Showing papers by "Naohiko Seki published in 2004"

Smad4-independent regulation of p21/WAF1 by transforming growth factor-beta.

Abstract: The transforming growth factor-β (TGF-β)–Smad signaling pathway inhibits the growth of human epithelial cells and plays a role in tumor suppression. The Smad4 gene is mutated or deleted in 50% of pancreatic cancers. In this study, the Smad4-null pancreatic cancer cell line BxPC-3 was transfected with either the Smad4 expression vector or the empty vector and incubated in the presence or absence of TGF-β. The cells were analysed using a cDNA microarray, which included 2280 named genes to screen for target genes regulated by TGF-β in either a Smad4-dependent or -independent manner. The microarray and subsequent quantitative RT–PCR analysis demonstrated that the Smad4-independent and -dependent signaling pathways driven by TGF-β upregulated only one of the 2280 genes, respectively, suggesting that Smad4-independent signaling downstream of TGF-β might be as widespread as Smad4-dependent signaling. In this study, we demonstrated that the cyclin-dependent kinase inhibitor p21/WAF1, which has been considered the major effector of the Smad-dependent growth inhibitory signal of TGF-β, is upregulated in a Smad4-independent manner. The upregulation occurs through Smad2/3-dependent transcriptional activation of the p21/WAF1 promoter region. These results suggest a novel mechanism of gene regulation, that is, a novel signal mediator other than Smad4.

S100A11 gene identified by in-house cDNA microarray as an accurate predictor of lymph node metastases of gastric cancer

Abstract: Gastric cancer is one of the most common malignancies in the world, and in Asian countries its incidence and mortality rates are very high. Worldwide, Japan ranks first in the incidence of this type of cancer for both sexes. To shed light on the mechanisms underlying the development and/ or progression of gastric cancer, we compared the expression profiles in gastric cancer cells obtained from surgical dissection of 20 gastric adenocarcinoma specimens with those in the corresponding non-cancerous mucosa, by cDNA microarray analysis. In total, 8,000 cDNA clones were randomly picked up and their 5'-end nucleotide sequences were determined. On the basis of sequence information, 4,608 independent clones were selected and used to produce the cDNA microarray. We identified 26 genes that were commonly up-regulated and 44 genes that were commonly down-regulated in cancerous tissues. To validate the cDNA microarray analysis, real-time PCR was performed. We found that gene S100A11 expression was associated with the development of lymph node metastases. S100A11 gene expression was clearly up-regulated in specimens from patients with lymph node metastases relative to those from patients without lymph node metastases. S100A11 gene expression status was useful to distinguish gastric cancers with lymph node metastases from those without lymph node metastasis. This genome-wide information contributes to an improved understanding of molecular changes during the development of gastric cancers. It may also help clinicians predict the development of lymph node metastases and assist researchers in identifying novel therapeutic targets for patients with gastric cancer.

Cloning of cDNA encoding a regeneration-associated muscle protease whose expression is attenuated in cell lines derived from Duchenne muscular dystrophy patients.

Abstract: In the dystrophin-mutant mdx mouse, an animal model for Duchenne muscular dystrophy (DMD), damaged skeletal muscles are efficiently regenerated and thus the animals thrive. The phenotypic differences between DMD patients and the mdx mice suggest the existence of factors that modulate the muscle wasting in the mdx mice. To identify these factors, we searched for mRNAs affected by the mdx mutation by using cDNA microarrays with newly established skeletal muscle cell lines from mdx and normal mice. We found that in the mdx muscle cell line, 12 genes, including l-arginine:glycine amidinotransferase and thymosin β4, are up-regulated, whereas 7 genes, including selenoprotein P and a novel regeneration-associated muscle protease (RAMP), are down-regulated. Northern blot analysis and in situ hybridization revealed that RAMP mRNA is predominantly expressed in normal skeletal muscle and brain, and its production is enhanced in the regenerating area of injured skeletal muscle in mice. RAMP expression was much lower in individual muscle cell lines derived from biopsies of six DMD patients compared to a normal muscle cell line. These results suggest that RAMP may play a role in the regeneration of skeletal muscle and that its down-regulation could be involved in the progression of DMD in humans.

Detection of Grb-2-related Adaptor Protein Gene (GRAP) and Peptide Molecule in Salivary Glands of MRL/lpr Mice and Patients with Sjögren's Syndrome

Abstract: The pathogenesis of Sjogren's syndrome (SS) is poorly understood. In this study we used an in-house mouse spleen cDNA microarray to analyse genes in spleens from MRL/lpr (an SS mouse model) mice. We have previously demonstrated that GRAP genes were up-regulated in salivary glands of the same mice. The microarray analysis showed that seven out of 2304 genes were highly expressed in spleens from the MRL/lpr mice, one of which was the GRAP gene. In other words, the GRAP gene is highly expressed in the salivary glands and spleen of MRL/lpr mice. We also carried out immunohistochemical studies. Mouse and human Grb-2-related adaptor protein (GRAP) antigens were expressed on ductal cells and infiltrating lymphocytes in salivary glands of MRL/lpr mice and SS patients, but only weakly in controls (MRL/+ mice and individuals with salivary cysts). These results suggest that the GRAP gene might have a role in the pathogenesis of SS.