Multinucleation of koilocytes is in fact multilobation and is related to aberration of the G2 checkpoint

Abstract

Aims: To clarify the fine structure of koilocytes and correlate this with genetic aberration of the G2 checkpoint Methods: Three dimensional reconstruction from confocal fluorescent images, together with functional assays for key molecules of the G2 checkpoint-cdc2 and cyclin B1-was performed in human uterine cervical samples. After confirming 22 human papillomavirus (HPV) types using a DNA chip from 30 cervical swabs, previously confirmed as 15 cervical low grade and 15 high grade intraepithelial lesions, the activity of molecules involved in the G2 checkpoint was evaluated using western blotting for cyclin B1, cdc2, and phospho-cdc2 (Y15 and T161), a nuclear extraction fractional assay, and a reverse transcription polymerase chain reaction assay. In addition, three dimensional confocal image restoration was performed on confirmed cervical intraepithelial neoplasia tissue samples. Results: T161 phospho-cdc2 and cyclin B1 expression was higher in HPV infected cervical lesions than in normal samples. Immunofluorescence, revealed that cyclin B1 was present predominantly in the nuclei of HPV infected cells, confirming the results of the nuclear fractional assay. On restoration of three dimensional confocal images, the multinucleation of koilocytes was revealed to be multilobation of a single nucleus, rather than true multinucleation. This multilobation appeared to be associated with chromosomal instability and aberration of the G2 checkpoint. Conclusions: The multiple nuclei of koilocytes are in fact multilobation of a single nucleus, and this phenomenon is associated with upregulation of gene products related to the G2 checkpoint.

PCR detection of multiple human herpesvirus DNA in saliva from HIV-infected individuals in Teresina, State of Piauí, Brazil

“…Two microliters of DNA were used in each PCR reaction. The integrity of the extracted DNA and the exclusion of PCR inhibitors in samples were confirmed by amplifying the human β-globin gene 17 . The primers for CMV genome detection (HSVF 5´-GACTTTGCCAGCCTSTACCC-3' and CMVR 5'-CTCTCCTCCGAATCCCCC-3') were based on the DNA polymerase gene of CMV (GenBank accession number AB329634.1) and amplified 499 base pairs PCR products.…”

“…The specificities of the primer pairs were validated by DNA sequencing of the PCR products. Genomic DNAs extracted from clinical HHV-8 orf64-orf65 [20] b-globin b-globin gene [17] HSV: human herpesvirus type 1, CMV: cytomegalovirus, EBV: Epstein-Barr virus, EBNA-1: Epstein-Barr virus nuclear antigen 1 samples were used as positive control standards for HSV-1, CMV and EBV previously confirmed by PCR 10 . The genomic DNA extracted from BCBL-1 cells was used as positive control standard for HHV-8.…”

Immunohistochemistry in the Diagnosis of Squamous Intraepithelial Lesions of the Uterine Cervix

“…The distinction of florid reactive changes, immature metaplastic patterns, and atrophic changes from HPVinduced alterations may cause difficulties. Att e m p t s h a v e b e e n m a d e t o r e d e f i n e t h e traditional criteria for lesion diagnosis, while other efforts aimed at the adoption of new, more objective methods, which might support the former (Bollmann et al, 2005;Cho et al, 2005;Guillaud et al, 2005;Prasad et al, 1994;Salvia et al, 2004;Scheurer et al, 2007). However, studies attempting to correlate HPV presence and replication to certain cytohistologic alterations are becoming less frequent and/or fruitful.…”

Human Papillomavirus and Related Diseases - From Bench to Bedside - A Clinical Perspective

“…The distinction of florid reactive changes, immature metaplastic patterns, and atrophic changes from HPVinduced alterations may cause difficulties. Attempts have been made to redefine the traditional criteria for lesion diagnosis, while other efforts aimed at the adoption of new, more objective methods, which might support the former (Bollmann et al, 2005;Cho et al, 2005;Guillaud et al, 2005;Prasad et al, 1994;Salvia et al, 2004;Scheurer et al, 2007). However, studies attempting to correlate HPV presence and replication to certain cytohistologic alterations are becoming less frequent and/or fruitful.…”