ABSTRACT
We have recently completed screening of the National Cancer Institute human tumor cell line panel and demonstrated that among four nucleotide excision repair proteins (XPA, XPB, XPD, and ERCC1), only the TFIIH subunit XPD endogenous protein levels correlate with alkylating agent drug resistance. In the present study, we extended this work by investigating the biological consequences of XPD overexpression in the human glioma cell line SK-MG-4. Our results indicate that XPD overexpression in SK-MG-4 cells leads to cisplatin resistance without affecting the nucleotide excision repair activity or UV light sensitivity of the cell. In contrast, in SK-MG-4 cells treated with cisplatin, XPD overexpression leads to increased Rad51-related homologous recombinational repair, increased sister chromatid exchanges, and accelerated interstrand cross-link removal. Moreover, we present biochemical evidence of an XPD-Rad51 protein interaction, which is modulated by DNA damage. To our knowledge, this is the first description of functional cross-talk between XPD and Rad51, which leads to bifunctional alkylating agent drug resistance and accelerated removal of interstrand cross-links.
Subject(s)
Antineoplastic Agents/pharmacology , Cisplatin/pharmacology , DNA Helicases , DNA Repair/physiology , Endonucleases , Proteins/physiology , Transcription Factors, TFII/physiology , Transcription Factors , Cell Cycle/physiology , Cell Nucleus/metabolism , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/physiology , Drug Resistance, Neoplasm , Glioma/drug therapy , Glioma/genetics , Glioma/metabolism , Humans , Melphalan/pharmacology , Precipitin Tests , Protein Biosynthesis , Proteins/metabolism , Rad51 Recombinase , Radiation Tolerance , S Phase/physiology , Sister Chromatid Exchange/drug effects , Sister Chromatid Exchange/physiology , Transcription Factor TFIIH , Transcription Factors, TFII/biosynthesis , Tumor Cells, Cultured , Ultraviolet Rays , Xeroderma Pigmentosum Group A Protein , Xeroderma Pigmentosum Group D ProteinABSTRACT
The nitrogen mustards are an important class of DNA cross-linking agents, which are utilized in the treatment of many types of cancer. Unfortunately, resistance often develops in the treatment of patients and the tumor either never responds to or becomes refractory to these agents. Resistance to the nitrogen mustards in murine and human tumor cells has been reported to be secondary to alterations in (i) the transport of these agents, (ii) their reactivity, (iii) apoptosis and (iv) altered DNA repair activity. In the present review, we will discuss the role of DNA repair in nitrogen mustard resistance in cancer. The nitrogen mustards' lethality is based on the induction of DNA interstrand cross-links (ICLs). Two DNA repair pathways are known to be involved in removal of ICLs: non-homologous DNA end-joining (NHEJ) and Rad51-related homologous recombinational repair (HRR). The reports discussed here lead us to hypothesize that low NHEJ activity defines a hypersensitive state, while high NHEJ activity, along with increased HRR activity, contributes to the resistant state in chronic lymphocytic leukemia. Studies on human epithelial tumor cell lines suggest that HRR rather than NHEJ plays a role in nitrogen mustard sensitivity.
Subject(s)
Antineoplastic Agents, Alkylating/pharmacology , DNA Repair/physiology , Drug Resistance, Neoplasm/genetics , Neoplasm Proteins/physiology , Nitrogen Mustard Compounds/pharmacology , Forecasting , Humans , Neoplasm Proteins/genetics , Recombination, GeneticABSTRACT
The objective of this study was to further define the role of homologous recombinational repair (HRR) in resistance to the nitrogen mustards in B-cell chronic lymphocytic leukemia (B-CLL). We have demonstrated previously that increased chlorambucil (CLB)-induced HsRad51 nuclear foci formation correlated with a CLB-resistant phenotype in B-CLL lymphocytes. In this report, we measured the protein levels of HsRad51 and Xrcc3 (an HsRad51 paralog) and correlated them with the in vitro CLB cytotoxicity (LD(50)) in lymphocytes from seventeen B-CLL patients. Both HsRad51 (r=0.75, P=0.0005) and Xrcc3 (r=0.52, P=0.03) protein levels correlated with the in vitro CLB LD(50). In addition, multiple linear regression analysis showed a significant correlation between Xrcc3 and Rad51 protein levels versus the CLB LD(50) (r=0.78, P=0.0014), suggesting that both proteins influence CLB cytotoxicity. Moreover, since HsRad51 expression varies in cell lines during the cell cycle, we determined proliferating cell nuclear antigen (PCNA) protein levels to assess possible differences in cell cycle progression. There was no correlation between PCNA protein levels and the CLB LD(50) (r=0.042, P=0.87) or with HsRad51/Xrcc3 protein levels. Our data suggest that HsRad51 and Xrcc3 protein expression may be predictive of the response in B-CLL patients to treatment with nitrogen mustards.
