Changes in O6-methylguanine-DNA methyltransferase expression during immortalization of cloned human fibroblasts

Suppressed expression of the DNA repair enzyme O⁶-methylguanine-DNAmethyltransferase (MGMT), characterized as the Mer^– phenotype,occurs only in malignant or transformed cell lines. To investigatethe relationship between the transformation process and lossof MGMT expression, we derived 20 cloned lines of IMR90 normalfibroblasts transfected with the plasmid pSV3neo expressingthe SV40 large-T antigen. Of the five lines that were grownuntil crisis phase, four emerged as continuously proliferatingimmortal lines. Of these, only one retained MGMT, the otherthree having become Mer^–. In every case the loss of MGMTcoincided with the final phase of immortalization followingcrisis. Because these were cloned cell lines it is clear thatthe phenotypic change to Mer^– is not merely due to selectionof a Mer^– cell from the initial population, but must involvea cellular change in MGMT regulation, but must involve a cellularchange in MGMT regulation. It is not clear if increased mutationrate associated with loss of MGMT results in increased frequencyof an immortalization event or if an immortalization event,such as telomere disruption, results in MGMT suppression. Inaddition, we have shown that, consistent with previous observations,both hypermethylation in promoter sequences and hypomethylationof downstream sequences in the body of the gene were closelyassociated with loss of MGMT expression. These studies alsoillustrate the utility of these new cloned cell lines for characterizingmolecular events associated with transformation and immortalization.