Inhibition of cervical carcinoma cell line proliferation by the introduction of a bovine papillomavirus regulatory gene.

Human papillomavirus (HPV) E6 and E7 oncogenes are expressed in the great majority of human cervical carcinomas, whereas the viral E2 regulatory gene is usually disrupted in these cancers. To investigate the roles of the papillomavirus E2 genes in the development and maintenance of cervical carcinoma, the bovine papillomavirus (BPV) E2 gene was acutely introduced into cervical carcinoma cell lines by infection with high-titer stocks of simian virus 40-based recombinant viruses. Expression of the BPV E2 protein in HeLa, C-4I, and MS751 cells results in specific inhibition of the expression of the resident HPV type 18 (HPV18) E6 and E7 genes and in inhibition of cell growth. HeLa cells, in which HPV gene expression is nearly completely abolished, undergo a dramatic and rapid inhibition of proliferation, which appears to be largely a consequence of a block in progression from the G1 to the S phase of the cell cycle. Loss of HPV18 gene expression in HeLa cells is also accompanied by a marked increase in the level of the cellular p53 tumor suppressor protein, apparently as a consequence of abrogation of HPV18 E6-mediated destabilization of p53. The proliferation of HT-3 cells, a human cervical carcinoma cell line devoid of detectable HPV DNA, is also inhibited by E2 expression, whereas two other epithelial cell lines that do not contain HPV DNA are not inhibited. Thus, a number of cervical carcinoma cell lines are remarkably sensitive to growth inhibition by the E2 protein. Although BPV E2-mediated inhibition of HPV18 E6 and E7 expression may contribute to growth inhibition in some of the cervical carcinoma cell lines, the BPV E2 protein also appears to exert a growth-inhibitory effect that is independent of its effects on HPV gene expression.     

Zinc finger arrays binding human papillomavirus types 16 and 18 genomic DNA: precursors of gene-therapeutics for in-situ reversal of associated cervical neoplasia

ABSTRACT: BACKGROUND: Human papillomavirus (HPV) types 16 and 18 are the high-risk, sexually transmitted infectious causes of most cervical intraepithelial neoplasias (CIN) or cancers. While efficacious vaccines to reduce the sexual acquisition of some high-risk HPVs have recently been introduced, no virus-targeted therapies exist for those already exposed and infected. Considering the oncogenic role of the transforming (E6 and E7) genes of high-risk HPVs in the slow pathogenesis of cervical cancer, we hypothesize that timely disruption or abolition of HPV genome expression within pre-cancerous lesions identified at screening may reverse neoplasia. We aimed to derive model zinc finger nucleases (ZFNs) for mutagenesis of the genomes of two high-risk HPV (types 16 & 18). Methods and results: Using ZiFiT software and the complete genomes of HPV types16 and 18, we computationally generated the consensus amino acid sequences of the DNA-binding domains (F1, F2, & F3) of (i) 296 & 327 contextually unpaired (or single) three zinc-finger arrays (sZFAs) and (ii) 9 & 13 contextually paired (left and right) three- zinc-finger arrays (pZFAs) that bind genomic DNA of HPV-types 16 and 18 respectively, inclusive of the E7 gene (s/pZFAHpV/E7). In the absence of contextually paired three-zinc-finger arrays (pZFAs) that bind DNA corresponding to the genomic context of the E6 gene of either HPV type, we derived the DNA binding domains of another set of 9 & 14 contextually unpaired E6 gene-binding ZFAs (sZFAE6) to aid the future quest for paired ZFAs to target E6 gene sequences in both HPV types studied (pZFAE6). This paper presents models for (i) synthesis of hybrid ZFNs that cleave within the genomic DNA of either HPV type, by linking the gene sequences of the DNA-cleavage domain of the FokI endonuclease FN to the gene sequences of a member of the paired-HPV-binding ZFAs (pZFAHpV/E7 +FN), and (ii) delivery of the same into precancerous lesions using HPV-derived viral plasmids or vectors. CONCLUSIONS: With further optimization, these model ZFNs offer the opportunity to induce target-mutagenesis and gene-therapeutic reversal of cervical neoplasia associated with HPV types 16 & 18.     

Antigen processing defects in cervical carcinomas limit the presentation of a CTL epitope from human papillomavirus 16 E6.

Human papillomavirus (HPV) infection, particularly type 16, is causally associated with the development of cervical cancer. The E6 and E7 proteins of HPV are constitutively expressed in cervical carcinoma cells making them attractive targets for CTL-based immunotherapy. However, few studies have addressed whether cervical carcinomas can process and present HPV E6/E7-derived Ags for recognition by CTL. We generated HLA-A*0201-restricted CTL clones against HPV16 E6(29-38) that recognized HPV16 E6 Ags transfected into B lymphoblastoid cells. These CTL were unable to recognize HLA-A*0201(+) HPV16 E6(+) cervical carcinoma cell lines even when the level of endogenous HPV16 E6 in these cells was increased by transfection. This defect in presentation of HPV16 E6(29-38) correlated with low level expression of HLA class I, proteasome subunits low molecular mass protein 2 and 7, and the transporter proteins TAP1 and TAP2 in the cervical carcinoma cell lines. The expression of all of these proteins could be up-regulated by IFN-gamma, but this was insufficient for CTL recognition unless the level of HPV16 E6 Ag was also increased by transfection. CTL recognition of the HPV16 E6(29-38) epitope in 721.174 B cells was dependent on TAP expression but independent of immunoproteasome expression. Collectively, these findings suggest that presentation of the HPV16 E6(29-38) epitope in cervical carcinoma cell lines is limited both by the level of TAP expression and by the low level or availability of the source HPV E6 oncoprotein. These observations place constraints on the use of this, and potentially other, HPV-derived CTL epitopes for the immunotherapy of cervical cancer.     

Triggering of death receptor apoptotic signaling by human papillomavirus 16 E2 protein in cervical cancer cell lines is mediated by interaction with c-FLIP

Human papillomavirus (HPV) E2 gene disruption is one of the key features of HPV-induced cervical malignant transformation. Though it is thought to prevent progression of carcinogenesis, the pro-apoptotic function of E2 protein remains poorly understood. This study shows that expression of HPV16 E2 induces apoptosis both in HPV-positive and -negative cervical cancer cell lines and leads to hyperactivation of caspase-8 and caspase-3. Activation of these signaling factors is responsible for the observed sensitivity to apoptosis upon treatment with anti-Fas antibody or TNF-α. In addition, immunoprecipitation experiments clearly show an interaction between HPV16 E2 and c-FLIP, a key regulator of apoptotic cell death mediated by death receptor signaling. Moreover, c-FLIP and a caspase-8 inhibitor protect cells from HPV16 E2-mediated apoptosis. Overexpression of c-FLIP rescues cervical cancer cells from apoptosis induced by HPV16 E2 protein expression. The data suggest that HPV16 E2 abrogates the apoptosis-inhibitory function of c-FLIP and renders the cell hypersensitive to the Fas/FasL apoptotic signal even below threshold concentration. This suggests a novel mechanism for deregulation of cervical epithelial cell growth upon HPV-induced transformation, which is of great significance in developing therapeutic strategies for intervention of cervical carcinogenesis.     

Tumourigenesis Driven by the Human Papillomavirus Type 16 Asian-American E6 Variant in a Three-Dimensional Keratinocyte Model

Infection with a transforming human papillomavirus (HPV) such as type 16 (of species Alphapapillomavirus 9) causes ano-genital and oral tumours via viral persistence in human squamous cell epithelia. Epidemiological studies showed that the naturally occurring HPV16 Asian-American (AA) variant (sublineage D2/D3) is found more often than the European Prototype (EP) (sublineage A1) in high-grade cervical neoplasia and tumours compared to non-cancer controls. Just three amino acid changes within the early gene, E6, of HPV16 AA have been linked to this augmented tumourigenicity. The AAE6 variant''s greater immortalizing and transforming potential over EPE6 has recently been confirmed in retrovirally-transduced keratinocytes expressing the E6 gene only. However, the tumourigenic role of the full-length viral genome of HPV16 has not yet been addressed with regard to these E6 variants. To investigate this process in the context of these two HPV16 E6 genotypes, an organotypic tissue culture model was used to simulate the HPV infectious life cycle. The AAE6 variant demonstrated an enhanced ability over EPE6 to drive the viral life cycle toward tumourigenesis, as evidenced phenotypically—by a more severe grade of epithelial dysplasia with higher proliferation and deregulated differentiation, and molecularly—by high viral oncogene E6 and E7 expression, but lack of productive viral life cycle markers. In contrast, EPE6 had low E6 and E7 but high E1∧E4 expression, indicative of a productive life cycle. We suggest increased viral integration into the host genome for AAE6 as one possible mechanism for these observed differences from EPE6. Additionally, we found downstream effects on immortalization and host innate immune evasion. This study highlights how minor genomic variations in transforming viruses can have a significant affect on their tumourigenic ability.     

RbAp48 is a critical mediator controlling the transforming activity of human papillomavirus type 16 in cervical cancer

Although human papillomavirus (HPV) infections are the primary cause of cervical cancer, the molecular mechanism by which HPV induces cervical cancer remains largely unclear. We used two-dimensional electrophoresis with mass spectrometry to study protein expression profiling between HPV16-positive cervical mucosa epithelial H8 cells and cervical cancer Caski cells to identify 18 differentially expressed proteins. Among them, retinoblastoma-binding protein 4 (RbAp48) was selected, and its differentiation expression was verified with both additional cervical cancer-derived cell lines and human tissues of cervical intraepithelial neoplasia and cervical cancer. Suppression of RbAp48 using small interfering RNA approach in H8 cells significantly stimulated cell proliferation and colony formation and inhibited senescence-like phenotype. Remarkably, H8 cells acquired transforming activity if RpAp48 was suppressed, because H8 cells stably transfected with RbAp48 small interfering RNA led to tumor formation in nude mice. In addition, overexpression of RbAp48 significantly inhibited cell growth and tumor formation. This RbAp48-mediated transformation of HPV16 is probably because of the regulation by RbAp48 of tumor suppressors retinoblastoma and p53, apoptosis-related enzymes caspase-3 and caspase-8, and oncogenic genes, including E6, E7, cyclin D1 (CCND1), and c-MYC. In brief, RbAp48, previously unknown in cervical carcinogenesis, was isolated in a global screen and identified as a critical mediator controlling the transforming activity of HPV16 in cervical cancer.     

TGF-beta signalling pathway factors in HPV-induced cervical lesions

Human papillomaviruses (HPV) are considered the etiological agents of cervical cancer, especially high-risk genotypes. TGF-beta (transforming growth factor-beta) is well known for its anti-proliferative effects but the neoplastic cells often lose their sensitivity to TGF-beta. A characteristic alteration associated with malignant progression is the loss of responsiveness to TGF-beta1-induced cell growth inhibition. The aim of the present study was to establish the possible role of some members of TGF-beta signalling pathway during cervical cancer development and the possible relationship with HPV infection. In order to establish TGF-beta gene expression levels in cervical oncogenesis, TGF-beta1, TGF-beta1 receptors and Smad2 were investigated in precancerous and cervical cancer samples (Quantitative Real-Time PCR). The study revealed that 84.5% of patients were positive for HPV DNA. The most prevalent HPV genotypes were high-risk HPV 16 and 18 in single or co-infections. Expression of TGF-beta1 decreased as tumor cells progressed from cervical intraepithelial neoplasia to cervical carcinoma. Furthermore, we observed that cervical lesions without HPV infection expressed significantly less TGF-beta1. TGF-betaRI and Smad2 gene expression levels were found to be decreased in SCC and AC samples in contrast with CIN1 and CIN2/3 samples. Our results showed that in human cervical cancer the disruption of TGF-beta/Smad signalling pathway might contribute to the malignant progression of cervical dysplasia. These data emphasize the importance of canonical TGF-beta pathway integrity in carcinogenesis.     

Organization of human papillomavirus productive cycle during neoplastic progression provides a basis for selection of diagnostic markers

The productive cycle of human papillomaviruses (HPVs) can be divided into discrete phases. Cell proliferation and episomal maintenance in the lower epithelial layers are followed by genome amplification and the expression of capsid proteins. These events, which occur in all productive infections, can be distinguished by using antibodies to viral gene products or to surrogate markers of their expression. Here we have compared precancerous lesions caused by HPV type 16 (HPV16) with lesions caused by HPV types that are not generally associated with human cancer. These include HPV2 and HPV11, which are related to HPV16 (supergroup A), as well as HPV1 and HPV65, which are evolutionarily divergent (supergroups E and B). HPV16-induced low-grade squamous intraepithelial lesions (CIN1) are productive infections which resemble those caused by other HPV types. During progression to cancer, however, the activation of late events is delayed, and the thickness of the proliferative compartment is progressively increased. In many HPV16-induced high-grade squamous intraepithelial lesions (CIN3), late events are restricted to small areas close to the epithelial surface. Such heterogeneity in the organization of the productive cycle was seen only in lesions caused by HPV16 and was not apparent when lesions caused by other HPV types were compared. By contrast, the order in which events in the productive cycle were initiated was invariant and did not depend on the infecting HPV type or the severity of disease. The distribution of viral gene products in the infected cervix depends on the extent to which the virus can complete its productive cycle, which in turn reflects the severity of cervical neoplasia. It appears from our work that the presence of such proteins in cells at the epithelial surface allows the severity of the underlying disease to be predicted and that markers of viral gene expression may improve cervical screening.     

Apoptosis induced by an antagonist peptide against HPV16 E7 in vitro and in vivo via restoration of p53

Human papilloma virus type 16 (HPV16) E7 is a viral oncoprotein that is believed to play a major role in cervical neoplasia. A novel antagonist peptide against HPV16 E7 was previously selected by phage display screening and the selected peptide was found to have anti-tumor efficacy against HPV16-positive cervical carcinoma through induction of cell cycle arrest. In the current study, to further elucidate the mechanisms of the antagonist peptide, the effects of the peptide on apoptosis are investigated by RT-PCR, Western blotting, MTT assay, TUNEL staining, Annexin V apoptosis assay, flow cytometry, and animal experiments. The antagonist peptide showed obvious anti-tumor efficacy through apoptosis induction, both in HPV16-positive cervical cancer cell lines and tumor xenografts. Our results also revealed that the peptide induced accumulation of cellular p53 and p21, and led to HPV16 E7 protein degradation. In the case of mRNA levels, it resulted in unaltered p53 and HPV16 E7 expression, but increased expression of p21. In contrast, the induction of apoptosis and p53 reactivation effects by the selected peptide were abolished after E7 knocked down with siRNA. These results demonstrate that the selected peptide can induce E7 degradation and lead to marked apoptosis in HPV16-related cancer cells by activating cellular p53 and its target genes, such as p21. Furthermore, the evident therapeutic efficacy obtained from the subcutaneous tumor model experiments in nude mice suggests a therapeutic potential for HPV16-related cancers of the selected peptide. Therefore, this specific peptide may be used to create specific biotherapies for the treatment of HPV 16-positive cervical cancers.     

Up-regulation of Foxp3 participates in progression of cervical cancer

Foxp3 was identified as a key protein in mediating inhibitory functions of regulatory T cell (Treg). Foxp3 was thought to express only in the T cell lineage until recently when some researches reported that Foxp3 was also expressed by cancer cells. In this study, we describe for the first time the expression of Foxp3 in cervical cancer. Progression from cervical intraepithelial neoplasia (CIN) to cervical cancer is a multistep process initiated by persistent infection with high-risk human papillomavirus (HPV). P16^INK4a is a crucial marker of HPV integration into host cells. In the present study, expressions of Foxp3 and P16^INK4a in CIN and cervical cancer were detected by immunohistochemistry. Our results found expression level of Foxp3 was increased during the progression of cervical neoplasia. Moreover, up-regulation of Foxp3 appeared to be correlated with the expression of P16^INK4a. Examination of the role of Foxp3 in differentiation by double immunostaining for cytokeratin 10 (CK10) showed significant association between Foxp3 expression and differentiation (Foxp3 vs CK10). Furthermore, positive expression of Foxp3 was correlated with tumor size. These data suggest that Foxp3 may play an important role in differentiation and growth of cervical cancer cells. Our findings provide new insights regarding the role of Foxp3 in differentiation and its association with HPV infection during the development of cervical cancer.     

Suppression in vivo of human papillomavirus type 18 E6-E7 gene expression in nontumorigenic HeLa X fibroblast hybrid cells.

The E6 and E7 genes of the cancer-associated human papillomavirus (HPV) types 16 (HPV16) and 18 (HPV18) can induce cell immortalization in vitro in normal human keratinocytes. This, however, is not associated with tumorigenicity in vivo. On the other hand, tumorigenicity of HPV18-positive HeLa cervical carcinoma cells can be suppressed by fusion of HeLa cells with normal human keratinocytes or fibroblasts. We have addressed the question of whether suppression of tumorigenicity in HeLa x fibroblast hybrid cells might be due to a reduced ability of these cells to express the HPV18 E6-E7 genes in vivo. Nontumorigenic hybrid cells and tumorigenic hybrid segregants were transplanted as organotypical cultures or injected subcutaneously into immunocompromised mice and were analyzed for HPV18 E6-E7 gene expression by RNA-RNA in situ hybridization. The tumorigenic hybrid cells showed a continuous and invasive growth that was associated with high levels of HPV18 E6-E7 mRNAs at all time points examined. In contrast, the nontumorigenic hybrid cells stopped cell proliferation approximately 3 days after transplantation. At this time they expressed the E6-E7 genes at low levels, whereas at day 2 high expression levels were observed. However, the mRNA levels of the cytoskeletal genes beta-actin and vimentin remained high for at least 14 days, demonstrating that inhibition of growth and of HPV18 E6-E7 gene expression was not due to cell death. These results suggest that growth inhibition of the nontumorigenic HeLa x fibroblast hybrid cells in vivo might be caused by suppression of HPV18 E6-E7 gene expression and are compatible with the idea of an intracellular surveillance mechanism for HPV gene expression existing in nontumorigenic cells.     

Interferon-β Induced microRNA-129-5p Down-Regulates HPV-18 E6 and E7 Viral Gene Expression by Targeting SP1 in Cervical Cancer Cells

Infection by human papillomavirus (HPV) can cause cervical intraepithelial neoplasia (CIN) and cancer. Down-regulation of E6 and E7 expression may be responsible for the positive clinical outcomes observed with IFN treatment, but the molecular basis has not been well determined. As miRNAs play an important role in HPV induced cervical carcinogenesis, we hypothesize that IFN-β can regulate the expressions of specific miRNAs in cervical cancer cells, and that these miRNAs can mediate E6 and E7 expression, thus modulate their oncogenic potential. In this study, we found that miR-129-5p to be a candidate IFN-β inducible miRNA. MiR-129-5p levels gradually decrease with the development of cervical intraepithelial lesions. Manipulation of miR-129-5p expression in Hela cells modulates HPV-18 E6 and E7 viral gene expression. Exogenous miR-129-5p inhibits cell proliferation in Hela cells, promotes apoptosis and blocks cell cycle progression in Hela cells. SP1 is a direct target of miR-129-5p in Hela cells. This study is the first report of a cellular miRNA with anti-HPV activity and provides new insights into regulatory mechanisms between the HPV and the IFN system in host cells at the miRNA level.     

Critical evaluation of HPV16 gene copy number quantification by SYBR green PCR

Background  

Human papilloma virus (HPV) load and physical status are considered useful parameters for clinical evaluation of cervical squamous cell neoplasia. However, the errors implicit in HPV gene quantification by PCR are not well documented. We have undertaken the first rigorous evaluation of the errors that can be expected when using SYBR green qPCR for quantification of HPV type 16 gene copy numbers. We assessed a modified method, in which external calibration curves were generated from a single construct containing HPV16 E2, HPV16 E6 and the host gene hydroxymethylbilane synthase in a 1:1:1 ratio.     

Human papillomavirus type 16 E7 peptide-directed CD8+ T cells from patients with cervical cancer are cross-reactive with the coronavirus NS2 protein

Human papillomavirus type 16 (HPV16) E6 and E7 oncoproteins are required for cellular transformation and represent candidate targets for HPV-specific and major histocompatibility complex class I-restricted CD8(+)-T-cell responses in patients with cervical cancer. Recent evidence suggests that cross-reactivity represents the inherent nature of the T-cell repertoire. We identified HLA-A2 binding HPV16 E7 variant peptides from human, bacterial, or viral origin which are able to drive CD8(+)-T-cell responses directed against wild-type HPV16 E7 amino acid 11 to 19/20 (E7(11-19/20)) epitope YMLDLQPET(T) in vitro. CD8(+) T cells reacting to the HLA-A2-presented peptide from HPV16 E7(11-19(20)) recognized also the HLA-A2 binding peptide TMLDIQPED (amino acids 52 to 60) from the human coronavirus OC43 NS2 gene product. Establishment of coronavirus NS2-specific, HLA-A2-restricted CD8(+)-T-cell clones and ex vivo analysis of HPV16 E7 specific T cells obtained by HLA-A2 tetramer-guided sorting from PBL or tumor-infiltrating lymphocytes obtained from patients with cervical cancer showed that cross-reactivity with HPV16 E7(11-19(20)) and coronavirus NS2(52-60) represents a common feature of this antiviral immune response defined by cytokine production. Zero of 10 patients with carcinoma in situ neoplasia and 3 of 18 patients with cervical cancer showed > or =0.1% HPV16 E7-reactive T cells in CD8(+) peripheral blood lymphocytes. In vivo priming with HPV16 was confirmed in patients with cervical cancer or preinvasive HPV16-positive lesions using HLA-A2 tetramer complexes loaded with the E6-derived epitope KLPQLCTEL. In contrast, we could not detect E6-reactive T cells in healthy individuals. These data imply that the measurement of the HPV16 E7(11-19(20)) CD8(+)-T-cell response may reflect cross-reactivity with a common pathogen and that variant peptides may be employed to drive an effective cellular immune response against HPV.