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.

Overexpression of human papillomavirus type 16 oncoproteins enhances hypoxia-inducible factor 1 alpha 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 1 alpha (HIF-1 alpha) 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-1 alpha 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-1 alpha small interfering RNA (siRNA) or nonspecific siRNA. The expression of HIF-1 alpha/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-1 alpha/VEGF expression and in vitro angiogenesis was investigated. RESULTS: HPV-16 E6- and E7-transfected cervical cancer cells express increased HIF-1 alpha protein and VEGF expression. These stimulatory effects were abrogated by cotransfection with either HIF-1 alpha 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-1 alpha 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-1 alpha siRNA or by treatment with resveratrol. CONCLUSION: HPV-16 oncoproteins contribute to enhanced angiogenesis in cervical cancer cells via HIF-1 alpha-dependent VEGF expression. Resveratrol suppresses 16 E6- and E7-induced HIF-1 alpha-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-kappaB activity. Overexpression of c-IAP2 in normal human oral keratinocyte conferred resistance to tumor necrosis factor-alpha (TNF-alpha)/cycloheximide (CHX)-induced apoptosis, suggesting the increased c-IAP2 expression in HOK16E6E7 may protect the cells from TNF-alpha-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.

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.

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 individual's 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.

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. STUDY DESIGN: A series of in vitro experiments were used to unearth differentially expressed genes among normal, immortalized and tumorigenic cell lines. Secondarily, a retrospective analysis was undertaken. METHODS: Differential display analysis was conducted to identify differentially expressed genes between human papillomavirus-infected immortalized HOK16B and benzo[ ]pyrene-derived tumorigenic cell line, HOK16B-BaP-T. The cell-specific expressions were examined by Northern blot analysis and compared with other known immortalized and cancer cell lines. Immunohistochemical staining was also conducted to localize metallothionein (MT I/II) protein expression among the different cell lines studied. A retrospective analysis of laryngeal specimens from archival tissues of 29 cancer patients who underwent primary surgical resection was also undertaken after immunohistochemical staining. RESULTS: Twenty-one differentially expressed complementary cDNA clones, both novel and known, were identified using the differential display analysis. Northern blot analysis confirmed that clone 6 hybridized to a 1.6-kb RNA in HOK16B-Bap-T cell line. Clone 4 showed decreased expression in immortalized and cancer cell compared with NHOK. MT I/II transcript was observed in HOK16B, which was further elevated in HOK16B-Bap-T. Retrospective analysis showed that high immunoreactivity to MT I/II in surgically resected laryngeal cancer specimen correlated with increased frequency of recurrence within 2 years of surgery. CONCLUSION: These findings suggest that clone 4 may potentially function as a tumor suppressor gene, which may be significant in tumor progression and invasion. Clone 6 may participate in viral-mediated oncogenic transformation of normal cells. Clone 6 may also have potential as a tumor maker differentiating normal from malignant tissue, as in the determination of surgical resection margins. MT I/II gene product may serve as a prognostic biomarker for laryngeal squamous cell carcinoma. The differentially expressed genes and gene products may serve as sensitive biomarkers for improved early detection, diagnosis, and prognosis of head and neck squamous cell carcinoma.

Human immunodeficiency virus type 1 infection and replication in normal human oral keratinocytes.

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.

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.

Identification of genes required for immortalization in human papillomavirus-infected human oral keratinocytes.

The early stages of head and neck cancer are presumed to require a senes of genetic alterations that are not represented by a distinct clinical phenotype. Therefore, genes with altered expression in the preneoplasia may be useful for the early detection of this highly recurrent cancer. In this study, we immortalized normal human oral keratinocytes (NHOK) by retroviral-mediated infection of HPV 16 transforming oncogenes, E6 and E7 (HOK16E6E7). Using the Affymetrix gene chip (U95Av2), we identified 177 known genes and EST that were overexpressed at least 3-fold or above in the immortalized cells, while 133 were down-regulated compared to NHOK. Northern blot analysis showed elevated levels of p55CDC in the immortalized cells, while NHOK showed high basal expression of small proline rich protein (SPRR2). The altered expression of these genes maybe associated with cellular proliferation or differentiation and the early stages of oral carcinogenesis.