(-)-Epigallocatechin-3-gallate inhibits human papillomavirus (HPV)-16 oncoprotein-induced angiogenesis in non-small cell lung cancer cells by targeting HIF-1α.

PURPOSE: To investigate the effects of (-)-epigallocatechin-3-gallate (EGCG) on human papillomavirus (HPV)-16 oncoprotein-induced angiogenesis in non-small cell lung cancer (NSCLC) cells and the underlying mechanisms. METHODS: NSCLC cells (A549 and NCI-H460) transfected with EGFP plasmids containing HPV-16 E6 or E7 oncogene were treated with different concentrations of EGCG for 16 h. The effects of EGCG on angiogenesis in vitro and in vivo were observed. The expression of HIF-1α, p-Akt, and p-ERK1/2 proteins in NSCLC cells was analyzed by Western blot. The levels of HIF-1α mRNA in NSCLC cells were detected by real-time RT-PCR. The concentration of VEGF and IL-8 in the conditioned media was determined by ELISA. HIF-1α, VEGF, and CD31 expression in A549 xenografted tumors of nude mice was analyzed by immunohistochemistry. RESULTS: HPV-16 E6 and E7 oncoproteins HIF-1α-dependently promoted angiogenesis in vitro and in vivo, which was inhibited by EGCG. Mechanistically, EGCG inhibited HPV-16 oncoprotein-induced HIF-1α protein expression but had no effect on HIF-1α mRNA expression in NSCLC cells. Additionally, 50 and 100 µmol/L of EGCG significantly reduced the secretion of VEGF and IL-8 proteins induced by HPV-16 E7 oncoprotein in NSCLC A549 cells. Meanwhile, HPV-16 E6 and E7 oncoproteins HIF-1α-dependently enhanced Akt activation in A549 cells, which was suppressed by EGCG. Furthermore, EGCG inhibited HPV-16 oncoprotein-induced HIF-1α and HIF-1α-dependent VEGF and CD31 expression in A549 xenografted tumors. CONCLUSIONS: EGCG inhibited HPV-16 oncoprotein-induced angiogenesis conferred by NSCLC through the inhibition of HIF-1α protein expression and HIF-1α-dependent expression of VEGF, IL-8, and CD31 as well as activation of Akt, suggesting that HIF-1α may be a potential target of EGCG against HPV-related NSCLC angiogenesis.

Human papillomavirus type 32 does not display in vitro transforming properties.

Human papillomavirus type 32 (HPV32) is one of the etiological agents of a benign oral condition, focal epithelial hyperplasia. However, the previously characterized properties of its E7 oncoprotein suggest a possible malignant nature for this virus. In this study we characterized the properties of HPV32 E6 and E7. Our data show that HPV32 E7, despite its high affinity for pRb, does not promote degradation of the cellular protein. In addition, HPV32 E6 does not prevent p53-mediated apoptosis and/or cell cycle arrest. Moreover, coexpression of HPV32 E6 and E7 in primary human fibroblasts or keratinocytes does not alter their proliferative state. Together, these data provide evidence of the benign nature of HPV32.

Pre-clinical safety and efficacy of TA-CIN, a recombinant HPV16 L2E6E7 fusion protein vaccine, in homologous and heterologous prime-boost regimens.

Human papillomavirus (HPV) E6 and E7 oncoproteins are attractive targets for T-cell-based immunotherapy of cervical intraepithelial neoplasia (CIN) and cancer. A newly designed vaccine, comprising the HPV16 L2, E6 and E7 as a single fusion protein (TA-CIN), was shown to elicit HPV16-specific CTL, T-helper cells and antibodies in a pre-clinical mouse model. These immune responses effectively prevented outgrowth of HPV16-positive tumour cells in a prophylactic setting as well as in a minimal residual disease setting. CTL immunity was optimally induced when TA-CIN was employed in heterologous prime-boost regimens in combination with TA-HPV, a clinical grade vaccinia-based vaccine. These data provide a scientific basis for the use of TA-CIN, alone or in combination with TA-HPV in future human trials.

The amino-terminus and membrane-spanning domains of LMP-1 inhibit cell proliferation.

The LMP-1 oncoprotein of EBV is required to maintain proliferation of infected B-cells and shares several features with CD40, TNF-R1, and related receptors. Members of this family can bind TRAF and TRADD molecules and activate NF-kappaB and AP-1, as can LMP-1. While CD40 and TNF-R1 are dependent on binding their ligands for their signaling, LMP-1 apparently is not. We have found that LMP-1 can act as a governor of cell proliferation and thereby limit its own activities. Its inhibition of proliferation is not mediated by apoptosis but results in cytostasis in four cell lines tested. The structural moiety of LMP-1 that distinguishes it from CD40 and TNF-R1, its amino-terminus and multiple membrane spanning segments, alone can mediate its cytostatic activity.

HPV-16 E7 but not E6 oncogenic protein triggers both cellular immunosuppression and angiogenic processes.

HPV-16 E6 and E7 oncoproteins impair the cell cycle in human uterine cervix carcinoma cells (HUCC) by acting on p53 and retinoblastoma proteins, respectively. We recently reported that E7 related into the extracellular compartment by HUCC SiHa cells could inhibit immune T-cell response to recall and alloantigens by a mechanism involving an overproduction of the immunosuppressive IFN alpha by antigen presenting cells (APCs). In this study, we found that besides E7, E6 protein and the vascular endothelium growth factor (VEGF) were released into the SiHa cell supernatants, and we further showed that extracellular E7 but not E6 oncoprotein 1) inhibits the immune cell response to recall and alloantigens, and 2) enhances the release of angiogenic cytokines, including TNF alpha, IL-1 beta and IL-6 by macrophages and/or dendritic cells. VEGF unexpectedly released by cancer cells could also contribute to angiogenesis. Thus in HUCC the same E7 oncoprotein which contributes to controlling the cancer cell cycle has the means in its extracellular configuration to contribute to microenvironmental immunosuppressive and angiogenic processes. Neutralizing anti-E7 antibodies either passively administered or induced by active immunization could represent a new immunotherapeutic endeavour to combat the immunosuppression and/or neoangiogenesis effects of extracellular E7 protein.

Immortalization of human cells and their malignant conversion by high risk human papillomavirus genotypes.

Papillomaviruses cause certain common human cancers, most notably carcinoma of the cervix. The viral oncoproteins E6 and E7 are essential components in malignant conversion, although, in spite of being necessary, they are not sufficient for the development of the malignant phenotype. High risk HPV oncogenes fulfill dual functions in genome-harboring cells: their derived oncoproteins stimulate cell growth by pleiotropic effects. At the same time they act as progression factors by inducing mutations in host cell DNA and aneuploidy. The mechanism underlying the process towards malignant conversion, usually covering a long latency period between primary infection and cancer emergence, is presently not fully understood. It emerges, however, that cancer development depends on the interruption of at least two signalling cascades (labeled as CIF I and CIF II) that interfere with the function of viral oncoproteins (CIF I) and with the transcription of viral oncogenes (CIF II). Further modifications of host cell genes most likely mediate the escape from immune surveillance mechanisms of the host and the development of metastases.

Transforming properties of the cottontail rabbit papillomavirus oncoproteins Le6 and SE6 and of the E8 protein.

Cottontail rabbit papillomavirus induces on cottontail and domestic rabbits papillomas which progress at a high frequency to carcinoma. The virus encodes three transforming proteins; one is translated from open reading frame (ORF) E7 and binds the retinoblastoma protein, and two, LE6 and SE6, are translated from the first and second ATGs of ORF E6, respectively. Here we show that neither of the E6 proteins coprecipitated with p53 in vitro, nor did they bind to a recently identified E6-binding protein (J. J. Chen, C. E. Reid, V. Band, and E. Androphy, Science 269:529-531, 1995). This protein was shown to bind to the E6 proteins of the high-risk human papillomairus types 16 and 18 but not to the low-risk human papillomavirus types VI and II. In-frame deletions cloned into the pZipNeo vector were used to identify structural features of SE6 and LE6 important for transformation of NIH 3T3 cells. Three deletions covering the amino-terminal half of SE6 did not transform cells. In two of the three deletions, two Cys-X-X-Cys motifs were deleted, each deletion preventing the formation of one of the potential small Zn fingers of SE6. Among the LE6 deletions, only one had a reduced transformation efficiency, while seven transformed cells at least as efficiently as wild-type LE6. In each of three of these seven mutants, two Cys-X-X-Cys motifs were deleted. None of the three amino acid deletions which abolished transformation by SE6 reduced transformation by LE6. Furthermore, transformation did not correlate with the level of SE6 or LE6 proteins detectable. ORF E8 colinear with ORF E6, which could generate a 50-amino-acid protein with a hydrophobic segment, did not transform cells when cloned into the pZipNeo vector. However, mutation of the E8 ATG, which did not alter the amino acid sequence of LE6, increased transformation by LE6 without affecting the level of LE6 expression. The data suggest that transformation by the E6 proteins is not mediated by interfering with p53 function or through binding to the E6-binding protein. Furthermore, different structural features are important to maintain transformation functions and protein stability of LE6 and SE6. Finally, E8 seems not to be a transforming protein but rather appears to modulate transformation bv LE6.

The v-ski oncogene cooperates with the v-sea oncogene in erythroid transformation by blocking erythroid differentiation.

The avian retrovirus oncogene v-ski was analysed for its ability to alter the differentiation program of erythroid cells and to cooperate with tyrosine kinase oncogenes in leukemogenesis. For this, a retrovirus combining v-ski with a temperature-sensitive version of the v-sea oncogene was constructed. In transformed erythroblasts, v-ski disturbed the concerted expression of several erythrocyte genes, leading to an abnormal erythroblast phenotype. Expression levels of hemoglobin and erythrocyte anion transporter (band 3) were elevated, while expression of the erythroid-specific histone H5 was strongly suppressed. v-ski could also be shown to repress or severely retard the temperature-induced erythroid differentiation of v-ski/ts-v-sea-transformed cells. The undifferentiated cells had an abnormal erythroblast or early reticulocyte phenotype characterized by unusually low levels of histone H5. In chicks, the v-ski/ts-v-sea virus displayed enhanced leukemogenicity compared with viruses containing just the single oncogenes. Thus, v-ski cooperates with tyrosine kinase oncogenes in a similar fashion to the v-erbA oncogene, however the pattern of genes affected by these two oncogenes is different.

Expression of adenovirus type 12 E1b 58-kDa protein in Escherichia coli and production of antibodies raised against a 58-kDa::beta-galactosidase fusion protein.

DNA fragments coding for the N-terminal 185 amino acids (aa) and for the entire coding region of the adenovirus (Ad)12 E1b 58-kDa protein have been cloned in a prokaryotic expression vector. The N-terminal region of the 58-kDa viral protein (aa 21-205) is expressed as a beta-galactosidase (beta Gal) fusion protein encoded by plasmid pB58Ngal. Escherichia coli strains transformed with this plasmid synthesize a full-length fusion protein of 150-kDa and two truncated proteins: a 140-kDa protein containing aa 64-205 and a 120-kDa polypeptide containing aa 158-205 of the E1b 58-kDa protein. Antibodies raised against purified fusion proteins specifically immunoprecipitate the E1b 58-kDa protein from Ad12-infected and transformed cells. Bacteria transformed with plasmid pB58 carrying the entire E1b 58-kDa coding region (minus the first N-terminal 20 aa which are replaced by 4 aa of beta Gal) showed dramatically reduced growth properties after induction of 58K gene expression. We have not been able to detect substantial amounts of the 58-kDa protein in these cells. However, the viral 58-kDa polypeptide could be synthesized in vitro from plasmid pB58 in a DNA-dependent translation system from E. coli.