Thomas P. Brent

A specific CpG methylation pattern of the MGMT promoter region associated with reduced MGMT expression in primary colorectal cancers.

The enzyme O6‐methylguanine‐DNA methyltransferase (MGMT) protects cells from the cytotoxic and mutagenic effects of alkylating agents. Approximately 20% of tumor cell lines lack MGMT activity and are highly sensitive to alkylating agents. In established cancer cell lines, MGMT expression appears to be correlated with methylation of residues in both the promoter and the body of the gene. The effect of methylation of the MGMT promoter on gene expression and carcinogenesis in primary tumors is unknown. We investigated methylation of the MGMT promoter region in primary colorectal cancers and normal colonic mucosa. We used five methylation‐sensitive restriction enzymes (BssHII, SacII, EagI, NaeI, and SmaI) and Southern blot analysis to assess methylation in 46 cancers and 22 controls. Methylation of EagI and NaeI sites was seen in 12 tumors but in none of the 22 normal colorectal mucosa specimens. This difference was statistically significant (P < 0.01). Methylation‐sensitive single‐nucleotide primer extension analysis of four additional cytosine residues confirmed methylation of the promoter region in the tumors identified by EagI and NaeI digestions and served to further quantitate the extent of methylation. Western blot analysis of 21 tumors revealed statistically significant lower MGMT expression in the eight tumors with methylation of the EagI and NaeI sites and nt −128 than in the 13 tumors lacking the methylation pattern (P < 0.05). MGMT activity was lower in tumors with methylation than in tumors that were not methylated. The difference was not, however, statistically significant. We conclude that a subset of colorectal tumors is characterized by a specific methylation pattern in the MGMT promoter associated with reduced MGMT expression. Mol. Carcinog. 24:90–98, 1999. ? 1999 Wiley‐Liss, Inc.

Methylation of selected CpGs in the human O6‐methylguanine‐DNA methyltransferase promoter region as a marker of gene silencing

O6‐Methylguanine‐DNA methyltransferase (MGMT) is a major determinant of susceptibility to methylating carcinogens and of tumor resistance to anticancer methylating and chloroethylating drugs. The silencing of MGMT expression that occurs in 20–30% of human tumor lines is tightly linked to methylation within the MGMT gene 5′ CpG island. Previous studies on a very limited number of cell lines showed that such methylation was uneven, with hot‐spots where methylation almost invariably occurred and intervening regions with very low incidences of methylation. To ascertain if such hot‐spot methylation is in fact a ubiquitous hallmark of MGMT‐silenced cells, we determined the methylation status of selected hot‐spot CpGs in an extensive panel of MGMT‐expressing and ‐silenced cell lines and xenografts. Using two simple and rapid bisulfite–polymerase chain reaction–based assays, we confirmed that in MGMT‐silenced cells, methylation occurred at these sites whereas it was essentially absent in MGMT‐expressing cells. Mol. Carcinog. 24:85–89, 1999.

Modulation of cyclophosphamide activity by O 6-alkylguanine-DNA alkyltransferase

Purpose: The human medulloblastoma cell line D283 Med (4-HCR), a line resistant to 4-hydroperoxycyclophosphamide (4-HC), displays enhanced␣repair of DNA interstrand crosslinks induced by phosphoramide mustard. D283 Med (4-HCR) cells are cross-resistant to 1,3-bis(2-chloroethyl)-1-nitrosourea, but partial sensitivity is restored after elevated levels of O6-alkylguanine-DNA alkyltransferase (AGT) are depleted by O6-benzylguanine (O6-BG). Studies were conducted to define the activity of 4-HC and 4-hydroperoxydidechlorocyclophosphamide against D283 Med (4-HCR) after AGT is depleted by O6-BG. Methods: Limiting dilution and xenograft studies were conducted to define the activity of 4-HC and 4-hydroperoxydidechlorocyclophosphamide with or without O6-BG. Results: The activity of 4-HC and 4-hydroperoxydidechlorocyclophosphamide against D283 Med (4-HCR) was increased after AGT depletion by O6-BG preincubation. Similar studies with Chinese hamster ovary cells, with or without stable transfection with a plasmid expressing the human AGT protein, revealed that the AGT-expressing cells were significantly less sensitive to 4-HC and 4-hydroperoxydidechlorocyclophosphamide. Reaction of DNA with 4-HC, phosphoramide mustard, or acrolein revealed that only 4-HC and acrolein caused a decrease in AGT levels. Conclusions: We propose that a small but potentially significant part of the cellular toxicity of cyclophosphamide in these cells is due to acrolein, and that this toxicity is abrogated by removal of the acrolein adduct from DNA by AGT.

Regulation of O6-methylguanine-DNA methyltransferase by methionine in human tumour cells.

Methionine (MET)-dependent cell lines require MET to proliferate, and homocysteine (HCY) does not act as a substitute for this requirement. From six O6-methylguanine-DNA methyltransferase (MGMT)-efficient (mer+) cell lines tested, two medulloblastomas (Daoy and D-341) and a lung non-small-cell adenocarcinoma with metastatic potential (H-1623) were most sensitive to MET deprivation, while two glioblastomas (U-138, D-263) and a small-cell lung carcinoma H-1944 were moderately to weakly dependent. Regardless of the degree of MET dependence, all of these lines down-regulated their MGMT activity within 48-72 h of transfer from MET+HCY- to MET-HCY+ media, long before the eradication of the culture. Reduction of MGMT activity was due to a decline of both MGMT mRNA and protein levels. However, the reduction was not related to the methylation status of the MGMT promoter at the SmaI site or the HpaII sites in the body of the gene; such sites have been shown to be associated in MGMT regulation and in defining the mer phenotype. MET-dependent, mer+ tumour cells cultured in MET-HCY+ were more sensitive to BCNU (IC50 = 5-10 microM) than those cultured in MET+HCY-(IC50 = 45-90 microM), while MET-independent or mer- cell lines were unaffected. This indicates that reduction of MGMT, imposed by the absence of MET, renders mer+ tumour cells more susceptible to alkylating agents. The relatively selective suppression of MGMT activity in mer+ MET-dependent tumour cells, in combination with the inability of such cells to proliferate in the absence of MET, may lead to the development of more effective treatment strategies for mer+ MET-dependent tumours.

Purification and characterization of human endonucleases specific for damaged DNA. Analysis of lesions induced by ultraviolet or x-radiation.

1. An endonuclease activity from a cultured human lymphoblast cell line, CCRF-CEM, was further purified by chromatography on phosphocellulose to remove a nonspecific acid endonuclease. 2. The purified enzyme acted quantitatively on apurinic sites in the DNA of PM2 phage. It showed optimum activity over a broad range of pH (7.0--8.5), was absolutely dependent on Mg2+ (optimum concentration 0.5 mM) and did not have detectable activity against intact DNA. 3. This enzyme was used as a probe to estimate the number of apurinic or apyrimidinic lesions induced in PM2 DNA by either ultraviolet or X-ray irradiation. High doses of ultraviolet radiation (2500 to 5000 J/m2) immediately induced 0.2 to 0.4 endonuclease-susceptible lesions per molecule of DNA. The lesions continued to increase for several hours after irradiation, reaching a level more than double that found initially. By contrast, in DNA exposed to 5000 rads of X-ray irradiation, the number of endonuclease-susceptible sites reached a maximum of about 0.6 per molecule immediately after exposure and did not increase further. It thus appears that ultraviolet-irradiated (but not X-ray irradiated) DNA contains damaged bases that are lost spontaneously after irradiation. 4. A second endonuclease was purified and was shown to act on ultraviolet-induced lesions that are distinct from either apurinic or apyrimidinic sites. These new lesions occur about ten times more frequently than ultraviolet-induced apurinic or apyrimidinic sites.

1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazine (VNP40101M): II. Role of O6-alkylguanine-DNA alkyltransferase in cytotoxicity

PurposeVNP40101M (1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazine) is a sulfonylhydrazine prodrug that possesses broad spectrum antitumor efficacy in murine models. VNP40101M activation generates chloroethylating species that alkylate DNA at the O6-position of guanine, and a carbamoylating agent, methyl isocyanate, which inhibits O6-alkylguanine-DNA alkyltransferase (AGT) in model systems. We determined whether expression of AGT in Chinese hamster ovary (CHO) cells decreased sensitivity to VNP40101M and explored the mechanism of VNP40101M cytotoxicity by employing analogs of VNP40101M that generate reactive intermediates with either carbamoylating or chloroethylating activity.MethodsAGT was overexpressed in CHO cells by transfection with an expression vector containing the human AGT gene. Cell lines expressing AGT were employed in clonogenic assays to determine the cytotoxicity of VNP40101M and its analogs.ResultsVNP40101M was more active against AGT-expressing CHO cells than 90CE (1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine), a chloroethylating generator devoid of carbamoylating activity. Furthermore, the greater the degree of AGT expression the more resistance to VNP40101M cytotoxicity. Combination chemotherapy experiments support the conclusions that methyl isocyanate and the chloroethylating species generated from the activation of VNP40101M function synergistically to kill cells.ConclusionsThe findings support the concept that alkylation of the O6-position of guanine residues in DNA is the predominant lesion created by VNP40101M, and that methyl isocyanate resulting from the base-catalyzed activation of VNP40101M inhibits AGT and presumably other enzymes involved in DNA repair, thereby enhancing the yield of the DNA G-C interstrand crosslinks responsible for the antitumor activity of this agent.

In vitro methylation of the human O6-methylguanine-DNA methyltransferase promoter reduces transcription.

Approx. 20% of human tumor cell lines (termed Mer-) are deficient in the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT; E.C.2.1.1.63). Such cells possess the MGMT gene and promoter sequences but have virtually no mRNA or protein. Cytosine methylation of gene sequences has been proposed as a mechanism by which MGMT could be suppressed in Mer- cells; however, the experimental evidence does not uniformly support this idea. We therefore investigated the effect of in vitro methylation of the MGMT promoter in a reporter gene construct transfected into cultured human cells. DNA methylation by HpaII or HhaI methylases suppressed the activity of the promoter, although the effect was not absolute. The occurrence of partial intracellular demethylation of promoter sequences may account for the incomplete inhibition of transcription. A model that attempts to reconcile the opposing views on the role of cytosine methylation in MGMT gene expression is presented.

Evidence that O6-alkylguanine-DNA alkyltransferase becomes covalently bound to DNA containing 1,3-bis(2-chloroethyl)-1-nitrosourea-induced precursors of interstrand cross-links

The reaction of partially purified human O6-alkylguanine-DNA alkyltransferase with 1,3-bis(2-chloroethyl)-1-nitrosourea-treated DNA resulted in formation of a DNA-protein covalent complex. Complex formation required active alkyltransferase and brief treatment of DNA with the drug. DNA lost its capacity to form the complex once drug-induced DNA interstrand cross-links were completely formed. These results are consistent with a model in which the transferase catalyzes cleavage at O6-guanine and transfer of the alkyl moiety in a putative O6, N1-ethanoguanine intermediate of cross-link formation. DNA-protein complex formation presumably results when the transferase accepts the N1-ethanoguanine-DNA structure, analogous to its acceptance of simple alkyl groups.

Partial purification of endonuclease activity from human lymphoblasts. Separation of activities for depurinated DNA and DNA irradiated with ultraviolet light.

Crude extracts of cultured human lymphoblasts (CCRF-CEM) contain endonuclease activity that cleaves ultraviolet-irradiated DNA in preference to untreated DNA. Purification of this activity was carried out using ultraviolet-irradiated PM2 phage DNA (5000 ergs/mm2) as substrate in the enzyme assay. Since endonuclease specific for depurinated or depyrimidinated DNA might account for the apparent ultraviolet-irradiated DNA-specific activity, fractions derived during purification were also assayed with partially depurinated DNA. Chromatography of a 45-60% (NH4)2SO4 fraction on a Sephadex G-100 column yielded a peak of activity (35 000 daltons) highly active against depurinated DNA but also active for ultraviolet-irradiated DNA. Further purification by DEAE-cellulose chromatography resolved two activities. One was highly specific for depurinated DNA with only minor activity for ultraviolet-irradiated DNA, and was strongly stimulated by Mg2+. The other was non-specifically active against ultraviolet-irradiated or untreated DNA and was independent of Mg2+. Additional studies suggest that neither of these two activities but a third enzyme is responsible for the ultraviolet-irradiated DNA-specific endonuclease activity observed in crude cell extracts.

Evidence that covalent complex formation between BCNU-treated oligonucleotides and E. coli alkyltransferases requires the O6-alkylguanine function.

Chloroethylnitrosoureas (CENUs) are thought to induce cytotoxic DNA interstrand cross-links via an initial reaction at O6-position of guanine, yielding a rearranged intermediate, O6,N1-ethanoguanine. Repair of these adducts by mammalian and bacterial DNA alkyltransferases blocks the formation of cross-links. Human alkyltransferase can form a covalent complex with DNA containing BCNU-induced cross-link precursors, but the nature of the DNA-protein linkage remains unknown. Using E. coli alkyltransferases expressed by the ada and ogt genes, we now demonstrate that both enzymes can form such complexes with CENU-treated DNA. We attribute this reaction to the O6-alkylguanine repair function, because an N-terminal fragment of the ada protein, which has only alkylphosphotriester repair activity, failed to form a similar complex. This result is consistent with the idea that complex formation requires an alkyltransferase reaction with a guanine adduct, such as O6,N1-ethanoguanine. It tends to exclude the possibility that such reactions simply involve alkylation of the enzyme by reactive DNA adducts such as chloroethylphosphate or chloroethylguanine.