Association of p16(INK4a) and pRb inactivation with immortalization of human cells.
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.
Tsutsui
Takeki
T
Department of Pharmacology, The Nippon Dental University, School of Dentistry at Tokyo, Japan.
Kumakura
Shin-Ichi
S
Yamamoto
Akito
A
Kanai
Hideaki
H
Tamura
Yukiko
Y
Kato
Takashi
T
Anpo
Masanori
M
Tahara
Hidetoshi
H
Barrett
J Carl
JC
D015112:4-Nitroquinoline-1-oxide; D016604:Aflatoxin B1; D002273:Carcinogens; D002453:Cell Cycle; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D003037:Cobalt Radioisotopes; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000378:metabolism*; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism; D009856:Oncogene Proteins, Viral / Q000378:metabolism; D010766:Phosphorylation; D012333:RNA, Messenger / Q000378:metabolism; D012097:Repressor Proteins; D016160:Retinoblastoma Protein / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019098:Telomerase / Q000378:metabolism; D016615:Telomere; D013997:Time Factors; D014407:Tumor Cells, Cultured; D014965:X-Rays
C493956:CDKN1A protein, human; D002273:Carcinogens; D003037:Cobalt Radioisotopes; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D012333:RNA, Messenger; D012097:Repressor Proteins; D016160:Retinoblastoma Protein; D015112:4-Nitroquinoline-1-oxide; D016604:Aflatoxin B1; D019098:Telomerase