Differential Inductive Signaling of CD90+ Prostate Cancer-Associated Fibroblasts Compared to Normal Tissue Stromal Mesenchyme Cells

Prostate carcinomas are surrounded by a layer of stromal fibroblastic cells that are characterized by increased expression of CD90. These CD90+ cancer-associated stromal fibroblastic cells differ in gene expression from their normal counterpart, CD49a+CD90lo stromal smooth muscle cells; and were postulated to represent a less differentiated cell type with altered inductive properties. CD90+ stromal cells were isolated from tumor tissue specimens and co-cultured with the pluripotent embryonal carcinoma cell line NCCIT in order to elucidate the impact of tumor-associated stroma on stem cells, and the ‘cancer stem cell.’ Transcriptome analysis identified a notable decreased induction of smooth muscle and prostate stromal genes such as PENK, BMP2 and ChGn compared to previously determined NCCIT response to normal prostate stromal cell induction. CD90+ stromal cell secreted factors induced an increased expression of CD90 and differential induction of genes involved in extracellular matrix remodeling and the RECK pathway in NCCIT. These results suggest that, compared to normal tissue stromal cells, signaling from cancer-associated stromal cells has a markedly different effect on stem cells as represented by NCCIT. Given that stromal cells are important in directing organ-specific differentiation, stromal cells in tumors appear to be defective in this function, which may contribute to abnormal differentiation found in diseases such as cancer

In vivo determination of substrate specificity of hepatitis C virus NS3 protease: Genetic assay for site-specific proteolysis

Hepatitis C virus (HCV) NS3 protease is responsible for the processing of the viral polyprotein and is considered as a primary target for the development of anti-HCV therapy, We have developed a genetic method in yeast to screen for good substrate sequences of the NS3 protease. A library of fusion proteins was constructed with a transcription factor, GAL4, linked to the intracellular domain of an integral membrane protein, STE2, by a randomized protease substrate sequence. In yeast cells expressing NS3 protease, the substrate sequences in the fusion proteins were specifically recognized and cleaved, This cleavage resulted in the release of GAL4 from the cytoplasmic membrane and the subsequent activation of reporter genes by GAL4, which was detected by the growth of yeast cells on selective media. Based on the analysis of 69 isolated substrate sequences, a consensus sequence was deduced: (Glu/Asp)-X-Val-Val-(Leu/Pro)-Cys down arrow (Ser/Ala), with the scissile bond being located between Cys and Ser or Ala and X not being determined. This is largely consistent with the previous results obtained by biochemical methods. An oligopeptide containing the deduced sequence was highly efficiently cleaved in vitro by the purified NS3 protease. These data demonstrated that the present genetic method could be used as an efficient tool for the in vivo determination of substrate specificity of proteases. (C) 2000 Academic Press.X1133sciescopu