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Vol. 59, Issue 5, 1181-1186, May 2001
Division of Clinical Virology, Karolinska Institute, Huddinge University Hospital, Stockholm, Sweden
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Abstract |
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 Top
 Abstract
 Introduction
Experimental Procedures
Results
Discussion
References
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Uridine-cytidine kinases (UCK) have important roles for the
phosphorylation of nucleoside analogs that are being investigated for
possible use in chemotherapy of cancer. We have cloned the cDNA of two
human UCKs. The 
30-kDa proteins, named UCK1 and UCK2, were expressed
in Escherichia coli and shown to catalyze the
phosphorylation of Urd and Cyd. The enzymes did not phosphorylate
deoxyribonucleosides or purine ribonucleosides. UCK1 mRNA was detected
as two isoforms of 1.8 and 2.7 kb. The 2.7-kb band was
ubiquitously expressed in the investigated tissues. The band of 1.8
kb was present in skeletal muscle, heart, liver, and kidney. The two
isoforms of UCK2 mRNA of 1.2 and 2.0 kb were only detected in placenta
among the investigated tissues. The genes encoding UCK1 and UCK2 were mapped to chromosome 9q34.2-9q34.3 and 1q22-1q23.2, respectively. We
tested 28 cytidine and uridine nucleoside analogs as possible substrates of the enzymes. The enzymes phosphorylated several of the
analogs, such as 6-azauridine, 5-fluorouridine, 4-thiouridine, 5-bromouridine, N4-acetylcytidine,
N4-benzoylcytidine, 5-fluorocytidine,
2-thiocytidine, 5-methylcytidine, and
N4-anisoylcytidine. The cloning and
recombinant expression of the two human UCKs will be important for
development of novel pyrimidine ribonucleoside analogs and the
characterization of their pharmacological activation.
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Introduction |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Uridine-cytidine
kinase (UCK) (EC 2.7.1.48) is a pyrimidine ribonucleoside kinase that
catalyzes the phosphorylation of uridine and cytidine to UMP and CMP.
The enzyme also catalyzes the phosphorylation of several cytotoxic
ribonucleoside analogs that have been investigated for possible
use as chemotherapeutic agents for treatment of cancer. The nucleoside
analogs are dependent on phosphorylation for their pharmacological
activity. Once phosphorylated, the compounds interfere with vital
cellular processes such as DNA or RNA synthesis or inhibit enzymes
involved in nucleotide synthesis (Cihak and Rada, 1976
). The
ribonucleoside analogs phosphorylated by UCK include 5-fluorouridine,
5-azacytidine, and cyclopentenyl cytosine/-uracil as well as the
recently developed
1-(3-C-ethynyl-
-D-ribo-pentofuranosyl)-cytosine/-uracil (Sköld, 1960; Lee et al., 1974; Vesely, 1985; Kang et al., 1989; Hattori et al., 1996; Tabata et al., 1997; Takatori et al., 1999; Verschuur et al., 2000). In addition to the ribonucleoside analogs, UCK
may also be important for the pharmacological activation of uridine and
cytidine base analogs, such as the clinically used analog
5-fluorouracil. This compound may be converted to 5-fluorouridine by
uridine phosphorylase that subsequently will be dependent on UCK
catalyzed phosphorylation (Reichard and Sköld, 1957; Reichard and
Sköld, 1958).
Most of the nucleoside analogs are dependent on phosphorylation to
their triphosphate form for pharmacological activity. After the first
phosphorylation catalyzed by UCK, pyrimidine ribonucleoside analogs are
further phosphorylated by UMP-CMP kinase (Van Rompay et al., 1999) and
nucleoside diphosphate kinases (Parks and Agarwal, 1973). However, the
first phosphorylation step catalyzed by UCK is considered rate-limiting
and the level of UCK activity may be correlated with the cellular
sensitivity to the nucleoside analogs (Reichard and Sköld, 1958;
Anderson and Brockman, 1964). Loss of UCK activity is also seen in
cells resistant to the nucleoside analogs (Reichard et al., 1959;
Vesely et al., 1971; Greenberg et al., 1977). Interestingly, several
studies suggest that UCK activity may increase in tumor cells compared
with normal tissues (Reichard et al., 1959; Herzfeld and Raper, 1979;
Shen et al., 1998).
Mammalian UCKs have been purified from several different tissues
(Reichard and Sköld, 1957; Krystal and Webb, 1971; Anderson, 1973; Cihak and Rada, 1976; Absil et al., 1980). However, the only mammalian UCK cDNA yet cloned is a cDNA isolated from mouse (Ropp
and Traut, 1996). We decided to identify and clone human uridine-cytidine kinases to study these enzymes for the pharmacological activation of nucleoside analogs. Our findings show that a UCK enzyme family with at least two members exists in humans cells. We have
further expressed the two enzymes recombinantly and have investigated
their ability to catalyze the phosphorylation of several pyrimidine
nucleoside analogs.
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Experimental Procedures |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Cloning and Expression of Human UCK1 and UCK2 cDNA.
We
searched the expressed sequence tag library of the GenBank database at
the National Institute for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) with the Basic Local
Alignment Search Tool (BLAST) to identify human cDNA clones that
encoded protein homologs to the cloned mouse UCK (Ropp and Traut,
1996). The expressed sequence tag cDNA clones were obtained from
Research Genetics Inc (Huntsville, AL). The DNA sequences of the
plasmids were determined with the ABI Prism 310 Genetic Analyzer
(PerkinElmer/Applied Biosystems, Norwalk, CT) and with the automatic
laser fluorescent sequencer (Amersham Pharmacia Biotech, Piscataway, NJ).
-D-galactoside at
A595 = 0.9 for 4 h. The bacteria were
harvested by centrifugation at 5,000g for 10 min and
resuspended in a buffer containing 50 mM
NaH2PO4 and 300 mM NaCl.
The bacteria were lysed by the addition of 1 mg/ml lysozyme and by
sonication. The protein extracts were cleared by centrifugation at
12,000g for 20 min. The crude extracts were loaded onto a
Talon Metal affinity resin column (CLONTECH). The purified recombinant
proteins were eluted in a 50 mM
NaH2PO4/300 mM NaCl buffer,
pH 7.0, supplemented with 150 mM imidazole (Sigma, St. Louis, MO). The
size and purity of the recombinant proteins were determined by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (PhastSystem;
Amersham Pharmacia Biotech). The protein concentrations were determined
with Bradford Protein Assay (Bio-Rad, Hercules, CA). Bovine serum
albumin (BSA) was used as the concentration standard. The proteins were
aliquoted and stored at 
80°C with 10 mM dithiothreitol and 0.5 mg/ml BSA.
Enzyme Assays.
The nucleosides, nucleoside analogs,
nucleoside triphosphates, and deoxynucleoside triphosphates were
obtained from Sigma and ICN Biomedicals Inc (Costa Mesa, CA).
Tetrahydrouridine was obtained from CN Biosciences, Inc. (San Diego,
CA). [
-32P]ATP (3000 Ci/mmol) was obtained
from Amersham Pharmacia Biotech. Enzyme assays were performed on fresh
aliquots of UCK1 and UCK2. For substrate screening, the nucleosides and
their analogs were added at a final concentration of 100 µM in a
10-µl reaction mixture containing 50 mM Tris-HCl, pH 7.6, 5 mM
MgCl2, 1 mM unlabeled ATP, 1 µCi of
[-32P]ATP and 5 ng of enzyme. The reaction
mixtures were incubated 30 min at 37°C. Two µl of the reaction
mixtures were spotted on poly(ethyleneimine)-cellulose F chromatography
sheets (Merck Inc., Whitehouse Station, NJ) and the nucleosides were
separated in a buffer containing NH4OH/isobuturic
acid/distilled H2O (1:66:33, v/v). The thin-layer
chromatography sheets were autoradiographed and quantified using a
FujiX BAS 1000 Bio-imaging Analyzer System (Fuji, Tokyo, Japan).
; Ahmed, 1981). Briefly, the assays were performed in 50 mM
Tris, pH 7.6, 100 mM KCl, 5 mM MgCl2, 15 mM NaF,
5 mM dithiothreitol, 0.5 mg/ml BSA, 5 mM ATP and 1 µM
[5-3H]Cyd (21.5 Ci/mmol; PerkinElmer Life
Sciences, Boston, MA) or 1 µM [5-3H]Urd (26 Ci/mmol; Amersham Pharmacia Biotech). Nucleoside and enzyme were added
to a total reaction volume of 50 µl. The amount of enzyme added to
the reaction was adjusted so that no more than 20% of the substrate
was consumed during the incubation period. The nucleoside concentration
ranged from 1 µM to 2 mM. At 0, 10, 20, and 30 min of incubation at
37°C, 10 µl of the reaction mixtures were spotted on Whatman DE-81
filters. The filters were dried and washed three times in 5 mM ammonium
formate, once in distilled water, and once with 95% ethanol. The
filter bound monophosphates were eluted with 0.1 M KCl and 0.1 M HCl.
Radioactivity was determined using a scintillation counter. All assays
were at least performed in triplicate. Donor specificity of UCK1 and
UCK2 was determined with the radiochemical method as described above.
The NTP concentrations were 5 mM and substrate concentrations were at
the Km values of Cyd and Urd.
Northern Blot.
cDNA probes of the human UCK1 (bp 5-836) and
UCK2 (bp 19-802) were labeled with
[
-32P]dCTP (6000 Ci/mmol, QuickPrime;
Amersham Pharmacia Biotech). The labeled probes were hybridized to a
human multiple tissue Northern blot (CLONTECH) with
poly(A)+ RNA of 12 different human tissues using
ExpressHyb hybridization solution (CLONTECH) as described in the
manufacturer's protocol.
Chromosome Mapping. We searched the sequence tagged sites library of the GenBank database at the National Institute for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) with the Basic Local Alignment Search Tool (BLAST) to identify gene markers for the human UCK1 and UCK2 sequences. The cytogenetic locations of UCK1 and UCK2 gene markers were obtained from the Genome database (http://www.gdb.org).
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Results |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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cDNA Cloning of Two Human UCK.
Human and mouse expressed
sequence tag cDNA clones homologs to the cloned mouse UCK cDNA (Ropp
and Traut, 1996) were identified in GenBank. Two distinct clusters of
cDNA sequences were identified as two separate enzymes present in both
human and mouse cDNA libraries. The cDNAs most similar to the cloned
mouse enzyme were named UCK1 (I.M.A.G.E. clone ID 2783059, 1401966, 877609, and 486302) and the remaining cDNA clones were named UCK2
(I.M.A.G.E. clone ID 1340011, 1160750, 1713319, 2178111, 642042, 1225388, 350272, and 846324) (Lennon et al., 1996). The longest open
reading frame of UCK1 encoded a 277-amino-acid protein with a predicted
molecular mass of 31 kDa. The enzyme was 92% identical to the cloned
mouse cDNA at the amino acid level. The longest open reading frame of human UCK2 encoded a 261-amino-acid protein with a predicted molecular mass of 29 kDa. The mouse homolog of UCK2 was 98% identical to the
human sequence at the amino acid level. We aligned the sequences of
human and mouse UCK1 and UCK2 with the UCKs of Caenorhabditis elegans and Escherichia coli (Fig.
1). Human UCK1 and UCK2 were 72%
similar, and the enzymes were 37 and 28% similar to UCK of
C. elegans and E. coli, respectively.
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Expression and Characterization of Recombinant Human UCK.
We
expressed the human UCK1 and UCK2 cDNA in E. coli and
purified the recombinant enzymes. SDS-polyacrylamide gel
electrophoresis showed a major band of 30 kDa for both enzymes (Fig.
2). To verify the enzymatic activity of
the recombinant enzymes, we tested the naturally occurring ribo- and
deoxyribonucleosides (Fig. 3). We used
ATP as the phosphate donor in the assays, because studies on tissue
purified UCK indicate that it is the preferred donor (Anderson, 1973;
Cihak and Rada, 1976; Ropp and Traut, 1998). Both enzymes
efficiently phosphorylated Urd and Cyd. No phosphorylation of Ado, Guo,
or any deoxyribonucleosides was detected for either enzyme.
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Phosphorylation of Nucleoside Analogs.
In addition to the
natural substrates, we also studied the phosphorylation of cytidine and
uridine nucleoside analogs (Fig. 4). We
tested 14 uridine and 14 cytidine base-substituted nucleoside analogs
for phosphorylation activity at 100 µM concentration. The
phosphorylation activity and a map of the base substituents are shown
in Table 2. We considered nucleoside
analogs that phosphorylated more than 5% of uridine to be substrates
of UCK.
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-D-arabinofuranosylcytosine, 2',2'-difluorodeoxycytidine and 2',3'-dideoxycytidine were not substrates of either enzyme.
Northern Blot Analysis.
We used a multiple tissue Northern
blot to study the expression pattern of human UCK1 and UCK2 mRNAs (Fig.
5). UCK1 mRNA was detected as two
isoforms of 1.8 and 2.7 kb. The 2.7-kb band was ubiquitously
expressed in the investigated tissues, with high level of expression in
liver, kidney, skeletal muscle, and heart, whereas low levels were
present in brain, placenta, small intestine, and spleen. The band of
1.8 kb was detected in skeletal muscle, heart, liver, and kidney. In
contrast, UCK2 mRNA was only detected in placenta as two transcripts of
1.2 and 2.0 kb. We were not able to detect UCK2 mRNA in any other
tissue even after extended exposure times (data not shown).
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Chromosome Mapping and Gene Structure.
Human sequence-tagged
site sequence markers identical to human UCK1 and UCK2 cDNAs were
identified. The marker for UCK1 (SHGC-10187) was localized to
chromosome 9q34.2-9q34.3. The UCK2 marker (SHGC-35183) was localized to
chromosome 1q22-1q23.2. We also identified partially sequenced human
genomic clones in the GenBank database, which contained the UCK1 and
UCK2 genes (clone RP11-40A7 and RP11-7G12, RP11-525G13). Sequence
analysis showed that the UCK1 gene was divided into seven exons
distributed over 7 kb (data not shown). The coding sequence of UCK2,
from bp 116 of the cDNA sequence, was similarly divided into seven
exons distributed over 19 kb (data not shown). The genomic sequence
corresponding to bp 1-116 of the UCK2 cDNA sequence was, however, not
available in the GenBank database.
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Discussion |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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We have cloned and recombinantly expressed the cDNA of two human
uridine-cytidine kinases and characterized the enzymes for phosphorylation of nucleosides and nucleoside analogs. The kinetic properties of both UCK1 and UCK2 for Cyd and Urd phosphorylation are
similar to those reported for enzymes purified from several sources
(Sköld, 1960; Anderson, 1973; Cihak and Rada, 1976), and
we are therefore convinced that the recombinant enzymes are useful
tools to study the phosphorylation of nucleoside analogs in vitro.
UCK activity has been detected in most investigated tissues (Herzfeld
and Raper, 1979; Shen et al., 1998). These data are in agreement with
ubiquitous expression of human UCK1 mRNA. In contrast to the expression
pattern of UCK1, we only detected UCK2 mRNA in placenta. Interestingly,
a fragment of human UCK2 cDNA has previously been cloned in a study
using differential display to isolate testis-specific transcripts
(Ozaki et al., 1996). A cDNA sequence similar to UCK2 has also been
shown to detect mRNA expression in tissue of rat brain (Yuh et al.,
1999), although we were unable to detect UCK2 expression in human
brain. We do not know the physiological importance of the
tissue-restricted expression of UCK2 or of the differences in
expression reported for different species. It is possible that UCKs may
be differentially expressed at different stages of development or that
other regulatory mechanisms influence the expression. Kinetic
properties have been reported for UCK purified from several sources
(Sköld, 1960; Anderson, 1973; Cihak and Rada, 1976). Most
studies have been performed on murine ascites tumors, calf thymus,
human colon, and murine liver and kidney; to our knowledge, however, no
study has been performed on such UCK2-expressing tissues as placenta and testis (Sköld, 1960; Lee et al., 1974; Fulchignoni-Lataud et
al., 1976; Ahmed, 1984).
Several of the nucleoside analogs tested in the present study were substrates of both UCK1 and UCK2, although for some of the compounds, there was a difference in phosphorylation efficiency between UCK1 and UCK2. 3-Deazauridine, 5-hydroxyuridine, and 6-azacytidine were phosphorylated >10-fold more efficiently by UCK2 compared with UCK1. This would indicate a higher toxicity for these compounds in tissues expressing UCK2 but, to our knowledge, there has been no report of nucleoside analog selectivity for a specific tissue expressing either UCK1 or UCK2.
Several pyrimidine ribonucleoside analogs like 6-azauridine (Pasternak
et al., 1961; Vesely and Cihak, 1973), 5-azacytidine (Cihak and
Broucek, 1972; Vesely and Cihak, 1973), 5-hydroxyuridine (Smith and
Visser, 1965), and 4-thiouridine (Lindsay and Yu, 1974) exert the
pharmacological effects in their monophosphate forms by inhibiting OMP
decarboxylase, in addition to their incorporation into RNA or DNA
(Cihak and Rada, 1976). In contrast, most deoxyribonucleoside analogs
are predominantly dependent on phosphorylation to their triphosphate
form, and incorporation into DNA, for pharmacological activity.
However, the deoxyribonucleoside analog 5-fluorodeoxyuridine is also
active as a monophosphate derivative through its inhibition of
thymidylate synthase, a target that subsequently causes inhibition of
DNA synthesis. In conclusion, the targets for deoxyribonucleoside analogs mainly affect DNA synthesis and are therefore different compared with the molecular targets for ribonucleoside analogs. The
different mode of action of ribonucleoside analogs makes this group of
compounds interesting for further studies to develop clinically useful
drugs. Promising results in animal tumor models have recently been
shown for
1-(3-C-ethynyl--D-ribo-pentofuranosyl)-cytosine and -uracil (Takatori et al., 1999). Cyclopentyl cytosine is another interesting compound with anti-tumor activity that presently is evaluated in clinical trials (Verschuur et al., 2000). Characterization of the UCKs involved in the pharmacological activation of the pyrimidine ribonucleoside analogs will be important for further development of this therapeutic strategy.
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Footnotes |
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Received November 20, 2000; Accepted January 31, 2001
This work was supported by grants from the Swedish Medical Research Council, the Swedish Cancer Foundation, the Swedish Foundation of Strategic Research and the European Commission.
The DNA sequence reported in this paper has been deposited in the GenBank database (accession no. AF236636, AF236637, and AF237290).
Send reprint requests to: Dr. Anna Karlsson, Division of Clinical Virology, Karolinska Institute, Huddinge University Hospital, S-141 86 Stockholm, Sweden. E-mail: anna.karlsson{at}mbb.ki.se
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Abbreviations |
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UCK, uridine-cytidine kinase; kb, kilobase pairs(s); bp, base pair(s); BSA, bovine serum albumin.
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References |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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