Multinucleation of Koilocytes Is in Fact Multilobation and Is Related to Aberration of the G2 Checkpoint

576 ORIGINAL ARTICLE J Clin Pathol: first published as 10.1136/jcp.2004.022152 on 25 May 2005. Downloaded from Multinucleation of koilocytes is in fact multilobation and is related to aberration of the G2 checkpoint N H Cho, S Kang, S Hong, G B Jeong, I W Choi, H J Choi, H K Choi ............................................................................................................................... J Clin Pathol 2005;58:576–582. doi: 10.1136/jcp.2004.022152 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 See end of article for authors’ affiliations 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 Correspondence to: Dr N H Cho, Department transcription polymerase chain reaction assay. In addition, three dimensional confocal image restoration of Pathology, was performed on confirmed cervical intraepithelial neoplasia tissue samples. Seodaemoon-ku, Sinchon- Results: T161 phospho-cdc2 and cyclin B1 expression was higher in HPV infected cervical lesions than in dong 134, Yonsei normal samples. Immunofluorescence, revealed that cyclin B1 was present predominantly in the nuclei of University College of Medicine 120–752, Seoul, HPV infected cells, confirming the results of the nuclear fractional assay. On restoration of three Korea; cho1988@yumc. dimensional confocal images, the multinucleation of koilocytes was revealed to be multilobation of a single yonsei.ac.kr nucleus, rather than true multinucleation. This multilobation appeared to be associated with chromosomal Accepted for publication instability and aberration of the G2 checkpoint. 28 October 2004 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. oilocytes are defined as squamous cells with a large, cdc2 complex accumulates in G2, and is negatively regulated well demarcated, clear perinuclear zone surrounded by before mitosis by phosphorylation at threonine 14 (T14) and Ka dense peripheral cytoplasmic rim. These changes tyrosine 15 (Y15) of cdc2. Dephosphorylation of both residues predominantly affect superficial and intermediate cells, and (T14 and Y15) is required for the activation of cyclin B–cdc2, provide the most reliable evidence of human papillomavirus and a dual specific phosphatase, cdc25, carries out this http://jcp.bmj.com/ (HPV) infection.12 One of the most distinctive features of dephosphorylation. G2 checkpoint aberrations may directly koilocytes is multinucleation, which is not specific for HPV result in abnormal centrosome numbers—aneuploidy—and infection, and be seen in reactive processes, including post- may lead to genomic instability.10–14 High risk HPV E6/E7 irradiation reactivity.2 Nevertheless, the presence of koilo- oncoproteins cooperate to induce centrosome abnormality cytes is a common feature in HPV infection, adding weight to and mitotic defects, which accumulate in parallel with the diagnosis, especially when extensive. However, it is nuclear atypia, predominantly multinucleation.13 Expression doubtful whether virally infected keratinocytes are truly of either E6 or E7 can independently bypass the mitotic on September 24, 2021 by guest. Protected copyright. multinucleated even in when the G2 checkpoint is aberrant. checkpoint. E6 can inhibit the checkpoint by degradation of HPV genomes are established as episomes at approximately p53, whereas E7 appears to bypass the checkpoint in the 50 copies/cell after infection of keratinocytes in the basal presence of high concentrations of p53, possibly through the layer, and they replicate in synchrony with cellular DNA loss of the retinoblastoma protein and/or the increased replication.34In contrast to normal differentiating suprabasal expression of the cellular oncoprotein MDM2 (mouse double cells, which exit the cell cycle, HPV infected cells remain minute 2).14 active in the cell cycle and re-enter S phase until they reach terminal differentiation, resulting in amplification of the viral ‘‘The mammalian cell division cycle is governed by the E1 and E2 genes and expression of late transcripts in a orchestrated activation and inactivation of cyclin depen- differentiation dependent manner.5–7 The mammalian cell dent kinases’’ division cycle is governed by the orchestrated activation and inactivation of cyclin dependent kinases (cdks).3 During the We aimed to clarify the fine structure of koilocytes and to G1 phase, cyclins D1, D2, and D3 form complexes with cdk4 correlate their presence with genetic aberrations of the G2 and cdk6, and cyclin E with cdk2; these complexes function checkpoint at the transcriptional and post-translational levels as retinoblastoma kinases, and modulate the activity of E F, 2 of the cyclin B1–cdc2 complex. We found that samples with hence modulating the expression of proliferative genes. HPV infection showed increased expression of T161-cdc2 and Cyclin A associates with cdk2 during the S phase, and with cyclin B1. Using three dimensional confocal reconstruction cdc2 (cdk1) at the S–G2 boundary and into G2. Progression through G2, culminating in mitosis, requires cdc2 to form complexes with cyclin B1 and B2. Threonine residue 161 of Abbreviations: cdk, cyclin dependent kinase; HPV, human papillomavirus; HSIL, high grade squamous intraepithelial lesion; PBS, mammalian cdc2 is implicated in cyclin binding, and phosphate buffered saline; PCR, polymerase chain reaction; PI, phosphorylation at this position may be a prerequisite for propidium iodide; SSPE, saline sodium phosphate–EDTA buffer; RT, the association between cdc2 and cyclin B1.89The cyclin B– reverse transcription www.jclinpath.com Multilobation of koilocytes 577 analyses, we also found that the multiple nuclei of koilocytes USA). HPV amplified products and b globin amplified are in fact the result of multilobation of one nucleus and that products were denatured by incubating with 3N sodium J Clin Pathol: first published as 10.1136/jcp.2004.022152 on 25 May 2005. Downloaded from this phenomenon is associated with upregulation of gene hydroxide solution (10% vol/vol) at room temperature for five products related to G2 checkpoint aberration in koilocytes, as minutes, neutralised by adding 1 mol/litre Tris/HCl (pH 7.2; assessed by the nuclear fractional assay and confocal double 5% vol/vol) then 3N hydrochloric acid (10% vol/vol), and immunofluorescence. finally cooled for five minutes on ice. The cervical samples were mixed with a hybridisation solution made of 6 ml SSPE MATERIALS AND METHODS (saline sodium phosphate–EDTA buffer; Sigma Chemical Co, Our study was conducted at an outpatient clinic of the St Louis, Missouri, USA) and 0.2% sodium dodecyl sulfate Yonsei University College of Medicine in Seoul, Korea. We and applied to the DNA chip. Hybridisation was performed at investigated 30 cervicovaginal swabs using conventional 40˚C for two hours and the samples were then washed with Papanicolaou smears and simultaneously performed HPV 3 ml SSPE for two minutes, 1 ml SSPE for two minutes, and genotyping analysis by DNA chip microarray, followed by L1 air dried at room temperature. Hybridised HPV DNA was consensus polymerase chain reaction (PCR). We used 15 visualised using a DNA chip scanner (ScanArray Lite; GSI cervical swabs from the ‘‘within normal limits’’ category as Lumonics, Ottawa, Canada). the control group. After confirmation of 22 HPV genotypes by DNA chip (Biomed Lab Co, Seoul, Korea), we looked at koilocytes from Western blotting Cervical swabs were washed twice in cold phosphate buf- the cervical swab and tissue specimens by three dimensional confocal restoration—we reconstructed stacks of two dimen- fered saline (PBS) and lysed in lysis buffer (50mM Tris/ sional images of koilocytes to produce three dimensional HCl, pH 7.5; 120mM NaCl; 5% Nonidet P400; 10mM NaF; images by confocal microscopy. We evaluated the functional activity of the G2 molecules in 30 cervical swabs by western blotting for cyclin B1, cdc2, and phospho-cdc2 (Y15), A HSIL 16 HSIL 16/18/58 HSIL 16/18 compared the relative amounts of G2 checkpoint molecules NIM HSIL 35 HSIL 18 in the nuclei and the cytoplasm using the nuclear fractional Cdc2 Cyclin B1 assay, and analysed transcript levels by reverse transcription Cdc2 (RT) PCR. Furthermore, we performed indirect immuno- pT161-cdc2 fluorescence for cyclin B1 and cdc2 in 30 paraffin wax pY15-cdc2 embedded cervical intraepithelial lesion samples correspond- Cyclin B1 ing to the cervical swab samples. pT161-cdc2 p53 HPV genotyping p53 HPV detection and genotyping were performed using an Actin HPVDNAChip and a PCR based DNA microarray system Actin provided by Microarray Centre, Biomed Lab Co. The B HPVDNAChip contains 22 type specific probes that consist Cyclin B1 of 15 high risk groups (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, β Globin 58, 59, 66, 68, and 69) and seven low risk groups (6, 11,

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