title: Immortalization of bovine capillary endothelial cells by hTERT alone involves inactivation of endogenous p16INK4A/pRb.
abstract: Recent studies show that stable expression of the human telomerase catalytic subunit, hTERT, alone can lead several types of normal human somatic cells to bypass replicative senescence and become immortal. The molecular mechanisms by which telomerase immortalizes cells are not fully understood, although a key function of telomerase is to maintain a critical length of telomeres in order to preserve the stability and integrity of the genome. Here we report that stable transfection of hTERT alone was sufficient to allow bovine capillary endothelial (BCE) cells to bypass senescence and acquire immortality. Surprisingly, telomere lengths in these stable transfectants were progressively shortened during an increasing number of population doublings (PDLs), despite high telomerase activity. The expression of the cyclin-dependent kinase inhibitors (CDKIs) p16INK4A and p21CIP1/WAF1 was concomitantly repressed, and the retinoblastoma protein (pRb) was maintained in a hyperphosphorylated state in the telomerase-expressing cells. Re-expression of p16INK4A in these cells by either treatment with a demethylating agent or by adenovirus-mediated expression reinduced a senescence-like phenotype, suggesting that the inactivation of p16INK4A was due to DNA methylation and was crucial for the immortalization process. In agreement with this finding, the expression levels of the prototypic DNA methyltransferase DNMT1 were elevated in the hTERT-positive cells.
authors.affiliation: Microbiology and Tumor Biology Center, Karolinska Institute, S-171 77 Stockholm, Sweden.
authors.forename: Niina
authors.initials: N
authors.lastname: Veitonmäki
authors.forename: Jonas
authors.initials: J
authors.lastname: Fuxe
authors.forename: Magnus
authors.initials: M
authors.lastname: Hultdin
authors.forename: Göran
authors.initials: G
authors.lastname: Roos
authors.forename: Ralf F
authors.initials: RF
authors.lastname: Pettersson
authors.forename: Yihai
authors.initials: Y
authors.lastname: Cao
mesh_terms: D000818:Animals; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D002417:Cattle; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000187:drug effects; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000378:metabolism*; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors; D004268:DNA-Binding Proteins; D000077209:Decitabine; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D016160:Retinoblastoma Protein / Q000378:metabolism*; D019098:Telomerase / Q000235:genetics / Q000378:metabolism*; D016615:Telomere / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: C493956:CDKN1A protein, human; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D016160:Retinoblastoma Protein; D000077209:Decitabine; D015254:DNA Modification Methylases; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619380:DNMT1 protein, human; D019098:Telomerase; D001374:Azacitidine