Abstract
DNA interstrand cross-links (ICLs) are critical cytotoxic lesions produced by cancer chemotherapeutic agents such as the nitrogen mustards and platinum drugs; however, the exact mechanism of ICL-induced cell death is unclear. Here, we show a novel mechanism of p53-independent apoptotic cell death involving prolonged cell-cycle (G2) arrest, ICL repair involving HR, transient mitosis, incomplete cytokinesis, and gross chromosomal abnormalities resulting from ICLs in mammalian cells. This characteristic ‘giant' cell death, observed by using time-lapse video microscopy, was reduced in ICL repair ERCC1- and XRCC3-deficient cells. Collectively, the results illustrate the coordination of ICL-induced cellular responses, including cell-cycle arrest, DNA damage repair, and cell death.
Highlights
Extensive genetic and biochemical studies suggest that interstrand cross-link (ICL) repair involves the initial incision and ‘unhooking’ of ICLs by Mus81-Eme[1] and XPF-ERCC1 endonucleases,[11] and subsequent processing by homologous recombination (HR)[2] and translesion synthesis[12] involving XRCC2, XRCC3, WRN, BRCA2, Fanconi anaemia (FA),[13,14,15] and DNA pol-z16 depending on the phase of the cell cycle.[17]
DNA ICL agents are widely used in cancer chemotherapy; an emergence of acquired drug resistance, due at least partly to ICL repair, is a major clinical problem.[6,7]
We have investigated cell death resulting from ICL in mammalian cells, and a connection between cell death and ICL repair associated with prolonged G2 arrest and incomplete cytokinesis has been indicated
Summary
Clinical inherent sensitivity of tumours to ICL agents may result from a defect in recognition or repair, and enhanced repair can result in clinical acquired resistance,[6,7] the precise mechanism of ICL repair in mammalian cells has still to be fully elucidated.[3,10] Extensive genetic and biochemical studies suggest that ICL repair involves the initial incision and ‘unhooking’ of ICLs by Mus81-Eme[1] and XPF-ERCC1 endonucleases,[11] and subsequent processing by homologous recombination (HR)[2] and translesion synthesis[12] involving XRCC2, XRCC3, WRN, BRCA2, Fanconi anaemia (FA),[13,14,15] and DNA pol-z16 depending on the phase of the cell cycle.[17]. The aim of this study was to elucidate the mechanism of cell death resulting from ICL by the clinically used nitrogen mustard mechlorethamine (HN2). This is evaluated in relation to cell-cycle response and repair of ICL damage using DNA-repair-deficient Chinese hamster ovary (CHO) cell lines and human cervix carcinoma (HeLa) cells. The fate of cells following ICL damage is investigated by using time-lapse video microscopy. These studies show the coordination of ICL-induced cell-cycle arrest, DNA damage repair, and cell death. Our data more fully define the mechanism of ICL-induced cell death following cell-cycle arrest and failure of ICL repair
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