Anita Szalmás

Epigenetic alterations in cervical carcinogenesis

During cervical carcinogenesis, the major etiologic factor, the persistent oncogenic HPV infection itself is not sufficient to immortalize and transform the epithelial host cells. Together with further genetic and epigenetic alterations disrupting the cell cycle control, the host cell acquires immortal phenotype and progresses further to an overt malignant and invasive phenotype. Here, we discuss how cancer-associated epigenetic alterations can affect the expression of papillomaviral as well as host genes in relation to stages representing the multistep process of carcinogenesis. Biomarker roles in clinical diagnosis and prognosis might be assigned to the epigenetic pattern of the involved genes.

The Human Papillomavirus E6 PDZ Binding Motif: From Life Cycle to Malignancy.

Cancer-causing HPV E6 oncoproteins are characterized by the presence of a PDZ binding motif (PBM) at their extreme carboxy terminus. It was long thought that this region of E6 had a sole function to confer interaction with a defined set of cellular substrates. However, more recent studies have shown that the E6 PBM has a complex pattern of regulation, whereby phosphorylation within the PBM can regulate interaction with two classes of cellular proteins: those containing PDZ domains and the members of the 14-3-3 family of proteins. In this review, we explore the roles that the PBM and its ligands play in the virus life cycle, and subsequently how these can inadvertently contribute towards the development of malignancy. We also explore how subtle alterations in cellular signal transduction pathways might result in aberrant E6 phosphorylation, which in turn might contribute towards disease progression.

Prevalence and Activity of Epstein-Barr Virus and Human Cytomegalovirus in Symptomatic and Asymptomatic Apical Periodontitis Lesions

INTRODUCTION Apical periodontitis is a polymicrobial inflammation with a dominant flora of opportunistic Gram-negative bacteria; however, a pathogenic role of human herpesviruses such as Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) has been implicated recently. The aims of this study were to determine the prevalence, activity, and disease association of EBV and HCMV in apical periodontitis in an Eastern Hungarian population. METHODS Forty samples with apical periodontitis (17 symptomatic and 23 asymptomatic) and 40 healthy pulp controls were collected. EBV and HCMV prevalences were measured by polymerase chain reaction (PCR) detection of the viral DNA and viral activity was tested by reverse-transcription PCR amplification of viral messenger RNA. RESULTS EBV DNA and EBNA-2 messenger RNA were found in apical periodontitis lesions at significantly (p < 0.0001) higher frequencies (72.5% and 50%, respectively) than in controls (both 2.5%). The occurrence of HCMV infection was rare in both apical lesions (10%) and controls (0%). The presence of EBV DNA in apical lesions was associated significantly with large (> or = 5 mm) lesion size (p = 0.02) but not with symptoms (p = 0.30). Symptomatic manifestation was significantly associated with the co-occurrence (odds ratio [OR], 8.80; 95% confidence interval [CI], 1.69-45.76) but not the sole occurrences of EBNA-2 messenger RNA (OR, 2.29; 95% CI, 0.48-11.06) and large lesion size (OR, 4.02; 95% CI, 0.81-19.89). CONCLUSION EBV infection is a frequent event in apical periodontitis, whereas the involvement of HCMV still remains to be elucidated. This study showed that symptomatic manifestation was likely to occur if a large-sized apical periodontitis lesion is aggravated with active EBV infection.

Detection of Osteoprotegerin and TNF‐alpha mRNA in Ankylotic Stapes Footplates in Connection With Measles Virus Positivity

Hypothesis: Otosclerosis is a bone remodeling disorder of the otic capsule causing conductive and sensorineural hearing loss. Persistent measles virus infection of the temporal bone with increased tumor necrosis factor (TNF)‐alpha and decreased osteoprotegerin mRNA expression is supposed to be the main etiologic factor in otosclerosis.

Codetection of Measles Virus and Tumor Necrosis Factor-Alpha mRNA in Otosclerotic Stapes Footplates†

Hypothesis: Otosclerosis is a disease of unknown etiology causing conductive or sensorineural hearing loss. Persistent measles virus infection of the otic capsule is considered to be one of the etiologic factors in otosclerosis.

Disease-Associated Novel CD46 Splicing Variants and Pathologic Bone Remodeling in Otosclerosis†

Objective/Hypothesis: Otosclerotic bone is supposed to show unique CD46 expression pattern because otosclerosis is an organ‐specific disease with viral etiology.

Elevated tumor necrosis factor-alpha expression in periapical lesions infected by Epstein-Barr virus.

INTRODUCTION In apical periodontitis, there is an intense inflammatory response to endodontopathogenic bacteria, an essential component of the pathogenic microbiota. The inflammation can be aggravated by herpesviruses acting as nonessential pathogens in periapical lesions. This study aimed to determine the levels of tumor necrosis factor-alpha (TNF-α) and transforming growth factor-beta (TGF-β) in periapical lesions in relation to local occurrence of Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), human herpesvirus 6 (HHV-6), and human herpesvirus 8 (HHV-8). METHODS Fifty-eight samples with apical periodontitis and 20 clinically healthy gingival control tissues were collected. Viral DNA was determined with nested polymerase chain reaction, and cytokine mRNA expression was detected with real-time polymerase chain reaction assays. RESULTS Periapical lesions harbored EBV (75.9%) and HHV-6 (22.4%) at significantly higher frequencies compared with controls (P < .000001 and P < .05, respectively), whereas HCMV (12%) and HHV-8 (0%) occurred rarely. The median TNF-α expression was 13 times higher (P < .001) and TGF-β expression was 5 times higher in periapical lesions than in controls (P < .001). TNF-α expression was significantly higher in EBV-positive lesions than in EBV-negative lesions (P = .032). Presence of symptoms, lesion size, and infection by HCMV or HHV-6 had no significant association with either TNF-α or TGF-β expression. CONCLUSIONS The herpesviral component of the endodontic microbiota did not correlate with TGF-β expression, whereas EBV infection was associated with a median 1.5 times further elevation of the high TNF-α expression characteristic for periapical lesions.

Effects of human papillomavirus (HPV) type 16 oncoproteins on the expression of involucrin in human keratinocytes

BackgroundThe human papillomavirus (HPV) life cycle is closely linked to keratinocyte differentiation. Oncogenic HPV infection has been shown to hamper the normal differentiation of keratinocytes; however, the underlying mechanisms responsible for this phenomenon are yet to be clarified. Here, we aimed to study the effects of HPV16 E6 and E7 oncogenes on the expression of involucrin (IVL), an established marker of keratinocyte differentiation, in human foreskin keratinocyte (HFK) cells.ResultsThe differentiation of HFK cells by serum and high calcium significantly increased both the mRNA and the protein levels of IVL. The E6 and E7 oncoproteins of HPV16 together caused strong down-regulation of IVL mRNA and protein both in proliferating and in differentiating HFK cells. To study the effects of HPV oncogenes on the IVL promoter, we made transient transfection assays and luciferase tests and found that HPV 16 E6 but not E7 repressed IVL promoter activity in proliferating HFK cells. The inhibitory effect of HPV 16 E6 on the human IVL promoter could be localised to the proximal regulatory region (PRR) of the gene.ConclusionsThese results suggest that the down-regulation of IVL promoter activity by HPV 16 E6 significantly contribute to the inhibition of endogenous IVL expression by the HPV 16 oncoproteins. In contrast, the down-regulation of endogenous IVL expression by HPV16 E7 is probably not caused by a direct and specific effect of E7 on the IVL promoter.

Lineage-specific silencing of human IL-10 gene expression by promoter methylation in cervical cancer cells

Epigenetic analysis was performed to demonstrate that the normal and neoplastic epithelial cells do not serve as the source of the locally elevated IL-10 production during cervical carcinogenesis. Bisulfite sequencing was used to correlate promoter CpG methylation with the transcription of the gene. Lack of IL-10 transcription in HeLa, SiHa, Caski, HT-3, C33-A, HaCaT cell lines and in primary human keratinocytes correlated consistently with the methylated state of the proximal CpG residues, particularly with the two most proximal CpGs at positions -185 and -110. These two sites were also highly methylated in normal and malignant cervical cells directly isolated from patient material. On the other hand, IL-10 producing peripheral blood mononuclear cells had unmethylated CpG residues in the proximal promoter associated with acetylated H3 and H4 histones as determined by chromatin immunoprecipitation. In HeLa carrying epigenetically silenced endogeneous IL-10 promoter, the transfected non-CpG methylated 1 kb and 0.6 kb proximal promoter fragments could drive reporter gene expression, which was reversed by cassette methylation of these promoter fragments. In conclusion, the CpG methylation pattern of the proximal promoter is implicated as a major determinant of transcriptional silencing of human IL-10 expression in cells of cervical epithelial origin.

IL-10 promoter nt -1082A/G polymorphism and human papillomavirus infection in cytologic abnormalities of the uterine cervix

The role of A/G polymorphism at nucleotide -1082 in the interleukin-10 (IL-10) promoter was assessed by following the disease course of 253 patients who had had a routine diagnostic Hybrid Capture human papillomavirus (HPV) test because of cytologic or colposcopic abnormalities of the uterine cervix. At baseline, 97 (78%) of the 125 high-risk HPV-positive and 83 (65%) of the 128 HPV-negative patients had equivocal cytologic atypia classified as P3 by the Papanicolaou classification, and the rest of the patients had mild colposcopic atypia with cytologic results of no oncogenic significance. In the high-risk HPV-infected patients, the frequency distribution of the nt -1082 genotypes (A/A: 28%; A/G: 52%; G/G: 20%) did not differ significantly from that in the controls (A/A: 25%; A/G: 51%; G/G: 24%; p = 0.70). On the other hand, the nt -1082 G allele tended to decrease susceptibility to equivocal cytologic atypia unrelated to HPV infection (A/G: OR = 0.56 [95% CI: 0.31-1.02], G/G: OR = 0.27 [95% CI: 0.11-0.63], p for trend = 0.05). With respect to the development of high-grade cervical intraepithelial neoplasia (CIN), the established risk factors, such as high-risk HPV infection (RR = 104.6, 95% CI: 14.2-769.9) and cytologic atypia (RR = 9.6, 95% CI: 2.34-39.7) but not the various nt -1082 genotypes (A/A: reference; A/G: RR = 1.11 [95% CI: 0.59-2.08]; G/G: RR = 0.62 [95% CI: 0.25-1.50]) were found to increase the risk for high-grade CIN. In conclusion, the nt -1082 polymorphism had no influence on the early phase of cervical carcinogenesis but may determine different susceptibilities to cervical abnormalities unrelated to HPV infection.