Evaluation of Serum Biomarkers (FGF-2, HGF, MIF and PTN) in Patients With Testicular Germ Cell Cancer.

BACKGROUND/AIM: FGF-2, HGF, MIF and PTN have been suggested as biomarkers for testicular germ cell cancer patients in earlier studies. Our study was designed to validate these potential novel tumor markers. MATERIALS AND METHODS: Serum FGF-2, HGF, MIF and PTN levels were analysed using an ELISA technique in a screening cohort of 20 testicular germ cell cancer patients and 10 healthy men. MIF levels were measured in a validation cohort of 84 patients with testicular cancer, 24 with non-malignant testicular tumors and 64 healthy men. RESULTS: Serum FGF-2, HGF and PTN levels did not differ in cancer patients and healthy males within the screening cohort, whereas MIF was significantly increased among cancer patients. Within the validation cohort, a modest but insignificant increase of serum MIF was observed in TGCT patients compared to healthy men. MIF levels were not correlated with adverse clinical-pathological parameters. CONCLUSION: FGF-2, HGF, MIF and PTN are not suitable as non-invasive biomarkers for testicular germ cell cancer patients.

Dominant-negative inhibition of Ets 1 suppresses tumor growth, invasion and migration in rat C6 glioma cells and reveals differentially expressed Ets 1 target genes.

We previously reported that the inactivation of the Ets 1 transcription factor by a specific decoy strategy reduces rat C6 glioma cell proliferation and mmp-9 expression. In the present study, we analysed the effects of the dominant-negative form of Ets 1 (Ets-DB) on rat C6 glioma cell proliferation, migration, invasion, in vivo tumor growth on the chicken chorioallantoic membrane (CAM) and mmp-9 expression. In addition, we examined differences in gene expression between Ets-DB expressing and control cells using suppression subtractive hybridization (SSH). We found that retrovirus mediated expression of Ets-DB inhibited cellular proliferation, migration, invasion, mmp-9 expression, cellular growth in soft agar, and in vivo growth in the chicken chorioallantoic membrane assay. SSH analysis revealed expression of different genes in Ets-DB expressing cells involved in basic cellular processes. Each of these genes contained binding sites for different Ets-factors within their promoters. Finally, we found that, in addition to Ets 1, Elk-1, Elf-1, Fli-1 and Etv-1 are further Ets family members expressed in rat C6 glioma cells. Our results indicate that Ets transcription factors play important roles for basic properties of rat C6 glioma cells. Targeting of these factors might therefore become a useful experimental tool for therapeutic strategies against malignant gliomas.

Tumor-stroma interactions of metastatic prostate cancer cell lines: analyses using microarrays.

Tumor-stroma interactions are of great importance not only for the development and progression of primary prostate carcinoma but probably also for the establishment of metastasis. Fibroblasts are an important stromal cell type encountered by metastatic tumor cells at different sites. In previous investigations, we had found that media conditioned by three metastatic prostate cancer cell lines (LNCaP, PC-3, and DU-145) induced cultured nonprostatic fibroblasts to proliferate or to express matrix-metalloproteinase-1 considered important for tumor invasion. Fibroblast-conditioned media in turn stimulate proliferation of DU-145 cells and migration of PC-3 cells. Both tumor cells and fibroblasts secrete VEGF suggesting that not only metastatic but also stromal cells at metastatic sites contribute to the vascularization of metastasis necessary for continuous growth. In order to better understand the reciprocal tumor-stroma cross-talk in molecular terms we used the mRNA extracted from stimulated and unstimulated neoplastic and fibroblastic stromal cells for cDNA array hybridization using Affymetrix chips. The three prostate cell lines influenced the fibroblasts nearly in the same manner. In particular proteins involved in cell adhesion, cell-cell contact, and cell cycle regulation were downregulated in stimulated fibroblasts. In contrast, fibroblasts affected every prostate cancer cell line in different ways, which may be because of the different origin of the metastatic prostate cancer cell lines.

Tumour-stroma interactions between metastatic prostate cancer cells and fibroblasts.

Previous work has shown the importance of tumour-stroma 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 cross- talk 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 TNFalpha 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 TNFalpha, 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.

Ets-1 expression promotes epithelial cell transformation by inducing migration, invasion and anchorage-independent growth.

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 integrinbeta2 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.

A novel component of the ubiquitin pathway, ubiquitin carboxyl extension protein 1 is overexpressed in prostate cancer.

Prostate cancer is among the most common tumors in industrialized nations. However, little is known about the molecular events underlying its development. In the present study we used suppressive subtraction hybridization (SSH) in combination with laser-assisted microdissection in order to compare gene expression between prostate carcinoma and the normal prostate proper. Both are mixed tissues which consist of an epithelial and a stromal compartment. We first compared mRNA (cDNA) expression by SSH and then used real-time quantitative RT-PCR analysis of microdissected tissue probes in order to verify differential expression of subtracted cDNA clones. We also used differentially expressed cDNAs for the synthesis of radiolabelled riboprobes in order to attribute differential expression to specific cell types in tissue sections by in situ hybridization. Using this approach we found an up-regulation of ubiquitin carboxyl extension protein 1 (UBCEP-1) mRNA in prostate carcinoma cells compared to the normal glandular epithelium of the prostate proper. UBCEP-1 mediated ubiquitin chain elongation may promote prostate carcinoma development by increasing via the proteasome pathway the degradation of proteins which are involved in growth inhibition or apoptosis.

Hydrogenases in green algae: do they save the algae's life and solve our energy problems?

Green algae are the only known eukaryotes with both oxygenic photosynthesis and a hydrogen metabolism. Recent physiological and genetic discoveries indicate a close connection between these metabolic pathways. The anaerobically inducible hydA genes of algae encode a special type of highly active [Fe]-hydrogenase. Electrons from reducing equivalents generated during fermentation enter the photosynthetic electron transport chain via the plastoquinone pool. They are transferred to the hydrogenase by photosystem I and ferredoxin. Thus, the [Fe]-hydrogenase is an electron 'valve' that enables the algae to survive under anaerobic conditions. During sulfur deprivation, illuminated algal cultures evolve large quantities of hydrogen gas, and this promises to be an alternative future energy source.

Differential regulation of the Fe-hydrogenase during anaerobic adaptation in the green alga Chlamydomonas reinhardtii.

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 non-algal 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.