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From
Received for publication, June 4, 2002, and in revised form, August 9, 2002
Mitomycin C (MC) is a cytotoxic chemotherapeutic
agent that causes DNA damage in the form of DNA cross-links as well as
a variety of DNA monoadducts and is known to induce p53. The various DNA adducts formed upon treatment of mouse mammary tumor cells with MC
as well as 10-decarbamoyl MC (DMC) and 2,7-diaminomitosene (2,7-DAM),
the major MC metabolite, have been elucidated. The cytotoxicity of DMC
parallels closely that of MC in a number of rodent cell lines tested,
whereas 2,7-DAM is relatively noncytotoxic. In this study, we
investigate the ability of MC, DMC, and 2,7-DAM to activate p53 at
equidose concentrations by treating tissue culture cell lines with the
three mitomycins. Whereas MC and DMC induced p53 protein levels and
increased the levels of p21 and Gadd45 mRNA, 2,7-DAM did not.
Furthermore, MC and DMC, but not 2,7-DAM, were able to induce apoptosis
efficiently in ML-1 cells. Therefore the 2,7-DAM monoadducts were
unable to activate the p53 pathway. Interestingly, DMC was able to
initiate apoptosis via a p53-independent pathway whereas MC was not.
This is the first finding that adducts of a multiadduct type
DNA-damaging agent are differentially recognized by DNA damage
sensor pathways.
Mitomycin C (MC)1 (Chart
1), a natural antibiotic and cytotoxic cancer
chemotherapeutic agent is used in the
clinical treatment of several human malignancies (1). MC induces DNA
damage in the form of DNA cross-links and monofunctional DNA alkylation products in various bacterial and mammalian cells and tissues (2-7).
Treatment of mammalian cells with MC causes an increase in the cellular
p53 level (8). In cells, MC is enzymatically reduced, yielding reactive
species that are capable of producing radicals through redox cycling,
as well as a variety of DNA adducts. Six major adducts are formed as
shown in mouse mammary tumor cells and their precise molecular
structures have been elucidated (4). Very recently, the MC derivative
decarbamoyl mitomycin C (DMC; Chart 1) was shown to generate an array
of DNA cross-links and monoadducts in EMT6 mouse mammary tumor cells
that had similar or identical structures to those formed with
MC.2 One of these, a
monoadduct that is a stereoisomer of one of the monoadducts
formed by MC is formed predominantly upon treatment with
DMC.2 In contrast, another mitomycin derivative,
2,7-diaminomitosene (2,7-DAM), the major metabolite of MC in tumor
cells (10), forms only monofunctional DNA adducts in the same cell line
(4, 11). A comparison of the three drugs with respect to their DNA
cross-linking and monoalkylating activities and their specific DNA
adducts formed in the EMT6 cells is summarized in Table I and the
corresponding DNA adducts are shown in Chart
2.
Comparative studies of the cytotoxicity of the same three mitomycins
indicated that MC and DMC have closely similar cytotoxicities (12-14),
whereas 2,7-DAM is essentially noncytotoxic in the cell lines tested
(11, 15). Because the cytotoxicity of these drugs correlates with the
frequencies of the DNA cross-link adducts but not with the DNA
monoadducts, these findings point to cross-links as being the lesions
primarily responsible for the cytotoxicities of the
mitomycins.2 It is therefore apparent that the specific
structures of the various DNA adducts play a differential role in the
cytotoxicity of MC. The basis for a differential cytotoxic activity of
the individual DNA adducts of MC is not known. However, p53 induction has been correlated in many instances with the cytotoxic activity of
chemotherapeutic drugs (16). Although it was known that treatment of
mammalian cells with MC results in an increase in the level of p53 (17)
the outcome on p53 induction by DMC and 2,7-DAM had not been
investigated. Little is known about how particular DNA adducts can
activate a signal transduction program that culminates in p53
accumulation. Furthermore, studies focused on the cytotoxic activities
of MC, DMC, or 2,7-DAM have not addressed the possible involvement of
the tumor suppressor p53 in mediating the cytotoxic or apoptotic
response. It may be hypothesized that the cytotoxic mechanism of MC is
mediated, at least in part, by the tumor suppressor protein p53 (17,
18). The ability of DNA adducts to induce p53 has been implicated by
the fact that agents that generate multiple type DNA adducts also cause
the accumulation of p53 (19, 20). However, it has been difficult to
recognize a general correlation between cytotoxicities and the
induction of p53 thus far (Ref. 21 and references therein). The lack of
cytotoxicity of 2,7-DAM in contrast to MC and DMC may be explained by
the hypothesis that 2,7-DAM-DNA monoadducts are unable to induce a
signal transduction pathway that culminates in the induction of p53. We
tested this hypothesis by comparing the induction of p53 in a wild-type
p53 containing myeloid leukemia cell line (ML-1) by MC, DMC, and
2,7-DAM. We report here a dramatic differential p53 response in ML-1
cells treated with MC and DMC versus treatment with 2,7-DAM.
We further show that a human myeloid leukemia cell line (K562), lacking
functional p53 is resistant to MC but not DMC cytotoxic effects.
Reagents--
Camptothecin, propidium iodide, MTT assay
reagents, and anti-actin were purchased from Sigma. Zeocin was
purchased from Invitrogen. RPMI 1640 and fetal bovine serum (FBS) were
purchased from Invitrogen. D. M. Vyas (Bristol-Myers Squibb
Co.) supplied MC. 2,7-DAM and DMC were synthesized from MC as
previously described (12, 22).
Cell Culture--
The ML-1 cells were a generous gift from
Michael Kastan. This myeloid leukemia cell line was grown in RPMI 1640 with 10% FBS and 5% CO2. The K562 leukemia cell line was
purchased from ATCC and does not contain p53 (23). The cells were
seeded at a density of 2.5 × 105/ml and exponentially
growing cells were used in all experiments.
MTT Cytotoxicity Assay--
ML-1 or K562 cells were grown in 1×
RPMI supplemented with 10% FBS. Exponentially growing cells were
seeded at 2.5 × 105/ml in 24-well plates and either
left untreated or treated with graded dosages of MC, DMC, or 2,7-DAM
for 24 h. Cells were spun down and re-suspended in 0.5 ml of MTT
containing medium (0.5 mg/ml), and incubated at 37 °C for
1 h. Cells were spun down and re-suspended in 1 ml of 0.04 N HCl in isopropyl alcohol to lyse the cells. After 5 min
at room temperature, samples were spun down and 250-µl aliquots were
used for absorbance measurements. Cell viability was measured as the
difference in the absorbance 550 and 620 nm. The assay detects living,
but not dead cells. Data are expressed as a percent of the control
healthy growing strain.
Flow Cytometry--
FACS analysis was carried out on a BD
Biosciences scan. Cells were spun down at 2300 rpm for 7 min,
washed twice with phosphate-buffered saline, and resuspended in
20 ml of phosphate-buffered saline containing 2% bovine serum albumin
and 0.1% NaN3. Ethanol (9 ml) was then added dropwise
while vortexing. Propidium iodide staining and RNase treatment were
carried out at 37 °C for 30 min 24 h prior to flow cytometry.
Nuclear Extract Preparation--
Nuclear extract was
prepared using a variation on the Dignam Protocol (24). Cells were spun
down and resuspended in 5 packed cell pellet volumes of buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM phenylmethylsulfonyl fluoride,
0.5 mM dithiothreitol). They were then put on ice for 10 min prior to centrifugation for 10 min at 2,000 rpm. The pellet was
resuspended in 2 packed cell pellet volumes of buffer A (volume prior
to the initial wash). The cells were run through a 25-gauge needle
twice and nuclei were then spun down at 2,000 rpm for 10 min followed
by an additional 20-min spin at the 15,000 rpm. The pellet was
resuspended at 109 cells per 3 ml of buffer B (20 mM HEPES, pH 7.9, 25% glycerol, 0.42 mM NaCl,
1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM
dithiothreitol) by running it through a 25-gauge needle twice. The
suspension was rocked gently for 30 min at 4 °C. The extract was
centrifuged for 30 min at 15,000 rpm and the supernatant aliquots were
stored at Western Blot Analysis--
Samples were electrophoresed on a
10% SDS-PAGE and electrotransferred to nitrocellulose. Blots were
probed with a mixture of monoclonal antibodies specific to p53 (pAbs
1801, 240, and 421). PARP cleavage was detected using a monoclonal
anti-PARP antibody from Pharmingen. The blots were probed with
anti-actin (Sigma) to normalize for gel loading. The signals were
visualized after incubation with goat anti-mouse or goat anti-rabbit
secondary antibody using ECL solutions.
Quantitative Real-time PCR with Molecular Beacon--
For each
sample, 3 µg of cytoplasmic RNA obtained with TRISOL (Sigma) was
incubated at 65 °C for 10 min with 200 pmol of
oligo(dT)15 primer (Roche Molecular Biochemicals) in
a total volume of 10 µl. After cooling on ice, 10 µl of reverse
transcriptase mixture was added: 2× AMV buffer, 12.5 units of avian
myeloblastosis virus (Roche Molecular Biochemicals), 5 mM dNTP, 40 units of RNase inhibitor RNasin (Promega).
Samples were incubated for 1 h at 37 °C. Heating samples at
95 °C for 1 min stopped reactions. Sample volumes were then adjusted
to 100 µl with H2O and samples were stored at
For PCR with molecular beacons, 10 µl of the diluted RT products were
used. The reactions were carried out under the following conditions.
1× TaqMan buffer (PerkinElmer Life Sciences), 2.5 mM MgCl2, 250 µM dNTP, 15 pmol of
each primers, 2.5 units of AmpliTaq Gold polymerase (PerkinElmer Life
Sciences), and 125 ng of the appropriate molecular beacon. Forty cycles
of amplification (94 °C denaturation for 30 s, 55 °C
annealing for 1 min, and 72 °C elongation for 30 s) were
carried out in sealed tubes in an Applied Biosystems 7700 Prism
spectrofluorometric thermal cycler (PerkinElmer Life Sciences).
Fluorescence was measured during the annealing step and plotted
automatically for each sample. The primer pairs used for the PCR
reactions were synthesized by Operon and were the following:
P21, forward primer, 5'-ACCTTCCAGCTCCTGTAACATACT-3', antisense primer, 5'-GTCTAGGTGGAGAAACGGGAA-3'; Gadd45,
forward primer, 5'-CCATGCAGGAAGGAAAACTATG-3', antisense primer,
5'-CCCAAACTATGGCTGCACACT-3'; GAPDH, forward primer,
5'-AGAGCACAAGAGGAAGAGAGAGACC-3', antisense primer,
5'-AACTGTGAGGAGGGGAGATTCAG-3'. The sequences of the molecular beacons
were the following: P21,
5'-CGCTGCAGGACACATGGGGAGCCGAGCAGCG-3'; Gadd45,
5'-CGCTGCAGAATGGTTGAGTTACATTAAAATAAACCGCAGCG-3'; and GAPDH, 5'-GGACGCGGTGGGGGACTGAGTGTGGCGTCC-3'.
Electrophoretic Mobility Shift Assays--
Synthetic
oligonucleotides were purchased for this study from Operon. The
superconsensus site contained three adjacent p53 half-sites (26).
The sequence of this oligonucleotide was
5'-TCGAGCCGGGCATGTCCGGGCATGTCCGGGCATGTC-3'. Labeling of the
oligonucleotides was performed with the large fragment of DNA
polymerase I and [32P]dCTP. Electrophoretic mobility
shift assay experiments (30 µl) were carried out in reaction mixtures
with 150 pmol of 32P-oligonucleotide. 10 µg of nuclear
extract was added and the reaction was incubated for 20 min at room
temperature in a reaction buffer containing 20 mM HEPES, pH
7.8, 100 mM KCl, 1 mM EDTA, pH 8.0, 1 mM dithiothreitol, 1 µg of sheared salmon sperm DNA, and
10% glycerol. Reactions were carried out in the presence or absence of
pAb421 as indicated. Samples were separated by 4% polyacrylamide gel
electrophoresis (gels were pre-run at 100 V for 15 min at 4 °C) at
200 V for 3-3.5 h. Gels were dried for 1 h at 55 °C and autoradiography was performed.
MC and DMC but Not 2,7-DAM Can Induce p53 Nuclear
Accumulation--
The noncytotoxic effect of 2,7-DAM suggested that
the DNA monoadducts produced in cells after treatment with this
compound were unable to induce a signal transduction pathway that
culminated in the induction of p53. We tested this hypothesis by
treating the ML-1 cell line (a well established line for its p53
response to various different DNA damaging drugs) with MC, DMC, and
2,7-DAM, as well as with two other drugs shown previously to induce p53 (17). Treatment was carried out with the mitomycins at a concentration of 5 µM for 3 and 6 h, and nuclear extracts were
prepared and analyzed by Western blot as shown in Fig.
1. Both MC and DMC induced a robust
stabilization of p53 (Fig. 1, lanes 2-5) at both time points, whereas no p53 stabilization was detected in cells that had
been treated with the same concentration of 2,7-DAM (Fig. 1,
lanes 6 and 7). Higher doses of 2,7-DAM (50-100
µM) also failed to induce detectable levels of p53 (data
not shown). It can be stated with high certainty that 2,7-DAM was
unable to induce p53 as the Western blot analysis was carried out using
a mixture of p53 monoclonal antibodies specific to the central domain
as well as the carboxyl and amino terminus (pAb240, pAb421, and
pAb1801, respectively). It is therefore highly unlikely that any p53
induced by 2,7-DAM would go undetected by the Western blot procedure
employed. Both MC and DMC were able to induce p53 to levels comparable
with those induced by treatment of the ML-1 cells with camptothecin (a
topoisomerase targeting poison) and Zeocin (a member of the family of
bleomycins) (Fig. 1, lanes 8-11).
MC and DMC Treatment but Not Treatment with 2,7-DAM Results in the
Transcriptional Activation of p53 Target Genes--
The tumor
suppressor p53 is a transcription factor that activates a multitude of
downstream target genes (27). Two p53 downstream target genes that are
well characterized are p21waf1 and gadd45 (28, 29).
We analyzed the transactivation of the endogenous p21 and
gadd45 genes in the ML-1 cell line in response to the
chemotherapeutic drug treatments shown above. The drugs able to result
in the stabilization of p53 (MC, DMC, Cpt, and Zeocin) also induced
significant transactivation of the endogenous p21 and gadd45
genes as monitored by RT-PCR with molecular beacons (Fig.
2, A and B,
respectively). The ML-1 cells treated with 2,7-DAM on the other hand
showed very limited transactivation of the endogenous p53 target genes
tested and this limited activation decreased at the longer time
point (Fig. 2, A and B).
The p53 Induced by MC and DMC Can Bind to DNA--
The treatment
of ML-1 cells with both MC and DMC was able to induce p53 and also
resulted in the activation of p21 and gadd45 transcription.
This suggested that treatment of the ML-1 cells with both drugs
resulted in stabilization of the p53 species that were able to bind to
DNA. We analyzed the DNA binding ability of p53 in nuclear extracts
derived from ML-1 cells treated with MC, DMC, and 2,7-DAM by EMSA (Fig.
3A). Although MC and DMC form different DNA adducts (Table I) both
resulted in an increase in the level of p53 (Fig. 1) and an increase in
the p53 DNA binding ability (Fig. 3A, lanes 2 and
3) when ML-1 cells were treated with a drug concentration of
5 µM for 6 h. Not surprisingly, no p53-specific gel
shift was detected in nuclear extract derived from ML-1 cells treated
with 5 µM 2,7-DAM for 6 h (Fig. 3A,
lane 4). The pAb421 induced gel shift species detected in
the ML-1 nuclear extract from drug-treated cells has been determined to be specific by competition analysis with nonlabeled specific and nonspecific oligonucleotide and a representative example is shown (Fig.
3B).
MC and DMC but Not 2,7-DAM Are Able to Induce Apoptosis Efficiently
in the ML-1 Cell Line Containing Wild-type p53--
Because treatment
of ML-1 cells with MC and DMC, but not 2,7-DAM, was able to result in
the stabilization of p53 we asked if this correlated with the ability
of MC and DMC, but not 2,7-DAM, to induce apoptosis. ML-1 cells were
treated with 5 µM MC, DMC, or 2,7-DAM for 24 h and
the cells were then fixed and stained with propidium iodide. The
samples were analyzed by FACS and compared with nondrug-treated cells
to assess the increase in sub-G1 DNA content (Fig.
4A). Both the MC- and
DMC-treated ML-1 cells showed a substantial increase in
sub-G1 DNA content (although not as dramatic as that seen
with Cpt), indicating that the ML-1 cells initiated apoptosis in
response to these drugs. The ML-1 cells treated with 2,7-DAM on the
other hand showed no increase in sub-G1 DNA content after
24 h of treatment, indicating that these cells were in fact not
undergoing cell death. Apoptosis was also monitored by PARP cleavage
and this further confirmed the induction of apoptosis by MC and DMC but
not by 2,7-DAM treatment (Fig. 4D).
DMC but Not MC or 2,7-DAM Is Able to Induce Apoptosis in the K562
Cell Line Lacking Functional p53--
To address the ability of MC
and DMC to induce apoptosis in the absence of p53, the K562 cell line
(a line without p53 (23)) was treated and analyzed in the same way. No
substantial increase in sub-G1 DNA content was observed
when the K562 cells were treated for 24 h with MC or 2,7-DAM (Fig.
4, B and E). Surprisingly, although K562
cells did not undergo apoptosis when treated with MC, a substantial increase in apoptotic cells resulted after DMC treatment as determined by FACS analysis and PARP cleavage (Fig. 4, B and
E). This demonstrates that DMC was able to induce
apoptosis in a p53-independent manner whereas MC was not. Therefore,
although DMC is able to induce p53, its entire cytotoxic
effect does not require p53.
Cytotoxicitities of MC, DMC, and 2,7-DAM to the ML-1 (Wild-type
p53) and K562 (p53 Null) Cell Lines--
Cytotoxicity can be observed
in the absence of apoptotosis (30) and therefore the cytotoxicities of
the panel of mitomycins were compared utilizing the ML-1 cells to
confirm that 2,7-DAM was not cytotoxic. Cytotoxicity of the three
mitomycins was monitored by the MTT cytotoxicity assay (Fig.
5) as well as by trypan blue exclusion
(data not shown). Cytotoxicity was observed when ML-1 cells were
treated with MC and DMC but not when they were treated with 2,7-DAM
(Fig. 5A). Therefore 2,7-DAM treatment of ML-1 cells did not
induce p53, did not induce apoptosis, and was not cytotoxic to the ML-1
cells. We also analyzed the cytotoxicity of these drugs in the p53 null
cell line K562 to investigate their p53 independent cytotoxicities.
Under these conditions 2,7-DAM was observed to be noncytotoxic, whereas
MC demonstrated limited cytotoxicity and DMC was cytotoxic at the 5 and
10 µM drug concentrations (Fig. 5B). This
correlated with the observation that DMC was able to induce PARP
cleavage in the K562 cell (Fig. 4E). Although Fig. 5
demonstrates that DMC is not as cytotoxic to K562 cells as it is to
ML-1 cells, it is clearly more cytotoxic to K562 cells than either MC
or 2,7-DAM.
DNA Adducts Do Not Always Activate the p53 Pathway--
It is
often stated that DNA damage activates the tumor suppressor p53.
Normally p53 is present at low levels and specific stimuli are able to
elicit signal transduction pathways that allow for the stabilization of
p53. Chemotherapeutic drugs that damage DNA function to a great extent
by activating pathways that result in p53 stabilization (17). MC is a
clinically relevant chemotherapeutic drug whose mode of action
continues to be investigated. Here we demonstrate that, in contrast to
the DNA adducts induced by MC and DMC, the monofunctional derivative of
MC 2,7-DAM allows for DNA adducts that are unable to activate p53. Thus
this form of DNA damage evades the p53 tumor suppressor pathway and is
not cytotoxic.
Previously it was shown that MC and DMC are equally cytotoxic in
various rodent cell lines (12-14). The present work extends this
finding to human myeloid leukemia cells. The cytotoxicity of MC has
been attributed mainly to the generation of lethal DNA cross-links. DMC
was initially thought to be devoid of DNA cross-linking activity, based
on chemical considerations and on tests carried out in cell-free
systems that employed chemical reductive drug activation (13, 31).
Consequently, DMC has traditionally been regarded as the monofunctional
counterpart of MC; however, this is not the case. We demonstrated
recently, using a mouse mammary tumor cell line, that DMC generates the
DNA cross-link adduct 3 (see Chart 2) at frequencies
comparable with those observed with MC under equidose drug treatment
conditions. In contrast, the frequencies of monoadducts, also generated
by both drugs, are widely different, with DMC displaying 20-50-fold
greater monoadduct frequencies than MC2 (see Table I and
Chart 2). These findings suggest that the DNA cross-links, common to
both drugs, are major determinants of the drugs cytotoxicity and
possibly of their abilities to induce the stabilization of p53.
A Relationship Exists between p53 Induction and Cytotoxicity of
Mitomycins--
We show here that the ability of the two cytotoxic
mitomycins to form DNA cross-links correlates with the ability of these drugs to induce the p53 pathway. The 2,7-DAM-DNA adducts do not contribute to cytotoxicity of MC (11, 15). In keeping with this lack of
cytotoxicity of 2,7-DAM we show here that this compound is also unable
to induce p53, suggesting that there is a connection between MC
cytotoxicity and the ability to induce p53 in ML-1 cells. Indeed, of
the three mitomycin drugs MC, DMC, and 2,7-DAM investigated here, only
MC and DMC are significantly cytotoxic and only the same two drugs
elicit the accumulation of p53. Therefore the ability of MC and DMC to
signal for the induction of p53 may require the formation of DNA
interstrand cross-links. However, the work presented here does not rule
out the possibility that monoadducts, as long as they are not the ones
produced by 2,7-DAM, might have the ability to activate the p53 pathway.
In fact, no direct relationship seems to exist between the structural
nature of the DNA damage and stabilization of p53. UV light-induced DNA
structural damage does not include DNA cross-links, yet p53 is
stabilized as a result (17). O6-Methylguanine
lesions of DNA, produced by the simple alkylating agent
N-methyl-N'-nitro-nitrosoguanidine induce p53
efficiently (32); antibenzo[a]pyrene-diolepoxide, which
forms bulky N2-guanine monoadducts also induces p53 (33).
Thus, p53 induction can occur by specific types of monofunctional DNA
lesions. It is likely to be mediated by the action of proteins specific
to the particular structure of the lesion (see Ref. 32). Highly relevant to the present findings, it has been shown that DNA
interstrand cross-links, formed by psoralen, induce "futile repair
synthesis" (34), and the authors suggest that this process, rather
than the cross-links per se, is responsible for inducing the
p53 pathway. It is conceivable that the differential effects of the
three mitomycin derivatives originate from the analogous phenomenon.
The fact that a mechanism exists in cells through reductive conversion
of MC to 2,7-DAM to evade the cytotoxic potential of MC (11, 15) should
be considered. It is likely that cells defective in this conversion
will be more sensitive to MC treatment. Furthermore, derivatives of MC
and/or MC analogues that cannot be metabolized (and thereby
inactivated) to 2,7-DAM may be more effective in treating malignancies.
Because of this DMC may be considered a more compelling clinical choice
for use as a chemotherapeutic. DMC is cytotoxic, can induce p53 and
does so in the absence of producing DNA adducts that can be evaded.
Cytotoxicity and Induction of Apoptosis by DMC in the K562 Myeloid
Leukemia Cell Line Lacking p53--
In contrast to MC, DMC was also
capable of inducing apoptosis in the absence of p53 (as observed by
drug treatment of the K562 myeloid leukemia cell line lacking p53).
Although DMC was not as cytotoxic to K562 cells as it was to ML-1
cells, DMC was more cytotoxic to K562 cells than either MC or 2,7-DAM.
This may be clinically relevant especially given the fact that over
50% of total human cancers have defective or mutated p53 genes (35). Perhaps this cytotoxicity results because DMC is able to activate an
alternative DNA damage pathway that utilizes either p73 or BRCA1 (9,
36). This may be a reason for considering DMC for use in cancer
chemotherapy. When choosing drugs to treat specific cancers it will be
important in general to consider alternate DNA damage-mediated pathways.
We acknowledge Shereaf Walid for technical
assistance and thank Sara Rockwell for comments on the manuscript.
*
This work was supported by National Institutes of Health
SCORE Grant 1S06 GM60754 and National Science Foundation Award
MCB-9722262 (to J. B.), National Institutes of Health NCI Grants
CA 28681 and CA 71961 (to M. T.), and National Institutes of
Health Research Centers in Minority Institutions Award RR-03037 from
the Division of Research Resources to Hunter College.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Present address: CRIC, Group of Molecular Carcinogenesis, 150 cours
Albert Thomas, 69372 Lyon, France.
¶
Supported by an MBRS grant to Hunter College.
§§
To whom correspondence should be addressed. E-mail:
bargonetti@genectr.hunter.cuny.edu.
Published, JBC Papers in Press, August 14, 2002, DOI 10.1074/jbc.M205495200
2
Palom, Y., Suresh Kumar, G., Tang, L.-Q.,
Paz, M. M., Musser, S. M., Rockwell, S., and Tomasz, M. (2002)
Chem. Res. Toxicol., in press.
The abbreviations used are:
MC, mitomycin
C;
DMC, decarbamoyl mitomycin C;
2, 7-DAM, 2,7-diaminomitosene;
RT, reverse transcriptase;
FBS, fetal bovine serum;
FACS, fluorescence-activated cell sorter;
pAb, polyclonal antibody;
PARP, poly(ADP-ribose)polymerase;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
Differential Activation of p53 by the Various Adducts of
Mitomycin C*
,§,¶,¶,
,
,§§
The Institute for Biomolecular Structure and
Function, and Department of Biological Sciences, Hunter College and
Graduate School and the ** Department of Chemistry, Hunter
College, CUNY, New York, New York 10021
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Chart 1.
Structure of mitomycins.
Frequencies of DNA monoadducts and cross-links formed in EMT6 mouse
mammary tumor cells treated with MC, DMC, or 2,7-DAM at 10 µM concentration
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Chart 2.
The major DNA adducts formed in MC-treated
mouse mammary tumor cells.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
80 °C.
80 °C.
PCR primer pairs were designed to anneal to their target at the same
temperature (55 °C) and to amplify DNA fragments of ~100 bp as
described previously (25).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
MC, DMC, but not 2,7-DAM induce p53 nuclear
accumulation. Exponentially growing ML-1 cells, grown in 1× RPMI
medium and supplemented with 10% fetal bovine serum were either left
untreated or treated with 5 µM MC, 5 µM
DMC, 5 µM DAM, 0.5 µM camptothecin
(CPT), or 50 µg/ml Zeocin for 3 (lanes 2,
4, 6, 8, and 10) or 6 (lanes 3, 5, 7, 9, and
11) hours. 100 µg of nuclear protein was resolved by
electrophoresis on a 10% SDS-PAGE, transferred to a nitrocellulose
membrane, and probed with either a mixture of p53-specific monoclonal
antibodies (pAb240, pAb421, and pAb1801) or anti-actin. Results are
representative of four independent experiments.
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Fig. 2.
MC and DMC treatment but not treatment with
2,7-DAM results in the transcriptional activation of p53 target
genes. Quantitative real-time RT-PCR with molecular beacons was
used to analyze p21waf1 (A) and
gadd45 (B) expression. Results were
normalized using the control samples and the glyceraldehyde-3-phosphate
dehydrogenase values to give relative units of mRNA induction.
Bars represent the -fold induction of p21 and
gadd45 as shown. Results are representative of two
independent experiments.
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Fig. 3.
MC- and DMC-induced p53 exhibits DNA binding
activity. A, gel mobility shift assay of p53
superconsensus site with nuclear proteins extracted from ML-1 cells.
ML-1 cells were grown in 1× RPMI medium, supplemented with 10% FBS.
Exponentially growing cells were either left untreated (lane
1), or treated with 5 µM MC (lane 2), 5 µM DMC (lane 3), or 5 µM DAM
(lane 4) for 6 h. Nuclear extracts were then incubated
with 32P-labeled DNA oligonucleotides corresponding to the
p53 superconsensus site (SCS), in the presence of
p53-specific monoclonal pAb421 antibody, electrophoresed on a 4%
polyacrylamide gel, and visualized by autoradiography. B,
competition of the pAb421-induced gel shift by unlabeled
oligonucleotide corresponding to the SCS and GADD45 p53-binding sites
as well as a nonspecific oligonucleotide were used as indicated.
Lanes 1 and 2 contain extract from untreated ML-1
cells. Lanes 3-10 contain nuclear extract from cells
treated with 20 µM camptothecin (CPT).
View larger version (37K):
[in a new window]
Fig. 4.
MC and DMC, but not 2,7-DAM induce apoptosis
in ML-1 cells whereas only DMC induces p53 independent apoptosis.
A and B, FACS analysis of ML-1 and K562 cells.
Cells were grown in 1× RPMI supplemented with 10% FBS. Exponentially
growing cells were either left untreated (control) or
treated with 5 µM MC, DMC, DAM, or 0.5 µM
camptothecin (CPT) for 24 h. Cells were fixed with 30%
ethanol, stained with propidium iodide for 24 h, and analyzed by
flow cytometry cell sorting. C, comparison of
sub-G1 DNA content in ML-1 and K562 cells. D,
Western blot analysis using anti-human PARP antibody. Nuclear proteins
extracted from ML-1 cells were resolved by 10% SDS-PAGE. Drug
treatment was for 3 and 24 h except in the case of camptothecin,
which was for 3 h as indicated. Anti-actin was used as a loading
control (data not shown). E, Western blot analysis of
nuclear extracts of K562 cells untreated or treated with camptothecin
(0.5 µM), MC (5 µM), DMC (5 µM), or 2,7-DAM (5 µM) for 24 h.
Extracts were resolved by 10% SDS-PAGE and probed with anti-human PARP
antibody.
View larger version (12K):
[in a new window]
Fig. 5.
MC, DMC, but not 2,7-DAM are cytotoxic in
ML-1 cells, and only DMC is cytotoxic in K562 cells. MTT
cytotoxicity assay of ML-1 (A) and K562 (B)
cells. Exponentially growing ML-1 and K562 cells were either left
untreated or treated with graded doses of MC, DMC, or 2,7-DAM for
24 h. Cells were then incubated in MTT containing medium
(0.5 mg/ml) at 37 °C for 1 h. Aliquots of cell lysate were used
for absorbance measurements at 550 and 620 nm and the difference was
calculated. Data are expressed as percent of control untreated cell
line sample. Bars represent the S.E. ± mean of two
independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
Present address: Cell Biology Program, Memorial
Sloan-Kettering Cancer Center, 430 E. 67th St. New York, NY 10021.
On leave of absence from Indian Institute of Chemical Biology.
ABBREVIATIONS
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MATERIALS AND METHODS
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