ABSTRACT
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.
Subject(s)
Gene Expression Regulation, Neoplastic , Prostatic Neoplasms/metabolism , Ribosomal Proteins/biosynthesis , Ribosomal Proteins/genetics , Ubiquitins/biosynthesis , Ubiquitins/genetics , Aged , Apoptosis , Cell Line, Tumor , Cloning, Molecular , DNA Primers/chemistry , DNA Primers/pharmacology , DNA, Complementary/metabolism , Densitometry , Humans , In Situ Hybridization , Lasers , Male , Middle Aged , Prostatic Neoplasms/pathology , RNA, Complementary/chemistry , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Spectrometry, Fluorescence , TATA-Box Binding Protein/metabolism , Time FactorsABSTRACT
Hereditary nonpolyposis colorectal cancers (HNPCCs) are an important subgroup of colorectal carcinomas. Compared to sporadic variants, they present several particular features, the most important of which are less invasive and metastatic properties linked to a more favorable prognosis. This contrasts to the generally poor differentiation of the epithelial tumor component. Since matrix-degrading proteases secreted by stromal fibroblasts contribute significantly to tumor invasion, we analyzed the stromal expression of 2 matrix metalloproteinases (MMP-1 and -9) and of one of their regulators, the Ets 1 transcription factor, by both immunohistochemistry and in situ hybridization in sporadic colorectal carcinomas and HNPCC tumors. We found that MMP-1 and -9 as well as Ets 1 are upregulated in the fibroblastic stroma during the development from sporadic adenomas to invasive carcinomas. HNPCC tumors exhibited a significantly lower expression of Ets 1, MMP-1 and -9. These findings on the basis of lower matrix-degrading properties of the fibroblastic tumor stroma in HNPCC tumors might help to explain why, in spite of their less differentiated phenotype, HNPCC tumors have a less invasive and metastatic potential compared to sporadic cancers.
Subject(s)
Colorectal Neoplasms, Hereditary Nonpolyposis/enzymology , Colorectal Neoplasms/enzymology , Matrix Metalloproteinase 1/metabolism , Matrix Metalloproteinase 9/metabolism , Neoplasm Invasiveness/pathology , Proto-Oncogene Proteins/metabolism , Stromal Cells/enzymology , Transcription Factors/metabolism , Adenoma/enzymology , Adenoma/genetics , Adenoma/pathology , Adult , Aged , Aged, 80 and over , Cell Differentiation , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Colorectal Neoplasms, Hereditary Nonpolyposis/genetics , Colorectal Neoplasms, Hereditary Nonpolyposis/pathology , Extracellular Matrix/metabolism , Fibroblasts/enzymology , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Humans , Immunoenzyme Techniques , In Situ Hybridization , Matrix Metalloproteinase 1/genetics , Matrix Metalloproteinase 9/genetics , Middle Aged , Neoplasm Invasiveness/genetics , Proto-Oncogene Protein c-ets-1 , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-ets , RNA, Messenger/metabolism , Transcription Factors/geneticsABSTRACT
Testicular germ cell tumours (TGCTs) are histologically heterogeneous neoplasms with variable malignant potential. Previously, we demonstrated frequent 3p allele loss in TGCTs, and recently we and others have shown that the 3p21.3 RASSF1A tumour suppressor gene (TSG) is frequently inactivated by promoter hypermethylation in a wide range of cancers including lung, breast, kidney and neuroblastoma. In order to investigate the role of epigenetic events in the pathogenesis of TGCTs, we analysed the promoter methylation status of RASSF1A and nine other genes that may be epigenetically inactivated in cancer (p16(INK4A), APC, MGMT, GSTP1, DAPK, CDH1, CDH13, RARbeta and FHIT) in 24 primary TGCTs (28 histologically distinct components). RASSF1A methylation was detected in four of 10 (40%) seminomas and 15 of 18 (83%) nonseminoma TGCT (NSTGCT) components (P=0.0346). None of the other nine candidate genes were methylated in seminomas, but MGMT (44%), APC (29%) and FHIT (29%) were frequently methylated in NSTGCTs. Furthermore, in two mixed germ cell tumours, the NSTGCT component for one demonstrated RASSF1A, APC and CDH13 promoter methylation, but the seminoma component was unmethylated for all genes analysed. In the second mixed germ cell tumour, the NSTGCT component was methylated for RASSF1A and MGMT, while the seminoma component was methylated only for RASSF1A. In all, 61% NSTGCT components but no seminoma samples demonstrated promoter methylation at two or more genes (P=0.0016). These findings are consistent with a multistep model for TGCT pathogenesis in which RASSF1A methylation occurs early in tumorigenesis and additional epigenetic events characterize progression from seminoma to NSTGCTs.
Subject(s)
Acid Anhydride Hydrolases , DNA Methylation , Neoplasm Proteins/genetics , Neoplasms, Germ Cell and Embryonal/genetics , Seminoma/genetics , Testicular Neoplasms/genetics , Tumor Suppressor Proteins , Adenomatous Polyposis Coli Protein/genetics , Cadherins/genetics , Gene Expression Regulation, Neoplastic , Gene Silencing , Genes, Tumor Suppressor , Humans , Male , O(6)-Methylguanine-DNA Methyltransferase/genetics , Promoter Regions, GeneticABSTRACT
Neovascularization of the inflamed synovium and pannus is one of the hallmarks of chronic rheumatoid arthritis. It contributes to disease progression by supplying blood to the inflamed tissues and by recruiting immune competent and inflammatory cells. Angiogenesis is tightly regulated at several levels, but of significant importance is transcription. The Ets 1 transcription factor has been intimately linked to the regulation of angiogenesis under both physiological and pathological conditions and is induced in endothelial cells by vascular endothelial growth factor, the most important angiogenic factor in rheumatoid arthritis. We investigated Ets 1 expression in synovial membranes of joints in patients with active rheumatoid arthritis and compared the results to those obtained in patients with degenerative joint disease, which is characterized by significantly less neoangiogenesis. Using quantitative densitometric and real-time RT-PCR approaches, we found a significant upregulation of Ets 1 transcripts in rheumatic, compared to osteoarthritic, synovial membranes. Moreover, we were able to attribute both Ets 1 mRNA and Ets 1 protein to capillary endothelial cells of newly formed blood vessels by in situ hybridization and immunohistochemistry. Finally, our data suggest important roles of the Ets 1 transcription factor in the regulation of inflammatory angiogenesis in rheumatoid arthritis.
