| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Experimental Therapeutics, Preclinical Pharmacology |
Istituto Superiore di Sanitá, 00161 Rome, Italy [G. A., S. C., S. M., M. C., M. B.]; Molecular Oncogenesis Laboratory, Regina Elena Cancer Institute, 00158 Rome, Italy [S. S., M. C.]; and Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms EN6 3LD, United Kingdom [P. B., P. K.]
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
repair
complex, the variants exhibited alterations in the expression of other
genes that influence drug sensitivity. Although A2780 cells possess a
wild-type p53 gene, all of the clones contained a
heterozygous G to T tranversion at codon 172. This change resulted in a
Val to Phe substitution and was associated with a constitutive
production of high levels of p53, which was inactive as a
transcriptional activator of bax and p21.
The hMLH1/p53 defective variants displayed a less prominent cell cycle
arrest and reduced apoptosis after CCNU treatment. In contrast,
MMR-defective A2780 variants, which had a similar hMutL defect but
retained a wild-type p53, did exhibit the expected CCNU sensitivity.
Expression of a dominant-negative p53val135 increased CCNU resistance
of both MMR-proficient and MMR-deficient A2780 cells. Thus, defective
MMR and p53 influence CCNU sensitivity in opposite directions. Their
effects are independent, and sensitization by defective MMR does not
require a functional p53 response. |
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
In addition to MGMT, base, and nucleotide excision repair, MMR can also
modulate sensitivity to CNUs. The first two steps of the MMR pathway
are catalyzed by the hMutS mismatch recognition heterodimer
(comprising hMSH2 and hMSH6) and the hMutL complex (hMLH1 and
hPMS2). Tumor cell lines defective in either of these complexes are
frequently 2- to 4-fold more sensitive to CCNU than proficient
cells (8)
. The extent of their sensitivity is similar to
that produced by loss of the base excision repair pathway in
Aag-/- cells.
Cellular stress responses may also influence the survival of cells with DNA damage. The responses include cell cycle arrest and apoptosis, and the p53 protein is intimately involved in their control. The p53 protein is a transcriptional activator of several target genes, including p21 and bax, the products of which regulate cell proliferation and survival (9) . Mouse thymocytes with an inactivated p53 gene are resistant to ionizing radiation and chemotherapeutic agents (10, 11, 12) . The enhanced survival of cells with abrogated p53 function is generally considered to reflect the central role of wild-type p53 in inducing apoptosis. Loss of p53 function leads to resistance to chemotherapy and radiotherapy in many tumor cell lines (13, 14, 15, 16) . In addition, p53 mutations in tumors are generally associated with a poorer clinical prognosis (for a review, see Ref. 17 ). On the other hand, hypersensitivity to similar chemotherapeutic drugs is correlated with p53 mutations in several different cell types (18, 19, 20, 21) . The reasons for this apparent discrepancy are not fully understood and may reflect the complexity of p53 functions in controlling cell growth and death. Thus, although it may not be possible to predict the outcome of loss of p53 functions, cell killing after DNA-damaging therapeutic drugs is likely to be influenced by p53 status.
In a previous study, we noted exceptions to a general correlation between MMR defects and sensitivity to CCNU. In view of the central role of p53 in modulating responses to drug treatment, we have examined the impact of both p53 and MMR on CCNU sensitivity. Cultures of the model ovarian carcinoma cell line A2780 were found to contain a subpopulation of drug-naïve cells that had lost both MMR and the p53 response. The latter was most likely because of a Val to Phe mutation in codon 172 of the p53 gene. These doubly defective cells were resistant to CCNU and underwent less prominent cell cycle arrest and apoptosis. When defects in MMR or p53 were analyzed separately in A2780 cells, they were found to contribute independently to the response of the cell to CCNU. We conclude that MMR and p53 both modulate CCNU sensitivity, but their effects are independent and in opposition. The presence of a subpopulation of A2780 cells with MMR and p53 defects indicates that multiple changes that modify drug responsiveness may occur in drug-naïve cells.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Cell Culture, Survival Determination, and Selection for Methylation
Tolerance.
A2780 cells, a kind gift of Dr. R. Brown (Glasgow University), were
cultured in -MEM (Life Technologies, Inc.) supplemented with
10% FCS, penicillin (100 units/ml), and streptomycin (100 µg/ml;
complete medium). Cultures were incubated at 37°C in 5%
CO2 and 95% relative humidity. The cloning
efficiency of the A2780 cell lines varied considerably: 30% for
the parental A2780 and SC1 cells, 15% for MNU-1A and MNU-1B, and
95–100% in the case of the eight A2780-MNU clones.
Survival after treatment with DNA-damaging agents was determined by clonogenic assay as previously described (8) . Cells at clonal density (100–400 cell/60 mm dish) were treated 18 h after seeding with MNU (30 min at 37°C in PBS/20 mM Hepes, pH 7.4), CCNU (1 h in complete medium), 6-TG (24 h in complete medium) or UV. Cultures were then washed and fed with complete medium, and 1–2 weeks later, surviving colonies were fixed with methanol, stained with Giemsa, and counted. For determination of cell growth, cells were harvested at daily intervals and counted microscopically. MNU survival experiments were carried out in the presence of 25 µM O6-bzGua. In this case cell cultures were pretreated for 2 h with the MGMT inhibitor, which was also included in the complete medium for the next 3 days.
Selection of MNU-resistant variants was carried out as previously described (22) . Briefly, separate cultures of exponentially growing A2780 cells were treated with a single dose of 2 mM MNU in the presence of O6-bzGua. Surviving cells were isolated by single cell cloning. Comparisons in resistance or sensitivity to CCNU were made at 10% survival (D10).
Western Blotting.
Exponentially growing cells were treated with CCNU or UV as described
above and cell extracts were prepared 24 h later from 2 x
107 cells resuspended in a buffer containing 50
mM Tris·HCl, pH 7.5/1 mM EDTA/10
mM DTT and 0.2% TritonX-100. Cell extracts (20–60 µg)
were denatured and separated on 7.5 or 12% SDS polyacrylamide gels
together with a prestained low molecular weight marker (Bio-Rad,
Hercules, CA). Proteins were transferred to nitrocellulose membranes
(Bio-Rad) using a Trans-Blot cell apparatus (Bio-Rad) at 30
mA overnight at 4°C. The remaining protein binding sites of the
nitrocellulose were blocked by immersion in TBS-Tween (10
mM Tris·HCl, pH 7.5, 150
mM NaCl, 0.05% Tween 20) for 30 min in the
presence of 0.1% gelatin and an additional 30 min in the presence of
3–7% powdered skim milk. The blocked filter was incubated with
primary antibody (1 µg/ml for the monoclonal antibodies
anti-PMS2, anti-hMLH1, and anti-p21 and 0.1 µg/ml for anti-p53 or
1:1000 diluted antiserum for hMSH6 and hMSH2) for 1 h at room
temperature on a rocker platform. After washing with TBS-Tween, the
appropriate secondary antibody was added for an additional 1 h.
The blots were developed using the ECL detection reagents (Amersham
Pharmacia Biotech).
Cell Cycle and TUNEL Analysis.
Cell cycle analysis was performed by flow cytometric measurements of
the DNA content of the cells. Cells were harvested at appropriate time
points from treatment and stained at 4°C for at least 3 h in PBS
containing 0.1% Triton X-100, 5 µg/ml propidium iodide (Sigma), and
20 µg/ml RNase A (Roche Molecular Biochemicals). Cell cycle
analyses were performed on an Epics XL analyzer (Coulter Corp.).
Cytospin preparations of 2 x 104 cells were air dried and fixed with formaldehyde (4% in PBS, pH 7.4) for 60 min at room temperature. Slides were incubated for 2 min on ice in a permeabilized solution containing 0.25% Triton-X 100 (Bio-Rad) and 0.1% sodium citrate. TUNEL assay was performed using the in situ cell death detection kit with fluorescein (Boehringer Mannheim) according to the manufacturer’s instructions. Hoechst 33342 (1 µg/ml in PBS) was used to counterstain the nuclei.
Sequencing of the p53 Gene.
Each exon of the p53 gene was amplified by 30 PCR cycles at
95°C for 2 min, 55°C for 2 min, and 72°C for 3 min using the
previously described oligonucleotide primers (23)
. The PCR
reaction contained 100 ng of DNA, 0.25 µM each
primer, 100 µM each deoxynucleotide
triphosphate, 1.5 mM MgCl2,
10 mM Tris-HCl (pH 8.3), 50
mM KCl, and 0.5 units of Taq
polymerase (Perkin-Elmer) in a volume of 50 µl. The PCR products were
extracted with Qiagen (M-Medical, Firenze, Italy) and
cycle-sequenced in both directions with AmpliTaqFS with a Dye
Deoxy terminator cycle sequencing kit (Perkin-Elmer) on the ABI
Prisma 310 DNA sequencer.
DNA Transfection with p53val135 and Selection of G418-resistant
Transfectants.
The plasmid pNp53cG(val135) contains a chimera of mouse p53 cDNA
and genomic DNA under the transcriptional control of a Harvey sarcoma
virus long terminal repeats, as well as a selectable marker gene
neo that confers resistance to G418. It encodes a mutant p53
protein with a substitution from alanine to valine at position 135
(24)
. To introduce this vector into A2780 cells,
exponentially growing cells were plated in complete medium into
100-mm-diameter dishes at 1 x 106
cells/dish. One day later, 5 µg of plasmid DNA was transfected by the
calcium phosphate precipitation procedure, and cell cultures were
diluted 24 h later in selective medium containing 1 mg/ml G418
(Geneticin, Life Technologies, Inc.). G418-resistant colonies were
formed after 15–20 days with one refeeding with selective medium.
Several colonies were isolated with 0.25% trypsin, transferred into
24-well plates, and grown in the absence of selection. After 2 weeks,
cells were transferred into flasks and grown in selective medium for
further study.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Eight methylation-tolerant A2780 variants that were isolated
independently exhibited an identical 100-fold increased resistance to
MNU and a high degree of cross-resistance to the base analogue 6-TG
(five examples are shown in Fig. 1, A and B
). These properties are consistent with
deficient MMR, and none of the eight clones expressed detectable hMLH1
or hPMS2 (Fig. 1C
and data not shown). The levels of hMSH2
and hMSH6 proteins were comparable to those of the parental A2780 cells
and data not shown). Extracts of three of the five
clones that were selected for further investigation (A2780-MNU clones
1, 2, and 5) were tested for the ability to repair a single C:T
mismatch in an in vitro MMR assay. None of the extracts
performed detectable repair (Ref. 27
and data not shown).
This provides further confirmation that these clones are defective in
MMR. It is likely that all eight share the same hMLH1 defect.
|
.
|
. One day after exposure to 50
µM CCNU, both the parental A2780 and the
variant cells accumulated in G2. This
accumulation persisted for at least 3 days in the parental cells,
whereas the cell cycle distribution of A2780-MNU clone 1 was
essentially normal 2–3 days after treatment. We also determined
whether the differential cell cycle perturbation after CCNU exposure
was correlated with changes in the levels of apoptosis. The proportion
of apoptotic cells, as measured by TUNEL, was significantly reduced in
A2780-MNU clone 1 in comparison to the parental cells (Fig. 2C)
. Although the qualitative difference between the variant
and the parental cells is maintained, the absolute amount of apoptosis
is moderate in both cell lines, suggesting that the majority of the
population may die by a different pathway.
The p53-dependent DNA damage response was defective in A2780-MNU clones
1, 2, and 5. Exposure to 20 J/m2 UV light
increased p53 levels in the parental A2780 cells (Fig. 3A)
. In contrast, p53 was
present at significant levels in all of the unirradiated A2780-MNU
clones, and there was no detectable increase after UV irradiation (Fig. 3A
and data not shown). The functional status of the p53
protein in the representatives A2780-MNU clones 1, 2, and 5 was
analyzed by measuring induction of the Bax and p21 proteins. The low
levels of p21 and Bax in unirradiated parental A2780 cells were
increased by UV irradiation. In contrast neither Bax nor p21 expression
was detectably increased by UV irradiation in MNU clones 1, 2, and 5
(Fig. 3A)
. The p53 in MNU clones 1, 2, and 5 is therefore
unable to activate its normal target genes.
|
. In contrast, the high constitutive levels of p53 in the
A2780-MNU clones were not further increased by CCNU exposure. In
addition, there was no detectable induction of p21 following CCNU
treatment of the A2780-MNU clones (Fig. 3B)
. Taken together,
the data indicate that the UV and CCNU-induced p53-dependent responses
are abrogated in the A2780-MNU clones and that their p53 is unable to
effect transcriptional activation of its downstream targets.
Genomic sequencing of all of the exons of the p53 gene
revealed that clones 1, 2, and 5 contained a single mutation, a
heterozygous G to T transversion at codon 172. This mutation, which
results in a replacement of valine with phenylalanine, was present in
all of the MNU-resistant clones but not in the parental A2780 cells, in
which a wild-type p53 sequence was confirmed (Fig. 3C
and
data not shown).
We conclude that the A2780-MNU clones are representatives of a preexisting hMLH1-deficient cell population that has a mutated p53 gene. The mutation is associated with a deficiency in the p53-dependent response to DNA-damaging agents that might contribute to the increased chemoresistance of these clones. The effects of the deficient p53 response appears to outweigh the sensitivity conferred by their hMLH1 deficiency.
CCNU Sensitivity in MMR-defective A2780 Cells with an Intact
p53-damage Response.
To rid A2780 of the hMLH1- and p53-defective subpopulation, two
subclones, SC1 and SC2, were isolated by single cell cloning. The MNU
sensitivities of SC1 and SC2 were indistinguishable from that of A2780
cells (Fig. 4A)
. A
methylation-tolerant variant was isolated from each of the subclones by
selection with MNU. These variants (MNU-1A and MNU-2A) were highly
resistant to MNU and cross-resistant to 6-TG
.
Western blotting indicated that the levels of hMLH1 and hPMS2 were
reduced to less than 10% in both MNU-1A and MNU-2A (Fig. 4B)
. Both clones expressed the other MMR proteins at levels
comparable to SC1 and SC2.
|
shows cell survival, as growth
curves or clonal assays, for CCNU-treated MNU-1A, MNU-2A, and their
parental SC1 and SC2 cells. Both hMLH1-deficient clones MNU-1A and
MNU-2A were hypersensitive to CCNU. Because the properties of MNU-1A
and MNU-2A appeared to be similar, only the former was characterized in
detail.
|
. This is consistent with a
functional p53 response in these cells.
|
Increased Resistance to CCNU Conferred by Expression of a
Dominant-negative p53.
The contribution of the p53 response to CCNU sensitivity was
investigated by expressing a dominant-negative p53 in MNU-1A
and the parental SC1 cells. This p53 protein contains an amino acid
change (Ala-135 
Val), which confers a dominant mutant phenotype
(24)
. Introduction of p53val135 into A2780 cells conferred
an approximately 2-fold CCNU resistance in both the hMLH1-proficient
SC1 and the hMLH1-deficient MNU-1A cells (Fig. 5B)
. Thus,
CCNU resistance is modulated by p53, and the p53 response acts
independently of MMR to increase survival after CCNU treatment.
As expected, high constitutive levels of p53 protein were observed in
both parental and hMLH1-defective cells expressing p53val135. The level
of p53 protein was not detectably altered by either UV or CCNU
treatment of SC1 or MNU-1A cells containing the dominant-negative
p53val135 (Fig. 6B)
. In contrast, both SC1 and MNU-1A in
which p53val135 was expressed retained their ability to substantially
increase p21 levels after CCNU treatment
. Thus,
p21 induction by CCNU appears to be at least partly independent of p53
in A2780 cells.
In summary, both MMR and p53 defects influence CCNU sensitivity in A2780 cells. The MMR defect in the clonal SC1 or SC2 cells, in which the p53 responses to DNA-damaging agents are intact, sensitizes them to the drug. In contrast, abrogation of these responses in the same cells by expression of a dominant-negative p53 protein confers sufficient CCNU resistance to overwhelm the MMR-related sensitivity. In addition, CCNU may induce a p53-independent increase in p21 expression. This response is also abrogated in the A2780-MNU clones. The absence of p21 induction by this route may add further to their drug resistance.
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
A2780 cells contain a MMR-deficient subpopulation of which the eight A2780-MNU clones were members. The hMLH1 gene in these clones is epigenetically silenced and could be reactivated by exposure to 5-azadeoxycytidine.4 An intrinsically drug-resistant subpopulation of A2780 cells has been described (28) , and it is likely that many of the previously established resistant variants of A2780 and the MNU-resistant hMLH1 defective clones that we isolated are derived from these same cells. This likelihood is strengthened by our observation of a mutation at codon 172 of the p53 gene in the MNU-resistant clones and the presence of the same mutation in an A2780 cell line selected previously for resistance to cisplatin (29) . The A2780-MNU clones that were representative of the subpopulation were more resistant to CCNU than A2780 cells because the deficient p53 response overrode the sensitizing effect of the silent hMLH1. The p53 in these cells was present at constitutively high levels and was inactive as a transcriptional activator. The p53 mutation was, however, heterozygous. This suggests that p53 phe172 mutation acts in a dominant-negative fashion. Significantly, in the tumors in which the p53 phe172 mutation has been found, the mutation is also heterozygous and is associated with a stabilization of the p53 protein (30 , 31) . This observation indicates that the p53 phe172 mutation in the A2780 subpopulation is indeed likely to act in a dominant-negative fashion. Thus, alterations in at least two genes (hMLH1 and p53), which can separately modulate sensitivity to cytotoxic drugs, had occurred spontaneously in this subpopulation.
The hMLH1 gene appears to be particularly susceptible to epigenetic down-regulation. Importantly, methylation-related silencing of hMLH1 has been demonstrated in colon, endometrial, and ovarian cancers (32, 33, 34, 35) . Loss of this key MMR gene product confers a substantial mutator phenotype that may provide a pool of cells upon which selection for enhanced growth may operate. A MMR defect will therefore facilitate the propagation of clones with mutations conferring a growth advantage (36 , 37) . Indeed, we noted that the A2780-MNU variants exhibited higher growth rates and cloning efficiencies than the parental A2780 or the A2780SC1 and SC2 clones. Although p53 mutations are relatively rare in MMR-defective tumors (see, for example, Ref. 38 ), the apparently clonal nature of the A2780 subpopulation is compatible with the p53 phe172 mutation arising as a consequence of the MMR defect. Codon 172 is not a mutational hot-spot in the p53 gene in human cancer (39) . It is interesting in this regard that the infrequent p53 phe172 mutations that are recorded in the IARC p53 database (39) are all in ovarian or colon cancers—two types of tumor in which MMR defects, and in particular hMLH1 silencing, are most common (32, 33, 34, 35) .
The systematic analysis of the effects of MMR and the p53 response on CCNU susceptibility in the A2780 model ovarian carcinoma cell line provides evidence for a general relationship between MMR deficiency and CCNU sensitivity. Both A2780SC1 and SC2, which retained a functional p53 response, were sensitized to CCNU by a MMR defect. In addition, p53 was also revealed as a significant and independent determinant of CCNU sensitivity in these cells. MMR-deficient, p53-proficient cells were rendered CCNU resistant by expression of a dominant-negative p53 protein. Thus, MMR and the p53 response have separate and opposing effects on CCNU sensitivity. Because the two pathways influence CCNU sensitivity independently, we conclude that MMR processing of CCNU-induced DNA damage is not required to provide a signal that triggers the p53 response. Furthermore, functional p53 is not essential for the protective interaction of MMR with CCNU DNA lesions and MMR can be coupled to a p53-independent death pathway after exposure to this drug. Comparison of our results to published data (15) indicates that a selective abrogation of the p53 response decreases the susceptibility of A2780 cells to CCNU, ionizing radiation, cisplatin, doxorubicin, and araC to similar extents.
CCNU induced p21 in A2780 cells expressing the dominant-negative mutant p53val135. A similar induction of p21 by a CNU has been reported in the p53-null human osteosarcoma Saos-2 and in a glioblastoma cell line with mutant p53 (40) . Whether the lack of p21 induction by CCNU in the eight A2780-MNU clones is due to the particular p53phe172 mutation or to additional genetic changes in these cells is unclear, as is the implication of this p53-independent induction of p21 for CNU sensitivity. It is tempting to speculate, however, that loss of the p53-independent p21 induction might also contribute to the extreme CCNU resistance of the A2780-MNU clones.
In summary, our data indicate that two alterations commonly found in human cancer, defective MMR, which is present in a significant minority of sporadic colorectal and other tumors, and loss of p53 function, separately modify the response to a CNU. Information on the MMR status of tumors is available through measurements of microsatellite instability, and p53 status is frequently determined in the clinic. Knowledge of these two parameters would be advantageous in designing an appropriate therapeutic strategy.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 This work was partially supported by the
Associazione Italiana Ricerca sul Cancro, the Imperial Cancer Research
Fund, and the Italy-USA Project. 
2 To whom requests for reprints should be
addressed, at Istituto Superiore di Sanitá, Section of Chemical
Carcinogenesis, Viale Regina Elena 299, 00161 Rome, Italy. Phone and
Fax: 39-06-49902355; E-mail: bignami{at}iss.it
3 The abbreviations used are: CNU,
chloroethylnitrosourea; MMR, mismatch repair; CCNU,
N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea;
MGMT,
O6-methylguanine-DNA-methyltransferase; Aag,
3-methyladenine DNA glycosylase; MNU,
N-methyl-N-nitrosourea;
O6-bzGua,
O6-benzylguanine; 6-TG, 6-thioguanine;
TUNEL, terminal deoxynucleotidyl transferase-mediated nick end
labeling.
4 P. Karran and P. Branch, unpublished data.
Received 6/14/99; revised 9/29/99; accepted 10/14/99.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  M. F. Blasi, I. Ventura, G. Aquilina, P. Degan, L. Bertario, C. Bassi, P. Radice, and M. Bignami A Human Cell-Based Assay to Evaluate the Effects of Alterations in the MLH1 Mismatch Repair Gene. Cancer Res., September 15, 2006; 66(18): 9036 - 9044. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. O'Brien and R. Brown Signalling cell cycle arrest and cell death through the MMR System Carcinogenesis, April 1, 2006; 27(4): 682 - 692. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Offman, G. Opelz, B. Doehler, D. Cummins, O. Halil, N. R. Banner, M. M. Burke, D. Sullivan, P. Macpherson, and P. Karran Defective DNA mismatch repair in acute myeloid leukemia/myelodysplastic syndrome after organ transplantation Blood, August 1, 2004; 104(3): 822 - 828. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Papouli, P. Cejka, and J. Jiricny Dependence of the Cytotoxicity of DNA-Damaging Agents on the Mismatch Repair Status of Human Cells Cancer Res., May 15, 2004; 64(10): 3391 - 3394. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Passagne, A. Evrard, J.-Y. Winum, P. Depeille, P. Cuq, J.-L. Montero, D. Cupissol, and L. Vian Cytotoxicity, DNA Damage, and Apoptosis Induced by New Fotemustine Analogs on Human Melanoma Cells in Relation to O6-Methylguanine DNA-Methyltransferase Expression J. Pharmacol. Exp. Ther., November 1, 2003; 307(2): 816 - 823. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Karran Mechanisms of tolerance to DNA damaging therapeutic drugs Carcinogenesis, December 1, 2001; 22(12): 1931 - 1937. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
), clone 1 (•), clone 2 (
), clone 3 (
), clone 4 (
), and
clone 5 (
) were treated with MNU in the presence of
O6-bzGua (A) or with 6-TG
(B). C, MMR proteins hMLH1, hPMS2, hMSH2,
and hMSH6 in extracts of A2780 and the methylation-tolerant variants.
Cell extracts were separated by SDS-PAGE and protein detected by
immunoblotting. Antisera to hMSH6 and hMSH2 were used together.
),
SC1 (
), MNU-1A (•), and MNU-2A (
