Junko Nishitani

Overexpression of Human Papillomavirus Type 16 Oncoproteins Enhances Hypoxia-Inducible Factor 1α Protein Accumulation and Vascular Endothelial Growth Factor Expression in Human Cervical Carcinoma Cells

Purpose: Human papillomavirus (HPV)-16 oncoproteins, E6 and E7, are associated with enhanced tumor angiogenesis in human cervical cancers. The purpose of this study was (a) to investigate whether expression of HPV-16 E6 and E7 oncoproteins induces hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor expression in cervical cancer cells; and (b) to assess the effect of resveratrol on 16 E6- and E7-induced HIF-1α and VEGF gene expression. Experimental Design: Human cervical cancer cell lines C-33A and HeLa were transiently cotransfected with pSG5-HPV-16 E6 or 16 E7 constructs along with HIF-1α small interfering RNA (siRNA) or nonspecific siRNA. The expression of HIF-1α/VEGF was measured using real-time PCR, Western blot analysis, or ELISA. The in vitro angiogenic activity induced by 16 E6- and E7-transfected cells was examined. The effect of resveratrol on oncoprotein-induced HIF-1α/VEGF expression and in vitro angiogenesis was investigated. Results: HPV-16 E6- and E7-transfected cervical cancer cells express increased HIF-1α protein and VEGF expression. These stimulatory effects were abrogated by cotransfection with either HIF-1α siRNA or treatment with resveratrol. Blocking extracellular signal-regulated kinase 1/2 (ERK 1/2) and phosphoinositide-3-kinase by PD98059 and LY294002, respectively, abolished 16 E6- and E7-induced HIF-1α and VEGF expression. Functionally, we showed that HPV-16 E6- and E7-transfected cervical cancer cells stimulated in vitro capillary or tubule formation, and these angiogenic effects could be abolished either by cotransfection with HIF-1α siRNA or by treatment with resveratrol. Conclusion: HPV-16 oncoproteins contribute to enhanced angiogenesis in cervical cancer cells via HIF-1α–dependent VEGF expression. Resveratrol suppresses 16 E6- and E7-induced HIF-1α–mediated angiogenic activity and, thus, is a promising chemotherapeutic agent for human cervical cancer.

Human papillomavirus type 16 E6 and E7 oncoproteins upregulate c-IAP2 gene expression and confer resistance to apoptosis

Inhibition of apoptosis plays an important role in the cellular immortalization and transformation induced by E6 and E7 oncoproteins of human papillomavirus (HPV). Here, we report that the transcription of the inhibitor of apoptosis gene, cellular inhibitor of apoptosis protein 2, (c-IAP2), is significantly upregulated in HPV16 E6/E7-immortalized human oral keratinocytes (HOK16E6E7). Overexpression of E6/E7 from the high-risk HPV16 or 18, but not from the low-risk HPV6, activated c-IAP2 promoter. E6 from HPV16 and 18 played a major role in the activation. In addition, the induction of c-IAP2 transcription required nuclear factor-κB activity. Overexpression of c-IAP2 in normal human oral keratinocyte conferred resistance to tumor necrosis factor-α (TNF-α)/cycloheximide (CHX)-induced apoptosis, suggesting the increased c-IAP2 expression in HOK16E6E7 may protect the cells from TNF-α-mediated cell death. Moreover, depletion of endogenous c-IAP2 using RNA interference in HOK16E6E7 induced apoptosis, indicating that c-IAP2 is necessary for HPV16 E6/E7-induced resistance to apoptosis and cell survival. Of note, high levels of c-IAP2 transcription were found in several HPV16- or HPV18-positive cancer cells, and depletion of c-IAP2 caused cell death in HPV18-positive HeLa cells. Thus, upregulation of c-IAP2 by E6 and E7 may confer resistance to apoptosis that is necessary for sustained growth of some HPV16- and HPV18-positive cancer cells.

Human Immunodeficiency Virus Type 1 Infection and Replication in Normal Human Oral Keratinocytes

ABSTRACT Recent epidemiologic studies show increasing human immunodeficiency virus type 1 (HIV-1) transmission through oral-genital contact. This paper examines the possibility that normal human oral keratinocytes (NHOKs) might be directly infected by HIV or might convey infectious HIV virions to adjacent leukocytes. PCR analysis of proviral DNA constructs showed that NHOKs can be infected by CXCR4-tropic (NL4-3 and ELI) and dualtropic (89.6) strains of HIV-1 to generate a weak but productive infection. CCR5-tropic strain Ba-L sustained minimal viral replication. Antibody inhibition studies showed that infection by CXCR4-tropic viral strains is mediated by the galactosylceramide receptor and the CXCR4 chemokine coreceptor. Coculture studies showed that infectious HIV-1 virions can also be conveyed from NHOKs to activated peripheral blood lymphocytes, suggesting a potential role of oral epithelial cells in the transmission of HIV infection.

Novel Transcriptional Potentiation of BETA2/NeuroD on the Secretin Gene Promoter by the DNA-Binding Protein Finb/RREB-1

ABSTRACT The basic helix-loop-helix protein BETA2/NeuroD activates transcription of the secretin gene and is essential for terminal differentiation of secretin-producing enteroendocrine cells. However, in heterodimeric complexes with its partner basic helix-loop-helix proteins, BETA2 does not appear to be a strong activator of transcription by itself. Mutational analysis of a proximal enhancer in the secretin gene identified several cis-acting elements in addition to the E-box binding site for BETA2. We identified by expression cloning the zinc finger protein RREB-1, also known to exist as a longer form, Finb, as the protein binding to one of the mutationally sensitive elements. Finb/RREB-1 lacks an intrinsic activation domain and by itself did not activate secretin gene transcription. Here we show that Finb/RREB-1 can associate with BETA2 to enhance its transcription-activating function. Both DNA binding and physical interaction of Finb/RREB-1 with BETA2 are required to potentiate transcription. Thus, Finb/RREB-1 does not function as a classical activator of transcription that recruits an activation domain to a DNA-protein complex. Finb/RREB-1 may be distinguished from coactivators, which increase transcription without sequence-specific DNA binding. We suggest that Finb/RREB-1 should be considered a potentiator of transcription, representing a distinct category of transcription-regulating proteins.

HIV-1 infection in peripheral blood lymphocytes (PBLs) exposed to alcohol.

Epidemiological and in vitro studies have implied that heavy alcohol consumption may increase an individuals risk of HIV-1 infection. To examine the role of alcohol in direct infection of T-cells, viral reverse transcripts and HIV-1 receptor expression were examined in infected peripheral blood lymphocytes (PBLs) pretreated with alcohol. PCR results showed that alcohol increased HIV-1 DNA in PBLs by at least 10-fold. Alcohol enhanced the expression of the CXCR4 chemokine co-receptor but not the major HIV-1 CD4 receptor. Pretreatment with alcohol was also associated with increased intracellular cAMP. Thus, alcohol may facilitate enhanced viral infection by increasing the availability of HIV-1 co-receptor. This effect is associated with increases in intracellular cAMP.

Alcohol Enhances HIV Type 1 Infection in Normal Human Oral Keratinocytes by Up-regulating Cell-Surface CXCR4 Coreceptor

Recent studies suggest that normal human oral keratinocytes (NHOKs) can be infected by HIV-1, and alcohol can enhance HIV infection and replication in lymphocytes. In this study, we examined the possibility that alcohol might facilitate HIV-1 infection of NHOKs by up-regulating cell surface expression of the coreceptor, CXCR4. Alcohol enhanced in vitro infection of NHOKs by CXCR4-tropic strains of HIV-1 as indicated by synthesis of viral reverse transcripts and production of p24gag protein. Alcohol had no effect on CXCR4 gene expression or on total cellular complements of CXCR4 protein. However, alcohol did enhance the fraction of total CXCR4 expressed on the cell surface relative to intracellular stores. Alcohol-induced up-regulation of cell surface CXCR4 expression and HIV-1 infectivity could be blocked by SDF-1alpha-mediated internalization. These data suggest that alcohol may influence oral HIV transmission by altering the cellular compartmentalization of CXCR4 in cells of the oral cavity.

Partnering to harmonize IRBs for community-engaged research to reduce health disparities.

Emerging advances in health disparities research include controlled trials and comparative effectiveness studies that are frequently conducted at multiple community and academic sites. Review by different institutional review boards (IRBs) presents a major impediment to the timely and effective conduct of such research. When research involves minority and underserved communities as well as multiple geographic regions, institutional requirements and interpretation of ethical standards may vary substantially. Such variations can complicate the informed consent process and research protocol, and may undermine participant respect and trial quality. In addition, multiple IRB review can lead to unnecessary delays, jeopardizing funding and capacity to perform collaborative projects. In response to these issues, the Research Centers in Minority Institutions (RCMI) Translational Research Network (RTRN) is developing a community-partnered approach to streamlining IRB review across its consortium of 18 RCMI grantee institutions that will ensure compliance while enhancing the quality of health disparities research.

Potential biomarkers for head and neck squamous cell carcinoma.

Objective The purpose of the study was twofold: 1) to search for potential biomarkers that were overexpressed in cell lines that could represent both a clinical premalignant (immortalized) and a malignant state, and 2) to attempt to correlate metallothionein gene expression with clinical outcome in laryngeal carcinoma.

Overexpression of galectin-7 in head and neck SCC cell lines increases apoptosis

Objectives: The purpose of this study was to determine if overexpression of galectin-7 in squamous cell carcinoma cell lines increases cell apoptosis. Our previous study showed that galectin-7 was expressed in normal human oral keratinocytes (NHOK), down-regulated more than 10-fold in its immortalized derivative (HOK 16E6E7), and not expressed in head and neck squamous cell carcinoma. This suggests that galectin-7 may play a role in tumor suppressor function in oral keratinocytes. Methods: A series of in vitro experiments were utilized to determine the expression of galectin-7 in various head and neck squamous cell carcinoma cell lines and to compare their rates of apoptosis by transfection with gal-7/PCDNA3.1 construct and by Hoechst 33342 staining and DNA ladder analysis. Results: Hoechst 33342 staining showed that 60% of transfected cells had nuclear condensation suggesting apoptosis. This compared to only 5% cell nuclear condensation in SCC4 and SCC9. A clear DNA ladder is visible in transfected cell lines confirming apoptosis. Conclusions: Overexpression of galectin-7 in squamous cell carcinoma cell lines, SCC4 and SCC9, was associated with increase apoptosis. This suggests that galectin-7 may play a role in tumor suppressor function by promoting apoptosis. This work was supported by NIH/NIDCR K23 DE00430 and NIH/RCMI p20 RR11145.

11:20: YC1 Induces Apoptosis in SCCA by Inhibiting PI3K/Akt Pathway

PROBLEM: Despite advances in cancer therapy, morbidity and mortality rates of head and neck cancer have remained unchanged. The use of compounds directed specifically to molecular targets is a promising modality for enhancing tumor response to chemotherapy and prevention of tumor progression/resistance to improve organ preservation and overall survival. Hypoxia-inducible factor-1alpha (HIF-1alpha), a potential molecular target which is over-expressed in many human cancers and their metastases, has been correlated with a more aggressive tumor phenotype and confers resistance to radioand chemotherapies. The goal of this study is to investigate whether YC-1, a specific inhibitor of HIF-1alpha, can inhibit proliferation and induce apoptosis in human head & neck squamous cell carcinoma cells. METHODS: Head & neck squamous cell carcinoma (SCC) cell lines including SCC9, SCC29B, and SCC116, were cultured in the presence or absence of different concentrations of YC-1. Cell viability was tested using MTT assay, while the protein levels of HIF-1alpha, bcl-2, caspase-3, and the phosphorylated signaling components related to PI-3K/Akt/mTOR signaling pathway were determined by Western blotting. RESULTS: Treatment with different concentrations of YC-1 for 24-48 hours resulted in a dose-dependent decrease in the percentage of viable cells. Western blot analysis showed that YC-1 suppressed HIF-1alpha protein and several mediators of thePI-3K/Akt/mTOR signaling pathway including the phosphorylated Akt, 70S6K, and 4E-BP-1. The results also showed a dose-dependent increase in activated caspase-3 and a decrease in Bcl-2 protein following treatment with different concentrations of YC-1 for 24 hours. CONCLUSION: YC-1 induces apoptosis in head & neck squamous cell carcinoma cells possibly by interfering with PI-3K/Akt/mTOR/4E-BP signaling pathways, suggesting that inhibition of HIF-1alpha is a potential target for the treatment of human head & neck cancer. SIGNIFICANCE: HIF-1alpha can be a potential target to render human squamous cell carcinoma sensitive to current chemoradiotherapeutics.