sign in sign up
<<
Home , Adenosine diphosphate, Inosine triphosphate

[CANCER RESEARCH 27, 646-«S3,April 1967] Utilization of L-Methionine and S-Adenosyl-L-methionine for Methylation of Soluble RNA by Mouse Liver and Hepatoma Extracts1

R. L. HANCOCK The Jackson Laboratory, Bar Harbor, Maine

SUMMARY following reaction: ATP + L-methionine = SAM + pyrophos The methyl group of L-methionine or S-adenosyl-L-methionine phate + monophosphate (7). SAM is used in the methylation of sRNA by sRNA methylases in the following reaction: sRNA + is used by nonparticulate mouse liver and hepatoma prepara SAM = methylated sRNA + S-adenosyl-L-homocysteine (10). tions to methylate soluble ribonucleic acid (sRNA). Esclierichia The two reactions are presented along with a representa coli K12 sRNA was a more active substrate than yeast sRNA. tion of the origin of the unmethylated sRNA (tp-RNA) in Chart Four different lots of E. coli B sRNA gave similar incorporations between lots. "Stripped" and "nonstripped" sRNA had similar 1. An array of RNA methylases exhibiting specificity for par ticular bases and for sRNA from different biologic sources, along amounts of methyl incorporation. Other triphosphates with other, less si^ecific RNA methylases, has been described besides triphosphate were capable of sup]x>rting the incorjioration of methyl groups from L-methionine into sRNA. (11, 15, 16, 17, 22). The most extensively methylated sRNA molecules known have been found in mammary adenocarcinoma The nonspecificity of the reaction was tissue (3), and recently Mittelman et al. (18) have found RNA shown to be due to an adenosine diphosphokinase reaction and methylase activity to be extremely high in certain viral-induced not to nucleoside tri : L-methionine nucleosidyl trans- tumor cells. Soluble and ribosomal RNA isolated from HeLa ferase activity. cells cultured in vitro were shown to have a large percentage of Mouse hepatoma BW7756 was found to have lower amounts of methylation activity than mouse liver if L-methionine-14CH3 methylated . A large proportion of these methyl groups was believed to be on the moiety (6). was used as the methyl donor, although hepatoma tissue had Studies on the enzymatic pathway of sRNA methylation were over twice as much sRNA methylase activity per se as liver on initiated to examine further the possibility of "hypermethyla- a gm tissue equivalent or protein weight basis. However, em tion" of hepatoma sRNA. In previous studies (12) it was found bryonic liver as well as other embryonic and neoplastic tissues that although mouse liver had a large amount of S-adenosyl- also had increased amounts of sRNA methylase activity over transferase activity, the activity of B\V 7756 mouse hepatoma that of their adult counterparts. was not measurable. The work described in the present report indicates that this mouse hepatoma apparently does possess INTRODUCTION trace amounts of S-adenosyltransferase activity since the labeled The methyl group of methionine is activated through a reac methyl group of L-methionine was found to be transferred to tion catalyzed by : L-methionine S-adeno- sRNA. Furthermore, the methyl group of SAM was used by syltransferase (E.G. 2.4.2.13), hereafter termed S-adenosyltrans- hepatoma extracts to methylate sRNA. Experiments with ferase. This enzyme catalyzes the formation of SAM2 by the SAM demonstrate that the BW 7756 hepatoma has twice as much sRNA methylase activity ¡«rgmwet weight or per mg 1This investigation was supported in part by an allocation protein as that found in mouse liver. from USPHS General Research Support Grant SO 1 FR-05545-03 and American Cancer Society Grant IN 19F to the Jackson Lab MATERIALS AND METHODS oratory, Bar Harbor, Maine. 2Abbreviations used are as follows: sRNA, soluble ribonucleic The following chemicals were obtained commercially: hy- acid; rRNA, ribosomal ribonucleic acid; tp-RNA, precursor trans droxylamine HC1 grade I, ATP, salmon sperm DNA, yeast fer ribonucleic acid; t"'-RNA, methylated transfer ribonucleic sRNA, L-methionine, Tris (Sigma, Inc., St. Louis, Mo.) ; Esch- acid; Ar, adenosine; AMP, ; ADP erichia coli K12, E. coli B, and yeast ("stripped"3 and "non- ; ATP, adenosine triphosphate; dATP, stripped"), rabbit and rat liver sRNA (General Biochemicals, triphosphate; UTP, triphosphate; GTP, Inc., Chagrin Falls, Ohio); UTP, GTP, CTP, ITP (Pabst Lab triphosphate; CTP, triphosphate; GSH, gluta- oratories, Milwaukee, \Vis.); L-methionine-14CH3 (10.3 me/ thione; SAM, «S-adenosyl-L-methionine; SAH, S-adenosyl-L- homocysteine; SAM-UCH3,jS-adenosyl-L-methionine-uCH3; Tris, mmole) and SAM-14CH3 (50.2 mc/mmole) (New England Nu- tris(hydroxymethyl)aminomethane; ITP, triphosphate; IDP, inosine diphosphate; UDP, ; NADPH, 3The term "stripped" is used to denote the removal of the reduced nicotinamide- dinucleotide phosphate. from transfer RNA, usually done by treating the RNA Received August 31, 1966;accepted November 16, 1966. with an alkaline procedure.

G46 CANCER RESEARCH VOL. 27

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1967 American Association for Cancer Research. Methylation of Soluble RNA

tDNA that the specific activity of the resultant SAM is not known because of SAM present in the supernatant fraction, and there fore these incorporations should not be considered to represent the absolute moles of methyl groups being attached to sRNA

tp-RNA molecules. The routine reaction mixture for studies using S-ADENOSYL - SAM-14CHs as a methyl source was as follows: 100 /¿molesof SAM « ATP+ L-METH. TRANSFERASE Tris (pH 7.5), 50 /¿molesofGSH, 30 /¿molesofMgCl2, 5 mg of RNA E. coli K12 sRNA, 0.5 /¿cof SAM-»CH3 (0.499 mc/mmole), METHYLASE and 1.0 ml of 100,000 X g (approximately 250 mg equivalents SAH of tissue) in a total volume of 2 ml. The reaction mixture was incubated for 12 min at 37°C."Soluble RNA methylase activity" t "'-RNA is used in this writing to denote the rate of the sRNA methylase reaction per se, that is, the transfer of methyl groups from SAM CHART 1. Pathway for sRNA methylation. Nonmethylated transfer RNA (tRNA) is synthesized upon a specific DNA tem to sRNA. plate by RNA polymerase. This precursor transfer RNA (t"-RNA) receives its methyl groups from S-adenosyl-L-methionine (SAM) RESULTS via sRNA methylase(s) action resulting in /S-adenosyl-L-homo- cysteine (SAH) and a methylated species of transfer RNA (t'n- Characteristics of the sRNA-methylating Reaction. RNA). SAM is synthesized by ATP:L-methionine (L-meth) The following studies were concerned with the overall reaction S-adenosyl transíerase. ATP, adenosine triphosphate; GTP, gua- that is made up of the S-adenosyltransf erase and sRNA methyl nosine triphosphate; UTP, ; CTP, cytidine ase reactions. The sRNA methylase activity determined may triphosphate. represent several specific sRNA methylases, as has been found to be the case in bacteria (16, 17). High-speed supernatant frac clear Corp., Boston, Mass.); polyinosinic acid and polyadenylic tions (100,000 X g for 1 hour) of female 129/Rr mouse liver were acid (Miles Chem. Co., Elkhart, Ind.); crystallized bovine albu assayed for their ability to incorporate the methyl groups from min (Pentex, Inc., Kankakee, 111.);yeast D--6-phosphate, L-methionine-14CH3or SAM-14CH3(Table 1). It was found that NADP oxidoreductase, ATP, and D-hexose-6-phosphotransferase E. coli sRNA served as an excellent substrate (see section on (Boehringer and Soehne, Mannheim, Germany). ]K>lymerspecificity). The reaction rate was linear for a 15-minute The 129/Rr and C57L/J strains of mice and BW 7756 hepa- period (Chart 2A), and incoqjoration of methyl groups was de toma, indigenous to C57L/J mice, were purchased from the Jack pendent upon E. coli K12 sRNA, although a small amount of son Laboratory. AXC and Buffalo strains of rats, and Buffalo endogenous activity was present in all enzyme preparations rats carrying the R3B (3rd generation) and AXC rats carrying (Table 2). Such endogenous activity was undoubtedly due to the the transplantable H35 hepatoma (30th generation) were kindly presence of mouse liver sRNA in crude supernatant fractions. donated for this work by Dr. Melvin D. Reuber of the Depart The reaction rate was linear over a range of sRNA concentra ment of Pathology at the NIH, Bethesda, Maryland. tions from 0 to about 7 mg. Although larger incoq>orations could Race III New Zealand white rabbits were procured from the have been obtained with 10 mg of sRNA, a concentration of 5 Hamilton Station section of the Jackson Laboratory. mg of sRNA was selected for reasons of economy (Chart 2B). iS-Adenosyltransferase was assayed according to the method The reaction rate increased linearly with increasing amounts of of Cantoni and Durell (8) and the sRNA methylase activity by enzyme up to about 25 mg of protein (Chart 2(7)- The optimal the method of Srinivasan and Borek (22). Protein determinations concentration of reduced GSH was 0.025 M. The portion of the were done by the spectral method of Warburg and Christian reaction catalyzed by S-adenosyltransferase reaches substrate (23) and the biuret method (9). All enzyme preparations were saturation at about 20 m\i of L-methionine (12). As can be seen from fresh tissues. Unless otherwise stated, the enzyme prepara in Chart 2£>,theoverall reaction appears to become saturated tions were obtained from female 129/Rr mouse liver. The en with L-methionine at the 0.1-mM range and therefore certainly zyme preparation was made by homogenizing 1 gm of fresh liver for 8 strokes in a glass homogenize!1in 4 volumes of a solution TABLE 1 containing 0.25 M sucrose and 0.01 M MgCl».The homogenate Mouse Liver Soluble RNA (sRNA) Methylase Activity was centrifuged for 15 minutes at 10,000 X g and then at 100,000 time "CHi6 X g for 1 hour and used immediately. The fractionation was sRNA+—+Incubation (min)001212SAM-»CHja(cpm)28282093060L-Methionine(cpm)1719135600 done at 4°C. The routine reaction mixture for studies using the L-methio- nine as a methyl source was as follows: the reaction mixture con taining 100 jumólesof Tris (pH 7.5), 50 /¿molesof GSH, 30 AmólesofMgCl2, 20 /¿molesofATP, 5 mg of E. coli K12 sRNA, 1 /¿cof L-methionine-^CHs (10.3 mc/mmole), and 1.0 ml of " Routine reaction mixture (see under Materials and Methods) 100,000 X g supernatant (approximately 250 mg equivalents of except for above-indicated omissions of Escherichia coli K12 129/Rr female liver) in a total volume of 2 ml. The reaction mix sRNA. SAM-14CH3, S-adenosyl-L-methionine-14CH,. ture was incubated for 12 min at 37°C.Aunit was defined as the 6 Routine reaction mixture (see under Materials and Methods) number of /i/miólesof methyl groups from methionine-14CH3 except for 100 AmólesMgCl2 and indicated omissions or additions incorporated per gm of tissue per 12 minutes. It is recognized of 2 mg E. coli K12 sRNA.

APRIL 1967 647

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1967 American Association for Cancer Research. R. L. Hancock reflects the rate-limiting aspect of the portion of the pathway way (Chart 2E). The pH optimum for the overall reaction ap catalyzed by sRNA methylase which also becomes saturated at peared to be about 7.5 (Chart 2F). the 0.1-mm range (Chart 3). The S-adenosyltransf erase portion Specificity of the sRNA Methylase System with Respect of the pathway is enhanced by large concentrations of magne to Polymers and Nucleoside Triphosphates. Polyinosinic sium (0.3 M) (7, 12). However, it became apparent in early ex acid, polyadenylic acid, and salmon sperm DNA did not serve periments that the portion of the pathway catalyzed by sRNA as substrates. Yeast sRNA was methylated but was a poor ac methylase was inhibited by the high concentrations of MgCU, ceptor compared with E. coli K12 sRNA (see Table 3). A study and 0.03 M MgCl? was found to be optimal for the overall path was made to make sure of the deacvlation of terminally added

2A •=300 c 300 Z) I" 200

U I IDO >* N C LJ O IO 20 30 40 5O IO 15 T (min.) mg sRNA

2D

100 200 IO 20 30 40 L-Methionine (//M mg Protein

500 500

400

-300

200

100

300 50 100 7 8 9 IO II pH

CHART2. The following experiments were done with L-methionine-"CHs as the methyl source. A, Incorporation of methyl groups with respect to time. The reaction mixture was routine except that it contained 50 Amólesof MgCl„and2 mg of Escherichia coli soluble RNA (sRNA). An 8-tube aliquot of the reaction mixture containing 8 ml of 100,000 X firsupernatant was made, and a blank was prepared in the same manner but contained no sRNA; 2-ml aliquots were removed at varying times for assay as described in the Method section. The zero-time assay was prepared by taking an appropriate quantity of the reaction mixture before the addition of the enzyme prepara tion. The discrepancy in the amounts of methylation shown in this figure is due to suboptimal amounts of which had been used in early experiments. B, Incorporation of methyl groups with respect to varying amounts of sRNA. The reaction mixture was routine except for 50 nmoles of MgClz, varying amounts of E. coli K12 sRNA, and 0.5 ml of the 100,000 X g supernatant fraction. C, Methyl incorporation with respect to varying amounts of enzyme protein. The reaction mixture was routine except for 2 mg of E. coli K12 sRNA and varying amounts of the 100,000 X g supernatant from 129/Rr female liver. D, Methyl incorporation with respect to varying amounts of L-methionine. The reaction mixture was routine except for the varying amounts of L-methionine. E, Methyl in corporation with respect to varying amounts of magnesium. The reaction mixture was routine except for the varying amounts of MgClj. F, Methyl incorporation with respect to varying pH. The reaction mixture was routine except that lOÛAimolesof Tris (hydroxymethyl) aminomethane buffer at varying pH's were introduced.

648 CANCER RESEARCH VOL. 27

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1967 American Association for Cancer Research. Methylation of Soluble RNA

TABLE 2 Hurwitz (11), one might not expect the rodent liver sRXA to be Endogenous Activity of the Reaction Mixture Using a very good substrat«.However, the i»orperformance of yeast L-Methionine-uCH,° sRNA was somewhat surprising. ATP*—++sRNA+—+cpm5040155818 In view of the specificity studies on the S-adenosyltransferase step (12), it was unexpected to find that several different nucleo- side tri were able to substitute for ATP in the reac tion resulting in the incorporation of the L-methionine methyl group into sRNA (Table 6). Attempts to isolate S-uridyl-L-

TABLE 3 0 Routine reaction mixture except for the above-indicated Differences of Various Soluble RNA (sRNA) Preparations to Serve omissions. as Substrate for Methyl Acceptor Activity Using 6 ATP, adenosine triphosphate; sRNA, soluble RNA.

sRNAEscherichia *65208165020163027765177865165225B-8530cpm618211814178110022194454 i BEscherichia coli 30

2YeastLot coli Kl 2O " Routine reaction mixture except for 5 mg of various stripped sRNA preparations. 6 Average of paired data. The correlation of the paired assay samples was 0.975.

TABLE 4 The Differences Between Stripped and Nonstripped Soluble (sRNA) as Methyl Acceptors Using L-Methionine-ltCHt"

O.I 0.2 Stripped_—++Hydroxylamine treated—+-+cpm65133692625492 S- Qdenosyl-L-methionine (mM ) CHART 3. Comparison of SAM utilization by hepatoma and liver extracts. The reaction mixture was routine except that 0.5 tic of SAM-14CHa (50.2 mc/mmole) and varying amounts of un- labeled SAM were added. SAM, S-adenosyl-L-methionine. " Routine reaction mixture except for 5 mg of stripped or non- L-methionine-I4CH3 which had been terminally added byaminoacyl stripped yeast sRNA preparations. sRNA synthetases. Such aminoacyl linkages can be hydrolyzed 6 Average of paired data. The correlation of the paired assay with the alkaline hydroxylamine treatment. As can be seen in samples was 0.999. Table 4, stripped, i.e., sRNA with the amino acid removed, and nonstripped yeast sRNA have similar incorporations of 14CHs TABLE 5 groups if both receive the hydroxylamine treatment. The Differences of Various Soluble RNA (sRNA) Preparations to Serve "stripped" yeast sRNA which had not received the hydroxyl as Substrate for Methyl Acceptor Activity Using amine step had eight times as much incorporation of 14CH3 S-Adenosyl-L-methionine-l>CH¡'> groups. Furthermore, stripped and nonstripped yeast sRNA sRNAEscherichia were similar in their efficiencies as substrates using SAM as the methyl donor (380 and 326 cpm/20 mg protein respectively). K12Escherichiacoli The use of SAM-14CH3eliminates the possibility of the terminal WBacillus coli addition of L-methionine-14CH3to sRNA by these crude enzyme 6009YeastYeastYeastsubtilis preparations. The hydroxylamine step was included to insure that any trace amounts of L-methionine that might be present due to hydrolysis of the SAM-MCH»wouldnot be attached in a cell)Rabbit(whole terminal position to sRNA. liverRat Table 5 shows the variation in methylation of sRNA's from liverStripped++++—-++cpmb183234218501822274 different sources by mouse liver sRNA methylase. The bacterial " Routine reaction mixture except for 5 mg of various sRNA sources of sRNA are far suprior to those of yeast. Rat and rabbit additions. liver sRNA are also very poor substrates. From the species 6 Average of paired data. The correlation of the paired assay specificity studies of Srinivasan and Borek (22) and Gold and samples was 0.995.

APRIL 1967 649

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1967 American Association for Cancer Research. R. L. Hancock

TABLE G Requirement Using L-Methionine-uCH3a

tissue)Xone26ATP*5110307049501420GTP99CTP101608UTP112281679571UTPtriphosphate (cpm/gm Tissue129/Hr +ADP1300ITP821

liver129/Rrfemale liverC57L/Jmale liverBW7750host hepatomaXucleoside

" Routine reaction mixture except for the substitution of 20 /umoles of various nucleoside triphos- phates for ATP. b ATP, adenosiue triphosphate; GTP, ; CTP, ; UTP, uridine triphosphate; ITP, .

TABLE 7 UTP: ADP Diphosphokinase Reaction with Protein from 100,000 X g Supernatant fraction

No.Reaction Tube

mixtureATP eraseGlucose:o-hexose-6-phosphotransf taseSupernatant-6-phosphate: N ADP-oxidoreduc fractionAmmonium fractionUTPADPATPAsulfate

O.D. at 340 mii/5 min1+02++03+++04++++0.1465++++0.0386+++++0.0457"+++++-f0.1368+++++0.1159+++++0 " The UTP-dependent NADPH formation had a 2-minute lag before any detectable change in the 340 HIMabsorption occurred. UTP, uridine triphosphate; XADPH, reduced form of nicotinamide- adenine diuucleotidc phosphate; ATP, adenosine triphosphate; UTP, uridine triphosphate; ADP, adenosine diphosphate; Tris, tris(hydroxymethyl)aminomethane. 6 Reaction mixture contained 130 jumólesof Tris, pH 7.6, 9 ¿»molesofMgCla, 16 Amólesof glucose, 0.2 jumóleof NADP, 0.05 /«moleof nucleoside tri- (or di-) phosphate where indicated, 1.4 units of yeast i>-glucose-6-phosphate:NADP oxidoreductase and 2.8 units of ATP:D-hexose-G-phosphotrans- ferase where indicated, and 0.05 ml of a 100,000 X g supernatant fraction (14 mg equivalents of 129 Rr/J female mouse liver) or 0.03 ml of a 0-70% ammonium sulfate protein fraction (1.98 mg protein). The (+) symbol indicates the presence of a particular reagent. methionine-14CH3 from reaction mixtures in which the UTP had found when L-methionine is used as a source of methyl groups been substituted for ATP were uniformly unsuccessful. The (Table 8). This is reasonable if one considers the extremely low facts that ADP enhances such UTP activity and that CTP and •S-adenosyltransferase activity present in hepatoma extract, ITP also can substitute for ATP are best explained by the oc compared with liver (Table 9). Preliminary studies (see Table 8) currence of a nucleoside diphosphokinase reaction. have shown that the "minimal deviation" rat hepatoma H35 The reaction UTP + ADP ^ ATP + UDP can be demon and the undifferentiated hepatoma R3B may have more «S-adeno- strated by using protein from the 100,000 X g supernatant syltransferase activity than mouse hepatoma BW 7756. (Table 7). Using the assay system of Berg and Joklik (1), the The BW 7756 mouse hepatoma is considered highly differenti 100,000 X g supernatant itself cannot be demonstrated to possess ated but is fast growing. The H35 rat hepatoma is also very dif a UTP-dependent reaction forming ATP. The amount of endoge ferentiated and even produces bile. It grows quite slowly and is classified as a "minimal deviation" type of hepatoma.4 The H35 nous ATP is probably sufficient in this sensitive assay to produce optimal amounts of NADPH through the formation of glucose-6- hepatoma is thought to be the least deviated of any hepatoma phosphate and its subsequent reduction to 6-phospho-glucoiiic (5). The H35 hepatoma was induced with diacetylaminofluorene, acid. However, if one uses the ammonium-sulfate-precipitated as was the R3B hepatoma, but R3B hepatoma is a fast growing, protein (0-70% saturated ammonium sulfate), then a UTP- undifferentiated tumor (19). The sRNA methylase activity of the mouse hepatoma using dependent reaction can be shown to occur only if ADP is added. SAM-14CHj as the methyl donor was over 200% of normal liver Quantitative Studies on sRNA Methylation in Liver and Itepatoma. A decrease in the rate of methyl incorporation into sRNA by the hepatoma extracts compared with liver is 4For a discussion of minimal deviation tumors see Reference 5.

600 CANCER RESEARCH VOL. 27

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1967 American Association for Cancer Research. Methylation of Soluble RNA based upon gm equivalents of tissue or mg of protein (1400 cpm cate that the rat hepatoma H35 may not have an increased per gm liver and 3120 cpm i>ergm hepatoma). Plots from the sRNA methylase activity (Table 8). data of Chart 3 indicated that the Km values for liver (6.76 X Fetal liver preparations used L-methionine for sRNA methyla- 10~5M) and hepatoma 3.75 X 10~5M approximate one another tion at a rate intermediate between rates found for tumor and (Chart 4). If the sRNA methylase activity is made up of a family liver (Table 8). Studies now in progress upon the changes in of methylases analogous to the bacterial system, this difference sRNA methylase activity of mouse and rabbit liver with age in Km values is not remarkable. Studies on sRNA methylases of show that the fetal liver has much higher activity than adult rat hepatomas are not complete, although first experiments indi- liver. However, like hepatoma, fetal liver has no perceptible (S-adenosyltransferase activity; this may be instrumental in de TABLE 8 creasing the efficiency of utilization of L-methionine for sRNA Methylalion of Soluble RNA (sRNA) with L-Methionine by Rat and methylation. Rabbit embryonic tissues, compared with their Mouse Liver and Hepatoma Tissue" adult counterparts (Chart 5), in general had an increase in the amount of sRXA methylase activity when calculated on a tissue group in of corporation cpm/ value for weight equivalent. When activities were computed on the basis livers or gmtissueAverage normal of protein instead of wet weight equivalent of tissue, all embry TissueMouse hepato liver of mas mouse or onic tissues except brain still demonstrated a sizable increase in assayed52511111Methyl rat100.059.432.7100.050.858.2100.021.7 value7440440524263440175020003280712S.E.±563±475±665%ofsRXA methylase activity. A comparison between transplanted neoplastic mouse tissues and 5 normal tissues showed large in- experiment:C57L/J 4months)C57L/J9 mouse liver (adult to TABLE 9 liver(fetal)»BW77569 and cF mouse Liver and Hepatoma ATP:L-Methionine S-Adenosyliransferase Activity" hepatomaRat experiment:AXC ofmethio-nine-dependent liverH359 rat of inorganic phos livers or phorus released of rathepatomaR3B hepato-assayed3411111Amólesper gm oftissue6aver normal TissueC57L/J liverofmouse rathepatomaC57L/J or liverBW77569 mouse rat100.00.7100.031.738.4 mouse hepatomaNo. age29.10.320.215.614.66.45.7S.E.±1.6±0.3%ofvalue

" All values are averages from duplicate assays. The "rat liverBW77569 mouse experiment" reaction mixture was conducted the same as the (C57L/J)AXChepatoma routine mouse assay but contained 100 Mmoles of tris(hydroxy- liverAXC9 rat methyl)aminomethane, pH 8.2, 100 Mmoles of , and liverBuffalo9 host rat 100 /¿molesof MgCl¡.Mouse liver and hepatoma were used here liverH35 9 host rat as controls, and their lower values, as compared with those used (AXC)R3Brat hepatoma in the "mouse experiment," reflect the inhibition of the sRNA rat hepatoma (Buffalo)No. methylase enzyme by the higher MgCl: concentration. 6 Fetal liver preparation represents approximately 35 near-term " ATP, adenosine triphosphate. mice and assayed in duplicate. 6 Assay was done as described in Ref. 12.

-3.0 -20 -1.0 0 .0 2.0 3.0 4.0 X IO"4

CHART4. A Lineweaver and Burk plot for the K„of liver and hepatoma sRXA methylase activity as described in Chart 3.

APRIL 1967 651

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1967 American Association for Cancer Research. R. L. Hancock creases of sRNA methylase activity in neoplastia cells over that 4000 in normal cells (Chart 6). •=Neoplastia Tissue O = Normal Tissue DISCUSSION The increase in sRNA methylase activity of hepatomas may •5 be explained as being a general requirement for, or property of, •4 growth, since fetal liver cells also display increased amounts. No 3000 studies have been made ujxm the percentage of methylated nucleotides found in sRNA from B\V 7756 mouse hepatoma tis sue. Although the overall rate of methylation of sRNA by hepa in4» toma extracts was found to be lower than that of liver, this is not necessarily in conflict with concepts of "hypermethylation" P •2 E of sRNA or "abnormal" methylase as a potentially important 0-20001- part of the carcinogenic process (4, 19). The number of methyla- •3 E tions on sRNA molecules would not necessarily be proportional o. to the amount of assayable sRNA methylase activity residing in u the cell. The final products of sRNA methylation may be deter 06 mined by the specificities of the reactions involving certain sRNA methylases and the secondary structure and/or tertiary configurations of particular sRNA molecules. Such differences in 1000 04

03 2000 01 CHART6. Comprison of sRNA methylase activity in normal and neoplastic mouse tissues. The reaction mixture was routine except for the enzyme extracts from various mouse tissues as indi cated. A range of tissue equivalents from 233 to 320 was used. • Neoplastic tissues: 1, melanoma B16; 2, rhabdomyosarcoma BW 10139; 3, neuroblastoma C1300; 4, hepatoma BW7756; 5, testicular interstitial cell tumor H10119; 6, lymphatic leukemia o> BW.5147.O, Normal tissues (129/Rr female): 1, skin; 2, skeletal v> muscle; 3, brain; 4, liver; 5, testis; 6, spleen. a» configurations between specific RXA molecules are suggested by E o» the work of Sarin et al. (21). 1000 The change in the amount of SAM synthesis in the hepatoma E cell may be an example of the economy shown by the neoplastic CL U cell. There is a retention of a particular level of S-adenosyltrans- ferase activity for sRNA methylation, among other methylations, which may be necessary for the correct function of transfer RNA in protein synthesis. Studies (to be presented elsewhere) showing an increase in the extent of methylation of E. coli sRNA by fetal mouse liver and hepatoma extracts have been made. If such "hypermethylated" sRNA molecules are formed in vivo in embryonic and neoplastic cells, then this will need to be examined in terms of differentiation. Possibly, hypermethyl ated sRNA molecules miscode and thereby the cell produces nonfunctional . This would be the state of an undiffer- entiated embryonic or dedifferentiated neoplastic cell. Liver Kidney BrainIN Lung Heart The problem of several nucleotides being able to substitute CHART5. Comparison of sRNA methylase activity in adult for ATP in the L-methionine utilization for the methylation reac and embryonic rabbit tissues. The reaction mixture was routine tion can be resolved in terms of a nucleoside diphosphokinase except for the enzyme extracts from various rabbit tissues as reaction. Such reactions have been described. For example, Berg indicated. A range of tissue equivalents from 201 to 278 mg was and Joklik reported on the following reaction (2) : used. E indicates embryonic (27-day-old gestation) and A indi cates adult (11 months old). ITP + ADP = IDP + ATP

652 CANCER RESEARCH VOL. 27

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1967 American Association for Cancer Research. Methylation of Soluble RNA

UTP + ADP = UDP + ATP 6. Brown, G., and Attardi, G. Methylation of Nucleic Acids in HeLa Cells. Biochem. Biophys. Res. Commun., W: 298-302, However, most of the nucleoside diphosphokinase of liver has 1965. been reported to occur in the mitochondrial fraction (13). Mouse 7. Cantoni, G. Activation of Methiouine for Transmethylation. liver supernatant was shown to possess adenosine diphospho J. Biol. Chem., 189: 745-754, 1951. activity. The )>ercentage of the total nucleoside diphospho 8. Cantoni, G., and Dnrell, J. Activation of Methionine for kinase activity present in the liver cell has not been studied Transmethylat Bion. J. Biol. Chem., giB: 1033-1048, 1957. here, but the supernatant activity probably represents a small 9. Colowick, S., and Kaplan, N. Methods in Enzymology, Vol. proportion of the total cellular activity. The system is completely Ill, 450 pp. New York: Academic Press, Inc., 1957. dependent upon a nucleoside tri phosphate (Table 7). The activ 10. Fleissner, E., and Borek, E. A New Enzyme of RNA Synthesis: RNA Methylase. Proc. Nati. Acad. Sei. U. S., 48: 1199-1203, ity cannot be demonstrated to be due to an endogenous supply of ATP. Studies upon the abilities of various RXA's from mouse 19G2. 11. Gold, M., and Hurwitz, J. The Enzymatic Methylation of the liver to act as a substrate for mouse sRNA methylase are in Nucleic Acids. Cold Spring Harbor Symp. Quant. Biol., 28: progress. Neither Ar, AMP, ADP, nor ATP were found upon 149-159, 1963. paper Chromatographie examination of the UTP used in these 12. Hancock, R. L. S-Adenosylmethionine Synthesizing Activity experiments. Experiments show, however, that the UTP- and of Normal and Neoplastic Mouse Tissues. Cancer Res., 26: ATP-dependent activity are both found in the fraction precipi 2425-2430, 1966. tated between 30% and 55% saturation with ammonium sulfate. 13. Herbert, E., Potter, V. R., and Takag, Y. Nucleotide Metab olism. IV. The of 5'-Uridine Nucleotides by Therefore, it is assumed that the nucleoside diphosphokinase is coprecipitated with the S-adenosyltransferase. Cell Fractions from Rat Liver. J. Biol. Chem., 213: 923-940, 1955. 14. Hoagland, M., and Zamecnik, P. Intermediate Reactions in ADDENDUM . Federation Proc., 16: 197, 1957. The above work was presented in part at the 152nd meeting of 15. Hurwitz, J., Anders, M., Gold, M., and Smith, I. The En the American Chemical Society, held during September 11-16, zymatic Methylation of Ribonucleic Acid and Deoxyribonu- I960 (Abstract #134). A paper has appeared from Dr. E. Borek's cleic Acid. VII. The Methylation of Ribosomal Ribonucleic laboratory describing studies on the extent of sRNA methylation Acid. J. Biol. Chem., 240: 1256-1266,1965. and the specific bases methylated by tumor preparations (E. 16. Hurwitz, J., Gold, M., and Anders, M. The Enzymatic Methyl Tsutsui, P. R. Srinivasan, and E. Borek. Proc. Nati. Acad. Sci. ation of Ribonucleic Acid and Deoxyribonucleic Acid. III. U. S., 56: 1003-1009,1966). Purification of Soluble Ribonucleic Acid Methylating En zymes. J. Biol. Chem., 239: 34G2-3473,1964. ACKNOWLEDGMENTS 17. Hurwitz, J., Gold, M., and Anders, M. The Enzymatic Methyl I wish to thank Dr. M. Reuber for AXC and Buffalo rats and ation of Ribonucleic Acid and Ueoxyribonucleic Acid. IV. rats transplanted with R3B and 1135hepatoma. I also am indebted The properties of the Soluble Hibonueleic Acid Methylating to Mrs. P. McFarland for her excellent technical assistance. Enzymes. J. Biol. Chem., 239: 3474-3482,1964. 18. Mittelman, A., Yohn, D., Hall, R., and Grace, J., Jr. The in REFERENCES Vitro sHNA Methylase Activity of SY-40 Induced Hamster Tumors. Proc. Am. Assoc. Cancer Res., 6: 45, 1965. 1. Berg, P., and Joklik, W. Transphosphorylation between 19. Morris, H. Studies on the Development, Biochemistry and Nucleoside . Nature, 172: 1008-1009,1953. of Experimental Hepatomas. Advan. Cancer Res., 9: 2. Berg, P., and Joklik, W. Enzymatic Phosphorylation of 227-302, 1965. Nucleoside Diphosphates. J. Biol. Chem., 210: 657-672, 1954. 20. Reuber, M. A Transplantable Bile-secreting Hepatocellular 3. Bergquist, P., and Matthews, R. Occurrence and Distribution Carcinoma in the Rat. J. Nati. Cancer Inst., 26: 891-899,1901. of Methylated in Hibonucleic Acids of Subcellular 21. Sarin, P., Zamecnik, P., Bergquist, P., and Scott, J. Con- Fractions. Biochem. J., 85: 305-313, 1962. formational Differences among Purified Samples of Transfer 4. Borek, E. The Methylation of Transfer RNA : Mechanism and Function. Cold Spring Harbor Symp. Quant. Biol., 28: 139- RNA from Yeast. Proc. Nati. Acad. Sei. U. S., 65: 579-585, 148, 1963. 1966. 5. Bottomley, R., Pitot, H., Potter, V., and Morris, H. Metabolic 22. Srinivasan, P., and Borek, E. The Species Variation of RNA Adaptations in Rat Hepatomas. IV. Reciprocal Relationship Methylase. Proc. Nati. Acad. Sei. U. S., 49: 529-533, 1963. between Threonine Dehydrase and Glucose-6-phosphate-de- 23. Warburg, O., and Christian, W. Isolation and Crystallization hydrogenase. Cancer Res., 23: 400-409, 1963. of Enolase. Biochem. Z., 310: 384-421, 1942.

APRIL 1967 (553

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1967 American Association for Cancer Research. Utilization of l-Methionine and S-Adenosyl-l-methionine for Methylation of Soluble RNA by Mouse Liver and Hepatoma Extracts

R. L. Hancock

Cancer Res 1967;27:646-653.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/27/4/646

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/27/4/646. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1967 American Association for Cancer Research.