Genomic differences between O6-methylguanine-DNA methyltransferase proficient (Mex+) and deficient (Mex-) cell lines: possible role of genetic and epigenetic changes in conversion of Mex+ into Mex-.

Cell lines that possess O6-methylguanine-DNA methyltransferase (MGMT) repair activity (Mex+ phenotype) or are deficient for MGMT (Mex-) are compared at genomic level. It is shown that 1) 208F rat cells do not contain the MGMT gene, as detected by Southern blot hybridization. 2) Mex- HeLa MR and CHO-9 cells express very low amounts of MGMT mRNA, as detected by PCR. The size of the MGMT specific PCR product was slightly smaller than that generated from Mex+ HeLa S3 cells. 3) HeLa MR, compared to various human Mex+ cell lines, shows a restriction fragment length polymorphism indicating mutational alteration of MGMT gene sequences. 4) Mex- cells (HeLa MR) and cells that express very low MGMT activity (GM637) exhibit hypomethylation of the MGMT gene as revealed by MspI/HpaII restriction digests. 5) Exposure of Mex- cells to 5-azacytidine and selection with N-hydroxyethyl-N-chloroethylnitrosourea (HeCNU) did not yield Mex+ revertants. With V79 cells treated with 5-azacytidine clones resistant to HeCNU were isolated. These cells were MGMT deficient and not cross-resistant to N-methyl-N'-nitro-N-nitrosoguanidine indicating the existance of a defence mechanism other than MGMT against chloroethylating agents. The data suggest down-regulation of MGMT transcription accompanied by decreasing CpG methylation, but in some cell lines also mutational alterations to be involved in extinction of the Mex+ phenotype.