Vol. 56, Issue 3, 478-484, September 1999

DNA Protein Cross-Links Produced by NSC 652287, a Novel Thiophene Derivative Active Against Human Renal Cancer Cells

Wilberto Nieves-Neira,¹ Maria I. Rivera,¹ Glenda Kohlhagen, Miriam L. Hursey, Philippe Pourquier, Edward A. Sausville, and Yves Pommier

Laboratory of Molecular Pharmacology, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (W.N-N., G.K., P.P., Y.P.); and Laboratory of Drug Discovery Research and Development, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland (M.I.R., M.L.H., E.A.S.)

2, 5-bis(5-Hydroxymethyl-2-thienyl)furan (NSC 652287), is a representative of a series of thiophene derivatives that exhibit potent and selective antitumor activity against several tumor cell lines in the National Cancer Institute Anticancer Drug Screen. NSC 652287 has noticeable activity for the renal cell lines and produces cures in certain corresponding xenografts. The cellular mechanisms of action of NSC 652287 were therefore investigated in this study in greater detail. The most sensitive renal carcinoma cell line, A498, exhibited cell cycle arrest in G₀-G₁ and G₂-M at 10 nM NSC 652287, with increased p53 and p21^WAF1 protein. At higher concentrations, NSC 652287 still induced p53 elevation but with p21^WAF1 reduction and massive apoptosis. These results collectively suggested that NSC 652287 induced DNA damage. Using alkaline elution techniques, we found that NSC 652287 induced both DNA-protein and DNA-DNA cross-links with no detectable DNA single-strand breaks. These DNA-protein cross-links (DPC) persisted for at least 12 h after drug removal and their frequency was correlated with cytotoxicity in the renal cell lines studied. The most sensitive cells (A498) produced the highest DPC followed by the cell line with intermediate sensitivity (TK-10). DPC were minimal in the two resistant cell lines, ACHN and UO-31. Nonetheless, a similar degree of DPC occurred at doses imparting equitoxic effects. These results indicate that DNA is a primary target for the novel and potent anticancer thiophene derivative, NSC 652287. NSC 652287 did not cross-link purified DNA or mammalian topoisomerase I suggesting the importance of active metabolite(s) for the cross-linking activity.