Amylin gene expression mediated by cAMP/PKA and transcription factors HNF-1 and NFY.

The regulation of amylin gene expression is not clearly understood. In this study, we used the pancreatic beta cell line, INS-1, to evaluate the regulation of amylin gene expression. When INS-1 cells were cultured in media containing IBMX, the ratio of amylin secretion to insulin secretion increased by 200%. This coincided with an increase in amylin mRNA content. Moreover, there was a three to four-fold increase in amylin promoter activity which was inhibited by the specific protein kinase A (PKA) inhibitor, H89. Electrophoretic mobility shift assays (EMSA) demonstrated that IBMX induced protein-DNA binding to the FLAT and CAAT elements of the amylin promoter. Competitive EMSA experiments revealed that these proteins are likely to be HNF-1 and NFY, respectively. IBMX-induced amylin promoter activity was inhibited by mutations in the FLAT and CAAT elements. These results indicate that amylin is positively regulated by cAMP and PKA through the transcription factors HNF-1 and NFY.

Multiple primary cancer, including transitional cell carcinoma of the upper uroepithelial tract in a multigeneration HNPCC family: molecular genetic, diagnostic, and management implications.

OBJECTIVE: We report a multigeneration family where colorectal cancer and cancer of multiple diverse anatomic sites, inclusive of transitional cell carcinoma of the upper uroepithelial tract, were manifested in several relatives. METHODS: A specific pattern of cancer of the colorectum, endometrium, ovary, small bowel, and transitional cell carcinoma, with a vertical distribution of this cancer phenotype through multiple generations, was consonant with a diagnosis of hereditary nonpolyposis colorectal cancer. RESULTS: Germline mutation testing identified the MSH2 mutation, which segregated with the cancer phenotype. This family study clearly demonstrates the value of genetic testing in the management and treatment decision process. CONCLUSIONS: We document, perhaps for the first time, how molecular genetic testing in hereditary nonpolyposis colorectal cancer can aid in the identification of a potential renal transplant donor for a relative with the MSH2 mutation who is experiencing renal insufficiency secondary to transitional cell carcinoma.

BVL-1-like VL30 promoter sustains long-term expression in erythroid progenitor cells.

Congenital blood disorders are common and yet clinically challenging globin disorders. Gene therapy continues to serve as a potential therapeutic method to treat these disorders. While tremendous advances have been made in vivo, gene delivery protocols and vector prototypes still require optimization. Alternative cis-acting promoter elements derived from VL30 retroelements have been effective in expressing tissue-specific transgene expression in vivo in nonerythroid cells. VL30 promoter elements were isolated from ELM-I-1 erythroid progenitor cells upon erythropoietin (epo) treatment. These promoters were inserted into a VL30-derived expression vector and reintroduced into the ELM-I-1 cells. beta-Galactosidase reporter gene activity from the ELM 5 clone, a BVL-1-like VL30 promoter, was capable of expressing sustained levels of the transgene expression over a 16-week assay period. These findings delineate the potential utility of these retroelement promoters as transcriptionally active, erythroid-specific, long terminal repeat (LTR) components for current globin vector constructs.

Conserved, erythropoietin-responsive VL30 promoters isolated from erythroid progenitor cells.

Virus-like 30S (VL30) elements are endogenous retro-elements of the mouse retrotransposon family. These elements are transcriptionally responsive in a temporal and tissue-specific manner due to the U3 promoter region of the elements' long terminal repeat (LTR). We have analyzed VL30 promoters from erythroid progenitor cell lines (MEL 585S and ELM-I-1) that contrasted in their response to erythropoietin (epo). Through RT-PCR-generated cDNAs, VL30 promoters were identified and showed homology to the third and fourth U3 subgroups, with GATA-1, Jak2/STAT5, and B10 RRE sites. One clone (ELM5) showed 97% homology to BVL-1, a putative epo-responsive VL30 element. In addition, a novel U3 promoter (MEL/ELM CONSTIT) showed complete sequence homology between both cell lines. Ribonuclease protection confirmed that epo-induced VL30 promoters were activated in ELM-I-1 cells, whereas the conserved VL30 MEL-ELM CONSTIT VL30 promoter showed no enhanced expression in the epo-unresponsive MEL cells. Identification of these U3 promoters suggests that VL30s are conserved and can be transcriptionally activated in an epo-specific manner.

The role of oxygen-derived free radicals and nitric oxide in cytokine-induced antiproliferation of pancreatic cancer cells.

INTRODUCTION: Pancreatic cancer cells are susceptible to antiproliferative effects of cytokines such as tumor necrosis factor-alpha (TNF-alpha). However, little is known about the mechanisms involved. AIM: To determine the mechanisms of the antiproliferative effects of TNF-alpha on pancreatic cancer cells. METHODOLOGY AND RESULTS: In the current study, four of five pancreatic cancer cell lines tested were responsive to the antiproliferative effects of TNF-alpha. In two cell lines, the effects of TNF-alpha were completely abolished by superoxide dismutase, suggesting that superoxide anion mediates the effects. Further, inhibition of nitric oxide (NO) synthase by L-NAME potentiated the TNF-alpha response, suggesting a protective role for endogenously produced NO in these two cell lines. MiaPaCa-2 cells, which were unresponsive to the antiproliferative effects of TNF-alpha, produced five times more nitrite than the other cell lines. Treatment of MiaPaCa-2 cells with interferon-gamma (IFN-gamma) increased inducible nitric oxide synthase mRNA as shown by reverse transcription-polymerase chain reaction. This induction was potentiated by TNF-alpha and further enhanced by a combination of three cytokines (INF-gamma, TNF-alpha, and interleukin 1-beta (IL-1beta). This combination of cytokines increased nitrite accumulation fourfold and inhibited the proliferation of this resistant cell line. These effects were prevented by L-NAME. CONCLUSIONS: These studies suggest that TNF-alpha inhibits proliferation of pancreatic cancer cells by increasing the production of superoxide anion and that endogenously produced NO protects against this effect. In addition, there is a direct correlation between the amount of NO produced and resistance to TNF-alpha in the only resistant cell line. In contrast, treatment with a combination of IFN-gamma, TNF-alpha and IL-1beta upregulates inducible nitric oxide synthase, and the resulting markedly enhanced NO production inhibits pancreatic cancer cell growth. This pathway may provide a valuable target for therapy of this devastating disease.