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Alterations in the transfer RNA population of hepatoma 9618A as compared with normal rat liver
BIOCHIMICA ET BIOPHYSICA ACTA
415
BBA 97087
A L T E R A T I O N S IN T H E T R A N S F E R RNA POPULATION OF HEPATOMA 9618A AS COMPARED W I T H NORMAL RAT L I V E R S. A. S. VOLKERS* AND MILTO~I W. T A Y L O R
Microbiology Department, Indiana University, Bloomington, Ind. 474oz ( U.S.A .) (Received July 23rd, 1971)
SUMMARY
Aminoacyl-tRNAs from hepatoma 9618A were compared with the corresponding aminoacyl-tRNAs from normal rat liver using reversed phase cochromatography. The hepatoma exhibited two more Lys-tRNA species and one more PhetRNA species than did rat liver. These species appeared to be due to new or altered species of tRNA.
Alterations in the tRNAs of systems undergoing, or having undergone, changes in metabolic control processes have been reported to occur in m a n y systems. The findings of TAYLOR et al. 1,2 suggested that, in general, there was a remarkable similarity between tR_NAs obtained from different mammalian organs, and also between different species. However, these workers did observe differences between the tRNAs of various tumor cells and normal tissues. Since then, differences have been observed in pre-neoplastic livers of ethionine-fed rats s, in various mouse plasma cell tumors 4,5 and in various hepatomas e-n. l~any of these studies have been carried out with relatively poorly differentiated tumors, for which a control tissue was difficult to find. Because of this, we decided to compare tRNAs obtained from three rat hepatomas of varying degrees of differentiation (namely 9618A, 5123D and 3924A)~with those obtained from normal rat liver. Our findings with hepatomas 5123 D (well differentiated) and 3924 A (poorly differentiated) have been published elsewhere 8,~. This communication presents evidence for the presence of new species of Lys- and Phe-tRNA in the highly differentiated hepatoma 9618A. Hepatoma 9618A was maintained b y serial transplantation into male Buffalo rats at intervals of IO months. When the hepatomas were required for experimental purposes, the tumors were removed and, either used immediately, or stored at --7 o°. Normal livers were removed from decapitated rats and treated in the same way as the tumors. Transfer RNA, aminoacyl-tRNA synthetase and aminoacyl-tRNAs were prepared as described previously 9. Mixtures of the labelled aminoacyl-tRNAs were applied to a reversed phase chromatography column TM previously equilibrated with Buffer A (o.oi M sodium acetate buffer (pH 4.5), containing o.oi IV[ magnesium * Present address: ~iochemistry Department, Glasgow University, Glasgow, W.2., Scotland.
Biochim. Biophys. Acta, 254 (1971) 415-518
416
S. A . S . VOLKERS, M. W. TAYLOR
acetate, o.oi IV[ mercaptq~thanol and o.ooi M EDTA) supplemented with o.35 M NaC1 , and eluted with a ~ a d i e n t (2.0 1) of 0.35-0.55 M NaC1 in Buffer A. The elution rate was I.O ml/min, and IO ml fractions were collected. The aminoacyl-tRNA in each fraction was precipitated b y the addition of an equal volume of a solution containing o.16 M E D T A and 0.2 % cetyltrimethyl ammonium bromide 18. After freezing and thawing to facilitate precipitation, the precipitates were collected on glass fiber filters (Whatman Type GF/C), washed with water, dried for I h at 60 ° and counted in a Beckman scintillation counter using a toluene based PPO cocktail. Transfer RNAs specific for six amino acids were compared in this manner. The elution patterns of four of them, namely His-, Leu-, Ser- and Tyr-tRNAs, showed little or no differences in elution profiles. Cochromatography of Lys-tRNA (Fig. Ia) from rat liver and hepatoma 9618A revealed the presence irl each source of three species which chromatographed at similar ionic strengths, one of these being present in relatively minor amounts. However, the Lys-tRNA9618A possessed two extra species which eluted at a higher ionic strength. In addition to this, quantitative differences were apparent in the three common species. The elution profiles obtained with Phe-tRNA are shown in Fig. Ib. An extra minor species was present in Phe-tRNA9818 A as compared with Phe-tRNAuver. Table I shows a compalison of the numbers of iso-accepting t R N A species observed in hepatoma 9618A as compared with normal liver. The results are also compared with those previously obtained 9 with hepatomas 5123D and 3924 A. 12
4
50
.C E
~6 ~2 ~_
60
70
80
90
100
110
120
130
140
~3~6
4o
1~o
~oo
b
8
4
%
, ---~_-80
90
100
110 i20 Froction No.
Fig. I. Reversed phase 2 c o c h r o m a t o g r a p h y of (a) [14C]Lys-tRNAD61s^ and [3H]Lys~tRNAnver and (b) [l~C]Phe-tRNAg.18A a n d [SH]Phe-tRNAnver. The chromatographic conditions were as described in the text. The recovery of 14C ( O - O ) and SH ( O - Q ) was greater t h a n 90 % in all cases.
Biochim. Biophys. Acta, 254 (1971) 415-418
t R N A FROM HEPATOMAAND NORMALLIVER
417
TABLE I N U M B E R S OF I S O - A C C E P T I N G
A mino acid
His Leu Lys Phe Ser Tyr
tRNAs
O B S E R V E D IN ~V~ORRIS H E P A T O M A S
Tissue Liver
3924`4
5123D
9618.4
3 3 3 I 2 3
3 3 3 I 2 3
i 3 3 2 4 3
3 3 5 2 2 3
The alterations we have observed are not due to alterations of the synthetase enzymes, as aminoacylating the tRNAs with homologous or heterologous enzymes gave similar profiles. Any artifacts due to minor impurities in the labelled amino acids were avoided b y reversing the labelled amino acids. A mixture containing I.O /,mole each of the other 19 amino acids was also added to the reaction mixture to prevent any spurious aminoacylation. The extra peaks were not due to aggregates, as heating the t R N A to 80 ° prior to aminoacylation caused no alteration in the elution patterns. This treatment is reported 14 to dissociate any tRNA aggregates which m a y have formed on standing. Also, these alterations are not due solely to the increased growth rate of the cells, as our unpublished observations, and those of AGARWALet al. 1~ indicate that aminoacyl-tRNAs prepared from regenerating liver cochromatograph at the same point as the corresponding aminoacyl-tRNAs prepared from normal liver. We conclude, therefore, that these differences observed in hepatoma 9618A are due to the neoplastic nature of the cells Furthermore, as these alterations occur in such a highly differentiated tumor, it is possible that they may be related to the alterations in control mechanisms which originally caused the cell to become malignant. TAYLOR et al. 1,2 suggested that tRNA alterations may play some part in differentiation and neoplasia, and it had been thought that these three tumors in various stages of differentiation might show a gradation in any tRNA alterations which might occur. That this is not so is evident from Table I. In the very poorly differentiated tumor (3924 A) no differences were observed in any of the aminoacyl-tRNAs examined; in the moderately well differentiated tumor (5213D), no differences were observed in Leu-, Lys- and Tyr-tRNAs, while in the highly differentiated tumor (9618A) differences were observed only in the Lys- and Phe-tRNAs. Thus no clear pattern of alterations has emerged. However, the fact remains that alterations in the elution profiles of tRNAs do occur, even in very highly differentiated hepatomas, while very few alterations have been observed in the patterns from different organs x,3. The molecular basis of these alterations is, as yet, unclear. They may be due to changes in structure produced, for example, by increased methylation, causing the t R N A to elute differently, or altering the ability of a certain species to recognise the corresponding aminoacyl-tRNA synthetase. An alteration of an isoprenoid base next to the anticodon m a y cause alterations in elution patterns and also alter ribosome binding properties. The alteration of the amino acid moiety after aminoacylation m a y also cause an alteration in elution properties. We are now in the process of examining some of these possibilities more closely. Biochim. Biophys. `4cra, 254 (I97 I) 415-418
418
S . A . S . VOLKERS, M. W. TAYLOR
ACKNOWLEDGEMENT
We thank Mrs. Suzanne Prather for excellent technical assistance, and Dr. H. P. Morris for the gift of tumor bearing rats. This work was supported by U.S. Public Health Service Grant No. CA-II496, Grant No. lO18 from the Damon Runyon Memorial Fund for Cancer Research and Grant No. IN 46K from the American Cancer Society. REFERENCES i M. W. TAYLOR, C. N. BUCK, G. A. GRANGER AND J. J. HOLLAND, J. Mol. Biol., 33 (1968) 809. 2 M. W. TAYLOR, G. A. GRANGER, C. A. BUCK AND J. J. HOLLAND, Proc. Nail. Acad. Sci. U.S., 57 (1967) 1712. 3 R. AXEL, I. ]3. WI~INSTEIN AND E. FARBER, Proc. Natl. Acad. Sci. U.S., 58 (I967) I255. 4 J. F. MUSHINSKI AND M. POTTER, Biochemistry, 8 (1969) 1684. 5 W. K. YANG AND G. D. NOVELLI, Biochem. Biophys. Res. Commun., 31 (1968) 534. 6 ]3. S. ]3ALIGA, E. ]3OREK, I. ]3. WEINSTEIN AND P. R. SRINIVASAN, Proc. Natl. Acad. Sci. U.S., 62 (I969) 899. 7 M. OOLDM~, W. 1~. JOHNSTON AND A. C. GRIFFIN, Cancer Res., 29 (1969) lO51. 8 M. W. TAYLOR, S. A. S. VOLKERS, ]3. K. CNOE AND J. G. ZEIKUS, Cancer Res., 3I (I97 I) 688. 9 S. A. S. VOLKIgRS AND 1~. W. TAYLOR,Biochemistry, io (i97 I) 488. io G. GONANO, V. P. CHIARUGI, G. PIRRO AND H. MARINI, Biochemistry, Io 1197 I) 9oo. 1I D. SRINIVASAN, P. R. SRINIVASAN, D. GRUNBERGER, I. B. WEINSTEIN AND H. P. MORRIS, Biochemistry, i o {i971) I966. 12 J. F. W ~ i s s AND A. D. KELMERS, Biochemistry, 6 (1967) 2507 . 13 A. SIBATANI, Anal. Biochem., 33 (197 o) 279. 14 A. ADAMS AND H. G. ZACHAU, Eur. J. Biochem., 5 (i968) 556. 15 M. K. AGARWAL, J. HANOUNE AND I. ]3. W~INSTEIN, Biochim. Biophys. Acta, 224 (197 o) 259.
Biochim. Biophys. Acta, 254 (I971) 415-418
415
BBA 97087
A L T E R A T I O N S IN T H E T R A N S F E R RNA POPULATION OF HEPATOMA 9618A AS COMPARED W I T H NORMAL RAT L I V E R S. A. S. VOLKERS* AND MILTO~I W. T A Y L O R
Microbiology Department, Indiana University, Bloomington, Ind. 474oz ( U.S.A .) (Received July 23rd, 1971)
SUMMARY
Aminoacyl-tRNAs from hepatoma 9618A were compared with the corresponding aminoacyl-tRNAs from normal rat liver using reversed phase cochromatography. The hepatoma exhibited two more Lys-tRNA species and one more PhetRNA species than did rat liver. These species appeared to be due to new or altered species of tRNA.
Alterations in the tRNAs of systems undergoing, or having undergone, changes in metabolic control processes have been reported to occur in m a n y systems. The findings of TAYLOR et al. 1,2 suggested that, in general, there was a remarkable similarity between tR_NAs obtained from different mammalian organs, and also between different species. However, these workers did observe differences between the tRNAs of various tumor cells and normal tissues. Since then, differences have been observed in pre-neoplastic livers of ethionine-fed rats s, in various mouse plasma cell tumors 4,5 and in various hepatomas e-n. l~any of these studies have been carried out with relatively poorly differentiated tumors, for which a control tissue was difficult to find. Because of this, we decided to compare tRNAs obtained from three rat hepatomas of varying degrees of differentiation (namely 9618A, 5123D and 3924A)~with those obtained from normal rat liver. Our findings with hepatomas 5123 D (well differentiated) and 3924 A (poorly differentiated) have been published elsewhere 8,~. This communication presents evidence for the presence of new species of Lys- and Phe-tRNA in the highly differentiated hepatoma 9618A. Hepatoma 9618A was maintained b y serial transplantation into male Buffalo rats at intervals of IO months. When the hepatomas were required for experimental purposes, the tumors were removed and, either used immediately, or stored at --7 o°. Normal livers were removed from decapitated rats and treated in the same way as the tumors. Transfer RNA, aminoacyl-tRNA synthetase and aminoacyl-tRNAs were prepared as described previously 9. Mixtures of the labelled aminoacyl-tRNAs were applied to a reversed phase chromatography column TM previously equilibrated with Buffer A (o.oi M sodium acetate buffer (pH 4.5), containing o.oi IV[ magnesium * Present address: ~iochemistry Department, Glasgow University, Glasgow, W.2., Scotland.
Biochim. Biophys. Acta, 254 (1971) 415-518
416
S. A . S . VOLKERS, M. W. TAYLOR
acetate, o.oi IV[ mercaptq~thanol and o.ooi M EDTA) supplemented with o.35 M NaC1 , and eluted with a ~ a d i e n t (2.0 1) of 0.35-0.55 M NaC1 in Buffer A. The elution rate was I.O ml/min, and IO ml fractions were collected. The aminoacyl-tRNA in each fraction was precipitated b y the addition of an equal volume of a solution containing o.16 M E D T A and 0.2 % cetyltrimethyl ammonium bromide 18. After freezing and thawing to facilitate precipitation, the precipitates were collected on glass fiber filters (Whatman Type GF/C), washed with water, dried for I h at 60 ° and counted in a Beckman scintillation counter using a toluene based PPO cocktail. Transfer RNAs specific for six amino acids were compared in this manner. The elution patterns of four of them, namely His-, Leu-, Ser- and Tyr-tRNAs, showed little or no differences in elution profiles. Cochromatography of Lys-tRNA (Fig. Ia) from rat liver and hepatoma 9618A revealed the presence irl each source of three species which chromatographed at similar ionic strengths, one of these being present in relatively minor amounts. However, the Lys-tRNA9618A possessed two extra species which eluted at a higher ionic strength. In addition to this, quantitative differences were apparent in the three common species. The elution profiles obtained with Phe-tRNA are shown in Fig. Ib. An extra minor species was present in Phe-tRNA9818 A as compared with Phe-tRNAuver. Table I shows a compalison of the numbers of iso-accepting t R N A species observed in hepatoma 9618A as compared with normal liver. The results are also compared with those previously obtained 9 with hepatomas 5123D and 3924 A. 12
4
50
.C E
~6 ~2 ~_
60
70
80
90
100
110
120
130
140
~3~6
4o
1~o
~oo
b
8
4
%
, ---~_-80
90
100
110 i20 Froction No.
Fig. I. Reversed phase 2 c o c h r o m a t o g r a p h y of (a) [14C]Lys-tRNAD61s^ and [3H]Lys~tRNAnver and (b) [l~C]Phe-tRNAg.18A a n d [SH]Phe-tRNAnver. The chromatographic conditions were as described in the text. The recovery of 14C ( O - O ) and SH ( O - Q ) was greater t h a n 90 % in all cases.
Biochim. Biophys. Acta, 254 (1971) 415-418
t R N A FROM HEPATOMAAND NORMALLIVER
417
TABLE I N U M B E R S OF I S O - A C C E P T I N G
A mino acid
His Leu Lys Phe Ser Tyr
tRNAs
O B S E R V E D IN ~V~ORRIS H E P A T O M A S
Tissue Liver
3924`4
5123D
9618.4
3 3 3 I 2 3
3 3 3 I 2 3
i 3 3 2 4 3
3 3 5 2 2 3
The alterations we have observed are not due to alterations of the synthetase enzymes, as aminoacylating the tRNAs with homologous or heterologous enzymes gave similar profiles. Any artifacts due to minor impurities in the labelled amino acids were avoided b y reversing the labelled amino acids. A mixture containing I.O /,mole each of the other 19 amino acids was also added to the reaction mixture to prevent any spurious aminoacylation. The extra peaks were not due to aggregates, as heating the t R N A to 80 ° prior to aminoacylation caused no alteration in the elution patterns. This treatment is reported 14 to dissociate any tRNA aggregates which m a y have formed on standing. Also, these alterations are not due solely to the increased growth rate of the cells, as our unpublished observations, and those of AGARWALet al. 1~ indicate that aminoacyl-tRNAs prepared from regenerating liver cochromatograph at the same point as the corresponding aminoacyl-tRNAs prepared from normal liver. We conclude, therefore, that these differences observed in hepatoma 9618A are due to the neoplastic nature of the cells Furthermore, as these alterations occur in such a highly differentiated tumor, it is possible that they may be related to the alterations in control mechanisms which originally caused the cell to become malignant. TAYLOR et al. 1,2 suggested that tRNA alterations may play some part in differentiation and neoplasia, and it had been thought that these three tumors in various stages of differentiation might show a gradation in any tRNA alterations which might occur. That this is not so is evident from Table I. In the very poorly differentiated tumor (3924 A) no differences were observed in any of the aminoacyl-tRNAs examined; in the moderately well differentiated tumor (5213D), no differences were observed in Leu-, Lys- and Tyr-tRNAs, while in the highly differentiated tumor (9618A) differences were observed only in the Lys- and Phe-tRNAs. Thus no clear pattern of alterations has emerged. However, the fact remains that alterations in the elution profiles of tRNAs do occur, even in very highly differentiated hepatomas, while very few alterations have been observed in the patterns from different organs x,3. The molecular basis of these alterations is, as yet, unclear. They may be due to changes in structure produced, for example, by increased methylation, causing the t R N A to elute differently, or altering the ability of a certain species to recognise the corresponding aminoacyl-tRNA synthetase. An alteration of an isoprenoid base next to the anticodon m a y cause alterations in elution patterns and also alter ribosome binding properties. The alteration of the amino acid moiety after aminoacylation m a y also cause an alteration in elution properties. We are now in the process of examining some of these possibilities more closely. Biochim. Biophys. `4cra, 254 (I97 I) 415-418
418
S . A . S . VOLKERS, M. W. TAYLOR
ACKNOWLEDGEMENT
We thank Mrs. Suzanne Prather for excellent technical assistance, and Dr. H. P. Morris for the gift of tumor bearing rats. This work was supported by U.S. Public Health Service Grant No. CA-II496, Grant No. lO18 from the Damon Runyon Memorial Fund for Cancer Research and Grant No. IN 46K from the American Cancer Society. REFERENCES i M. W. TAYLOR, C. N. BUCK, G. A. GRANGER AND J. J. HOLLAND, J. Mol. Biol., 33 (1968) 809. 2 M. W. TAYLOR, G. A. GRANGER, C. A. BUCK AND J. J. HOLLAND, Proc. Nail. Acad. Sci. U.S., 57 (1967) 1712. 3 R. AXEL, I. ]3. WI~INSTEIN AND E. FARBER, Proc. Natl. Acad. Sci. U.S., 58 (I967) I255. 4 J. F. MUSHINSKI AND M. POTTER, Biochemistry, 8 (1969) 1684. 5 W. K. YANG AND G. D. NOVELLI, Biochem. Biophys. Res. Commun., 31 (1968) 534. 6 ]3. S. ]3ALIGA, E. ]3OREK, I. ]3. WEINSTEIN AND P. R. SRINIVASAN, Proc. Natl. Acad. Sci. U.S., 62 (I969) 899. 7 M. OOLDM~, W. 1~. JOHNSTON AND A. C. GRIFFIN, Cancer Res., 29 (1969) lO51. 8 M. W. TAYLOR, S. A. S. VOLKERS, ]3. K. CNOE AND J. G. ZEIKUS, Cancer Res., 3I (I97 I) 688. 9 S. A. S. VOLKIgRS AND 1~. W. TAYLOR,Biochemistry, io (i97 I) 488. io G. GONANO, V. P. CHIARUGI, G. PIRRO AND H. MARINI, Biochemistry, Io 1197 I) 9oo. 1I D. SRINIVASAN, P. R. SRINIVASAN, D. GRUNBERGER, I. B. WEINSTEIN AND H. P. MORRIS, Biochemistry, i o {i971) I966. 12 J. F. W ~ i s s AND A. D. KELMERS, Biochemistry, 6 (1967) 2507 . 13 A. SIBATANI, Anal. Biochem., 33 (197 o) 279. 14 A. ADAMS AND H. G. ZACHAU, Eur. J. Biochem., 5 (i968) 556. 15 M. K. AGARWAL, J. HANOUNE AND I. ]3. W~INSTEIN, Biochim. Biophys. Acta, 224 (197 o) 259.
Biochim. Biophys. Acta, 254 (I971) 415-418