RhoC and guanine nucleotide exchange factor Net1 in androgen-unresponsive mouse mammary carcinoma SC-4 cells and human prostate cancer after short-term endocrine therapy.

BACKGROUND: Endocrine resistance is a critical issue in managing patients with prostate cancer. This study is undertaken to search for a potential molecular target connected with this process using a model system of androgen-dependent and androgen-unresponsive SC-3 and SC-4 cells. METHODS: Expression profiles, actin stress fiber organization, and the levels of activated Rho GTPases were compared between SC-4 and SC-3 cells using an oligonucleotide microarray, phalloidin staining, and a Rho activation assay. The cell viability was analyzed with a Rho inhibitor or by stable transfection with either a dominant-negative (DN) form of RhoC or a mutant form of NET1 (mutNET1). The expressions of RhoC, NET1, and epithelial-mesenchymal transition (EMT) markers were immunohistochemically analyzed in human prostate cancer specimens after short-term endocrine therapy and in an untreated condition. RESULTS: SC-4 cells exhibited mesenchymal phenotypes with activation of Rho signals. Treatment with a Rho inhibitor suppressed the cell viability in SC-4 cells, but not in SC-3 cells. The cell viability of SC-4 cells stably expressing DN-RhoC and mutNET1 was also attenuated. In the immunohistochemical analysis, NET1 and the EMT marker of N-cadherin were expressed at higher levels in prostate cancers after short-term endocrine therapy than in untreated tumors, and RhoC expression was maintained after short-term endocrine therapy. CONCLUSIONS: Rho signaling is involved in the cell survival of SC-4 cells. The higher expressions of RhoC and NET1 in human prostate cancers after short-term endocrine therapy suggest that RhoC and NET1 may become therapeutic targets during endocrine therapy.

Complete sequencing and characterization of 21,243 full-length human cDNAs.

As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.

Identification of highly expressed genes in peripheral blood T cells from patients with atopic dermatitis.

BACKGROUND: Analysis of genes that are differentially expressed in patients with atopic dermatitis (AD) and normal individuals will provide important information on the underlying molecular pathogenetic mechanisms of AD. METHODS: Transcript of freshly isolated peripheral blood T cells from 59 individuals were analyzed with a fluorescent differential display (FDD) method. Ninety-two differentially expressed genes were identified in this manner. Additionally, real-time quantitative RT-PCR was employed to investigate the expression of the FDD-selected genes and also genes related to T cell function. RESULTS: A number of genes, including CC chemokine receptor 4, T cell-specific tyrosine kinase (Emt/Itk), integrin beta1, integrin alpha6, IQGAP1 and MAR/SAR DNA-binding protein (SATB1), were shown to be more highly expressed in patients with moderate and/or severe AD than in controls or patients with mild AD. Because the products of these upregulated genes influence chemotaxis, adhesion, migration and Th2 polarization, it is suggested that in more severe AD, circulating T cells may function differently in this regard. Several other genes, the role of which in T cell function is currently unknown, were also found to be differentially expressed in AD. These included the heat shock protein 40 and vasopressin-activated calcium-mobilizing receptor 1. CONCLUSION: The upregulated genes identified in this work may serve as useful markers for moderate to severe AD as opposed to normal or mild AD and also as markers indicating progression to more severe AD. Further functional characterization will provide a better understanding of the pathophysiology of circulating T cells in AD.

Transcriptome analysis of acetate metabolism in Corynebacterium glutamicum using a newly developed metabolic array.

Following the determination of the whole-genome sequence of Corynebacterium glutamicum, we have developed a DNA array to extensively investigate gene expression and regulation relevant to carbon metabolism. For this purpose, a total of 120 C. glutamicum genes, including those in central metabolism and amino acid biosyntheses, were amplified by PCR and printed onto glass slides. The resulting array, designated a "metabolic array", was used for hybridization with fluorescently labeled cDNA probes generated by reverse transcription from total RNA samples. As the first demonstration of transcriptome analysis in this industrially important microorganism, we applied the metabolic array to study differential transcription profiles between cells grown on glucose and on acetate as the sole carbon source. The changes in gene expression observed for the known acetate-regulated genes (aceA, aceB, pta, and ack) were well consistent with the literature data of northern analyses and enzyme assays, indicating the utility of the metabolic array in transcriptome analysis of C. glutamicum. In addition to the known responses, many previously unrecognized co-regulated genes were identified. For example, several TCA cycle genes, such as gltA, sdhA, sdhB, fumH, and mdh, and the gluconeogenic gene pck were up-regulated in the acetate medium. On the other hand, a few genes involved in glycolysis and the pentose phosphate pathway, as well as many amino acid biosynthetic genes, were down-regulated in acetate. Furthermore, two gap genes, gapA and gapB, were found to be inversely regulated, suggesting the presence of a new regulatory step for carbon metabolism between glycolysis and gluconeogenesis.