CHANGES IN O-6-METHYLGUANINE-DNA METHYLTRANSFERASE EXPRESSION DURING IMMORTALIZATION OF CLONED HUMAN FIBROBLASTS

Suppressed expression of the DNA repair enzyme O-6-methylguanine-DNA m ethyltransferase (MGMT), characterized as the Mer(-) phenotype, occurs only in malignant or transformed cell lines, To investigate the relat ionship between the transformation process and loss of MGMT expression , we derived 20 cloned lines of IMR90 normal fibroblasts transfected w ith the plasmid pSV3neo expressing the SV40 large-T antigen, Of the fi ve lines that were grown until crisis phase, four emerged as continuou sly proliferating immortal lines, Of these, only one retained MGMT, th e other three having become Mer(-). In every case the loss of MGMT coi ncided with the final phase of immortalization following crisis, Becau se these were cloned cell lines it is clear that the phenotypic change to Mer(-) is not merely due to selection of a Mer(-) cell from the in itial population, but must involve a cellular change in MGMT regulatio n, It is not clear if increased mutation rate associated with loss of MGMT results in increased frequency of an immortalization event or if an immortalization event, such as telomere disruption, results in MGMT suppression, In addition, we have shown that, consistent with previou s observations, both hypermethylation in promoter sequences and hypome thylation of downstream sequences in the body of the gene were closely associated with loss of MGMT expression, These studies also illustrat e the utility of these new cloned cell lines for characterizing molecu lar events associated with transformation and immortalization.