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

Growth constraint of bacterial and human cells has been shown to trigger genetic 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 methylation in select regions of three CPG islands [estrogen receptor (ER), E-cadherin (ECAD) and O-6-methylguanine-DNA methyltransferase (MGMT)] examined. NHF grown to and left at confluence for 2-21 days showed little (<10%) CpG methylation in the ER and ECAD CPG islands, These confluent, growth-arrested cells, however, displayed extensive (similar to 50%) methylation of the MGMT CPG island. CpG methylation in the MGMT CpG island was not associated with cellular senescence. The methylation was, however, heritable, but not permanent, 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 logarithmically growing cells by 10 population doublings following replating. These results suggest that growth constraint can trigger transient epigenetic change even in normal non-senescent human cells.