Chlamydomonas reinhardtii, a unicellular green alga, contains a hydrogenase enzyme, which is induced by anaerobic adaptation of the cells. Using the suppression subtractive hybridization (SSH) approach, the differential expression of genes under anaerobiosis was analyzed. A␣PCR fragment with similarity to the genes of bacterial Fe‐hydrogenases was isolated and used to screen an anaerobic cDNA expression library of C. reinhardtii. The cDNA sequence of hydA contains a 1494‐bp ORF encoding a protein with an apparent molecular mass of 53.1 kDa. The transcription of the hydrogenase gene is very rapidly induced during anaerobic adaptation of the cells. The deduced amino‐acid sequence corresponds to␣two␣polypeptide sequences determined by sequence analysis of the isolated native protein. The Fe‐hydrogenase contains a short transit peptide of 56 amino acids, which routes the hydrogenase to the chloroplast stroma. The isolated protein belongs to a new class of Fe‐hydrogenases. All four cysteine residues and 12 other␣amino acids, which are strictly conserved in the active site (H‐cluster) of Fe‐hydrogenases, have been identified. The N‐terminus of the C. reinhardtii protein is markedly truncated compared to other nonalgal Fe‐hydrogenases. Further conserved cysteines that coordinate additional Fe–S‐cluster in other Fe‐hydrogenases are missing. Ferredoxin PetF, the natural electron donor, links the hydrogenase from C. reinhardtii to the photosynthetic electron transport chain. The hydrogenase enables the survival of the green algae under anaerobic conditions by transferring the electrons from reducing equivalents to the enzyme.
Ets-1 is the prototype of the family of ETS transcription factors. In human tumors, Ets-1 is expressed in endothelial cells and fibroblasts of the tumor stroma and is proposed to play a role in tumor vascularization and invasion by upregulating expression of matrix-degrading proteases. In human carcinomas, Ets-1 is also expressed by neoplastic cells, but little is known about the functional implications of this observation. We have addressed the role of Ets-1 in epithelial HeLa tumor cells by selecting stably Ets-1 over and underexpressing HeLa cells. Ets-1 expression increases the transformed phenotype of HeLa cells, by promoting cell migration, invasion and anchorage-independent growth, while Ets-1 downregulation reduces cell attachment. In correlation with these results, Ets-1 upregulation increases integrinb2 expression but not that of other integrins. These results suggest that, in addition to its role in the tumor stroma, Ets-1 may also promote tumor development and progression by increasing neoplastic transformation.
Abstract.Previous work has shown the importance of tumourstroma interactions for prostate cancer development at the primary site. The aim of the present study was to find out whether evidence can be found for a tumour-stroma crosstalk also between metastatic prostate cancer cell lines and non-prostatic stromal fibroblasts which are encountered by metastatic cells at most sites. We addressed this issue in cell culture systems using 3 metastatic human prostate cancer cell lines (LnCaP, PC-3 and DU-145) on the one hand, and a human fibroblast line (HFF, human foreskin fibroblasts) on the other. We incubated fibroblasts with tumour cell-and tumour cells with fibroblast-conditioned media and evaluated several parameters important for the establishment of metastases such as cell proliferation, migration and expression of matrix degrading proteases. We also determined in the conditioned media the concentrations of several growth factors and cytokines which might be responsible for the observed effects. We found that media conditioned by all 3 metastatic prostate cancer cell lines stimulated fibroblast proliferation which corresponds to fibrous stroma induction in vivo. DU-145 cell conditioned media induced in fibroblasts expression of mmp-1 mRNA known to be important for tumour invasion. ELISA assays revealed that tumour cells secrete bFGF, PDGF and TNF· known to stimulate fibroblast proliferation and/or MMP-1 expression. Cultivation of DU-145 carcinoma cells in fibroblast conditioned medium resulted in an enhanced proliferation and anchorage-independent growth of this cell line in soft agar. Fibroblast conditioned medium also increased migration of PC-3 cells in the wound assay and slightly augmented mmp-1 expression. KGF (able to stimulate proliferation of normal and neoplastic prostate epithelial cells) was secreted by fibroblasts at higher concentrations than by all 3 tumour cell lines. In addition, fibroblasts secreted TNF·, bFGF, PDGF, HGF and also VEGF, the most important factor for tumour vascularization. Our results provide evidence that tumour-stroma interactions do not only exist at the primary site but also between metastatic prostate cancer cell lines and their fibroblastic microenvironment. These interactions, which are mediated through secreted factors, affect several steps of the metastatic cascade including proliferation, anchorage-independent growth, migration and the secretion of matrix-degrading proteases.
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