[Show abstract][Hide abstract] ABSTRACT: The life cycle of human papillomaviruses (HPVs) is strictly linked to the differentiation of their natural host cells. The HPV E6 and E7 oncoproteins can delay the normal differentiation program of keratinocytes; however, the exact mechanisms responsible for this have not yet been identified. The goal of this study was to investigate the effects of HPV16 oncoproteins on the expression of genes involved in keratinocyte differentiation. Primary human keratinocytes transduced by LXSN (control) retroviruses or virus vectors expressing HPV16 E6, E7 or E6/E7 genes were subjected to gene expression profiling. The results of microarray analysis showed that HPV 16 E6 and E7 have the capacity to downregulate the expression of several genes involved in keratinocyte differentiation. Quantitative real-time polymerase chain reaction (qRT-PCR) assays were performed to confirm the microarray data. To investigate the effects of the HPV oncoproteins on the promoters of selected keratinocyte differentiation genes, luciferase reporter assays were performed. Our results suggest that the HPV 16 E6 and/or E7 oncogenes are able to downregulate the expression of several genes involved in keratinocyte differentiation (such as desmocollin 1, keratin 4, S100 calcium-binding protein A8 and small proline-rich protein 1A), at least partially by downregulating their promoter activity. This activity of the HPV oncoproteins may have a role in the productive virus life cycle, and also in virus-induced carcinogenesis.
Archives of Virology 12/2014; 160(2). DOI:10.1007/s00705-014-2305-y · 2.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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).
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.
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.
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.
Journal of endodontics 04/2013; 39(4):456-60. DOI:10.1016/j.joen.2012.12.028 · 3.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
The Src family tyrosine kinases (SFK) are cellular regulatory proteins that influence cell adhesion, proliferation, invasion and survival during tumor development. Elevated activity of Src was associated with increased cell proliferation and invasivity in human papillomavirus (HPV)-associated malignancies; therefore, transduced human foreskin keratinocytes (HFK) were used to investigate whether SFK activation is a downstream effect of papillomaviral oncoproteins. Activation of ubiquitously expressed SFKs, namely Src, Yes and Fyn, was investigated in both proliferating and differentiating keratinocytes.
In proliferating keratinocytes, Src, Yes and Fyn mRNA levels were not affected by HPV 16 E6 or E7 oncoproteins, while at the protein level as detected by western blot, the presence of both E6 and E7 resulted in substantial increase in Src and Yes expression, but did not alter the high constitutive level of Fyn. Phospo-kinase array revealed that all ubiquitously expressed SFKs are activated by phosphorylation in the presence of HPV 16 E7 oncoprotein. Keratinocyte differentiation led to increased Yes mRNA and protein levels in all transduced cell lines, while it did not influence the Src transcription but resulted in elevated Src protein level in HPV16 E7 expressing lines.
This study revealed that HPV 16 oncoproteins upregulate Src family kinases Src and Yes via posttranscriptional mechanisms. A further effect of HPV 16 E7 oncoprotein is to enhance the activating phosphorylation of SFKs expressed in keratinocytes.
[Show abstract][Hide abstract] ABSTRACT: The 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.
The 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.
These 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.
[Show abstract][Hide abstract] ABSTRACT: The occurrence of human herpesvirus (HHV) 6 subtypes A and B in apical periodontitis was determined. The relationship of HHV-6 subtypes to other disease associated herpesviruses, i.e., Epstein-Barr virus (EBV) and human cytomegalovirus, was also investigated.
Forty apical periodontitis samples (17 symptomatic and 23 asymptomatic) and 40 healthy pulp control samples were collected. Nested polymerase chain reaction was used to detect HHV-6 DNA.
HHV-6 DNA was observed in significantly higher frequencies in apical periodontitis samples than in control samples (20% vs. 2.5%; P = .03). Further classification of apical lesions revealed that subtype B of HHV-6 was significantly associated with large-sized and symptomatic lesions (P < .01). Thirty-one apical lesions (77%) harbored ≥1 of the tested herpesviruses: EBV was the most frequent herpesvirus (72.5%) in apical periodontitis, followed by HHV-6 (20%).
Our findings suggest that EBV and HHV-6B infections can be associated with symptomatic apical periodontitis.