Quantitation and mapping of integrated human papillomavirus on human metaphase chromosomes using a fluorescence microscope imaging system.
Integrated human papillomavirus type 16 (HPV-16) DNA was directly visualized on metaphase chromosomes in the two human cervical carcinoma cell lines SiHa and CaSki by fluorescence in situ hybridization with a biotinylated DNA probe (7.9 kb). The fluorescence intensities of hybridization signals from single copies and dispersed clusters of integrated HPV-16 DNA were quantified using a microscope equipped with a cooled-CCD camera that was interfaced to an image processor and host computer. Hybridization signals were localized on chromosomes using separate, registered images of 4',6-diamidino-2-phenylindole (DAPI) or propidium iodide stained metaphase chromosome spreads. In both SiHa and CaSki spreads, a single fluorescein signal was observed on one or both chromatids of chromosome 13, which was identified by simultaneous hybridization with a biotinylated centromere probe specific for chromosomes 13 and 21. Ratios of the distance from 13pter to the HPV-16 signals to the entire chromosome length were approximately 0.63 +/- 0.05 in both SiHa and CaSki cells, indicating the possibility of a common integration domain on chromosome 13. In SiHa cells, no additional signals were observed on other chromosomes. This observation, taken together with literature reports that SiHa cells contain 1 to 2 copies of the HPV-16 genome in this region of chromosome 13, suggests that each fluorescein signal on chromosome 13 represents one equivalent of the HPV-16 genome. The total integrated fluorescence intensity in isolated CaSki metaphase chromosome spreads was approximately two orders of magnitude greater than that of a single copy of HPV-16 DNA in SiHa cells, indicating an increase in HPV-16 copy number.(ABSTRACT TRUNCATED AT 250 WORDS)
Callahan
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Department of Biochemistry, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, Maryland 21205.
Karim
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Zheng
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Tso
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Lesko
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D000328:Adult; D002294:Carcinoma, Squamous Cell / Q000235:genetics / Q000473:pathology; D002874:Chromosome Mapping; D002882:Chromosomes, Human, Pair 13; D015344:DNA Probes, HPV; D004279:DNA, Viral / Q000235:genetics; D005260:Female; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008677:Metaphase; D008856:Microscopy, Fluorescence; D008875:Middle Aged; D009693:Nucleic Acid Hybridization; D027383:Papillomaviridae / Q000235:genetics*; D014407:Tumor Cells, Cultured / Q000473:pathology; D002583:Uterine Cervical Neoplasms / Q000235:genetics / Q000473:pathology
D015344:DNA Probes, HPV; D004279:DNA, Viral