Analysis of telomere length variation and Shelterin complex subunit gene expression changes in ethanol-exposed human embryonic stem cells.

Telomeres protect chromosome ends from degradation. Telomere length (TL) can be altered by aging and environmental stress. Shortened TL has been observed in peripheral blood leukocytes of alcohol dependent subjects and ethanol-exposed somatic cells. To understand the impact of ethanol on telomeres in pluripotent stem cells, we investigated the influence of ethanol on TL and the expression of six Shelterin complex subunit or telomere-regulating genes (POT1, RAP1, TIN2, TPP1, TRF1, and TRF2) in human embryonic stem cells (hESCs), which were exposed to 0, 25, 50, or 100 mM of ethanol for 3, 7, or 14 days. Ethanol-induced TL and Shelterin complex subunit gene expression changes were examined by quantitative polymerase chain reactions. Two-way ANOVA tests indicated that TL variation and expression changes of four associated Shelterin complex subunit genes (POT1, TPP1, TIN2, and TRF2) were mainly dependent on the length of ethanol exposure, while TRF1 and RAP1expression was influenced by ethanol concentration, exposure time, and the interaction of ethanol concentration and exposure time. Tukey's multiple comparison tests showed that TL and the expression of POT1, RAP1, TIN2, TPP1, and TRF1 were decreased after a 7-day (versus a 3-day) ethanol exposure. However, the decreased expression of all six Shelterin complex subunit genes was recovered and TL was not further shortened after a 14-day (versus a 7-day) ethanol exposure, likely due to the adaptation of hESCs to ethanol-induced stress. Our study provided further evidence that TL is regulated and maintained by telomere-regulating genes in stem cells under ethanol stress.