Faculty Publications

PURPOSE: Squamous cell carcinoma (SCC) of the tongue is a common malignancy of the oral cavity. Furin convertase activates several precursor matrix metalloproteinases involved in the degrdn. of the extracellular matrix. The pattern of expression of furin and vascular endothelial growth factor-C (VEGF-C), two key mols. in neoplasm development, was examd. during the progression from normal epithelium to invasive SCC. Exptl. Design: We evaluated furin and VEGF-C expression and microvessel d. (MVD) by immunohistochem. in human tongue sections harboring normal epithelium, dysplastic epithelium, and/or SCC. Sections from 46 glossectomy specimens were assessed for furin expression. A selected group of 15 cases, each contg. normal epithelium, precursor lesions, and invasive SCC, were further studied for furin and VEGF-C expression and MVD quantification. We also evaluated the pattern of furin expression and VEGF-C processing by Western blot anal. in three SCC cell lines with different degrees of aggressiveness. RESULTS: Furin and VEGF-C expression was notably higher in most precursor lesions and SCCs than in normal epithelia. Approx. 60% (n = 26) and 100% (n = 15) of the normal epithelia showed low-intensity staining for furin and VEGF-C, resp. Intense staining for furin and VEGF-C was detected in .apprx.80% (n = 34) and 100% (n = 15) of the SCCs, resp. A significant correlation was seen between the expression of these two markers (Spearman's test, P < 0.00002). We found a statistically significant increase in MVD when either dysplasia (432 +- 19.06; P < 0.05) or SCC (546 +- 17.24) was compared with normal epithelium (315 +- 17.27; P < 0.0001). SCC71, the most aggressive cell line analyzed, was the one with the highest furin expression. This cell line totally processed the VEGF-C proform, whereas the less aggressive line SCC9, exhibiting the least furin expression, did not. SCC15, of intermediate aggressiveness and furin expression, showed intermediate pro-VEGF-C processing. CONCLUSIONS: These findings suggest that furin is a useful marker of tumor progression and is responsible for VEGF-C processing. This in turn would enhance angiogenesis, leading to increased MVD assocd. with preinvasive and invasive neoplasia. [on SciFinder (R)]

The chromosomal features that influence retroviral integration site selection are not well understood. Here, we report the mapping of 226 avian sarcoma virus (ASV) integration sites in the human genome. The results show that the sites are distributed over all chromosomes, and no global bias for integration site selection was detected. However, RNA polymerase II transcription units (protein-encoding genes) appear to be favored targets of ASV integration. The integration frequency within genes is similar to that previously described for murine leukemia virus but distinct from the higher frequency observed with human immunodeficiency virus type 1. We found no evidence for preferred ASV integration sites over the length of genes and immediate flanking regions. Microarray analysis of uninfected HeLa cells revealed that the expression levels of ASV target genes were similar to the median level for all genes represented in the array. Although expressed genes were targets for integr ation, we found no preference for integration into highly expressed genes. Our results provide a more detailed description of the chromosomal features that may influence ASV integration and support the idea that distinct, virus-specific mechanisms mediate integration site selection. Such differences may be relevant to viral pathogenesis and provide utility in retroviral vector design.

More than 25% of head and neck squamous cell carcinomas (HNSCC) and 99% of cervical cancers (CxCa) are positive for high-risk human papillomaviruses (HPVs). Furthermore, the type I tyrosine kinase receptor ErbB-2 is overexpressed in at least 30% of HNSCC and CxCa. Recently, we demonstrated that E6/E7 of HPV type 16 cooperate with ErbB-2 to induce cell transformation of human normal oral epithelial ( NOE) cells. This is accompanied by overexpression of cyclin D1 in NOE cells. To determine the role of cyclin D1 in E6/E7/ErbB-2 cooperation, we examined the independent effects of E6/E7 and ErbB-2, and the combined effect of E6/E7 and ErbB-2 in mouse normal embryonic. broblast (NEF), wild type (wt), and knockout cyclin D1 ( D1(-/-)) cells. We report that NEF-wt cells transduced with E6/E7 alone and E6/E7/ ErbB-2 together form small and large tumors in nude mice, respectively, as well as different sized colonies in soft agar; whereas ErbB-2 alone elicits neither tumor formation in vivo nor colony formation in soft agar. More importantly, E6/E7, ErbB-2 and E6/E7/ ErbB-2 together all fail to induce neoplastic transformation of cyclin D1(-/-) cells in vivo and in vitro. Furthermore, using antisense cyclin D1 we completely inhibited tumor and colony formation of NEF-wt-E6/E7 and wt-E6/E7-ErbB-2 as well as human NOE-E6/E7-ErbB-2-transformed cells. These analyses reveal that cyclin D1 is the downstream target of the neoplastic transformation induced by E6/E7 or E6/E7/ ErbB-2 cooperation in normal cells. Our data suggest that anti-cyclin D1 therapy may be highly specific in the treatment of all human cancers expressing high-risk HPVs or HPVs/ErbB-2.

BACKGROUND: The enzyme porphobilinogen synthase (PBGS), which is central to the biosynthesis of heme, chlorophyll and cobalamins, has long been known to use a variety of metal ions and has recently been shown able to exist in two very different quaternary forms that are related to metal ion usage. This paper reports new information on the metal ion independence and quaternary structure of PBGS from the photosynthetic bacterium Rhodobacter capsulatus. RESULTS: The gene for R. capsulatus PBGS was amplified from genomic DNA and sequencing revealed errors in the sequence database. R. capsulatus PBGS was heterologously expressed in E. coli and purified to homogeneity. Analysis of an unusual phylogenetic variation in metal ion usage by PBGS enzymes predicts that R. capsulatus PBGS does not utilize metal ions such as Zn2+, or Mg2+, which have been shown to act in other PBGS at either catalytic or allosteric sites. Studies with these ions and chelators confirm the predictions. A broad pH optimum was determined to be independent of monovalent cations, approximately 8.5, and the Km value shows an acidic pKa of ~6. Because the metal ions of other PBGS affect the quaternary structure, gel permeation chromatography and analytical ultracentrifugation experiments were performed to examine the quaternary structure of metal ion independent R. capsulatus PBGS. The enzyme was found to be predominantly hexameric, in contrast with most other PBGS, which are octameric. A protein concentration dependence to the specific activity suggests that the hexameric R. capsulatus PBGS is very active and can dissociate to smaller, less active, species. A homology model of hexameric R. capsulatus PBGS is presented and discussed. CONCLUSION: The evidence presented in this paper supports the unusual position of the R. capsulatus PBGS as not requiring any metal ions for function. Unlike other wild-type PBGS, the R. capsulatus protein is a hexamer with an unusually high specific activity when compared to other octameric PBGS proteins.

The role of quantitative viral load in development of hepatocellular carcinoma (HCC) among chronic hepatitis B virus (HBV) carriers was evaluated using real-time PCR (TaqMan PCR), a highly sensitive method for quantitative detection of HBV DNA. Serum samples collected at study entry from HCC cases and matched controls were chosen separately from ongoing prospective cohort studies in Senegal, West Africa, and Haimen City, China. For 14 HCC cases and 28 controls from Senegal, the relative risk (RR, 95% CI) of HCC was 15.6 (2.0-124.3) for those positive by the TaqMan PCR assay. Average length of follow-up (study entry to death from HCC) among cases was 2.8 (+/-1.6) years. The paired median difference between cases and controls was 3.8 x 10(4) virions/ml, with cases higher (P = 0.09). In order to clarify the relationship with lower-titer viremia, we selected 55 cases and 55 matched controls from the Chinese cohort all negative for serum HBV DNA by conventional dot blot hybridiza! tion. In this group, the RR associated with HBV DNA positivity by TaqMan PCR was 3.1 (1.1-9.2), with an average duration of follow-up of 3.3 (+/-2.1) years. The median difference in quantitative viremia between cases and controls was 6.0 x 10(4) virions/ml, with cases higher (P < 0.0001). Increased risk appeared to be confined to subjects with viral loads >2.3 x 10(4) virions/ml. In conclusion, HBV viremia, except perhaps at extremely low levels, is associated with increased risk for HCC in prospective studies of chronic carriers in two disparate populations.

Kidney cancer is curable by surgical resection and therapy, if detected at an early stage. Using sensitive methylation-specific polymerase chain reaction, we screened matched tumor DNA and preoperative urine DNA from 50 kidney cancer patients, for hypermethylation of a panel of six normally unmethylated tumor suppressor genes: VHL, p16/CDKN2a, p14ARF, APC, RASSF1A, and Timp-3. When compared to the tumor DNA, an identical pattern of gene hypermethylation was found in the matched urine DNA from 44 of 50 patients (88% sensitivity) including 27 of 30 cases of stage I disease. By contrast, hypermethylation was not observed in normal and benign disease controls (100% specificity). We conclude that promoter hypermethylation is a common and early event in kidney tumorigenesis and can be detected in the urine DNA from patients with organ-confined renal cancer of all histologic types.

The mitotic spindle checkpoint protein Bub1 has been found to be mutated at low frequency in certain human cancers characterized by aneuploidy. Simian virus 40 large T antigen efficiently immortalizes rodent cells and occasionally transforms them to tumorigenicity. T antigen can also cause genomic instability, inducing chromosomal aberrations and aneuploidy. Here, we report an interaction between Bub1 and T antigen. T antigen coimmunoprecipitates with endogenous Bub1 and Bub3, another component of the spindle checkpoint complex. Genetic analysis demonstrates that the interaction of T antigen with Bub1 is not required for immortalization but is closely correlated with transformation. T antigen induces an override of the spindle checkpoint dependent on Bub1 binding. This interaction with proteins of the spindle checkpoint machinery suggests another role for T antigen and provides insight into its ability to cause chromosomal aberrations, aneuploidy, and transformation.