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Camptothecin and Adriamycin are clinically important inhibitors for topoisomerase (Topo) I and Topo II, respectively. The ataxia-telangiectasia mutated (ATM) product is essential for ionizing radiation-induced DNA damage responses, but the role of ATM in Topo poisons-induced checkpoints remains unresolved. We found that distinct mechanisms are involved in the activation of different cell cycle checkpoints at different concentrations of Adriamycin and camptothecin. Adriamycin promotes the G(1) checkpoint through activation of the p53-p21(CIP1/WAF1) pathway and decrease of pRb phosphorylation. Phosphorylation of p53(Ser20) after Adriamycin treatment is ATM dependent, but is not required for the full activation of p53. The G(1) checkpoint is dependent on ATM at low doses but not at high doses of Adriamycin. In contrast, the Adriamycin-induced G(2) checkpoint is independent on ATM but sensitive to caffeine. Adriamycin inhibits histone H3(Ser10) phosphorylation through inhibitory phosphorylation of CDC2 at low doses and down-regulation of cyclin B1 at high doses. The camptothecin-induced intra-S checkpoint is partially dependent on ATM, and is associated with inhibitory phosphorylation of cyclin-dependent kinase 2 and reduction of BrdUrd incorporation after mid-S phase. Finally, apoptosis associated with high doses of Adriamycin or camptothecin is not influenced by the absence of ATM. These data indicate that the involvement of ATM following treatment with Topo poisons differs extensively with dosage and for different cell cycle checkpoints.

St. John's wort (SJW), a widely used herbal product, has been implicated in drug interactions resulting from the induced expression of the cytochrome P450 CYP3A4 isoform. In this study, we determined the effect of SJW on the metabolism of irinotecan, a pro-drug of SN-38 and a known substrate for CYP3A4. Five cancer patients were treated with irinotecan (350 mg/m(2), intravenously) in the presence and absence of SJW (900 mg daily, orally for 18 days) in an unblinded, randomized crossover study design. The plasma levels of the active metabolite SN-38 decreased by 42% (95% confidence interval [CI] = 14% to 70%) following SJW cotreatment with 1.0 micro M x h (95% CI = 0.34 micro M x h to 1.7 micro M x h) versus 1.7 micro M x h (95% CI = 0.83 micro M x h to 2.6 micro M x h) (P =.033, two-sided paired Student's t test). Consequently, the degree of myelosuppression was substantially worse in the absence of SJW. These findings indicate that patients on irinotecan treatment should refrain from taking SJW because plasma levels of SN-38 were dramatically reduced, which may have a deleterious impact on treatment outcome.

DNA topoisomerase I (top1) is a ubiquitous enzyme that forms reversible DNA single-strand breaks (cleavage complexes) and plays a role in transcription, DNA replication, and repair. Top1 is the target of camptothecins which selectively trap top1 cleavage complexes and represent a novel class of anticancer drugs active against human solid tumors. The present study demonstrates that recombinant large T antigen (T-Ag), a virus encoded helicase with strong affinity for tumor suppressors and cell cycle- and replication-related proteins, suppresses top1 cleavage complexes and top1 catalytic activity. This top1 suppressive activity is probably not due to T-Ag binding to DNA, as a T-Ag truncation mutant containing only the first 246 amino acids and deficient in DNA binding also inhibited top1, and the inhibition was independent of ATP. T-Ag also antagonized and reversed the trapping of top1 cleavage complexes by camptothecin. These results demonstrate a functional interaction between T-Ag and top1: they also suggest the importance of top1-protein interactions for the regulation of DNA replication and modulation of camptothecin activity.

Two classes of drugs interact with DNA topoisomerase (topo) I, namely topoI poisons such as the camptothecins, which create DNA single-strand breaks and the catalytic inhibitors, which do not. Here, we demonstrate that the antimalarial agent chloroquine is a catalytic inhibitor of eukaryote topoI, as the drug inhibited topoI-mediated DNA relaxation. Chloroquine is known to be a topoII catalytic inhibitor and as such is able to inhibit the activity of a topoII poison, i.e. etoposide. We now show that chloroquine also inhibits the topol poison camptothecin as camptothecin-stimulated nicking of plasmid DNA was inhibited by chloroquine. These observations also apply to endogenous topoI in whole cells. Accordingly, camptothecin-induced single-strand breaks as well as cytotoxicity were antagonised by chloroquine. Further, in a band depletion assay in whole cells, chloroquine prevented camptothecin-mediated topoI trapping, indicating that chloroquine inhibits topoI by interfering with the DNA binding step of the enzyme. In contrast to camptothecin, chloroquine is a weak base and therefore does not enter the cell if the extracellular fluid is acidic, as is the case in most solid tumors. This leads to the possibility of directing cytotoxicity to solid tumors with low extracellular pH by combining a neutral anticancer agent, i.e. camptothecin with a weak base antagonist, i.e. chloroquine. To test the feasibility of this principle, we investigated the drug combination at varying extracellular pH. We found that the antagonising effect of chloroquine on camptothecin-mediated trapping of topoI and DNA single-strand break formation was abolished at acidic extracellular pH. In a clonogenic assay, camptothecin in combination with chloroquine selectively killed cells at low pH (6.2), while camptothecin cytotoxicity was antagonised by chloroquine at normal pH (7.2). In conclusion, we show that the topoI catalytic inhibitor chloroquine inhibits camptothecin and that chloroquine can target the cytotoxic effect of camptothecin to tumor cells in acidic environments.

TAN-1518 A is a cytotoxic agent with suppressive activity against Meth A fibrosarcoma in vivo. This compound inhibits calf thymus DNA topoisomerase I (Topo I) but does not stimulate cleavable complex formation in the nuclei of Chinese hamster ovary (CHO)-K1 cells, suggesting that it inhibits Topo I in a manner different from that of camptothecin (CPT). To clarify the mode of action of TAN-1518 A, we examined its effects on the eukaryotic microorganism Schizosaccharomyces pombe (S. pombe), which does not require Topo I as an essential factor for growth. TAN-1518 A inhibited purified S. pombe Topo I as potently as did CPT. TAN-1518 A, unlike CPT, did not stimulate Topo I-induced DNA cleavage; instead, it inhibited CPT-induced cleavable complex formation. We constructed a S. pombe strain, IR9, that produced excess Topo I. IR9 was hypersensitive to CPT, but its growth was not affected by TAN-1518 A. The CPT-mediated death of IR9 cells was reduced dramatically in the presence of TAN-1518 A. These findings clearly demonstrate that TAN-1518 A is a specific inhibitor of Topo I in eukaryotic cells and also suggest that this agent inhibits some earlier step(s) that occurs before the formation of cleavable complex on DNA strands in the catalytic cycle of this enzyme.