Cells Expressing HPV16 E6 Research Articles

Oral administration of human papillomavirus type 16 E7 displayed on Lactobacillus casei induces E7‐specific antitumor effects in C57/BL6 mice

The mounting of a specific immune response against the human papillomavirus type 16 E7 protein (HPV16 E7) is important for eradication of HPV16 E7-expressing cancer cells from the cervical mucosa. To induce a mucosal immune response by oral delivery of the E7 antigen, we expressed the HPV16 E7 antigen on the surface of Lactobacillus casei by employing a novel display system in which the poly-gamma-glutamic acid (gamma-PGA) synthetase complex A (PgsA) from Bacillus subtilis (chungkookjang) was used as an anchoring motif. After surface expression of the HPV16 E7 protein was confirmed by Western blot, flow cytometry and immunofluorescence microscopy, mice were orally inoculated with L. casei-PgsA-E7. E7-specific serum IgG and mucosal IgA productions were enhanced after oral administration and significantly enhanced after boosting. Systemic and local cellular immunities were significantly increased after boosting, as shown by increased counts of lymphocytes (SI = 9.7 +/- 1.8) and IFN-gamma secreting cells [510 +/- 86 spot-forming cells/10(6)cells] among splenocytes and increased IFN-gamma in supernatants of vaginal lymphocytes. Furthermore, in an E7-based mouse tumor model, animals receiving orally administered L. casei-PgsA-E7 showed reduced tumor size and increased survival rate versus mice receiving control (L. casei-PgsA) immunization. These results collectively indicate that the oral administration of E7 displayed on lactobacillus induces cellular immunity and antitumor effects in mice.

Alteration of cell-cycle regulatory proteins in human oral epithelial cells immortalized by HPV16 E6 and E7

E6 and E7 oncoproteins from high-risk human papillomavirus (HPV) can transform cells in tissue culture and induce tumors in vivo by abrogating the cell-cycle checkpoint. To investigate the impact of HPV16 E6 and E7 on the cell-cycle regulatory machinery in oral epithelial cells, normal human oral epithelial cells were transfected with HPV16 E6 and E7 open reading frames, and alterations in cell-cycle regulatory proteins in cells expressing HPV16 E6 and E7 were analyzed. E6 and E7 expression results in immortalization of oral epithelial cells. E7 inactivates retinoblastoma protein (Rb) by forming complexes with hypophosphorylated Rb in immortalized oral epithelial cells. P53 and P21 protein levels were increased in immortalized cells compared to normal primary oral epithelial cells. Cyclin D1–cell-cycle-dependent kinase 4 binary association is disrupted in immortalized oral epithelial cells. These results indicate that E7 plays an important role in abrogation of cell-cycle regulation in oral epithelial cells, with E6 having a smaller impact. This suggests that the pathogenesis of HPV in oral epithelial cells differs from that in cervical epithelial cells.

Efficient expression of modified human papillomavirus 16 e6/e7 fusion protein and the antitumor efficacy in a mouse model.

Infection with human papillomavirus, particularly type 16 (HPV16), is highly associated with the development of cervical intraepithelial neoplasia and cervical cancer. The two early viral oncogenes, E6 and E7, are selectively retained and constitutively expressed in tumor cells and are therefore attractive immunotherapeutic targets. Thus a vaccine strategy based on recombinant HPV16 E6/E7 fusion protein represents an efficient approach against HPV16-associated tumors. Although the expression level of HPV16 E6/E7 fusion protein was presumed to be low, direct experimental proof in vivo was lacking. To enhance the expression level and investigate its antitumor efficacy in vivo, we constructed a modified HPV16 E6/E7 fusion gene with three point-mutations and expressed it in Escherichia coli. The encoded protein, denoted mE6(1-120)/mE7(1-60), comprises 120 N-terminus amino acids of E6 and 60 N-terminus amino acids of E7 plus a histine tag, was purified on an affinity column, and subsequently characterized by Western blotting. Immunization of mice with mE6(1-120)/mE7(1-60) completely protected them against subsequent challenge and rechallenge with TC-1 tumor cells expressing HPV16 E6 and E7 proteins. In the therapeutic experiments, most mice eliminated the preexisting tumors and had a long-term protection. Consistent with the results of in vivo experiments, the splenocytes from immunized mice elicited cytotoxic T lymphocytes and specifically lysed TC-1 cells in vitro. More importantly, the expression level of mE6(1-120)/mE7(1-60) was significantly improved, meeting the necessary quantity required for a vaccine clinical trial. In conclusion, these data provide a scientific basis for the use of modified mE6(1-120)/mE7(1-60) in future human trials.

Expression of the potential novel gene E6DG1 downregulated by the E6 protein of human papillomavirus type 16 is correlated with anchorage-independent growth

A novel gene transcript that is downregulated by HPV16 E6 protein was identified in mouse cells using differential hybridization and designated E6DG1. The cloned cDNA of E6DG1 was 1.3 kb in length and contained a small ORF potentially encoding a polypeptide of 45 amino acids. In vitro transcription and translation of E6DG1 cDNA resulted in a product of approximately 7 kDa and Western blot analysis using antibodies for E6DG1 peptide detected 7 and 14 kDa proteins. Downregulation of E6DG1 mRNA levels in cells expressing HPV16 E6 protein was observed at subconfluent cell densities, but not in confluent cells. Repression of E6DG1 protein enhanced the anchorage-independent growth and weakened the cell adhesion in Panc1 cells. Immunofluorescence analysis revealed the localization of E6DG1 within the nucleus. These results indicate that the E6DG1 protein may function in a signaling pathway related to anchorage-independent growth and adhesion control.

Recombinant DNA vaccines protect against tumors that are resistant to recombinant vaccinia vaccines containing the same gene

Antigen-specific cancer immunotherapy involves the delivery of tumor-associated antigen to the host for the generation of tumor-specific immune responses and antitumor effects. We hypothesized that different delivery systems may influence the pattern of antigen-specific immune response and the outcome of antitumor effect. We therefore evaluated recombinant vaccinia virus and naked DNA for the generation of antigen-specific immune responses and antitumor effects. We previously found that recombinant vaccinia and naked DNA vaccines containing the chimeric Sig/E7/LAMP-1 gene were capable of controlling the growth of HPV-16 E7-expressing tumor cells (TC-1). In this study, we performed a head-to-head comparison of optimized delivery of Sig/E7/LAMP-1 vaccinia and DNA vaccines using dose-escalating tumor challenge. At a dose of 1 x 10(6) TC-1 cells per mouse, Sig/E7/LAMP-1 DNA provided 100% protection against subcutaneous growth of tumors, while Vac-Sig/E7/LAMP-1 protected only 40% of the mice. Furthermore, Sig/E7/LAMP-1 DNA vaccines are capable of protecting against challenge with a more stringent subclone of TC-1 (TC-1 P2) established from TC-1 tumors that survived initial Sig/E7/LAMP-1 vaccinia vaccination. Immunological assays revealed that both vaccines induced comparable levels of CD8(+) T cell precursors and anti-E7 antibody titers. Interestingly, Sig/E7/LAMP-1 vaccinia induced both E7-specific IFN-gamma- and IL4-secreting CD4(+) T cell precursors while Sig/E7/LAMP-1 DNA induced only E7-specific IFN-gamma-secreting CD4(+) T cell precursors. We also found that IL-4 knockout C57BL/6 mice vaccinated with Sig/E7/LAMP-1 vaccinia exhibited a more potent antitumor effect than vaccinated wild-type C57BL/6 mice in our tumor protection experiments. These results suggest that IL-4 may play a detrimental role in the antitumor effect mediated by vaccinia vaccines. Our findings suggested that DNA vaccines may provide better tumor protection than vaccinia vaccines employing the same gene, which may have implications in the future design of antigen-specific cancer immunotherapy.

Induction of apoptosis in human keratinocytes containing mutated p53 alleles and its inhibition by both the E6 and E7 oncoproteins.

Induction of apoptosis is a function of external stimuli and cellular gene expression. Many cells respond to DNA damage by the induction of apoptosis, which depends on a functional p53 protein and is signaled by elevation of p53 levels. We have investigated the response of immortalized human keratinocytes (HaCaT) bearing mutated alleles of p53 to genotoxic stress and the effect of human papillomavirus (HPV) 16 E6 and E7 on this response. UVC irradiation triggered HaCaT's cell death with several characteristics of apoptosis, including DNA laddering, chromatin condensation and fragmentation, and the appearance of cells with a low content of DNA (categorized as sub-G1 by cell sorter analysis). This response was accompanied by accumulation of cells in S phase of the cell cycle. HaCaT cells infected with retroviruses carrying HPV16 E6 or E7 showed a significant reduction in their apoptotic response, which was not observed in cells infected with the LXSN vector DNA-carrying virus. Reduced apoptosis in HaCaT cells expressing E6 or E7 also was observed after treatment with the alkylating agent mitomycin C. Western blot analysis of p53 and p21/WAF-1/CIP-1, a downstream effector of p53, did not reveal any changes in the levels of these proteins after UVC irradiation in either HaCaT cells or HaCaT cells expressing HPV16 E6 or E7.

The human papillomavirus-16 E6 oncoprotein decreases the vigilance of mitotic checkpoints.

The E6 and E7 proteins of the high risk human papillomaviruses (HPVs) are consistently expressed in HPV-positive cervical carcinomas. We investigated the ability of HPV-16 E6 and E7 to disrupt mitotic checkpoints in normal diploid human cells. Acute expression of HPV-16 E6, but not HPV-16 E7, decreased the fidelity of multiple checkpoints controlling entry into and exit from mitosis. After irradiation, nearly 50% of cells containing HPV-16 E6 readily entered mitosis as opposed to less than 10% of control cells. Consistent with this, asynchronous populations of cells expressing HPV-16 E6 had increased cdc2-associated histone H1 kinase activity relative to control populations. In addition, HPV-16 E6 increased sensitivity to chemically-induced S-phase premature mitosis and decreased mitotic spindle assembly checkpoint function relative to control populations. HPV-16 E6 mutants with a reduced ability to target p53 for degradation were unable to abrogate mitotic checkpoints, suggesting a possible mechanism by which HPV-16 E6 disrupts mitotic checkpoints. Expression of a mutant p53 gene yielded an intermediate phenotype relative to HPV-16 E6, generating moderate increases in sensitivity to chemically-induced S-phase PCC and mitotic spindle disruption and a heightened propensity to enter mitosis after irradiation.

Identification of a naturally processed HLA A0201‐restricted viral peptide from cells expressing human papillomavirus type 16 E6 oncoprotein

Human papillomavirus (HPV) DNA encoding the oncogenic proteins E6 and E7 is usually retained in cervical carcinomas, implicating these proteins as potential target antigens for immune recognition in this virally associated tumor. We have characterized endogenously processed peptides eluted from major histocompatibility complex class I molecules in cells infected with a recombinant vaccinia expressing the HPV-16 E6 oncoprotein. The reverse-phase chromatography profile of peptides eluted from isolated HLA-A0201 molecules in cells expressing the E6 oncoprotein differs from that of cells not expressing E6. Sequential Edman degradation of novel peaks found in the peptide profiles from cells expressing HPV-16 E6 led to the identification of a naturally processed HLA-A0201-restricted E6 peptide of sequence KLPQLCTEL. This approach has allowed the identification of a viral peptide which is processed and presented by cells expressing the E6 oncoprotein and is a likely target for cytotoxic T lymphocyte recognition in HLA-A0201-positive patients.

Growth arrest by induction of p53 in DNA damaged keratinocytes is bypassed by human papillomavirus 16 E7.

Cellular tumor suppressors p53 and Rb play an important role in controlling cell proliferation. Inactivation of these tumor suppressor proteins can occur by gene mutation or by association with oncoproteins from the small DNA tumor viruses. One function of p53 is in regulating cell cycle check-point control after DNA damage. To dissect the pathways by which p53 and Rb may act, the E6 and E7 oncogenes of human papillomavirus (HPV) types 6 and 16 were introduced into primary human epithelial cells by retroviral transfer vector, and cells were assayed for growth arrest after DNA damage induced by actinomycin D. The E6 or E7 oncogenes from the low-risk HPV6 had no affect on growth arrest, p53 protein levels increased, Rb protein levels decreased, and Rb was predominantly in the hypophosphorylated state similar to vector-infected cells. Either the E6 or the E7 oncogene from the high-risk HPV16 abrogated growth arrest. Cells expressing HPV16 E6 (16E6) were severely reduced in p53 protein levels that did not increase detectably after DNA damage, Rb protein levels did not decrease, and hyperphosphorylated Rb was present. After DNA damage in cells expressing 16E7 p53 levels increased, and Rb protein levels decreased; however, Rb was predominantly in the hyperphosphorylated state. Even though p53 protein levels increased in response to DNA damage in cells expressing 16E7, G1 growth arrest was bypassed. This suggests that the circuitry controlling the growth arrest signal after DNA damage may be interconnected between the p53 and Rb pathways.