Journal Article

Association of p16<sup>INK4a</sup> and pRb inactivation with immortalization of human cells

Takeki Tsutsui, Shin-ichi Kumakura, Akito Yamamoto, Hideaki Kanai, Yukiko Tamura, Takashi Kato, Masanori Anpo, Hidetoshi Tahara and J.Carl Barrett

in Carcinogenesis

Volume 23, issue 12, pages 2111-2117
Published in print December 2002 | ISSN: 0143-3334
Published online December 2002 | e-ISSN: 1460-2180 | DOI:
Association of p16INK4a and pRb inactivation with immortalization of human cells

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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.

Keywords: ALT, alternative lengthening of telomeres; CDK, cyclin-dependent kinase; ERK 1, extracellular-signal related kinase 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HPV, human papillomavirus; hTERT, human telomerase catalytic subunit; MSP, methylation-specific PCR; ppRb, hyperphosphorylated pRb; pRb, retinoblastoma gene product; RT–PCR, reverse transcription–PCR

Journal Article.  5422 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics

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