Yeou-Ping Tsao

The Expression of HPV-16 E5 Protein in Squamous Neoplastic Changes in the Uterine Cervix

To investigate the expression of human papillomavirus type 16 (HPV-16) E5 protein in squamous neoplastic changes in the uterine cervix, the specific E5 antibody was generated and used to identify the expression of E5 protein in 40 cases of HPV-16-positive tissues and 5 previously identified HPV-negative normal cervical tissues. The results revealed that E5 protein was primarily expressed in the lower third of the epithelium in low-grade squamous intraepithelial lesions (SILs) and throughout the whole epithelium in high-grade SILs. In invasive squamous carcinoma, 60% of HPV-16-infected cancers which contained the episomal viral genome had the E5 gene, and could express E5 protein which was located throughout the whole epithelium. Previously, we documented the expression of type I growth factor receptors [ERBB1/EGFR (epidermal growth factor receptor), ERBB2, ERBB3 and ERBB4] in the full range of cervical neoplasias by immunohistochemistry assay. Hence, in this study, we extensively analyzed the correlation between the expression of E5 protein and the expression of type I growth factor receptors. Among 40 HPV-16- infected cervical neoplasias, we found that the expression of E5 protein was significantly correlated with either the expression of the ERBB1 or the ERBB4 receptor.

Characterization and analysis of human papillomaviruses of skin warts

We analysed human papillomavirus (HPV) infections in 61 tissue specimens of skin warts of Taiwanese patients by DNA hybridization. The prevalence of HPV infection was 69% by Southern blot hybridization. The typing of HPVs was performed by dot blot hybridization under highly stringent conditions with each probe separately. The prevalence of HPV-1, 2/3, 4, 5, 8, 11, 16 and 18 in skin warts was 13, 7, 16, 2, 0, 5, 2 and 8%, respectively. Chi-squared analysis revealed that there was a correlation between HPV type and copy number. Most HPV-4-induced warts were verruca vulgaris. HPV-1 DNA was detected in verruca plantaris and verruca vulgaris. No specific histopathological features were found to be indicative of the presence or absence of HPV, or of the various types of HPV infection.

The regulation mechanism of c-jun and junB by human papillomavirus type 16 E5 oncoprotein

SummaryIn this study, we show that HPV-16 E5 induced anchorage-independent growth in immortalized human epidermal keratinocytes and that HPV-16 E5 in human keratinocytes had higher expression of c-jun and junB; also, we investigated the role of transcriptional initiation pathways in the expression elevation. In addition, Ras-dependent pathway, as well as PKC-dependent pathway, leads to HPV-16 E5-induced c-jun gene expression.

ErbB4 (JM-b/CYT-1)-induced expression and phosphorylation of c-Jun is abrogated by human papillomavirus type 16 E5 protein

Human papillomavirus type 16 E5 (HPV-16 E5) is a highly hydrophobic membrane protein with weak-transforming activity, which is associated with ErbB4 receptor in HPV-16-infected cervical lesions. Presently, we investigated the transforming mechanisms of E5 involving ErbB4 signaling. Firstly, we report a role for ErbB4 (JM-b/CYT-1) receptor that activates c-jun gene expression and phosphorylating at Ser63 and Ser73 of the c-Jun protein in ligand-independent and Ras-c-jun NH2-terminal kinase-dependent pathway. Secondly, we show that HPV-16 E5 protein can form a complex with ErbB4 via binding to the extracellular and transmembrane domains of ErbB4 (JM-b/CYT-1). When co-expressing HPV-16 E5 and ErbB4 in cells, E5 can abrogate ErbB4-induced c-Jun protein expression and phosphorylation resulted in increasing cell proliferation compared to ErbB4-expressing cells. The interaction between of HPV-16 E5 and ErbB4 provides more insight into the mechanisms of HPV-16 E5 transformation induction.

Co-vaccination with adeno-associated virus vectors encoding human papillomavirus 16 L1 proteins and adenovirus encoding murine GM-CSF can elicit strong and prolonged neutralizing antibody

Non‐infectious human papillomavirus‐like particles (VLPs), encoded by the major capsid gene L1, have been shown to be effective as vaccines to prevent cervical cancer. We have developed the genetic immunization of the L1 gene to induce a neutralizing antibody. We constructed and generated a recombinant adeno‐associated virus encoding human papillomavirus (HPV) 16 L1 protein that could form virus‐like particles in transduced cells. Previous reports have demonstrated that the formation of VLP is necessary to induce high titers of neutralizing antibodies to protect an animal from viral challenge. Therefore, we carried out a single intramuscular (i.m.) injection with recombinant adeno‐associated virus encoding HPV‐16 L1 protein (rAAV‐16L1) in BALB/c mice, which ultimately produced stronger and more prolonged neutralizing L1 antibodies, when compared to the DNA vaccine. Immunohistochemistry showed that the accumulation of antigen presenting cells, such as macrophages and dendritic cells, in rAAV‐16L1 and L1 DNA‐injected muscle fibers may be due to the L1 protein expression, but not to AAV infection. When compared to the L1 VLP vaccine, however, the titers of neutralizing L1 antibodies induced by VLP were higher than those induced by rAAV‐16L1. Co‐vaccinating with rAAV‐16L1 and adenovirus encoding murine GM‐CSF (rAAV‐16L1/rAd‐mGM‐CSF) induced comparable higher levels of neutralizing L1 antibodies with those of VLP. This implies that a single i.m. co‐injection with rAAV‐16L1/rAd‐mGM‐CSF can achieve the same vaccine effect as a VLP vaccine requiring 3 booster injections.

Sequence variants and functional analysis of human papillomavirus type 16 E5 gene in clinical specimens.

Summary.u2002Previously, we found that the E5 protein can be expressed in HPV-16 infected precancerous lesions and cervical cancer [4]. In this study, we investigated the presence of sequence variants of E5 in HPV-16 infected tissues. Toward this end, we amplified the E5 gene by polymerase chain reaction from 29 HPV-16 infected tissues including eight normal tissues, seven high grade neoplastic tissues (high grade squamous intraepithelial lesions (HSIL) and 14 cervical cancer tissues. Sequence analysis demonstrated that there were three mutational hot spots at positions 3979, 4042, and 4077 of the HPV-16 DNA; these and other mutations resulted in six variants in the E5 sequence. This resulted in four E5 protein mutants, named WTE5 [wild type E5 protein], 14E5, 21E5 and 56E5. Functional analysis of these four mutant proteins revealed that the transforming activities of 14E5, 21E5 and 56E5 were 0.95, 0.59, and 0.89 fold of WTE5, respectively. Although E5 was expressed in all of the HSIL and cervical cancer tissues, but in only one of the eight normal tissues tested, only WT E5 protein was found in HSIL while in cervical cancer tissues both WT and mutant E5 proteins were detected. Since these E5 proteins exhibited the same in vitro transforming activity, these data suggest that expression of E5 is important in development and progression toward malignancy but mutation of E5 does not affect the transformation process.

Differential induction and regulation of c-jun, junB, junD and c-fos by human papillomavirus type 11 E5a oncoprotein

The E5a gene of human papillomavirus type 11 (HPV-11) is a transforming oncogene. In this study, we investigated the mechanism of E5a induced transformation. Our results show that the expression of c-jun and junB, but not junD, was activated by HPV-11 E5a in NIH 3T3 cells and human epidermal keratinocytes. However, the expression of c-fos was activated by E5a in NIH 3T3 cells, but not in keratinocytes. We further investigated the mechanism of c-jun and junB induction by E5a. The amount of c-jun and junB RNAs correlated with the amount of E5a RNA in the heavy metal inducible system. E5a constitutively activated the expression of c-jun and junB at the initiation of transcription level. In addition, analyses of the effect of serum on c-jun expression in E5a transformed human epidermal keratinocytes show that EGF might have a stimulatory effect on c-jun gene expression in E5a expressing keratinocytes.

E5a gene of human papillomavirus type 11 is required for initiation but not for maintenance of transformation in NIH 3T3 cells

We have previously shown that the E5a gene of human papilloma virus type 11 (HPV-11/HPV-6c) is a transforming oncogene. In order to dissect the biological consequences of E5a gene expression we utilized the lac operator/repressor system to manipulate E5a gene expression. Cells were cotransfected with the lac repressor gene and the E5a gene that had been inserted downstream of a simian virus 40 (SV40) promoter containing the lac operator sequence. The expression of E5a gene could therefore be repressed by binding of lac repressor to the lac operator sequence in proximity to this SV40 regulatory region. The transfected cells were cultured in the presence of the inducer IPTG and under G418 selection. IPTG derepressed E5a gene expression by binding to the repressor and reducing its affinity for the lac operator sequence. In these studies, we found that E5a-transformed cells still maintained the transformed phenotype as judged by growth density, cell morphology and anchorage-independent growth when E5a gene expression was repressed. We also found that c-jun expression was induced 3 h after E5a expression was induced by IPTG and c-jun expression was not shut down after repression of E5a expression. This is the first demonstration that the E5a gene of HPV-11 initiates transformation of NIH 3T3 cells but is dispensable for maintenance of the transformed phenotype.

Antisense oligodeoxynucleotides to c-jun inhibits proliferation of transformed NIH 3T3 cells induced by E5a of HPV-11.

E5a of HPV-11 is a transforming oncogene. Previously, we had shown that the E5a gene is required only for the initiation of transformation; c-jun might be involved in the maintenance of transformation. In this study, we exposed E5a transformed NIH 3T3 cells to antisense oligodeoxynucleotides complementary to the 24 nucleotides corresponding to the translation initiation site of the c-jun gene, and examined the effects of this treatment on cell proliferation. Results show that antisense c-jun oligodeoxynucleotides could repress c-jun production and inhibit cell proliferation in E5a transformed cells.

Protein kinase C- and ras-dependent activation of c-jun gene by human papillomavirus type 11 E5a oncoprotein

E5a of HPV-11 is a transforming oncogene. Previously, we have shown that E5a constitutively activates the expression of protooncogene c-jun by transcriptional regulation through the AP-1 binding site in the c-jun promoter. In the present study, we used two different types of cells: the E5a transfected NIH 3T3 cells and human epidermal keratinocytes, and selectively inhibited different signal transduction pathways to investigate effects of E5a on c-jun expression. We find that protein kinase C and ras-dependent pathways are important for the c-jun induction by E5a, but not the cAMP-dependent pathway.