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Prolonged Culture of Telomerase-Immortalized Human Fibroblasts Leads to a Premalignant Phenotype^{1 ,,2}

Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel [M. M., I. S., N. E., X. T., S. S., A. M., Y. T., N. G., D. G., V. R.], and the Laboratory of Human Carcinogenesis, National Cancer Institute, NIH, Bethesda, Maryland 20892 [C. C. H.]

Telomere shortening in primary human fibroblasts results in replicative senescence, which can be overcome by telomerase (hTERT) overexpression. However, because immortalization is one of the hallmarks of malignant transformation, careful analysis of hTERT-immortalized cells is of crucial importance for understanding both processes. To this end, we infected WI-38 fibroblasts with a retrovirus carrying the hTERT cDNA and analyzed their proliferative behavior during 600 days [500 population doublings (PDLs)] of continuous culture. Growth of three independent mass cultures was uniform for 150 PDLs after telomerase infection, followed by a progressive acceleration of growth in two of three cultures. Expression of p16^INK4A was significantly elevated in the immortalized cells but gradually disappeared during the accelerated growth phase. This alteration correlated with loss of the contact inhibition response and conferred the cells with sensitivity to H-Ras-induced transformation. In contrast, the p53- and pRb-mediated checkpoints such as the DNA damage response, chromosomal stability and entry into quiescence remained intact, irrespective of INK4A locus expression. Importantly, detailed examination of one of the WI-38/hTERT cultures during the accelerated growth phase revealed overexpression of the c-myc and Bmi-1 oncogenes, as well as loss of p14^ARF expression. Collectively, our results indicate that although hTERT-immortalized cells behave similarly to primary cells during the first 150 PDLs, long-term growth in culture may favor the appearance of clones carrying potentially malignant alterations.

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