Ultrastructural localization of human papillomavirus sequences by in situ hybridization and image analysis

Genital infection caused by human papillomavirus (HPV) is of great sanitary importance because of the role it can play in the development of squamous intraepithelial lesions and carcinomas. Because not all lesions have a mali gnant transformation, it is important that clinical and biological paramete rs contributing to the evolution of a specific lesion be determined. We used CaSki cells with 400-600 integrated copies of HPV-16 genome for in situ hybridization (ISH) with electron microscopy (EM) to locate HPV sequen ces. Three biotinylated DNA probes for HPV types 6/11, 16/18 and 31/33/51 w ere used, and hybrid detection was done with an anti-biotin antibody conjug ated with 15 nm gold colloids. Electron microphotographs with positive labe ling were studied by image analysis to skeletonize them to highlight the he terochromatin fibers and to determinate the spatial localization of colloid al-gold particles. Electron microscopy and ISH technique revealed 2 differe nt distribution patterns: colloidal-gold particles spread over the whole nu clear surface, individually or in small clusters, and large accumulations w ith many gold particles in the nuclear periphery, next to the membrane. Ima ge analysis revealed there was a spa tial association between the gold part icles and heterochromatin fibers in all cases. We believe this association is the consequence of viral genome integration. With these results and knowledge that integration of viral DNA is linked t o a worse prognosis, we propose the application of this methodology for the future study of HPV infected biopsies to obtain prognostic information in lesions caused by this virus.