| ABSTRACT: |
|
Alkylation of DNA by chloroethylnitrosourea (CNU) at the guanine N7
position has been shown to occur in a sequence-selective fashion. In this
report we find that the depurination of these alkylated sites occurs with
two distinct kinetic components--GG sequences depurinate within 30 min of
exposure to CNU, while depurination at GT sequences is first observed
after 1 h and continues to increase 16 h after drug exposure. These
apurinic sites are converted to DNA strand breaks and constitute less than
10% of the total sites of guanine N7 alkylation. Spermidine was found to
decrease alkylation in 5'-GG-3' sequences but increases alkylation at
5'-GTC-3' sequences. These findings suggest that the majority of the
guanine N7 alkylations formed by CNU are stable, with a minor adduct being
responsible for the slow depurination event. We propose that the rapid
depurination induced by CNU occurs from an initial guanine O6 alkylation,
which then depurinates via a guanine O6-N7 cyclized intermediate. We also
propose that the resulting apurinic sites may lead to DNA interstrand
cross-linking (ISC). In support of these hypotheses we show that (i) DNA
modified with the monoalkylating agent dimethylsulfate forms DNA ISC upon
depurination; (ii) ellagic acid enhances the level of guanine N7
alkylation and alters the pattern of sequence selectivity shown by three
bifunctional chloroethylating agents CNU, mitozolomide and methyl
3-(2-chloroethyl)-4-oxoimidazo[5,1-d]-1,2,3,5-tetrazine-8-ca rboxylate but
not with nitrogen mustard; (iii) ellagic acid has no effect upon the
frequency of alkylation observed with the monofunctional alkylators
N-methyl-N-nitrosourea, N-ethyl-N-nitrosourea and methylmethanesulfonate;
(iv) ellagic acid increases the frequency of depurination and strand break
formation induced by CNU without affecting the sequence-selective pattern of depurination. |