Global effects of human papillomavirus type 18 E6/E7 in an organotypic keratinocyte culture system.
ABSTRACT: The effects of human papillomavirus type 18 (HPV-18) E6 and E7 proteins on global patterns of host gene expression in primary human keratinocytes grown in organotypic raft culture system were assessed. Primary human keratinocytes were infected with retroviruses that express the wild-type HPV-18 E6 and E7 genes from the native differentiation-dependent HPV enhancer-promoter. Total RNA was isolated from raft cultures and used to generate probes for querying Affymetrix U95A microarrays, which contain >12,500 human gene sequences. Quadruplicate arrays of each E6/E7-transduced and empty vector-transduced samples were analyzed by 16 pairwise comparisons. Transcripts altered in > or =12 comparisons were selected for further analysis. With this approach, HPV-18 E6/E7 expression significantly altered the expression of 1,381 genes. A large increase in transcripts associated with DNA and RNA metabolism was observed, with major increases noted for transcription factors, splicing factors, and DNA replication elements, among others. Multiple genes associated with protein translation were downregulated. In addition, major alterations were found in transcripts associated with the cell cycle and cell differentiation. Our study provides a systematic description of transcript changes brought about by HPV-18 E6/E7 in a physiologically relevant model and should furnish a solid source of information to guide future studies.
Project description:HPV-16 E6 and E7 genes are required to efficiently immortalize a broad spectrum of cell types including cervical keratinocytes. Therefore, the E6/E7 genes can be considered relevant targets for anti-cancer therapy. We produced several engineered hairpin (HP) ribozymes to specifically disrupt HPV-16 E6/E7 mRNA. After extensive biochemical characterization, one anti-E6 HP ribozyme (R434) was selected for in vivo testing because of its superior catalytic capabilities. When expressed in cis, R434 efficiently inhibited E6 in vitro translation. Cis-expression of the HP ribozyme with HPV-16 E6/E7 genes in normal human keratinocytes reduced the growth rate and prevented immortalization. RNA analysis by reverse transcription-PCR showed that E6/E7 transcripts were cleaved in post-transfected cells and virtually were eliminated after long term expression. Of interest, an inactive version of the HP also was able to significantly affect the immortalizing ability of E6/E7, probably through passive hybridization. The combination of passive and cleaving antisense RNA therefore is established as an effective inhibitor of HPV-16 E6/E7 immortalization.
Project description:The life cycle of human papillomaviruses (HPV) 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 down-regulate the expression of several genes involved in keratinocyte differentiation. Quantitative real-time polymerase chain reaction (qRT-PCR) assays were performed to confirm 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 down-regulate the expression of several genes involved in keratinocyte differentiation, at least partially by down-regulating 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. Primary human foreskin keratinocytes were transduced by retrovirus vectors containing HPV 16 E6, E7, E6/E7 or the control vector LXSN. The global gene expression patterns of transduced keratinocytes were analyzed on Affymetrix microarrays
Project description:Human Papillomavirus (HPV) infection is the main risk factor for the development and progression of cervical cancer. HPV-16 E6 and E7 expression is essential for induction and maintenance of the transformed phenotype. These oncoproteins interfere with the function of several intracellular proteins, including those controlling the PI3K/AKT/mTOR pathway in which Phospolipase D (PLD) and Phosphatidic acid (PA) play a critical role.PLD activity was measured in primary human keratinocytes transduced with retroviruses expressing HPV-16 E6, E7 or E7 mutants. The cytostatic effect of rapamycin, a well-known mTOR inhibitor with potential clinical applications, was evaluated in monolayer and organotypic cultures.HPV-16 E7 expression in primary human keratinocytes leads to an increase in PLD expression and activity. Moreover, this activation is dependent on the ability of HPV-16 E7 to induce retinoblastoma protein (pRb) degradation. We also show that cells expressing HPV-16 E7 or silenced for pRb acquire resistance to the antiproliferative effect of rapamycin.This is the first indication that HPV oncoproteins can affect PLD activity. Since PA can interfere with the ability of rapamycin to bind mTOR, the use of combined strategies to target mTOR and PLD activity might be considered to treat HPV-related malignancies.
Project description:Infection by human papillomavirus (HPV) is a major risk factor for human cervical carcinoma. However, the HPV infection alone is not sufficient for cancer formation. Cervical carcinogenesis is considered a multistep process accompanied by genetic alterations of the cell. Ras is activated in approximately 20% of human cancers, and it is related to the metastatic conversion of tumor cells. We investigated how Ras activation was involved in the malignant conversion of HPV-infected lesions. The active form of H-ras was introduced into human primary keratinocytes expressing the HPV type 18 (HPV18) oncoproteins E6 and/or E7. We analyzed the keratinocytes' growth potentials and found that the activation of the Ras pathway induced senescence-like growth arrest. Senescence could be eliminated by high-risk E7 expression, suggesting that the pRb pathway was important for Ras-induced senescence. Then we analyzed the effect of Ras activation on epidermis development by using an organotypic "raft" culture and found that the E7 and H-ras coexpressions conferred invasive potential on the epidermis. This invasiveness resulted from the upregulation of MT1-MMP and MMP9 by H-ras and E7, respectively, in which the activation of the MEK/extracellular signal-regulated kinase pathway was involved. These results indicated that the activation of Ras or the related signal pathways promoted the malignant conversion of HPV-infected cells.
Project description:Keratinocyte infection with high-risk human papillomavirus genotypes has been linked to cancer development. In cervix, the alpha HPV16 and HPV18 have been reported as the mayor causative agents of cervical cancer. Oncogenic progression and chronic inflammation are closely related processes, with IL-6 as one of the main pro-inflammatory cytokines involved. However, there are limited studies about the regulation of IL-6 by low and high risk HPVs and the HPV proteins implicated in this modulation. In this work, we report the overexpression of IL-6 in HPV infected cervical cancer derived cell lines (HeLa and SiHa) compared to non-tumorigenic keratinocytes (HaCaT), and in Cervical Intraepithelial Neoplasia grade 1 HPV16 and HPV18 positive cervical samples compared to HPV negative samples without lesions. Moreover, we generated HaCaT keratinocytes that express E5, E6, and E7 from high risk (16 or 18) or low risk (62 and 84) HPVs. E5 proteins do not modify IL-6 expression, while E7 modestly increase it. Interestingly, E6 proteins in HaCaT cells upregulate IL-6 mRNA expression and protein secretion. Indeed, in HaCaT cells that express high risk HPV16E6 or HPV18E6 proteins, only the truncated E6* isoforms were expressed, showing the stronger IL-6 overexpression, while in HaCaT cells that express low risk HPV62 and HPV84 full length E6 proteins, IL-6 was also upregulated but not so drastically. Since HaCaT cells have a mutated p53 form that is not degraded by the introduction of E6 or E6/E7, it seems that E6/E7 regulate IL-6 by an additional mechanism independent of p53. In addition, basal keratinocytes showed a strong expression of IL-6R using immunohistochemistry, suggesting an autocrine mechanism over proliferative cells. Altogether, IL-6 cytokine expression in keratinocytes is upregulated by E6 and E7 proteins from HPVs 16, 18, 62, and 84, especially by high risk HPV16 and HPV18 E6*, which may contribute to promote a pro-inflammatory and highly proliferative microenvironment that can persist over time and lead to cervical tumorigenesis.
Project description:Recurrent respiratory papillomatosis (RRP) is a chronic, debilitating disease of the upper airway caused by human papillomavirus type 6 (HPV-6) or HPV-11. We describe responses of peripheral blood mononuclear cells (PBMC) and T cells from RRP patients and controls to the HPV-11 early proteins E6 and E7. PBMC were exposed in vitro to purified E6 or E7 proteins or transduced with fusion proteins containing the first 11 amino acids of the human immunodeficiency virus type 1 protein tat fused to E6 or E7 (tat-E6/tat-E7). T(H)1-like (interleukin-2 [IL-2], gamma interferon [IFN-gamma], IL-12, and IL-18), and T(H)2-like (IL-4 and IL-10) cytokine mRNAs were identified by reverse transcription-PCR, and IFN-gamma and IL-10 cytokine-producing cells were identified by enzyme-linked immunospot assay. These studies show that HPV-11 E6 skews IL-10-IFN-gamma expression by patients with RRP toward greater expression of IL-10 than of IFN-gamma. In addition, there is a general cytokine hyporesponsiveness to E6 that is more prominent for T(H)1-like cytokine expression by patients with severe disease. Patients showed persistent IL-10 cytokine expression by the nonadherent fraction of PBMC when challenged with E6 and tat-E6, and, in contrast to controls, both T cells and non-T cells from patients expressed IL-10. However, E7/tat-E7 cytokine responses in patients with RRP were similar to those of the controls. In contrast, E6 inhibited IL-2 and IL-18 mRNA expression that would further contribute to a cytokine microenvironment unfavorable to HPV-specific, T-cell responses that should control persistent HPV infection. In summary, E6 is the dominant inducer of cytokine expression in RRP, and it induces a skewed expression of IL-10 compared to the expression of IFN-gamma.
Project description:E7 protein from cutaneous as well as mucosal HPV types can alter NF-?B activity. Conflicting literature data show a HPV-induced up- or down-regulation of the NF-?B pathway in different cell lines. In a previous study we detected the expression of E7 gene of HPV15 in a subungual tumor of a patient affected by incontinentia pigmenti (IP). IP is a rare X-linked genodermatosis in which the IKK? gene is altered. From observations in transgenic IKK? defective mice, it was suggested that IKK-deficient cells may undergo rapid hyper-proliferation and apoptosis/necrosis, leading to increased pro-inflammatory cytokine production in the neighboring IKK-positive cells. The objective of this study was to ascertain if beta HPV 15 can alter apoptosis and NF-?B pathway in normal and IKK?-deficient keratinocytes. The human immortalized keratinocyte cell line (HaCaT), and human primary keratinocyte (HPK) cells were transduced with a retrovirus expressing E6-E7 proteins of HPV 15 and IKK? was successful silenced mimicking the HPV15 infection and IP. HPV15 E6-E7 gene expression improved NF-?B activity in human keratinocytes even when IKK? was silenced by siRNA. In IKK? silenced keratinocyte cells, TNF-?-induced apoptosis was strongly reduced by the expression of HPV15 E6-E7 genes. Beta HPV15 exerted this anti-apoptotic activity by decreasing pro-apoptotic BAK and cleaved Caspase 3 proteins. In conclusion, we can speculate that presence of persistent infection by beta papillomavirus might influence the biological fate of IP by altering NF-?B activation and apoptosis in IKK? mutated cells, favoring their survival and possibly the development of tumors in the late stage of disease. Taken together, our data reinforce the importance of host genetic background in the pathogenesis of HPV-associated skin lesions.
Project description:We used freshly established immortalized human keratinocytes with a well-defined HPV16 E6 E7 expression cassette to get a more complete and less biased overview about global changes induced by HPV16 using RNA-seq. We identified novel factors regulated by HPV oncogenes that could serve an essential role in cancer development. Overall design: mRNA profiles of human Keratinocytes transduced with HPV16-E6/E7 constructs and empty vectors in triplicates, sequenced with Illumina Hiseq 2000.
Project description:Telomerase activation is critical for the immortalization of primary human keratinocytes by the high-risk HPV E6 and E7 oncoproteins, and this activation is mediated in part by E6-induction of the hTERT promoter. E6 induces the hTERT promoter via interactions with the cellular ubiquitin ligase, E6AP, and with the c-Myc and NFX-1 proteins, which are resident on the promoter. In the current study we demonstrate that E6 protein interacts directly with the hTERT protein. Correlating with its ability to bind hTERT, E6 also associates with telomeric DNA and with endogenous active telomerase complexes. Most importantly, E6 increases the telomerase activity of human foreskin fibroblasts transduced with the hTERT gene, and this activity is independent of hTERT mRNA expression. Unlike its ability to degrade p53, E6 does not degrade hTERT protein in vitro or in vivo. Our studies of E6/hTERT interactions also reveal that the C-terminal tagged hTERT protein, although incapable of immortalizing fibroblasts, does immortalize keratinocytes in collaboration with the viral E7 protein. Thus, E6 protein mediates telomerase activation by a posttranscriptional mechanism and these findings provide a model for exploring the direct modulation of cell telomerase/telomere function by an oncogenic virus and suggest its potential role in both neoplasia and virus replication.
Project description:Human papillomaviruses (HPVs) amplify in differentiated strata of a squamous epithelium. The HPV E7 protein destabilizes the p130/retinoblastoma susceptibility protein family of tumor suppressors and reactivates S-phase reentry, thereby facilitating viral DNA amplification. The high-risk HPV E6 protein destabilizes the p53 tumor suppressor and many other host proteins. However, the critical E6 targets relevant to viral DNA amplification have not been identified, because functionally significant E6 mutants are not stably maintained in transfected cells. Using Cre-loxP recombination, which efficiently generates HPV genomic plasmids in transfected primary human keratinocytes, we have recapitulated a highly productive infection of HPV-18 in organotypic epithelial cultures. By using this system, we now report the characterization of four HPV-18 E6 mutations. An E6 null mutant accumulated high levels of p53 and amplified very poorly. p53 siRNA or ectopic WT E6 partially restored amplification, whereas three missense E6 mutations that did not effectively destabilize p53 complemented the null mutant poorly. Unexpectedly, in cis, two of the missense mutants amplified, albeit to a lower extent than the WT and only in cells with undetectable p53. These observations and others implicate p53 and additional host proteins in regulating viral DNA amplification and also suggest an inhibitory effect of E6 overexpression. We show that high levels of viral DNA amplification are critical for late protein expression and report several previously undescribed viral RNAs, including bicistronic transcripts predicted to encode E5 and L2 or an alternative form of E1^E4 and L1.