Ecteinascidin 743 (Et743) is a novel anticancer agent presently in Phase II/III clinical trials. Responses have been observed in sarcomas, which is very interesting because these poor prognosis tumors are notoriously resistant to previously used treatments. Et743 (NSC 648766) differs from other anticancer drugs in clinical use because it forms covalent adducts at specific guanines in the minor groove of DNA and because it selectively block transcription of genes containing transcription factors that bind to guanine-rich sequences (such as NF-Y transcription factor). To further elucidate the mechanism of action of Et743, we have generated an Et743-resistant cell line (HCT116/ER5). The HCT116/ER5 cells exhibit enhanced microsatellite instability and aneuploidy. Complete lack of expression of the DNA repair gene XPG was found in HCT116/ER5 cells as a result of chromosome 13q DNA copy-number loss, due to an unbalanced t(13; 14) translocation, and loss of heterozygosity at 13q33.3, due to a frameshift mutation of XPG at codon 240 resulting in a stop codon at position 243. Transfection of XP-G cDNA restored the sensitivity of the HCT116/ER5 cells to Et743 demonstrating that proteins involved in the nucleotide excision repair pathway such as the endonuclease XPG are essential for the antiproliferative activity Et743. Xeroderma pigmentosum cells deficient in the NER genes XPG, XPA, XPD, or XPF were resistant to Et743, and sensitivity was restored by complementation with wild-type genes. Moreover, studies of cells deficient in XPC or in the Cockayne Syndrome genes (CSA and CSB) indicated that the drug sensitivity is dependent specifically on the transcription-coupled pathway of NER. Thus, we found that Et743 interacts with the transcription-coupled NER machinery to induce lethal DNA strand breaks. Et743 defines a novel class of anticancer drugs in which enhanced antiproliferative activity parallels enhanced cellular DNA-repair capability. Because most cancers have alterations in the cell cycle checkpoint pathways (p53, pRb) and cell cycle machinery (cyclins, cyclin-dependent kinase inhibitors - such as p16), we are exploring inhibitors of cell cycle checkpoints as novel anticancer agents. 7-hydroxystaurosporine (UCN-01) is a novel anticancer agent in phase II/III clinical trials. We found that UCN-01 is synergistic with DNA damaging agents such as topoisomerase inhibitors and drugs that act during the S-phase of the cell cycle. This synergism has been related to an abrogation of the S-phase checkpoint, which is controlled by 2 protein kinases, Chk1 and Chk2. UCN-01 was found to inhibit Chk1 and we are examining the effects of UCN-01 on Chk2 and the role of Chk2 in cell cycle checkpoint in cancer cells.
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