Sustained activation of p53 in confluent nucleotide excision repair-deficient cells resistant to ultraviolet-induced apoptosis

Carvalho, Helotônio; Ortolan, Tatiana G.; dePaula, Tomás; Leite, Ricardo A.; Weinlich, Ricardo; Amarante-Mendes, Gustavo P.; Menck, Carlos Frederico Martins

doi:10.1016/j.dnarep.2008.03.003

Cited by 16 publications

(12 citation statements)

References 39 publications

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“…H1299 is p53 deficient while H1355 is a heterozygous mutant of p53. Following DNA damage, p53 is normally activated to induce cell cycle arrest and facilitate DNA repair [52]. The differences in p53 expression could affect cell survival and DNA repair efficiency following XPF–ERCC1 knockdown.…”

Section: Discussionmentioning

confidence: 99%

Downregulation of XPF–ERCC1 enhances cisplatin efficacy in cancer cells☆

et al. 2010

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Bulky cisplatin lesions are repaired primarily by nucleotide excision repair (NER), in which the structure specific endonuclease XPF–ERCC1 is a critical component. It is now known that the XPF–ERCC1 complex has repair functions beyond NER and plays a role in homologous recombination (HR). It has been suggested that expression of ERCC1 correlates with cisplatin drug resistance in non-small cell lung cancer (NSCLC). In our study, using NSCLC, ovarian, and breast cancer cells, we show that the XPF–ERCC1 complex is a valid target to increase cisplatin cytotoxicity and efficacy. We targeted XPF–ERCC1 complex by RNA interference and assessed the repair capacity of cisplatin intrastrand and interstrand crosslinks by ELISA and alkaline comet assay, respectively. We also assessed the repair of cisplatin-ICL-induced double-strand breaks (DSBs) by monitoring γ-H2AX focus formation. Interestingly, XPF protein levels were significantly reduced following ERCC1 downregulation, but the converse was not observed. The transcript levels were unaffected suggesting that XPF protein stability is likely affected. The repair of both types of cisplatin-DNA lesions was decreased with downregulation of XPF, ERCC1 or both XPF–ERCC1. The ICL-induced DSBs persist in the absence of XPF–ERCC1. The suppression of the XPF–ERCC1 complex significantly decreases the cellular viability which correlates well with the decrease in DNA repair capacity. A double knockdown of XPF–ERCC1 displays the greatest level of cellular cytotoxicity when compared with XPF or ERCC1 alone. The difference in cytotoxicity observed is likely due to the level of total protein complex remaining. These data demonstrate that XPF–ERCC1 is a valid target to enhance cisplatin efficacy in cancer cells by affecting cisplatin-DNA repair pathways.

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Section: Discussionmentioning

confidence: 99%

Downregulation of XPF–ERCC1 enhances cisplatin efficacy in cancer cells☆

et al. 2010

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“…Primary human fibroblasts were obtained from skin biopsies of a normal individual (FHN) [33], a Xeroderma Pigmentosum group C patient (XP17VI) [34] and a Cockayne Syndrome group B patient (GM00739, Coriell Cell Repositories). The cells, kindly provided by Dr. Alain Sarasin (IGR, Villejuif, France) and Dr. Claudimara Lofti, were cultivated in a Dulbeccós Modified Eagle Medium (LGC Biotecnologia, São Paulo, SP, Brazil) supplemented with 15% fetal bovine serum (FBS) (Cultilab, Campinas, SP, Brazil), 100 U/ml of penicillin G sodium, 100 μg/ml of streptomycin and 0.25 mg/ml of amphotericin B (Life Technologies, Carlsbad, CA).…”

Section: Methodsmentioning

confidence: 99%

UVB-Induced Cell Death Signaling Is Associated with G1-S Progression and Transcription Inhibition in Primary Human Fibroblasts

Ortolan

Menck

2013

PLoS ONE

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DNA damage induced by ultraviolet (UV) radiation can be removed by nucleotide excision repair through two sub-pathways, one general (GGR) and the other specific for transcribed DNA (TCR), and the processing of unrepaired lesions trigger signals that may lead to cell death. These signals involve the tumor suppressor p53 protein, a central regulator of cell responses to DNA damage, and the E3 ubiquitin ligase Mdm2, that forms a feedback regulatory loop with p53. The involvement of cell cycle and transcription on the signaling to apoptosis was investigated in UVB-irradiated synchronized, DNA repair proficient, CS-B (TCR-deficient) and XP-C (GGR-deficient) primary human fibroblasts. Cells were irradiated in the G1 phase of the cell cycle, with two doses with equivalent levels of apoptosis (low and high), defined for each cell line. In the three cell lines, the low doses of UVB caused only a transient delay in progression to the S phase, whereas the high doses induced permanent cell cycle arrest. However, while accumulation of Mdm2 correlated well with the recovery from transcription inhibition at the low doses for normal and CS-B fibroblasts, for XP-C cells this protein was shown to be accumulated even at UVB doses that induced high levels of apoptosis. Thus, UVB-induced accumulation of Mdm2 is critical for counteracting p53 activation and apoptosis avoidance, but its effect is limited due to transcription inhibition. However, in the case of XP-C cells, an excess of unrepaired DNA damage would be sufficient to block S phase progression, which would signal to apoptosis, independent of Mdm2 accumulation. The data clearly discriminate DNA damage signals that lead to cell death, depending on the presence of UVB-induced DNA damage in replicating or transcribing regions.

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“…Accordingly, loss of their function has consequences for DNA repair, cellular proliferation and survival. [6][7][8] Simple model organisms are very useful to decipher complex DNA damage responses. In Caenorhabditis elegans, the effects of IR have been studied extensively.…”

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confidence: 99%

NER and HR pathways act sequentially to promote UV-C-induced germ cell apoptosis in Caenorhabditis elegans

et al. 2010

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Ultraviolet (UV) radiation-induced DNA damage evokes a complex network of molecular responses, which culminate in DNA repair, cell cycle arrest and apoptosis. Here, we provide an in-depth characterization of the molecular pathway that mediates UV-C-induced apoptosis of meiotic germ cells in the nematode Caenorhabditis elegans. We show that UV-C-induced DNA lesions are not directly pro-apoptotic. Rather, they must first be recognized and processed by the nucleotide excision repair (NER) pathway. Our data suggest that NER pathway activity transforms some of these lesions into other types of DNA damage, which in turn are recognized and acted upon by the homologous recombination (HR) pathway. HR pathway activity is in turn required for the recruitment of the C. elegans homolog of the yeast Rad9-Hus1-Rad1 (9-1-1) complex and activation of downstream checkpoint kinases. Blocking either the NER or HR pathway abrogates checkpoint pathway activation and UV-C-induced apoptosis. Our results show that, following UV-C, multiple DNA repair pathways can cooperate to signal to the apoptotic machinery to eliminate potentially hazardous cells.

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Sustained activation of p53 in confluent nucleotide excision repair-deficient cells resistant to ultraviolet-induced apoptosis

Cited by 16 publications

References 39 publications

Downregulation of XPF–ERCC1 enhances cisplatin efficacy in cancer cells☆

Downregulation of XPF–ERCC1 enhances cisplatin efficacy in cancer cells☆

UVB-Induced Cell Death Signaling Is Associated with G1-S Progression and Transcription Inhibition in Primary Human Fibroblasts

NER and HR pathways act sequentially to promote UV-C-induced germ cell apoptosis in Caenorhabditis elegans

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