Burkholderia pseudomallei kills the nematode Caenorhabditis elegans using an endotoxin-mediated paralysis.

We investigated a non-mammalian host model system for fitness in genetic screening for virulence-attenuating mutations in the potential biowarfare agents Burkholderia pseudomallei and Burkholderia mallei. We determined that B. pseudomallei is able to cause 'disease-like' symptoms and kill the nematode Caenorhabditis elegans. Analysis of killing in the surrogate disease model with B. pseudomallei mutants indicated that killing did not require lipopolysaccharide (LPS) O-antigen, aminoglycoside/macrolide efflux pumping, type II pathway-secreted exoenzymes or motility. Burkholderia thailandensis and some strains of Burkholderia cepacia also killed nematodes. Manipulation of the nematode host genotype suggests that the neuromuscular intoxication caused by both B. pseudomallei and B. thailandensis acts in part through a disruption of normal Ca2+ signal transduction. Both species produce a UV-sensitive, gamma-irradiation-resistant, limited diffusion, paralytic agent as part of their nematode pathogenic mechanism. The results of this investigation suggest that killing by B. pseudomallei is an active process in C. elegans, and that the C. elegans model might be useful for the identification of vertebrate animal virulence factors in B. pseudomallei.

Correlation of aromatase and cyclooxygenase gene expression in human breast cancer specimens.

Aromatase, the enzyme system catalyzing estrogen biosynthesis, is found in stromal tissue in the breast. The increased expression of the aromatase CYP19 gene in breast cancer tissues was recently associated with a promoter region regulated through cAMP-mediated pathways. PGE2, derived from cyclooxygenase, increases intracellular cAMP levels and stimulates estrogen biosynthesis. This association suggest that local production of PGE2 via cyclooxgenase isozymes may influence estrogen biosynthesis. The present study represents the first to examine the levels of mRNA expression of CYP19, COX-1, and COX-2 genes in human breast cancer specimens and normal breast tissue samples using semi-quantitative RT-PCR methods. Positive correlations were observed between CYP19 and COX-2 and the greater extent of breast cancer cellularity. Linear regression analysis using a bivariate model shows a strong linear association between CYP19 expression and the sum of COX-1 and COX-2 expression. This significant relationship between the aromatase and cyclooxygenase enzyme systems suggests that autocrine and paracrine mechanisms may be involved in hormone-dependent breast cancer development via growth stimulation from local estrogen biosynthesis.

Effects of matrix components on aromatase activity in breast stromal cells in culture.

Local estradiol production within breast tissue is maintained by the aromatase cytochrome P450arom complex, which has been localized primarily to the stromal component of tumors but also has been detected in the breast epithelial cells. Paracrine interactions between stromal and epithelial components of the breast are critical to the sustained growth and progression of breast tumors. Maintenance of the differentiated state, including hormone and growth factor responsiveness, requires extracellular matrix proteins as substrata for cells. This research has focused on developing a cell culture system that more closely mimics in vivo interactions in order to dissect actual paracrine signaling between these two cell types. Human fibroblasts were isolated from breast tissue and were maintained in a cell culture system grown on plastic support or on a collagen I support matrix. The collagen I matrix model supports cell maintenance and subsequent differentiation on collagen rather than maximal proliferation, therefore allowing for a more accurate environment for the study of hormonal control and cellular communication. Initial experiments compared aromatase activity of patient fibroblasts grown on plastic versus collagen I using the tritiated water release method. Constitutive aromatase activity was found to be lower when cells were grown on a collagen gel for 4-7 days (7.7 fold lower) using DMEM/F12 containing 10% dextran coated charcoal stripped serum. However, fibroblasts grown on collagen I appeared to be significantly more responsive to stimulation by 100 nM dexamethasone (plastic: 6.0 fold induction, collagen: 33.2 fold induction) when pretreated for 12 h prior to measurement of aromatase activity. In an effort to examine paracrine interactions between the stromal and epithelial cells in breast tissue, experiments using conditioned media from fibroblast cultures were performed. Testosterone administration to fibroblasts results in the production of estradiol into the media in sufficient concentrations to elicit an increase in pS2 expression when the conditioned media is administered to MCF-7 cells. The addition of a potent aromatase inhibitor resulted in a complete suppression of fibroblast-derived estrogens and showed only a modest increase in pS2 expression. Culturing breast fibroblasts and epithelial cells on extracellular matrix allows for a more meaningful examination of the paracrine interactions between these cell types within the context of an appropriate extracellular environment. This study highlights the need for evaluation of gene expression in cell culture systems that accurately reflect the tissue microenvironment.

Androgens influence estrogen-induced responses in human breast carcinoma cells through cytochrome P450 aromatase.

The aromatase cytochrome P450 complex is responsible for the in vivo conversion of androgens to estrogens. Although breast cancer epithelial cells have been reported to have appreciable aromatase activity, its biologic significance remains uncertain. To address this, the effect of androgens on the expression of the estrogen-regulated gene pS2 in hormone-dependent human breast carcinoma cells in vitro was examined. Steroid-deprived MCF-7 cells were exposed to varying concentrations (1 nM, 10 nM, and 100 nM of androstenedione or testosterone for 2,4, and 6 days. Baseline aromatase activity was 4.9 (+/-3.1) fmol 3H2O/hour/microgram DNA [34.3 (+/-21.3) fmol/hr/10(6) cells] and was not influenced by the androgens. As an indication of estrogen biosynthesis, northern analysis was performed to quantitate pS2 mRNA expression. Although no significant pS2 induction was observed at 2 days, both 4 and 6 day exposure to 100 nM testosterone resulted in a 3-fold increase in pS2 mRNA expression. 5 alpha-dihydrotestosterone (5 alpha-DHT) failed to elicit a similar pS2 response. This testosterone-induced response was inhibited with the aromatase inhibitor 7 alpha (4'-amino) phenylthio-1,4-androstadiene-3,17-dione (7 alpha-APTADD) and with 10 microM tamoxifen. MCF-7 breast cancer cells possess endogenous aromatase activity at high enough levels to convert androgens to estrogens and elicit an estrogen-induced response. The expression of aromatase may offer a potential advantage to hormone-responsive cells, providing an additional autocrine growth pathway which may be exploited.

Biochemistry and pharmacology of 7alpha-substituted androstenediones as aromatase inhibitors.

The inhibition of aromatase, the enzyme responsible for converting androgens to estrogens, is therapeutically useful for the endocrine treatment of hormone-dependent breast cancer. Research by our laboratory has focused on developing competitive and irreversible steroidal aromatase inhibitors, with an emphasis on synthesis and biochemistry of 7alpha-substituted androstenediones. Numerous 7alpha-thiosubstituted androst-4-ene-3,17-diones are potent competitive inhibitors, and several 1,4-diene analogs, such as 7alpha-(4'-aminophenylthio)-androsta-1,4-diene-3,17-di one (7alpha-APTADD), have demonstrated effective enzyme-activated irreversible inhibition of aromatase in microsomal enzyme assays. One focus of current research is to examine the effectiveness and biochemical pharmacology of 7alpha-APTADD in vivo. In the hormone-dependent 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary carcinoma model system, 7alpha-APTADD at a 50 mg/kg/day dose caused an initial decrease in mean tumor volume during the first week, and tumor volume remained unchanged throughout the remaining 5-week treatment period. This agent lowers serum estradiol levels and inhibits ovarian aromatase activity. A second research area has focused on the synthesis of more metabolically stable inhibitors by replacing the thioether linkage at the 7alpha position with a carbon-carbon linkage. Several 7alpha-arylaliphatic androst-4-ene-3,17-diones were synthesized by 1,6-conjugate additions of appropriate organocuprates to a protected androst-4,6-diene or by 1,4-conjugate additions to a seco-A-ring steroid intermediate. These compounds were all potent inhibitors of aromatase with apparent Kis ranging between 13 and 19 nM. Extension of the research on these 7alpha-arylaliphatic androgens includes the introduction of a C1-C2 double bond in the A-ring to provide enzyme-activated irreversible inhibitors. The desired 7alpha-arylaliphatic androsta-1,4-diene-3,17-diones were obtained from their corresponding 7alpha-arylaliphatic androst-4-ene-3,17-diones by oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). These inhibitors demonstrated enzyme-mediated inactivation of aromatase with apparent k(inact)s ranging from 4.4 x 10(-4) to 1.90 x 10(-3) s(-1). The best inactivator of the series was 7alpha-phenpropylandrosta-1,4-diene-3,17-dione, which exhibited a T(1/2) of 6.08 min. Aromatase inhibition was also observed in MCF-7 human mammary carcinoma cell cultures and in JAr human choriocarcinoma cell cultures, exhibiting IC50 values of 64-328 nM. The 7alpha-arylaliphatic androgens thus demonstrate potent inhibition of aromatase in both microsomal incubations and in choriocarcinoma cell lines expressing aromatase enzymatic activity. Additionally, the results from these studies provide further evidence for the presence of a hydrophobic binding pocket existing near the 7alpha-position of the steroid in the active site of aromatase. The size of the 7alpha-substituent influences optimal binding of steroidal inhibitors to the active site and affects the extent of enzyme-mediated inactivation observed with androsta-1,4-diene-3,17-dione analogs.