Differential resistance of human embryonic stem cells and somatic cell types to hydrogen peroxide-induced genotoxicity may be dependent on innate basal intracellular ROS levels.

Previously, we demonstrated that undifferentiated human embryonic stem cells (hESC) displayed higher resistance to oxidative and genotoxic stress compared to somatic cells, but did not further probe the underlying mechanisms. Using H2O2-induced genotoxicity as a model, this study investigated whether higher resistance of hESC to oxidative and genotoxic stress could be due to lower innate basal intracellular levels of reactive oxygen species (ROS), as compared to their differentiated fibroblastic progenies (H1F) and two other somatic cell types - human embryonic palatal mesenchymal (HEPM) cells and peripheral blood lymphocytes (PBL). Comet assay demonstrated that undifferentiated hESC consistently sustained lower levels of DNA damage upon acute exposure to H2O2 for 30 min, compared to somatic cells. DCFDA and HE staining with flow cytometry showed that undifferentiated hESC had lower innate basal intracellular levels of reactive oxygen species compared to somatic cells, which could lead to their higher resistance to genotoxic stress upon acute exposure to H2O2.

Evaluation of human embryonic stem cells and their differentiated fibroblastic progenies as cellular models for in vitro genotoxicity screening.

This study evaluated human embryonic stem cells (hESC) and their differentiated fibroblastic progenies as cellular models for genotoxicity screening. The DNA damage response of hESCs and their differentiated fibroblastic progenies were compared to a fibroblastic cell line (HEPM, CRL1486) and primary cultures of peripheral blood lymphocytes (PBL), upon exposure to Mitomycin C, gamma irradiation and H2O2. It was demonstrated that hESC-derived fibroblastic progenies (H1F) displayed significantly higher chromosomal aberrations, micronuclei formation and double strand break (DSB) formation, as compared to undifferentiated hESC upon exposure to genotoxic stress. Nevertheless, H1F cell types displayed comparable sensitivities to genotoxic challenge as HEPM and PBL, both of which are representative of somatic cell types commonly used for genotoxicity screening. Subsequently, transcriptomic and pathways analysis identified differential expression of critical genes involved in cell death and DNA damage response upon exposure to gamma irradiation. The results thus demonstrate that hESC-derived fibroblastic progenies are as sensitive as commonly-used somatic cell types for genotoxicity screening. Moreover, hESCs have additional advantages, such as their genetic normality compared to immortalized cell lines, as well as their amenability to scale-up for producing large, standardized quantities of cells for genotoxicity screening on an industrial scale, something which can never be achieved with primary cell cultures.

Inhibition of poly (ADP-Ribose) polymerase-1 in telomerase deficient mouse embryonic fibroblasts increases arsenite-induced genome instability.

BACKGROUND: The telomerase enzyme is a viable target for anti-cancer therapy given the innate differences in telomerase activity between tumour cells and normal somatic cells. However, the time lag between telomerase inhibition and telomeres becoming critically short to trigger cell death, allows cancer cells to acquire drug resistance. Inhibition of DNA repair pathways along with telomerase could be an alternative strategy to enhance anti-tumour effects and circumvent the possibility of drug resistance. Poly (ADP-Ribose) Polymerase-1 (PARP-1), an important DNA damage sensor and a DNA repair factor, has important roles in maintaining telomeres and chromosomal stability. In this study, the effects of combined inhibition of PARP-1 and telomerase in mouse embryonic fibroblasts (MEFs) following sodium arsenite exposure (a carcinogen and potent DNA damaging agent), were evaluated. RESULTS: Inhibition of PARP in telomerase deficient MEFs induced an increase in arsenite-induced DNA damage as compared to control cells. Combined inhibition also resulted in enhanced genomic instability, demonstrated by elevated micronuclei induction and chromosomal aberrations with decreased cell survival. In addition, telomerase inhibition in PARP-1 deficient MEFs led to greater telomere shortening and increased genomic instability. CONCLUSIONS: Our study demonstrated that the co-inhibition of PARP-1 and telomerase in MEFs rendered cells more susceptible to DNA damaging agents. Hence, these results offer support for the use of combined inhibition of PARP-1 and telomerase as a strategy to minimise the problems associated with long-term telomerase inhibition in cancer therapeutics.

Human embryonic stem cells may display higher resistance to genotoxic stress as compared to primary explanted somatic cells.

The use of human embryonic stem (hES) cells in genotoxicity screening can potentially overcome the deficiencies associated with using immortalized cell lines, primary explanted somatic cells, and live animal models. Hence this study sought to compare the responses of hES cells and primary explanted somatic cells (IMR-90 cells, human fetal lung fibroblasts) to genotoxic stress, to evaluate whether hES cells can accurately reflect the normal physiology of human somatic cells. The effects of mitomycin C (MMC) on the chromosomal stability of hESC and IMR-90 was assayed and compared by fluorescence in situ hybridization (FISH) with telomere-specific peptide nucleic acid and multicolor (m) FISH techniques. The results showed that, the percentage of aberrant cells increased from 6% in the untreated control to 57.5% at the higher dose of 0.06 microg/ml MMC (9.6-fold increase) group in the case of IMR-90 cells, whereas hES cells displayed a corresponding increase from 6% to 28% (4.6-fold increase). Telomere FISH ascertained that the main types of damage induced by MMC are chromosomal breaks and the loss of telomeric signals. No fusions were observed in all samples analyzed. This was further confirmed by mFISH, which showed that fusions and translocations were not the type of aberration induced by MMC, with no such aberrations being observed in all samples analyzed. Hence, hES cells of the H1 line are apparently more resistant to MMC-induced DNA damage, as compared to the IMR-90 cells. These results highlight possible intrinsic differences in response to damaging agents between hES cells and normal somatic cells.

Short dysfunctional telomeres impair the repair of arsenite-induced oxidative damage in mouse cells.

Telomeres and telomerase appear to participate in the repair of broken DNA ends produced by oxidative damage. Arsenite is an environmental contaminant and a potent human carcinogen, which induces oxidative stress on cells via the generation of reactive oxygen species affecting cell viability and chromosome stability. It promotes telomere attrition and reduces cell survival by apoptosis. In this study, we used mouse embryonic fibroblasts (MEFs) from mice lacking telomerase RNA component (mTERC(-/-) mice) with long (early passage or EP) and short (late passage or LP) telomeres to investigate the extent of oxidative damage by comparing the differences in DNA damage, chromosome instability, and cell survival at 24 and 48 h of exposure to sodium arsenite (As3+; NaAsO2). There was significantly high level of DNA damage in mTERC(-/-) cells with short telomeres as determined by alkaline comet assay. Consistent with elevated DNA damage, increased micronuclei (MN) induction reflecting gross genomic instability was also observed. Fluorescence in situ hybridization (FISH) analysis revealed that increasing doses of arsenite augmented the chromosome aberrations, which contributes to genomic instability leading to possibly apoptotic cell death and cell cycle arrest. Microarray analysis has revealed that As3+ treatment altered the expression of 456 genes of which 20% of them have known functions in cell cycle and DNA damage signaling and response, cell growth, and/or maintenance. Results from our studies imply that short dysfunctional telomeres impair the repair of oxidative damage caused by arsenite. The results will have implications in risk estimation as well as cancer chemotherapy.

Lack of poly(ADP-ribose) polymerase-1 gene product enhances cellular sensitivity to arsenite.

Arsenite (As3+) has long been known to induce cancer and other degenerative diseases. Arsenite exerts its toxicity in part by generating reactive oxygen species. Identification of genetic factors that contribute to arsenic mutagenicity and carcinogenicity is critical for the treatment and prevention of arsenic exposure in human population. As poly(ADP-ribose) polymerase (PARP) is critical for genomic DNA stability, role of PARP-1 was evaluated in arsenic-induced cytotoxic and genotoxic effects. Our study revealed that telomere attrition, probably owing to arsenite-induced oxidative stress, was much more pronounced in PARP-1-/- mouse embryonic fibroblasts (MEF; 40%) compared with PARP-1+/+ MEFs (10-20%). Correlation observed between telomere reduction and apoptotic death in PARP-1 null cells strongly indicates that the telomere attrition might be a trigger for enhanced apoptotic death after arsenite treatment. Elevated DNA damage detected by alkaline comet assay points to an impaired repair ability of arsenite-induced DNA lesions in PARP-1-/- MEFs. Consistent with elevated DNA damage, increased micronuclei induction reflecting gross genomic instability was also observed in arsenite-treated PARP-1-/- MEFs. Microarray analysis has revealed that arsenite treatment altered the expression of about 311 genes majority of which have known functions in cellular responses to stress/external stimulus and cell growth and/or maintenance. Our results suggest an important role for PARP-1 gene product in the maintenance of chromosome-genome stability in response to arsenite-induced DNA damage.