Cell cycle association and hypoxia regulation of excision repair cross complementation group 1 protein (ERCC1) in tumor cells of head and neck cancer

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Abstract

Excision repair cross complementation group 1 (ERCC1) is a key component of homologous recombination-based repair of interstrand DNA cross-links (ICLs). As a consequence, ERCC1 mediates resistance to mitomycin C (MMC) and platinum chemotherapeutic agents and may predict treatment failure. Clinical response to MMC or cisplatin (CDDP)-based radiochemotherapy (RCT) was assessed in 106 head and neck squamous cell carcinoma (HNSCC) patients and correlated with cell nuclear immunoreactivity of the mouse monoclonal (clone: 8 F1) ERCC1 antibody in tumor tissue samples. BEAS-2B epithelial and Detroit 562 pharyngeal squamous carcinoma cells were treated with CDDP, MMC, and 5-fluorouracil (5-FU) at 50 % growth inhibitory (IC-50) concentrations. ERCC1 protein synthesis was compared with cell cycle distribution using combined immunocytochemistry and flow cytometry. ERCC1 messenger RNA (mRNA) and protein expression was investigated in normoxic and hypoxic conditions in Detroit 562 cells. Clinically, the nonresponder revealed significantly lower HNSCC tissue ERCC1 immunoreactivity than the responder ( p = 0.0064) or control normal mucosa, which led to further mechanistic investigations. In vitro, control cells and cells treated with cytotoxic agents showed increasing ERCC1 levels from the G1 through S and G2 phases of the cell cycle. In CDDP-treated cells, ERCC1 mRNA and protein expression increased. Under hypoxic conditions, ERCC1 gene expression significantly decreased. Although ERCC1 ⁺ cells show increased chemoresistance, they might be particularly radiosensitive, representing G2 cell cycle phase and less hypoxic. ERCC1 expression might be indirectly related with some conditions important for RCT treatment, but it is not a clear predictor for its failure in HNSCC patients.

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Down-regulation of Rad51 and decreased homologous recombination in hypoxic cancer cells.

Kårstein Måseide, Ranjit Bindra, P. Schaffer … (2004)

There is an emerging concept that acquired genetic instability in cancer cells can arise from the dysregulation of critical DNA repair pathways due to cell stresses such as inflammation and hypoxia. Here we report that hypoxia specifically down-regulates the expression of RAD51, a key mediator of homologous recombination in mammalian cells. Decreased levels of Rad51 were observed in multiple cancer cell types during hypoxic exposure and were not associated with the cell cycle profile or with expression of hypoxia-inducible factor. Analyses of RAD51 gene promoter activity, as well as mRNA and protein stability, indicate that the hypoxia-mediated regulation of this gene occurs via transcriptional repression. Decreased expression of Rad51 was also observed to persist in posthypoxic cells for as long as 48 h following reoxygenation. Correspondingly, we found reduced levels of homologous recombination in both hypoxic and posthypoxic cells, suggesting that the hypoxia-associated reduction in Rad51 expression has functional consequences for DNA repair. In addition, hypoxia-mediated down-regulation of Rad51 was confirmed in vivo via immunofluorescent image analysis of experimental tumors in mice. Based on these findings, we propose a novel mechanism of genetic instability in the tumor microenvironment mediated by hypoxia-induced suppression of the homologous recombination pathway in cancer cells. The aberrant regulation of Rad51 expression may also create heterogeneity in the DNA damage response among cells within tumors, with implications for the response to cancer therapies.

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The structure-specific endonuclease Ercc1-Xpf is required to resolve DNA interstrand cross-link-induced double-strand breaks.

Laura Niedernhofer, Arjan F. Theil, Jan H. J. Hoeijmakers … (2004)

Interstrand cross-links (ICLs) are an extremely toxic class of DNA damage incurred during normal metabolism or cancer chemotherapy. ICLs covalently tether both strands of duplex DNA, preventing the strand unwinding that is essential for polymerase access. The mechanism of ICL repair in mammalian cells is poorly understood. However, genetic data implicate the Ercc1-Xpf endonuclease and proteins required for homologous recombination-mediated double-strand break (DSB) repair. To examine the role of Ercc1-Xpf in ICL repair, we monitored the phosphorylation of histone variant H2AX (gamma-H2AX). The phosphoprotein accumulates at DSBs, forming foci that can be detected by immunostaining. Treatment of wild-type cells with mitomycin C (MMC) induced gamma-H2AX foci and increased the amount of DSBs detected by pulsed-field gel electrophoresis. Surprisingly, gamma-H2AX foci were also induced in Ercc1(-/-) cells by MMC treatment. Thus, DSBs occur after cross-link damage via an Ercc1-independent mechanism. Instead, ICL-induced DSB formation required cell cycle progression into S phase, suggesting that DSBs are an intermediate of ICL repair that form during DNA replication. In Ercc1(-/-) cells, MMC-induced gamma-H2AX foci persisted at least 48 h longer than in wild-type cells, demonstrating that Ercc1 is required for the resolution of cross-link-induced DSBs. MMC triggered sister chromatid exchanges in wild-type cells but chromatid fusions in Ercc1(-/-) and Xpf mutant cells, indicating that in their absence, repair of DSBs is prevented. Collectively, these data support a role for Ercc1-Xpf in processing ICL-induced DSBs so that these cytotoxic intermediates can be repaired by homologous recombination.

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Equivalence test in quantitative reverse transcription polymerase chain reaction: confirmation of reference genes suitable for normalization.

Anja von Au, S Schwager, Bastian Gunawan … (2004)

In quantitative reverse transcription-polymerase chain reaction (qRT-PCR), normalization using reference genes is a common useful approach, but the validation of suitable reference genes remains a crucial problem. Use of unconfirmed reference genes may lead to misinterpretation of the expression of target genes. The aim of this study was to adapt an adequate statistical approach to identify and validate reference genes suitable for normalization in qRT-PCR assays. We introduce the equivalence test for the identification of stably expressed reference genes. To evaluate the advantages of this test, the expression of five genes widely used as reference genes (18S, B2M, HPRT1, LMNB1, and SDHA), and of two target genes (TP53 and MMP2), was determined with qRT-PCR in different tissues (clear cell renal cell carcinoma, colon carcinoma, and gastrointestinal stromal tumors). We demonstrate that a stable expression of a reference gene in one tumor type does not predict a stable expression in another tumor type. In addition, we found that even within one tumor type, the expression of a reference gene was not stable for different biological groupwise comparisons. These observations confirm that there is no universal reference gene and underline the importance of specific validation of potential reference genes for any experimental condition.

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Contributors

József Dudás: +43-512-50482475 , jozsef.dudas@i-med.ac.at

Volker H. Schartinger: volker.schartinger@i-med.ac.at

Angela Romani: angela.romani@i-med.ac.at

Gabriele Schweigl: gabriele.schweigl@uki.at

Kristian Kordsmeyer: kristian.kordsmeyer@student-i-med.ac.at

Patricia Irina Marta: patricia.marta@i-med.ac.at

Christoph Url: christopher.url@i-med.ac.at

Florian Kral: florian.kral@i-med.ac.at

Herbert Riechelmann: herbert.riechelmann@i-med.ac.at

Journal

Journal ID (nlm-ta): Tumour Biol

Journal ID (iso-abbrev): Tumour Biol

Title: Tumour Biology

Publisher: Springer Netherlands (Dordrecht )

ISSN (Print): 1010-4283

ISSN (Electronic): 1423-0380

Publication date (Electronic): 12 May 2014

Publication date PMC-release: 12 May 2014

Publication date (Print): August 2014

Volume: 35

Issue: 8

Pages: 7807-7819

Affiliations

[ ]Department of Otorhinolaryngology and Head and Neck Surgery, Medical University Innsbruck, Anichstrasse 35, 6020 Innsbruck, Austria

[ ]Institute of Pathology, Medical University Innsbruck, Müllerstrasse 44, 6020 Innsbruck, Austria

Article

Publisher ID: 2001

DOI: 10.1007/s13277-014-2001-2

PMC ID: 4158184

PubMed ID: 24817012

SO-VID: 66bc6b36-6163-4e45-baf9-3cdb9214eb79

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Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.

History

Date received : 5 January 2014

Date accepted : 22 April 2014

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ScienceOpen disciplines: Oncology & Radiotherapy

Keywords: genetic instability,oropharynx,hypopharynx,dna repair,homologue recombination

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ScienceOpen disciplines: Oncology & Radiotherapy

Keywords: genetic instability, oropharynx, hypopharynx, dna repair, homologue recombination

Cell cycle association and hypoxia regulation of excision repair cross complementation group 1 protein (ERCC1) in tumor cells of head and neck cancer

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Most cited references 45

Down-regulation of Rad51 and decreased homologous recombination in hypoxic cancer cells.

The structure-specific endonuclease Ercc1-Xpf is required to resolve DNA interstrand cross-link-induced double-strand breaks.

Equivalence test in quantitative reverse transcription polymerase chain reaction: confirmation of reference genes suitable for normalization.

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