methylnitronitrosoguanidine and Glioma

Methylating drugs such as temozolomide (TMZ) are widely used in the treatment of brain tumours (malignant gliomas). The mechanism of TMZ-induced glioma cell death is unknown. Here, we show that malignant glioma cells undergo apoptosis following treatment with the methylating agents N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and TMZ. Cell death determined by colony formation and apoptosis following methylation is greatly stimulated by p53. Transfection experiments with O(6)-methylguanine-DNA methyltransferase (MGMT) and depletion of MGMT by O(6)-benzylguanine showed that, in gliomas, the apoptotic signal originates from O(6)-methylguanine (O(6)MeG) and that repair of O(6)MeG by MGMT prevents apoptosis. We further demonstrate that O(6)MeG-triggered apoptosis requires Fas/CD95/Apo-1 receptor activation in p53 non-mutated glioma cells, whereas in p53 mutated gliomas the same DNA lesion triggers the mitochondrial apoptotic pathway. This occurs less effectively via Bcl-2 degradation and caspase-9, -2, -7 and -3 activation. O(6)MeG-triggered apoptosis in gliomas is a late response (occurring >120 h after treatment) that requires extensive cell proliferation. Stimulation of cell cycle progression by the Pasteurella multocida toxin promoted apoptosis whereas serum starvation attenuated it. O(6)MeG-induced apoptosis in glioma cells was preceded by the formation of DNA double-strand breaks (DSBs), as measured by gammaH2AX formation. Glioma cells mutated in DNA-PK(cs), which is involved in non-homologous end-joining, were more sensitive to TMZ-induced apoptosis, supporting the involvement of DSBs as a downstream apoptosis triggering lesion. Overall, the data demonstrate that cell death induced by TMZ in gliomas is due to apoptosis and that determinants of sensitivity of gliomas to TMZ are MGMT, p53, proliferation rate and DSB repair.

Topics: Antineoplastic Agents, Alkylating; Apoptosis; Blotting, Western; Brain Neoplasms; Caspases; Cell Cycle; Cell Proliferation; Dacarbazine; DNA Breaks, Double-Stranded; DNA Damage; Fas Ligand Protein; Fas-Associated Death Domain Protein; Flow Cytometry; Glioma; Guanine; Humans; Methylnitronitrosoguanidine; O(6)-Methylguanine-DNA Methyltransferase; Proto-Oncogene Proteins c-bcl-2; RNA, Small Interfering; Temozolomide; Tumor Cells, Cultured; Tumor Stem Cell Assay; Tumor Suppressor Protein p53

The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) has been implicated in resistance of human brain tumors to alkylating agents. We observed that 14 human medulloblastoma- and glioma-derived cell lines differ in sensitivity to the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), as shown by their 28-fold range in 10% survival dose (LD10). By using the substrate analogue inhibitor O6-benzylguanine (O6-BG), we showed that the contribution of MGMT to resistance varies widely, as evidenced by 3- to 30-fold reductions in LD10 among the lines, and varies up to 20-fold among subpopulations of individual lines. Importantly, variability in resistance, manifested as a 20-fold range in LD10, persists after measurable MGMT is eliminated, disclosing differential contributions of other resistance mechanisms to survival. Cells exposed to MNNG while suspended in growth medium are more resistant than cells alkylated as subconfluent monolayers, and MGMT accounts for a smaller proportion of their resistance. Notably, the MGMT content of the lines is not statistically correlated with MNNG resistance or with potentiation of killing by O6-BG, even though MGMT is a biochemically demonstrated determinant of resistance. In contrast, the same lines vary less in resistance to the ethylating agent N-ethylnitrosourea (ENU), and MGMT makes only a small contribution to resistance. Our results strongly indicate that resistance to both MNNG and ENU is multifactorial.

Topics: Alkylating Agents; Antineoplastic Agents; Brain Neoplasms; Cell Adhesion; Colonic Neoplasms; Culture Media; Drug Resistance; Drug Screening Assays, Antitumor; Ethylnitrosourea; Glioma; Medulloblastoma; Methylation; Methylnitronitrosoguanidine; Methyltransferases; O(6)-Methylguanine-DNA Methyltransferase; Tumor Cells, Cultured

The ethyl-N-nitrosourea-induced rat glioma N32 was treated with the mutagenic compound N-methyl-N'-nitro-N-nitrosoguanidine and the surviving cells cloned by limited dilution. Out of 20 clones tested 8 did not produce tumors subcutaneously even after challenge doses 3 log units above the minimal tumor dose for N32. All of 5 clones grew in a retarded manner intracerebrally but produced tumors in some animals. Preimmunizations with three of the rejected clones (tum-) gave protection against subcutaneous and intracerebral isografts of the unmutated N32. This effect could be enhanced if the cells used for immunizations were pretreated with interferon gamma (IFN gamma) for 48 h. If immunizations were started subsequent to challenge, only immunization with one of two tested tum- clones pretreated with IFN gamma induced significant rejection against intracerebral N32 isografts. Both N32 and its tum- clones were MHC class I positive and MHC class II negative. IFN gamma treatment enhanced the MHC class I expression with 20%-90% on the tum- clones and with 40% on N32. MHC class II expression could be induced on N32 cells after 7 days of IFN gamma treatment but not on any of the tum- clones tested. We conclude that the enhancing effect of IFN gamma treatment on tumor isograft rejection may depend on up-regulation of MHC class I but not of MHC class II. This investigation demonstrates that it is possible to induce rejection of weakly immunogenic intracerebral brain tumors by immunization with selected highly immunogenic tumor cell mutants. In conjunction with relevant cytokines, the cross-protective effect of these tum- variants might be further enhanced and serve as a model for immunotherapy against malignant human brain tumors.

Topics: Animals; Brain Neoplasms; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Glioma; Graft Rejection; Histocompatibility Antigens Class I; Histocompatibility Antigens Class II; Immunization; Immunotherapy; Interferon-gamma; Male; Methylnitronitrosoguanidine; Neoplasm Transplantation; Rats; Rats, Inbred F344; Survival Analysis; Survival Rate; Time Factors; Transplantation, Isogeneic; Treatment Outcome; Tumor Cells, Cultured

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.

Topics: Base Composition; Base Sequence; Binding, Competitive; Colonic Neoplasms; Deoxyribonuclease I; DNA; DNA Repair; Glioma; Guanine; Humans; Kinetics; Methylnitronitrosoguanidine; Methyltransferases; Molecular Sequence Data; O(6)-Methylguanine-DNA Methyltransferase; Thymine; Tumor Cells, Cultured

Alkylating agents, mechlorethamine and N-methyl-N'-nitro-N-nitrosoguanidine, induce the production of plasminogen activator in U-87MG cells, an alkylation DNA repair deficient (Mer-) human glioblastoma strain. Enzyme induction was not observed, however, in U-178MG and SH-101 cells, alkylation repair proficient (Mer+) glioblastoma strains, or in HeLa cells, which reactivated and supported well the growth of alkylation damaged adenovirus 3. In the alkylation repair defective U-87MG strain, enhanced production of plasminogen activator occurred in a narrow concentration range of treatment with either alkylating agent, causing a 20 to 50% inhibition of [3H]thymidine incorporation. Maximum plasminogen activator induction was observed between 32 and 48 h after alkylation treatment and the levels of enzyme produced were 5 to 10 times those of untreated control levels. This alkylation dependent enzyme induction required protein synthesis for it did not occur in the presence of cycloheximide. It was hence concluded that plasminogen activator induction in alkylation repair deficient human cells is caused by unrepaired DNA damage and that it may represent an eukaryotic SOS-like function. In addition, plasminogen activator induction may be useful as a sensitive assay for the identification of alkylation repair defective human tumors for which the susceptibility to alkylation chemotherapy should be expected to increase.

Topics: Alkylating Agents; Cell Line; Cycloheximide; DNA Repair; Enzyme Induction; Glioma; Humans; Mechlorethamine; Methylnitronitrosoguanidine; Plasminogen Activators

Topics: Brain Neoplasms; Glioma; Humans; Methylnitronitrosoguanidine; Occupational Diseases

Topics: Adenoviruses, Human; Astrocytoma; Brain Neoplasms; Cell Line; DNA Repair; Glioma; Humans; Methylnitronitrosoguanidine; Ultraviolet Rays; Virus Cultivation