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

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 p16(INK4A), 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.

Kang MK ，Kameta A ，Shin KH ，Baluda MA ，Park NH ... -  《JOURNAL OF CELLULAR PHYSIOLOGY》

Keratinocyte growth conditions modulate telomerase expression, senescence, and immortalization by human papillomavirus type 16 E6 and E7 oncogenes.

Fu B ，Quintero J ，Baker CC 《CANCER RESEARCH》

Replicative senescence of normal human oral keratinocytes is associated with the loss of telomerase activity without shortening of telomeres.

Kang MK ，Guo W ，Park NH 《-》

Retinoic acid extends the in vitro life span of normal human oral keratinocytes by decreasing p16(INK4A) expression and maintaining telomerase activity.

Retinoic acid (RA) plays an important role in the regulation of cell growth and differentiation. To investigate whether RA extends in vitro the life span of human epithelial cells, we examined the effect of all-trans RA on both the cumulative population-doubling level (PDL) and the replicative senescence of cultured oral keratinocytes. When proliferating oral keratinocytes were cultured in medium containing 1 nM of all-trans RA, the in vitro life span of the cells was increased 1.5- to 1.8-fold compared to the vehicle control and the replicative senescence of the cells was significantly inhibited. Since the replicative senescence of human epithelial cells is associated with a steady increase of p16(INK4A) and a loss of telomerase activity, we expected that RA could delay the replicative senescence of oral keratinocytes by decreasing p16(INK4A) expression and/or inhibiting the loss of telomerase activity. To test this possibility, we examined the expression of replicative senescence-associated genes and the telomerase activities of different PDL numbers of oral keratinocytes exposed to 1 nM of all-trans RA. The protein level of cellular p16(INK4A) in the RA-treated oral keratinocytes was gradually but significantly enhanced by an increased PDL number; however, the level was significantly lower than that of the vehicle control at all of the same PDL numbers. In contrast, the telomerase activity was maintained in oral keratinocytes with increasing PDL numbers induced by RA treatment. Summarizing, these results indicate that RA induces the in vitro life-span extension of oral keratinocytes, which is linked to a decreased cellular level of p16(INK4A) and the maintenance of telomerase activity.

You YO ，Lee G ，Min BM 《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》

Mass cultured human fibroblasts overexpressing hTERT encounter a growth crisis following an extended period of proliferation.

During the process of immortalization, at least two mortality checkpoints, M1 and M2, must be bypassed. Cells that have bypassed M1 (senescence) have an extended life span, but are not necessarily immortal. Recent studies have shown that ectopic expression of the catalytic subunit of telomerase (hTERT) enables normal human cells to bypass senescence (M1) and oncogene transformed cells to avert crisis (M2) and become immortal. However, it is unclear whether hTERT expression is sufficient for normal human fibroblasts to overcome both M1 and M2 and become immortal. We have investigated the role of telomerase in immortalization by maintaining mass cultures of hTERT-transduced primary human fetal lung fibroblasts (MRC-5 cells) for very long periods of time (more than 2 years). In the present studies, up to 70% of MRC-5 cells were transduced with retroviral vectors that express hTERT. hTERT-transduced cells exhibited high levels of telomerase activity, elongation of telomeres, and proliferation beyond senescence. However, after proliferating for more than 36 population doublings (PDLs) beyond senescence, the overall growth rate of hTERT-expressing cells declined. During theses periods of reduced growth, hTERT-transduced MRC-5 cells exhibited features typical of cells in crisis, including an increased rate of cell death and polyploidy. In some instances, very late passage cells acquired a senescence-like phenotype characterized by arrest in the G1 phase of the cell cycle and greatly reduced DNA synthesis. At the onset of crisis, hTERT-transduced cells expressed high levels of telomerase and had very long telomeres, ranging up to 30 kb. Not all cells succumbed to crisis and, consequently, some cultures have proliferated beyond 240 PDLs, while another culture appears to be permanently arrested at 160 PDLs. Late passage MRC-5 cells, including postcrisis cells, displayed no signs of malignant transformation. Our results are consistent with the model in which telomerase and telomere elongation greatly extends cellular life span without inducing malignant changes. However, these investigations also indicate that hTERT-expressing cells may undergo crisis following an extended life span and that immortality is not the universal outcome of hTERT expression in normal diploid fibroblasts.

MacKenzie KL ，Franco S ，May C ，Sadelain M ，Moore MA ... -  《EXPERIMENTAL CELL RESEARCH》