In vivo trafficking of endothelial progenitor cells their possible involvement in the tumor neovascularization

Circulating endothelial progenitor cell (EPCs) have been reported to contribute to vasculogenesis in adult organisms. To investigate the possible recruitment of EPCs and organization to form tumor vasculature, we investigated the in vivo real-time trafficking of EPCs non-invasively by using positron emission tomography (PET). A conditionally immortalized endothelial cell line derived from rat bone marrow (TR-BME1) was labeled with [2-(18)F] 2-fluoro-2-deoxy-D-glucose (FDG) and chased the accumulation in the rat tumor with PET. TR-BME1 cells were accumulated in the tumor tissues time-dependently. To investigate that the accumulation of the cells is specific or not, rats were previously irradiated with gamma-ray to suppress the influence of non-labeled EPCs derived from its bone marrow and used for PET analysis. The accumulation of TR-BME1 cells in the tumor was enhanced in gamma-ray-irradiated rats compared with that of non-irradiated ones, suggesting that TR-BME1 cells accumulated in the tumor specifically like as EPCs. Then the involvement of matrix metalloproteinases (MMPs) in EPC recruitment was examined. An inhibitor of MMP, MMI270, which suppressed invasion and tube formation abilities of TR-BME1 cells, only slightly suppressed the accumulation of TR-BME1 cells in the tumor of rats. These results suggest that EPCs are recruited in the tumor tissues for formation of tumor vasculature, and demonstrate the usefulness of TR-BME1 cells for studies on EPC related phenomena.     

Dynamic regulation of gene expression by the Flt-1 kinase and Matrigel in endothelial tubulogenesis

A nontubulogenic endothelial cell line, NP31, can be transformed by the active form of the Flt-1 kinase (BCR-FLTm1) into Tb3 cells, which show a tubulogenic property only when cultured in Matrigel. By utilizing this strict dependence of NP31 on BCR-FLTm1 and Matrigel for experimental angiogenesis, we performed microarray analyses under several conditions and found 97 genes whose dynamically regulated profiles of gene expression are divided into nine groups, in two major clusters. In one major cluster, gene expression is interdependently regulated by BCR-FLTm1 or Matrigel. The second major cluster contains genes whose expression patterns under BCR-FLTm1 influence are reversed by Matrigel. Based on these gene expression patterns in NP31 driven by BCR-FLTm1 and/or Matrigel, we propose a model in which sequential and alternate stimulation by BCR-FLTm1 and Matrigel induces cooperative regulation of subsets of genes. Microarray analyses of Tb3 under 11 different conditions revealed 5 candidate genes whose gene expression regulation is most closely associated with tubulogenesis.     

Small molecule antagonists of the CCR2b receptor. Part 2: Discovery process and initial structure-activity relationships of diamine derivatives

Structure-activity relationships (SAR) of a weakly active class of CCR2b inhibitors were utilized to initiate a lead evolution program employing the Drug Discovery Engine. Several alternative structural series have been discovered that display nanomolar activity in the CCR2b binding and CCR2b-mediated chemotaxis assays.     

The CAP-Gly domain of CYLD associates with the proline-rich sequence in NEMO/IKKgamma

CYLD was originally identified as the human familial cylindromatosis tumor suppressor. Recently, it was reported that CYLD directly interacts with NEMO/IKKgamma and TRAF2 in the NF-kappaB signaling pathway. The two proteins bind to a region of CYLD that contains a Cys-box motif and the third cytoskeleton-associated protein-glycine conserved (CAP-Gly) domain. Here we report that the third CAP-Gly domain of CYLD specifically interacts with one of the two proline-rich sequences of NEMO/IKKgamma. The tertiary structure of the CAP-Gly domain shares the five-stranded beta sheet topology with the SH3 domain, which is well known as a proline-rich sequence-recognition domain. However, chemical shift mapping revealed that the peptide binding site of the CAP-Gly domain is formed without the long peptide binding loop characteristic of the SH3 domain. Therefore, CAP-Gly is likely to be a novel proline-rich sequence binding domain with a mechanism different from that of the SH3 domain.     

Effect of TNF-alpha on human osteosarcoma cell line Saos2--TNF-alpha regulation of bone sialoprotein gene expression in Saos2 osteoblast-like cells

Tumor necrosis factor-alpha (TNF-alpha) is a major mediator of inflammatory response in many diseases. It inhibits bone formation and stimulates bone resorption. To determine the molecular mechanisms involved in the regulation of gene expression of osteoblast-like cells, we analyzed the effects of TNF-alpha on the human osteosarcoma cell line Saos2. We used RT-PCR to examine the effects of TNF-alpha on bone sialoprotein (BSP), core binding factor a1 (Cbfa1), osterix, alpha 1 (I) collagen, cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), cathepsin B, cathepsin L and tissue inhibitors of metalloproteinase-1 (TIMP-1). TNF-alpha (10ng/ml) increased BSP, IL-6 and COX-2 mRNA levels after 3h, reaching maximal levels at 12 h. Cbfa1 mRNA levels increased after 3 h, but decreased by 24 h. Osterix, cathepsin B, cathepsin L and TIMP-1 mRNA levels did not change after stimulation with TNF-alpha. On the other hand, alpha 1 (I) collagen mRNA expression was suppressed by TNF-alpha at 24 h. Transient transfection analyses were performed using chimeric constructs of the rat BSP gene promoter linked to a luciferase reporter gene. TNF-alpha (10 ng/ml) had no effect on the promoter activities of BSP transfected into Saos2 cells. The results of gel mobility shift assays using radiolabeled double-stranded cAMP response element (CRE) and FGF2 response element (FRE) oligonucleotides in the proximal promoter of the rat BSP gene showed increased binding of nuclear proteins at 6 h. Gel mobility shift assays with radiolabelled COX-2-CRE and COX-2-NF kappa B oligonucleotides revealed an increase in the binding of nuclear proteins from TNF-alpha-stimulated Saos2 cells. These studies, therefore, showed that TNF-alpha indirectly increased BSP expression, and that it could be mediated through COX-2 and Cbfa1 expression in Saos2 osteoblast-like cells.     

Identification of a new point mutation in the human xanthine dehydrogenase gene responsible for a case of classical type I xanthinuria

A 60-year-old Japanese man was diagnosed as having hypouricemia at an annual health check-up. The routine laboratory data was not remarkable except that the patient's hypouricemia and plasma levels of xanthine and hypoxanthine were much higher than those of normal subjects. Furthermore, the patient's daily urinary excretion of xanthine and hypoxanthine was markedly increased compared with reference values. The xanthine dehyrogenase activity of the duodenal mucosa was below the limits of detection. Nevertheless, allopurinol was metabolized to oxypurinol in vivo. Based on these findings, a subtype of classical xanthinuria (type I) was diagnosed. The xanthine dehyrogenase protein was detected by Western blotting analysis. Sequencing of the cDNA of the xanthine dehyrogenase obtained from the duodenal mucosa revealed that a point mutation of C to T had occurred in nucleotide 445. This changed codon 149 from CGC (Arg) to TGC (Cys), a finding that has not been previously reported in patients with classical xanthinuria type I.     

Fine localization of a major disease-susceptibility locus for diffuse panbronchiolitis

Diffuse panbronchiolitis affecting East Asians is strongly associated with the class I human leukocyte antigen (HLA) alleles. Recent observations suggest that a major disease-susceptibility gene may be located between the HLA-B and HLA-A loci in the class I region of the major histocompatibility complex on chromosome 6. To test this possibility, we analyzed 14 polymorphic markers in 92 Japanese patients and 93 healthy controls. Of these, seven marker alleles, including HLA-B54 and HLA-A11, were significantly associated with the disease. Maximum-likelihood haplotype analysis and subsequent direct determination of individual haplotypes identified a group of disease-associated haplotypes, one of which contained all seven disease-associated marker alleles. Another haplotype, containing HLA-B*5504, was also associated with the disease. All these haplotypes seem to have diverged from a common ancestral haplotype in East Asians and share a specific segment containing three consecutive markers between the S and TFIIH loci in the class I region. Furthermore, one of the markers within the candidate region showed the highest delta value, indicating the strongest association. Of 20 Korean patients with diffuse panbronchiolitis, 17 also shared the combination of the disease-associated marker alleles within the candidate region. These results indicate that an HLA-associated major susceptibility gene for diffuse panbronchiolitis is probably located within the 200 kb in the class I region 300 kb telomeric of the HLA-B locus on the chromosome 6p21.3.