Tsutsui Takeki, Kumakura Shin-Ichi, Yamamoto Akito, Kanai Hideaki, Tamura Yukiko, Kato Takashi, Anpo Masanori, Tahara Hidetoshi, Barrett J Carl
Department of Pharmacology, The Nippon Dental University, School of Dentistry at Tokyo, Japan.
Carcinogenesis. 2002 Dec;23(12):2111-7. doi: 10.1093/carcin/23.12.2111.
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 p16(INK4a). These cell lines had a hyperphosphorylated pRb and reduced expression of p21(WAF1/CIP1). All of seven fibroblast cell lines immortalized either spontaneously or by (60)Co, X-rays, 4-nitroquinoline 1-oxide or aflatoxin B(1), maintaining their telomeres by the ALT (alternative lengthening of telomeres) pathway, displayed loss of expression of p16(INK4a) and hyperphosphorylation of pRb. Levels of p21(WAF1/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 p16(INK4a) 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 p16(INK4a) and hyperphosphorylation of pRb. These demonstrate the association of inactivation of both p16(INK4a) and pRb with immortalization of human cells including fibroblasts and epithelial cells and telomerase-positive cells and ALT-positive cells.
为了研究细胞周期调节基因失活与人类细胞永生化之间的关联，我们测定了11种体外永生化的人类细胞系（包括成纤维细胞和角质形成细胞）中INK4a、Rb和WAF1/CIP1的表达状态。两种具有端粒酶活性的人乳头瘤病毒16型E6表达细胞系（包括一个成纤维细胞系和一个角质形成细胞系）未检测到p16(INK4a)表达。这些细胞系的pRb发生了过度磷酸化，且p21(WAF1/CIP1)表达降低。7种通过自发或经(60)Co、X射线、4-硝基喹啉1-氧化物或黄曲霉毒素B(1)永生化的成纤维细胞系，通过端粒替代延长（ALT）途径维持其端粒，均表现出p16(INK4a)表达缺失和pRb过度磷酸化。p21(WAF1/CIP1)的表达水平在这些细胞系中各不相同。两种感染编码人端粒酶催化亚基（hTERT）cDNA的逆转录病毒载体后永生化的成纤维细胞系，也伴随着p16(INK4a)和pRb途径的失活。仅获得端粒酶活性不足以使这些细胞系永生化。综上所述，所有包括成纤维细胞和角质形成细胞在内的细胞系，无论具有端粒酶活性还是通过ALT途径维持端粒，均表现出p16(INK4a)表达缺失和pRb过度磷酸化。这些结果证明了p16(INK4a)和pRb的失活与人类细胞（包括成纤维细胞、上皮细胞、端粒酶阳性细胞和ALT阳性细胞）的永生化之间存在关联。
