In vivo sequencing of camptothecin-induced topoisomerase I cleavage sites in human colon carcinoma cells.
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.
Pondarré
C
C
Laboratory of Molecular Pharmacology, Division of Basic Sciences, Bldg 37, Rm 5C25, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4255, USA.
Strumberg
D
D
Fujimori
A
A
Torres-León
R
R
Pommier
Y
Y
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D001483:Base Sequence; D001665:Binding Sites; D002166:Camptothecin / Q000494:pharmacology*; D003110:Colonic Neoplasms / Q000235:genetics*; D004249:DNA Damage; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004273:DNA, Neoplasm / Q000737:chemistry* / Q000378:metabolism; D004275:DNA, Ribosomal / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012337:RNA, Ribosomal, 18S / Q000737:chemistry; D017422:Sequence Analysis, DNA; D059004:Topoisomerase I Inhibitors; D014407:Tumor Cells, Cultured
D000972:Antineoplastic Agents, Phytogenic; D004273:DNA, Neoplasm; D004275:DNA, Ribosomal; D004791:Enzyme Inhibitors; D012337:RNA, Ribosomal, 18S; D059004:Topoisomerase I Inhibitors; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin