Table Of ContentJOURNAL OF CHROMATOGRAPHY LIBRARY- volume 45C
chromatography and
modification of
nucleosides
C:
pat? modified nucleosides in cancer
and normal metabolism
methods and applications
edited by
Charles W. Gehrke and Kenneth C. T. Kuo
Department of Biochemistry, University of Missouri-Columbia, and Cancer
Research Center, P. 0. Box 1268, Columbia, MO 65205- 1268, US.A .
ELSEVIER
Amsterdam - Oxford - New York - Tokyo 1990
ELSEVIER SCIENCE PUBLISHERS B.V.
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Distributors for the United States and Canada:
ELSEVIER SCIENCE PUBLISHING COMPANY INC.
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Library of Congress Catalogingin-PublicationD ata
(Revised for added vol., part C)
Chromatography and modification of nucleosides.
(Journal of chromatography library ; v. 45A- )
Includes bibliographical references.
Contents: pt. A. Analytical methods for major and
modified nucleosides, HPLC. GC, MS, NMR, UV, and FT-IR
-- pt. B. Biological roles and function of modification
-- pt. C. Modified nucleosides in cancer and normal
metabolism.
1. Nucleosides--Analysis. 2. Nucleosides--Metabolism.
3. Nucleosides--Derivatives--Synthesis. 4. Chromatogra-
phy. I. Gehrke, Charles W. 11. Kuo. Kenneth C. T.,
.
1936- 111. Series.
q~m.~aac471 989 2547.7i9046 89-25973
ISBN 0-444-88598-6
0 Elsevier Science Publishers B.V., 1990
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VII
PREFACE
This volume addresses the role of modified nucleosides as
biomarkers in the early detection of cancer and the clinical
management of the cancer patient. The introduction by Waalkes and
Gehrke describes in some detail the initial concepts, beginning
methods used, and applications of the methodology. The modified
nucleosides have been thought to be derived predominantly from
transfer ribonucleic acid (tRNA) and have been known to be excreted
in abnormal amounts in the urine of the patients with various
malignant diseases (ref. 1-4) and tumor-bearing animals (ref. 5).
Since these modified nucleosides have no salvage pathway, they are
excreted in the urine as intact molecules (ref. 6).
In 1966, Borek @t al. reported that tRNA methyltransferase
activity was higher in cancer tissue than in corresponding normal
tissue (ref. 7) and that the concentration of methylated nucleosides
in tRNA of cancer tissue was higher than that in corresponding
normal tissue (ref. 8). Thus, urinary elevation of modified
nucleosides has been suggested to be caused by the increased tRNA
turnover in tumor tissue rather than by cell death (ref. 9).
The tRNA is a highly complex biomacromolecule and as many as
20% of its approximately 85 bases may be modified. These
modifications may be as simple as the introduction of a methyl
group or extremely complex, require the action of many enzymes and
the modifications occur after the synthesis of the primary sequence
of the tRNA (ref. 10). In part A of this series, many contributing
scientists have reported that the enzymes involved have been shown
to be species specific, base specific, site specific and sequence
specific. Some of the modifications require the action of only one
enzyme, whereas more complex modifications such as the
introduction of a 'Q-base' require the sequential intervention of
several enzymes.
It has also been shown that the mammalian cell lacks the
kinases to convert the modified nucleosides that are released on
metabolism into their corresponding nucleoside triphosphates and
VIII
therefore their incorporation into the macromolecule. Consequently,
the modified nucleosides are released into the blood and finally
excreted in the urine.
With this background of investigations and the central role of
nucleic acids, involving the important biomacromolecules of tRNA,
rRNA, mRNA and DNA in protein synthesis, metabolism, and
biological regulation have resulted in intense and continued research
on the structure, sequence, and conformation of these
macromolecules by scientists representing a wide spectrum of
disciplines, from biochemistry to molecular and subcellular biology
to clinical research. With new findings many fundamental
investigations are continuing on the chemical structure of
nucleoside modifications in nucleic acids; the establishment of
additional primary sequences and three dimensional conformation of
the polymers; and research on the dynamic properties of nucleic
acids under physiological conditions, all of which are essential for
increasing our understanding of their complex biological and
functional relationships of the tRNAs and other nucleic acids. To
this end, the uses of chemical, chromatographic, biochemical,
genetic, and molecular biological tools and systems, ranging from E.
coli, yeast, and cyanobacteria to drosophila, provide the model
investigative systems for the researcher. These subjects are
discussed in depth by many scientists in their disciplines from
molecular biology, microbiology, biochemistry, to chromatographic
separation sciences. Please refer to parts A and B of this series on
"Chromatography and Modification of Nucleosides" for the latest
findings from research on the role and function of nucleic acids.
This volume presents a comprehensive treatment of
ribonucleoside analysis by reversed-phase high performance liquid
chromatography and other chromatographic techniques and
application of the methods in many clinical studies. In the early
1970s, the National Cancer Institute (NCI) made the decision to
study biochemical materials produced by the body as potential
"biological markers" of cancer. Such markers would either indicate
the presence of cancer or they would reflect changes in tumor mass
and would be useful in following cancer therapy. These markers
IX
might be found in the body fluids, for example, in blood and urine of
patients with cancer.
Initially, part of the NCI program was devoted to the study of
minor components of nucleic acids, known as modified nucleosides.
These modified nucleosides appear in body fluids mainly as the
result of the destruction of transfer ribonucleic acid (RNA). Since
there is no mechanism in the body for re-use of these modified
nucleosides, the presence of these compounds in body fluids is a
measure of the destruction rate of transfer RNA. Transfer RNA
performs critical functions in protein synthesis, and has important
roles in regulation, transcription, inhibiting enzyme activity, and
protein degradation. Therefore, measurement of the amount of
modified nucleosides in body fluids could indicate changes in the
body as the result of cancer, and changes in the amount of these
compounds might be a measure of the effectiveness of the therapy
given the patient.
First, however, it was most important to develop new
chemical methods for measuring the modified nucleosides as the
components are present at only very low levels in body fluids. The
conventional techniques of the 1970's of ion-exchange and paper
chromatography lacked the speed, sensitivity, resolution, and
quantitation for measuring a number of nucleosides simultaneously.
These obvious problems in analysis, as well as the cumbersome
nature of the methods, made it necessary to develop new
chromatographic-analytical techniques. It was also shown by
Gehrke and Kuo that the nucleosidekreatinine ratios were
remarkably similar in random or "spot" samples as compared to
obtaining a total 24 hour urine collection, and therefore the
excretion of the nucleosides relative to creatinine is constant, not
episodic. Thus, "spot samples" provided data as valid as data from
24 hour collections. Development of methods for the analysis of
modified nucleosides has been a major thrust of Dr. Charles W.
Gehrke and Kenneth Kuo's research for a number of years. Earlier,
gas-liquid chromatography was utilized to search for and measure
cancer markers in body fluids, then their research group pioneered
high performance liquid chromatographic methods. The development
X
of these methods of measurement has allowed studies with Dr. T.P.
Waalkes at the Oncology Center at Johns Hopkins University in
Baltimore. Also, these studies revealed that the use of multiple
markers may be useful in staging patients, and in detecting minimal,
residual, and recurrent cancer.
In the late 70s, the development of high performance liquid
chromatographic instrumentation, reliable pumps, new detectors and
efficient reversed-phase columns offered a new analytical approach,
and the development of a highly specific affinity chromatography
sample cleanup method for isolation of ribonucleosides, based on the
work of Dr. Uziel at Oak Ridge National Labortory (ref. 11) promised
to provide a much improved method for studying ribonucleosides in
complex matrices such as physiological fluids (ref. 12, 13, 14). At
the same time, parallel protocols were being investigated for
improved nucleic acid isolation, quantitative enzymatic hydrolysis
of RNAs, high resolution preparative RPLC, and affinity
chromatography to obtain pure known and unidentified nucleosides
for UV absorption spectroscopy and interfaced mass spectrometry
investigations for characterization and identification of
nucleosides. In addition, nucleoside structure-spectrum
relationships, composition, and conformation using the techniques of
HPLC-UV, FT-IR, NMR, and MS were developed as well as structure
UV-RPLC retention relationships (part A).
Nucleotides, nucleosides, and bases are of major importance in
biological systems e.g. in the formation and function of nucleic
acids. Disorders in purine and pyrimidine metabolism are believed
to be involved in diseases such as cancer. Borek's expectation "of
finding some unique metabolic products or components of malignant
cells circulating in body fluids which can be measured" (ref. 15) was
met during the last decade as specific excretion patterns of
modified nucleosides in urine were found to be related to distinct
metabolic disorders.
The Introduction on "Nucleoside Markers for Cancer: and the
Chapters that follow in part C, present the advanced experimental
approaches and technologies for measuring modified nucleosides in
biological fluids. Also, the progress made since 1980 and future
XI
prospects of modified nucleosides as biologic markers of cancer are
discussed. New findings are presented and conclusions drawn by the
leading investigators from many countries on modified nucleosides
as "biochemical signals" in cancer and as "selective markers" in
metabolism for whole body turnover of tRNA, rRNA, and mRNA.
Columbia, Missouri Charles W. Gehrke
1989 Kenneth C.T. Kuo
References:
1. Waalkes, T.P., Gehrke, C.W., Zumwalt, R.W., d.The urinary
excretion of nucleosides of ribonucleic acid by patients with
advanced cancer. Cancer 36 (1 975) 390-398.
2. Gehrke C.W., Kuo K.C., Waalkes T.P., Borek E. tRNA breakdown
products as markers of cancer. Cancer 39 (1979) 1150-1153.
3. Speer J., Gehrke, C.W., Kuo, K.C., Waalkes, T.P., Borek, E., tRNA
breakdown products as markers for cancer. Cancer 44 (1979)
21 20-21 23.
4. Fischbein, A., Sharma, O.K., Selikoff, I.J., Borek, E., Urinary
excretion of modified nucleosides in patients with malignant
mesothelioma. Cancer Res 43 (1 983) 2971 -2974.
5. M. Shimizu, S. Fujimura, Studies on the abnormal excretion of
pyrimidine nucleosides in the urine of Yoshida ascites
sarcoma-bearing rats: Increased excretion of deoxycytidine,
pseudouridine and cytidine, Biochim Biophys Acta 51 7 (1 978)
277-286.
6. Weissman, SM., Eisen, AZ, Lenrio, M., Pseudouridine
metabolism-Ill: studies with isotopically labeled pseudo-
uridine. J. Lab. Clin. Med. 60 (1962) 40-47.
7. Srinivasan, P.R., Borek, E., tRNA methylases in tumors of
animal and human origin. Proc USA 56 (1966)1003-1009.
8. Borek, E., Introduction to symposium. tRNA and tRNA
modification in differentiation and neoplasia. Cancer Res
31 (1971) 596-597.
9. Borek, E., Baliga, B.S., Gehrke, C.W., U. High turnover rate of
transfer RNA in tumor tissue. Cancer Res 37 (1977) 3362-
3366.
XI1
10. Vreken, P., Tavenier, P., Urinary excretion of six modified
nucleosides by patients with breast carcinoma. Am. Clin.
Biochem. 24 (1 987) 598-603.
11. Uziel, M. Smith, L.H., and Taylor, S.A., Modified nucleosides in
urine: Selective removal and analysis, Clin. Chem., 22 (1976)
1451-1455.
12. Gehrke, C.W., Zumwalt, R.W., McCune, R.A., and Kuo, K.C.,
Quantitative high-performance liquid chromatography analysis
of modified nucleosides in physiological fluids, tRNA, and DNA,
Recent results in Cancer Research 84 (1983) 344-359.
13. Kuo, K.C., Esposito, F., McEntire, J.E., Gehrke, C.W., Nucleoside
profiles by HPLC-UV in serum and urine of Controls and cancer
patients, in: F. Cimino and F. Salvatore (Eds.) Human Tumor
Markers, Walter de Gruyter Berlin and New York, (1987) 519-
544.
14. Gehrke, C.W., and Kuo, K.C., Ribonucleoside analysis by
reversed-phase high performance liquid chromatography, J.
Chromatogr. 471 (1989) 3-36.
15. E., Borek, the Morass of tumor markers, Trends Biochem. Sci.,
1985; 10: 182-184.
XI11
ERNEST BOREK
1911-1986
A Pioneer in Methylation of Nucleic Acids
XIV
Dedication
ERNEST BOREK
19 11-1986
Ernest Borek, pioneer i n nucleic acids methylation, was born
i n Nyircsaszari, Hungary, and moved with his family to New York
City at the age of 14. He graduated at City College of New York
City and took his Ph.D. i n biochemistry at Columbia University.
Ernest's interests covered many areas of biochemistry. He
was on the faculty of the Department of Chemistry of City
University of New York from 1934-1969 and was a professor i n the
Department of Biochemistry of Columbia University from 1959-1969,
where his research efforts included such diverse areas as studies
on the biosynthesis of cholesterol, the demonstration of a bio-
1o gi cal ly active ir radi atio n product as the basis for indir ect
induction or the "Borek-Ryan" Effect, the demonstration of
"re1a xed control over RNA synthesis i n bacteria, the discovery
"
of the enzymes which methylate nucleic acids at the polymer
level, the discovery of aberrant tRNA methylation i n tumor
tissues, the demonstration of unique tRNA species i n tumor
tissues, and the development of the concept of urinary analysis
of modified nucleosides as biological markers for cancer.
Borek felt that a turning point i n his career was the year
1951. By the late 1940s, he had become bored with biochemists'
preoccupation with tracing, atom by atom, the origins of various
intermediary metabolites. He continued some research perfunc-
torily, but his main interest was writing a book envisioned as
'Biochemistry for the Millions' (It turned out to be, i n those
pre-Sputnik days, for the dozens.). He wrote: "So far the
biochemists' efforts have been directed mainly toward the
completion of the atlas of molecular anatomy. We must begin to
patiently assemble still another science, molecular physiology-
-the study of the relation between molecular structure and
cellular function" (Man . &t Chemical Machine, 1952). He was
