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Carcinogenesis |
Curriculum in Toxicology, University of North Carolina, Chapel Hill, North Carolina 27514 [R. A. H., J. C. B.], and Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 [R. A. H., C. A. A., J. C. B.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recent studies in Saccharomyces cerevisiae have shown the involvement of MSH2/MSH6-dependent MMR in the repair of adenines misincorporated opposite the oxidatively damaged base 8-oxoguanine (11) . Experiments with mutS-deficient Escherichia coli strains demonstrated the ability of the MMR system to recognize and repair exocylic DNA adducts such as those formed during endogenous lipid oxidation (12) . Furthermore, mouse embryonic stem cells with defective Msh2 alleles are more resistant to oxidative damage generated by low-level radiation (13) . On the basis of this evidence, it is possible that at least a portion of the differential cytotoxicity induced by certain DNA-damaging agents is a result of variations in oxidative stress responses between MMR+ and MMR- cells. This becomes especially important considering that endogenous generation of oxidatively damaged bases is a continuous occurrence in the DNA of normal, healthy individuals (14) and also can be influenced by numerous environmental factors (15) .
We designed the experiments described in this report to test the hypothesis that absence of MLH1 may provide cells with a selective growth advantage when exposed to specific forms of oxidative stress. We used both human and murine cell line pairs as model systems to study the effects of MLH1 in our experiments. The human HCT116 colon carcinoma cells have a mutant hMLH1 gene, whereas the HCT116+3 cells contain a wild-type hMLH1 gene introduced by the transfer of a normal human chromosome 3. The mouse MC2 embryonic fibroblasts were derived from Mlh1-/- knockout mice, whereas the MC5 fibroblasts have wild-type Mlh1. HCT 116 and MC2 cells both are deficient in MMR activity, resistant to treatment with 6-TG and MNNG and demonstrate increased microsatellite instability (5 , 16 , 17) . The HCT 116+3 cells and the MC5 cells both have proficient MMR activity and significantly reduced microsatellite instability and also exhibit greatly increased sensitivity to both MNNG and 6-TG (17 , 18) .
To assess the role of MLH1 in mediating the effects of oxidative stress on cultured cells, we chose to use two well-characterized mediators of oxidative stress, H2O2 and TBH. H2O2 and TBH are known to generate a variety of different types of oxidative DNA lesions, including base modifications, frameshift mutations, and DNA strand breaks (19, 20, 21) . Recent studies in Escherichia coli have demonstrated the ability of H2O2 to induce frameshift mutations preferentially in microsatellite sequences, and further show that this damage is enhanced in cells lacking MMR activity (22) . Here we demonstrate that MLH1- and Mlh1- cell lines exhibit increased resistance to the cytotoxic effects of H2O2 and TBH compared with the MLH1+ and Mlh1+ cell lines, suggesting the involvement of MLH1 in the cellular survival response to oxidative stress. Moreover, our experiments also suggest that this resistance is the result of a requirement for MLH1 in the transduction of a mitochondrial-mediated apoptotic signal in cells exposed to peroxides.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cytotoxicity Assessment.
Cells were seeded at 4000 cells/well into 96-well plates 24 h
before treatment. H2O2 and
TBH treatments were for 1 h, and IR treatments were for <10 min
to achieve 2-, 4-, 8-, 16-, and 32-Gy doses using a
Cs137 source irradiator (J. L. Sheppard
Associates). Viability was assessed 72 h after treatment using the
MTS-based CellTiter96
AQueous Non-Radioactive
Cell Proliferation Assay (Promega, Madison WI) according to the
manufacturer’s instructions.
Cell Cycle Analysis (Dual-parameter Flow Cytometry).
Cells were pulse-labeled with 10 µM BrdUrd for 20 min,
harvested and processed with FITC-conjugated Anti-BrdUrd (250 ng;
Becton Dickinson, San Jose, CA) according to the manufacturer’s
instructions, then stained with PI (final concentration 5 µg/ml) and
analyzed using a FACScan flow cytometer (Becton Dickinson).
Quantitative analyses and cell cycle histograms were generated using
the CellQuest 3.1 software program (Becton Dickinson).
DNA Fragmentation Analysis.
The floating cell population from each sample was collected at 24 h after treatment. Genomic DNA was prepared and the entire sample
analyzed for laddering on a 0.8% agarose gel as described previously
(23)
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Microscopic Analysis of Nuclear Morphology.
Cells were plated at equal densities on glass slide chambers. At
24 h after H2O2
treatment, the cells were fixed in methanol:acetic acid (3:1) and then
stained with 20 µg/ml PI for 15 min before being analyzed using a
Zeiss Axiophot fluorescent microscope coupled with a Spot CCD camera
system (Olympus).
Active Caspase 3 Measurement.
The entire cell population from each sample was gently harvested at
24 h after treatment, fixed, and incubated with 20 µl of
PE-conjugated primary antibody to active caspase 3 (PharMingen)
according to the manufacturer’s instructions. The cells were rinsed in
PBS and immediately analyzed on a FACSort flow cytometer using the FL1
detector with standard PE settings.
Measurement of DNA Lesion Frequency.
Damage induced in nuclear DNA following a 1-h exposure to
H2O2 and TBH was evaluated
using a quantitative long PCR assay as described by Van Houten et
al. (24)
, with minor modifications. Briefly, 30 ng of
DNA were amplified using primers for a 13.5-kb human
ß-globin gene fragment (sense,
5'-CGAGTAAGAGACCATTGTGGCAG-3'; antisense,
5'-GCACTGGCTTAGGAGTTGGACT-3'). The single-band end products of each PCR
reaction were measured using PicoGreen dsDNA quantitation reagent
(Molecular Probes, Eugene, OR). The results of treated sample
amplifications are presented as relative to the untreated control
amplifications.
Cytochrome C Release Analysis.
Cells were treated with
H2O2 for 1 h. At
24 h after treatment, cells were gently resuspended in cytoplasmic
extract buffer [1 mM EDTA, 60 mM KCl, 10
mM HEPES (pH 7.5), 0.5% NP40, 1 mM
phenylmethylsulfonyl fluoride, 1 mM DTT, and
1 x pepstatin/leupeptin], iced for 30 min, and
homogenized by pulling 10 times through a 25-gauge needle. Nuclei and
cell debris were pelleted by centrifugation at 14,000 x g, and 25 µg of each extract was separated on a 15%
SDS-PAGE gel for Western analysis using anti-cytochrome c
antibody (PharMingen) at a 1:200 dilution with enhanced
chemiluminescence (Amersham) detection.
Assessment of Mitochondrial Permeability.
At 24 h after 1-h treatment with
H2O2, each sample was
resuspended in 5 ml of prewarmed (37°C) medium. A 1-ml aliquot was
incubated with 1 µg/ml of the cationic dye JC-1 (Molecular Probes,
Eugene, OR) for 30 min at 37°C and then analyzed using a conventional
flow cytometer with simultaneous FL1 (green/525 nm) and FL2 (red/590
nm) fluorescence detection. Both parameters are represented on a
density contour plot of each sample population. Quantitative
estimations of JC-1 monomer formation based on gating of the population
gaining FL1 fluorescence in three separate experiments are given in the
lower right corner of each frame in Fig. 7
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. However, the level of sensitivity was significantly
greater in the HCT116+3 (MLH1+) and MC5 (Mlh1+) cell lines when
compared with that of their repair-deficient counterparts. At a dose of
400 µM
H2O2, the mean viability of
the HCT116+3 cell line was 51%, whereas the mean viability of the
HCT116 cell line was 78%. Confirmation of these results was obtained
using a colony-forming assay in which the HCT116+3 cells had 
10%
and HCT116 cells had 60% colony forming at the 400
µM dose (data not shown).
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. Because current reports
of MMR involvement in the repair of IR-induced lesions are
contradictory, we also evaluated the responses in our model system
to IR, a commonly used model for the induction of oxidative stress. All
four cell lines exhibited a similar response to IR, regardless of MMR
status. This is perhaps because IR induces a much higher level of
double-strand breaks and comparatively less base damage than do either
of the peroxides. From these results, we conclude that the presence of
MLH1 or Mlh1 increases sensitivity to peroxide-induced oxidative
stress, but has a negligible effect on survival after high-dose IR.
We next wanted to eliminate the possibility that differences in DNA
damage dosing were mediating the observed cytotoxicity differences. To
determine the relative amounts of DNA damage delivered to each cell
line during treatment with peroxides, we used a quantitative long PCR
assay designed to measure induced levels of polymerase-blocking DNA
damage (24)
. As shown in Fig. 2
, there were no significant differences observed between the HCT116 and
HCT116+3 cell lines in the initial amounts of DNA damage generated
during a 1-h exposure to increasing doses of either
H2O2 or TBH. Furthermore,
analysis of damage levels at several time points after treatment showed
no significant differences, suggesting that the repair of
polymerase-blocking DNA damage between the two cell lines is similar
(data not shown). This suggests that the greater sensitivity of the
HCT116+3 cell line to peroxides is not attributable to an increased
initial DNA damage load placed on the cell.
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, the results indicated that, regardless of
MLH1 status, a significant portion of the treated cells exhibited
reduced DNA synthesis and accumulated in the late S and
G2-M compartments of the cell cycle in a
dose-dependent manner. For example, gating of cells on BrdUrd/PI
dot plots of the 200 µM
H2O2 samples showed that
HCT116 contained an average of 54% of cells in
G2-M and HCT116+3 contained an average of 48% of
cells in G2-M. The reduction of
G2-M content in the HCT116+3 cells at higher
doses of each peroxide correlated with cell death, as evidenced by the
increased sub-2N DNA content.
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, the HCT116+3 cells exhibited a
significant dose-dependent increase at 24 h in the percentage of
sub-2N DNA content after a 1-h exposure to either peroxide. The HCT116
cells only show elevated sub-2N DNA content after high doses of TBH,
but still significantly less than that seen in the HCT116+3 cells. This
is in agreement with the cytotoxicity of TBH seen at these doses (Fig. 1B)
. Overall, the analysis of cell cycle profiles provided
little proof of variations in the integrity of cell cycle checkpoints
after peroxide exposure. The quantitative analysis of cellular
degradation, coupled with simple visual observation of treated cell
cultures, suggested instead that levels of cell death were the most
likely factor contributing to the observed differences in cytotoxicity.
Sensitivity to Peroxides in MLH1-proficient Cells Is Attributable
to Induction of an Apoptotic Pathway.
In an effort to understand better the basis for the differential
cytotoxicity, we set out to determine the mode of cell death incurred
after peroxide exposure in both the MLH1+ and MLH1- cell lines. DNA
was isolated from floating HCT116 and HCT116+3 cells grown in 100
mM dishes after exposure to
H2O2 and TBH and analyzed
for the presence of ladder-like 180-bp DNA fragmentation characteristic
of cells undergoing apoptosis. DNA ladders and floating cell
populations were observed in the HCT116+3 cells, both increasing in a
dose-dependent manner with each peroxide (Fig. 4A)
. No evidence of ladders was seen in the HCT116 cells after
H2O2 or TBH treatment, and
only the highest doses of TBH initiated significant levels of floating
cells. As shown in Fig. 4B
, additional data supporting the
presence of apoptotic cells in the HCT116+3 cell line was obtained
through observation of nuclear morphology in each cell line after
exposure to H2O2. By
staining the cells with PI, we observed condensed and fragmented
nuclei indicative of apoptotic cellular events in the HCT116+3 cells,
whereas the HCT116 cells displayed little if any evidence of morphology
characteristic of apoptotic cell death.
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. Likewise, the resistant HCT116 cells demonstrated little activation
of caspase 3, only being induced at the highest doses of TBH. This data
suggests a role for MLH1 in triggering apoptosis that includes, but
also is upstream of, caspase 3 signaling.
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shows the detection of increased cytochrome c in the
HCT116+3 extracts at both the 400- and 800-µM
H2O2 doses at 24 h
post-treatment. Similar results exhibiting weaker bands were also
observed at 6 h after treatment with the same doses (data not
shown). No significant increases in cytochrome c were
detected in cytoplasmic extracts from identically treated HCT116 cells
at either time point. These results suggest that cytochrome
c release may be an important part of the peroxide-induced
apoptotic pathway in MLH1+ cells.
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m) through the formation of pores transversing both the inner
and outer mitochondrial membranes (30
, 31)
. To determine
the role of mitochondrial permeability in the release of cytochrome
c in HCT116+3 cells, we incubated live cells with the
cationic dye JC-1 and observed the resulting fluorescence using
dual-parameter flow cytometry. JC-1 selectively accumulates in
mitochondria in a m dependent manner where it forms red
fluorescing J-aggregates. In untreated cells from both cell lines, the
majority of JC-1 appears in its red (590 nm) fluorescing dimer form
after being taken up by the mitochondria (Fig. 7)
. A significant dose-dependent appearance of green (525 nm)
fluorescing (nondimerized) cytoplasmic JC-1 molecules was observed in
the HCT116+3 cells after
H2O2 exposure, indicating
an increase in the permeability of the mitochondria. Across each of the
doses tested, the cytoplasmic JC-1 fluorescence in HCT116+3 cells was
significantly higher than in the HCT116 cells. This indicates that loss
of m in MLH1+ cell lines may be an important factor in the
initiation or execution of peroxide-induced apoptosis. |
DISCUSSION |
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Previous studies have indicated a role for Msh2 in the apoptotic response to protracted low-level radiation-induced oxidative stress in murine embryonic fibroblasts (13) . Mice with homozygous deletions of Msh2 were used to demonstrate a role in vivo for Msh2 in the regulation of apoptosis in the small intestine (32) . Also, microinjection of plasmids containing hMLH1 or hMSH2, but not hPMS2, hMSH3, and hMSH6, induces apoptosis in both repair-proficient and -deficient cell lines (11) . Our results support and extend the previous findings by demonstrating the involvement of the MLH1 protein in mediating cellular sensitivity to peroxide-induced oxidative stress via regulation of apoptosis. An important observation supported throughout our work is that with normal background damage levels, the MLH1+ cells exhibit higher levels of apoptosis when compared with MLH1- cells. Therefore, altered regulation of MLH1 expression could be a factor in triggering cell death even under steady-state levels of DNA damage.
A growing body of evidence links the MMR proteins MLH1 and MSH2 to the apoptotic process, yet little is known about the mechanisms through which this might occur. Recent data suggests that MMR competency is required for initiation of an apoptotic response to methylating agents through a pathway thought to involve the p53 tumor suppressor protein (6 , 32, 33, 34) . Much less is known about the pathways involved in the lower-level resistance to such agents as cisplatin and oxidative stress, but they appear to differ from those of the methylators (35, 36, 37) . Key initiators of the apoptotic cascade in most mammalian cell types include the loss of mitochondrial membrane integrity and the subsequent release of cytochrome c. Cytochrome c release from mitochondria after the exposure of cultured cells to such DNA-damaging agents as IR, cisplatin, and methyl methanesulfonate has been documented previously (38) . In our study, we observed both increased mitochondrial permeability and cytochrome c release after H2O2 treatment preferentially in the more sensitive MLH1+ cell line. This suggests that sensitivity to peroxides may be mediated by MLH1 through an apoptotic signal transduction pathway involving the mitochondria.
JNK/SAPK signal transduction pathways are activated after exposure to oxidative stressors, including peroxides (39) . Recent evidence has shown that JNK/SAPK can be activated by genotoxic stressors to translocate to the mitochondria and phosphorylate Bcl-xL at specific Thr-Pro sites (38) . Phosphorylation of Bcl-xL is subsequently hypothesized to promote cytochrome c translocation and lead to the induction of apoptosis. Nehmé et al. (37) reported that MMR-proficient cells activate JNK more efficiently after cisplatin treatment than MMR-deficient cells, and only MMR-proficient cells are able to activate c-Abl. On the basis of these observations, it is intriguing to speculate that the difference in ability of MLH1+ and MLH1- cell lines to undergo apoptosis after exposure to peroxides might be linked to the activation of JNK/SAPK pathways, possibly through their ability to regulate Bcl-2 family members.
Collectively, our work demonstrates that MLH1- and Mlh1- cell lines exhibit increased resistance to the cytotoxic effects of H2O2 and TBH compared with their MLH1+ and Mlh1+ counterparts, suggesting the involvement of MLH1 in the cellular survival response to oxidative stress. Moreover, our experiments also suggest that this resistance is the result of a requirement for MLH1 in the transduction of a mitochondrial-mediated apoptotic signal in cells exposed to peroxides. Given the extensive nature of endogenously generated oxidative DNA damage (15) , this work may hold important implications for understanding the evolution of both sporadic and hereditary tumors of the MMR- phenotype and might prove useful in the development of targeted chemotherapeutic strategies for patients with MMR-deficient tumors.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Laboratory of Molecular Carcinogenesis, National
Institute of Environmental Health Sciences, Research Triangle Park, NC
27709. E-mail: barrett{at}mail.nih.gov 
2 Abbreviations: MMR, mismatch repair; 6-TG,
6-thioguanine; H2O2, hydrogen peroxide; TBH,
tert-butyl hydroperoxide; HCT116+3, HCT 116 + chromosome 3;
BrdUrd, 5-bromo-2'-deoxyuridine; IR, ionizing radiation; JC-1,
5,5',6,6'-tetrachloro-1,1'3,3'tetraethylbenzimidazilylcarbocyanine
iodide; MNNG,
N-methyl-N'-nitro-N-nitrosoguanidine;
PI, propidium iodide; PE, phycoerythrin; JNK/SAPK, c-Jun
N-terminal kinase/stress-activated protein kinase. MTS,
[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,
inner salt].
Received 10/ 5/00. Accepted 12/13/00.
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and mismatch repair deficiency in an ovarian tumor cell line.. J. Biol. Chem., 271: 19645-19648, 1996.- and hMutL-dependent phosphorylation of p53 in response to DNA methylator damage.. Proc. Natl. Acad. Sci. USA, 96: 12384-12388, 1999.This article has been cited by other articles:
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