REPLICATIVE SENESCENCE OF NORMAL HUMAN ORAL KERATINOCYTES IS ASSOCIATED WITH THE LOSS OF TELOMERASE ACTIVITY WITHOUT SHORTENING OF TELOMERES

Telomerase activity was analyzed in 7 different cultures of secondary normal human oral keratinocytes (NHOKs), 1 normal human oral epithelia l tissue specimen, 1 immortalized human oral keratinocyte (HOK) cell l ine, and 10 human oral cancer cell lines using the PCR-based telomeric repeat amplification protocol assay. Telomerase activity was found in all tested cells and tissue, but the activity in NHOKs and epithelial tissue was lower than that in other tested cell lines, inasmuch as co ntinued subculture of NHOKs results in replicative senescence, we inve stigated the association between telomerase activity and replicative s enescence by evaluating the enzyme activity in NHOK cultures with diff erent population doubling levels, Three different NHOK cultures were i ndependently subcultured until these cells reached the postmitotic sta ge, Unlike in fibroblasts derived from the human oral cavity, signific ant telomerase activity was detected in rapidly proliferating NHOKs, a nd telomerase activity was barely detectable in the keratinocytes near and at senescence, However, the terminal restriction fragment consist ing of telomeric DNA was found to be constantly maintained at similar to 6.0 kilobases in NHOKs without any detectable shortening of telomer es by subcultures, Intracellular p53 and p21(WAF1/CIP1) protein levels in NHOKs were gradually and significantly diminished by the passage o f cells, These data indicate that actively proliferating NHOKs contain telomerase activity and that replicative senescence of NHOKs is assoc iated with the loss of telomerase activity without shortening of telom eres, However, replicative senescence of NHOKs is apparently not linke d to an accumulation of wild-type p53 and/or p21(WAF1/CIP1) proteins i n these cells.