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Find video protocols related to scientific articles indexed in Pubmed.
Congenital diseases and semaphorin signaling: Overview to date of the evidence linking them.
Congenit Anom (Kyoto)
PUBLISHED: 10-31-2014
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Semaphorins and their receptors, neuropilins and plexins, were initially characterized as a modulator of axonal guidance during development, but are now recognized as a regulator of a wide range of developmental events including morphogenesis and angiogenesis, and activities of the immune system. Owing to the development of next-generation sequencing technologies together with other useful DNA assays, it has also become clear that semaphorin signaling plays a crucial role in many congenital diseases such as retinal degeneration and congenital heart defects. This review summarizes the recent knowledge about the relationship between a variety of congenital diseases and semaphorin signaling.
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Immunohistochemistry for histone h3 lysine 9 methyltransferase and demethylase proteins in human melanomas.
Am J Dermatopathol
PUBLISHED: 03-25-2014
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Methylation and demethylation of histone H3 lysine 9 (H3K9) play a role in the transcriptional regulation of several cancer-related genes and are closely associated with malignant tumor behavior. A novel study has recently demonstrated that SETDB1, a member of the H3K9 methyltransferases, accelerates tumor formation significantly in a zebrafish melanoma model. However, the expression of H3K9 methyltransferases including SETDB1 and demethylases has not been systematically examined in samples of human melanoma. Here, we used immunohistochemistry to examine the expression of the H3K9 methyltransferases, EHMT2 and SETDB1, and a H3K9 demethylase, LSD1, in 67 patients with melanoma. Overexpression of EHMT2, SETDB1, and LSD1 was observed in 14 (21%), 38 (57%), and 53 (79%) of the 67 patients, respectively. A significant relationship was observed between overexpression of EHMT2 or SETDB1 and aggressive tumor behavior such as lymph node metastasis and/or distant metastasis (P < 0.05), whereas no significant relationship was evident for LSD1 immunoreactivity. Univariate log-rank tests demonstrated that patients with melanoma overexpressing EHMT2 had a poorer outcome (P < 0.001), whereas overexpression of SETDB1 or LSD1 had no prognostic impact. These results suggest that overexpression of EHMT2 might be a prognostic marker in patients with melanoma.
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Follistatin-like 5 is expressed in restricted areas of the adult mouse brain: Implications for its function in the olfactory system.
Congenit Anom (Kyoto)
PUBLISHED: 03-05-2014
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Follistatin-like 5 (Fstl5), a member of the follistatin family of genes, encodes a secretory glycoprotein. Previous studies revealed that other members of this family including Fstl1 and Fstl3 play an essential role in development, homeostasis, and congenital disorders. However, the in vivo function of Fstl5 is poorly understood. To gain insight into the function of Fstl5 in the mouse central nervous system, we examined the Fstl5 expression pattern in the adult mouse brain. The results of in situ hybridization analysis showed a highly restricted pattern of Fstl5, namely, with localization in the olfactory system, hippocampal CA3 area and granular cell layer of the cerebellum. Restricted expression in the olfactory system suggests a possible role for Fstl5 in maintaining odor perception.
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Pioneering axons regulate neuronal polarization in the developing cerebral cortex.
Neuron
PUBLISHED: 02-25-2014
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The polarization of neurons, which mainly includes the differentiation of axons and dendrites, is regulated by cell-autonomous and non-cell-autonomous factors. In the developing central nervous system, neuronal development occurs in a heterogeneous environment that also comprises extracellular matrices, radial glial cells, and neurons. Although many cell-autonomous factors that affect neuronal polarization have been identified, the microenvironmental cues involved in neuronal polarization remain largely unknown. Here, we show that neuronal polarization occurs in a microenvironment in the lower intermediate zone, where the cell adhesion molecule transient axonal glycoprotein-1 (TAG-1) is expressed in cortical efferent axons. The immature neurites of multipolar cells closely contact TAG-1-positive axons and generate axons. Inhibition of TAG-1-mediated cell-to-cell interaction or its downstream kinase Lyn impairs neuronal polarization. These results show that the TAG-1-mediated cell-to-cell interaction between the unpolarized multipolar cells and the pioneering axons regulates the polarization of multipolar cells partly through Lyn kinase and Rac1.
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A disintegrin and metalloproteinase 17 (ADAM17) mediates epidermal growth factor receptor transactivation by angiotensin II on hepatic stellate cells.
Life Sci.
PUBLISHED: 01-08-2014
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Epidermal growth factor receptor (EGFR) transactivation induced by angiotensin II (Ang II) participates in the progression of various diseases. A disintegrin and metalloproteinase 17 (ADAM17) is thought to promote renal fibrosis, cardiac hypertrophy with fibrosis and atherosclerosis by activation of the EGFR through secretion of EGFR ligands. The purpose of this study was to investigate whether Ang II-induced EGFR transactivation occurs on hepatic stellate cells (HSCs) and whether the reaction is mediated via ADAM17.
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Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) with hemophagocytosis (HPS): successful treatment using high-dose chemotherapy (BFM-NHL & ALL-90) and autologous peripheral blood stem cell transplantation.
J Clin Exp Hematop
PUBLISHED: 09-03-2013
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Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a rare form of non-Hodgkin lymphoma, in which lymphoma cells infiltrate preferentially into subcutaneous adipose tissue. Although various treatment trials for SPTCL have been attempted, no standardized therapy has been established. Here, we report a case of ?/?(+) T-cell-phenotype SPTCL (SPTCL-AB) with hemophagocytosis (HPS) in a 14-year-old girl, who presented with low-grade fever, general fatigue and chest swelling. Laboratory examinations revealed leukocytopenia, and bone marrow aspiration cytology showed HPS. The diagnosis of SPTCL-AB was made by biopsy on the basis of thickened subcutaneous tissue in the chest wall. Following high-dose chemotherapy (HDT) of BFM-NHL & ALL-90, autologous peripheral blood stem cell transplantation (auto-PBSCT) was performed. The patient responded to the treatment and has remained asymptomatic for 2 years. Our results suggest that a combination of HDT of BFM-NHL & ALL-90 and auto-SCT treatment is effective for SPTCL associated with HPS.
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BCL2 and BCLxL are key determinants of resistance to antitubulin chemotherapeutics in melanoma cells.
Exp. Dermatol.
PUBLISHED: 05-29-2013
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Malignant melanoma is refractory to various chemotherapeutics including antitubulin agents such as paclitaxel. Previous studies have suggested a link between ?III-tubulin overexpression and paclitaxel resistance through alterations in the properties of the mitotic spindle. We found that paclitaxel treatment induced temporary mitotic arrest in 7 melanoma cell lines irrespective of the ?III-tubulin level, suggesting that ?III-tubulin had no significant influence on spindle properties. On the other hand, the amount of BCL2, an anti-apoptotic protein, was well correlated with paclitaxel resistance. Treatment of the paclitaxel-resistant cell lines with ABT-737, an inhibitor of BCL2 and BCLxL, or simultaneous knock-down of BCL2 and BCLxL dramatically increased the cells sensitivity, while knock-down of MCL1, another member of the BCL2 family, had only a minimal effect. Our results suggest that the paclitaxel sensitivity of melanoma cells is attributable to apoptosis susceptibility rather than a change in spindle properties and that BCL2 and BCLxL play a pivotal role in the former.
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Development of the dorsal ramus of the spinal nerve in the mouse embryo: involvement of semaphorin 3A in dorsal muscle innervation.
Congenit Anom (Kyoto)
PUBLISHED: 03-10-2013
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The spinal nerve, which is composed of dorsal root ganglion (DRG) sensory axons and spinal motor axons, forms the dorsal ramus projecting to the dorsal musculature. By using the free-floating immunohistochemistry method, we closely examined the spatiotemporal pattern of the formation of the dorsal ramus and the relationship between its projection to the myotome/dorsal musculature and semaphorin 3A (Sema3A), which is an axonal guidance molecule. In embryonic day (E) 10.5-E11.5 wild-type mouse embryos, we clearly showed the existence of a waiting period for the dorsal ramus projection to the myotome. In contrast, in E10.5-E11.5 Sema3A-deficient embryos, the dorsal ramus fibers projected beyond the edge of the myotome without exhibiting the waiting period for projection. These results strongly suggest that the delayed innervation by dorsal ramus fibers may be caused by Sema3A-induced axon repulsion derived from the myotome. Next, by performing culture experiments, we confirmed that E12.5 mouse axons responded to Sema3A-induced repulsion. Together, our results imply that Sema3A may play a key role in the proper development of the dorsal ramus projection.
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Loss of HOXD10 expression induced by upregulation of miR-10b accelerates the migration and invasion activities of ovarian cancer cells.
Int. J. Oncol.
PUBLISHED: 02-22-2013
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Small and large non-coding RNAs (ncRNAs) contribute to the acquisition of aggressive tumor behavior in diverse human malignancies. Two types of ncRNAs, miRNA?10b (miR-10b) and homemobox (HOX) transcript antisense RNA (HOTAIR), can suppress the translation of the HOXD10 gene, an mRNA encoding a transcriptional repressor that inhibits the expression of cell migration/invasion-associated genes. Using epithelial ovarian cancer cell lines and primary tumors, we investigated whether miR?10b and/or HOTAIR can regulate the expression of HOXD10, and whether it permits gain of pro?metastatic gene products, matrix metallopeptidase 14 (MMP14) and ras homolog family member C (RHOC). Overexpression of miR-10b induced a decrease in HOXD10 protein expression, and upregulated the migration and invasion abilities in ovarian cancer cell lines (P<0.05). In these cells, a significant increase of MMP14 and RHOC protein was observed. No significant upregulation of the HOXD10 protein was observed in cells with the treatment of HOTAIR-siRNA. Positive signals for HOXD10 and MMP14 proteins were observed in 47 (69%) and 25 (37%) of 68 patients with epithelial ovarian cancers. An inverse correlation between HOXD10 and MMP14 immunoreactivities was observed (P<0.05), and miR-10b expression was also inversely correlated with HOXD10 protein expression (P<0.05). These results suggested that downregulation of HOXD10 expression by miR-10b overexpression may induce an increase of pro-metastatic gene products, such as MMP14 and RHOC, and contribute to the acquisition of metastatic phenotypes in epithelial ovarian cancer cells.
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Elucidation of target muscle and detailed development of dorsal motor neurons in chick embryo spinal cord.
J. Comp. Neurol.
PUBLISHED: 02-15-2013
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The avian cervical spinal cord includes motoneurons (MNs) that send their axons through the dorsal roots. They have been called dorsal motoneurons (dMNs) and assumed to correspond to MNs of the accessory nerve that innervate the cucullaris muscle (SAN-MNs). However, their target muscles have not been elucidated to date. The present study sought to determine the targets and the specific combination of transcription factors expressed by dMNs and SAN-MNs and to describe the detailed development of dMNs. Experiments with tracing techniques confirmed that axons of dMNs innervated the cucullaris muscle. Retrogradely labeled dMNs were distributed in the ventral horn of C3 and more caudal segments. In most cases, some dMNs were also observed in the C2 segment. It was also demonstrated that SAN-MNs existed in the ventral horn of the C1-2 segments and the adjacent caudal hindbrain. Both SAN-MNs and dMNs expressed Isl1 but did not express Isl2, MNR2, or Lhx3. Rather, these MNs expressed Phox2b, a marker for branchial motoneurons (brMNs), although the intensity of expression was weaker. Dorsal MNs and SAN-MNs were derived from the Nkx2.2-positive precursor domain and migrated dorsally. Dorsal MNs remain in the ventral domain of the neural tube, unlike brMNs in the brainstem. These results indicate that dMNs and SAN-MNs belong to a common MN population innervating the cucullaris muscle and also suggest that they are similar to brMNs of the brainstem, although there are differences in Phox2b expression and in the final location of each population. J. Comp. Neurol. 521: 2987-3002, 2013. © 2013 Wiley Periodicals, Inc.
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Coexpression of Runx1 and Runx3 in mechanoreceptive dorsal root ganglion neurons.
Dev Neurobiol
PUBLISHED: 01-22-2013
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Runt-related transcription factors (Runx) regulate the development of various cells. It has been reported that Runx1 and Runx3 are expressed in distinct subpopulations of primary sensory neurons in the dorsal root ganglion (DRG), and play important roles in the differentiation of nociceptive and proprioceptive neurons, respectively. In the present study, we examined the developmental changes of the expression of Runx1 and Runx3 in the mouse DRG during embryonic and postnatal stages. We found that the expression of Runx3 preceded that of Runx1, but dramatically decreased before birth, whereas the Runx1 expression was maintained during postnatal periods. These results suggest that roles of Runx1 and Runx3 may change dynamically in the differentiation and maturation of DRG neurons. In addition, several DRG neurons expressed both Runx1 and Runx3 throughout embryonic and postnatal stages and many Runx3-expressing DRG neurons coexpressed Runx1 at postnatal day 28. Double and triple labeling studies suggest that some of the Runx1/Runx3-double expressing neurons coexpressed TrkB, c-ret, and TrkC, which have been shown in the mechanoreceptive DRG neurons. These results suggest that Runx1/Runx3-double expressing neurons may represent mechanoreceptive properties in the DRG
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Predisposition for borderline personality disorder with comorbid major depression is associated with that for polycystic ovary syndrome in female Japanese population.
Neuropsychiatr Dis Treat
PUBLISHED: 11-01-2011
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Polycystic ovary syndrome (PCOS) is a common lifestyle-related endocrinopathy in women of reproductive age and is associated with several mental health problems. We examined the genotypic distributions of IRS-1 Gly972Arg and CYP11B2 -344T/C, which were previously described as influencing PCOS, and assayed the serum levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-?), in a set of female patients with borderline personality disorder (BPD) with comorbid major depressive disorder (MDD) (n = 50) and age-matched control subjects (n = 100), to investigate the predisposition for BPD with MDD. The results showed that the patients were more frequently IRS-1 972Arg variant allele carriers (P = 0.013; OR 6.68; 95% CI = 1.30-34.43) and homozygous for the CYP11B2 -344C variant allele (P = 0.022; OR = 3.32; 95% CI = 1.18-9.35) than the control subjects. The IL-6 level was significantly higher in the patients than in the controls (P < 0.0001). There was no significant difference in the serum TNF-? level between patients with BPD with MDD and the healthy comparison group (P = 0.5273). In conclusion, the predisposition for BPD with MDD is associated with that for PCOS, in the female Japanese population. An elevated serum IL-6 level is considered to be a possible biomarker of BPD with MDD.
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Transcriptional and post-transcriptional regulation of ?III-tubulin protein expression in relation with cell cycle-dependent regulation of tumor cells.
Int. J. Oncol.
PUBLISHED: 09-01-2011
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The expression of ?III-tubulin (TUBB3) is generally restricted to neurons, but its mRNA is often expressed at low levels in non-neuronal cells. Interestingly, however, a number of non-neural tumors occasionally express high levels of TUBB3 protein, leading to a significant resistance to taxane derivatives. However, the molecular mechanisms controlling TUBB3 expression and its turnover during normal cell growth are largely unknown. Here, we present evidence that TUBB3 expression occurs in a cell cycle-dependent manner, and that its protein levels are controlled by the ubiquitin-proteasome system. Both mRNA and protein of TUBB3 accumulated around the G2/M stage of the cell cycle, and reduction of TUBB3 expression by siRNA resulted in partial inhibition of cell growth. Furthermore, the cell cycle-dependent expression of TUBB3 was mediated by the RE-1-silencing transcription factor REST through its binding to the RE-1 element that is present in the first intron of the TUBB3 gene. These results demonstrate a novel role of TUBB3 in cell cycle progression in non-neuronal cells, and further suggest that dysregulation of the REST-TUBB3 system could be a primary cause of the TUBB3 overexpression.
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Nucleus accumbens-associated 1 contributes to cortactin deacetylation and augments the migration of melanoma cells.
J. Invest. Dermatol.
PUBLISHED: 05-12-2011
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We investigated the prognostic significance and post-transcriptional acetylation-modification of cortactin (CTTN) via the nucleus accumbens-associated 1 (NACC1)-histone deacetylase 6 (HDAC6) deacetylation system in primary melanomas and melanoma cell lines. Overexpression of CTTN protein was observed in 56 (73%) of 77 stage I-IV melanomas, and was significantly correlated with tumor thickness, lymph node metastasis, distant metastasis, and disease outcome. The patients whose tumors exhibited CTTN overexpression had a poorer outcome than patients without this feature (P=0.028, log-rank test). NACC1 and CTTN proteins, but not HDAC6, were overexpressed in four melanoma cell lines in comparison with a primary culture of normal human epidermal melanocytes. Knockdown of both NACC1 and HDAC6 markedly downregulated the migration activity of all melanoma cell lines (P<0.05), and induced a gain of CTTN protein acetylation status. Confocal microscopy showed that hyperacetylation of CTTN modulated by depletion of both NACC1 and HDAC6 induced disappearance of CTTN protein at the leading edge of migrating cells, resulting in stabilization of the focal adhesion structure and development of actin stress fibers. These data suggest that the acetylation status of CTTN modulated by the NACC1-HDAC6 deacetylation system induces acceleration of melanoma cell migration activity via an actin-dependent cellular process, possibly contributing to aggressive behavior (invasion/metastasis) of the melanoma cells.
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Downregulation of microRNA-211 is involved in expression of preferentially expressed antigen of melanoma in melanoma cells.
Int. J. Oncol.
PUBLISHED: 02-22-2011
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MicroRNAs (miRNAs) are small non-coding RNAs whose aberrations are involved in the initiation and progression of human cancers. To seek unique miRNAs contributing to melanoma tumorigenesis, we investigated the global miRNA expression profile of 7 melanoma cell lines and 3 primary cultures of neonatal human epidermal melanocytes (NHEMs) using the stem-loop real-time PCR method. We found 7 miRNAs that were commonly downregulated and 18 that were upregulated in all of the melanoma cell lines in comparison with the 3 primary cultures of NHEMs. We focused on one commonly downregulated miRNA (miR-211), and analyzed its relationship to the expression of preferentially expressed antigen of melanoma (PRAME) protein, which is a potential target of miR-211. We found that all melanoma cell lines exhibited marked down--regulation of miR-211 and upregulation of PRAME mRNA/protein expression in comparison with NHEMs (P<0.05). A significant inverse correlation between miR-211 and PRAME protein expression was found in melanoma cell lines and primary cultures of NHEMs (correlation coefficient of -0.733, P<0.05). We demonstrated that overexpression of miR-211 induced a reduction of PRAME protein levels, and confirmed the target specificity between miR-211 and PRAME by luciferase reporter assay. These results suggest that downregulation of miR-211 may be partly involved in aberrant expression of the PRAME protein in melanoma cells.
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Rho/Rho-kinase signaling pathway controls axon patterning of a specified subset of cranial motor neurons.
Eur. J. Neurosci.
PUBLISHED: 01-11-2011
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Cranial motor neurons, which are divided into somatic motor (SM), branchiomotor (BM) and visceral motor (VM) neurons, form distinct axonal trajectories to innervate their synapse targets. Rho GTPase regulates various neuronal functions through one of the major effector proteins, Rho-kinase. Here, we addressed the in vivo role of the Rho/Rho-kinase signaling pathway in axon patterning of cranial motor neurons. We performed conditional expression of a dominant-negative mutant for RhoA or Rho-kinase in transgenic mice by using the Cre-loxP system to suppress the activity of these molecules in developing cranial motor neurons. Blockade of the Rho/Rho-kinase signaling pathway caused defects in the patterning of SM axons but not in that of BM/VM axons, in which defects were accompanied by reduced muscle innervation and reduced synapse formation by SM neurons. In addition, blockade of the signaling pathway shifted the trajectory of growing SM axons in explant cultures, whereas it did not appear to affect the rate of spontaneous axonal outgrowth. These results indicate that the Rho/Rho-kinase signaling pathway plays an essential role in the axon patterning of cranial SM neurons during development.
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Non-invasive evaluation of liver fibrosis using acoustic radiation force impulse imaging in chronic hepatitis patients with hepatitis C virus infection.
Hepatogastroenterology
PUBLISHED: 10-12-2010
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Acoustic radiation force impulse (ARFI) imaging is a new technology for performing liver elastography. However, use of this technique to estimate the degree of fibrosis in chronic liver disease (CLD) related to hepatitis C virus (HCV) infection has not yet been established. We evaluated the validity, accuracy and flexibility of the ARFI method in CLD.
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Identification and characterization of a novel zebrafish semaphorin.
Neurosci. Lett.
PUBLISHED: 08-31-2010
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The semaphorin gene family contains numerous secreted and transmembrane proteins. Some of them function as the repulsive and attractive axon guidance molecules during development. Herein, we report the cloning and characterization of a novel member of zebrafish semaphorin gene, semaphorin 6E (sema6E). Sema6E is expressed predominantly in the nervous system during embryogenesis. Results also show that Sema6E binds Plexin-A1, but not other Plexins. Sema6E chemorepels not only dorsal root ganglion axons but also sympathetic axons. Therefore, Sema6E might utilize Plexin-A1 as a receptor to repel axons of the specific types during development.
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Immunohistochemical study of HER2 and TUBB3 proteins in extramammary Paget disease.
Am J Dermatopathol
PUBLISHED: 06-11-2010
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Metastatic extramammary Paget disease (EMPD) is a potentially fatal malignancy for which effective chemotherapy and good biomarkers are desirable for management. We investigated the status of human epidermal growth factor receptor (HER2) and neuronal ?-tubulin isotype (class III ?-tubulin; TUBB3), whose overexpression is a factor involved in resistance of tumor cells to taxane derivatives) in 32 patients with EMPD. HER2 status was evaluated by immunohistochemistry followed by fluorescence in situ hybridization, and TUBB3 status was evaluated by immunohistochemistry. On the basis of the US Food and Drug Administration-approved criteria, 20 (63%) of the 32 EMPD tumors were found to overexpress HER2. Positive immunoreactivity for TUBB3 was observed in 7 (22%) of the 32 patients. Although some clinicopathologic variables (nodule formation, depth of tumor cells, presence of lymph node metastasis, and serum carcinoembryonic antigen level) were significantly associated with disease outcome (P < 0.05), HER2 gain or aberrant TUBB3 expression showed no significant correlation. However, the higher incidence of HER2 gain and the relatively lower incidence of aberrant TUBB3 expression suggested that HER2-targeted immunotherapy combined with taxane derivatives is warranted for metastatic EMPD, and that HER2 and TUBB3 status might be a good biomarker for determining the most appropriate therapeutic modality.
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Application of a new histological staging and grading system for primary biliary cirrhosis to liver biopsy specimens: Interobserver agreement.
Pathol. Int.
PUBLISHED: 04-21-2010
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Recently the authors proposed a new staging and grading system for primary biliary cirrhosis (PBC) that takes into account necroinflammatory activity and histological heterogeneity. Herein is proposed a convenient version of this system. Scores for fibrosis, bile duct loss, and chronic cholestasis were combined for staging: stage 1, total score of 0; stage 2, score 1-3; stage 3, score 4-6; and stage 4, score 7-9. Cholangitis activity (CA) and hepatitis activity (HA) were graded as CA0-3, and HA0-3, respectively. Analysis of interobserver agreement was then conducted. Digital images of 62 needle liver biopsy specimens of PBC were recorded as virtual slides on DVDs that were sent to 28 pathologists, including five located overseas. All participants were able to apply this version in all 62 cases. For staging, kappa was 0.385 (fair agreement) and the concordance rate was 63.9%. For necroinflammatory activity, the kappa and concordance rate were 0.110 (slight agreement) and 36.9% for CA, and 0.197 (slight agreement) and 47% for HA, respectively. In conclusion, this new staging and grading system for PBC seems to be more convenient and practical than those used at present, but more instruction and guidance are recommended for the grading of necroinflammatory activity in practice.
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Detection of human papillomavirus in a urothelial carcinoma mimicking urethral caruncle.
Int. J. Urol.
PUBLISHED: 04-10-2010
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Primary carcinoma of the female urethra is an uncommon diagnosis, accounting for less than 0.02% of all carcinomas in women. Urothelial carcinomas occupying the distal urethra in young females are considered to be extremely rare. Here we report what we believe to be the sixth case of primary urothelial carcinoma in the published English-language literature. The patient, a 26-year-old woman, presented with a distal urethral lesion that resembled a caruncle, but which was proved to be a urothelial carcinoma on histopathological examination of the resected specimen. Human papillomavirus type 51 DNA was detected in the tumor by polymerase chain reaction and in situ hybridization. These findings suggest that human papillomavirus might be involved in a subset of urothelial carcinomas of the urethra.
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Role for netrin-1 in sensory axonal guidance in higher vertebrates.
Fukushima J Med Sci
PUBLISHED: 12-17-2009
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During development, dorsal root ganglion (DRG) neurons in higher vertebrates extend their axons centrally to the spinal cord through the dorsal root entry zone (DREZ) and peripherally to muscle and skin targets. After entering the spinal cord, DRG axons project into the dorsal mantle layer. In this review, we focus on evidence showing the role for netrin-1 in forming sensory axonal trajectories. Netrin-1 is a diffusible axonal guidance molecule that chemorepels developing DRG axons. When DRG axons project toward the DREZ, ventral spinal cord-derived netrin-1 prevents DRG axons from projecting aberrantly toward the ventral spinal cord. At later stages, the dorsal spinal cord cells transiently express netrin-1. This dorsal spinal cord-derived netrin-1 prevents DRG axons from invading the dorsal spinal cord during the waiting period. Together, the data reviewed provide strong evidence that netrin-1 plays a crucial role in sensory axon projection during development.
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Association of the TNF-{alpha}-C-857T polymorphism with resistance to the cholesterol-lowering effect of HMG-CoA reductase inhibitors in type 2 diabetic subjects.
Diabetes Care
PUBLISHED: 12-10-2009
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OBJECTIVE An association of the C-857T polymorphism of the tumor necrosis factor-alpha (TNF-alpha) gene promoter region with LDL cholesterol levels has been reported. This study was designed to evaluate the relationship between the TNF-alpha-C-857T polymorphism and LDL cholesterol levels according to statin treatment in subjects with type 2 diabetes. RESEARCH DESIGN AND METHODS DNA was obtained from 322 Japanese subjects (160 male and 162 female) with type 2 diabetes, and TNF-alpha-C-857T polymorphisms were determined by direct sequencing. Serum LDL cholesterol was measured by a direct method. RESULTS Although serum LDL cholesterol levels were significantly higher in the T carriers (C/T + T/T) than in the non-T carriers (C/C) (3.14 +/- 0.86 vs. 2.89 +/- 0.75 mmol/l, P < 0.05), there was no difference in LDL cholesterol levels between the non-T carriers and the T carriers in statin-untreated subjects (2.87 +/- 0.73 vs. 2.89 +/- 0.76 mmol/l, NS), whereas in statin-treated subjects, LDL cholesterol levels were significantly higher in the T carriers than in the non-T carriers (3.43 +/- 0.89 vs. 2.90 +/- 0.78 mmol/l, P = 0.0007). There were no differences in HDL cholesterol and triglyceride levels between the non-T carriers and the T carriers in both statin-treated and -untreated subjects. The percent decrease in LDL cholesterol levels after administration of statins was significantly smaller in the T carriers compared with the non-T carriers (27.6 vs. 36.4%, P = 0.031). CONCLUSIONS The mutant allele of the C-857T promoter polymorphism of the TNF-alpha gene may predispose to resistance to the LDL cholesterol-lowering effect of statins and could be one of the markers used to predict the efficacy of statins.
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Expression profiles of nestin in vascular smooth muscle cells in vivo and in vitro.
Exp. Cell Res.
PUBLISHED: 09-02-2009
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Nestin is an intermediate filament protein expressed in neural and mesenchymal stem cells. Here, we investigated the expression of nestin in vascular smooth muscle cells (VSMCs) in vivo and in vitro. In the developing arteries, medial VSMCs were found to express nestin; its expression was prominent in embryos but was down-regulated after birth (3-6 weeks) in a region-dependent manner; its expression was abolished in the adult. Thus, the expression of nestin is specific to developing VSMCs. In primary VMSC cultures, nestin expression was induced by serum, but was independent of cell-cycle progression. Signaling analyses revealed that the serum-induced nestin expression depended on the extracellular signal-regulated kinase (ERK) and protein kinase B (PKB)(Akt) pathways, via the platelet derived growth factor (PDGF) and epidermal growth factor (EGF) receptors. Nestin expression was closely related to the up-regulation and activation of Sp1 and Sp3. Among major serum growth factors and cytokines, PDGF-BB was the most potent inducer of nestin expression. Nestin was also up-regulated in arteries undergoing vascular remodeling following balloon injury. Its expression was particularly strong in the cells lining the lumen of the neointima, suggesting a possible correlation between nestin expression and the progression of vascular remodeling.
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DNA hypomethylation at the CpG island is involved in aberrant expression of the L1 cell adhesion molecule gene in colorectal cancer.
Int. J. Oncol.
PUBLISHED: 07-30-2009
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The L1 cell adhesion molecule (L1CAM) has been identified as a target gene of beta-catenin-TCF signaling in colorectal cancer (CRC) and associated with aggressive tumor behavior such as invasion and metastasis. We investigated the methylation status at the L1CAM gene promoter and/or L1CAM mRNA/protein expression in 4 CRC cell lines and 71 primary CRCs. Aberrant L1CAM expression was immuno histochemically observed in 31 (43.7%) of 71 cases, and correlated with advanced stage and presence of lymph node and distant metastases (P<0.05). Treatment with a demethylating agent induced L1CAM mRNA/protein expression in two cell lines lacking L1CAM expression. Bisulfite-modified genome sequencing suggested that DNA methylation status at core promoter and putative TCF-binding sites within the L1CAM promoter was correlated with L1CAM mRNA/protein expression in 4 CRC cell lines. Using the crypt isolation followed by bisulfite-modified genome sequencing and methylation-specific PCR methods, we confirmed that the DNA hypomethylation at core promoter and putative TCF-binding sites was well correlated with the aberrant L1CAM protein expression in primary CRC samples. These results suggest that DNA hypomethylation at the L1CAM CpG islands might induce L1CAM aberrant expression and contribute to the acquisition of aggressive tumor behavior in CRC.
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Differential microRNA expression between bone marrow side population cells and hepatocytes in adult mice.
Int. J. Mol. Med.
PUBLISHED: 06-11-2009
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Hematopoietic stem cells (HSCs) can differentiate into many kinds of parenchymal cells populating several organs. Nevertheless, the differentiation mechanism of HSCs toward hepatocytes remains poorly understood. To identify specific microRNAs (miRNAs) contributing to the mechanism, we investigated the differential expression of miRNAs between side population (SP) cells of bone marrow and hepatocytes in adult mice. We used a miRNA microarray followed by stem-loop-mediated reverse transcription real-time PCR to identify 12 SP-specific and 2 hepatocyte-specific miRNAs. Of these, 3 (miR-451, -150 and -223) were strongly expressed (>10-fold relative enrichment) in SP cells. Two of these miRNAs (miR-451 and -223) were strongly associated with the hematic cell lineage but not with progenitor characteristics. Two-thirds (6/9) of the miRNAs that were moderately expressed in SP cells in comparison with hepatocytes were also up-regulated in potential hepatic stem cells (HSCEs). The single miRNA (miRNA-127) that was up-regulated in SP cells compared with lineage-positive bone marrow cells might be an SP marker, since it was markedly down-regulated in HSCEs. These results suggest that SP cells and HSCE share a common profile of miRNA expression and that miRNA-127 may contribute to the maintenance of a quiescent state in SP cells.
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Netrin-1 signaling for sensory axons: Involvement in sensory axonal development and regeneration.
Cell Adh Migr
PUBLISHED: 04-14-2009
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During development, dorsal root ganglion (DRG) neurons extend their axons toward the dorsolateral part of the spinal cord and enter the spinal cord through the dorsal root entry zone (DREZ). After entering the spinal cord, these axons project into the dorsal mantle layer after a waiting period of a few days. We revealed that the diffusible axonal guidance molecule netrin-1 is a chemorepellent for developing DRG axons. When DRG axons orient themselves toward the DREZ, netrin-1 proteins derived from the ventral spinal cord prevent DRG axons from projecting aberrantly toward the ventral spinal cord and help them to project correctly toward the DREZ. In addition to the ventrally derived netrin-1, the dorsal spinal cord cells adjacent to the DREZ transiently express netrin-1 proteins during the waiting period. This dorsally derived netrin-1 contributes to the correct guidance of DRG axons to prevent them from invading the dorsal spinal cord. In general, there is a complete lack of sensory axonal regeneration after a spinal cord injury, because the dorsal column lesion exerts inhibitory activities toward regenerating axons. Netrin-1 is a novel candidate for a major inhibitor of sensory axonal regeneration in the spinal cord; because its expression level stays unchanged in the lesion site following injury, and adult DRG neurons respond to netrin-1-induced axon repulsion. Although further studies are required to show the involvement of netrin-1 in preventing the regeneration of sensory axons in CNS injury, the manipulation of netrin-1-induced repulsion in the CNS lesion site may be a potent approach for the treatment of human spinal injuries.
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Laminin peptide YIGSR and its receptor regulate sensory axonal response to the chemoattractive guidance cue in the chick embryo.
J. Neurosci. Res.
PUBLISHED: 03-26-2009
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During early development, centrally projecting dorsal root ganglion (DRG) neurons extend their axons toward the dorsal spinal cord. We previously reported the involvement of dorsal spinal cord-derived chemoattraction in this projection (Masuda et al. [ 2007] Neuroreport 18:1645-1649). However, the molecular nature of this attraction is not clear. Here we show that laminin-1 (alpha1beta1gamma1) is expressed strongly along the pathway of DRG axons and that its 67-kDa receptor (67LR) is present on DRG cells. This evidence suggests that laminin-1-67LR signaling may be involved in DRG axonal guidance. By employing culture assays, we show that laminin-1 or the YIGSR peptide, a soluble peptide of the laminin beta1 chain, promotes the DRG axonal response to dorsal spinal cord-derived chemoattraction. By using a function-blocking antibody against 67LR, we show that the anti-67LR antibody blocks the modulation of DRG axonal response by the YIGSR peptide in vitro. Furthermore, the in ovo injection of the anti-67LR antibody inhibits the DRG axonal growth toward the dorsal spinal cord. These results provide evidence that the YIGSR peptide promotes dorsal spinal cord-derived chemoattraction via 67LR to contribute to the formation of the initial trajectories of developing DRG axons.
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Laser capture microdissection and cDNA array analysis for identification of mouse KIAA/FLJ genes differentially expressed in the embryonic dorsal spinal cord.
Brain Res.
PUBLISHED: 01-31-2009
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During early development, centrally projecting dorsal root ganglion (DRG) neurons extend their axons toward the dorsal spinal cord. We previously reported that this projection is achieved by dorsal spinal cord-derived chemoattraction. However, the molecular nature of the chemotrophic cue is not yet fully understood. To identify novel genes differentially expressed in the dorsal spinal cord in the embryonic day 10.5 mouse, we used the Kazusa cDNA array system comprising approximately 1700 mouse KIAA/FLJ (mKIAA/mFLJ) cDNA clones and laser capture microdissection (LCM) in combination with PCR-based cDNA amplification. We observed that a certain population of genes showed significantly increased expression in the dorsal spinal cord. In situ hybridization analysis verified the expression of mRNAs of 6 genes (Hip1r, Nav2, Fstl5, Cacna1h, Bcr, and Bmper) in the cells that constitute the dorsal spinal cord. The dorsal spinal cord-specific genes identified in this study provide a basis for studying the molecular nature of the neural development including the axonal guidance of DRG neurons. These results also demonstrate that the combined use of LCM coupled with the Kazusa cDNA array technology will be useful for the identification of large proteins expressed in the restricted small regions of embryos.
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Ontogeny of reticular framework of white pulp and marginal zone in human spleen: immunohistochemical studies of fetal spleens from the 17th to 40th week of gestation.
Cell Tissue Res.
PUBLISHED: 01-10-2009
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The antigenic heterogeneity of the reticular framework of the white pulp (WP) and marginal zone (MZ) is well documented in the human adult spleen. The ontogeny of the WP and MZ of human fetal spleens was examined with special reference to the heterogeneity of the reticular framework. In the spleen of the 17th gestational week (gw), alpha-smooth muscle actin (alpha-SMA)-positive reticulum cells were scattered around the arterioles. From the 20th to 23rd gw, alpha-SMA-positive reticulum cells increased in number and began to form a reticular framework. An accumulation of T and B lymphocytes occurred within the framework, and a primitive WP was observed around the arterioles. At the 24th gw, antigenic diversity of the reticular framework was observed, and T and B lymphocytes were segregated in the framework. T lymphocytes were sorted into the alpha-SMA-positive reticular framework, and the periarteriolar lymphoid sheath (PALS) was formed around the arteriole. B lymphocytes aggregated in eccentric portions to the PALS and formed the lymph follicle (LF). The reticular framework of the LF was alpha-SMA-negative. MZ appeared in the alpha-SMA-positive reticular framework around the WP at the 26th gw. The PALS, LF, and MZ developed with gestational time. The reticular framework of the PALS, LF, and MZ is thus heterogeneous in the fetal spleen, and the development of the heterogeneity is related to the ontogeny of the PALS, LF, and MZ.
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Loss of class III beta-tubulin induced by histone deacetylation is associated with chemosensitivity to paclitaxel in malignant melanoma cells.
J. Invest. Dermatol.
PUBLISHED: 01-01-2009
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Overexpression of class III beta-tubulin (TUBB3) is an important mechanism of taxane resistance. Using 7 melanoma cell lines, 2 normal neonatal human epidermal melanocyte (NHEM) cultures, and 49 primary melanomas, we investigated TUBB3 expression, its relationship to chemosensitivity to taxane derivatives, and the epigenetic mechanism controlling TUBB3 gene expression. Normal melanocytes in vitro and in vivo strongly expressed TUBB3 protein. NHEMs exhibited marked chemoresistance to paclitaxel-induced apoptosis. A subset (10 of 49, 20%) of primary malignant melanomas was TUBB3 negative. The incidence of TUBB3-negative melanomas increased with stage of progression. TUBB3 protein expression varied among cell lines; one (HMV-I) of the seven cell lines exhibited an extremely low endogenous level. TUBB3 protein expression correlated well with chemosensitivity to paclitaxel-induced apoptosis (P<0.05). Treatment with a histone deacetylase (HDAC) inhibitor restored TUBB3 expression in HMV-I. Chromatin immunoprecipitation assays revealed that histones H3 and H4 were hypoacetylated at the TUBB3 gene in HMV-I as compared with a TUBB3-overexpressing cell type (HMV-II). Treatment with the HDAC inhibitor induced gain of histone acetylation only in HMV-I. These results suggest that loss of TUBB3 protein may be induced by histone deacetylation in a subset of malignant melanomas, and may be associated with chemosensitivity to taxane.
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Development of the dorsal ramus of the spinal nerve in the chick embryo: a close relationship between development and expression of guidance cues.
Brain Res.
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The spinal nerve, which is composed of dorsal root ganglion (DRG) axons and spinal motor axons, divides into ventral and dorsal rami. Although the development of the ventral ramus has been examined in considerable detail, that of the dorsal ramus has not. Therefore, we first examined the spatial-temporal pattern of the dorsal ramus formation in the chick embryo, with special reference to the projection to the dermamyotome and its derivatives. Next, we focused on two guidance molecules, chick semaphorin 3A (SEMA3A) and fibroblast growth factor 8 (FGF8), because these are the best candidates as molecules for controlling the dorsal ramus formation. Using in situ hybridization and immunohistochemistry methods, we clearly showed a close relationship between the spatial-temporal expression of SEMA3A/FGF8 and the projection of dorsal ramus fibers to the dorsal muscles. We further examined the axonal response of motor and DRG neurons to SEMA3A and FGF8. We showed that motor axons responded to both SEMA3A-induced repulsion and FGF8-induced attraction. On the other hand, DRG axons responded to SEMA3A-induced repulsion but not to FGF8-induced attraction. These findings suggest that FGF8-induced attraction may guide early motor axons beneath the myotome and that SEMA3A-induced repulsion may prevent these early motor axons from entering the myotome. Our results also imply that the loss of SEMA3A expression in the dorsal muscles may lead to the gross projection of the dorsal ramus fibers into the dorsal muscles. Together, SEMA3A and FGF8 may contribute to the proper formation of the dorsal ramus.
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Overexpression of histone deacetylase 6 contributes to accelerated migration and invasion activity of hepatocellular carcinoma cells.
Oncol. Rep.
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Histone deacetylase 6 (HDAC6) is a cytoplasmic enzyme that regulates many important biological processes, including cell migration, viral infection and autophagy. The aim of this study was to investigate the significance of HDAC6 in the invasion and metastasis activities of hepatocellular carcinoma (HCC). Three HCC cell lines and two primary cultures of hepatocytes were used for biological experiments. Immunohistochemistry for HDAC6 protein was also examined in 70 resected primary HCCs. Knockdown of the HDAC6 gene in the HCC cell lines was carried out by treatment with siRNA, and their migration and invasion activities were examined by the scratch assay and Matrigel invasion assay, respectively. HDAC6 expression was greater in all of the HCC cell lines compared to the primary cultures of hepatocytes. Knockdown of HDAC6 markedly downregulated the migration and invasion activities of all HCC cell lines (P<0.05). Overexpression of HDAC6 protein to a level higher than that in the corresponding normal hepatocytes was observed in 14 (20%) of the 70 primary HCCs, and was significantly correlated with high clinical stage, number of tumors, vascular invasion and intrahepatic metastasis (P<0.05). These results suggest that overexpression of the HDAC6 protein is involved in the migration and invasion activities of HCC cells, and may be a good biomarker for prediction of intrahepatic metastasis.
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Blastic plasmacytoid dendritic cell neoplasm : report of two cases.
J Clin Exp Hematop
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Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a clinically aggressive tumor derived from the precursor of plasmacytoid dendritic cells. We describe two cases of BPDCN. In case 1, the patient presented with multiple erythema on the trunk and arms. Histopathology of a skin biopsy specimen and immunohistochemistry demonstrated that the tumor cells were small to medium-sized with a blastoid morphology and positive for CD4, CD56, CD123 and T-cell leukemia-1 (TCL-1). In case 2, the patient presented with a solitary skin nodule and rapidly developed involvement of the bone marrow and peripheral blood. Although immunohistochemistry of the infiltrating tumor cells demonstrated positivity for CD4, CD56, CD123 and TCL-1, the cells were large with a distinct nucleolus, and different from those of typical BPDCN. The atypical morphological features of BPDCN may be diagnostically problematic, and should therefore be recognized correctly.
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Sensor and effector kinases in DNA damage checkpoint regulate capacity for homologous recombination repair of fission yeast in G2 phase.
DNA Repair (Amst.)
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Although the G2/M DNA damage checkpoint is currently viewed as a set of coordinated cellular responses affecting both cell cycle progression and non-cell cycle targets, the relative contributions of the two target categories to DNA repair and cell survival after exposure to ionizing radiation have not been clearly addressed. We investigated how rad3 (ATR ortholog) or chk1/cds1 (CHK1/CHK2 orthologs) null mutations change the kinetics of double-strand break (DSB) repair in Schizosaccharomyces pombe cells under conditions of forced G2 arrest. After 200-Gy ?-ray irradiation, DSBs were repaired in rad3? cdc25-22 or chk1? cds1? cdc25-22 cells, almost as efficiently as in cdc25-22 cells at the restrictive temperature. In contrast, little repair was observed in the checkpoint-deficient cells up to 4h after higher-dose (500Gy) irradiation, whereas repair was still efficient in the control cdc25-22 cells. Immediate loss of viability appeared not be responsible for the repair defect after the higher dose, since both checkpoint-proficient and deficient cells with cdc25-22 allele synchronously resumed cycling with a similar time course when released to the permissive temperature 4h after irradiation. Recruitment of repair proteins Rad11 (Rpa1 ortholog), Rad22 (Rad52 ortholog), and Rhp54 (Rad54 ortholog) to the damage sites was not significantly impaired in the checkpoint-deficient cells, whereas their release was profoundly delayed. Our results suggest that sensor and effector kinases in the damage checkpoint machinery affect the efficiency of repair downstream of, or in parallel with the core repair reaction.
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Downregulation of cylindromatosis gene, CYLD, confers a growth advantage on malignant melanoma cells while negatively regulating their migration activity.
Int. J. Oncol.
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The cylindromatosis gene (CYLD) encodes a deubiquitinase that was initially identified as a tumor suppressor and has recently been investigated in connection with a variety of normal physiological processes. In contrast to its cell-proliferative activity, the effect of CYLD protein on cell migration has been a matter of debate. We investigated the effect of CYLD-siRNA on the migration activity of malignant melanoma cells. Expression of CYLD mRNA/protein was lower in 6 of 8 malignant melanoma cell lines than in 3 sets of primary-cultured normal human epidermal melanocytes. Knockdown of CYLD significantly increased the proliferation activities of two melanoma cell lines (p<0.05), along with BCL3 nuclear translocation followed by CCND1 overexpression. In contrast to the proliferation-related activity, CYLD knockdown significantly decreased the cell migration of all the melanoma cell lines (n=7, p<0.05), and we demonstrated that the mechanism regulating melanoma cell migration was activation of RAC1 through the action of CYLD. Our findings provide new insight into the role of CYLD-induced RAC1 activation in melanoma cell migration.
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