Analysis of telomeric instability in the progeny of mammalian cells exposed in vitro to different chemical mutagens

PAVIOLO NS; BOLZÁN A

Campinas

Workshop; 2nd Course on Advanced Topics in Human Molecular Genetics; 2011

Universidade Estadual de Campinas (UNICAMP)

Telomeres are DNA protein complexes located at the very ends of eukaryotic chromosomes, which preserve their integrity and stability, protecting them against exonucleotic degradation and fusion. Telomeric maintenance is fundamental for genomic stability and cell viability. Since a high proportion of chemical mutagens are used as antitumor agents, the study of the damage induced by these compounds at the genetic level is of great interest. This project will address the study of telomeric instability -which involves the analysis of chromosomal damage at telomeres, telomere length and the activity of telomerase- in the progeny of mammalian cells exposed to different chemical mutagens. Fluorescence in situ hybridization (FISH) with a telomeric peptide nucleic acid probe was employed to analyze the effect induced by the radiomimetic compounds bleomycin (BLM) and streptonigrin (STN) in two mammalian cell lines as a function of subculture time. Chinese hamster embryo cells (CHE 2n=22-23) and domestic rabbit cells (CPC 2n=44). CHE cells were treated with 2.5 µg/ml of BLM or 100 ng/ml of STN during the log phase of growth and harvested at different times between 18 h and 30 days after treatment. Preliminary data shows chromosomal aberrations and chromatid telomere loss in the first mitosis and four days after STN treatment. Absence of FISH signal at the terminal regions of some CHE chromosomes and differences in signal intensity were also observed in control metaphases owing to the fact that the telomeric sequences are either completely missing from the chromosomal ends or, more likely, that the copy number of these sequences is too low to be effectively detected by FISH, due to extensive telomere loss occurring during in vitro proliferation and immortalization of these cells. The overall purpose of this project is to offer a better understanding of the effects of chemical mutagens on the telomeres of eukaryotic chromosomes, and in particular, to determine whether chemical mutagens have delayed effects on telomeres of the exposed cells.