Deficiencies in DNA repair (mismatch repair deficiencies in colon cancers, nucleotide excision repair in ovarian and testicular cancers, and in melanomas), deficiencies in cell cycle checkpoints (Rb, p53, BRCA1, BRCA2, and Chk2 deficiencies in solid tumors), and apoptosis (APC mutations in colon cancers, Bcr-Abl recombinations in leukemia, Bcl-2 overexpression in lymphomas) promote cancers. They also contribute to therapeutic responses and resistance to chemotherapy by preventing a normal apoptotic response of tumor cells bearing the above mutations.To integrate these alterations with the emerging cellular network of molecular pathways, we have developed a mapping convention that can be visually represented as molecular interaction maps (MIMs). These maps are being presented by different members of the LMP (Dr. Kohn, Dr. Aladjem, and Dr. Pommier) at different meetings. They are published in international journals with high impact factors and we have developed an interactive Website in collaboration with the LMP Bioinformatic group (Dr. Weisntein) (http://discover.nci.nih.gov/mim).Our studies in DNA repair include studies with Ecteinascidin 743 (Et-743 - Yondelis) (NSC 648766), DNA alkylating agents, and topoisomerase inhibitors. Et-743 is a novel anticancer agent in Phase II/III clinical trials. Et743 is remarkable because of its clinical activity and its unique mechanism of action. Responses have been observed in sarcomas, which are notoriously resistant to other known treatments, as well as in ovarian and breast cancer. Et-743 differs from other clinically used anticancer agents because it forms covalent adducts at specific guanines in the DNA minor groove and because it selectively Et743 traps the transcription-coupled NER (TC-NER) machinery to induce lethal DNA strand breaks. Thus, Et-743 defines a novel class of anticancer drugs in which enhanced antiproliferative activity parallels enhanced cellular DNA-repair capability. These findings led us to study the NER-dependence for cisplatin. We found that defective TC-NER sensitizes cells to cisplatin, whereas defective global genome repair (GG-NER) did not affect cisplatin response. The complementary between the activities of Et-743 and cisplatin with respect to TC-NER suggests the use of Et743 in cisplatin-resistant tumors and vice-versa. A clinical protocol has been proposed for a Phase I clinical trial of Et-743 in ovarian cancers resistant to cisplatin (Collaboration with Dr. Elise Kohn, Pathology Branch, CCR, NCI). Further molecular studies are planned to determine the transcription- and the strand-specific-dependence of the DNA single-strand breaks induced by Et-743. We are also looking at TC-NER-dependent transcription inhibition by microarray analyses using NER-deficient, XPD, and XPD-complemented cells. We also found that Et-743 induces the degradation of RNA polymerase II as Et-743 blocks transcription. This degradation is related to ubiquitinin ligase activity of the tumor suppressor gene VHL and BRCA1.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. We found that UCN-01 inhibits both Chk1 and Chk2, and we are investigating the role of Chk2 in cell cycle checkpoint response in cancer cells. We have expressed Chk2 as a recombinant protein and preliminary experiments are ongoing to discover Chk2 inhibitors using a high throughput screen (collaboration with Drs. Shoemaker and Scudiero, DTP, NCI).Our studies on apoptosis are focused on the apoptotic chromatin modifications. We found that APE-1 (APEX), which is also implicated in oxidative stress response and Jun-Fos transcriptional activity (REF-1) is directly involved in apoptotic DNA fragmentation. Proteolytic cleavage of APE-1 by caspase 3 activates the endo- and 3'-exo-nuclease of APEX during apoptosis. We also found that one of the early events in apoptosis is the induction of apoptotic topoisomerase I-DNA complexes. The apoptotic topoisomerase I-DNA complexes are induced by a variety of apoptotic stimuli: arsenic trioxide, etoposide, camptothecin, platinum derivatives. We propose that these apoptotic topoisomerase I-DNA complexes are produced by oxidative lesion of genomic DNA, which trap topoisomerase I bound to chromatin. Apoptotic topoisomerase I-DNA complexes in turn activate additional apoptotic responses/pathways and might represent an irreversible apoptotic activation loop.Finally, we are studying the molecular and cellular pharmacology of novel anticancer drugs selected by and in collaboration with NCI the Developmental Therapeutics Program (DTP). We have determined the DNA damaging effects of dimethane sulfonate analogues with alkylating activity and selective renal cell carcinoma cytotoxicity. We have also studied three drugs in early clinical development: tetrandrine, benzothiazole and aminoflavone.
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