Effective intra-S checkpoint responses to UVC in primary human melanocytes and melanoma cell lines.

The objective of this study was to assess potential functional attenuation or inactivation of the intra-S checkpoint during melanoma development. Proliferating cultures of skin melanocytes, fibroblasts, and melanoma cell lines were exposed to increasing fluences of UVC and intra-S checkpoint responses were quantified. Melanocytes displayed stereotypic intra-S checkpoint responses to UVC qualitatively and quantitatively equivalent to those previously demonstrated in skin fibroblasts. In comparison with fibroblasts, primary melanocytes displayed reduced UVC-induced inhibition of DNA strand growth and enhanced degradation of p21Waf1 after UVC, suggestive of enhanced bypass of UVC-induced DNA photoproducts. All nine melanoma cell lines examined, including those with activating mutations in BRAF or NRAS oncogenes, also displayed proficiency in activation of the intra-S checkpoint in response to UVC irradiation. The results indicate that bypass of oncogene-induced senescence during melanoma development was not associated with inactivation of the intra-S checkpoint response to UVC-induced DNA replication stress.

Cyclobutane Pyrimidine Dimer Density as a Predictive Biomarker of the Biological Effects of Ultraviolet Radiation in Normal Human Fibroblast.

This study compared biological responses of normal human fibroblasts (NHF1) to three sources of ultraviolet radiation (UVR), emitting UVC wavelengths, UVB wavelengths, or a combination of UVA and UVB (solar simulator; emission spectrum, 94.3% UVA and 5.7% UVB). The endpoints measured were cytotoxicity, intra-S checkpoint activation, inhibition of DNA replication and mutagenicity. Results show that the magnitude of each response to the indicated radiation sources was best predicted by the density of DNA cyclobutane pyrimidine dimers (CPD). The density of 6-4 pyrimidine-pyrimidone photoproducts was highest in DNA from UVC-irradiated cells (14% of CPD) as compared to those exposed to UVB (11%) or UVA-UVB (7%). The solar simulator source, under the experimental conditions described here, did not induce the formation of 8-oxo-7,8-dihydroguanine in NHF1 above background levels. Taken together, these results suggest that CPD play a dominant role in DNA damage responses and highlight the importance of using endogenous biomarkers to compare and report biological effects induced by different sources of UVR.

Gene silencing associated with SWI/SNF complex loss during NSCLC development.

UNLABELLED: The SWI/SNF chromatin-remodeling complex regulates gene expression and alters chromatin structures in an ATP-dependent manner. Recent sequencing efforts have shown mutations in BRG1 (SMARCA4), one of two mutually exclusive ATPase subunits in the complex, in a significant number of human lung tumor cell lines and primary non-small cell lung carcinoma (NSCLC) clinical specimens. To determine how BRG1 loss fuels tumor progression in NSCLC, molecular profiling was performed after restoration of BRG1 expression or treatment with a histone deacetylase inhibitor or a DNA methyltransferase (DNMT) inhibitor in a BRG1-deficient NSCLC cells. Importantly, validation studies from multiple cell lines revealed that BRG1 reexpression led to substantial changes in the expression of CDH1, CDH3, EHF, and RRAD that commonly undergo silencing by other epigenetic mechanisms during NSCLC development. Furthermore, treatment with DNMT inhibitors did not restore expression of these transcripts, indicating that this common mechanism of gene silencing did not account for their loss of expression. Collectively, BRG1 loss is an important mechanism for the epigenetic silencing of target genes during NSCLC development. IMPLICATIONS: Inactivation of the SWI/SNF complex provides a novel mechanism to induce gene silencing during NSCLC development.

Is activation of the intra-S checkpoint in human fibroblasts an important factor in protection against UV-induced mutagenesis?

The ATR/CHK1-dependent intra-S checkpoint inhibits replicon initiation and replication fork progression in response to DNA damage caused by UV (UV) radiation. It has been proposed that this signaling cascade protects against UV-induced mutations by reducing the probability that damaged DNA will be replicated before it can be repaired. Normal human fibroblasts (NHF) were depleted of ATR or CHK1, or treated with the CHK1 kinase inhibitor TCS2312, and the UV-induced mutation frequency at the HPRT locus was measured. Despite clear evidence of S-phase checkpoint abrogation, neither ATR/CHK1 depletion nor CHK1 inhibition caused an increase in the UV-induced HPRT mutation frequency. These results question the premise that the UV-induced intra-S checkpoint plays a prominent role in protecting against UV-induced mutagenesis.