Confluence-induced alterations in CpG island methylation in cultured normal human fibroblasts

Growth constraint of bacterial and human cells has been shown to trigger ge netic mutation. We questioned whether growth constraint might also trigger epigenetic mutation in the form of CpG island methylation. Logarithmically growing normal human fibroblasts (NHF) displayed little (0-15%) CpG methyla tion in select regions of three CPG islands [estrogen receptor (ER), E-cadh erin (ECAD) and O-6-methylguanine-DNA methyltransferase (MGMT)] examined. N HF grown to and left at confluence for 2-21 days showed little (<10%) CpG m ethylation in the ER and ECAD CPG islands, These confluent, growth-arrested cells, however, displayed extensive (similar to 50%) methylation of the MG MT CPG island. CpG methylation in the MGMT CpG island was not associated wi th cellular senescence. The methylation was, however, heritable, but not pe rmanent, as the level of CpG methylation in the MGMT CPG island of cells 4 population doublings following replating after confluence were no different from those in confluent cultures, but returned to levels noted in logarith mically growing cells by 10 population doublings following replating. These results suggest that growth constraint can trigger transient epigenetic ch ange even in normal non-senescent human cells.