Nucleic Acids Research, Vol 25, Issue 20 4111-4116, Copyright © 1997 by Oxford University Press
C Pondarre, D Strumberg, A Fujimori, R Torres-Leon and Y Pommier
Camptothecin (CPT) is a specific topoisomerase I (top1) poison which traps
top1 cleavable complexes; e.g. top1-linked DNA single-strand breaks with
5'-hydroxyl and 3'-top1 linked termini. CPT is also a potent anticancer
agent and several of its derivatives have recently shown activity in the
chemotherapy of solid tumors. Our aim was to apply the ligation-mediated
polymerase chain reaction (LM-PCR) method to DNA extracted from CPT-treated
cells in order to: (i) evaluate LM- PCR as a sensitive technique to detect
in vivo CPT-induced cleavable complexes; (ii) investigate the frequency and
distribution of CPT- induced DNA damage in vivo ; and (iii) compare the
distribution and intensity of cleavage sites in vivo and in vitro. This
report describes a protocol allowing the sequencing of top1-mediated DNA
strand breaks induced by CPT in the coding strand of the 18S rRNA gene of
human colon carcinoma cells. CPT or its clinical derivatives, topotecan,
CPT-11, SN- 38, and 9-aminocamptothecin differed in their potency and
exhibited differences in their DNA cleavage pattern, which is consistent
with our previous in vitro studies [Tanizawa et al . (1995) Biochemistry ,
43, 7200-7206]. CPT-induced DNA cleavages induced in the presence of
purified top1 were induced at the same sites in the human 18S rDNA.
However, the relative intensity of the cleavages were different in vivo and
in vitro. Because mammalian cells contain approximately 300 copies of the
rDNA gene per genome, rDNA could be used to monitor CPT-induced DNA
cleavage in different cell lines and possibly in tumor samples.
ARTICLES
In vivo sequencing of camptothecin-induced topoisomerase I cleavage sites in human colon carcinoma cells
Laboratory of Molecular Pharmacology, Division of Basic Sciences, Bldg 37, Rm 5C25, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4255, USA.
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