Genes up- and down-regulated by dermcidin in breast cancer: a microarray analysis

Dermcidin (DCD) is a human gene mapped to chromosome 12q13 region, which is co-amplified with multiple oncogenes with a well-established role in the growth, survival and progression of breast cancers. Here, we present a summary of a DNA microarray-based study that identified the genes that are up- and down-regulated in a human MDA-361 pLKO control clone and three clones expressing short hairpin RNA against three different regions of DCD mRNA. A list of 235 genes was differentially expressed among independent clones (> 3-fold change and p < 0.005). The gene expression of 208 was reduced and of 27 was increased in the three DCD-RNAi clones compared to pLKO control clone. The expression of 77 genes (37%) encoding for enzymes involved in amino acid metabolism, glucose metabolism and oxidoreductase activity and several genes required for cell survival and DNA repair were decreased. The expression of EGFR/ErbB-1 gene, an important predictor of outcome in breast cancer, was reduced together with the genes for betacellulin and amphiregulin, two known ligands of EGFR/ErbB receptors. Many of the 27 genes up-regulated by DCD-RNAi expression have not yet been fully characterized; among those with known function, we identified the calcium-calmodulin-dependent protein kinase-II delta and calcineurin A alpha. We compared 132 up-regulated and 12 down-regulated genes in our dataset with those genes up- and down-regulated by inhibitors targeting various signaling pathway components. The analysis showed that the genes in the DCD pathway are aligned with those functionally influenced by the drugs sirolimus, LY-294002 and wortmannin. Therefore, DCD may exert its function by activating the PI3K/AKT/mTOR signaling pathway. Together, these bioinformatic approaches suggest the involvement of DCD in the regulation of genes for breast cancer cell metabolism, proliferation and survival.     

Natural Human Gene Correction by Small Extracellular Genomic DNA Fragments

Classical gene targeting employs natural homologous recombination for a gene correction using a specially designed and artificially Delivered DNA construct but the method is very inefficient. On the other hand, small DNA fragments in the form of tiny chromatin-like articles naturally present in blood plasma can spontaneously penetrate into human cells and cell nuclei. We hypothesized that these natural DNA nanoparticles with recombinagenic free ends might be effective agents for gene replacement therapy. We demonstrate that a mixture of small fragments of total human chromatin from non-mutant cells added to a culture medium without transfection agents efficiently repaired a 47 base pair deletion in the CASP3 gene in 30% of treated human MCF7 breast cancer cells, as shown by restoration of caspase-3 apoptotic function and CASP3 DNA and mRNA structure. Such an innate gene replacement mechanism might function naturally in an organism using its own apoptotic DNA fragments. This mechanism might enable human cancer cell phenotype normalization in the presence of excess normal Cells.     

Postsecretory processing generates multiple cathelicidins for enhanced topical antimicrobial defense

The production of antimicrobial peptides and proteins is essential for defense against infection. Many of the known human antimicrobial peptides are multifunctional, with stimulatory activities such as chemotaxis while simultaneously acting as natural antibiotics. In humans, eccrine appendages express DCD and CAMP, genes encoding proteins processed into the antimicrobial peptides dermcidin and LL-37. In this study we show that after secretion onto the skin surface, the CAMP gene product is processed by a serine protease-dependent mechanism into multiple novel antimicrobial peptides distinct from the cathelicidin LL-37. These peptides show enhanced antimicrobial action, acquiring the ability to kill skin pathogens such as Staphylococcus aureus and Candida albicans. Furthermore, although LL-37 may influence the host inflammatory response by stimulating IL-8 release from keratinocytes, this activity is lost in subsequently processed peptides. Thus, a single gene product encoding an important defense molecule alters structure and function in the topical environment to shift the balance of activity toward direct inhibition of microbial colonization.     

3',5'-Cyclic diguanylic acid (c-di-GMP) inhibits basal and growth factor-stimulated human colon cancer cell proliferation

The novel cyclic dinucleotide, 3',5'-cyclic diguanylic acid, cGpGp (c-di-GMP), is a naturally occurring small molecule that regulates important signaling mechanisms in prokaryotes. Recently, we showed that c-di-GMP has "drug-like" properties and that c-di-GMP treatment might be a useful antimicrobial approach to attenuate the virulence and pathogenesis of Staphylococcus aureus and prevent or treat infection. In the present communication, we report that c-di-GMP (50 microM) has striking properties regarding inhibition of cancer cell proliferation in vitro. c-di-GMP inhibits both basal and growth factor (acetylcholine and epidermal growth factor)-induced cell proliferation of human colon cancer (H508) cells. Toxicity studies revealed that exposure of normal rat kidney cells and human neuroblastoma cells to c-di-GMP at biologically relevant doses showed no lethal cytotoxicity. Cyclic dinucleotides, such as c-di-GMP, represent an attractive and novel "drug-platform technology" that can be used not only to develop new antimicrobial agents, but also to develop novel therapeutic agents to prevent or treat cancer.     

Interaction of Restin with transcription factors

The melanoma-associated antigen (MAGE) genewas first isolated from a melanoma cell in 1987. Up tonow more than 30 members of MAGE have beenidentified. These members build up a super gene fam-ily and were classified into MAGE-A, B, C, D, E, F, Gand H subfamilies by homolog analysis[1]. On the basisof tissue distribution, these antigens could be catego-rized into two groups, CT antigen (Cancer/Testis An-tigen) and non-CT antigen. The MAGE-A, B, C and Ewere considered to be the CT …     

TMPRSS4 induces cancer cell invasion through pro-uPA processing

TMPRSS4 is a novel type II transmembrane serine protease that is highly expressed on the cell surface in pancreatic, thyroid, colon, and other cancer tissues. Previously, we demonstrated that TMPRSS4 mediates cancer cell invasion, epithelial-mesenchymal transition, and metastasis and that increased TMPRSS4 expression correlates with colorectal cancer progression. We also demonstrated that TMPRSS4 upregulates urokinase-type plasminogen activator (uPA) gene expression to induce cancer cell invasion. However, it remains unknown how proteolytic activity of TMPRSS4 contributes to invasion. In this study, we report that TMPRSS4 directly converted inactive pro-uPA into the active form through its proteolytic activity. Analysis of conditioned medium from cells overexpressing TMPRSS4 demonstrated that the active TMPRSS4 protease domain is released from the cells and is associated with the plasma membrane. Furthermore, TMPRSS4 could increase pro-uPA-mediated invasion in a serine proteolytic activity-dependent manner. These observations suggest that TMPRSS4 is an upstream regulator of pro-uPA activation. This study provides valuable insights into the proteolytic function of TMPRSS4 as well as mechanisms for the control of invasion.     

Effect of dimerization of a β-turn antimicrobial peptide, PST13-RK, on antimicrobial activity and mammalian cell toxicity

PST13-RK (KKKFPWWWPFKKK-NH₂) is an improved derivative of tritrpticin adopting a β-turn structure. In order to investigate the effect of dimerization of PST13-RK on antimicrobial activity and mammalian cell toxicity, we designed and synthesized its Cys- and Lys-linked dimers. The dimerization of PST13-RK resulted in a 2–4 fold decreased antimicrobial activity against Gram-positive and Gram-negative bacteria. However, the dimers showed a large increase in mammalian cell toxicity against mouse NIH-3T3, human MDA-MB-361, and human A549 cells. These results suggested that PST13-RK is active as a monomer to bacterial cells but as an oligomer to mammalian cells. Since the dimeric PST13-RK is much more effective against the cancer cells than the monomer, it might be an attractive candidate for anticancer chemotherapeutic drugs.     

Pro-apoptotic ASY/Nogo-B protein associates with ASYIP

We have previously shown that ectopic expression of the ASY/Nogo-B gene induced apoptosis in various cancer cell lines. Nogo-A, a splice variant of the ASY, has been reported to have an inhibitory effect on neuronal regeneration in the central nervous system. To investigate the mechanism of ASY-induced apoptosis or inhibition of neuronal regeneration, we cloned a cDNA for the ASY-interacting protein from the human cDNA library using the yeast two-hybrid method, and obtained a cDNA we designated as ASYIP. The ASYIP protein contains two hydrophobic regions and a double lysine endoplasmic reticulum (ER) retrieval motif at its C-terminus, which was shown to be identical to RTN3, a reticulon family protein of unknown function. We showed that ASY and ASYIP proteins formed a complex also in human cells. Mutational analysis indicated that both of the hydrophobic regions of the ASYIP protein were required for the association. By immunofluorescence analysis, the ASYIP protein was shown to be co-localized with ASY in the ER. Characterization of the ASYIP gene may be very useful in clarifying the mechanism of ASY-induced apoptosis or Nogo-involved inhibition of neuronal regeneration in the central nervous system.     

TCTP Is an Androgen-Regulated Gene Implicated in Prostate Cancer

TCTP has been implicated in a plethora of important cellular processes related to cell growth, cell cycle progression, malignant transformation and inhibition of apoptosis. In addition to these intracellular functions, TCTP has extracellular functions and plays an important role in immune cells. TCTP expression was previously shown to be deregulated in prostate cancer, but its function in prostate cancer cells is largely unknown. Here we show that TCTP expression is regulated by androgens in LNCaP prostate cancer cells in vitro as well as human prostate cancer xenografts in vivo. Knockdown of TCTP reduced colony formation and increased apoptosis in LNCaP cells, implicating it as an important factor for prostate cancer cell growth. Global gene expression profiling in TCTP knockdown LNCaP cells showed that several interferon regulated genes are regulated by TCTP, suggesting that it may have a role in regulating immune function in prostate cancer. In addition, recombinant TCTP treatment increased colony formation in LNCaP cells suggesting that secreted TCTP may function as a proliferative factor in prostate cancer. These results suggest that TCTP may have a role in prostate cancer development.     

Characterization of human placental beta-hexosaminidase I2. Proteolytic processing intermediates of hexosaminidase A

The lysosomal hydrolase beta-hexosaminidase (beta-N-acetylhexosaminidase, EC 3.2.1.52) exists as two major isozymes in normal human tissue: an acidic A-form and a basic B-form. There are also minor forms of intermediate pI known as I-forms. Increases in one or more of these intermediates have been associated with various disease states. Although the two major isozymes have been extensively studied, the structure and biosynthetic origins of the I-forms are unknown. Characterization of a placental hexosaminidase I-form, presented in this report, demonstrates that it is composed of two forms of partially processed hexosaminidase A. The major form contains an intact pro-alpha chain and a pro-beta chain lacking 2 residues from its amino terminus (Ala and Arg). The minor form also contains an alpha and a beta subunit, but each has undergone further proteolytic processing. The amino terminus of each of these partially processed polypeptide chains matches one of those previously found on stable processing intermediates in a single normal human fibroblast cell line. These data confirm that similar processing intermediates exist in human placenta, suggesting that this I-form lacks a unique enzymatic function in vivo. A sequence of normal proteolytic processing events is postulated.     

整合素连接激酶与肿瘤

整合素连接激酶（integrin—linked kinase,ILK）是一种Ser／Thr蛋白激酶。ILK可以与整合素结合,以依赖于P13K的方式激活,参与多种信号传导通路,包括整合素、生长因子及Wnt信号传导通路。ILK在调节细胞黏附、凋亡、铺展、迁移、生长、细胞周期、肿瘤形成等过程中发挥重要的作用。目前已经发现ILK在多种肿瘤中高表达,包括：神经胶母细胞瘤、黑色素瘤、胃癌、甲状腺肿瘤、卵巢癌、结肠癌、非小细胞肺癌和前列腺癌等。目前国内外大量实验证明ILK和肿瘤密切相关,所以ILK将成为肿瘤基因治疗和肿瘤药物的理想靶位点。     

Isolation of endothelial cells from human placental microvessels: effect of different proteolytic enzymes on releasing endothelial cells from villous tissue

Approaches for the isolation of human placental microvascular endothelial cells (HPMEC) using proteolytic enzymes have been described recently. However, the isolation procedure and enzyme composition most suitable for optimal disaggregation of placental tissue and isolation of HPMEC has not yet been established. We tested different proteolytic enzymes and enzyme mixtures for their capabilities of releasing endothelial cells from human term placental villous tissue. Best results were obtained with a mixture of collagenase/dispase/deoxyribonuclease I (0.28%/0.25%/0.01%). By adding a discontinuous Percoll gradient centrifugation step to the enzymatic dispersion, about 1 x 10(6) cells/g tissue with more than 30% von Willebrand factor (vWf)-positive cells were obtained. However, the total cell number and number of vWf-positive cells were highly dependent on the lot of collagenase used. A perfusion step prior to mincing of villous tissue did not increase the amount of vWf-positive cells. We conclude that the methods described in this study are suitable to isolate high yields of HPMEC and that the composition of the collagenase preparation is crucial to the successful release of endothelial cells from placental tissue. To obtain pure HPMEC, further separation steps, e.g., cell sorting with antibodies against endothelial specific cell surface antigens are necessary.