Senescence occurs with hTERT repression and limited telomere shortening in human oral keratinocytes cultured with feeder cells

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Abstract
We investigated the phenotypic and molecular alterations in normal human oral keratinocytes (NHOK) during in vitro replication in two different culture conditions. The cells were cultured either in chemically defined Keratinocyte Growth Medium (KGM) without feeder layers or in serum‐containing flavin‐adenine dinucleotide (FAD) medium with feeder layers. Primary NHOK underwent 22 ± 3 population doublings (PDs) in KGM and 42 ± 4 PDs in FAD medium, reflecting 52% increase in replication capacity with feeder layers. In both culture conditions, exponentially replicating NHOK demonstrated telomerase activity and expression of human telomerase reverse transcriptase (hTERT) gene. Telomerase activity and hTERT expression were rapidly diminished in senescing NHOK, which exhibited small decrease of telomere length for the remaining limited cellular replications until the complete arrest of cell division. However, telomere length in senescent NHOK was 6.7 ± 0.5 kilobase pairs (kbps), significantly longer than that (5.12 kbps) of senescent human fibroblasts. The onset of senescence was accompanied with marked induction of p16INK4A, and this occurred in both culture systems using either KGM or FAD medium. These results indicate that replicative senescence of NHOK is associated with loss of telomerase activity followed by limited telomere shortening.