNA contained only 31 ±8 pmoles of 6-TG in nucleo tide form per mg of DNA. At 24 hr after partial hepatectomy, however, during peak synthesis of DNA in regenerating liver, DNA contained 494 ±55 pmoles of 6-TG nucleotide per mg of DNA. Quantities of 6-TG equal to or greater than those incorporated into the nucleic acids were associated with these macromolecules, particularly poby(A)@RNA, demonstrating the need for isolation of 6-TG nucleotide from both DNA and RNA to insure that the antimetabolite was incorporated as part of the structure of these mole cubes. The dose of 6-TG used in these studies would appear to be reasonable, since the degree of incorporation into RNA correlates well with the cytotoxic level reported by Tidd and Paterson (18). DISCUSSION The previous report (4) demonstrated that 6-TG had major effects on the 1st wave of partial hepatectomy-induced en JUNE 1977 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1977 American Association for Cancer Research. 1879 C. K. Carrico and A. C. Sartorelli J 800 • I I I CONTROL CELL SAP 6-TG POLYSOMES 6OO@ I - U - z 300 E 0 I- ‘4 0. U 0 a. z 0 0 200 z “a z 100 U 0 a. 0 C.) z “a z U, 6-TG CELL SAP 6-TG POLYSOMES 0 800 . - 600 - - 400 - U) >- I- 0 I- 0 a. C.) 200 z 300 0 I-. ‘4 DISTANCE MIGRATED (cm) Chart 4. Gradient polyacrylamide gel electrophoretic scans of products from a recombined rat liver in vitro protein-synthesizing system in which polysomes from 6-TG-treated, partially hepatectomized animals and cell sap from untreated control rats were used. The data were obtained by proce dunes identical to those described in Chart 2, except that in both cases polysomes were obtained from 6-TG-treated, partially hepatectomized rat liver and cell sap from either 6-TG treated- or untreated regenerating liver. 0 a. 200 0 U z z“a100 U) 0 >- zyme synthesis and DNA replication in regenerating matliver and that the biosynthesis of poly(A)@RNA was depressed in 6-TG-treated regenerating liver before the synthesis of DNA. To provide evidence for a connection between these 2 phe nomena, an in vitro protein-synthesizing system was used which allowed a close inspection of the translational proc ess. A rat liver system was chosen for the initial studies because evidence is available which indicates that the initi ation factors are species and, possibly, tissue specific (1, 19). The 2 major separable components of this sytem are the polysomes and the cell sap. The polysome portion consists of nibosomes, mRNA, bound tRNA, and bound factors. The cell sap component contains the majority of the tRNA, the ammnoacyl-tRNAsynthetases, and the free factors (i.e., EF1, EF2, and R). Gray and Rachmelen (6) have reported that the acceptor activity of tymosyb-tRNAin E. co!i is susceptible to the action of 6-TG and have equated this sensitivity with the incorpora tion of the analog into these tRNA molecules. For this reason, radioactive tyrosine was chosen as one of the Ia beled precursors to be used in the in vitro protemn-synthesiz ing system. In addition, since leucine had been used to assess the effects of 6-TG on total protein synthesis in vivo (4), radioactive beucine was also used in the in vitro protein synthesizing system. The profiles of peptides labeled in the presence of radioactive tyrosine and those labeled in the presence of radioactive leucine differed markedly, implying that different peptides (on groups of peptides) are being 1880 l@ 0 2 4 6 DISTANCE 8 2 MIGRATED 4 6 8 (cm) Chant 5. Gradient polyacrylamide gel electrophoretic scans of products derived from the wheat germ in vitro protein-synthesizing system. 6-TG was injected i.p. into rats 12 hr after partial hepatectomy at a dose of 40 mg/kg body weight. The control group received an equivalent volume of 0.9% NaCI solution. At 18 hr after partial hepatectomy, the rats were sacrificed, and the cytoplasmic poly(A)@RNA was isolated from their livers. Equal amounts of poby(A)@RNA from control or 6-TG-treated regenerating livers were added to a wheat germ in vitro protein-synthesizing system containing wheat germ 530 fraction, dithiothreitol, ATP, GTP, creatine phosphate, magnesium ace tate, KCI, creatine phosphokinase, [‘H]tyrosine,and a cold amino acid mix lacking tyrosine as described under “Materials and Methods.―The synthe sized products were processed as described in Chart 1. represented. These peptides appear to contain quite differ ent amounts of these 2 amino acids, and some of the pep tides high in tyrosine content are sensitive to the action of 6TG, whereas those containing more beucine are not. Thus, in profiles of tyrosine-containing peptides synthesized in vitro from 6-TG-denived systems, a decreased tyrosine in corporation occurred into 1 particular region with no appar ent shifting of the position of the peaks in that region. This result bendssupport to the concept, derived from studies in viVo (4), that the protein synthetic machinery per se is not altered by 6-TG, since at beast 1 group of peptides (i.e., those high in leucine content) are apparently synthesized in the same fashion in the in vitro protein-synthesizing sys tems derived from the livers of 6-TG-tneated and control mats. CANCER RESEARCHVOL. 37 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1977 American Association for Cancer Research. 6-TG Action on RNA Biosynthesis 1Incorporation Table tional system allows the addition of equivalent amounts of poly(A)@ RNA from control and 6-TG-treated regenerating (3mCi/mmole) 12 hr after partial hepatectomy,rats were given 6@[US]TG livers, no difference would be expected in the polyacryl ratswere at a dose of 40 mg/kg body weight. Six hr later, amide gel profiles of peptides synthesized in vitro if the sole andhydrolyzed sacrificed, and their liver RNA fractions were isolated in 0.3 N KOH.The neutralized hydrolysateswere chro effect of 6-TG were to decrease the amount of mRNA syn thesized in vivo. That the functional ability of the mRNA matographedon Cellex-Danion-exchangecolumns to separate 6-TG from its nucleotide, as described under “Materials and Meth from the livers of 6-TG-treated animals to be translated in ods.―RNA vitro was altered is evidenced by the fact that significantly more peptides high in tyrosine content were synthesized pmoles/mgbNucleus source RNAfractiona from 6-TG-containing poly(A)@RNA than from an equivalent (4)CPoIy(A)@ PoIy(A) 142 ±57 amount of control poby(A)@RNA. This finding might be the (4)Cytoplasm 7203 ±2603 result of incorporation of the punine antimetabolite into (6)Poby(A)@ Poby(A) 37 ±11 mRNA in a manner that allows more efficient translation. 124 ±50 (5) Since both the initiator and the terminator codons contain a a Poly(A)@ and poly(A) RNA were isolated from total nuclear and guanine moiety, one possibility is that substitution of 1 or cytoplasmic RNA on pobyuridylate-Sephanose as described previ both of these guanine residues by a 6-TG molecule might ousby(4). drastically alter either the mateof initiation or termination. b Incorporation of 6-TG is expressed as pmobes 6-TG occurring as the nucleotide per mg of RNA±S.E. The fact that 6-TG-containing poly(A)@RNA is translated C Numbers in parentheses, number of animals in each detenmi more efficiently in the wheat germ system cannot be extrap nation. olated to the liver in vitro translational system because it is doubtful whether the products synthesized in the 2 systems are identical; thus, the polyacrylamide gel profiles of these 2 The polysome component of the in vitro protein-synthe sets of products differ. Since different mRNA molecules sizing machinery appears to be responsible for the inhibi probably incorporate different amounts of 6-TG and in dif tory effect of 6-TG seen in profiles of high-tyrosine-contain fementboci within the molecule, the ultimate effects on pep ing peptides synthesized in vitro. This implies that the nibo tide synthesis that one observes are probably the result of a somes, the mRNA, or the bound factors are being affected by 6-TG, rather than the tRNA as reported for E. co!i by Gray combination of actions by 6-TG on mRNA. and Rachmeler (6). Although 6-TG, incorporated into cer The data presented in this paper are supportive of the tam tRNA molecules, may alter their amino acid acceptor postulated mechanism presented in the earlier report (4) in activity, this effect does not appear to be significant in the in which 6-TG, given before the onset of S phase, interferes vitro protein-synthesizing system used in these expeni with the synthesis of certain proteins by depressing the ments. Data presented in the previous paper (4), on the synthesis of their mRNA's. In addition, 6-TG is presumably effects of 6-TG on the synthesis of poIy(A)@RNA in regenem incorporated into the mRNA's for these enzymes; use of the ating liver, indicate that the most likely site of action of 6-TG wheat germ system has allowed the detection of an altered on the polysomes responsible for inhibition of protein syn efficiency in the mateof translation of these 6-TG-containing thesis in vitro is the mRNA. mRNA molecules. The occurrence of such an effect in vivo Since the time at which 6-TG was administered to mats might result in either an altered amount of enzyme on en corresponded to a time after partial hepatectomy when a zyme molecules with an impaired function on both. 2nd burst of RNA synthesis occurs (see, e.g., Ref. 16), it This entire sequence of events occurs before the 1st wave seemed reasonable to assume that at least some of the of mitosis after partial hepatectomy. The initiating events, analog was incorporated into this newly synthesized RNA. which may be related, occur in G1, before the onset of DNA That this assumption was indeed true was demonstrated by synthesis, and therefore, the action of 6-TG on these meta the finding that 6-TG was incorporated into RNA at this time bolic events would appear to be divorced from the incompo to a significant extent. Under the conditions used, the ration of the punine antimetabolite into DNA. poly(A)@RNA incorporated significantly more 6-TG per mg of RNA than did the poly(A) RNA. In addition, the poly(A)@ ACKNOWLEDGMENTS RNA fraction exhibited a greater response to the action of 6TG in terms of diminished incorporation of radioactive on capable assistance of Vow-Mci Huang and Paula A. Wilson is grate otic acid and decreased A260than did the poly(A) RNA (4). fullyTheacknowledged. 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