[CANCER RESEARCH 31, 599-604, May 1971]
The Possible Role of Nucleic Acid Methylases in the Induction of
Cancer
P. N. Magee
The Courtauld institute of Biochemistry, The Middlesex Hospital Medical School, London, WIP 5PR, England
The possible importance
of tRNA methylases
in
carcinogenesis was suggested by Srinivasan and Borek (48,
49). This hypothesis has stimulated a considerable amount of
subsequent work and is currently under investigation in several
laboratories. The hypothesis was based originally on the
observation by Bergquist and Mathews (3) that some tumors
contained higher levels of methylated RNA than their
corresponding normal tissues and on the reports by Farber and
Magee (14, 31) that the potent hepatic carcinogens ethionine
(13) and dimethylnitrosamine
(29) methylated the RNA of
the liver after administration to rats. Thus Borek (4) stated,
"If, as it seems likely, these imposed alkylations by chemical
carcinogens have a causal relation to the tumours they
produce,
then such chemical alkylations
may find a
counterpart in aberrant or excessive methylations of RNA or
DNA by naturally occurring methylating enzymes." The
hypothesis was extended to include viral carcinogenesis as
follows: "We have postulated
that, since all nucleic
acid-methylating enzymes are species specific, an oncogenic
virus might introduce a capacity for the synthesis of nucleic
acid-methylating enzymes which are foreign to the host.
Should this occur, then the host would harbor enzymes which
might methylate its nucleic acids aberrantly, just as alkylating
carcinogens do."
Subsequent work by Borek and his colleagues and by other
workers has given support for this hypothesis. Increased levels
of tRNA methylases were reported in human mammary tumor
and rat hepatoma (57), in mouse hepatoma (20), in hamster
tumors induced by SV40 virus (37, 40), and in liver tumors
from chicks with Marek's disease (19, 34). The work with
hamsters by McFarlane and Shaw (37) is of particular interest
because the tRNA methylase activity of the SV40 tumor cells
was not only higher than that of liver, lung, kidney, spleen,
and some other tissues, but no differences were found between
the capacities of normal and tumor cell extracts to methylate
DNA or ribosomal RNA. Increased urinary excretion of
methylated purines was found in human subjects with
leukemia (43), in tumor-bearing rats and mice (35), and in
hamsters with tumors induced by SV40 virus (36). In contrast
to these essentially confirmatory findings, however, Kaye and
Leboy (22), using different conditions of enzyme assay (46),
concluded that neither the rate, nor the extent, nor the
pattern of methylation
could be used to distinguish
unequivocally between extracts from tumor and from normal
organs. The essential difference in the conditions used was
based on the observation that the rates and extents of
methylation of tRNA by organ and tumor extracts could be
considerably stimulated by the presence of ammonium ions.
More recently, however, Stewart and Corrance (52), using
assay procedures similar to those of Kaye and Leboy, have
reported that levels of methylase activity with Escherichia coli
tRNA as substrate were higher in extracts of malignant,
newborn, and regenerating tissue than in extracts of normal
tissue when comparison was made at optimum concentration
of ammonium ions. When liver tRNA was used as substrate,
there was no incorporation of methyl groups by various liver
extracts, but a small amount of incorporation was observed
with extracts from ethionine-induced
liver tumors. The
authors conclude that there are some differences between the
complements of tRNA methylases in malignant and normal
tissue (52). Gantt and Evans (18) have compared the RNA
methylase capacity of an in vitro long-term nonneoplastic
mouse embryo cell line with a neoplastic subline and also with
tumor tissue arising from another subline or cells implanted
into syngeneic animals. Cell-free extracts of the neoplastic
tissues had much greater capacity to methylate E. coli soluble
RNA than the cultured nonneoplastic control line, and the
authors conclude that these results support the hypothesis that
aberrant alkylation of nucleic acids may be a necessary event
in some types of neoplasia.
The work discussed above gave evidence of increased
methylase activity in a variety of tumors but did not give any
indication of increased biosynthesis of the methylated bases in
the intact animal during carcinogenesis. This aspect of the
problem was investigated by Craddock (10), who studied
methylation of tRNA and rRNA in normal animals and in
animals fed carcinogenic diets containing dimethylnitrosamine,
aflatoxin, and ethionine. The rats were given injections of
methionine-14C, and the labeled nucleic acids were analyzed
for the presence of labeled methylated bases. The level of
labeling of each methylated base in tRNA was found to be
increased by each carcinogen, and there appeared to be a
change in composition of the major bases of tRNA during
carcinogenesis by dimethylnitrosamine and by aflatoxin, giving
support to the view that there is a change towards more highly
methylated species of tRNA during carcinogenesis.
It seems, therefore, that the balance of evidence does favor
some form of aberrant nucleic acid methylation during
carcinogenesis as postulated by Borek and his colleagues, and
an attempt will be made here to assess its significance and to
compare these aberrant methylations caused by mtethylases
with those induced by alkylating carcinogens.
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599
P. N. Magee
The Role of Alkylation in the Induction of Cancer
Certain alkylating agents, including sulfur and nitrogen
mustards, were reported to induce tumors in experimental
animals some years ago, and the suggestion was made that
carcinogenesis resulted from alkylation of some component or
components of the cell. The question of carcinogenesis by
alkylating agents has been discussed by Brookes and Lawley
(6), who concluded that alkylation can result in carcinogenesis
but that, in general, alkylating agents cannot be regarded as
powerful carcinogens. With the discovery, of the carcinogenic
action of dimethylnitrosamine
by Magee and Barnes (29),
however, and the subsequent very extensive studies on
structure-activity
relationships by Druckrey et al. (11), it
appeared
possible
that alkylation,
and in particular
methylation, could, under some circumstances, be powerfully
carcinogenic since dimethylnitrosamine
and subsequently
other nitrosamines were shown to act as alkylating agents in
vivo
(30-32).
The
evidence
for
alkylation
by
dimethylnitrosamine was based on the presence of methylated
components of proteins and nucleic acids in the tissues of
animals treated with the labeled nitrosamine, the methyl
groups being derived from it. These presumably represented
aberrant methylations either in excess of or at sites other than
those utilized by the normal metabolic transfer of methyl
groups from S-adenosylmethionine. Since, however, the nitroso
compounds appear to be very considerably more powerful
carcinogens than the known alkylating agents, doubts were
expressed as to whether alkylation could explain the whole or
perhaps any of the carcinogenic activities of the nitrosamines.
Since the original hypothesis of Borek was partly based on the
reported capacities of dimethylnitrosamine
and ethionine to
alky late in vivo, it is clearly important for this hypothesis to
know whether the carcinogenic action of dimethylnitrosamine
is mediated via alkylation.
An early objection to the alkylation hypothesis of
nitrosamine
carcinogenesis was based on the powerful
tumor-inducing
capacity
of some cyclic nitrosamines,
including jV-nitrosomorpholine,
TV-nitrosopiperidine,
and
/V-nitrosopyrrolidine, and their supposed inability to give rise
to alkylating intermediates in vivo (11, 30). This question has
been studied experimentally by Lijinsky and Ross (25), who
failed to find evidence of alkylated bases in the liver DNA's
after treatment of rats with tritium-labeled nitrosoazetidine,
nitrosohexamethyleneimine,
nitrosomethylaniline,
and
nitrosomethylcyclohexylamine.
Evidence for an alkylated base
was found in liver RNA of rats given nitrosoazetidine but not
after treatment with nitrosohexamethyleneimine.
On the other
hand, nitrosomethylcyclohexylamine,
although not a liver
carcinogen, did give rise to an alkylated base in liver RNA,
identified as 7-methylguanine by mass spectrometry. It is not
clear whether all possible alkylated bases would have been
detected by the methods used in this work. Nevertheless, some
doubt is thrown on the alkylation hypothesis of nitrosamine
carcinogenesis. It is possible, of course, that dimethylnitro
samine and other methylating carcinogens might be active by
virtue of methylation in the cell while the cyclic compounds
might act by some other mechanism, but this does not seem
600
likely. Additional contrary evidence has recently been
published by Krügeret al. (23), who could find no evidence of
methylation of nucleic acids or proteins by dimethylnitro
samine in the rainbow trout, a species highly susceptible to
liver tumor induction by this carcinogen.
If nitrosamine carcinogenesis was the only evidence for
aberrant
methylation
as a mechanism
of chemical
carcinogenesis, the methylase hypothesis of Borek might be
more readily open to challenge. There is now, however,
evidence that, under some conditions, methylating and
ethylating agents that are not nitroso compounds may show
carcinogenic
potencies approaching
that of the latter.
Repeated s.c. injections of dimethyl sulfate were shown some
years ago to give rise to sarcomas at the injection site (12).
These findings, however, to some extent lacked force because
of the high susceptibility of rat s.c. tissue to carcinogenesis by
a variety of compounds that do not induce tumors elsewhere.
An indication, however, appeared in a brief report by
Alexander and Connell (1) that the simple ethylating agent,
ethylmethanesulfonate,
could give rise to a high incidence of
kidney tumors in mice when given in 3 relatively high separate
doses. The same compound, under similar conditions of
dosage, was shown by Swann and Magee (54) to induce in the
rat an incidence of about 50% renal tumors, the histological
structure of which was identical with those induced by
dimethylnitrosamine.
In
comparable
experiments,
methylmethanesulfonate
induced about a 10% incidence of
tumors of the nervous system (54) and the same compound,
administered to pregnant rats, has induced nervous tumors in
their progeny (P. Kleihues, personal communication). Unless
these alkylalkanesulfonates, which do alkylate in vivo (53, 55),
owe their carcinogenic action to some mechanism other than
alkylation, it appears that alkylation, and in particular
aberrant methylation, can be a potent carcinogenic stimulus.
The relatively weak carcinogenic actions of some alkylating
agents reported in the past may have been explicable by their
chemical reactivity with nonspecific body components and
their failure to react at a sufficient intensity with crucial
cellular receptors. These latter findings support the suggestion
that methylations by aberration of normal metabolic processes
might have the same result as those of externally administered
methylating agents.
The Significance
Carcinogenesis
of
Methylation
of
Nucleic
Acids
in
The observations discussed above suggest that methylation
of some cellular constituent may result in carcinogenesis but
do not in themselves give any indication of the nature of this
component. Most authors agree that the crucial cellular target
must be a macromolecule, either nucleic acid or protein, but
there is no absolutely compelling evidence for this. It is now
known that all the major groups of chemical carcinogens,
including polycyclic hydrocarbons, aromatic amines, nitroso
compounds, and alkylating agents, either react directly or give
rise to chemically reactive intermediates (proximate and
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Role of Nucleic Acid Methy lases in Cancer Induction
ultimate carcinogens) that react with DNA, RNA, and was administered to rats in doses of 500 mg/kg, and the liver
proteins. A large part of the recent research effort in chemical DNA appeared to contain radioactivity that was confined to
7-ethylguanine. If this result can be substantiated it will
carcinogenesis has been devoted to attempts to determine
which one of these macromolecules is the crucial target for support an earlier report by Stekol et al. (50, 51 ) that DNA
malignant transformation (38, 39). As pointed out by Miller was ethylated on the 7-position of guanine by ethionine in
and Miller (39), all carcinogenic agents must alter, directly or vivo (50, 51). Assessment of the significance of these findings
indirectly,
the content
and/or expression of heritable must await further study. From the standpoint of Borek's
information, governing growth and replication of the cell hypothesis, however, the important point arising from this
destined to become neoplastic. Such an alteration could be ethionine work is that the tRNA is much more highly
brought about directly by a change in DNA or indirectly by a ethylated than other RNA species, but it should not be
change in RNA or protein, as suggested by Pilot and overlooked that nuclear proteins were also relatively highly
Heidelberger (44). The original observations on interactions of labeled (15).
carcinogens with cellular macromolecules involved proteins,
An interesting further conclusion from the studies of
but more recently increasing attention has been paid to nucleic Ortwerth and Novelli (42) is that ethylation of tRNA by
acid interactions, with emphasis on possible relationships
ethionine in vivo may not entirely proceed in a manner similar
between carcinogenesis and mutagenesis (5, 24). Perhaps to normal methylation because S-adenosylethionine does not
because of this, DNA has been favored as the most likely seem to be the active intermediate. This conclusion receives
cellular target, partly because the cellular change must be support from recent unpublished work by Dr. A. E. Pegg at
heritable and partly on the basis of correlations between the Courtauld Institute of Biochemistry, London. He has
quantitative measurements of binding of carcinogens with compared the methylation and ethylation of E. coli tRNA in
nucleic acids and proteins. Brookes and Lawley (7), for vitro after administration of methionine and ethionine. All the
example, found no positive correlation between the extent of methylated and ethylated bases found in vivo were formed in
binding of a series of polycyclic hydrocarbons to the RNA or the in vitro system with bacterial tRNA as an acceptor of alkyl
proteins of mouse skin with their carcinogenic potency in this groups, although the relative proportions were not exactly
tissue, but the binding to DNA did show a strong positive similar. In the in vitro experiments, 7-methylguanine and
correlation. In similar experiments, Colburn and Boutwell (9) 7-ethylguanine both represented 6% of the total incorporation
compared the binding of /3-propiolactone and some related into guanine, but in vivo 7-methylguanine was 12% of the total
alkylating agents to DNA, RNA, and protein of mouse skin incorporation whereas 7-ethylguanine represented about 24%
with their capacity to initiate and promote the induction of of the total. One explanation of this result would be that
tumors. Again there was a correlation between capacity' to another means of producing 7-ethylguanine in tRNA in vivo,
initiate tumors and the extent of binding to DNA but no such not involving S-adenosylethionine as an intermediate, can take
correlation with binding to RNA or protein. The recent work place.
of Cleaver (8) and Reed et al.(45) on xeroderma pigmentosum
The significance of alkylation of nucleic acids, and of
gives support of another kind for DNA as the critical target for reaction on the 7-position of guanine in particluar, is far from
carcinogenesis.
clear. Under some circumstances 7-alkylguanine apparently
On the other hand, reasons for preferring tRNA rather than may be transcribed as guanine. Ludlum (26) has shown that
DNA as the primary target for chemical carcinogens have been 7-alkylguanine in synthetic polynucleotide templates has no
put forward in the papers of Weinstein (58) and Weinstein et apparent effect on the incorporation of adenylic acid by RNA
al. (59). These include the lack of evidence for a change in DNA polymerase but that the presence of 3-methylcytidylic acid
analogous to mutation and the apparent normality of tumor leads to mispairing of uridylic acid (27, 28). Similar mispairing
DNA together with the evidence that tumor cells can, in some has recently been observed with polycytidylic acid templates
circumstances, revert to normal. If carcinogenesis involves treated with the potent carcinogens A^-nitrosomethyl- and
(D. B. Ludlum and P. N. Magee,
changes in the pattern of gene expression rather than in the A'-nitrosoethylurea
genetic material itself, loss of a tRNA or a change in its unpublished results).
specificity might result in cancer. The hepatic carcinogen
Although the circumstantial evidence discussed above tends
ethionine is of interest in this context since it has been clearly to favor the importance of DNA alteration in carcinogenesis,
shown to ethylate RNA (2, 13, 15, 41, 42, 47, 51), while it there is no doubt that RNA must, in some situations at least,
has been claimed that DNA is ethylated only to a very small play an essential part since the Rous sarcoma virus and some
extent or not at all (16, 42). This difference between the other oncogenic viruses contain RNA but no DNA. In this
behavior of ethionine and other alkylating carcinogens could context, the recent suggestion by Huebner and Todaro (21)
clearly be of significance in determining
the relative that vertically transmitted oncogenes of RNA tumor viruses
importance
of cellular macromolecules
as targets for may be determinants of cancer is particularly interesting.
carcinogens. In the experiments of Ortwerth and Novelli (42), Freeman et al. (17) have shown that morphological
in which no significant labeling of DNA was observed, the transformation of rat embryo cells in culture can be induced
radioactive ethionine was given in tracer doses but recent by the combined action of diethylnitrosamine
and murine
experiments by Swann, Pegg, Hawks, Farber, and Magee (56) leukemia viruses but not by either agent acting alone. Since
have suggested that DNA may become labeled when the diethylnitrosamine is known to ethylate RNA in vivo (33, 55),
ethionine is given in larger doses. Tritium-labeled ethionine
it may be that alkylation can activate a latent RNA tumor
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601
P. N. Magee
virus, if this is so, a similar activation might be mediated by an
excessive or aberrant action of a cellular RNA methylase.
Conclusions
From the above consideration
of carcinogenesis by
alkylating carcinogens and by ethionine, is is concluded that
the possibility of the induction of cancer by the aberrant
action of nucleic acid methylases must be recognized. Apart
from the greater degree of methylation of tRNA than of other
types of RNA, however, there seems to be no strong reason, in
the present rate of knowledge, for implicating aberrant tRNA
in the initiation of cancer. The possibility of aberrant action
by DNA methylases and the possibility that latent oncogenic
viral RNA might be activated by methylase action should also
be considered.
REFERENCES
1. Alexander, P., and Connell, D. I. The Failure of the Potent
Mutagenic Chemical, Ethyl Methane Sulphonate to Shorten the
Life-span of Mice. Cellular Basis and Late Somatic Effects of
Ionizing Radiation. Symposium, London, pp. 259-265, 1962.
2. Axel, R., Weinstein, I. B., and Farber, E. Patterns of Transfer RNA
in Normal Rat Liver and during Hepatic Carcinogenesis. Proc. Nati.
Acad. Sei. U. S., 58: 1255-1260, 1967.
3. Bergquist, P. L., and Mathews, R. E. F. Occurrence and
Distribution of Methylated Purines in the Ribonucleic Acid of
Subcellular Fractions. Biochem. ¡.,85: 305-313, 1962.
4. Borek, E. The Methylation of Transfer RNA: Mechanism and
Function. Cold Spring Harbor Symp. Quant. Biol., 18: 139-148,
1963.
5. Brookes, P. Quantitative Aspects of the Reaction of Some
Carcinogens with Nucleic Acids and the Possible Significance of
Such Reactions in the Process of Carcinogenesis. Cancer Res., 26:
1994-2003, 1966.
6. Brookes, P., and Lawley, P. D. Alkylating Agents. Brit. Med. Bull.,
20. 91-95, 1964.
7. Brookes, P., and Lawley, P. D. Evidence for the Binding of
Polynuclear Aromatic Hydrocarbons to the Nucleic Acids of Mouse
Skin: Relation between Carcinogenic Power of Hydrocarbons and
Their Binding to Deoxyribonucleic Acid. Nature, 202: 781-784,
1964.
8. Cleaver, J. E. Defective Repair Replication of DNA in Xeroderma
Pigmentosum. Nature, 218: 652-656, 1968.
9. Colburn, N. H., and Boutwell, R. K. The Binding of
0-Propiolactone and Some Related Alkylating Agents to DNA,
RNA and Protein of Mouse Skin; Relation between
Tumor-initiating Power of Alkylating Agents and Their Binding to
DNA. Cancer Res., 28: 653-660, 1968.
10. Craddock, V. M. Methylation of Transfer RNA and of Ribosomal
RNA in Rat Liver in the Intact Animal and the Effect of
Carcinogens. Biochim. Biophys. Acta, 195: 351-369, 1969.
11. Druckrey, H., Preussmann, R., Ivankovic, S., and Schmähl,D.
Organotrope
Carcinogene Wirkungen bei 65 verschiedenen
yV-Nitroso-Verbindungen an BD-Ratten. Z. Krebsforsch., 69:
103-201, 1967.
12. Druckrey, H., Preussmann, R., Nashed, N., and Ivankovic, S.
Carcinogene
Alkylierende
Substanzen.
I. Dimethylsulfat,
Carcinogene Wirkung an Ratten und warscheinliche Ursache von
Berufskrebs. Z. Krebsforsch., 68: 103-107, 1966.
602
13. Farber, E. Ethionine Carcinogenesis. Advan. Cancer Res., 7:
383-474, 1963.
14. Farber, E., and Magee, P. N. The Probable Alkylation of Liver
Ribonucleic Acid by the Hepatic Carcinogens Dimethylnitrosamine
and Ethionine. Biochem. J., 76: 58P, 1960.
15. Farber, E., McConomy, J., Franzen, B., Marroquin, F., Stewart,
G. A., and Magee, P. N. Interaction between Ethionine and Rat
Liver Ribonucleic Acid and Protein in Vivo. Cancer Res., 27:
1761-1772, 1967.
16. Färber,E., McConomy, J., and Frumanski, B. Relative Degrees of
Labelling of Liver DNA and RNA with Ethionine. Proc. Am.
Assoc. Cancer Res., 8: 16. 1967.
17. Freeman, A. E., Price, P. J., Igel, H. J., Young, J. C., Maryak, J. M.,
and Huebner, R. J. Morphological Transformation of Rat Embryo
Cells Induced by Diethylnitrosamine and Murine Leukaemia
Viruses. J. Nati. Cancer Inst., 44: 65-78, 1970.
18. Gantt, R., and Evans, V. J. Comparison of Soluble RNA Methylase
Capacity in Paired Neoplastic and Nonneoplastic Cell Lines in
Vitro. Cancer Res., 29: 536-541, 1969.
19. Hacker, B., and Mandel, L. R. Altered Transfer RNA Methylase
Patterns Induced by an Avian Oncogenic Virus. Biochim. Biophys.
Acta, 790. 38-51, 1969.
20. Hancock,
R.
L.
Utilization
of
L-Methionine
and
5-adenosyl-L-methionine for Methylation of Soluble RNA by
Mouse Liver and Hepatoma Extracts. Cancer Res., 27: 646-653,
1967.
21. Huebner, R. J., and Todaro, G. J. Oncogenes of RNA Tumor
Viruses as Determinants of Cancer. Proc. Nati. Acad. Sei. U. S., 64:
1087-1094, 1969.
22. Kaye, A. M., and Leboy, P. S. Methylation of Ribonucleic Acid by
Mouse Organs and Tumors, Ionic Stimulation in Vitro. Biochim.
Biophys. Acta, 757: 289-301, 1968.
23. Krüger,F. W., Walker, G., and Wiessler, M. Carcinogenic Action of
Dimethylnitrosamine in Trout Not Related to Methylation of
Nucleic Acids and Protein in Vivo. Experientia, 26: 520-522,
1970.
24. Lawley, P. D. Effects of Some Chemical Mutagens and Carcinogens
on Nucleic Acids. Progr. Nucleic Acid Res. Mol. Biol.,5: 89-131,
1966.
25. Lijinsky, W., and Ross, A. E. Alkylation of Rat Liver Nucleic Acids
not Related to Carcinogenesis by A'-Nitrosamines. J. Nati. Cancer
Inst., 42: 1095-1100, 1969.
26. Ludlum, D. B. The Properties of 7-Methylguanine-containing
Templates for Ribonucleic Acid Polymerase. J. Biol. Chem., 245:
477-482, 1970.
27. Ludlum, D. B. Alkylated Polycytidylic Acid Templates for RNA
Polymerase. Biochim. Biophys. Acta, 213: 142-148, 1970.
28. Ludlum, D. B., and Wilhelm, R. C. Ribonucleic Acid Polymerase
Reactions with Methylated Polycytidylic Acid Templates. J. Biol.
Chem., 243: 2750-2753, 1968.
29. Magee, P. N., and Barnes, J. M. The Production of Malignant
Primary Hepatic Tumours in the Rat by Feeding Dimethylnitro
samine. Brit. J. Cancer, 10: 114-122, 1956.
30. Magee, P. N., and Barnes, J. M. Carcinogenic Nitroso Compounds.
Advan. Cancer Res., 10: 163-246, 1967.
31. Magee, P. N., and Farber, E. Toxic Liver Injury and Carcinogenesis.
Methylation of Rat Liver Nucleic Acids by Dimethylnitrosamine in
Vivo. Biochem. J., 83: 114-124, 1962.
32. Magee, P. N., and Hultin, T. Toxic Liver Injury and Carcinogenesis.
Methylation
of
Proteins
of
Rat
Liver
Slices
by
Dimethylnitrosamine m Vitro. Biochem. J., 83: 106-114,1962.
33. Magee, P. N., and Lee, K. Y. Cellular Injury and Carcinogenesis.
Alkylation of Ribonucleic Acid of Rat Liver by Diethylnitrosamine
and n-butylmethylnitrosamine in Vivo. Biochem. J., 91: 35-42,
1964.
CANCER RESEARCH VOL.31
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1971 American Association for Cancer Research.
Role of Nucleic Acid Methy lases in Cancer Induction
34. Mandel, L. R., Hacker, B., and Maag, T. A. Increased Transfer RNA
Methylase Activity in a Liver Tumor from Chicks with Marek's
Disease. Cancer Res., 29: 2229-2231, 1969.
35. Mandel, L. R., Srinivasan, P. R., and Borek, E. Origin of Urinary
Methylated Purines. Nature, 209. 586-588, 1966.
36. McFarlane, E. S., and Shaw, G. H. Observed Increase in Methylated
Purines Excreted by Hamsters Bearing Adenovirus-12 Induced
Tumours. Can. J. Microbiol., 14: 185-187, 1968.
37. McFarlane, E. S., and Shaw, G. H. Nucleic Acid Methylase Activity
in Hamsters Bearing Tumours Induced by Adenovirus-12. Can. J.
Microbiol., 14: 499-502, 1968.
38. Miller, E. C., and Miller, J. A. Mechanisms of Chemical
Carcinogenesis: Nature of Proximate Carcinogens and Interactions
with Macromolecules. Pharmacol. Rev., 18: 805-838, 1966.
39. Miller, J. A., and Miller, E. C. A Survey of Molecular Aspects of
Chemical Carcinogenesis. Lab. Invest., 75; 217-239, 1966.
40. Mittelman, A., Hall, R. H., Yohn, D. S., and Grace, J. T. The in
Vitro Soluble RNA Methylase Activity of SV40-induced Hamster
Tumors. Cancer Res., 27: 1409-1414, 1967.
41. Ortwerth, B. J., DelMonte, U., Rosen, L., and Novelli, G. D.
Changes in Rat Liver tRNA During Ethionine Feeding. Federation
Proc., 27: 803, 1968.
42. Ortwerth, B. J., and Novelli, G. D. Studies on the Incorporation of
L-Ethionine-ethyl-l-'4C
into the Transfer RNA of Rat Liver.
Cancer Res., 29: 380-390, 1969.
43. Park, R. W., Holland, J. F., and Jenkins, A. Urinary Purines in
Leukaemia. Cancer Res., 22: 469-477, 1962.
44. Pilot, H. C., and Heidelberger, C. Metabolic Regulatory Circuits.
Cancer Res., 23: 1694-1700, 1963.
45. Reed, W. B., Landing, B., Sugarman, G., Cleaver, J. E., and Melnyk,
J. Xeroderma Pigmentosa: Clinical and Laboratory Investigation of
Its Basic Defect. J. Am. Med. Assoc., 207: 2073-2079, 1969.
46. Rodeh, R., Feldman, M., and Littauer, U. Z. Properties of Soluble
Ribonucleic Acid Methylases from Rat Liver. Biochemistry, 6:
451-460, 1967.
47. Rosen, L. Ethylation in Vivo of Purines in Rat Liver tRNA by
L-ethionine. Biochem. Biophys. Res. Commun., 33: 546-550,
1968.
48. Srinivasan, P. R., and Borek, E. The Species Variation of RNA
Methylase. Proc. Nati. Acad. Sei. U. S., 49: 529-533, 1963.
49. Srinivasan, P. R., and Borek, E. Enzymatic Alteration of Nucleic
Acid Structure. Science, 145: 548-553, 1964.
50. Stekol, J. A. Formation and Metabolism of 5-Adenosyl Derivatives
of S-Alkyl Homocysteines in the Rat and Mouse. In: S. K. Shapiro
and F. Schlenk (eds.), Transmethylation and Biosynthesis of
Methionine, pp. 235-252. Chicago: University of Chicago Press,
1965.
51. Stekol, J. A., Mody, U., and Perry, J. The Incorporation of the
Carbon of the Ethyl Group of Ethionine into Liver Nucleic Acids
and the Effect of Ethionine Feeding on the Content of Nucleic
Acids in Rat Liver. J. Biol. Chem., 235: PC59-PC60, 1960.
52. Stewart, M. J., and Corrance, M. H. The Methylation of Transfer
RNA in Vitro by Extracts of Normal and Malignant Tissue. Cancer
Res., 29: 1642-1646, 1969.
53. Swann, P. F., and Magee, P. N. Nitrosamine-induced
Carcinogenesis. The Alkylation of Nucleic Acids of the Rat by
jV-Methyl-yV-nitrosourea Dimethylnitrosamine, Dimethylsulphate
and Methyl Methanesulphonate. Biochem. J., 110: 39-47, 1968.
54. Swann, P. F., and Magee, P. N. Induction of Rat Kidney Tumours
by Ethyl Methanesulphonate and Nervous Tissue Tumours by
Methyl Methanesulphonate and Ethyl Methanesulphonate. Nature,
22.?: 947-948, 1969.
55. Swann, P. F., and Magee, P. N. Comparison between the Ethylation
of Nucleic Acids by Diethylnitrosamine, Ethyl Methanesulphonate,
and ¿V-ethyl-jV-nitrosoureaand the Carcinogenic Activity of Each
56.
57.
58.
59.
Compound, p. 3. Abstracts, Tenth International Cancer Congress,
Houston, 1970, in press.
Swann, P. F., Pegg, A. E., Hawks, A., Farber, E., and Magee, P. N.
Evidence for Ethylation of Rat Liver DNA Following Ethionine
Administration to Rats. Biochem. J., in press.
Tsutsui, E., Srinivasan, P. R., and Borek, E. tRNA Methylases in
Tumors of Animal and Human Origin. Proc. Nati. Acad. Sci. U.S.,
56: 1003-1009, 1966.
Weinstein, I. B. A Possible Role of Transfer RNA in the Mechanism
of Carcinogenesis. Cancer Res., 28: 1871-1874, 1968.
Weinstein, I. B., Grunberger, D., Fujimura, S., and Fink, L. M.
Chemical Carcinogens and Cancer. Cancer Res., 31: 651-655,
1971.
Discussion
Dr. Gefter: Did you mention what the primary effect is,
using chemical ethylation in DNA?
Dr. Magee: What do you mean by the primary effect?
Dr. Gefter: What is the major compound you find in DNA
after treatment?
Dr. Magee: 7-Ethylguanine
Dr. Gefter: With er/zy/methanesulfonate.
Dr. Magee: Yes.
Dr. Gefter: Would you like to comment on its possible
mutagenic effect. In bacteria, it is quite clear that it is a
mutagen, and is clearly having an effect on DNA and
producing a subsequent change.
Dr. Magee: Yes.
Dr. Gefter: So I don't quite understand, then, the idea that
it doesn't have any effect on RNA synthesis or that it is copied
like guanine by RNA polymerase. Clearly, it must have some
effect on some nucleic acid.
Dr. Magee: That is true, but you see, there is ethylation at
other sites as well. And it may be that these are important
ones. But because they are quantitatively smaller, it doesn't
mean to say they are not the biologically significant ones. That
is what I am suggesting.
Dr. Gefter: So with chemical ethylation, there are other
compounds produced in DNA other than 7-ethylguanine.
Dr. Magee: Oh, yes, definitely. In fact, the slide that I
showed of the distribution of bases produced in the intact
animal by dimethylnitrosamine is very closely copied by the
action of methylmethanesulfonate
in vitro. This was shown by
Brookes and Lawley. The proportions of the different
methylated bases are very similar, indeed.
Dr. Mandel: Dr. Magee, could you comment on the
appearance of the methylated bases in the urine following the
administration of carcinogens? Do you find compounds other
than 7-methylguanine, for example? Do you find them in
DNA?
Dr. Magee: That is something we should do. We haven't
looked for them. We have looked for 7-methylguanine in the
urine, and we find, when we give dimethylnitrosamine in toxic
doses, that the level of 7-methylguanine in the urine goes up
by a factor of about 2, 2.5, that's all. But when we give it in
carcinogenic doses for the liver, that is to say continued
feeding over long periods, 50 mg/kg, then you can't detect it
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603
P. N. Magee
because there is a scatter, an increase in the urine of
7-methylguanine.
But equally, I know that under some circumstances the
7-methylguanine does go up with carcinogens. And I think this
might indicate the action of the methylases because in the
intact animal, 7-methylguanine
put in by a chemical
carcinogen like this is not very stable. It comes out very
quickly. A large part of it comes out from both DNA and
RNA in the first 24 hr after the application of the carcinogen.
Dr. Carbon: Unless I have made a miscalculation, the
experiment in which you measured the amount of label
604
incorporated from ethionine into DNA corresponded to 1 in
1,000,000 bases. This seems negligible.
Dr. Magee: 1 would think so, yes. But how can you tell? I
don't know.
Dr. Carbon: Well, DNA as we all prepare it, is not really
100% pure. This level of 1 base in 1,000,000 could easily be in
RNA.
Dr. Borek: Is ethionine a mutagen? If it isn't, this would be
the first instance of a carcinogen which is not a mutagen. In
view of its slight interaction with DNA, I would be surprised if
it is a mutagen.
CANCER RESEARCH VOL.31
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The Possible Role of Nucleic Acid Methylases in the Induction
of Cancer
P. N. Magee
Cancer Res 1971;31:599-604.
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