Exploring new potential role of DDB2 by host cell reactivation assay in human tumorigenic cells

Background The Host Cell Reactivation assay (HCR) allows studying the DNA repair capability in different types of human cells. This assay was carried out to assess the ability in removing UV-lesions from DNA, thus verifying NER efficiency. Previously we have shown that DDB2, a protein involved in the Global Genome Repair, interacts directly with PCNA and, in human cells, the loss of this interaction affects DNA repair machinery. In addition, a mutant form unable to interact with PCNA (DDB2PCNA-), has shown a reduced ability to interact with a UV-damaged DNA plasmid in vitro. Methods In this work, we have investigated whether DDB2 protein may influence the repair of a UV-damaged DNA plasmid into the cellular environment by applying the HCR method. To this end, human kidney 293 stable clones, expressing DDB2Wt or DDB2PCNA-, were co-transfected with pmRFP-N2 and UV-irradiated pEGFP-reported plasmids. Moreover, the co-localization between DDB2 proteins and different NER factors recruited at DNA damaged sites was analysed by immunofluorescence and confocal microscopy. Results The results have shown that DDB2Wt recognize and repair the UV-induced lesions in plasmidic DNA transfected in the cells, whereas a delay in these processes were observed in the presence of DDB2PCNA-, as also confirmed by the different extent of co-localization of DDB2Wt and some NER proteins (such as XPG), vs the DDB2 mutant form. Conclusion The HCR confirms itself as a very helpful approach to assess in the cellular context the effect of expressing mutant vs Wt NER proteins on the DNA damage response. Loss of interaction of DDB2 and PCNA affects negatively DNA repair efficiency.

XPD/ERCC2 mutations interfere in cellular responses to oxidative stress

Nucleotide excision repair (NER) is a conserved, flexible mechanism responsible for the removal of bulky, helix-distorting DNA lesions, like ultraviolet damage or cisplatin adducts, but its role in the repair of lesions generated by oxidative stress is still not clear. The helicase XPD/ERCC2, one of the two helicases of the transcription complex IIH, together with XPB, participates both in NER and in RNA pol II-driven transcription. In this work, we investigated the responses of distinct XPD-mutated cell lines to the oxidative stress generated by photoactivated methylene blue (MB) and KBrO3 treatments. The studied cells are derived from patients with XPD mutations but expressing different clinical phenotypes, including xeroderma pigmentosum (XP), XP and Cockayne syndrome (XP-D/CS) and trichothiodystrophy (TTD). We show by different approaches that all XPD-mutated cell lines tested were sensitive to oxidative stress, with those from TTD patients being the most sensitive. Host cell reactivation (HCR) assays showed that XP-D/CS and TTD cells have severely impaired repair capacity of oxidised lesions in plasmid DNA, and alkaline comet assays demonstrated the induction of significantly higher amounts of DNA strand breaks after treatment with photoactivated MB in these cells compared to wild-type cells. All XPD-mutated cells presented strong S/G2 arrest and persistent γ-H2AX staining after photoactivated MB treatment. Taken together, these results indicate that XPD participates in the repair of lesions induced by the redox process, and that XPD mutations lead to differences in the response to oxidatively induced damage.

p21Waf1 deficiency does not decrease DNA repair in E1A+cHa-Ras transformed cells by HDI sodium butyrate

This study aimed to explore a role of p21Waf1 in γH2AX foci formation and DNA repair as assessed by a Host-Cell Reactivation Assay in wild-type (p21Waf+/+) and p21Waf1-deficient E1A+Ras-transformed cells. p21Waf1+/+cells have low γH2AX background compared to p21Waf1−/−cells. The treatment with histone deacetylase inhibitor (HDI) sodium butyrate (NaBut) causes to accumulation of γH2AX in p21Waf+/+cells with little effect in p21Waf−/−cells. Moreover, NaBut inhibits DNA repair in wt cells but not in p21Waf1−/−cells. This could be explained by the weakening of GADD45 and PCNA proteins binding in NaBut-treated p21Waf1-expressing cells but not in p21Waf1−/−cells. We suggest that in wt-ERas cells NaBut activates both p21Waf1 expression and a release of p21Waf1 from the complexes with E1A that leads to suppression of DNA repair and promotes γH2AX persistency. The absence of p21Waf1 is by itself considered by the cell as stressful factor with formation of γH2AX. But the lack of p21Waf1 interferes with an inhibitory effect of NaBut to inhibit DNA repair and thereby to stop concomitant accumulation of harmful mutations. We conclude that p21Waf1 is directly involved in control of genome integrity and DNA repair acting through modulation of the components of the DNA repair machinery.

Host Cell Reactivation and Transcriptional Activation of Carboplatin-Modified BRCA1

The breast cancer susceptibility gene 1 ( BRCA1) has been shown to maintain genomic stability through multiple functions in the regulation of DNA damage repair and transcription. Its translated BRCT (BRCA1 C-terminal domain) acts as a strong transcriptional activator. BRCA1 damaged by carboplatin treatment may lead to a loss of such functions. To address the possibility of the BRCA1 gene as a therapeutic target for carboplatin, we investigated the functional consequences of the 3′-terminal region of human BRCA1 following in vitro platination with carboplatin. A reduction in cellular BRCA1 repair of carboplatin-treated plasmid DNA, using a host cell reactivation assay, was dependent on the platination levels on the reporter gene. The transcriptional transactivation activity of the drug-modified BRCA1, assessed using a one-hybrid GAL4 transcriptional assay, was inversely proportional to the carboplatin doses. The data emphasized the potential of the BRCA1 gene to be a target for carboplatin treatment.