The two acyl-homoserine lactones (AHLs) N-(butyryl)-L-homoserine lactone and N-[3-oxododecanoyl]-L-homoserine lactone (3-oxo-C 12 -HSL) are required for quorum sensing in Pseudomonas aeruginosa. These AHLs derive their invariant lactone rings from S-adenosylmethionine and their variable acyl chains from the cellular acyl-acyl carrier protein (ACP) pool. This reaction is catalysed by specific AHL synthases, which exhibit acyl chain specificity. Culture supernatants of P. aeruginosa contain multiple 3-oxo-AHLs that differ in their acyl chain lengths but their physiological role, if any, remains unknown. An in vitro fatty acid-3-oxo-AHL synthesis system was established utilizing purified P. aeruginosa Fab proteins, ACP and the LasI 3-oxo-AHL synthase. In the presence of excess protein, substrates and cofactors, this system produced almost exclusively 3-oxo-C 12 -HSL. When the β-ketoacyl-ACP reductase (FabG) catalysed step was made rate-limiting by switching from the preferred NADPH cofactor to NADH, increased levels of short chain 3-oxo-AHLs were produced, presumably because shorter-chain ketoacyl-ACPs accumulated and thus became LasI substrates. Consistent with these in vitro observations, a fabG(Ts) mutant produced increased amounts of 3-oxo-AHLs in vivo. Thus, in vitro and in vivo evidence indicated that modulation of FabG activity of the fatty acid biosynthetic pathway may determine the acyl chain lengths of these 3-oxoAHLs and that the LasI 3-oxo-AHL synthase is sufficient for their synthesis.

RELT is a recently identified Tumor Necrosis Factor Receptor that posesses two homologues in humans named RELL1 and RELL2. We investigated whether RELT and its homologues could induce cellular death when transiently transfected into HEK 293 epithelial cells. Transfection of RELT family members into HEK 293 epithelial cells induced cell death characterized by rounding and lifting of cells accompanied by DNA fragmentation, characteristics that are consistent with the activation of an apoptotic pathway. Overexpression of RELT in COS-7 cells resulted in cell rounding and lifting without DNA fragmentation, suggesting that the effects of RELT signaling may vary among different cell types. In summary, we report that overexpression of RELT or its homologues RELL1 and RELL2 in HEK 293 epithelial cells results in cell death with morphological characteristics consistent with the activation of an apoptotic pathway.

Prostate-specific antigen (PSA) is a member of the kallikrein family and has been an important biological marker for prostate cancer. The mechanisms regulating PSA expression in prostatic cancer cells are unclear. The present study was designed to elucidate the role of 13-cis-retinoic acid (RA) in regulation of PSA and the tumorigenic potential of the human prostate cancer cell line LNCaP. The growth regulation of LNCaP cells was examined by DNA synthesis and doubling time. The tumorigenic potential of prostate cancer cells was analyzed by soft agar colony-forming assay, in vitro invasion assay, type IV collagenase assay and binding to extracellular matrix assay. The nuclear receptors for retinoic acid (RAR alpha, -beta, -gamma and RXR alpha, -beta, -gamma) as well as PSA mRNA were determined by Northern blot using specific oligonucleotide probes. Our results suggest that 13-cis-RA significantly inhibits PSA secretion and expression both at the mRNA and protein levels compared with untreated cells. Electron microscopic studies suggest that after 13-cis-RA treatment, cells become more differentiated as they contain lumina, lined by plasma membrane and microvilli. Prostate cancer cell growth and tumorigenic potential after 13-cis-RA treatment was significantly decreased compared with controls. Nude mice tumorigenicity studies showed that 13-cis-RA-treated cells produced significantly smaller tumors compared with untreated cell tumors. There was also a significant increase in the expression of RXRa mRNA after 13-cis-RA treatment compared with untreated cells.

Receptor Expressed in Lymphoid Tissues (RELT) is a human Tumor Necrosis Factor Receptor (TNFR) family member that has two identified homologous binding partners, RELL1 and RELL2. This study sought to further understand the pattern of RELT expression, the functional role of RELT family members, and the mechanism of RELT-induced apoptosis. RELT protein expression was detected in the spleen, lymph node, brain, breast and peripheral blood leukocytes (PBLs). A smaller than expected size of RELT was observed in PBLs, suggesting a proteolytically cleaved form of RELT. RELL1 and RELL2 overexpression activated the p38 MAPK pathway more substantially than RELT in HEK-293 cells, and this activation of p38 by RELT family members was blocked by dominant-negative mutant forms of OSR1 or TRAF2, implicating these molecules in RELT family member signaling. RELT was previously shown to induce apoptosis in human epithelial cells despite lacking the characteristic death domain (DD) found in other TNFRs. Seven deletion mutants of RELT that lacked differing portions of the intracellular domain were created to assess whether RELT possesses a novel DD. None of the deletion mutants induced apoptosis as efficiently as full-length RELT, a result that is consistent with a novel DD being located at the carboxyl-terminus. Interestingly, induction of apoptotic morphology by RELT overexpression was not prevented when signaling by FADD or Caspase-8 was blocked, indicating RELT induces apoptosis by a pathway distinct from other death-inducing TNFRs such as TNFR1. Collectively, this study provides more insights into RELT expression, RELT family member function, and the mechanism of RELT-induced death.