Role of CAGE, a novel cancer/testis antigen, in various cellular processes, including tumorigenesis, cytolytic T lymphocyte induction, and cell motility

A cancer-associated antigen gene (CAGE) was identified by serological analysis of a recombinant cDNA expression library (SEREX). The gene was identified by screening cDNA expression libraries of human testis and gastric cancer cell lines with sera from patients with gastric cancer. CAGE was found to contain a D-E-A-D box domain and encodes a putative protein of 630 amino acids with possible helicase activity. The CAGE gene is widely expressed in various cancer tissues and cancer cell lines. Demethylation plays a role in the activation of CAGE in certain cancer cell lines where the gene is not expressed. The functional roles of CAGE in tumorigenesis, the molecular mechanisms of CAGE expression, and cell motility are also discussed.     

Pyruvate dehydrogenase kinase-4 deficiency lowers blood glucose and improves glucose tolerance in diet-induced obese mice

The effect of pyruvate dehydrogenase kinase-4 (PDK4) deficiency on glucose homeostasis was studied in mice fed a high-fat diet. Expression of PDK4 was greatly increased in skeletal muscle and diaphragm but not liver and kidney of wild-type mice fed the high-fat diet. Wild-type and PDK4(-/-) mice consumed similar amounts of the diet and became equally obese. Insulin resistance developed in both groups. Nevertheless, fasting blood glucose levels were lower, glucose tolerance was slightly improved, and insulin sensitivity was slightly greater in the PDK4(-/-) mice compared with wild-type mice. When the mice were killed in the fed state, the actual activity of the pyruvate dehydrogenase complex (PDC) was higher in the skeletal muscle and diaphragm but not in the liver and kidney of PDK4(-/-) mice compared with wild-type mice. When the mice were killed after overnight fasting, the actual PDC activity was higher only in the kidney of PDK4(-/-) mice compared with wild-type mice. The concentrations of gluconeogenic substrates were lower in the blood of PDK4(-/-) mice compared with wild-type mice, consistent with reduced formation in peripheral tissues. Diaphragms isolated from PDK4(-/-) mice oxidized glucose faster and fatty acids slower than diaphragms from wild-type mice. Fatty acid oxidation inhibited glucose oxidation by diaphragms from wild-type but not PDK4(-/-) mice. NEFA, ketone bodies, and branched-chain amino acids were elevated more in PDK4(-/-) mice, consistent with slower rates of oxidation. These findings show that PDK4 deficiency lowers blood glucose and slightly improves glucose tolerance and insulin sensitivity in mice with diet-induced obesity.     

miR-326-Histone Deacetylase-3 Feedback Loop Regulates the Invasion and Tumorigenic and Angiogenic Response to Anti-cancer Drugs

Histone modification is known to be associated with multidrug resistance phenotypes. Cancer cell lines that are resistant or have been made resistant to anti-cancer drugs showed lower expression levels of histone deacetylase-3 (HDAC3), among the histone deacetylase(s), than cancer cell lines that were sensitive to anti-cancer drugs. Celastrol and Taxol decreased the expression of HDAC3 in cancer cell lines sensitive to anti-cancer drugs. HDAC3 negatively regulated the invasion, migration, and anchorage-independent growth of cancer cells. HDAC3 conferred sensitivity to anti-cancer drugs in vitro and in vivo. TargetScan analysis predicted miR-326 as a negative regulator of HDAC3. ChIP assays and luciferase assays showed a negative feedback loop between HDAC3 and miR-326. miR-326 decreased the apoptotic effect of anti-cancer drugs, and the miR-326 inhibitor increased the apoptotic effect of anti-cancer drugs. miR-326 enhanced the invasion and migration potential of cancer cells. The miR-326 inhibitor negatively regulated the tumorigenic, metastatic, and angiogenic potential of anti-cancer drug-resistant cancer cells. HDAC3 showed a positive feedback loop with miRNAs such as miR-200b, miR-217, and miR-335. miR-200b, miR-217, and miR-335 negatively regulated the expression of miR-326 and the invasion and migration potential of cancer cells while enhancing the apoptotic effect of anti-cancer drugs. TargetScan analysis predicted miR-200b and miR-217 as negative regulators of cancer-associated gene, a cancer/testis antigen, which is known to regulate the response to anti-cancer drugs. HDAC3 and miR-326 acted upstream of the cancer-associated gene. Thus, we show that the miR-326-HDAC3 feedback loop can be employed as a target for the development of anti-cancer therapeutics.     

Glucosamine increases vascular contraction through activation of RhoA/Rho kinase pathway in isolated rat aorta

Diabetes is a well-known independent risk factor for vascular disease. However, its underlying mechanism remains unclear. It has been reported that increased influx of the hexosamine biosynthesis pathway (HBP) induces O-GlcNAcylation of proteins, leading to insulin resistance. In this study, we determined whether or not O-GlcNAc modification of proteins could increase vessel contraction. Using an endothelium-denuded aortic ring, we observed that glucosamine induced OGlcNAcylation of proteins and augmented vessel contraction stimulated by U46619, a thromboxane A(2) agonist, via augmentation of the phosphorylation of MLC(20), MYPT1(Thr855), and CPI17, but not phenylephrine. Pretreatment with OGT inhibitor significantly ameliorated glucosamine-induced vessel constriction. Glucosamine treatment also increased RhoA activity, which was also attenuated by OGT inhibitor. In conclusion, glucosamine, a product of glucose influx via the HBP in a diabetic state, increases vascular contraction, at least in part, through activation of the RhoA/Rho kinase pathway, which may be due to O-GlcNAcylation.     

Embryonic Demise Caused by Targeted Disruption of a Cysteine Protease Dub-2

Background

A plethora of biological metabolisms are regulated by the mechanisms of ubiquitination, wherein this process is balanced with the action of deubiquitination system. Dub-2 is an IL-2-inducible, immediate-early gene that encodes a deubiquitinating enzyme with growth regulatory activity. DUB-2 presumably removes ubiquitin from ubiquitin-conjugated target proteins regulating ubiquitin-mediated proteolysis, but its specific target proteins are unknown yet.

Methodology/Principal Findings

To elucidate the functional role of Dub-2, we generated genetically modified mice by introducing neo cassette into the second exon of Dub-2 and then homologous recombination was done to completely abrogate the activity of DUB-2 proteins. We generated Dub-2+/− heterozygous mice showing a normal phenotype and are fertile, whereas new born mouse of Dub-2−/− homozygous alleles could not survive. In addition, Dub-2−/− embryo could not be seen between E6.5 and E12.5 stages. Furthermore, the number of embryos showing normal embryonic development for further stages is decreased in heterozygotes. Even embryonic stem cells from inner cell mass of Dub-2−/− embryos could not be established.

Conclusions

Our study suggests that the targeted disruption of Dub-2 may cause embryonic lethality during early gestation, possibly due to the failure of cell proliferation during hatching process.     

Analysis of spinal cord proteome in the rats with mechanical allodynia after the spinal nerve injury

Proteome analysis was carried out to identify the proteins associated with neuropathic pain after peripheral nerve injury. Five proteins displayed different expression levels among three groups of rats. Among these proteins, creatine kinase B expression level was lower in the pain-positive rats compared to the sham or pain-negative rats. Therefore, a lower creatine kinase B expression level may be important in the development and maintenance of neuropathic pain.     

Identification of auto-antibodies in the sera of patients with rheumatoid arthritis

Serological analysis of a recombinant cDNA expression library was carried out and a number of auto-antibodies were found that were highly prevalent in the sera of such patients.