The control of O6-methylguanine-DNA methyltransferase (MGMT) activity in mammalian cells: a pre-molecular view

Human peripheral blood lymphocytes (PBLs) can have a range of O⁶-methylguanine-DNA methyltransferase (MGMT) activities. PBLs from some individuals may have almost no MGMT activity. Such individuals have most often been subject to malignancy or to immunodeficiency disease. Long-term lymphoblastoid lines (LCLs) prepared from PBLs of normal subjects by Epstein-Barr virus (EBV) transformation have MGMT activities which are in general somewhat higher than the PBLs from which they derive. Such cultures are therefore generally MGMT-positive. Only in rare cases, and generally from patients with low MGMT activity, are freshly obtained lines with very low activity obtained. There is however a 4-fold range of MGMT activity over which multiple lines derived from the same PBL sample can be found. Long-term cultivation can lead to LCLs with low activity as well as to lines of high activity. On rare occasions an MGMT-positive line may, within a few divisions, give a negative line. Some (but not all) MGMT-negative (or very low) lines have been known to gain (some) activity. Chinese hamster ovary (CHO) cell lines are in general very low in MGMT activity. Lines of higher activity can be selected by treatment with mutagenic crosslinking alkylating agents. Chinese hamster lines with high MGMT activity can be obtained by transfection with human DNA from MGMT-positive cells. Lines with significant activity can also be obtained by transfection of CHO cells with human DNA from MGMT-negative (or very low) cells. Resistance to MNNG treatment can be acquired without the acquisition of significant MGMT activity. Crosses of lines of high and low MGMT activity give equivocal results. Hybrids of low × low activity have no activity. Crosses of positive × positive strains give varied results. It has not been possible to identify MGMT-positive hybrids as including one particular chromosome by this type of experiment. There is no evidence for a general adaptive effect on MGMT synthesis greater than the variation within the cell cycle.     

The origin of O6-methylguanine-DNA methyltransferase in Chinese hamster ovary cells transfected with human DNA

Transfection of Chinese hamster ovary (CHO) cells with human DNA has been shown in several laboratories to produce clones which stably express the DNA-repair protein, O6-methylguanine-DNA methyltransferase (MGMT), that is lacking in the parent cell lines (Mex- phenotype). We have investigated the genetic origin of the MGMT in a number of such MGMT-positive (Mex+) clones by using human MGMT cDNA and anti-human MGMT antibodies as probes. None of the five independently isolated Mex+ lines has human MGMT gene sequences. Immunoblot analysis confirmed the absence of the human protein in the extracts of these cells. The MGMT mRNA in the lines that express low levels of MGMT (0.6-1.4 x 10(4) molecules/cell) is of the same size (1.1 kb) as that present in hamster liver. One cell line, GC-1, with a much higher level of MGMT (4 x 10(4) molecules/cell) has two MGMT mRNAs, a major species of 1.3 kb and a minor species of 1.8 kb. It has also two MGMT polypeptides (32 and 28 kDa), both of which are larger than the 25 kDa MGMT present in hamster liver and other Mex+ transfectants. These results indicate that the MGMT in all Mex+ CHO cell clones is encoded by the endogenous gene. While spontaneous activation of the MGMT gene cannot be ruled out in the Mex+ cell clones, the intervention of human DNA sequences may be responsible for activation of the endogenous gene in the GC-1 line.     

Induction of repair enzyme O6-methylguanine-DNA methyltransferase gene expression under the influence of cytokine EMAP II in human cells in vitro

The aim of our study was to investigate the effect of recombinant human cytokine EMAP II (endothelial monocyte-activating polypeptide II) on the expression of MGMT gene, encoding repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) in human cell cultures. The influence of EMAP II on cell proliferation was performed using routine MTT assay. Identification of MGMT in cell extracts was performed using Western blot analysis. We used cell lines: A102 (fibroblasts), CB-1 (umbilical cord blood stromal cells), 4BL6 (cells derived from peripheral blood). It was shown that cytokine EMAP II caused induction of MGMT expression in studied human cell lines. There was a decrease in cell number at high concentrations of this cytokine. It was found that the presence of cytokine EMAP II in serum-free growth medium leads to increasing of repair enzyme MGMT expression level in human cells in vitro.     

O6-methylguanine DNA methyltransferase and p53 status predict temozolomide sensitivity in human malignant glioma cells

Temozolomide (TMZ) is a methylating agent which prolongs survival when administered during and after radiotherapy in the first-line treatment of glioblastoma and which also has significant activity in recurrent disease. O6-methylguanine DNA methyltransferase (MGMT) is a DNA repair enzyme attributed a role in cancer cell resistance to O6-alkylating agent-based chemotherapy. Using a panel of 12 human glioma cell lines, we here defined the sensitivity to TMZ in acute cytotoxicity and clonogenic survival assays in relation to MGMT, mismatch repair and p53 status and its modulation by dexamethasone, irradiation and BCL-X(L). We found that the levels of MGMT expression were a major predictor of TMZ sensitivity in human glioma cells. MGMT activity and clonogenic survival after TMZ exposure are highly correlated (p < 0.0001, r2 = 0.92). In contrast, clonogenic survival after TMZ exposure does not correlate with the expression levels of the mismatch repair proteins mutS homologue 2, mutS homologue 6 or post-meiotic segregation increased 2. The MGMT inhibitor O6-benzylguanine sensitizes MGMT-positive glioma cells to TMZ whereas MGMT gene transfer into MGMT-negative cells confers protection. The antiapoptotic BCL-X(L) protein attenuates TMZ cytotoxicity in MGMT-negative LNT-229 but not in MGMT-positive LN-18 cells. Neither ionizing radiation (4 Gy) nor clinically relevant concentrations of dexamethasone modulate MGMT activity or TMZ sensitivity. Abrogation of p53 wild-type function strongly attenuates TMZ cytotoxicity. Conversely, p53 mimetic agents designed to stabilize the wild-type conformation of p53 sensitize glioma cells for TMZ cytotoxicity. Collectively, these results suggest that the determination of MGMT expression and p53 status will help to identify glioma patients who will or will not respond to TMZ.     

MGMT is a molecular determinant for potency of the DNA-EGFR-combi-molecule ZRS1

To enhance the potency of current EGFR inhibitors, we developed a novel strategy that seeks to confer them an additional DNA damaging function, leading to the design of drugs termed combi-molecules. ZRS1 is a novel combi-molecule that contains an EGFR tyrosine kinase targeting quinazoline arm and a methyltriazene-based DNA damaging one. We examined its effect on human tumor cell lines with varied levels of EGFR and O6-methylguanine DNA methyltransferase (MGMT). ZRS1 was more potent than the clinical methylating agent temozolomide in all cell lines, regardless of their MGMT status. However, its potency was in the same range as or less than that of Iressa, an EGFR inhibitor, against MGMT-proficient cells. In the MGMT-deficient or in MGMT-proficient cells exposed to the MGMT inhibitor O6-benzylguanine, its potency was superior to that of Iressa and temozolomide or a temozolomide+Iressa combination. Cell signaling analysis in A549 (MGMT(+)) and A427 (MGMT(-)) showed that ZRS1 strongly inhibited EGFR phosphorylation and related signaling pathways. In addition, the p53 pathway was activated by DNA damage in both cell lines, but apoptosis was significantly more pronounced in A427 cells. Using MGMT shRNA to block endogenous MGMT protein expression in A549 resulted in significant sensitization to ZRS1. Furthermore, transfection of MGMT into A427 greatly decreased the potency of ZRS1. These results conclusively show that MGMT is a critical molecular determinant for the full-blown potency of the dual EGFR-DNA targeting combi-molecule.     

甲基鸟嘌呤甲基转移酶表达调节在肿瘤发生和治疗中的作用

甲基鸟嘌呤甲基转移酶（O⁶-methylguanine-DNA methyltransferase,MGMT）是从细菌到哺乳类机体中存在的一种独特的DNA修复蛋白,其作用是在DNA损伤的修复过程,催化DNA分子鸟嘌呤O⁶位上的烷基从鸟嘌呤碱基转移至MGMT蛋白的半胱氨酸残基上,而使DNA分子鸟嘌呤复原.因此,机体中MGMT适当的表达有利于修复由烷化剂诱导而形成的O⁶烷基鸟嘌呤DNA加合物.MGMT蛋白的含量和活性不但在基因水平受到各种因素的调控,并且与某些药物的直接作用有关.调节MGMT在细胞内的活性,对于防御肿瘤的发生及某些肿瘤的治疗过程中克服肿瘤耐药性和克服骨髓毒性具有重要的意义.     

Incision at O6-methylguanine:thymine mispairs in DNA by extracts of human cells.

Human cell-free extracts were used to detect activities specifically incising O6-methylguanine (m6G) paired with C or T in DNA. A 45-bp double-stranded DNA containing one m6G across from a T (m6G:T) was the test substrate. Extracts from glioblastoma cell lines A172 and A1235 (lacking the m6G-specific repair protein m6G-DNA methyltransferase, MGMT) and colon carcinoma cell line HT29, containing MGMT, showed incision activities specific for the T strand of m6G:T [and G:T, as reported previously by Wiebauer and Jiricny (1989)] substrates, but did not cleave m6G:C (or G:C) substrates. Competition experiments showed that the activity was similar to, if not identical with, the activity in human cells that incises G:T mismatches. The incision sites were similar to those recognized by human G:T- or G:A-specific mismatch enzymes, i.e., the phosphodiester bonds both 3' and 5' to the poorly matched T, suggesting the glycolytic removal of the poorly matched T followed by backbone incisions by class I or II AP endonucleases. Three experiments in which MGMT was inactivated showed that the m6G:T incision activity was not simply due to a two-step mechanisms in which MGMT would first mediate conversion of the m6G:T substrate to a G:T substrate which would serve as a substrate for G:T incision. Extracts from HT29 contained a DNA-binding factor, possibly DNA sequence-specific, that inhibited incision of the m6G:T (but not the G:T) substrate, that was removed by the addition of synthetic DNA to the reaction.     

Characterization of cDNA encoding mouse DNA repair protein O6-methylguanine-DNA methyltransferase and high-level expression of the wild-type and mutant proteins in Escherichia coli.

A mouse cDNA clone encoding O6-methylguanine-DNA methyltransferase (MGMT), responsible for repair of mutagenic O6-alkylguanine in DNA, was cloned from a lambda gt11 library. On the basis of an open reading frame in cDNA, the mouse protein contains 211 amino acids with a molecular mass of 22 kDa. The size and the predicted N-terminal sequence of the mouse protein were confirmed experimentally. The deduced amino acid sequence of the mouse MGMT is 70% homologous to that of the human MGMT. Cysteine-149 was shown to be the only alkyl acceptor residue in the mouse protein, in confirmation of the prediction based on conserved sequences of different MGMTs. Mouse MGMT protein is recognized by some monoclonal antibodies specific for human MGMT. Site-directed mutagenesis was utilized to reclone the mouse cDNA in a T7 promoter-based vector for overexpression of the native repair protein in Escherichia coli. The mouse protein has a tetrapeptide sequence, Pro-Glu-Gly-Val at positions 56-59, absent in the human protein. Neither deletion of this tetrapeptide nor substitution of valine-169 with alanine affected the activity of the mutant proteins.     

Reduction of BCNU toxicity to lung cells by high-level expression of O(6)-methylguanine-DNA methyltransferase

1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) is an important cause of pulmonary toxicity. BCNU alkylates DNA at the O(6) position of guanine. O(6)-methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that removes alkyl groups from the O(6) position of guanine. To determine whether overexpression of MGMT in a lung cell reduces BCNU toxicity, the MGMT gene was transfected into A549 cells, a lung epithelial cell line. Transfected A549 cell populations demonstrated high levels of MGMT RNA, MGMT protein, and DNA repair activity. The overexpression of MGMT in lung epithelial cells provided protection from the cytotoxic effects of BCNU. Control A549 cells incubated with 100 microM BCNU had a cell survival rate of 12.5 +/- 1.2%; however, A549 cells overexpressing MGMT had a survival rate of 71.8 +/- 2.7% (P < 0.001). We also demonstrated successful transfection of MGMT into human pulmonary artery endothelial cells and a primary culture of rat type II alveolar epithelial cells with overexpression of MGMT, resulting in significant protection from BCNU toxicity. These data suggest that overexpression of DNA repair proteins such as MGMT in lung cells may protect the lung cells from cytotoxic effects of cancer chemotherapy drugs such as BCNU.     

O6-methylguanine-DNA methyltransferase deficiency in developing brain: implications for brain tumorigenesis

The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) is a cardinal defense against the mutagenic and carcinogenic effects of alkylating agents. We have reported evidence that absence of detectable MGMT activity (MGMT(-) phenotype) in human brain is a predisposing factor for primary brain tumors that affects ca. 12% of individuals [J.R. Silber, A. Blank, M.S. Bobola, B.A. Mueller, D.D. Kolstoe, G.A. Ojemann, M.S. Berger, Lack of the DNA repair protein O(6)-methylguanine-DNA methyltransferase in histologically normal brain adjacent to primary brain tumors, Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 6941-6946]. We report here that MGMT(-) phenotype in the brain of children and adults, and the apparent increase in risk of neurocarcinogenesis, may arise during gestation. We found that MGMT activity in 71 brain specimens at 6-19 weeks post-conception was positively correlated with gestational age (P相似文献   

Effects of temozolomide (TMZ) on the expression and interaction of heat shock proteins (HSPs) and DNA repair proteins in human malignant glioma cells

We previously reported the association of HSPA1A and HSPB1 with high-grade astrocytomas, suggesting that these proteins might be involved in disease outcome and response to treatment. With the aim to better understand the resistance/susceptibility processes associated to temozolomide (TMZ) treatment, the current study was performed in three human malignant glioma cell lines by focusing on several levels: (a) apoptotic index and senescence, (b) DNA damage, and (c) interaction of HSPB1 with players of the DNA damage response. Three human glioma cell lines, Gli36, U87, and DBTRG, were treated with TMZ evaluating cell viability and survival, apoptosis, senescence, and comets (comet assay). The expression of HSPA (HSPA1A and HSPA8), HSPB1, O₆-methylguanine-DNA methyltransferase (MGMT), MLH1, and MSH2 was determined by immunocytochemistry, immunofluorescence, and Western blot. Immunoprecipitation was used to analyze protein interaction. The cell lines exhibited differences in viability, apoptosis, and senescence after TMZ administration. We then focused on Gli36 cells (relatively unstudied) which showed very low recovery capacity following TMZ treatment, and this was related to high DNA damage levels; however, the cells maintained their viability. In these cells, MGMT, MSH2, HSPA, and HSPB1 levels increased significantly after TMZ administration. In addition, MSH2 and HSPB1 proteins appeared co-localized by confocal microscopy. This co-localization increased after TMZ treatment, and in immunoprecipitation analysis, MSH2 and HSPB1 appeared interacting. In contrast, HSPB1 did not interact with MGMT. We show in glioma cells the biological effects of TMZ and how this drug affects the expression levels of heat shock proteins (HSPs), MGMT, MSH2, and MLH1. In Gli36 cells, the results suggest that interactions between HSPB1 and MSH2, including co-nuclear localization, may be important in determining cell sensitivity to TMZ.     

Targeting of O6-MeG DNA methyltransferase (MGMT) to mitochondria protects against alkylation induced cell death

Mitochondrial DNA (mtDNA) mutations are implicated in pathogenesis of human diseases including cancer. To prevent mutations cells have developed repair systems to counteract harmful genetic changes caused by DNA damaging agents. One such DNA repair protein is the O(6)-Methylguanine-DNA methyltransferase (MGMT) that prevents certain types of alkylation damage. Yet, the role of MGMT in preventing alkylation induced DNA damage in mtDNA is unclear. We explored the idea of increasing cell survival after alkylation damage by overexpressing MGMT in mitochondria. We show that overexpression of this repair protein in mitochondria increases cell survival after treatment with the DNA damaging agent MNNG.     

Effects of Some Growth Factors and Cytokines on the Expression of the Repair Enzyme MGMT and Protein MARP in Human Cells In Vitro

The inducible repair enzyme O⁶-methylguanine-DNA methyltransferase (MGMT) eliminates O⁶-methylguanine adducts in DNA and protects the cells from damaging effects of alkylating agents. We have found that anti-MGMT antibodies recognize both the MGMT protein with a mol. weight?~?24 kDa and a protein with a mol. weight?~?48 kDa, which was named MARP (anti-methyltransferase antibody recognizable protein). A number of growth factors and cytokines were shown to regulate the expression of MGMT and MARP proteins. The ranges of concentrations of several growth factors and cytokines that caused increasing or decreasing protein amounts in human cell cultures were determined. The results of special biological experiments have allowed us to assume a possible role of MARP in the repair of alkyl adducts in human cells.     

Temozolomide Resistance in Glioblastoma Cell Lines: Implication of MGMT,MMR, P-Glycoprotein and CD133 Expression

Background

The use of temozolomide (TMZ) has improved the prognosis for glioblastoma multiforme patients. However, TMZ resistance may be one of the main reasons why treatment fails. Although this resistance has frequently been linked to the expression of O6-methylguanine-DNA methyltransferase (MGMT) it seems that this enzyme is not the only molecular mechanism that may account for the appearance of drug resistance in glioblastoma multiforme patients as the mismatch repair (MMR) complex, P-glycoprotein, and/or the presence of cancer stem cells may also be implicated.

Methods

Four nervous system tumor cell lines were used to analyze the modulation of MGMT expression and MGMT promoter methylation by TMZ treatment. Furthermore, 5-aza-2’-deoxycytidine was used to demethylate the MGMT promoter and O(6)-benzylguanine to block GMT activity. In addition, MMR complex and P-glycoprotein expression were studied before and after TMZ exposure and correlated with MGMT expression. Finally, the effect of TMZ exposure on CD133 expression was analyzed.

Results

Our results showed two clearly differentiated groups of tumor cells characterized by low (A172 and LN229) and high (SF268 and SK-N-SH) basal MGMT expression. Interestingly, cell lines with no MGMT expression and low TMZ IC₅₀ showed a high MMR complex expression, whereas cell lines with high MGMT expression and high TMZ IC₅₀ did not express the MMR complex. In addition, modulation of MGMT expression in A172 and LN229 cell lines was accompanied by a significant increase in the TMZ IC₅₀, whereas no differences were observed in SF268 and SK-N-SH cell lines. In contrast, P-glycoprotein and CD133 was found to be unrelated to TMZ resistance in these cell lines.

Conclusions

These results may be relevant in understanding the phenomenon of TMZ resistance, especially in glioblastoma multiforme patients laking MGMT expression, and may also aid in the design of new therapeutic strategies to improve the efficacy of TMZ in glioblastoma multiforme patients.     

Chemotherapeutic resistance in anaplastic astrocytoma cell lines treated with a temozolomide–lomeguatrib combination

The treatment of anaplastic astrocytoma (AA) is controversial. New chemotherapeutic approaches are needed for AA treatment. Temozolomide (TMZ) is one of the chemotherapeutic drugs for the treatment of AA. The cytotoxic effects of TMZ can be removed by the MGMT (O(6)-methylguanine-DNA methyltransferase) enzyme. Then, chemotherapeutic resistance to TMZ occurs. MGMT inhibition by MGMT inactivators (such as lomeguatrib) is an important anticancer therapeutic approach to circumvent TMZ resistance. We aim to investigate the effect of TMZ–lomeguatrib combination on MGMT expression and TMZ sensitivity of SW1783 and GOS-3 AA cell lines. The sensitivity of SW1783 and GOS-3 cell lines to TMZ and to the combination of TMZ and lomeguatrib was determined by a cytotoxicity assay. MGMT methylation was detected by MS-PCR. MGMT and p53 expression were investigated by real-time PCR after drug treatment, and the proportion of apoptotic cells was analyzed by flow cytometry. When the combination of TMZ–lomeguatrib (50 μM) was used in AA cell lines, IC₅₀ values were reduced compared to only using TMZ. MGMT expression was decreased, p53 expression was increased, and the proportion of apoptotic cells was induced in both cell lines. The lomeguatrib–TMZ combination did not have any effect on the cell cycle and caused apoptosis by increasing p53 expression and decreasing MGMT expression. Our study is a pilot study investigating a new therapeutic approach for AA treatment, but further research is needed.     

Purification of human DNA (cytosine-5-)-methyltransferase

We have developed a facile procedure for the purification of DNA methyltransferase activity from human placenta. The procedure avoids the isolation of nuclei and the dialysis and chromatography of large volumes. A purification of 38,000-fold from the whole cell extract has been achieved. The procedure employs ion exchange, affinity, and hydrophobic interaction chromatography coupled with preparative glycerol gradient centrifugation. A protein of 126,000 daltons was found to copurify with the activity and was the major band seen in the most highly purified material after SDS gel electrophoresis. This observation, coupled with an observed sedimentation coefficient of 6.3S, suggests that the enzyme is composed of a single polypeptide chain of this molecular weight. Hemimethylated DNA was found to be the preferred substrate for the enzyme at each stage in the purification. The ratio of the activity of the purified product on hemimethylated to that on unmethylated M13 duplex DNA was about 12 to 1. Thus, the purified activity has the properties postulated for a maintenance methyltransferase. The availability of highly purified human DNA methyltransferase should facilitate many studies on the structure, function, and expression of these activities in both normal and transformed cells.