N-(2-chloroethyl)-N-nitrosourea tethered to lexitropsin induces minor groove lesions at the p53 cDNA that are more cytotoxic than mutagenic.
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 GC-->AT transitions, mainly at 5' purine G sites; (d) all AT-targeted mutations but one were AT-->TA transversions; (e) the distribution of the CENU-lex mutations along the p53 cDNA was not random, with position 273 (codon 91), where only GC-->AT 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.
Inga
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A
CSTA-Mutagenesis Laboratory, National Cancer Institute (IST), Genova, Italy.
Chen
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FX
Monti
P
P
Aprile
A
A
Campomenosi
P
P
Menichini
P
P
Ottaggio
L
L
Viaggi
S
S
Abbondandolo
A
A
Gold
B
B
Fronza
G
G
D000478:Alkylation; D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000633:toxicity; D001483:Base Sequence; D018076:DNA, Complementary / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D005038:Ethylnitrosourea / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology / Q000633:toxicity; D016158:Genes, p53 / Q000187:drug effects*; D006801:Humans; D008969:Molecular Sequence Data; D009153:Mutagens / Q000494:pharmacology* / Q000633:toxicity; D009429:Netropsin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology / Q000633:toxicity; D013329:Structure-Activity Relationship; D014162:Transfection