Immortalization in a normal foreskin fibroblast culture following transduction of cyclin A2 or cdk1 genes in retroviral vectors.
Human diploid fibroblasts (HDF) rarely, if ever, undergo spontaneous transformation to an immortalized cell type. Here we report the immortalization of an HDF cell line following transduction with cyclin A2 or cdk1 human genes via retroviral vectors. Fluorescence in situ hybridization (FISH) studies using the retroviral vector as a probe indicate that these cell lines are monoclonal. No telomerase activity could be detected in these cell lines, and the telomere length in the immortalized cells was observed to be 10-20 kb longer than that in low-passage cells from the parental fibroblast line. Cytogenetic studies revealed that the immortal lines share common chromosomal aberrations. FISH studies with a probe for p53 revealed loss of one copy of this gene which was associated with reduced steady-state levels of both p53 and p53-regulated p21(WAF1/Sdi1/CIP1) messages in both quiescent and proliferating immortalized cultures relative to the parental cells. Additional FISH studies with probes for p16(INK4a) and Rb, carried out after the immortalized cells proliferated in excess of 100 population doublings, also revealed loss of one copy of these genes in both cell lines. These cell lines, together with the well-characterized parental cells, could provide useful research material for the study of the mechanisms of immortalization and of regulation of proliferative senescence in HDF.
Luo
Ping
P
Department of Pathology, University of Washington Seattle, Seattle, WA 98195, USA.
Tresini
Maria
M
Cristofalo
Vincent
V
Chen
Xiaoqin
X
Saulewicz
Anthony
A
Gray
Matthew D
MD
Banker
Deborah E
DE
Klingelhutz
Aloysius L
AL
Ohtsubo
Motoaki
M
Takihara
Yoshihiro
Y
Norwood
Thomas H
TH
D016203:CDC2 Protein Kinase / Q000096:biosynthesis / Q000235:genetics*; D002455:Cell Division / Q000235:genetics; D002461:Cell Line, Transformed; D016922:Cellular Senescence / Q000235:genetics*; D002869:Chromosome Aberrations; D002999:Clone Cells / Q000378:metabolism; D019925:Cyclin A / Q000096:biosynthesis / Q000235:genetics*; D056751:Cyclin A2; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics; D005347:Fibroblasts / Q000201:enzymology / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007231:Infant, Newborn; D019656:Loss of Heterozygosity / Q000235:genetics; D008297:Male; D016160:Retinoblastoma Protein / Q000235:genetics; D012190:Retroviridae / Q000235:genetics; D019098:Telomerase / Q000378:metabolism; D014161:Transduction, Genetic / Q000379:methods*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
C503721:CCNA2 protein, human; C493956:CDKN1A protein, human; D019925:Cyclin A; D056751:Cyclin A2; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D016160:Retinoblastoma Protein; D016159:Tumor Suppressor Protein p53; D016203:CDC2 Protein Kinase; D019098:Telomerase