Quantitation and mapping of integrated human papillomavirus on human metaphase chromosomes using a fluorescence microscope imaging system

Abstract

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 dis- Key terms: Microscopy, fluorescence/image processing, computer-assistedhucleic acid hybridizatiodDNA probes, HPV The technique of in situ hybridization allows one to detect, localize, and quantify unique nucleic acid sequences within single cells and upon individual chromosomes. Radioactive probes and autoradiographic techniques have been required for quantitation of target sequence copy number and are often necessary in order to detect specific nucleic acid sequences present in low abundance (4,12). However, recent advances have been made in the labeling and detection of hybridized nonradioactive probes; thus it is now possible to detect specific nucleic acid sequences only a few kilobases in length on metaphase chromosomes (3,10,16,17,26,30). In physical mapping studies, fluorescence detection of hybridization signals on chromosomes provides better microscopic spatial resolution and greater precision than microautoradiography (6,18,20). In addition, fluorescence detection of nonra-

Optimization of biotinyl-tyramide-based in situ hybridization for sensitive background-free applications on formalin-fixed, paraffin-embedded tissue specimens

“…By comparison with previous studies [10,11] and because of the consistency of this result, it is concluded that this signal represents the integrated HPV 16 on chromosome 13. There is no commercially available centromeric probe for chromosome 13 to enable additional support for this interpretation.…”

“…HPV 16 has been shown to be integrated at 13q21-q31 by in situ hybridization analysis with 3 H-labeled probe and chromosome identification by Geimsa staining [10] and by detection of biotin-labeled HPV probe detected with avidin-FITC, which enabled detection of integrated HPV in metaphase spreads as a 'pin-prick' signal [11]. The SiHa cell line is aneusomic and has been found to contain 66 – 72 chromosomes, but to be diploid for the acrocentric chromosome 13 [10].…”

Rolling circle replication of human papillomavirus type 16 DNA in epithelial cell extracts

“…Finally, high-risk HPV DNA is frequently integrated into the chromosome of cervical cancer cells and is occasionally found amplified as head-to-tail tandem repeats Pett & Coleman 2007;Lace et al 2009), as typically observed in CaSki cells, which harbor approximately 500 tandem repeats of the HPV16 genome (Callahan et al 1992;Van Tine et al 2004). Although the underlying mechanism behind this type of viral DNA integration is elusive, a plausible explanation is that a linear concatemeric HPV genome is synthesized in cells by a rolling circle mechanism, followed by integration into the host chromosome through cellular pathways for double-strand break repair.…”

Human papillomavirus DNA integration and messenger RNA transcription in cervical low- and high-risk squamous intraepithelial lesions in Austrian women

“…We continued our former study (25) to achieve the following: 1) collect more information on the physical state and the frequency of integration of HPV6, 11, 16, 18, 31, 33, 52b, and 58 DNA in benign and reactive cervical cells and SILs of 442 patients, 2) examine the integration profiles of different cancer‐associated HPV types in 125 of these patients, and 3) amplify E6/E7 mRNA transcripts by RT‐PCR analysis and use 2D gel electrophoresis to determine the physical state of viral DNA and the HPV16 DNA copy number in relation to the SiHa cell line. As described in several studies (30–35) , in this cervical carcinoma cell line, a single copy of HPV16 DNA is integrated in its genome and is therefore suitable for routine clinical determination of integration. We found that the viral loads of HPV16 E6/E7 are no prerequisite for the physical state of the integrated form of cervical lesion grades (Table 6).…”

“…The Hybrid Capture (HC II) System (Digene) for LR-HPV types (6, 11, 42, 43, and 44) and HR-HPV types (16,18,31,33,35,39,45, 51, 52b, 56, 58, 59, and 68), based on hybridization of the target HPV DNA to label RNA probes in solution (26) , was used.…”