The long-term goal of our research is to understand the mechanism of cellular senescence and differentiation in human epithelial cells. As the first step toward our goal, we have established an in vitro model of cellular senescence and differentiation with serially subcultured normal human oral keratinocytes (NHOK). NHOK underwent a maximum of 22+3 population doublings (PDs) through distinct phases of replication: exponential, senescing, senescent and terminal differentiation. Using our in vitro model, we have investigated the mode of senescence of NHOK in comparison to other human somatic cells, such as human diploid fibroblasts (HDF). In contrast to HDF, exponentially replicating NHOK showed telomerase activity and entered the senescent phase in the absence of telomere shortening. Also, in contrast to HOF, whose senescence program is triggered by p53 and p21WAF1/CIP1, senescent NHOK exhibited decreased levels of these proteins. These findings indicate that NHOK enter senescence in a telomere length-independent manner and through a distinct mechanism from that of HDF. We also found that subcultured NHOK exhibited the features of senescence and differentiation in a sequentially distinct manner, suggesting that separate mechanisms elicit these processes during in vitro passage. Accordingly, we raise a question: What genes constitute the molecular pathways of replicative senescence and differentiation in NHOK? This question will be addressed in the present proposal with the following specific aims: (1) To investigate the profile and the kinetics of gene expression in exponentially replicating, senescing, and senescent and terminally differentiating NHOK during serial subculture, (2) to identify the gene(s) associated with senescence and/or differentiation of NHOK in vitro, and (3) to determine whether the ectopic expression of the identified genes can trigger the senescence and differentiation programs in exponentially replicating NHOK and in immortalized human oral keratinocytes. With these specific aims, we expect to address the following questions: Which genes are involved in senescence and/or differentiation in NHOK? Are same type of gene(s) involved in both senescence and differentiation pathways or not? Can we induce senescence and/or differentiation of cells with specific genes identified by our specific aims? It is expected that the outcome of this proposal will develop into novel hypotheses with which we can further study the molecular mechanisms of cellular replication, senescence, and differentiation of human epithelial cells.
