© 2005 Foundation for Promotion of Cancer Research
Inverse Correlation Between the Expression of O6-Methylguanine-DNA Methyl Transferase (MGMT) and p53 in Breast Cancer
Surgical Oncology, Tokyo Medical and Dental University, Graduate School, Tokyo, Japan
For reprints and all correspondence: Takayuki Osanai, 1-5-45, Yushima, Bunkyo-ku, Tokyo, Japan. E-mail: t-osanai.srg2{at}tmd.ac.jp
Received August 28, 2004; accepted January 2, 2005
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Abstract |
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 TOP Abstract INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONReferences |
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Background: Expression of O6-methylguanine-DNA methyl transferase (MGMT), a DNA repair protein, has been associated with tumor resistance to alkylating agents such as cyclophosphamide. Promoter hypermethylation of MGMT is a strong predictor of survival, and expression of mutant p53 protein may be associated with downregulated MGMT expression in brain tumors. In order to clarify further the correlation between MGMT and p53 expression, we investigated the expression levels of both MGMT and p53 in breast cancer.
Methods: MGMT and p53 expression was examined in tissues from 48 consecutive cases of primary breast cancer patients using immunohistochemical staining with antibodies specific for MGMT and p53. The prognosis for survival was analyzed based on MGMT and p53 immunoreactivity and clinicopathological characteristics.
Results: Expression of either MGMT or p53 was classified as negative or positive based on immunohistochemical staining. The expression of MGMT showed a significant inverse correlation with three clinicopathological characteristics, the status of the estrogen receptor, local recurrence and distant metastasis. There was a significant correlation between the expression level of p53 and the lymphatic involvement, estrogen receptor status and distant metastasis. MGMT and p53 were correlated with distant metastasis and local recurrence, but Kaplan–Meier curves did not show a significant difference. MGMT immuno-negative specimens showed a significantly higher expression of p53 (P = 0.026, 
2 test).
Conclusion: p53 may be associated with the regulation of MGMT expression in breast cancers.
Key Words: O6-methylguanine • DNA methyl transferase (MGMT) • breast cancer • p53
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INTRODUCTION |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONReferences |
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Alkylating agents cause gene mutations through the formation of the promutagenic base, O6-methylguanine. These mutagenic and cytotoxic adducts are removed from the O6-guanine by O6-methylguanine-DNA methyl transferase (MGMT) (1). MGMT is present in all normal tissues. However, in some human tumors, MGMT is absent (2–4).
MGMT repairs the potentially cytotoxic antitumor lesions induced in DNA by certain cancer chemotherapeutic alkylating agents such as cyclophosphamide (CPM) (1). Tumors with little or no MGMT will thus predictably be responsive to drug therapy; conversely, tumors with high MGMT levels will probably be drug resistant (5). Some authors reported that tumor MGMT expression was not predictive of response to alkylating agents in breast cancers (6). Nevertheless, MGMT is reportedly a strong predictor of survival in brain and other solid tumors, including breast tumors (5–10).
The reason why MGMT is a predictive marker of survival is unclear. Some authors suggested a carcinogenic mechanism induced by the disruption of MGMT, as was demonstrated in transgenic or knockout mouse models (11). According to this hypothesis, abnormal MGMT expression in humans could result in activation of oncogenes or inactivation of tumor suppressor genes.
Tumor suppressor gene p53 plays an important role in many cellular processes, including the cell cycle, response of cells to DNA damage, cell death, cell differentiation and neovascularization, and it is commonly mutated in human tumors. Several studies have provided conflicting results about the relationship between the expression of p53 and MGMT (12–14). In vitro studies have shown that wild-type p53 acts as an inhibitor of MGMT gene expression (15). To clarify further the relationship between MGMT and p53 expression, we examined the expression of both MGMT and p53 in breast cancer.
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PATIENTS AND METHODS |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONReferences |
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PATIENTS AND TUMOR SPECIMENS
Between February 2000 and February 2002, 48 breast cancer patients were enrolled. These patients received no systemic chemotherapy before surgery. Follow-up data for retrospective analyses were obtained by reviewing patient records. The mean follow-up period was 34 months (range 24–48 months). Representative formalin-fixed, paraffin-embedded tumor specimens from patients who underwent curative resection were selected for this study (Table 1). The clinical and pathological characteristics of patients were classified according to the UICC TNM Classification of Malignant Tumors.
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PATHOLOGICAL REVIEW
Histological sections from the 48 surgical specimens were reviewed without information on clinical outcome. The sections were routinely prepared in paraffin, and then stained with hematoxylin and eosin. To compare the significance of p53 and MGMT expression on prognosis with other known predictive parameters, the following morphological details were recorded: grade of tumor differentiation (grading), tumor diameter (of invasive area), lymph node involvement, and venous and lymphatic infiltration. The status of the hormone receptors, estrogen receptor (ER) and progesterone receptor (PgR), was estimated by immunohistochemical staining. All histological slides were reviewed by two of the authors without knowledge of the clinical outcome.
The follow-up examinations were performed at our hospital. The diagnosis of recurrent disease was based on clinical and radiological examinations, and was performed by pathological findings.
ANTIBODIES
The mouse anti-MGMT monoclonal antibody was purchased from Chemicon International, Inc. (CA, USA), and the monoclonal mouse anti-human p53 protein (Clone DO-7) from DAKO A/S (Denmark).
IMMUNOHISTOCHEMICAL STAINING
Paraffin sections (4 µm thick) were dewaxed in xylene, rinsed in graded ethanol, and finally rehydrated in double-distilled water. For antigen retrieval, the sections were autoclaved for 10 min at 121°C in 10 mmol/l citrate buffer, pH 6.0, and treated with 3% hydrogen peroxidase for 15 min at room temperature to block endogenous peroxidase activity. After blocking with 10% normal goat serum for 30 min at room temperature, sections were incubated for 60 min at room temperature with a 1:100 dilution of anti-MGMT mouse monoclonal antibody, or with a 1:50 dilution of anti-human p53 mouse monoclonal antibody. Next, the sections were incubated with Histofine Simple Stain Max PO (MULTI) (Nichirei) for 30 min at room temperature. Peroxidase activity was visualized with 0.02% 3,3'-diaminobenzine tetrahydrochloride and 0.03% (v/v) H2O2 in Tris-buffered saline (TBS), and the sections were counterstained with Mayer's hematoxylin.
The sample was considered positive when immunoreactivity was detected in >10% of the cells in nuclei.
STATISTICAL ANALYSIS
The clinicopathological characteristics were compared with MGMT- and p53-positive and -negative groups, and the significance of associations was determined with the Mann–Whitney U-test or Student's test. Survival data were used to generate Kaplan–Meier curves that were compared on the basis of MGMT status or p53 status by using the log-rank test. Statistical significance was judged as P < 0.05.
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RESULTS |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONReferences |
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EXPRESSION OF MGMT
For MGMT, positive staining was present predominantly in the nuclei, although some weak cytoplasmic staining was also observed (Fig. 1). There was no clear pattern of staining intensity that could be related to histological classification. The sample was considered positive when immunoreactivity was detected in >10% of the cells in nuclei.
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EXPRESSION OF P53
In both tumor and normal breast, p53 was present predominantly in the nuclei (Fig. 2). The staining pattern of p53 was heterogeneous within tumors: in some tumors, most of the cells expressed the protein in a relatively homogenous pattern, whereas in others only a few of the cells were stained either as isolated regions within the tumors or as isolated cells.
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The sample was considered positive when immunoreactivity was detected in >10% of the cells in nuclei.
CORRELATION BETWEEN MGMT EXPRESSION STATUS AND CLINICOPATHOLOGICAL CHARACTERISTICS
The expression level of MGMT shows a significant inverse correlation with three clinicopathological characteristics, the status of the ER (P = 0.0414), local recurrence (P = 0.0004) and distant metastasis (P = 0.0004). However, there was no relationship between MGMT expression and any other clinicopathological characteristics (Table 2). Kaplan–Meier curves did not show a significant difference.
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CORRELATION BETWEEN P53 EXPRESSION STATUS AND CLINICOPATHOLOGICAL FEATURES
There was a significant correlation between the expression level of p53 and the lymphatic involvement (P = 0.0319), ER status (P = 0.0182) and distant metastasis (P = 0.0043). However, there was no relationship between p53 expression and any other clinicopathological characteristics (Table 3). Kaplan–Meier curves did not show a significant difference.
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CORRELATION BETWEEN MGMT AND P53 EXPRESSION
Specimens immuno-negative for MGMT expression showed a significantly higher expression of p53 (P = 0.026,
2 test, Table 4).
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DISCUSSION |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONReferences |
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Recently, it has been suggested that carcinogenesis must be understood not only as a process at the gene level, but also as an epigenetic process. The genetic and epigenetic pathways are not isolated, but rather a complex network of cross-talk between genetic and epigenetic factors may exist.
Hypermethylation of the MGMT CpG island as the cause of MGMT transcriptional silencing in cell lines defective in O6-methylguanine repair has been recognized (13). Similarly, Esteller et al. reported that the MGMT gene is epigenetically inactivated by promoter hypermethylation in many primary tumor types (14,16). Esteller et al. also reported a direct relationship between MGMT aberrant methylation and G:C to A:T transition mutations of p53 in colorectal tumors (14).
Several studies have suggested the involvement of p53 protein in the expression of the MGMT gene. Rolhion et al. reported that MGMT gene expression was significantly lower in p53 altered tumors, and mutant p53 was clearly correlated with poor survival of patients in glioblastomas (12). Because transition mutations of p53 in human cancer are common and occur in all malignant cell types, the relationship observed between MGMT expression and p53 is highly relevant. Some of these tumors, such as those derived from the brain, lung, head and neck, and lymphomas, also have hypermethylation-associated inactivation of MGMT. Thus, the epigenetic defect of the MGMT gene may predispose these cells to acquire p53 transition mutations. In the present study, we examined the expression of both MGMT and p53 in breast cancer to clarify the correlation between MGMT and p53 expression.
Several lines of evidence suggest that MGMT-deficient cell lines are more sensitive to alkylating agents, such as CPM metabolites, than cells expressing relatively high levels of MGMT (17). In brain tumors, tumor MGMT gene expression was found to be predictive of response to chemotherapy (5). In contrast, MGMT gene expression was not predictive of chemotherapy response in breast tumors (6). Cayre et al. reported that low MGMT expression in breast cancer was significantly related to poor survival, but not predictive of response to the alkylating agents (6). Matsukura et al. (10) reported that expression of MGMT was a good prognostic factor in hepatocellular, gastric and breast cancer.
In conclusion, we showed that expression of p53 may be associated with the regulation of MGMT expression in breast tumors, and that MGMT immuno-negativity and p53 immuno-positivity may be strong predictors of breast cancer survival.
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References |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONReferences |
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