SummaryAlthough cell density-dependent regulation of the luminescence genes in Vibrio fischeri is a model for quorum sensing in Gram-negative bacteria, relatively little is known about the promoter of the luminescence operon. The luminescence operon is activated by the LuxR protein, which requires a diffusible acylhomoserine lactone signal. The lux box, a 20 bp inverted repeat, is located in the luxI promoter region and is required for LuxR-dependent induction of the luminescence genes. Using primer extension, we mapped the LuxR-dependent transcriptional start site of the lux operon to 19 bp upstream of the luxI start codon. This indicates that the lux box is centred at ¹42.5 bp from the start of transcription. To gain evidence about the location of the ¹10 sequence, we placed a consensus ¹35 hexamer at different locations relative to the luxI transcriptional start site and measured constitutive levels of luminescence in recombinant Escherichia coli. The strongest constitutive promoter contained a TATAGT hexamer 17 bp from the ¹35 consensus sequence and 6 bp from the transcriptional start site. We propose that this is the ¹10 hexamer. Also in recombinant E. coli, both halfsites of the lux box were required for LuxR-dependent gene activation and for activation by an autoinducerindependent, monomeric LuxR deletion protein. LuxRdependent activation of luminescence was eliminated when the lux box was centred at ¹47.5, ¹52.5 and ¹62.5 with respect to the luxI transcriptional start site. Our evidence, taken together with other information, points to a model in which a LuxR dimer overlaps the ¹35 region of the luxI promoter and functions as an ambidextrous activator with each LuxR subunit interacting with a different region of RNA polymerase.

Routinely used therapies are not adequate to treat the heterogeneity of breast cancer, and consequently, more therapeutic targets are desperately needed. To identify novel targets, we generated a breast cancer cDNA library enriched for genes that encode membrane and secreted proteins. From this library we identified SUSD2 (Sushi Domain Containing 2), which encodes an 822 amino acid protein containing a transmembrane domain and functional domains inherent to adhesion molecules. Previous studies describe the mouse homolog, Susd2, but there are no studies on the human gene associated with breast cancer. Immunohistochemistry analysis of human breast tissues showed weak or no expression of SUSD2 in normal epithelial cells, with the endothelial lining of vessels staining positive for SUSD2. However, staining was observed in pathological breast lesions and in lobular and ductal carcinomas. SUSD2 interacts with Galectin-1 (Gal-1), a 14-kDa secreted protein that is synthesized by carcinoma cells and promotes tumor immune evasion, angiogenesis and metastasis. Interestingly, we found that localization of Gal-1 on the surface of cells is dependent on the presence of SUSD2. Various phenotype assays indicate that SUSD2 increases the invasion of breast cancer cells and contributes to a potential immune evasion mechanism through induction of apoptosis of Jurkat T cells. Using a syngeneic mouse model, we observed accelerated tumor formation and decreased survival in mice with tumors expressing Susd2. We found significantly fewer CD4 tumor infiltrating lymphocytes in mice with tumors expressing Susd2. Together, our findings provide evidence that SUSD2 may represent a promising therapeutic target for breast cancer.

The Vibrio fischeri luminescence (lux) operon is regulated by a quorum-sensing system that involves the transcriptional activator (LuxR) and an acyl-homoserine lactone signal. Transcriptional activation requires the presence of a 20-base inverted repeat termed the lux box at a position centered 42.5 bases upstream of the transcriptional start of the lux operon. LuxR has proven difficult to study in vitro. A truncated form of LuxR has been purified, and together with 70 RNA polymerase it can activate transcription of the lux operon. Both the truncated LuxR and RNA polymerase are required for binding to lux regulatory DNA in vitro. We have constructed an artificial lacZ promoter with the lux box positioned between and partially overlapping the consensus ؊35 and ؊10 hexamers of an RNA polymerase binding site. LuxR functioned as an acyl-homoserine lactone-dependent repressor at this promoter in recombinant Escherichia coli. Furthermore, multiple lux boxes on an independent replicon reduced the repressor activity of LuxR. Thus, it appears that LuxR can bind to lux boxes independently of RNA polymerase binding to the promoter region. A variety of LuxR mutant proteins were studied, and with one exception there was a correlation between function as a repressor of the artificial promoter and activation of a native lux operon. The exception was the truncated protein that had been purified and studied in vitro. This protein functioned as an activator but not as a repressor in E. coli. The data indicate that the mutual dependence of purified, truncated LuxR and RNA polymerase on each other for binding to the lux promoter is a feature specific to the truncated LuxR and that full-length LuxR by itself can bind to lux box-containing DNA.Acyl-homoserine lactone (acyl-HSL)-dependent quorum sensing is common to a number of different genera and species of gram-negative bacteria. This type of cell density-dependent control of gene expression was first discovered in the marine luminescent bacterium Vibrio fischeri. The V. fischeri system remains one of the best-studied examples of quorum sensing. Quorum sensing in V. fischeri involves the interaction of the signal molecule N-3-(oxohexanoyl)homoserine lactone (3-oxo-C 6 -HSL) with LuxR, the transcriptional activator of the luminescence (lux) operon. Over 20 LuxR homologs have been described in the past 10 years (for recent reviews on quorum sensing and the LuxR family of transcription factors, see references 13 to 15 and 24).The V. fischeri lux operon consists of seven genes of which the first is luxI (12, 36). The luxI gene codes for an enzyme required for synthesis of 3-oxo-C 6 -HSL. The luxI gene has a 70 RNA polymerase (RNAP)-dependent promoter with a lux box, a 20-bp inverted repeat, centered at position Ϫ42.5 from the transcriptional start site (11, 34). The lux box and its location with respect to the other promoter elements are critical for LuxR-dependent activation of the lux genes (7, 11).

A genome-wide phenotype screen was used to identify factors and pathways that induce proliferation of human umbilical vein endothelial cells (HUVEC). HUVEC proliferation is a recognized marker for factors that modulate vascularization. Screening ''hits'' included known proangiogenic factors, such as VEGF, FGF1, and FGF2 and additional factors for which a direct association with angiogenesis was not previously described. These include the kinase TBK1 as well as Toll-like receptor adaptor molecule and IFN regulatory factor 3. All three proteins belong to one signaling pathway that mediates induction of gene expression, including a mixture of secreted factors, which, in concert, mediate proliferative activity toward endothelial cells. TBK1 as the ''trigger'' of this pathway is induced under hypoxic conditions and expressed at significant levels in many solid tumors. This pattern of expression and the decreased expression of angiogenic factors in cultured cells upon RNA-interference-mediated ablation suggests that TBK1 is important for vascularization and subsequent tumor growth and a target for cancer therapy.cancer ͉ cDNA͞libraries ͉ expression cloning ͉ human umbilical vein endothelial cells ͉ screening