P53-INDEPENDENT DOWN-REGULATION OF CYCLIN D1 AND P21(WAF1) IN THE PROCESS OF IMMORTALIZATION OF HUMAN ESOPHAGEAL EPITHELIAL-CELLS

Immortalization is considered to be an initial critical step in the pr ocess of multistage cell transformation. However, the molecular mechan isms underlying this event are not well understood. Our laboratory pre viously established the immortalized human esophageal epithelial cell line, HET-1A, by SV40 T-antigen transfection. In the present study, we investigated the role of G1 cyclins and cyclin dependent kinase inhib itors, in the process of immortalization. By using immunoprecipitation and Western blot analysis, sequential changes in the expression of bo th cyclin D1 and p21(Waf1) were detected during the conversion of prec risis esophageal epithelial cells to immortalized HET-1A cells. Reduce d expression levels of both cyclin D1 and p21(Waf1) were found in earl y passage and late passage immortalized cells when compared to levels in precrisis cells. In addition, continued subculture of the immortali zed cells led to increased expression levels of both cyclin D1 and p21 (Waf1). No significant changes in the expression of either cyclin E or p53 were observed in early or late passage immortalized cells when co mpared to precrisis cells. These results suggest that changes in the e xpression levels of cyclin D1 and p21(Waf1) but not cyclin E, may be i mportant for immortalization and continued propagation of human esopha geal epithelial cells, and these changes are not dependent on regulati on by p53.