Association of p16INK4a and pRb inactivation with immortalization of human cells

Abstract

To examine the association of cell cycle regulatory gene inactivation with human cell immortalization, we determined the expression status of INK4a, Rb, and WAF1/CIP1, in eleven in vitro immortalized human cell lines, including fibroblasts and keratinocytes. Two human papillomavirus type 16 E6 expressing cell lines with telomerase activity, including a fibroblast cell line and a keratinocyte cell line, expressed no detectable p16INK4a. These cell lines had a hyperphosphorylated pRb and reduced expression of p21WAF1/CIP1. All of seven fibroblast cell lines immortalized either spontaneously or by 60CO, X-rays, 4-nitroquinoline 1-oxide or aflatoxin B1, maintaining their telomeres by the ALT (alternative lengthening of telomeres) pathway, displayed loss of expression of p16INK4a and hyperphosphorylation of pRb. Levels of p21WAF1/CIP1 expression varied among the cell lines. Two fibroblast cell lines that became immortalized following infection with a retrovirus vector encoding human telomerase catalytic subunit (hTERT) cDNA were also accompanied by inactivation of p16INK4a and pRb pathways. Acquisition of telomerase activity alone was not sufficient for immortalization of these cell lines. Taken together, all the cell lines including fibroblasts and keratinocytes, with either telomerase activity or the ALT pathway for telomere maintenance showed loss of expression of p16INK4a and hyperphosphorylation of pRb. These demonstrate the association of inactivation of both p16INK4a and pRb with immortalization of human cells including fibroblasts and epithelial cells and telomerase-positive cells and ALT-positive cells.