Enzymatic methylation of transfer RNA by extracts of polyoma virus-transformed cells

BIOCHIMICA ET BIOPHYSICA ACTA

365

BBA 96573

ENZYMATIC M E T H Y L A T I O N OF T R A N S F E R RNA BY EXTRACTS OF POLYOMA V I R U S - T R A N S F O R M E D CELLS B. B R E I E R * AND R. W. H O L L E Y * "

Cornell University, Ithaca, N . Y . (U.S.A.) (Received March I3th, I97 o)

SUMMARY

A study of the enzymatic methylation of Escherichia coli t R N A and rat liver t R N A in vitro by extracts of five mammalian cell lines failed to provide evidence of a polyoma virus-induced t R N A methylase.

INTRODUCTION

It is known that the induction of new antigens and the induction of enzyme activities concerned with nucleic acid metabolism occur during cell transformation by oncogenic viruses J,2. Suggestions that tRNA m a y be involved in cell transformation have been put forth 3A. The proposal by SRINIVASAN AND BOREK5 that oncogenic viruses m a y transform cells by a mechanism involving altered methylation of t R N A has led to a number of studies of the methylation of t R N A by tumor cell extracts. The tRNA methylases are known to be species specific 6-s and their activity tlas been shown to change during changes in differentiation 9-n. Increased levels of t R N A methylase activity in tumor tissue compared with normal tissue have been found by two groups 1~,1~. In studies using viral-induced tumors, three groups 14-16 also found higher t R N A methylase levels in tumor tissue compared to control tissue. In their work with SV-4o-induced hamster tumor, MITTELMAN el al. 14 showed that by using homologous t R N A as substrate the tumor tissue extract catalyzed the formation of N~-methylguanosine although extracts of normal tissues could not. Since the SV-4 o viral genome is small, it would not be expected to code for very m a n y enzymes 1. The SV-4 o and the polyoma viruses appear to transform cells in parallel ways 1,2. Prompted by the implication of the existence of a SV-4o virus-specific t R N A methylase, we attempted to demonstrate an analogous enzyme in polyoma virus-transformed cells. MATERIALS AND METHODS

Cell cultures. Five cell lines were studied. One cell line (HPy), obtained from Flow Laboratories Inc., originated from a tumor induced in a b a b y hamster with polyoma virus. The tissue of origin for these cells is not known. The mouse cell line * To w h o m r e p r i n t requests should be sent to at D e p a r t m e n t ' o f Biochemistry, Mount Sinai School of Medicine, New York, N. Y. 10029, U.S.A. "* Present address: The Salk I n s t i t u t e for Biological Studies, San Diego, Calif., U.S.A.

Biochim. Biophys. Acta, 213 (197 o) 365-37 °

366

B. BREIER, R. W. HOLLEY

3T3 (ref. 17) and the p o l y o m a virus-transformed cell line derived from it, 3T3Py (ref. 18) were obtained from the laboratory of Dr. R. Dulbecco. The B H K (ref. i9) cell line of hamster cells was obtained from Flow Laboratories Inc., and the polyoma virus-transformed cell line derived from it, B H K P y (ref. 2o), was obtained from the laboratory of Dr. R. Dulbecco. The H P y , 3T3, and 3 T 3 P y cell lines were grown in Eagles minimum essential medium 21 containing IO °/o calf serum, 2 mM glutamine, plus 125 u n i t s / m l of both penicillin and streptomycin. All of these components were purchased from Flow Laboratories Inc. The B H K and the B H K P y cell lines were grown in the same medium after addition of IO ~{~ Tryptose phosphate broth (purchased from Difco Laboratories). Homogenizing solution. Contents were IO mM Tris-HC1 (Schwarz BioResearch Ultrapure Biological grade), p H 7.5; IO mM MgC12; I mM E D T A ; and 12 #M mercaptoethanol. Buffer solution. Contents were 0.50 M Tris-HC1; 0.05 M MgC12; i mM E D T A ; and 5 #1 of mereaptoethanol per ml of solution. S-Adenosyl-L-rnethionine. Obtained labeled in the methyl group with ~4C with a specific activity of 3 ° mC/mmole from Tracerlab Inc. The commercial product was diluted with distilled water to give a solution t h a t contained 5/~C/ml. Standard purine and pyrimidim bases. All bases (Cyclo Chemical Co.) were employed as solutions of IO mg/ml in concentrated HC1. Substrate tRNA. Rat liver and E. coli B t R N A were obtained from General Biochemicals. Procedure employed to enzyrnatically methylate tRNA. A typical experiment employed the following procedure. T w e n t y four 75-cm 2 flasks of cells were harvested. This involved discarding the culture medium and adding IO ml of Puck's Saline A (ref. 2z) to each flask. The cells were scraped off the flasks with a rubber policeman and the mixture was centrifuged 5 min at 2000 y.g at room temperature. The cells were then homogenized with 2.0 ml of ice-cold homogenizing solution, and the suspension was centrifuged at 3000 × g in a cold room for 15 min. The supernatant fraction was centrifuged at lO5 ooo × g for 60 rain in the 5 ° Ti rotor of a Spinco Model L ultracentrifuge maintained at zero degrees. The resulting supernatant fraction was used as the enzyme source. Two reaction inixtures were prepared from 2.0 ml of buffer solution, I.O ml (5 #C) of S-adenosyl-L-methionine, 0. 9 nil of enzyme solution, 0.5 ml of t R N A solution (2 mg/ml), and distilled water to give a 5-ml total volume. The two reaction mixtures were incubated in a 37 ° water bath. After 9 ° min, the reaction mixtures were extracted twice with an equal volume of water-saturated phenol and 6 times with ether. To eliminate a n y remaining S-adenosyl-L-methionine, use was made of the observation that S-adenosyl-L-methionine is soluble in 5 ~ trichloroacetic acid while t R N A is not 22. The t R N A was precipitated with an equal volume of IO o/.o trichloroaeetic acid and the precipitate was redissolved in 0.2 M Tris-HC1 (pH 7.5). The precipitation was repeated two times, after which the t R N A solution was washed 6 times with ether. A final precipitation of the t R N A was done with two volumes of cold 95 ~}~,ethanol.

Procedure employed /or the degradation o/ the enzyrnatically methylated tRNA. After the ethanol-precipitated t R N A was dried over P205 in a v a c u m n desiccator, it was treated with ioo#1 of 7 ° ~ HC104 for 2 h at Ioo ° (ref. 23). After cooling of the hydrolysate, 300 #1 of saturated KC1 were added. The tube was centrifuged and

Biochim. Biophys. Acta, 213 (~97o) 365-37 °

POLYOMA VIRUS-INDUCED t R N A

367

METHYLASE

the KC1Q was then further washed with 0.2 M HC1. The hydrolysate solutions were dried over P205 in the vacuum desiccator.

Procedure employed/or the thin-layer chromatography and counting o/the products o/ degraded enzymatically methylated tRNA. To each sample tube was added 200/zl of 0.2 M HC1. The standard bases were applied (lO-15 nmoles) to each of two 20 cm ×20 em "Avicel" plates (500 # thickness, Analtech Company). Then 30 #1 of hydrolysate were applied over the standards. Each plate was run in the first direction in the solvent system 2-propanol-conc. HCl-water (85:22:18, by vol.) 24 and in the second direction in the solvent system n-butanol-water-formic acid (77:13:1o, by vol.) ~5. A wick of Whatman 3 MM paper was attached to the end of the plate for each development. This two-directional chromatography was sufficient for the resolution of all compounds of interest except the pyrimidines and N6-methyladenine. These latter two compounds were resolved using a 20 cm ×2o cm "Avicel" plate (250# thickness) and the solvent system methanol-conc. HCl-water ( 7 : 2 : 1 , by vol.) 26. The sample was applied as three bands with IO#1 of hydrolysate on each, plus the appropriate standards. In this case a wick of Whatman No. I paper was used. All three solvent systems were prepared just before use and were preequilibrated for I h. When the chrolnatograms were finished and dried, the portions of the chromatograms containing ultraviolet light-absorbing materials were cut out and the cellulose was placed in scintillation vials. The remaining areas of the plate were also coupted to avoid missing an unanticipated base. Into each vial IO ml of modified BRAY'S~7 solution was added. RESULTS

The first line to be studied was the H P y line. The use of buffers of three different pH values with E. coli tRNA as substrate revealed fluctuations in the activities of at least five different tRNA methylases (Table I). With rat liver tRNA as the substrate, at pH 8.8, the tRNA methylase specific for Na-methylguanine was the only enzyme which retained appreciable activity. TABLE I METHYLATION

UTILIZING

THE

HPy

CELL LINE

AS E N Z Y M E

SOURCE

Counts~rain E. coli t R N A

N2-Methylguanine N1-Methylguanine N2,N2-Dilnethylguanine NX-Meth yladenine 5-Methyluracil

Rat liver t R N A

pH 7.2

p H 8.o

p H 8.8

235o 8 35 ° 500 15oo

28oo 45 i ioo i io 2300

37oo 63 19oo 18 195

p H 8.8 13.6 53 6.9 o o

The second cell line studied was the 3T3Py line. As in the case of the H P y cell line, an attempt was made to find a pH that optimized the yield of Nl-methyl guanine, because of the work of MITTELMANet al?4. The results are shown in Table II. The results are essentially the same as with the H P y cells as enzyme source. Biochim. Biophys. Acta, 213 (197 o) 365-37 o

368 TABLE

B. B R E I E R , R. W . H O L L E Y II

METHYLATION

UTILIZING THE

3TBPy

AND

3T3

C E L L L I N E S AS E N Z Y M E S O U R C E S

Counts/rain 3 T 3 P y enzyme

3T3 enzyme

E. coli IRNA

N2-Methylguanine N1-Methylg uanine Ne,N~-Dimethylguanine N1-Methyladenine 5-Methylcytosine 5-Methyluracil

p H 7.2

p H 8.8

Rat liver t R N A p H 8.8

3300 5-3 960 500 2 i4 ° 28

2800 25 16oo 15 92 o

9.8 62 o o o o

E. coli tRNA p H 8.8

Rat liver t R N A p H 8.8

3000 3° 2000 16 13 ° o

6.6 22 o o o o

The 3T 3 cell line was used as a control for the 3T3Py cell line. As can be seen in Table II, the results obtained using 3T3 and 3T3Py as enzyme source were sindlar. Such results do not support the presence of a viral-induced tRNA methylase in tile mouse system such as that described by MITTELMANet al. 14 using the SV-4o virusinduced hamster tumor. It seemed possible, however, that viral induction of a tRNA methylase is species specific. The work of two groups 28,29 demonstrated a species specificity in the induction of the enzyme deoxythymidine kinase by SV-4o virus. In order to test for such a species specificity, the B H K P y / B H K system was studied. These results are shown in Table III. TABLE

IlI

METHYLATION

UTILIZING THE

BHKPy

A N D B H t ~ _ C E L L L I N E S AS E N Z Y M E

S O U R C E AT p H

~.8

Counts~rain B H K P y enzyme

N2-Methylguanine Nl-Methylguanine N2,N2- Dimethylguanine N1-Methyladenine 5-Methylcytosine

IqHK enzyme

E. coli tRNA

Rat liver tRNA

E. eoli tRNA

Rat liver tRNA

16 160 115 ~2 OOO 88 560

156 62 72 o o

20 OOO 96 9 OOO 82 620

74 42 29 o o

DISCUSSION

The results of these studies of the tRNA methylase activities of several cell lines in vitro do not support the hypothesis of an oncogenic virus-induced alteration in tRNA methylation. Similar findings have been published by two other groups a°'31. A limitation to these studies is the attempt to study several different enzymes under one set of conditions. The stabilities and optimum reaction conditions of purified tRNA methylases of E. coli are known to vary considerably from enzyme to enzyme a2. Biochim. Biophys. Acta, 213 (197 o) 3 6 5 - 3 7 °

POL¥OMA VIRUS-INDUCED

tRNA

METHYLASE

36 9

The NH4 + concentration can have a considerable effect on t R N A methylase activity from extracts of rat liver 33 and from various mouse organs 31. The concentration of mercaptoethanol ~t and the strain of E. coli from which the substrate t R N A is obtained 34,~5 have strong effects on the activity of t R N A methylases obtained from human cells grown in tissue culture. The conditions used here are, however, not greatly different from those used by MITTELMAN et al. 14. Comparison of overall tRNA methylase activity between tissues is not a valid approach, since it has been shown 31 that, although leukemia spleen tissue tRNA methylase activity is greater than normal spleen tissue tRNA methylase activity, both extracts methylated t R N A to the same extent. Using 3o-4o-fold purified preparations of N'-methyladenine t R N A methylase, it has been shown 31 that, although the leukemia spleen tissue enzyme is increased in activity compared to the normal spleen tissue enzyme, the two enzymes had identical substrate specificity. Upon transformation by oncogenic viruses, cells experience heritable changes in their growth properties. In immunologically tolerant hosts the transformed cells usually form tumors. It is our opinion that the two lines of polyoma-transformed cells represent a more reliable system than that used by other workers. For example, in the work reported by MITTELMAN et al. ~, a study was made of a serially transplantable tumor line obtained after the injection of SV-4o virus into newborn hamsters. The major problem with this approach is the lack of a proper control tissue, since in tumors derived in the fashion described above, the tissue of origin is unknown. In the work described in this study, the 3T3 and the B H K cell lines are the cells of origin of the transformed cell lines and are therefore suitable controls. Our conclusion is that at present the evidence is unconvincing for the presence of new tRNA methylase activity in tumor tissue.

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