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One approach to the effective treatment of cancer requires the continued development of novel chemotherapeutic agents to kill tumor cells. Additionally, an element of cancer research has been devoted to understanding DNA repair pathways in hopes of defining the factors that confer resistance to anticancer drugs and developing strategies for modulating repair capacity as a means of overcoming resistance or enhancing sensitivity to cancer treatments. Historically, yeast, particularly Saccharomyces cerevisiae, has been used as a model system for DNA repair analyses. Additionally, it has been used to evaluate drug efficacy and selectivity, and to identify new targets for antitumor drugs. The usefulness of yeast for these types of analyses has been primarily because of it being considered to have well-conserved DNA repair processes among eukaryotes. However, as more information has accumulated in mammalian DNA repair, and particularly in DNA base excision repair (BER), a number of striking differences have emerged between yeast and mammalian (human) repair processes. The BER pathway is essential for the repair of damaged DNA induced by oxidizing and alkylating agents, which are the majority of chemotherapeutic drugs used currently in the clinic. The importance of this pathway in processing DNA damage makes its members potential targets for novel chemotherapeutic agents. However, because the BER process and its main players are remarkably divergent from S. cerevisiae to humans, it is worth keeping these differences in mind if yeast continues to be used as a model or primary system in the screening for potential new human therapeutics.

Only two DNA repair enzymes, DNA polymerase beta and O6-methylguanine-DNA methyltransferase, have been shown to be inducible in mammalian cells by genotoxic agents. We show here that crocidolite asbestos induces the DNA repair enzyme, apurinic/apyrimidinic (AP)-endonuclease, in isolated mesothelial cells, the progenitor cells of malignant mesothelioma. Asbestos at nontoxic concentrations of 1.25 and 2.5 microg/cm2 significantly increased AP-endonuclease mRNA and protein levels as well as enzyme activity (P < 0.05) in a dose-dependent manner in rat pleural mesothelial cells. These increases were persistent from 24 to 72 h after initial exposure to fibers. Changes were not observed with glass beads, a noncarcinogenic particle. Confocal scanning laser microscopy showed that AP-endonuclease was primarily localized in the nucleus but also in mitochondria. Our data are the first to demonstrate the inducibility of AP-endonuclease by a human class I carcinogen associated with oxidant stress in normal cells of the lung.

One of the most prevalent lesions in DNA is the apurinic/apyrimidinic (AP) site, which is derived from the cleavage of the N-glycosyl bond by DNA glycosylase or by spontaneous depurination. AP sites are repaired by AP endonucleases during the process of base excision repair; however, an imbalance in this DNA repair system may cause mutations as well as cell death. We have established a sensitive and convenient slot-blot method to detect AP sites in genomic DNA using a novel aldehyde reactive probe (ARP), which reacts with the aldehydic group of ring-opened AP sites. The reaction of 1 mM of ARP with 15 microg of genomic DNA containing AP sites at 37 degrees C was completed within 1 min. The AP site-ARP complex was remarkably stable during incubation in TE buffer, even at 100 degrees C for 60 min. The sensitivity of this assay enables detection of 2.4 AP sites per 10(7) bases. By using this ARP-slot-blot assay, the rate of spontaneous depurination of calf thymus DNA was determined. Under physiological conditions, AP sites were increased at 1.54 AP sites/10(6) nucleotides/day (9000 AP sites/cell/day). This highly sensitive assay allows us to determine the endogenous level of AP sites in genomic DNA, as well as to investigate whether DNA-damaging agents cause imbalances of base excision/AP endonuclease repair in vivo and in vitro.