Development of a DNA vaccine targeting human papillomavirus type 16 oncoprotein E6

Human papillomavirus (HPV), particularly type 16 (HPV-16), is present in more than 99% of cervical cancers. The HPV oncoproteins E6 and E7 are constantly expressed and therefore represent ideal targets for HPV vaccine development. We previously developed DNA vaccines encoding calreticulin (CRT) linked to HPV-16 E7 and generated potent E7-specific CD8(+) T-cell immune responses and antitumor effects against an E7-expressing tumor. Since vaccines targeting E6 also represent an important strategy for controlling HPV-associated lesions, we developed a DNA vaccine encoding CRT linked to E6 (CRT/E6). Our results indicated that the CRT/E6 DNA vaccine, but not a wild-type E6 DNA vaccine, generated significant E6-specific CD8(+) T-cell immune responses in vaccinated mice. Mapping of the immunodominant epitope of E6 revealed that an E6 peptide comprising amino acids (aa) 48 to 57 (E6 aa48-57), presented by H-2K(b), is the optimal peptide and that the region of E6 comprising aa 50 to 57 represents the minimal core sequence required for activating E6-specific CD8(+) T lymphocytes. We also demonstrated that E6 aa48-57 contains cytotoxic T-lymphocyte epitopes naturally presented by E6-expressing TC-1 cells. Vaccination with a CRT/E6 but not a CRT/mtE6 (lacking aa 50 to 57 of E6) DNA vaccine could protect vaccinated mice from challenge with E6-expressing TC-1 tumors. Thus, our data indicate that E6 aa48-57 contains the immunodominant epitope and that a CRT/E6 DNA vaccine may be useful for control of HPV infection and HPV-associated lesions.     

Human Papillomavirus E7 Induces Rereplication in Response to DNA Damage

Human papillomavirus (HPV) infection is necessary but not sufficient for cervical carcinogenesis. Genomic instability caused by HPV allows cells to acquire additional mutations required for malignant transformation. Genomic instability in the form of polyploidy has been demonstrated to play an important role in cervical carcinogenesis. We have recently found that HPV-16 E7 oncogene induces polyploidy in response to DNA damage; however, the mechanism is not known. Here we present evidence demonstrating that HPV-16 E7-expressing cells have an intact G₂ checkpoint. Upon DNA damage, HPV-16 E7-expressing cells arrest at the G₂ checkpoint and then undergo rereplication, a process of successive rounds of host DNA replication without entering mitosis. Interestingly, the DNA replication initiation factor Cdt1, whose uncontrolled expression induces rereplication in human cancer cells, is upregulated in E7-expressing cells. Moreover, downregulation of Cdt1 impairs the ability of E7 to induce rereplication. These results demonstrate an important role for Cdt1 in HPV E7-induced rereplication and shed light on mechanisms by which HPV induces genomic instability.     

Low-dose radiation enhances therapeutic HPV DNA vaccination in tumor-bearing hosts

Current therapeutic approaches to treatment of patients with bulky cervical cancer are based on conventional in situ ablative modalities including cisplatin-based chemotherapy and radiation therapy. The 5-year survival of patients with nonresectable disease is dismal. Because over 99% of squamous cervical cancer is caused by persistent infection with an oncogenic strain of human papillomavirus (HPV), particularly type 16 and viral oncoproteins E6 and E7 are functionally required for disease initiation and persistence, HPV-targeted immune strategies present a compelling opportunity in which to demonstrate proof of principle. Sublethal doses of radiation and chemotherapeutic agents have been shown to have synergistic effect in combination with either vaccination against cancer-specific antigens, or with passive transfer of tumor-specific cytotoxic T lymphocytes (CTLs). Here, we explored the combination of low-dose radiation therapy with DNA vaccination with calreticulin (CRT) linked to the mutated form of HPV-16 E7 antigen (E7(detox)), CRT/E7(detox) in the treatment of E7-expressing TC-1 tumors. We observed that TC-1 tumor-bearing mice treated with radiotherapy combined with CRT/E7(detox) DNA vaccination generated significant therapeutic antitumor effects and the highest frequency of E7-specific CD8⁺ T cells in the tumors and spleens of treated mice. Furthermore, treatment with radiotherapy was shown to render the TC-1 tumor cells more susceptible to lysis by E7-specific CTLs. In addition, we observed that treatment with radiotherapy during the second DNA vaccination generated the highest frequency of E7-specific CD8⁺ T cells in the tumors and spleens of TC-1 tumor-bearing mice. Finally, TC-1 tumor-bearing mice treated with the chemotherapy in combination with radiation and CRT/E7(detox) DNA vaccination generate significantly enhanced therapeutic antitumor effects. The clinical implications of the study are discussed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Human papillomavirus type 16 E7 oncoprotein associates with the cullin 2 ubiquitin ligase complex, which contributes to degradation of the retinoblastoma tumor suppressor

Human papillomavirus type 16 (HPV16) and other high-risk HPVs are etiologically linked to the development of cervical carcinomas and contribute to a number of other tumors of the anogenital tract, as well as oral cancers. The high-risk HPV E6 and E7 oncoproteins are consistently expressed in cervical cancer cells and are necessary for the induction and maintenance of the transformed phenotype. An important aspect of HPV16 E7's oncogenic activities is destabilization of the retinoblastoma tumor suppressor (pRB) through a ubiquitin/proteasome-dependent mechanism, although the exact molecular mechanism is unknown. Here, we report that HPV16 E7 is associated with an enzymatically active cullin 2 ubiquitin ligase complex and that the HPV16 E7/pRB complex contains cullin 2. Depletion of cullin 2 by RNA interference causes increased steady-state levels and stability of pRB in HPV16 E7-expressing cells, and ectopic expression of HPV16 E7 and the cullin 2 complex leads to pRB ubiquitination in vivo. Hence, we propose that the HPV16 E7-associated cullin 2 ubiquitin ligase complex contributes to aberrant degradation of the pRB tumor suppressor in HPV16 E7-expressing cells.     

人乳头瘤病毒16型早期基因E6、E7的克隆及表达

高危人乳头瘤病毒16型的感染与宫颈癌的发病密切相关。HPV16E6和E7蛋白在大多数HPV16相关宫颈癌及其癌前病变中持续表达,因此E6和E7蛋白可作为制备HPV16相关肿瘤及其癌前病变治疗性疫苗的靶抗原〔2〕。采用套式PCR方法,从宫颈癌患者组织中扩增出E6、E7基因并成功构建了包含E6、E7的表达质粒PET-E6、PET-E7。SDS聚丙烯酰胺凝胶电泳和Western-blotting分析结果表明,外源基因E6,E7在T7启动子控制下可获得稳定表达。     

Skin hyperproliferation and susceptibility to chemical carcinogenesis in transgenic mice expressing E6 and E7 of human papillomavirus type 38

The oncoproteins E6 and E7 of human papillomavirus type 38 (HPV38) display several transforming activities in vitro, including immortalization of primary human keratinocytes. To evaluate the oncogenic activities of the viral proteins in an in vivo model, we generated transgenic mice expressing HPV38 E6 and E7 under the control of the bovine homologue of the human keratin 10 (K10) promoter. Two distinct lines of HPV38 E6/E7-expressing transgenic mice that express the viral genes at different levels were obtained. In both lines, HPV38 E6 and E7 induced cellular proliferation, hyperplasia, and dysplasia in the epidermis. The rate of occurrence of these events was proportional to the levels of HPV38 E6 and E7 expression in the two transgenic lines. Exposure of the epidermis of nontransgenic mice to UV led to p21WAF1 accumulation and cell cycle arrest. In contrast, keratinocytes from transgenic mice continued to proliferate and were not positive for p21WAF1, indicating that cell cycle checkpoints are altered in keratinocytes expressing the viral genes. Although the HPV38 E6/E7-expressing transgenic mice did not develop spontaneous tumors during their life span, two-stage carcinogen treatment led to a high incidence of papillomas, keratoacanthomas, and squamous-cell carcinomas in HPV38 mice compared with nontransgenic animals. Together, these data show that HPV38 E6 and E7 display transforming properties in vivo, providing further support for the role of HPV38 in carcinogenesis.     

Intratumoral injection of therapeutic HPV vaccinia vaccine following cisplatin enhances HPV-specific antitumor effects

Despite the conventional treatments of radiation therapy and chemotherapy, the 5-year survival rates for patients with advanced-stage cervical cancers remain low. Cancer immunotherapy has emerged as an alternative, innovative therapy that may improve survival. Here, we utilize a preclinical HPV-16 E6/E7-expressing tumor model, TC-1, and employ the chemotherapeutic agent cisplatin to generate an accumulation of CD11c+ dendritic cells in tumor loci making it an ideal location for the administration of therapeutic vaccines. Following cisplatin treatment, we tested different routes of administration of a therapeutic HPV vaccinia vaccine encoding HPV-16 E7 antigen (CRT/E7-VV). We found that TC-1 tumor-bearing C57BL/6 mice treated with cisplatin and intratumoral injection of CRT/E7-VV significantly increased E7-specific CD8+ T cells in the blood and generated potent local and systemic antitumor immune responses compared to mice receiving cisplatin and CRT/E7-VV intraperitoneally or mice treated with cisplatin alone. We further extended our study using a clinical grade recombinant vaccinia vaccine encoding HPV-16/18 E6/E7 antigens (TA-HPV). We found that intratumoral injection with TA-HPV following cisplatin treatment also led to increased E7-specific CD8+ T cells in the blood as well as significantly decreased tumor size compared to intratumoral injection with wild type vaccinia virus. Our study has strong implications for future clinical translation using intratumoral injection of TA-HPV in conjunction with the current treatment strategies for patients with advanced cervical cancer.     

Human Papillomavirus-16 E7 Interacts with Glutathione S-Transferase P1 and Enhances Its Role in Cell Survival

Background

Human Papillomavirus (HPV)-16 is a paradigm for “high-risk” HPVs, the causative agents of virtually all cervical carcinomas. HPV E6 and E7 viral genes are usually expressed in these tumors, suggesting key roles for their gene products, the E6 and E7 oncoproteins, in inducing malignant transformation.

Methodology/Principal Findings

By protein-protein interaction analysis, using mass spectrometry, we identified glutathione S-transferase P1-1 (GSTP1) as a novel cellular partner of the HPV-16 E7 oncoprotein. Following mapping of the region in the HPV-16 E7 sequence that is involved in the interaction, we generated a three-dimensional molecular model of the complex between HPV-16 E7 and GSTP1, and used this to engineer a mutant molecule of HPV-16 E7 with strongly reduced affinity for GSTP1.When expressed in HaCaT human keratinocytes, HPV-16 E7 modified the equilibrium between the oxidized and reduced forms of GSTP1, thereby inhibiting JNK phosphorylation and its ability to induce apoptosis. Using GSTP1-deficient MCF-7 cancer cells and siRNA interference targeting GSTP1 in HaCaT keratinocytes expressing either wild-type or mutant HPV-16 E7, we uncovered a pivotal role for GSTP1 in the pro-survival program elicited by its binding with HPV-16 E7.

Conclusions/Significance

This study provides further evidence of the transforming abilities of this oncoprotein, setting the groundwork for devising unique molecular tools that can both interfere with the interaction between HPV-16 E7 and GSTP1 and minimize the survival of HPV-16 E7-expressing cancer cells.     

Induction of apoptosis in cervical carcinoma cells by peptide aptamers that bind to the HPV-16 E7 oncoprotein.

Human papillomaviruses (HPV) of the high-risk type are causally involved in human tumors, in particular cervical carcinoma. Expression of the viral oncogenes E6 and E7 is maintained in HPV-positive tumors, and it was shown that E6 and E7 of HPV-16 can immortalize human keratinocytes, the natural host cells of the virus. Expression of the viral genes is also required for maintenance of the transformed phenotype. The oncogenic activity of the E6 and E7 oncoproteins is mediated by their physical and functional interaction with cellular regulatory proteins. To knock out the function of the E7 protein in living cells, we have developed peptide aptamers with high specific binding activity for the E7 protein of HPV-16. We show here that E7-binding peptide aptamers induce programmed cell death (apoptosis) in E7-expressing cells, whereas E7-negative cells are not affected. Furthermore, E7-binding peptide aptamers induce apoptosis in HPV-16-positive tumor cells derived from cervical carcinoma. The data suggest that E7-binding peptide aptamers may be useful tools to specifically eliminate HPV-positive tumors.     

Langerhans cells and dendritic cells are cytotoxic towards HPV16 E6 and E7 expressing target cells

Dendritic cells (DC) can be cytotoxic towards tumor cells by means of TNF family molecules expressed on the cell surface of activated DCs. Tumor cells expressing appropriate receptors are killed by DC, generating a source of antigen to be presented to the immune system. It has not been investigated whether Langerhans cells (LC) are selectively cytotoxic to tumor cells. This is of particular interest for epithelial tumor cells that physically interact with LC in vivo. Among epithelial tumors, the oncogenic process of cervical tumors is relatively well defined by their Human Papillomavirus (HPV) mediated etiology. To study whether HPV16 E6 and E7 expressions, otherwise observed in cervical tumor cells, can sensitize normal cervical epithelial cells to DC and LC mediated killing, the E6 and E7 genes were introduced by retroviral transfection, and cells were subsequently used as targets in cytotoxicity assays. Expression of cytotoxic molecules by effector cells was measured in response to the pro-inflammatory cytokine IFN-γ; cytotoxicity was established and concomitant expression of receptor molecules was assessed on target cells. A correlation between the shrinkage of HPV16 E6 and E7+ tumors versus DC and LC infiltration was evaluated in a murine model of cervical cancer. DC and LC proved to be equally cytotoxic towards E6 and E7 expressing cervical epithelial cells. IFN-γ induced TRAIL expression by DC and LC, and inhibition of TRAIL partially blocked cytotoxic effects. Expression of TRAIL decoy receptors was reduced following introduction of E6 and E7 into host cells. Shrinkage of HPV16 E6 and E7 expressing tumors correlated with infiltration by S100+ DC and LC, co-localizing with apoptotic mouse tumor cells. In conclusion, DC and LC mediated killing may be exploitable for anti-tumor treatment. I. Caroline Le Poole and W.M. ElMasri have contributed equally to this paper.     

Antitumor effect of therapeutic HPV DNA vaccines with chitosan-based nanodelivery systems

Background

Cervical cancer is the second-most-common cause of malignancies in women worldwide, and the oncogenic activity of the human papilloma virus types (HPV) E7 protein has a crucial role in anogenital tumors. In this study, we have designed a therapeutic vaccine based on chitosan nanodelivery systems to deliver HPV-16 E7 DNA vaccine, considered as a tumor specific antigen for immunotherapy of HPV-associated cervical cancer. We have developed a Nano-chitosan (NCS) as a carrier system for intramuscular administration using a recombinant DNA vaccine expressing HPV-16 E7 (NCS-DNA E7 vaccine). NCS were characterized in vitro for their gene transfection ability.

Results

The transfection of CS-pEGFP NPs was efficient in CHO cells and the expression of green fluorescent proteins was well observed. In addition, NCS-DNA E7 vaccine induced the strongest E7-specific CD8+ T cell and interferon γ responses in C57BL/6 mice. Mice vaccinated with NCS-DNA E7 vaccine were able to generate potent protective and therapeutic antitumor effects against challenge with E7-expressing tumor cell line, TC-1.

Conclusions

The strong therapeutic effect induced by the Chitosan-based nanodelivery suggest that nanoparticles may be an efficient carrier to improve the immunogenicity of DNA vaccination upon intramuscular administration and the platform could be further exploited as a potential cancer vaccine candidate in humans.     

Immunotherapy of a human papillomavirus type 16 E7-expressing tumor by administration of fusion protein comprised of Mycobacterium bovis BCG Hsp65 and HPV16 E7

Human papillomavirus type 16 (HPV16) infection has been linked to the development of cervical and anal dysplasia and cancer. One hallmark of persistent infection is the synthesis of the viral E7 protein in cervical epithelial cells. The expression of E7 in dysplastic and transformed cells and its recognition by the immune system as a foreign antigen make it an ideal target for immunotherapy. Utilizing the E7-expressing murine tumor cell line, TC-1, as a model of cervical carcinoma, an immunotherapy based on the administration of an adjuvant-free fusion protein comprised of Mycobacterium bovis BCG Hsp65 linked to HPV16 E7 (HspE7) has been developed. Initial in vitro analyses indicate that immunization with HspE7 results in the induction of a type 1 immune response based on the pattern of secreted cytokines and the presence of cytolytic activity following antigenic recall. It has been previously shown that prophylactic immunization with HspE7 protected mice against challenge with TC-1 cells and that these tumor-free animals are also protected against rechallenge with TC-1 cells. The present report shows that a single therapeutic immunization with HspE7 induces regression of palpable tumors, confers protection against tumor rechallenge, and is associated with long-term survival (>253 days). In vivo studies using mice with targeted mutations in CD8 or MHC class II or depleted of CD8 or CD4 lymphocyte subsets demonstrate that tumor regression following therapeutic HspE7 immunization is CD8 dependent and CD4 independent. These studies extend previous observations on the induction of CTL by Hsp fusion proteins and are consistent with the clinical application of HspE7 as an immunotherapy for human cervical and anal dysplasia and cancer.     

Analysis of the p53-mediated G1 growth arrest pathway in cells expressing the human papillomavirus type 16 E7 oncoprotein.

Cells expressing human papillomavirus type 16 (HPV-16) E7, similar to those which express HPV-16 E6, are resistant to a p53-mediated G1 growth arrest. We examined the p53-mediated DNA damage response pathway in E7-expressing cells to determine the mechanism by which E7-containing cells continue to cycle. In response to DNA damage, no dramatic difference was detected in G1- or S-phase cyclin or cyclin-dependent kinase (Cdk) levels when E7-expressing cells were compared to the parental cell line, RKO. Furthermore, Cdk2 kinase activity was inhibited in both RKO cells and E7-expressing cells, while Cdk2 remained active in E6-expressing cells. However, the steady-state levels of pRB and p107 protein were substantially lower in E7-expressing cells than in the parental RKO cells or E6-expressing cells. There was no reduction in pRB mRNA levels, but the half-life of pRB in E7-expressing cells was markedly shorter. Infection of primary human foreskin keratinocytes with recombinant retroviruses expressing HPV-16 E7 resulted in a decrease in pRB protein levels, indicating this phenomenon is a consequence of E7 expression, not of immortalization or transformation. These data strongly suggest E7 interferes with the stability of pRB and p107 protein. We propose that the removal of these components of the p53-mediated G1 growth arrest pathway in E7-expressing cells contributes to the ability of E7 to overcome a p53-mediated G1 growth arrest.     

Characterization of HPV-16 E6 DNA vaccines employing intracellular targeting and intercellular spreading strategies

Summary Human papillomavirus (HPV) E6 and E7 are consistently expressed and are responsible for the malignant transformation of HPV-associated lesions. Thus, E6 and E7 represent ideal targets for therapeutic HPV vaccine development. We have previously used the gene gun approach to test several intracellular targeting and intercellular spreading strategies targeting HPV-16 E7. These strategies include the use of the sorting signal of lysosome-associated membrane protein (LAMP-1), Mycobacterium tuberculosis heat shock protein 70 (HSP70), calreticulin (CRT) and herpes simplex virus type 1 (HSV-1) VP22 proteins. All of these strategies have been shown to be capable of enhancing E7-DNA vaccine potency. In the current study, we have characterized DNA vaccines employing these intracellular targeting or intercellular spreading strategies targeting HPV-16 E6 for their ability to generate E6-specific CD8⁺ T cell immune responses and antitumor effects against an E6-expressing tumor cell line, TC-1, in C57BL/6 mice. We found that all the intracellular targeting strategies (CRT, LAMP-1, HSP70) as well as the intercellular spreading strategy (VP22) were able to enhance E6 DNA vaccine potency, although the orientation of HSP70 linked to E6 antigen in the E6 DNA vaccine appears to be important for the HSP70 strategy to work. The enhanced E6-specific CD8⁺ T cell immune response in vaccinated mice also translated into potent antitumor effects against TC-1 tumor cells. Our data indicate that all of the intracellular targeting and intercellular spreading strategies that have been shown to enhance E7 DNA vaccine potency were also able to enhance E6 DNA vaccine potency.     

In vitro biological activities of the E6 and E7 genes vary among human papillomaviruses of different oncogenic potential.

Human papillomavirus type 16 (HPV-16) and HPV-18 are often detected in cervical carcinomas, while HPV-6, although frequently present in benign genital lesions, is only rarely present in cancers of the cervix. Therefore, infections with HPV-16 and HPV-18 are considered high risk and infection with HPV-6 is considered low risk. We found, by using a heterologous promoter system, that expression of the E7 transforming protein differs between high- and low-risk HPVs. In high-risk HPV-16, E7 is expressed from constructs containing the complete upstream E6 open reading frame. In contrast, HPV-6 E7 was efficiently translated only when E6 was deleted. By using clones in which the coding regions of HPV-6, HPV-16, and HPV-18 E7s were preceded by identical leader sequences, we found that the ability of the E7 gene products to induce anchorage-independent growth in rodent fibroblasts correlated directly with the oncogenic association of the HPV types. By using an immortalization assay of normal human keratinocytes that requires complementation of E6 and E7, we found that both E6 and E7 of HPV-18 could complement the corresponding gene from HPV-16. However, neither E6 nor E7 from HPV-6 was able to substitute for the corresponding gene of HPV-16 or HPV-18. Our results suggest that multiple factors, including lower intrinsic biological activity of E6 and E7 and differences in the regulation of their expression, account for the low activity of HPV-6, in comparison with HPV-16 and HPV-18, in in vitro assays. These same factors may, in part, account for the apparent difference in oncogenic potential between these viruses.     

Immunological ignorance of an E7-encoded cytolytic T-lymphocyte epitope in transgenic mice expressing the E7 and E6 oncogenes of human papillomavirus type 16.

Certain human papillomaviruses (HPV) have been implicated in the etiology of cervical malignancies, and the E7 and E6 gene products of HPV type 16 are frequently expressed in these lesions. However, cytolytic T-lymphocyte (CTL)-mediated responses to HPV are rarely detectable in patients with cervical cancer. To examine whether the T-cell response is deficient during the HPV-induced transformation, we produced lines of transgenic (Tg) mice that expressed the E6 and E7 oncogenes in keratinized epithelia. The mice developed severe hypertrophy of all keratinized epithelia, but no malignancies were observed. Although epithelial cells from Tg mice could present at least an E7-encoded CTL epitope (E7 49-57), CTLs from these mice were neither primed to nor made tolerant of this epitope. No quantitative or qualitative differences were seen in the CTL responses of the Tg mice compared to those of their littermates following immunization with the peptide E7 49-57. Immunization of Tg mice with the E7 49-57 peptide protected them against a subcutaneous challenge with tumor cells expressing a transfected E7 gene, yet the skin was unaffected, although the cultured skin epithelial cells from Tg mice expressed E7. Our results suggest that the Tg mice were immunologically ignorant of HPV oncoproteins with respect to a CTL response and that a similar type of ignorance may explain why HPV-associated cervical cancer cells can escape immunological destruction.     

Human papillomavirus type 16 E6 and HPV-16 E6/E7 sensitize human keratinocytes to apoptosis induced by chemotherapeutic agents: roles of p53 and caspase activation

We and others have previously reported that human papillomavirus (HPV)-16 E6 protein expression sensitizes certain cell types to apoptosis. To confirm that this sensitization occurred in HPV's natural host cells, and to explore the mechanism(s) of sensitization, we infected human keratinocytes (HKCs) with retroviruses containing HPV-6 E6, HPV-16 E6, HPV-16 E7, or HPV-16 E6/E7. Apoptosis was monitored by DNA fragmentation gel analysis and direct observation of nuclei in cells stained with DAPI. Exposure of HKCs to etoposide, cisplatin, mitomycin C (MMC), atractyloside, and sodium butyrate, resulted in a time and dose-dependent induction of apoptosis. Expression of HPV-16 E6 and HPV-16 E6/E7, but not HPV-6 E6 or HPV-16 E7, enhanced the sensitivity of HKCs to cisplatin-, etoposide- and MMC-, but not atractyloside- or sodium butyrate-induced apoptosis. Expression of both HPV-16 E6 and HPV-16 E6/E7 decreased, but did not abolish, p53 protein levels relative to normal HKCs, and resulted in cytoplasmic localization of wt p53. P53 induction occurred in HPV-16 E6 and HPV-16 E6/E7 expressing cells after exposure to cisplatin or MMC, though never to levels found in normal untreated HKCs. P21 levels were decreased in HPV-16 E6 and HPV-16 E6/E7 expressing HKCs, and no induction of p21 was seen in these cells following exposure to cisplatin or MMC. Caspase-3 activity was found to be elevated in HPV-16 E6-expressing HKCs following exposure to cisplatin and MMC as documented by fluorometric and Western Blot analysis. Expression of wt CrmA or treatment of HPV-16 E6 expressing HKCs with the caspase-3 inhibitor DEVD.fmk prevented HPV-16 E6-induced sensitization in HKCs. These results suggest that HPV-16 E6 and HPV-16 E6/E7 expression sensitizes HKCs to apoptosis caused by cisplatin, etoposide and MMC, but not atractyloside or sodium butyrate. The data also suggest that wt p53 and caspase-3 activity are required for HPV-16 E6 and HPV-16 E6/E7-induced sensitization of HKCs to DNA damaging agents.     

Molecular Analysis of the Interaction between Human PTPN21 and the Oncoprotein E7 from Human Papillomavirus Genotype 18

Human papillomaviruses (HPVs) cause cellular hyperproliferation-associated abnormalities including cervical cancer. The HPV genome encodes two major viral oncoproteins, E6 and E7, which recruit various host proteins by direct interaction for proteasomal degradation. Recently, we reported the structure of HPV18 E7 conserved region 3 (CR3) bound to the protein tyrosine phosphatase (PTP) domain of PTPN14, a well-defined tumor suppressor, and found that this intermolecular interaction plays a key role in E7-driven transformation and tumorigenesis. In this study, we carried out a molecular analysis of the interaction between CR3 of HPV18 E7 and the PTP domain of PTPN21, a PTP protein that shares high sequence homology with PTPN14 but is putatively oncogenic rather than tumor-suppressive. Through the combined use of biochemical tools, we verified that HPV18 E7 and PTPN21 form a 2:2 complex, with a dissociation constant of 5 nM and a nearly identical binding manner with the HPV18 E7 and PTPN14 complex. Nevertheless, despite the structural similarities, the biological consequences of the E7 interaction were found to differ between the two PTP proteins. Unlike PTPN14, PTPN21 did not appear to be subjected to proteasomal degradation in HPV18-positive HeLa cervical cancer cells. Moreover, knockdown of PTPN21 led to retardation of the migration/invasion of HeLa cells and HPV18 E7-expressing HaCaT keratinocytes, which reflects its protumor activity. In conclusion, the associations of the viral oncoprotein E7 with PTPN14 and PTPN21 are similar at the molecular level but play different physiological roles.     

Human Papillomavirus E6E7-Mediated Adenovirus Cell Killing: Selectivity of Mutant Adenovirus Replication in Organotypic Cultures of Human Keratinocytes

Replication-competent adenoviruses are being investigated as potential anticancer agents. Exclusive virus replication in cancer cells has been proposed as a safety trait to be considered in the design of oncolytic adenoviruses. From this perspective, we have investigated several adenovirus mutants for their potential to conditionally replicate and promote the killing of cells expressing human papillomavirus (HPV) E6 and E7 oncoproteins, which are present in a high percentage of anogenital cancers. For this purpose, we have employed an organotypic model of human stratified squamous epithelium derived from primary keratinocytes that have been engineered to express HPV-18 oncoproteins stably. We show that, whereas wild-type adenovirus promotes a widespread cytopathic effect in all infected cells, E1A- and E1A/E1B-deleted adenoviruses cause no deleterious effect regardless of the coexpression of HPV18 E6E7. An adenovirus deleted in the CR2 domain of E1A, necessary for binding to the pRB family of pocket proteins, shows no selectivity of replication as it efficiently kills all normal and E6E7-expressing keratinocytes. Finally, an adenovirus mutant deleted in the CR1 and CR2 domains of E1A exhibits preferential replication and cell killing in HPV E6E7-expressing cultures. We conclude that the organotypic keratinocyte culture represents a distinct model to evaluate adenovirus selectivity and that, based on this model, further modifications of the adenovirus genome are required to restrict adenovirus replication to tumor cells.     

Expression of the human papillomavirus E7 oncogene during cell transformation is sufficient to induce susceptibility to lysis by activated macrophages.

Human papillomaviruses (HPV), and in particular HPV type 16, are etiologic agents in the development of cervical cancer, which is the second most common form of cancer in women worldwide. Mammalian cells are susceptible to transformation in vitro by the E6 and E7 oncogenes derived from the HPV-16 genome. NIH-3T3 cells transfected with the HPV-16 E7 oncogene were found to exhibit cytolytic susceptibility to murine-activated macrophages. In comparison, E6 oncogene-expressing cells were not susceptible to lysis by activated macrophages. The E7 oncoprotein is multifunctional, being capable of complexing with the retinoblastoma tumor suppressor gene (anti-oncogene) product, stimulating DNA synthesis, and causing cell transformation in vitro. Macrophage killing assays performed on cell lines expressing E7 mutants revealed that the ability to complex the retinoblastoma tumor suppressor gene product and stimulate DNA synthesis did not induce susceptibility to activated macrophages, whereas the ability of E7 to cause transformation was required to induce susceptibility to activated macrophages. These data suggest that cell transformation is a more important prerequisite for inducing susceptibility to activated macrophages than is the loss of tumor suppressor gene function. This study also provides an initial link between HPV-16 oncogene expression and the ability of activated macrophages to selectively recognize and destroy HPV-16-associated neoplastic cells.