The base excision repair (BER) pathway is essential for the removal of DNA bases damaged by alkylation or oxidation. A key step in BER is the processing of an apurinic/apyrimidinic (AP) site intermediate by an AP endonuclease. The major AP endonuclease in human cells (APE1, also termed HAP1 and Ref-1) accounts for >95% of the total AP endonuclease activity, and is essential for the protection of cells against the toxic effects of several classes of DNA damaging agents. Moreover, APE1 overexpression has been linked to radio- and chemo-resistance in human tumors. Using a newly developed high-throughput screen, several chemical inhibitors of APE1 have been isolated. Amongst these, CRT0044876 was identified as a potent and selective APE1 inhibitor. CRT0044876 inhibits the AP endonuclease, 3′-phosphodiesterase and 3′-phosphatase activities of APE1 at low micromolar concentrations, and is a specific inhibitor of the exonuclease III family of enzymes to which APE1 belongs. At non-cytotoxic concentrations, CRT0044876 potentiates the cytotoxicity of several DNA base-targeting compounds. This enhancement of cytotoxicity is associated with an accumulation of unrepaired AP sites. In silico modeling studies suggest that CRT0044876 binds to the active site of APE1. These studies provide both a novel reagent for probing APE1 function in human cells, and a rational basis for the development of APE1-targeting drugs for antitumor therapy.

Summary In mammalian cells, there are two isoforms of DNA topoisomerase 11, designated a (170-kDa form) and ,B (180-kDa form). Previous studies using cell lines have shown that the topoisomerase Ila and [ isoforms are differentially regulated during the cell cycle and in response to changes in growth state. Moreover, both isoforms can act as targets for a range of anti-tumour drugs. Here, we have analysed the normal tissue distribution in humans of topoisomerase Ila and f using isoform-specific antibodies. In addition, we have studied expression of these isoforms in 69 primary tumour biopsies, representative either of tumours that are responsive to topoisomerase 11-targeting drugs (breast, lung, lymphoma and seminoma) or of those that show de novo drug resistance (colon). Topoisomerase lla was expressed exclusively in the proliferating compartments of all normal tissues, and was detectable in both the cell nucleus and cytoplasm. In biologically aggressive or rapidly proliferating tumours (e.g. high-grade lymphomas and seminomas), there was a high level of topoisomerase Ila, although expression was still detectable in colon tumours, indicating that expression of this isoform is not sufficient to explain the intrinsic drug resistance of colon tumours. Topoisomerase Ill was expressed ubiquitously in vivo and was localized in both the nucleoli and the nucleoplasm. This isoform was present in quiescent cell populations, but was expressed at a generally higher level in all tumours and proliferating cells than in normal quiescent tissues. We conclude that topoisomerase Ila is a strict proliferation marker in normal and neoplastic cells in vivo, but that topoisomerase ll[ has a much more general cell and tissue distribution than has topoisomerase Ila. The apparent up-regulation of topoisomerase II,B in neoplastic cells has implications for the response of patients to anti-tumour therapies that include topoisomerase Il-targeting drugs.Keywords: topoisomerase Ila; topoisomerase P; immunochemistry Topoisomerase II is a homodimeric nuclear protein with many different roles in DNA metabolism, including relief of torsional stress and mitotic chromosome condensation and segregation (reviewed in Wang, 1985(reviewed in Wang, , 1991Watt and Hickson, 1994). Topoisomerase II is also one of the most important determinants of cellular sensitivity to a range of clinically important anti-tumour drugs. For example, topoisomerase II is the primary cellular target for several intercalating agents, including doxorubicin, mitoxantrone and epirubicin, as well as for the non-intercalating epipodophyllotoxins, etoposide and teniposide (reviewed in Osheroff et al, 1991;Pommier, 1993;Beck et al, 1993). Topoisomerase II is a so-called type II enzyme, defined as acting via the creation of double-stranded breaks in DNA, through which an intact DNA duplex is passed, before the break is resealed. As part of this breakage and religation process, a transient reaction intermediate is generated, termed the cleavage complex, consisting of a topoisomerase...

Summary Topoisomerase II is a key target for many anti-cancer drugs used to treat breast cancer. In human cells there are two closely related, but differentially expressed, topoisomerase II isoforms, designated topoisomerase Ila and P. Here, we report the production of a new polyclonal antibody raised against a fragment of the C-terminal domain of the 180 kDa form of topoisomerase II (the P isoform), which does not cross-react with the 170 kDa form (the a isoform). Using this antibody, together with a polyclonal antibody specific for the 170 al., 1991;Capranico and Zunino, 1992;Pommier, 1993). DNA topoisomerase II is a nuclear enzyme which alters DNA tertiary structure through transient double-stranded breakage of the DNA backbone and subsequent passage of a second intact DNA duplex through the break (reviewed in Osheroff et al., 1991;Wang, 1985;Austin and Fisher, 1990;Watt and Hickson, 1994). The aforementioned drugs, as well as several other intercalating agents, including amsacrine (Nelson et al., 1984), trap the enzyme in a covalently bound reversible complex with DNA, termed the cleavable complex. The stabilisation of this complex prevents religation of the broken DNA and produces lesions which are thought to be cytotoxic by virtue of their ability to inhibit the passage of the replication fork. There is evidence that the cellular level of topoisomerase II determines the extent of cleavable complex formation after drug treatment and, therefore, the degree of drug toxicity. Low levels of topoisomerase II are associated with the induction of a reduced number of DNA lesions and hence increased drug resistance (Beck et al., 1993;Pommier, 1993). The converse relationship has been shown in mutant cell lines hypersensitive to topoisomerase II inhibitors (Davies et al., 1988) and also in testicular teratoma cell lines compared with bladder cell lines (Fry et al., 1991).There are two isoforms of topoisomerase II in mammalian cells that are products of different genes (Drake et al., 1989;Jenkins et al., 1992;Tan et al., 1992;Austin et al., 1993).These isoforms are termed a (170 kDa form) and , ((180 kDa form) and have different patterns of expression, suggesting that they might perform different functions. The a isoform is produced primarily in late S-phase and during the G2/M phase of the cell cycle (Woessner et al., 1991), and is apparently more sensitive to teniposide and merbarone than is the (Drake, et al., 1989). The gene encoding the a isoform has been mapped to chromosome 17q21-22 in humans (Tsai-Pflugfelder et al., 1988). The P isoform is expressed throughout the cell cycle, with higher levels seen in non-proliferating cells (Woessner et al., 1991) and is encoded on chromosome 3p24 in humans (Jenkins et al., 1992;Tan et al., 1992).Drug resistance is a major clinical problem in the treatment of solid tumours. Tumours often become resistant to multiple, structurally unrelated drugs as a result of expression of the membrane efflux pump, P-glycoprotein (reviewed in Bradley and Ling, 1994). This is the classi...

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