Masaharu Terashima

Epigenetic inactivation of wnt inhibitory factor-1 plays an important role in bladder cancer through aberrant canonical Wnt/β-catenin signaling pathway

Purpose: Aberrant activation of the Wingless-type (Wnt) pathway plays a significant role in the pathogenesis of several human cancers. Wnt inhibitory factor-1 (Wif-1) was identified as one of the secreted antagonists that can bind Wnt protein. We hypothesize that Wif-1 plays an important role in bladder cancer pathogenesis. Experimental Design: To test this hypothesis, epigenetic and genetic pathways involved in the Wif-1 gene modulation and expression of Wnt/β-catenin-related genes were analyzed in 4 bladder tumor cell lines and 54 bladder tumor and matched normal bladder mucosa. Results: Wif-1 mRNA expression was significantly enhanced after 5-aza-2′-deoxycytidine treatment in bladder tumor cell lines. Wif-1 promoter methylation level was significantly higher and Wif-1 mRNA expression was significantly lower in bladder tumor samples than in bladder mucosa samples. In the total bladder tumor and bladder mucosa samples, an inverse correlation was found between promoter methylation and Wif-1 mRNA transcript levels. However, loss-of-heterozygosity at chromosome 12q14.3 close to the Wif-1 gene loci was a rare event (3.7%). Nuclear accumulation of β-catenin was significantly more frequent in bladder tumor than in bladder mucosa and inversely correlated with Wif-1 expression. In addition, known targets of the canonical Wnt/β-catenin signaling pathway, such as c-myc and cyclin D1, were up-regulated in bladder tumor compared with bladder mucosa, and this up-regulation was associated with reduced Wif-1 expression at both mRNA and protein levels. Furthermore, transfection of Wif-1 small interfering RNA into bladder tumor cells expressing Wif-1 mRNA transcripts had increased levels of c-myc and cyclin D1 and accelerated cell growth. Conclusion: This is the first report showing that CpG hypermethylation of the Wif-1 promoter is a frequent event in bladder tumor and may contribute to pathogenesis of bladder cancer through aberrant canonical Wnt/β-catenin signaling pathway. The present study elucidates novel pathways that are involved in the pathogenesis of bladder cancer.

CpG Hypermethylation of MDR1 Gene Contributes to the Pathogenesis and Progression of Human Prostate Cancer

Multidrug resistance 1 (MDR1) gene encodes for P-glycoprotein (P-gp), a Mr 170,000 transmembrane calcium-dependent efflux pump that is inactivated in prostate cancer. We hypothesize that inactivation of the MDR1 gene through CpG methylation contributes to the pathogenesis and progression of prostate cancer. To test this hypothesis, CpG methylation status of the MDR1 promoter and its correlation with clinicopathological findings were evaluated in 177 prostate cancer samples and 69 benign prostate hypertrophy (BPH) samples. Cellular proliferation index and apoptotic index were determined by proliferating cell nuclear antigen (PCNA) and single-strand DNA immunostaining, respectively. After 5-aza-2′-deoxycytidine treatment, increased expression of MDR1 mRNA transcript was found in prostate cancer cell lines (DU145, DuPro, and ND1). MDR1 methylation frequency was significantly higher in prostate cancer samples compared with BPH samples (54.8 versus 11.6%, respectively, P < 0.001). Logistic regression analysis revealed that PC patients are 11.5 times more likely to have MDR1 methylation than BPH patients (95% confidence interval 4.87–27.0) and that MDR1 methylation is independent of the age. Significant correlation of MDR1 methylation was observed with high pT category (P < 0.001), high Gleason sum (P = 0.008), high preoperative prostate-specific antigen (P = 0.01), and advancing pathological features. In addition, PCNA-labeling index were significantly higher in methylation-specific PCR (MSP)-positive than in MSP-negative prostate cancer samples (P = 0.048). In contrast, no significant difference in apoptotic index was found between MSP-positive and -negative prostate cancer samples. These findings suggest that CpG hypermethylation of MDR1 promoter is a frequent event in prostate cancer and is related to disease progression via increased cell proliferation in prostate cancer cells.

Promoter CpG hypomethylation and transcription factor EGR1 hyperactivate heparanase expression in bladder cancer

Heparanase plays a critical role in the degradation of extracellular matrix and cell membrane and is frequently upregulated in malignant tumors. Transcription factor, early growth response 1 (EGR1), is closely associated with inducible transcription of the heparanase gene. We hypothesized that promoter CpG hypomethylation with increased EGR1 expression could determine heparanase expression during the pathogenesis of bladder cancer. Bladder cancer cell lines (J82, T24 and transitional cell carcinoma) significantly restored heparanase expression after 5-Aza-dC treatment. Transfection of EGR1 siRNA with T24 bladder cancer cell line significantly downregulated heparanase expression compared to the control siRNA transfection. In 54 bladder cancer and paired normal bladder samples, heparanase expression was significantly higher in bladder cancer than in normal bladder (P<0.01). We performed methylation-specific PCR targeting the CpG sites within the core-binding consensus motifs of EGR1 (GGCG) and Sp1 (GGGCGG). Methylation prevalence was significantly higher in normal bladder than in bladder cancer (P<0.05) and inversely correlated with heparanase expression (P=0.055). In the total series of bladder cancer and normal bladder samples, the combination of promoter CpG methylation and EGR1 expression regulated heparanase expression in a stepwise manner, where heparanase expression was the lowest in methylation-positive and EGR1-negative samples and the highest in methylation-negative and EGR1-positive samples. To our knowledge, this is the first study demonstrating that increased heparanase expression during the pathogenesis of bladder cancer is due to promoter hypomethylation and transcription factor EGR1.

Smoking influences aberrant CpG hypermethylation of multiple genes in human prostate carcinoma

Aberrant CpG methylation profiles of gene promoters and their correlation with advanced pathologic features have been well investigated in prostate carcinoma (PC). Several case–control and prospective studies have revealed a positive association between current smoking and PC. The authors hypothesized that smoking influences both progression and prognosis of PC through CpG hypermethylation of related genes.

Cathepsin B and H activities and cystatin C concentrations in cerebrospinal fluid from patients with leptomeningeal metastasis

BACKGROUND Cysteine proteases are involved in the extension of cancer into the subarachnoid space. The presence of cathepsins B and H along with their potent inhibitor cystatin C in the cerebrospinal fluid (CSF) was investigated in patients with leptomeningeal metastasis of cancer (LM). MATERIALS AND METHODS CSF samples were obtained in 16 cases of LM (10 solid tumors and 6 leukemia or lymphoma) and compared with 11 cancer cases without involvement of the central nervous system, 12 multiple sclerosis cases and 34 healthy volunteers. The activity of the enzymes was measured, their molecular forms were analyzed by the Western blotting, and the concentration of cystatin C was measured by ELISA. Immunohistochemistry of the leptomeningeal tissues was also performed in six autopsy cases of LM. RESULTS High activities of cathepsins B and H along with decreased cystatin C concentration were observed in CSF of LM as compared with three control groups. Western blot analysis revealed higher concentration of the enzyme protein as well as its active forms in samples with higher enzyme activity. Cells metastasizing leptomeningeal tissue were clearly positive in immunohistochemical staining of cathepsins, indicating active production by tumor cells. CONCLUSION Production of cathepsins B and H by tumor cells and their high activity along with concomitant decrease of their potent inhibitor, cystatin C, in the CSF might contribute in the process of metastasis and spread of the cancer cells in the leptomeningeal tissues. A high enzyme activity/cystatin C concentration ratio in the CSF could be useful when diagnosing LM in combination with other parameters.

Functional Loss of the γ-Catenin Gene through Epigenetic and Genetic Pathways in Human Prostate Cancer

γ-Catenin is a cell adhesion molecule and a candidate mediator of Wnt signal transduction. We hypothesized that impaired regulation of γ-catenin through genetic and epigenetic pathways is associated with the pathogenesis of prostate cancer. To test this hypothesis, cytosine-phosphate-guanine methylation, loss of heterozygosity (LOH), and mutation status of the γ-catenin gene were analyzed in cultured prostate cancer cell lines, 180 localized prostate cancers, 69 benign prostatic hyperplasias, and 11 hormone refractory prostate cancers (HRPC). In prostate cancer cell lines (DuPro, LNCaP, ND-1, and PC3), γ-catenin mRNA transcripts were increased after 5-aza-2′-deoxycytidine treatment. In localized prostate cancer, γ-catenin expression was lower but prevalence of γ-catenin methylation was higher compared with benign prostatic hyperplasia. However, γ-catenin methylation did not correlate with Gleason sum, pT category, or capsular penetration. Among localized prostate cancers with positive γ-catenin methylation, the presence of LOH at chromosome 17q21 was closely related to down-regulation of γ-catenin mRNA expression. The γ-catenin mutations were not found in localized prostate cancers, whereas six mutations were found in five HRPCs within or close to the GSK-3β consensus motif phosphorylation site, among which four HRPCs showed strong nuclear γ-catenin accumulation. In these four HRPCs, Bcl-2 expression was increased, whereas the target of the Wnt signal, c-myc , was only expressed in one HRPC. Therefore, although epigenetic γ-catenin methylation is an early event in the development of prostate cancer, simultaneous events of epigenetic cytosine-phosphate-guanine methylation and genetic LOH may be responsible for functional loss of γ-catenin. The γ-catenin mutation related to Bcl-2 overexpression has a significant effect on the pathogenesis of HRPC. This is the first report to characterize the epigenetic and genetic regulation of γ-catenin in human prostate cancer.

Neuronal cell death induced by cystatin C in vivo and in cultured human CNS neurons is inhibited with cathepsin B.

Cystatin C, a cysteine protease inhibitor, is implicated in pathogenesis of late-onset Alzheimers disease and other neurological disorders. Our recent study showed that cystatin C injection into rat hippocampus induced neuronal cell death in granule cell layer of dentate gyrus in vivo. We further confirmed that cystatin C neurotoxicity was inhibited by simultaneous coapplication of cathepsin B, a cysteine protease. In vitro cytotoxicity was also studied in cultures of human CNS neurons, mixed cultures with astrocytes and A1 human hybrid neurons. Cystatin C induced neuronal cell death in a dose-dependent manner, which accompanied increased number of TUNEL (+) cells, up-regulation of active caspase-3 and DNA ladder. The results of the present study indicate that cystatin C participates in the process of apoptotic neuronal cell death in experimental conditions by means of inhibitory activity of cysteine proteases, and that cystatin C might be involved in the pathogenesis in human neurological disorders including Alzheimers disease.

Nitric oxide regulates actin reorganization through cGMP and Ca2+/calmodulin in RAW 264.7 cells

Abstract Nitric oxide (NO) has been reported to be involved in the regulation of pseudopodia formation, phagocytosis and adhesion in macrophages through the reorganization of actin. In the present study, we directly separated the globular (G) and filamentous (F) actin from quiescent or NO-stimulated macrophage-like cell line RAW 264.7 cells in order to investigate the dynamic redistribution of actin pools. We also focused on the regulatory mechanisms of actin assembly, induced by NO and its possible subsequent signaling pathway. We showed that predominant G-actin coexisted with Triton X-100-insoluble filamentous (TIF) and Triton X-100-soluble filamentous actin in resting RAW 264.7 cells. The exogenous NO produced by (±)-( E )-2-[( E )-hydroxyimino]-6-methoxy-4-methyl-5-nitro-3-hexenamide (NOR1), the endogenous NO induced by lipopolysaccharide (LPS) plus interferon-γ (IFNγ), and dibutyryl-cGMP increased the contents of TIF-actin in dose- and time-dependent manners and altered its morphology. The increase in the TIF-actin contents induced by NOR1 or LPS plus IFNγ was efficiently blocked by the radical scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide and the soluble guanylate cyclase inhibitor 1 H -[1,2,4]oxadiazolo[4,3- a ]quinoxalin-1-one or the arginine analogue N G -monomethyl- l -arginine acetate, respectively. Preincubation with the calmodulin antagonist W-7 almost completely blocked the NO-induced TIF-actin increase and morphological change. On the other hand, preincubation with C3 transferase, an inhibitor of Rho protein, efficiently prevented the change in cell morphology, but had no effect on the TIF-actin increase. We postulate that cGMP and subsequent Ca 2+ /calmodulin may be key regulators of actin reorganization in NO-stimulated RAW 264.7 cells.

Molecular Identification of Human Glutamine- and Ammonia-dependent NAD Synthetases CARBON-NITROGEN HYDROLASE DOMAIN CONFERS GLUTAMINE DEPENDENCY

NAD synthetase catalyzes the final step in the biosynthesis of NAD. In the present study, we obtained cDNAs for two types of human NAD synthetase (referred as NADsyn1 and NADsyn2). Structural analysis revealed in both NADsyn1 and NADsyn2 a domain required for NAD synthesis from ammonia and in only NADsyn1 an additional carbon-nitrogen hydrolase domain shared with enzymes of the nitrilase family that cleave nitriles as well as amides to produce the corresponding acids and ammonia. Consistent with the domain structures, biochemical assays indicated (i) that both NADsyn1 and NADsyn2 have NAD synthetase activity, (ii) that NADsyn1 uses glutamine as well as ammonia as an amide donor, whereas NADsyn2 catalyzes only ammonia-dependent NAD synthesis, and (iii) that mutant NADsyn1 in which Cys-175 corresponding to the catalytic cysteine residue in nitrilases was replaced with Ser does not use glutamine. Kinetic studies suggested that glutamine and ammonia serve as physiological amide donors for NADsyn1 and NADsyn2, respectively. Both synthetases exerted catalytic activity in a multimeric form. In the mouse, NADsyn1 was seen to be abundantly expressed in the small intestine, liver, kidney, and testis but very weakly in the skeletal muscle and heart. In contrast, expression of NADsyn2 was observed in all tissues tested. Therefore, we conclude that humans have two types of NAD synthetase exhibiting different amide donor specificity and tissue distributions. The ammonia-dependent synthetase has not been found in eucaryotes until this study. Our results also indicate that the carbon-nitrogen hydrolase domain is the functional domain of NAD synthetase to make use of glutamine as an amide donor in NAD synthesis. Thus, glutamine-dependent NAD synthetase may be classified as a possible glutamine amidase in the nitrilase family. Our molecular identification of NAD synthetases may prove useful to learn more of mechanisms regulating cellular NAD metabolism.

Synergistic effect of ubiquitin on lipopolysaccharide-induced TNF-α production in murine macrophage cell line RAW 264.7 cells

Ubiquitin synergistically augmented the production of tumor necrosis factor alpha (TNF-alpha) in the presence of lipopolysaccharide (LPS) in murine macrophage cell line RAW 264.7. To investigate the mechanism of this augmentation, we analyzed the effect of ubiquitin during TNF-alpha mRNA synthesis and degradation, and TNF-alpha degradation on RAW 264.7 cells stimulated by LPS. It is found that ubiquitin augmented TNF-alpha mRNA synthesis. Ubiquitin did not affect the degradation of TNF-alpha mRNA and TNF-alpha. In the presence of LPS, extracellular accumulation of TNF-alpha by ubiquitin was twice than those by LPS, but intracellular accumulation of TNF-alpha produced by ubiquitin with LPS or by LPS had no difference. These data indicate that ubiquitin might induce TNF-alpha accumulation mainly by up-regulation of the TNF-alpha gene transcription. Although extracellular functions of ubiquitin remain largely unknown, we postulate that ubiquitin might be involved in the modulatory mechanisms of immune response.