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N-(2-Chloroethyl)-N-nitrosourea Tethered to Lexitropsin Induces Minor Groove Lesions at the p53 cDNA That Are More Cytotoxic than Mutagenic¹

CSTA-Mutagenesis Laboratory, National Cancer Institute (IST), 16132-Genova, Italy [A. I., P. Mo., A. Ap., P. C., P. Me., L. O., S. V., A. Ab., G. F.]; Eppley Institute for Research in Cancer and Allied Diseases and Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, Nebraska 69198-6805 [F-X. C., B. G.]; and Department of Clinical and Experimental Oncology, University of Genova, 16132 Genova, Italy [S. V., A. Ab.]

Many different N-chloroethyl-N-nitrosourea (CENU) derivatives have been synthesized in an attempt to minimize carcinogenic activity while favoring antineoplastic activity. CENU derivatives linked to the dipeptide lexitropsin (lex) showed significant changes in groove- and sequence- selective DNA alkylation inducing thermolabile N³-alkyladenines (N3-Alkyl-As) at lex equilibrium binding sites. CENU-lex sequence specificity for DNA alkylation was determined using ³²P-end-labeled restriction fragments of the p53 cDNA. The adducted sites were converted into single-strand breaks by sequential heating at neutral pH and exposure to piperidine. To establish the mutagenic and lethal properties of CENU-lex-specific lesions, a yeast expression vector harboring a human wild-type p53 cDNA was treated in vitro with CENU-lex and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. p53 mutants were isolated from independent ade^- transformants. The results revealed that: (a) CENU-lex preferentially induces N3-Alkyl-A at specific lex equilibrium binding sites, the formations of which are strongly inhibited by distamycin; (b) reactivity toward Gs is still present, albeit to a lesser extent when compared to N-(2-chloroethyl)-N-cyclohexyl-N-nitrosourea and to CENU; (c) 91% of the 49 CENU-lex p53 mutations (45 of 49) were bp substitutions, 29 of which were GCAT transitions, mainly at 5' purine G sites; (d) all AT-targeted mutations but one were ATTA transversions; (e) the distribution of the CENU-lex mutations along the p53 cDNA was not random, with position 273 (codon 91), where only GCAT transitions were observed, being a real (n = 3, P < 0.0002) CENU-lex mutation hot spot; and (f) a shift in DNA alkylation sites between lesion spectra induced by CENU-lex and N-(2-chloroethyl-N-cyclohexyl-N-nitrosourea was associated with an increased lethality and a decreased mutagenicity, whereas no dramatic change in mutational specificity was observed. Hence, it is tempting to conclude that, in this experimental system, N3-Alkyl-A is more lethal than mutagenic, whereas O⁶-alkylguanine is a common premutational lesion formed at non-lex binding sites. These results suggest that CENU derivatives with virtually absolute specificity for A residues would make targeting of lethal, nonmutagenic lesions at A+T-rich regions possible, and this may represent a new strategy for the development of new chemotherapeutic agents with a higher therapeutic index.

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				J. D. Kelly, A. Inga, F.-X. Chen, P. Dande, D. Shah, P. Monti, A. Aprile, P. A. Burns, G. Scott, A. Abbondandolo, et al. Relationship between DNA Methylation and Mutational Patterns Induced by a Sequence Selective Minor Groove Methylating Agent J. Biol. Chem., June 25, 1999; 274(26): 18327 - 18334. [Abstract] [Full Text] [PDF]