Cisplatin is one of the most effective and widely used anticancer agents for the treatment of several types of tumors. The cytotoxic effect of cisplatin is thought to be mediated primarily by the generation of nuclear DNA adducts, which, if not repaired, cause cell death as a consequence of DNA replication and transcription blockage. However, the ability of cisplatin to induce nuclear DNA (nDNA) damage per se is not sufficient to explain its high degree of effectiveness nor the toxic effects exerted on normal, post-mitotic tissues. Oxidative damage has been observed in vivo following exposure to cisplatin in several tissues, suggesting a role for oxidative stress in the pathogenesis of cisplatin-induced dose-limiting toxicities. However, the mechanism of cisplatin-induced generation of ROS and their contribution to cisplatin cytotoxicity in normal and cancer cells is still poorly understood. By employing a panel of normal and cancer cell lines and the budding yeast Saccharomyces cerevisiae as model system, we show that exposure to cisplatin induces a mitochondrial-dependent ROS response that significantly enhances the cytotoxic effect caused by nDNA damage. ROS generation is independent of the amount of cisplatin-induced nDNA damage and occurs in mitochondria as a consequence of protein synthesis impairment. The contribution of cisplatin-induced mitochondrial dysfunction in determining its cytotoxic effect varies among cells and depends on mitochondrial redox status, mitochondrial DNA integrity and bioenergetic function. Thus, by manipulating these cellular parameters, we were able to enhance cisplatin cytotoxicity in cancer cells. This study provides a new mechanistic insight into cisplatin-induced cell killing and may lead to the design of novel therapeutic strategies to improve anticancer drug efficacy.

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SUMMARY DNA double-strand break (DSB) repair by homologous recombination (HR) is initiated by CtIP/MRN-mediated DNA end resection to maintain genome integrity. SAMHD1 is a dNTP triphosphohydrolase, which restricts HIV-1 infection, and mutations are associated with Aicardi-Goutières syndrome and cancer. We show that SAMHD1 has a dNTPase-independent function in promoting DNA end resection to facilitate DSB repair by HR. SAMHD1 deficiency or Vpx-mediated degradation causes hypersensitivity to DSB-inducing agents, and SAMHD1 is recruited to DSBs. SAMHD1 complexes with CtIP via a conserved carboxyl-terminal domain and recruits CtIP to DSBs to facilitate end resection and HR. Significantly, a cancer-associated mutant with impaired CtIP interaction but not dNTPase-inactive SAMHD1 fails to rescue the end resection impairment of SAMHD1 depletion. Our findings define a dNTPase-independent function for SAMHD1 in HR-mediated DSB repair by facilitating CtIP accrual to promote DNA end resection, providing insight into how SAMHD1 promotes genome integrity and prevents disease, including cancer.

Cells are exposed to both endogenous and exogenous sources of reactive oxygen species (ROS). At high levels, ROS can lead to impaired physiological function through cellular damage of DNA, proteins, lipids, and other macromolecules, which can lead to certain human pathologies including cancers, neurodegenerative disorders, and cardiovascular disease, as well as aging. We have employed Saccharomyces cerevisiae as a model system to examine the levels and types of ROS that are produced in response to DNA damage in isogenic strains with different DNA repair capacities. We find that when DNA damage is introduced into cells from exogenous or endogenous sources there is an increase in the amount of intracellular ROS which is not directly related to cell death. We have examined the spectrum of ROS in order to elucidate its role in the cellular response to DNA damage. As an independent verification of the DNA damage-induced ROS response, we show that a major activator of the oxidative stress response, Yap1, relocalizes to the nucleus following exposure to the DNA alkylating agent methyl methanesulfonate. Our results indicate that the DNA damageinduced increase in intracellular ROS levels is a generalized stress response that is likely to function in various signaling pathways.

two pathways are completely distinct and their major and Tomas Lindahl 1,5 forms in mammalian cells share no enzymes or other 1 Imperial Cancer Research Fund protein factors (Wood, 1996;). De-Clare Hall Laboratories fects in key activities of the BER process, such as AP South Mimms, Hertfordshire EN6 3LD endonuclease, DNA polymerase ␤, or the XRCC1-DNA United Kingdom ligase III heterodimer, lead to embryonic lethal pheno-2 Department of Genetics and Microbiology types in the mouse, indicating that repair of endogenous Centre Me ´dical Universitaire (CMU) DNA lesions is essential during development (Wilson 1211 Geneva 4 and Thompson, 1997). In contrast, NER defects gener-Switzerland ally are nonlethal, and mutations in any of the 7 key 3 Department of Biochemistry genes XPA to XPG can be the cause of the inherited and Division of Cancer Biology cancer-prone disease xeroderma pigmentosum in man. Department of Radiation Oncology Deamination of cytosine to uracil in DNA is counter-Emory University School of Medicine acted by BER; the repair process involves DNA polymer-Atlanta, Georgia 30322 ase ␤-catalyzed substitution of a single dCMP residue 4 Department of Chemistry in DNA to replace the excised uracil and deoxyribose Wesleyan University phosphate moieties and has been reconstituted with Middletown, Connecticut 06459 purified human factors (Kubota et al., 1996; Nicholl et al., 1997; Srivastava et al., 1998). Oxidized DNA bases such as thymine glycol (Tg) and 8-oxoguanine are be-Summary lieved to be repaired in a similar way, although the initial step is carried out by bifunctional enzymes, which can Oxidized pyrimidines in DNA are removed by a distinct both release a damaged base by DNA glycosylase activbase excision repair pathway initiated by the DNA glyity and cleave the DNA chain at the abasic site by AP cosylase-AP lyase hNth1 in human cells. We have lyase activity. Excision of various ring-saturated and reconstituted this single-residue replacement pathring-fragmented oxidized derivatives of thymine and cytosine is due to a Tg-DNA glycosylase-AP lyase activity, way with recombinant proteins, including the AP endothe human counterpart of E. coli endonuclease III or nuclease HAP1/APE, DNA polymerase ␤, and DNA li-Nth. The three-dimensional structure of the bacterial gase III-XRCC1 heterodimer. With these proteins, the enzyme has been established (Kuo et al., 1992); the nucleotide excision repair enzyme XPG serves as a homologous human enzyme hNth1 retains relevant key cofactor for the efficient function of hNth1. XPG profeatures and has been expressed in active form from a tein promotes binding of hNth1 to damaged DNA. The cloned cDNA (Aspinwall et al., 1997; Hilbert et al., 1997). stimulation of hNth1 activity is retained in XPG cata-Characteristic structural properties include a conserved lytic site mutants inactive in nucleotide excision repair. helix-hairpin-helix region that accounts for binding of The data support the model that development of Cockthe damaged pyrimidine and also contains an active ayn...