High glucose decreases intracellular glutathione concentrations and upregulates inducible nitric oxide synthase gene expression in intestinal epithelial cells

Diabetes is associated with oxidative stress and increased levels of inflammatory cytokines. The aim of the study was to assess the effects of inflammatory cytokines and oxidative stress associated with raised glucose levels on inducible nitric oxide synthase (iNOS) promoter activity in intestinal epithelial cells. High glucose (25 mmol/l) conditions reduced glutathione (GSH) levels in the human intestinal epithelial cell line, DLD-1. Addition of the antioxidant alpha-lipoic acid resulted in the restoration of GSH levels to normal. Upregulation of basal iNOS promoter activity was observed when cells were incubated in high glucose alone. This effect was significantly reduced by the addition of the antioxidant, alpha-lipoic acid and completely blocked with inhibition of NFkappa B activity. Cytokine stimulation [interleukin-1 beta, tumor necrosis factor-alpha, interferon-gamma] induced iNOS promoter activity in all conditions and this was accompanied by an increase in nitric oxide (NO) production. Inhibition of NFkappa-B activity decreased but did not completely inhibit cytokine-induced iNOS promoter activity and subsequent NO production. In conclusion, high glucose-induced iNOS promoter activity is mediated in part through intracellular GSH and NFkappa-B.

Pilot-scale evaluation of the application of low pH-inducible polyphosphate accumulation to the biological removal of phosphate from wastewaters.

To investigate the possible biotechnological application of the phenomenon of low pH-inducible phosphate uptake and polyphosphate accumulation, previously reported using pure microbial cultures and under laboratory conditions, a 2000 L activated sludge pilot plant was constructed at a municipal sewage treatment works. When operated as a single-stage reactor this removed more than 60% of influent phosphate from primary settled sewage at a pH of 6.0, as opposed to approximately 30% at the typical operational pH for the works of 7.0-7.3-yet without any deleterious effect on other treatment parameters. At these pH values the phosphorus content of the sludge was, respectively, 4.2% and 2.0%. At pH 6.0 some 33.9% of sludge microbial cells were observed to contain polyphosphate inclusions; the corresponding value at pH 7.0 was 18.7%. Such a process may serve as a prototype for the development of alternative biological and chemical options for phosphate removal from wastewaters.

Cybr, a cytokine-inducible protein that binds cytohesin-1 and regulates its activity

Cytokines regulate lymphocyte development and differentiation, but precisely how they control these processes is still poorly understood. By using microarray technology to detect cytokine-induced genes, we identified a cDNA encoding Cybr, which was increased markedly in cells incubated with IL-2 and IL-12. The mRNA was most abundant in hematopoietic cells and tissues. The predicted amino acid sequence is similar to that of GRP-1-associated protein (GRASP), a recently identified retinoic acid-induced cytohesin-binding protein. Physical interaction, dependent on the coiled-coil domains of Cybr and cytohesin-1, was demonstrated by coimmunoprecipitation of the overexpressed proteins from 293T cells. Cytohesin-1, in addition to its role in cell adhesion, is a guanine nucleotide-exchange protein activator of ARF GTPases. Acceleration of guanosine 5'-O-(thiotriphosphate) binding to ARF by cytohesin-1 in vitro was enhanced by Cybr. Because the binding protein modified activation of ADP ribosylation factor by cytohesin-1, we designate this cytokine-inducible protein Cybr (cytohesin binder and regulator).

BRCA1 and c-Myc Associate to Transcriptionally Repress Psoriasin, a DNA Damage-Inducible Gene

Evidence is accumulating to suggest that some of the diverse functions associated with BRCA1 may relate to its ability to transcriptionally regulate key downstream target genes. Here, we identify S100A7 (psoriasin), S100A8, and S100A9, members of the S100A family of calcium-binding proteins, as novel BRCA1-repressed targets. We show that functional BRCA1 is required for repression of these family members and that a BRCA1 disease–associated mutation abrogates BRCA1-mediated repression of psoriasin. Furthermore, we show that BRCA1 and c-Myc form a complex on the psoriasin promoter and that BRCA1-mediated repression of psoriasin is dependent on functional c-Myc. Finally, we show that psoriasin expression is induced by the topoisomerase IIA poison, etoposide, in the absence of functional BRCA1 and increased psoriasin expression enhances cellular sensitivity to this chemotherapeutic agent. Therefore, we identified a novel transcriptional mechanism that is likely to contribute to BRCA1-mediated resistance to etoposide.

A common polymorphism in the oxygen-dependent degradation (ODD) domain of hypoxia inducible factor-1alpha (HIF-1alpha) does not impair Pro-564 hydroxylation

The hypoxia-inducible factor (HIF) transcription complex, which is activated by low oxygen tension, controls a diverse range of cellular processes including angiogenesis and erythropoiesis. Under normoxic conditions, the alpha subunit of HIF is rapidly degraded in a manner dependent on hydroxylation of two conserved proline residues at positions 402 and 564 in HIF-1alpha in the oxygen-dependent degradation (ODD) domain. This allows subsequent recognition by the von Hippel-Lindau (VHL) tumor suppressor protein, which targets HIF for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, prolyl hydroxylation of HIF is inhibited, allowing it to escape VHL-mediated degradation. The transcriptional regulation of the erythropoietin gene by HIF raises the possibility that HIF may play a role in disorders of erythropoiesis, such as idiopathic erythrocytosis (IE).

Characterization of a thymidylate synthase (TS)-inducible cell line: a model system for studying sensitivity to TS- and non-TS-targeted chemotherapies

Thymidylate synthase (TS) is responsible for the de novo synthesis of thymidylate, which is required for DNA synthesis and repair and which is an important target for fluoropyrimidines such as 5-fluorouracil (5-FU), and antifolates such as Tomudex (TDX), ZD9331, and multitargeted antifolate (MTA). To study the importance of TS expression in determining resistance to these agents, we have developed an MDA435 breast cancer-derived cell line with tetracycline-regulated expression of TS termed MTS-5. We have demonstrated that inducible expression of TS increased the IC(50) dose of the TS-targeted therapeutic agents 5-FU, TDX, and ZD9331 by 2-, 9- and 24-fold respectively. An IC(50) dose for MTA was unobtainable when TS was overexpressed in these cells, which indicated that MTA toxicity is highly sensitive to increased TS expression levels. The growth inhibitory effects of the chemotherapeutic agents CPT-11, cisplatin, oxaliplatin, and Taxol were unaffected by TS up-regulation. Cell cycle analyses revealed that IC(50) doses of 5-FU, TDX and MTA caused an S-phase arrest in cells that did not overexpress TS, and this arrest was overcome when TS was up-regulated. Furthermore, the S-phase arrest was accompanied by 2- to 4-fold increased expression of the cell cycle regulatory genes cyclin E, cyclin A, and cyclin dependent kinase 2 (cdk2). These results indicate that acute increases in TS expression levels play a key role in determining cellular sensitivity to TS-directed chemotherapeutic drugs by modulating the degree of S-phase arrest caused by these agents. Moreover, CPT-11, cisplatin, oxaliplatin, and Taxol remain highly cytotoxic in cells that overexpress TS.

DUB-3, a Cytokine inducible Deubiquitinating Enzyme That Blocks Profileration

Previous studies have identified the DUB family of cytokine-regulated murine deubiquitinating enzymes, which play a role in the control of cell proliferation and survival. Through data base analyses and cloning, we have identified a human cDNA (DUB-3) that shows significant homology to the known murine DUB family members. Northern blotting has shown expression of this gene in a number of tissues including brain, liver, and muscle, with two transcripts being apparent (1.6 and 1.7 kb). In addition, expression was observed in cell lines including those derived from a number of hematopoietic tumors such as the Burkitt's lymphoma cell line RAJI. We have also demonstrated that DUB-3, which was shown to be an active deubiquitinating enzyme, is induced in response to interleukin-4 and interleukin-6 stimulation. Finally, we have demonstrated that constitutive expression of DUB-3 blocks proliferation and can initiate apoptosis in both IL-3-dependent Ba/F3 cells and NIH3T3 fibroblasts. These findings suggest that human DUB-3, like the murine DUB family members, is transiently induced in response to cytokines and can, when constitutively expressed, block growth factor-dependent proliferation.

Identification of 5-Fluorouracil-inducible Target Genes Using cDNA Microarray Profiling

The fluoropyrimidine 5-Fluorouracil (5-FU) is widely used in the treatment of cancer. To identify novel downstream mediators of tumor cell response to 5-FU, we used DNA microarray technology to identify genes that are transcriptionally activated by 5-FU treatment in the MCF-7 breast cancer cell line. Of 2400 genes analyzed, 619 were up-regulated by >3-fold. Highly up-regulated genes (>6-fold) with signal intensities of >3000 were analyzed by Northern blot. Genes that were consistently found to be up-regulated were spermine/spermidine acetyl transferase (SSAT), annexin II, thymosin-beta-10, chaperonin-10, and MAT-8. Treatment of MCF-7 cells with the antifolate tomudex and DNA-damaging agent oxaliplatin also resulted in up-regulation of each of these targets. The 5-FU-induced activation of MAT-8, thymosin-beta-10, and chaperonin-10 was abrogated by inactivation of p53 in MCF-7 cells, whereas induction of SSAT and annexin II was significantly reduced in the absence of p53. Moreover, each of these genes contained more than one potential p53-binding site, suggesting that p53 may play an important regulatory role in 5-FU-induced expression of these genes. In addition, we found that basal expression levels of SSAT, annexin II, thymosin beta-10, and chaperonin-10 were increased (by approximately 2-3-fold), and MAT-8 expression dramatically increased (by approximately 10-fold) in a 5-FU-resistant colorectal cancer cell line (H630-R10) compared with the parental H630 cell line, suggesting these genes may be useful biomarkers of resistance. These results demonstrate the potential of DNA microarrays to identify novel genes involved in mediating the response of tumor cells to chemotherapy.

Cyclooxygenase-2 and inducible nitric oxide synthase gene polymorphisms and risk of reflux esophagitis, Barrett's Esophagus, and esophageal adenocarcinoma

The incidence of esophageal adenocarcinoma has increased in recent years, and Barrett's esophagus is a recognized risk factor. Gastroesophageal reflux of acid and/or bile is linked to these conditions and to reflux esophagitis. Inflammatory disorders can lead to carcinogenesis through activation of "prosurvival genes," including cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). Increased expression of these enzymes has been found in esophageal adenocarcinoma, Barrett's esophagus, and reflux esophagitis. Polymorphic variants in COX-2 and iNOS genes may be modifiers of risk of these conditions. In a population-based case-control study, we examined associations of the COX-2 8473 T>C and iNOS Ser 608 Leu (C>T) polymorphisms with risk of esophageal adenocarcinoma, Barrett's esophagus, and reflux esophagitis. Genomic DNA was extracted from blood samples collected from cases of esophageal adenocarcinoma (n = 210), Barrett's esophagus (n = 212), and reflux esophagitis (n = 230) and normal population controls frequency matched for age and sex (n = 248). Polymorphisms were genotyped using TaqMan allelic discrimination assays. Odds ratios and 95% confidence intervals were obtained from logistic regression models adjusted for potential confounding factors. The presence of at least one COX-2 8473 C allele was associated with a significantly increased risk of esophageal adenocarcinoma (adjusted odds ratio, 1.58; 95% confidence interval, 1.04-2.40). There was no significant association between this polymorphism and risk of Barrett's esophagus or reflux esophagitis or between the iNOS Ser 608 Leu polymorphism and risk of these esophageal conditions. Our study suggests that the COX-2 8473 C allele is a potential genetic marker for susceptibility to esophageal adenocarcinoma.

The radiation-inducible pE9 promoter driving inducible nitric oxide synthase radiosensitizes hypoxic tumour cells to radiation

Driving high-level transgene expression in a tumour-specific manner remains a key requirement in the development of cancer gene therapy. We have previously demonstrated the strong anticancer effects of generating abnormally high levels of intracellular NO• following the overexpression of the inducible nitric oxide synthase (iNOS) gene. Much of this work has focused on utilizing exogenously activated promoters, which have been primarily induced using X-ray radiation. Here we further examine the potential of the pE9 promoter, comprising a combination of nine CArG radio-responsive elements, to drive the iNOS transgene. Effects of X-ray irradiation on promoter activity were compared in vitro under normoxic conditions and various degrees of hypoxia. The pE9 promoter generated high-level transgene expression, comparable with that achieved using the constitutively driven cytomegalovirus promoter. Furthermore, the radio-resistance of radiation-induced fibrosarcoma-1 (RIF-1) mouse sarcoma cells exposed to 0.1 and 0.01% O2 was effectively eliminated following transfection with the pE9/iNOS construct. Significant inhibition of tumour growth was also observed in vivo following direct intratumoural injection of the pE9/iNOS construct compared to empty vector alone (P<0.001) or to a single radiation dose of 10?Gy (P<0.01). The combination of both therapies resulted in a significant 4.25 day growth delay compared to the gene therapy treatment alone (P<0.001). In summary, we have demonstrated the potential of the pE9/iNOS construct for reducing radio-resistance conferred by tumour cell hypoxia in vitro and in vivo, with greater tumour growth delay observed following the treatment with the gene therapy construct as compared with radiotherapy alone.