Preferential incorporation of 3'-azido-2',3'-dideoxythymidine into telomeric DNA and Z-DNA-containing regions of Chinese hamster ovary cells.
3'-Azido-2',3'-dideoxythymidine (azidothymidine; AZT) induces bone marrow toxicity in patients chronically given therapeutic doses of drug and is tumorigenic in rodents, inducing squamous cell tumors in vaginal tissues of mice and rats. In the study reported here, we explored the incorporation of AZT into specific regions of mammalian chromosomal DNA. CHO cells were exposed to AZT for 4 h, allowed to complete at least one cell cycle, and then arrested in metaphase with colchicine. Regions of concentrated AZT incorporation were identified in individual metaphase chromosomes by immunohistochemistry using antiserum specific for AZT and a secondary antiserum with a streptavidin--Texas red end point. These studies demonstrated that most of the intensely staining regions were chromosomal ends or telomeres. When 18 metaphases were examined, all telomeres but one (39 of 40) were positive at least once. Using an anti-Z-DNA antibody, chromosomal regions containing DNA in Z conformation were also localized by immunohistochemistry using a rhodamine-conjugated secondary antibody. When metaphase chromosome spreads were stained for either AZT or Z-DNA, ideograms showing localization of AZT (18 metaphases) and DNA in Z configuration (26 metaphases) were drawn for every chromosome of each metaphase examined. These ideograms demonstrated that 60% of the regions that stained positive for AZT were also positive for Z-DNA. Furthermore, slides incubated with both antibodies, using streptavidin--Texas red to identify AZT and fluorescein to identify Z-DNA, confirmed colocalization of the two markers. Additional experiments exploring the induction of chromatin bridges in AZT-treated cells suggest that the analogue may be able to bind to and disrupt the normal functioning of telomeric DNA.
Olivero
O A
OA
Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, NIH, Bethesda, MD 20892.
Poirier
M C
MC
D000818:Animals; D016466:CHO Cells; D002843:Chromatin / Q000737:chemistry; D002874:Chromosome Mapping; D006224:Cricetinae; D004247:DNA / Q000378:metabolism*; D005260:Female; D008677:Metaphase; D008856:Microscopy, Fluorescence; D016615:Telomere / Q000378:metabolism*; D015215:Zidovudine / Q000378:metabolism*
D002843:Chromatin; D015215:Zidovudine; D004247:DNA