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Vol. 61, Issue 1, 222-229, January 2002
Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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(E)-2'-deoxy-2'-(fluoromethylene)cytidine (FMdC) is a
new analog of deoxycytidine with promising anticancer activity. We
investigated the action of FMdC on DNA metabolism by evaluating its
incorporation into DNA, its excision from DNA in vitro, and the role of
the incorporation of FMdC into DNA in causing cytotoxicity. In vitro DNA primer extension demonstrated that FMdC nucleotides were
incorporated with relatively high substrate efficiency into the C sites
of the elongating DNA strand. Once incorporated, FMdC became a poor substrate for further chain elongation by DNA polymerases, resulting in
a termination of DNA synthesis at the sites of incorporation. Furthermore, the 3' 
5' exonuclease activity of DNA polymerase 
or wild-type p53 protein was ineffective in removing the incorporated FMdC from DNA in vitro. FMdC also showed potent cytotoxic activity against human leukemia and solid tumor cells. Incubation with a low
concentration of FMdC (10 nM) induced cell cycle arrest at S or
G1 phases, but the cells eventually died as the time of incubation increased. Compared with HL-60 cells, human myeloid ML-1
cells with wild-type p53 were more sensitive to FMdC, but the S or
G1 phase arrest did not seem to depend on the presence or
absence of p53. Inhibiting the incorporation of FMdC into cellular DNA
by aphidicolin suppressed the cytotoxic effect of the compound. We
conclude that the incorporated FMdC nucleotide profoundly disrupts DNA
synthesis and resists excision by exonucleases, and that incorporation of this analog into DNA is a key molecular event responsible for the
drug's cytotoxicity.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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(E)-2'-Deoxy-2'-(fluoromethylene)cytidine
(FMdC) is a novel deoxycytidine analog developed as a specific
inhibitor of ribonucleotide reductase (McCarthy et al., 1991
). Early
studies demonstrated that the diphosphate of this compound affects
ribonucleotide reductase through a mechanism-based inhibition (van der
Donk et al., 1996) similar to that of gemcitabine. However, unlike
gemcitabine and 1-
-D-arabinofuranosylcytosine, FMdC is
relatively resistant to deamination by cytidine deaminase (Takahashi et
al., 1998). This favorable metabolic property may contribute in part to
the analog's potent anticancer activity. In vivo pharmacology study in
animals demonstrated that at the dose of 200 and 400 mg/kg, FMdC is
eliminated from the plasma and normal tissue with a plasma half-life
(t1/2) of about 60 min. The kidney is shown
to be the major site of initial distribution of FMdC (Adams et al.,
1996). After entering the cells, FMdC is first converted to its
5'-monophosphate and is then further phosphorylated to diphosphate and
triphosphate. The nucleotides of FMdC are thought to be the active
metabolites of this drug. The diphosphate of FMdC has been shown to
inhibit ribonucleotide reductase, whereas the triphosphate seems to
directly interfere with DNA polymerization (for review, see Seley,
2000). It has been demonstrated that FMdC displays a strong cytotoxic
activity against a variety of human solid tumor cell lines in culture
and shows a potent antitumor activity against murine tumor models and
human tumor xenografts in nude mice (Bitonti et al., 1994, 1995;
Snyder, 1994; Piepmeier et al., 1996; Wright et al., 1996; Sun et al.,
1997, 1998; Takahashi et al., 1998; Kotchetkov et al., 1999). Clinical
trials of FMdC, known as tezacitabine, have recently begun in patients
with hematological malignancies and solid tumors (Noriyuki et al.,
1999; Rodriguez et al., 1999; Faderl et al., 2000).
Many therapeutic nucleoside analogs share similar metabolic pathways
and pharmacological actions. After entering the cells, nucleoside
analogs are metabolically converted to nucleotides through a series of
sequential phosphorylation steps. The analog nucleotides are believed
to be the active forms of the drugs, which in many cases have multiple
sites of action in the cells. For example, ribonucleotide reductase and
DNA polymerases are inhibited by the diphosphate and triphosphate of
gemcitabine and fludarabine (Gandhi et al., 1994; Hui and Reitz, 1997;
Storniolo et al., 1997). Fludarabine also inhibits DNA ligation,
DNA-dependent RNA primer formation, and RNA synthesis (Huang and
Plunkett, 1991; Yang et al., 1992; Catapano et al., 1993).
Despite these multiple sites of action, incorporation of the analogs
into DNA, which results in the termination of DNA synthesis, seems to
be the most prominent event and is closely associated with the
cytotoxic action of the analogs. Inhibition of the incorporation of
analogs into DNA usually leads to a significant reduction of the
analog-induced cell death (Huang et al., 1995).
Like other nucleoside analogs, FMdC seems to have multiple sites of
action. The inhibition of ribonucleotide reductase by FMdC diphosphate
has been well characterized (van der Donk et al., 1996; Kanazawa et
al., 1998). In vitro studies suggest that the triphosphate of FMdC may
inhibit human DNA polymerase (pol) 
and cause a pause in further DNA
synthesis (Yonetani and Mizukami, 1996). However, the incorporation of
FMdC triphosphate into DNA and its biological significance have not
been systemically evaluated. In this study, we investigated the
incorporation of FMdC triphosphate into DNA and its consequences in
vitro and in whole cells. We also examined the ability of DNA pol and p53, two cellular molecules with intrinsic 3' 5' exonuclease
activity, to remove the incorporated FMdC from DNA in vitro.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Chemicals and Reagents.
FMdC and its triphosphate (FMdCTP)
were provided by Dr. H. S. Gill (Hoechst Marion Roussel, Inc.,
Cincinnati, OH). Propidium iodide was obtained from Sigma Chemical (St.
Louis, MO), RNase and proteinase K from Roche Molecular Biochemicals
(Indianapolis, IN), and ethidium bromide from Bio-Rad (Hercules, CA).
The 17-base M13 sequencing primer (5'-GTAAAACGACGGCCAGT-3') and
high-performance liquid chromatography-purified dATP, dCTP, dGTP, and
dTTP were obtained from Amersham Biosciences, Inc. (Piscataway, NJ).
The 25-base template (5'-CACACACGACTGGCCGTCGTTTTAC-3') was synthesized by Genosys Biotechnologies, Inc. (The Woodlands, TX).
[
-32p]ATP (specific activity 4500 Ci/mmol)
was purchased from ICN Radiochemicals, Inc. (Irvine, CA). T4
polynucleotide kinase and the large fragment of Escherichia
coli DNA polymerase I (Klenow fragment) were obtained from United
States Biochemical Corp. (Cleveland, OH). DNA polymerases (pol )
and (pol ) were purified from human T-lymphoblastoid cells and
characterized as described previously (Huang et al., 1990). The
wild-type (wt) p53 protein was purified from human leukemia ML-1 cells
by precipitation of the protein extracts with 50 mM
(NH4)2SO4,
followed by fractionation with the Pharmacia fast-performance liquid
chromatography system using a MonoQ column with a linear gradient of 20 to 700 mM NaCl in 50 mM Tris buffer (1 ml/min/fraction, 40 min). The
p53 protein in the fractions was characterized as described previously
(Huang, 1998). Fraction 25 was found to contain the highest level of
p53 protein with 3' 5' exonuclease activity. Immunodepletion of p53
by anti-p53 antibody (Ab-6; Oncogene Science, Cambridge, MA) also
removed the 3' 5' exonuclease activity from this fraction. Thus,
fraction 25 was used for the excision assays in this study.
Cell Culture. Human leukemia cell lines HL-60 and ML-1 were maintained in suspension culture RPMI 1640 medium supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) at 37°C in a humidified atmosphere containing 5% CO2. During exponential growth, the population doubling time was approximately 22 h. All experiments were carried out using exponentially growing cell cultures. The cell lines were tested periodically to ensure that they were free from Mycoplasma species.
Cytotoxicity Assays. Cell growth inhibition was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay. HL-60 or ML-1 cells were seeded onto 96-well plates at the initial density of 4000 cells/well. After incubation with various concentrations of FMdC for 72 h, 50 µl of MTT reagent (3 mg/ml) was added to each well, and incubation was continued for another 4 h. The cells were then centrifuged (1500 rpm, 10 min), and the medium was removed. The cell pellets were dissolved in 200 µl of dimethyl sulfoxide. Absorbance was measured at 570 nm within 1 h, using the Dynatech MR 5000 plate reader (Dynatech Labs, Chantilly, VA). The percentage of growth inhibition was calculated by dividing the absorbance of each FMdC-treated well by that of the untreated control. The antiproliferative activity was expressed as the drug concentration that induced 50% growth inhibition (IC50).
To examine the change in cell morphology, the FMdC-treated cells were centrifuged (550 rpm, 5 min) onto glass slides, using a Shandon-Elliot cytospin (London, UK). The slides were fixed with 100% methanol for 45 min, air-dried, and then stained with Wright's Giemsa stain solution (Biochemical Sciences Inc., Swedesboro, NJ). Cells were examined for morphological changes characteristic of apoptosis. Photomicrographs were taken using a 20× objective (Nikon, Tokyo, Japan). Apoptosis was further confirmed by a DNA fragmentation assay. After treatment with FMdC, approximately 5 × 106 cells were collected, and the cell pellets were digested in 1 ml of buffer containing 10 mM Tris-HCl, pH 7.8, 100 mM NaCl, 25 mM EDTA, 0.5% SDS, and 10 µl of proteinase K (1 mg/ml, added fresh) at 45°C overnight. The samples were analyzed on 1.8% agarose gel in 1× Tris-borate/EDTA buffer (100 mM Tris-borate, pH 8.3, 2 mM EDTA). After electrophoresis, the gel was incubated overnight in 400 ml of 0.1× Tris-borate/EDTA buffer containing 20 µl of RNase (500 µg/ml) and then photographed. DNA bands were quantitated using the ChemiImager 4400 imager system (Alpha Innotech Corporation, San Leandro, CA).Cell Cycle Analysis by Flow Cytometry.
Exponentially growing
cells were exposed to 10 nM FMdC for 6, 12, 24, 36, or 48 h,
washed with cold phosphate-buffered saline (PBS; 135 mM NaCl, 2.7 mM
KCl, 1.5 mM KH2PO4, 8 mM
Na2HPO4, pH 7.4), and
resuspended in PBS at 106 cells/ml. The cells
were fixed in 70% ethanol at 
20°C overnight, washed twice with
cold PBS, and stained with 1 ml of PBS containing propidium iodide (15 µg/ml), Tween 20 (0.5%), and 5 µl of RNase (500 µg/ml). The DNA
content was measured using a FACSVantage flow cytometer (BD
Biosciences, San Jose, CA). Data acquisitions and analyses were
performed using Cell-Quest software (BD Biosciences).
Preparation of 18-Base Oligomer with an Incorporated FMdC
Nucleotide at 3' End.
The 17-base M13 universal sequencing primer
was labeled at the 5' end with [-32P]ATP
using the T4 polynucleotide kinase and then annealed to the 25-base
template as described previously (Huang et al., 1990). The sequence of
32P-labeled 17-base primer/25-base template
hybrid was as follows: (5') 32P-GTAAAACGACGGCCAGT
(3') CATTTTGCTGCCGGTCAGCACACAC.
DNA Primer Extension Assay.
The 17-base primer/template and
18-base primer/template hybrids were used as the substrates for DNA
primer extension assays by DNA pol and pol I. The reaction mixtures
contained 20 mM Tris-HCl, pH 7.5, 8 mM MgCl2, 0.5 mM dithiothreitol, 10 mM NaCl, bovine serum albumin (20 µg/ml), and
the labeled DNA substrates at various concentrations, dNTPs, FMdCTP, as
well as pol I or pol . The reactions were incubated at 37°C for 30 min and analyzed by electrophoresis through a 10% polyacrylamide
sequencing gel. After autoradiography, the radioactivity of each DNA
band in the gel was quantitated using a gel analyzer (InstantImager;
Packard Instrument Co., Meriden, CT).
DNA Excision Assay.
The 17- and 18-base primer/template
hybrids were used as the substrates for excision by 3' 5'
exonuclease associated with human pol and wt p53 protein. The
reaction mixtures contained 20 mM Tris-HCl, pH 7.5, 8 mM
MgCl2, 0.5 mM dithiothreitol, 10 mM NaCl, bovine
serum albumin (20 µg/ml), and various concentrations of the labeled
DNA substrates, as well as pol or p53. The reactions were incubated
at 37°C for 30 min and analyzed by electrophoresis through a 10%
polyacrylamide sequencing gel. After autoradiography, the radioactivity
of the DNA bands in the gel was quantitated by the InstantImager. The
Km and Vmax
values from three separate experiments were calculated based on the
Michaelis-Menten equation, using the Winzyme computer program (Biosoft,
Ferguson, MO).
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Incorporation of FMdC Nucleotide into DNA.
Because most
nucleoside analogs cause cytotoxicity after being incorporated into
cellular DNA, we first tested the ability of human DNA pol to
incorporate FMdCTP into DNA in vitro, using a primer extension assay
with the 32P-labeled 17-/25-base hybrid as the
primer/template. As illustrated in Fig.
1A, FMdCTP was incorporated into the C
site of the primer strand at position 18 by pol in the absence of
dNTPs (lanes 2 and 3). In the presence of each of the 4 dNTPs at a
concentration of 0.5 µM, increasing concentrations of FMdCTP competed
with dCTP for incorporation, resulting in a concentration-dependent
pause of DNA extension at position 18 and a reduction of the
full-length products (lanes 4-7). Evaluation of DNA pol I indicated
that FMdCTP competed poorly with dCTP, as evidenced by the efficient
elongation of the strand to the full-length product in the presence of
various concentrations of FMdCTP (Fig. 1B, lanes 4-7). Even at the
FMdCTP/dCTP ratio of 20:1 (10:0.5 µM), no significant reduction of
the full-length product was seen (lane 7). Thus, FMdCTP seemed to be a
better substrate for human DNA pol than for the E. coli
enzyme, which strongly preferred normal dNTPs as the substrates.
However, in the absence of dNTPs, pol I was still able to incorporate
FMdCTP into the DNA (Fig. 1B, lanes 2 and 3).
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, we
further compared its incorporation kinetics with that of dCTP catalyzed
by pol . Various concentrations of FMdCTP or dCTP were incubated
with pol and the 32P-labeled primer/template,
and the reaction products were analyzed. As shown in Fig. 1C, we
observed a concentration-dependent incorporation of FMdCMP or dCMP into
position 18. When the radioactivity of each band was quantitated (Fig.
1D), dCTP seemed to be a somewhat better substrate for pol than was
FMdCTP.
DNA Chain Termination by FMdC Nucleotide.
To further
characterize the chain termination properties of FMdC, we prepared an
18-base primer containing a FMdCMP at the 3' end of the oligomer and
annealed it to the 25-base template as described under Materials
and Methods. This analog-primer/template hybrid was used as the
substrate for extension by pol and pol I, in comparison with the
normal 17-base primer/template. As shown in Fig.
2A, both pol and pol I extended the
normal DNA substrate efficiently in the presence of four normal dNTPs.
In contrast, the presence of FMdCMP at the 3' end of the primer
substantially inhibited strand elongation by each enzyme. Small amounts
of extension products were detected in samples incubated with pol I for
prolonged periods (30-40 min); no significant extension was observed
in samples incubated with pol . The time courses of primer extension of both normal and analog-terminated primers by pol and pol I are
shown in Fig. 2B.
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. We
observed a concentration-dependent increase of extension products in
samples with the normal DNA substrate (Fig. 2C, lanes 2-8), whereas
the extension of the analog primer was hardly detectable (lanes
10-16). The quantitative data are shown in Fig. 2D. Kinetic analysis
revealed an apparent Km value of 76.9 nM
for the normal 17-base primer as the substrate for pol . Because of
inadequate primer extension, we were unable to determine the
Km value for the analog-containing primer.
Excision of FMdC from 3' End of DNA by pol and p53.
Because FMdC showed potent DNA chain termination activity in vitro
(Fig. 2), we sought to determine whether the incorporated analog could
be removed from DNA by enzymes. Two proteins, DNA pol and the wt
p53, each with intrinsic 3' 5' exonuclease activity (Mummenbrauer
et al., 1996; Burgers, 1998), were tested for their abilities to
remove FMdCMP from the 3' terminus of the 18-base primer when
annealed to the 25-mer template. The 3'-FMdCMP primer was prepared as
described above; the normal 17-base primer was processed through the
same procedures and used as a control. As shown in Fig.
3A, pol effectively removed the
normal nucleotides from the 17-base primer; most of the 17-mers were
degraded to shorter oligonucleotides within 15 min. The p53
protein exhibited moderate activity in excising nucleotides from the
17-base primer; about 50% of the 17-mers still remained intact after a
45-min incubation. When the 18-base primer containing 3'-terminal
FMdCMP was used as the substrate, pol and p53 each showed a
substantially reduced ability to remove the analog from the 18-base
primer. Figure 3B shows a quantitative comparison of the time courses of excision of the normal 17-base primer and the FMdCMP-18-mer by pol
and p53. Clearly, the FMdCMP-containing DNA was a poor substrate
for excision by either enzyme.
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and p53 was further characterized using a kinetic assay. Various
concentrations of each primer/template were incubated with each enzyme,
and the excision products were quantitated (Fig. 3, C and D). The
generation of excision products by each enzyme was
concentration-dependent with both substrates, although the 18-base
primer with FMdCMP incorporated at its 3' end was much more resistant
to excision by both enzymes than was the normal 17-base primer. The
apparent kinetic parameters were calculated based on the
primer/template substrate concentrations and the respective
radioactivity associated with the excision products. As shown in Table
1, in the case of pol , the apparent
Km value for the normal 17-base primer (4.7 nM) was substantially greater than that for the FMdCMP-primer
(0.4 nM), whereas the Vmax for excision of
the FMdCMP was only 3% of that for excision of dCMP. Similarly,
for p53, there was a much greater affinity for the analog-containing
oligonucleotide, as measured by the apparent Km values of the normal 17-base primer
(26.1 nM) and the 18-base primer (2.0 nM). As with pol , the
Vmax for removal of FMdCMP was less than
6% of that for the removal of dCMP. Thus, although the affinity of the
repair activities was substantially greater for the analog-terminated
DNA than for normal primers, the rates at which pol and p53 were
able to excise the fraudulent nucleotide was greatly diminished
relative to the deoxynucleotide.
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Cellular Responses to FMdC Treatment.
Incubation of human
leukemia HL-60 and ML-1 cells in various concentrations of FMdC in
vitro caused a potent growth inhibition in both cell lines, although it
seemed that the ML-1 cells were more sensitive to FMdC than HL-60 cells
(Fig. 4A). The drug concentrations required to achieve IC50 during a continuous 72-h
incubation were 10.4 nM for HL-60 cells and 3.5 nM for ML-1 cells. A
colony formation assay showed that FMdC also exhibited potent activity
in human pancreatic cancer cells. Incubation of Panc-1 cells with FMdC substantially inhibited the ability of the cells to form colonies, with
IC50 values of 148.4 nM after a 24-h incubation
and 11.8 nM after continuous (10-d) exposure (data not shown).
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Incorporation into DNA is the most prominent action of nucleoside
analogs and is closely correlated with cytotoxicity (Kufe et al., 1984;
Huang et al., 1990, 1991, Xie and Plunkett, 1995). In many cases, such
incorporation causes a disturbance of DNA synthesis that is
characterized by in vitro assays as a kinetic delay of DNA chain
elongation at or near the sites of the incorporated analogs. The
relative potency of the chain termination effect varies among different
analogs, and there may be an apparent discrepancy in chain termination
observed in vitro and in whole cells. For instance, the deoxyadenosine
analog fludarabine shows potent DNA chain termination activity both in
vitro and in whole cells (Huang et al., 1990), whereas the
deoxycytidine analog 1--D-arabinofuranosylcytosine was
effective in terminating DNA synthesis in vitro but was incorporated mainly internally into cellular DNA in cell culture (Kufe et al., 1984;
Grant, 1998). Another deoxycytidine analog, gemcitabine, is
incorporated into the C sites of the elongating DNA strand and can be
extended by a normal deoxynucleotide in vitro before DNA synthesis
pauses (Huang et al., 1991). Consistent with this molecular
pharmacological behavior, gemcitabine nucleotide is also found
internally in DNA extracted from cells after incubation with this
nucleoside analog.
The present study demonstrated that FMdCTP was incorporated into the C
sites by DNA pol (Fig. 1). Once FMdCTP was incorporated into the
DNA, it potently terminated further DNA strand elongation (Fig. 2).
These findings are in contrast to the action of gemcitabine nucleotide,
which can be extended by pol and pol , and which is found
predominantly (>95%) in phosphodiester linkage in DNA extracted from
whole cells (Huang et al., 1991). In our study, the competing normal
nucleotide dCTP seemed to be preferred to FMdCTP by pol (Fig. 1A)
and was selected over FMdCTP almost exclusively by pol I (Fig. 1B).
Therefore, it is likely that the ratio of dCTP to FMdCTP in cells can
be used to predict the amount of analog incorporation into DNA. The
extent to which FMdCDP can inhibit ribonucleotide reductase and thereby
decrease the dCTP levels in whole cells may significantly alter the
cellular FMdCTP/dCTP ratio to enhance the incorporation of FMdCTP. In
designing a therapeutic strategy, a biochemical modulation approach
that decreases dCTP levels, such as those using either fludarabine
(Gandhi et al., 1993) or chlorodeoxyadenosine (Gandhi et al., 1996) may
be usefully applied to FMdC. It should be pointed out that although
FMdC nucleotide seemed to be a potent DNA chain terminator in vitro
when DNA pol is used as the catalyzing enzyme, it is possible that
other DNA polymerases in the cells might be able to extend the DNA
substrate containing FMdCMP at 3' end. This possibility can be tested
both in vitro by using various types of purified DNA polymerases, and in whole cells by using radioactive labeled FMdC to determine the
location of the incorporated analog in DNA (terminal or internal incorporation).
Because incorporation of nucleotide analogs into DNA is a critical
event in causing cytotoxicity, their excision from cellular DNA by a 3'
5' exonuclease activity is presumably an important mechanism in
cell resistance to nucleoside analogs. Several cellular molecules,
including DNA pol 
, pol , and wt p53, have intrinsic 3' 5'
exonuclease activity (Mummenbrauer et al., 1996; Burgers, 1998; Huang,
1998). The present study demonstrated that neither pol nor p53
effectively removed the incorporated FMdCMP from DNA in vitro (Fig. 3).
The kinetic analysis revealed an interesting interaction between the
drug-containing DNA and pol or p53. As shown in Table 1, p53 and
pol were each able to bind with a much higher affinity to the
FMdCMP-containing oligomer than to the normal oligodeoxynucleotide.
However, the rate of analog excision was slow, as evidenced by the
Vmax values, which were lower for the
FMdCMP oligodeoxynucleotide than the normal oligodeoxynucleotide. These
kinetic data suggest that once incorporated into the DNA, FMdCMP might
have caused a change in the DNA configuration at the 3' terminus so
that the analog-DNA was preferentially recognized by the proofreading
exonuclease of pol or p53. Importantly, this preferential binding
did not result in an effective excision; this failure also may have
been due to an analog-induced structural change at the 3' terminus that
was unfavorable for the excision process. This interesting phenomenon
has also been observed with the purine nucleoside analog fludarabine
(Kamiya et al., 1996). The findings that 3'-terminal FMdCMP is a poor
substrate for extension, and that it is resistant to excision indicate
that the analog is likely to be a strong DNA chain-terminating
nucleotide in whole cells. This attribute is likely to have
implications for mechanism-based combination strategies aimed at
increasing nucleotide analog incorporation into the DNA of cells
undergoing excision repair processes (Sandoval et al., 1996; Yang et
al., 2000).
The wt p53 is known to signal apoptosis in response to DNA damage. It
is possible that the interaction of p53 with the analog-containing DNA
serves as a signal to initiate apoptosis, thus making the cells more
sensitive to nucleoside analogs. The high binding affinity of p53 to 3'
FMdCMP-oligodeoxynucleotide and low excision rate make this possibility
more likely. Compared with the p53-null HL-60 cells, the wt p53 cells
(ML-1) seemed to be more sensitive to FMdC (Fig. 4A). In fact, previous
studies demonstrated that transfection of p53-null (H1299) cells with
wt p53 increased the sensitivity of the cells to the deoxycytidine
analog gemcitabine (Feng et al., 2000). In a separate experiment, we
showed that the levels of p53 protein in ML-1 cells increased as early
as 4 h after FMdC incubation (10 nM), and the p53 protein
accumulation increased in a time-dependent manner (up to 24 h
after drug treatment; data not shown). Thus, it is possible that the
binding of p53 to the FMdCMP-containing DNA may serve as a signal to
accelerate apoptosis. However, the greater sensitivity observed in ML-1
cells should not be solely attributed to their wild-type p53 status, because there may be other differences between ML-1 and HL-60 cells.
Factors including drug metabolism, pharmacodynamic factors, and DNA
repair capacity may potentially affect the cellular response to FMdC treatment.
Interestingly, in the HL-60 cell line, incubation with FMdC caused a moderate accumulation of cells in the S-phase before the cells succumbed to apoptosis (Fig. 4B). This pause of cells in the S phase may reflect the early cellular response to the drug-induced inhibition or chain termination of DNA synthesis. The concomitant appearance of apoptotic cells (i.e., sub-G1 population) and the decrease of S or G1 phase cells suggests that the apoptotic cells were derived from the S-phase cell population. The moderate accumulation of cells in the G1 phase observed in ML-1 cells (wt p53) might reflect the activation of p53 in response to the drug treatment. As described above, incubation of ML-1 cells with 10 nM FMdC led to a time-dependent accumulation of p53 protein, suggesting this molecule is likely to be involved in the cellular response to FMdC. Interestingly, under the same drug incubation conditions (10 nM FMdC for up to 24 h), no significant change in p21 protein was observed (data not shown). Further studies are needed to assess the molecular mechanisms responsible for the different cell-cycle disturbances caused by FMdC in various cell lines.
The important role of FMdC incorporation into DNA in causing
cytotoxicity was further demonstrated in our experiments with aphidicolin, a potent inhibitor of DNA replication. Although inhibition of DNA synthesis by aphidicolin alone did not affect cell viability, incubation of cells with aphidicolin before the addition of FMdC to
prevent incorporation of the analog substantially reduced FMdC-induced apoptosis (Fig. 5C). This result suggests that incorporation of FMdC
into DNA is a critical event in triggering apoptosis. This observation
was consistent with the reported actions of other nucleoside analogs,
such as fludarabine and gemcitabine, whose incorporation into cellular
DNA is also essential in causing cell death (Huang et al., 1990, 1991,
1995).
In summary, our study indicates that incorporation of FMdC into DNA is
a key biochemical event responsible for the cytotoxic action of this
analog against tumor cells. FMdC was incorporated into the C site of
the DNA strain. Once FMdC nucleotide was incorporated into DNA, further
extension of the 3'-FMdCMP-DNA strand by DNA polymerases was difficult.
The incorporated analog seemed to be resistant to excision by the 3'
5' exonuclease activity of pol and p53. The chain-termination
activity of FMdC is likely to cause cells to pause in the S phase of
the cell cycle and ultimately lead the initiation of processes that
promote cell death. It is also possible that if the wild-type p53
protein is present in the cells, the terminally incorporated FMdC
residues in DNA may serve as a mechanism to activate p53, trigger cell
cycle arrest in G1/S boundary, and ultimately
lead to cell death due to failure in removing the incorporated analog
from DNA.
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Footnotes |
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Received June 19, 2001; Accepted October 5, 2001
This work was funded in part by grants CA77339, CA28596, and Cancer Center Support grant CA16672 from the National Cancer Institute, Department of Health and Human Services, and by Matrix Pharmaceutical, Inc., Freemont, CA.
Peng Huang, M.D., Ph.D., Department of Molecular Pathology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. E-mail: phuang{at}mdanderson.org
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Abbreviations |
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FMdC, (E)-2'-deoxy-2'-(fluoromethylene)cytidine (tezacitabine); pol, DNA polymerase; FMdCMP, (E)-2'-deoxy-2'-(fluoromethylene)cytidine-5'-monophosphate; FMdCTP, (E)-2'-deoxy-2'-(fluoromethylene)cytidine-5'-triphosphate; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; PBS, phosphate-buffered saline; wt, wild-type.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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, FMdCTP; 
, dCTP. Data are
presented as the mean ± S.D. from three separate experiments.
, 18-mer, pol 
, 18-mer, pol I. Data are
presented as the mean ± S.D. from three separate experiments. C,
kinetic assay was performed for 17-base or 18-base primer extension by
pol 
and p53
Various concentrations of the normal 17-base primer/template and
3'-FMdCMP-18-base primer/template were incubated with purified either
pol 
) and HL-60 (