RESUMEN
High-risk human papillomavirus (HPV) types 16 and 18 are involved in the multistep process of cervical cancer. Transfection of normal keratinocytes with high-risk HPV-DNA generally gives rise to immortal cultures. This may be explained by the loss of senescence genes as a consequence of HPV-induced genetic instability. On the basis of the dominance of cellular senescence over immortality, fusion of normal keratinocytes with HPV-immortalized cells results in complementation of these putative gene defects. In a previous study, we showed that underrepresentation of chromosome 10 is a characteristic phenomenon during the early phase of immortalization. Here we show that introduction of a normal copy of chromosome 10 into HPV16-immortalized cells (HPKII) by Microcell-mediated chromosome transfer resulted in senescence of a significant number of hybrids. By using several derivatives of chromosome 10 for further fusion experiments, the chromosomal region responsible for senescence could be assigned to 10p14-p15. The potential significance of loss of gene function in this region is underlined by the high frequency (38.7%) of loss of heterozygosity in cervical cancers including early stage tumors.
Asunto(s)
Senescencia Celular/genética , Cromosomas Humanos Par 10/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/virología , Línea Celular Transformada , Transformación Celular Viral , Mapeo Cromosómico , ADN Viral/genética , Femenino , Eliminación de Gen , Técnicas de Transferencia de Gen , Humanos , Queratinocitos/citología , Queratinocitos/fisiología , Queratinocitos/virología , Pérdida de Heterocigocidad , Papillomaviridae/genética , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/virología , Displasia del Cuello del Útero/genética , Displasia del Cuello del Útero/patología , Displasia del Cuello del Útero/virologíaRESUMEN
Telomerase activity can be detected in most human cancers and immortal cell lines. In contrast, the lack of telomerase activity in normal diploid fibroblasts has been correlated with progressive reduction of telomere lengths to critically short sizes followed by the cessation of cell division and the onset of senescence. Several investigators have provided evidence for the localization of a telomerase suppressor gene on chromosome 3. The aim of our study was to determine whether other chromosomes are involved in telomerase repression. Beside human chromosome 3 (serving as positive control), chromosomes 4, 6, and 11 were introduced into HeLa cells via microcell-mediated chromosome transfer. Telomerase activity from different hybrid cell lysates was determined at an early time point after fusion using a Telomerase ELISA kit. Strong repression of telomerase activity was only found in a subset of HeLa hybrids in which chromosome 3 or chromosome 4 had been introduced. Telomerase suppression induced by chromosome 3 or 4 transfer was paralleled by a high frequency (30% or 43%, respectively) of a senescent-like phenotype. Chromosomes 6 and 11, the functional loss of which is also implicated in cervical cancer, had no effect. These results indicate that normal human chromosomes 3 and 4 carry a gene or genes that suppress telomerase activity and induce cellular senescence in HeLa cells.
