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Background: Estrogen is known to decrease the risk of colon cancer in postmenopausal women, and may exert its actions by decreasing interleukin-6 (IL6) production via stabilization of the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB). Estrogens are biosynthesized by CYP19A1 (aromatase), so it is possible that genetic variations in CYP19A1 influences the risk of colon cancer by altering expression of CYP19A1. Further, studies on gene-gene interactions suggest that single nucleotide polymorphisms in one gene may affect expression of other genes. The current study aims to explore the role of CYP19A1 single nucleotide polymorphisms on CYP19A1, NFκB1 and IL6 gene expression. Methods: Phenotype-genotype associations, cross-associations between genes, and haplotype analyses were performed in both normal human colon (n=82) and liver (n=238) samples. Results: CYP19A1 rs10459592, rs1961177, and rs6493497 were associated with CYP19A1 expression in colon samples (P=0.042, P=0.041, and P=0.013, respectively). CYP19A1 single nucleotide polymorphisms (rs12908960, rs730154, rs8025191, and rs17523880) were correlated with NFκB1 expression (P=0.047, P=0.04, P=0.05, and P=0.03, respectively), and CYP19A1 rs11856927, rs2470152, and rs2470144 (P=0.049, P=0.025, P=0.047, respectively) were associated with IL6 expression in the colon. While rs730154 and rs17523880 could not be analyzed in the liver samples, none of the other associations with the colon were replicated in the liver samples. Haplotype analysis revealed three separate haplotypes of the CYP19A1 single nucleotide polymorphism that were significantly associated with CYP19A1, NFκB1, and IL6 gene expression. Conclusion: CYP19A1 single nucleotide polymorphisms are associated not only with CYP19A1 expression but also with NFκB1 and IL6 expression. These data demonstrate the possible functional consequences of genetic variation within the CYP19A1 gene on other genes in a biologically plausible pathway. [ABSTRACT FROM AUTHOR]

Background Sulfotransferase 1A1 (SULT1A1) gene expression is tissue specific, with little to no expression in normal breast epithelia. Expression in breast tumors has been documented, but the transcriptional regulation of SULT1A1 in human breast tissue is poorly understood. We identified Nuclear Factor I (NFI) as a transcription factor family involved in the regulation of SULT1A1 expression. Methods Transcription Factor Activation Profiling Plate Array assay was used to identify the possible transcription factors that regulate the gene expression of SULT1A1in normal breast MCF-10A cells and breast cancer ZR-75-1 cells. Expression levels of NFI-C and SULT1A1 were determined by real-time RT-PCR using total RNA isolated from 84 human liver samples. Expression levels of SULT1A1, NFI-A, NFI-B, NFI-C, and NFI-X were also determined in different human breast cancer cell lines (MCF-7, T-47D, ZR-75-1, and MDA-MB-231), in the transformed human epithelial cell line MCF-10A, and in ZR-75-1 cells that were transfected with siRNAs directed against NFI-A, NFI-B, NFI-C, or NFI-X for 48 h. The copy numbers of SULT1A1 in cell lines ZR-75-1, MCF-7, T-47D, MDA-MB-231, and MCF-10A were determined using a pre-designed Custom Plus TaqMan® Copy Number kit from Life Technologies. Results In normal human liver samples, SULT1A1 mRNA level was positively associated with NFIC. In different human breast cancer and normal epithelial cell lines, SULT1A1 expression was positively correlated with NFI-B and NFI-C. SULT1A1 expression was decreased 41% and 61% in ZR-75-1 cells treated with siRNAs against NFI-A and NFI-C respectively. SULT1A1 gene expression was higher in cells containing more than one SULT1A1 copy numbers. Conclusions Our data suggests that SULT1A1 expression is regulated by NFI, as well as SULT1A1 copy number variation in human breast cancer cell lines. These data provide a mechanistic basis for the differential expression of SULT1A1 in different tissues and different physiological states of disease. [ABSTRACT FROM AUTHOR]

Trehalose (α,α-1,1-glucosyl-glucose) is essential for the growth of mycobacteria, and these organisms have three different pathways that can produce trehalose. One pathway involves the enzyme described in the present study, trehalose synthase (TreS), which interconverts trehalose and maltose. We show that TreS from Mycobacterium smegmatis, as well as recombinant TreS produced in Escherichia coli, has amylase activity in addition to the maltose ↔ trehalose interconverting activity (referred to as MTase). Both activities were present in the enzyme purified to apparent homogeneity from extracts of Mycobacterium smegmatis, and also in the recombinant enzyme produced in E. coli from either the M. smegmatis or the Mycobacterium tuberculosis gene. Furthermore, when either purified or recombinant TreS was chromatographed on a Sephacryl S-200 column, both MTase and amylase activities were present in the same fractions across the peak, and the ratio of these two activities remained constant in these fractions. In addition, crystals of TreS also contained both amylase and MTase activities. TreS produced both radioactive maltose and radioactive trehalose when incubated with [3H]glycogen, and also converted maltooligosaccharides, such as maltoheptaose, to both maltose and trehalose. The amylase activity was stimulated by addition of Ca2+, but this cation inhibited the MTase activity. In addition, MTase activity, but not amylase activity, was strongly inhibited, and in a competitive manner, by validoxylamine. On the other hand, amylase, but not MTase activity, was inhibited by the known transition-state amylase inhibitor, acarbose, suggesting the possibility of two different active sites. Our data suggest that TreS represents another pathway for the production of trehalose from glycogen, involving maltose as an intermediate. In addition, the wild-type organism or mutants blocked in other trehalose biosynthetic pathways, but still having active TreS, accumulate 10- to 20-fold more glycogen when grown in high concentrations (≥ 2% or more) of trehalose, but not in glucose or other sugars. Furthermore, trehalose mutants that are missing TreS do not accumulate glycogen in high concentrations of trehalose or other sugars. These data indicate that trehalose and TreS are both involved in the production of glycogen, and that the metabolism of trehalose and glycogen is interconnected. [ABSTRACT FROM AUTHOR]

Trehalose is a nonreducing disaccharide of glucose (alpha,alpha-1,1-glucosyl-glucose) that is essential for growth and survival of mycobacteria. These organisms have three different biosynthetic pathways to produce trehalose, and mutants devoid of all three pathways require exogenous trehalose in the medium in order to grow. Mycobacterium smegmatis and Mycobacterium tuberculosis also have a trehalase that may be important in controlling the levels of intracellular trehalose. In this study, we report on the purification and characterization of the trehalase from M. smegmatis, and its comparison to the trehalase from M. tuberculosis. Although these two enzymes have over 85% identity throughout their amino acid sequences, and both show an absolute requirement for inorganic phosphate for activity, the enzyme from M. smegmatis also requires Mg(2+) for activity, whereas the M. tuberculosis trehalase does not require Mg(2+). The requirement for phosphate is unusual among glycosyl hydrolases, but we could find no evidence for a phosphorolytic cleavage, or for any phosphorylated intermediates in the reaction. However, as inorganic phosphate appears to bind to, and also to greatly increase the heat stability of, the trehalase, the function of the phosphate may involve stabilizing the protein conformation and/or initiating protein aggregation. Sodium arsenate was able to substitute to some extent for the sodium phosphate requirement, whereas inorganic pyrophosphate and polyphosphates were inhibitory. The purified trehalase showed a single 71 kDa band on SDS gels, but active enzyme eluted in the void volume of a Sephracryl S-300 column, suggesting a molecular mass of about 1500 kDa or a multimer of 20 or more subunits. The trehalase is highly specific for alpha,alpha-trehalose and did not hydrolyze alpha,beta-trelalose or beta,beta-trehalose, trehalose dimycolate, or any other alpha-glucoside or beta-glucoside. Attempts to obtain a trehalase-negative mutant of M. smegmatis have been unsuccessful, although deletions of other trehalose metabolic enzymes have yielded viable mutants. This suggests that trehalase is an essential enzyme for these organisms. The enzyme has a pH optimum of 7.1, and is active in various buffers, as long as inorganic phosphate and Mg(2+) are present. Glucose was the only product produced by the trehalase in the presence of either phosphate or arsenate.