Comprehensive analysis of protein-expression changes specific to immunoglobulin G4-related disease.

BACKGROUND AND AIMS: Immunoglobulin 4 (IgG4)-related disease (IgG4-RD) is a lymphoproliferative disorder characterized by elevated serum IgG4 levels and tissue infiltration of IgG4-positive plasma cells. We analyzed the serum proteins, whose levels varied based on the disease state and treatment. MATERIALS AND METHODS: Serum proteins from patients with IgG4-related disease and healthy subjects were resolved using two-dimensional electrophoresis, silver-stained, and scanned. Alternatively, the proteins were labeled with Cy2, Cy3, and Cy5 before electrophoresis. The proteins, whose expression differed significantly between patients and healthy individuals, and between before and after steroid treatment, were identified and validated using enzyme-linked immunosorbent assays. RESULTS: Pre-treatment sera from patients with IgG4-related disease was characterized by increased levels of immunoglobulins such as IgG1, IgG4; inflammatory factors such as α-1 antitrypsin (A1AT); and proteins associated with immune system regulation such as clusterin and leucine-rich α-2-glycoprotein (LRG-1). The serum levels of A1AT, LRG-1 and clusterin, during treatment with prednisolone for up to 12 months revealed that LRG-1 levels were halved after 1 month of treatment, comparable to those in healthy subjects; LRG-1 levels remained normal until the end of treatment. CONCLUSION: LRG-1 could serve as a novel biomarker of IgG4-related diseases.

Characterization of JC Polyomavirus Derived from COS-IMRb Cells.

JC polyomavirus (JCPyV) causes progressive multifocal leukoencephalopathy (PML), a demyelinating disease of the central nervous system affecting immunocompromised patients. The study of PML-type JCPyV in vitro has been limited owing to the inefficient propagation of the virus in cultured cells. In this study, we carried out long-term culture of COS-7 cells (designated as COS-IMRb cells) transfected with PML-type M1-IMRb, an adapted viral DNA with a rearranged non-coding control region (NCCR). The JCPyV derived from COS-IMRb cells were characterized by analyzing the viral replication, amount of virus by hemagglutination (HA), production of viral protein 1 (VP1), and structure of the NCCR. HA assays indicated the presence of high amounts of PML-type JCPyV in COS-IMRb cells. Immunostaining showed only a small population of JCPyV carrying COS-IMRb cells to be VP1-positive. Sequencing analysis of the NCCR of JCPyV after long-term culture revealed that the NCCR of M1-IMRb was conserved in COS-IMRb cells without any point mutation. The JCPyV genomic DNA derived from a clone of COS-IMRb-3 cells was detected, via Southern blotting, as a single band of approximately 5.1 kbp without deletion. These findings suggest the potential of using COS-IMRb-3 cells as a useful tool for screening anti-JCPyV drugs.

Epidemiological study on Japanese encephalitis virus distribution in Ishikawa prefecture, Japan, by serological investigation using wild boar sera.

Thirty-seven specimens of wild boar sera were collected from August 2016 to March 2018 in Ishikawa prefecture, Japan. Thirty-two specimens (86.5%) were positive for neutralizing antibodies against Japanese encephalitis virus (JEV). Eight specimens (21.6%) were positive for IgM antibodies against JEV. One sample was obtained from a wild boar captured in February during the winter season. Four other serum specimens obtained during the winter season were positive using a JEV gene-specific PCR assay. Based on IgM and PCR assays, wild boars were infected with JEV during the winter season, suggesting that the prevalence of JEV antibodies in wild boars in Ishikawa is high and JEV activity is possible during winter in this region. In addition, wild boars may play an important role in the infection cycle of JEV.

Establishment of COS-JC cells persistently producing archetype JC polyomavirus.

JC polyomavirus (JCPyV) is the causative agent of progressive multifocal leukoencephalopathy (PML), a demyelinating disease of the central nervous system in immunocompromised patients. Archetype JCPyV circulates in the human population. There have been several reports of archetype JCPyV replication in cultured cells, in which propagation was not enough to produce high titers of archetype JCPyV. In this study, we carried out cultivation of the transfected cells with archetype JCPyV DNA MY for more than 2 months to establish COS-7 cells (designated COS-JC cells) persistently producing archetype JCPyV. Moreover, JCPyV derived from COS-JC cells was characterized by analyzing the viral propagation, size of the viral genome, amount of viral DNA, production of viral protein, and structure of the non-coding control region (NCCR). Southern blotting using a digoxigenin-labeled JCPyV probe showed two different sizes of the JCPyV genome in COS-JC cells. For molecular cloning, four of five clones showed a decrease in the size of complete JCPyV genome. Especially, clone No. 10 was generated the large deletion within the Large T antigen. On the other hand, the archetype structure of the NCCR was maintained in COS-JC cells, although a few point mutations occurred. Quantitative PCR analysis of viral DNA in COS-JC cells indicated that a high copy number of archetype JCPyV DNA was replicated in COS-JC cells. These findings suggest that COS-JC cells could efficiently propagate archetype JCPyV MY and offer a useful tool to study persistent infection of archetype JCPyV in a kidney-derived system.

High FDG uptake on PET is associated with negative cell-to-cell adhesion molecule E-cadherin expression in lung adenocarcinoma.

OBJECTIVES: E-cadherin is a main cell-to-cell adhesion molecule. A negative expression of E-cadherin correlates with distant metastasis in lung cancer. Recently, it was reported that there is an association between FDG uptake on PET and epithelial-mesenchymal transition (EMT) in non-small cell lung cancer. Downregulation of E-cadherin is one of the best markers of EMT. The purpose of this study was to compare E-cadherin expression with FDG uptake on PET, cell differentiation, aggressiveness and post-operative recurrence in patients with lung adenocarcinoma, and to investigate whether FDG uptake on PET is associated with E-cadherin expression. METHODS: We retrospectively reviewed 40 lung adenocarcinoma patients who underwent thoracotomy and FDG PET before thoracotomy. These patients were evaluated FDG PET metrics such as standardized uptake value (SUV), the immunohistochemical expression of E-cadherin in surgical specimens, clinicopathological features, including tumor size, pathologic stage, cell differentiation, aggressiveness and post-operative recurrence. RESULTS: High FDG uptake correlated with negative E-cadherin expression (P = 0.043). SUVmax was higher in a negative E-cadherin expression lung adenocarcinoma than in a positive E-cadherin expression lung adenocarcinoma (P = 0.033). Patients with moderately poorly differentiated adenocarcinoma had frequent negative E-cadherin expression or high FDG uptake (P = 0.004, P = 0.0001, respectively). Patients with aggressive adenocarcinoma had frequent negative E-cadherin expression or high FDG uptake (P = 0.004, P = 0.001, respectively). Kaplan-Meier analysis revealed that negative E-cadherin expression or high FDG uptake were strongly correlated with shortened disease-free survival (P = 0.0153, P = 0.0001, respectively). CONCLUSION: High FDG uptake on PET was associated with negative E-cadherin expression in patients with lung adenocarcinoma. Both high FDG uptake and negative E-cadherin expression were strongly correlated with poor differentiation, aggressiveness, and post-operative recurrence. These findings may cause the association between high FDG uptake and negative E-cadherin expression.

CPT11 prevents virus replication in JCI cells persistently infected with JC polyomavirus.

JC polyomavirus (JCPyV) is the causative agent of the demyelinating disease of the central nervous system known as progressive multifocal leukoencephalopathy (PML), which occurs in immunocompromised patients. Moreover, patients treated with natalizumab for multiple sclerosis or Crohn disease can develop PML, which is then termed natalizumab-related PML. Because few drugs are currently available for treating PML, many antiviral agents are being investigated. It has been demonstrated that the topoisomerase I inhibitors topotecan and ß-lapachone have inhibitory effects on JCPyV replication in IMR-32 cells. However, both of these drugs have marginal inhibitory effects on virus propagation in JC1 cells according to RT-PCR analysis. In the present study, the inhibitory effect of another topoisomerase I inhibitor, 7-ethy-10-[4-(1-piperidino)-1-piperidino] carbonyloxy camptothecin (CPT11), was assessed by investigating viral replication, propagation, and viral protein 1 (VP1) production in cultured cells. JCPyV replication was assayed using real-time PCR combined with Dpn I treatment in IMR-32 cells transfected with JCPyV DNA. It was found that JCPyV replicates less in IMR-32 cells treated with CPT11 than in untreated cells. Moreover, CPT11 treatment of JCI cells persistently infected with JCPyV led to a dose-dependent reduction in JCPyV DNA and VP1 production. Additionally, the inhibitory effect of CPT11 was found to be stronger than those of topotecan and ß-lapachone. These findings suggest that CPT11 may be a potential anti-JCPyV agent that could be used to treat PML.

An Ecological Survey of Mosquitoes and the Distribution of Japanese Encephalitis Virus in Ishikawa Prefecture, Japan, between 2010 and 2014.

Japanese encephalitis virus (JEV) is a flavivirus, responsible for over 30,000 annual cases of encephalitis worldwide, with a mortality rate of approximately 30%. Therefore, it is important to examine the distribution of mosquitos carrying JEV in the fields, even though recently, the number of Japanese encephalitis cases has been approximately 5 per year in Japan. We report the seasonal dynamics of mosquitoes between 2010 and 2014 in Ishikawa Prefecture, Japan. We collected 39,308 female adult mosquitoes, 98.2% of which were classified as Culex tritaeniorhynchus Giles. We identified JEV genomic RNA belonging to genotype 1 from the homogenate of Cx. tritaeniorhynchus, collected during our study using reverse transcription-PCR and nucleotide sequencing techniques. Our results indicate that mosquito vectors for JEV are distributed not only in areas in Ishikawa, but also throughout Japan, and the results suggest that we must be careful regarding JEV outbreaks in Japan in the future.

Sphingomyelin generated by sphingomyelin synthase 1 is involved in attachment and infection with Japanese encephalitis virus.

Japanese encephalitis virus (JEV) is a mosquito-borne RNA virus which infects target cells via the envelope protein JEV-E. However, its cellular targets are largely unknown. To investigate the role of sphingomyelin (SM) in JEV infection, we utilized SM-deficient immortalized mouse embryonic fibroblasts (tMEF) established from SM synthase 1 (SMS1)/SMS2 double knockout mice. SMS deficiency significantly reduced both intracellular and extracellular JEV levels at 48 h after infection. Furthermore, after 15 min treatment with JEV, the early steps of JEV infection such as attachment and cell entry were also diminished in SMS-deficient tMEFs. The inhibition of JEV attachment and infection were recovered by overexpression of SMS1 but not SMS2, suggesting SMS1 contributes to SM production for JEV attachment and infection. Finally, intraperitoneal injection of JEV into SMS1-deficient mice showed an obvious decrease of JEV infection and its associated pathologies, such as meningitis, lymphocyte infiltration, and elevation of interleukin 6, compared with wild type mice. These results suggest that SMS1-generated SM on the plasma membrane is related in JEV attachment and subsequent infection, and may be a target for inhibition of JEV infection.

Correlation of HIF-1α/HIF-2α expression with FDG uptake in lung adenocarcinoma.

OBJECTIVES: Hypoxia is a key element involved in the development and progression of tumors. HIF-1α may transiently induce and mediate the response to acute and severe hypoxia, while HIF-2α may induce a longer response and may control the response to chronic and moderate hypoxia. Hypoxia increases the cellular uptake of FDG. Therefore, HIF may play an important role in the process of the cellular uptake of FDG. The aim of this study was to compare HIF-1α/HIF-2α expression with FDG uptake, Glut-1 expression, and prognosis in the patients with lung adenocarcinoma and to investigate the role of HIF-1α/HIF-2α in the uptake of FDG in lung adenocarcinoma. METHODS: In the current work, we compared the immunohistochemical expression of HIF-1α and HIF-2α in surgical specimens of 44 patients with lung adenocarcinoma. The relationships between HIF-α expression and Glut-1 expression, FDG uptake, and clinicopathological factors, including prognosis, were analyzed. RESULTS: There was a marginal association between HIF-1α and HIF-2α expressions (P = 0.076). We found a significant correlation between HIF-2α expression and FDG uptake (P = 0.0001). HIF-1α expression showed a marginal association with FDG uptake (P = 0.066). FDG uptake correlated more significantly with HIF-2α expression than with HIF-1α expression. A significant correlation was noticed between Glut-1 expression and both HIF-1α and HIF-2α expressions (P = 0.005 and P = 0.003, respectively). Univariate analysis of disease-free survival demonstrated that FDG uptake and HIF-2α expression, but not HIF-1α expression, were related to recurrence (P < 0.0001). CONCLUSION: FDG uptake correlated more significantly with HIF-2α expression than with HIF-1α expression, and both FDG uptake and HIF-2α expression, but not HIF-1α expression was correlated with post-operative recurrence in the patients with lung adenocarcinoma. These results suggest that both FDG uptake and HIF-2α expression may represent a more aggressive phenotype and that HIF-2α may play a more important role than HIF-1α in the uptake of FDG in lung adenocarcinoma.

Impaired Japanese encephalitis virus replication in p62/SQSTM1 deficient mouse embryonic fibroblasts.

The role of the autophagy adaptor protein p62/SQSTM1 in Japanese encephalitis virus (JEV) replication in mouse embryonic fibroblasts (MEFs) was investigated. Amounts of JEV RNA and E protein were significantly smaller in p62-deficient cells than wild-type cells at 24 hr post-infection (p.i.). JEV RNA quantitation and viral plaque assays showed significant reductions in viral titers in p62-deficient cell culture fluid. Our results indicate that JEV replication is impaired in p62-deficient MEFs, suggesting that p62 positively regulates JEV replication in host cells.

Suppressive effect of topoisomerase inhibitors on JC polyomavirus propagation in human neuroblastoma cells.

JC polyomavirus (JCPyV) causes progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease of the central nervous system, in immunocompromised patients. Because no drugs have been approved for treating PML, many antiviral agents are currently being investigated for this purpose. The inhibitory effects of the topoisomerase I inhibitors topotecan and ß-lapachone were assessed by investigating viral replication, propagation and viral protein 1 (VP1) production in cultured cells. JCPyV replication was assayed using the human neuroblastoma cell line IMR-32 transfected with the JCPyV plasmid and RT- PCR combined with Dpn I treatment. Dpn I digests the input plasmid DNA containing methylated adenosine, but not newly replicated JCPyV DNA, in IMR-32 cells. It was found that JCPyV replicates less in IMR-32 cells treated with topotecan or ß-lapachone than in untreated cells. Moreover, drug treatment of JCI cells, which are IMR-32 cells persistently infected with JCPyV, led to a reduction in the amount of JCPyV DNA and population of VP1-positive cells. These results demonstrate that topotecan and ß-lapachone affects JCPyV propagation in human neuroblastoma cell lines, suggesting that topotecan and ß-lapachone could potentially be used to treat PML.

Hepatitis C virus NS3 protein modulates the biological behaviors of malignant hepatocytes by altering the expression of host cell microRNA.

Chronic hepatitis C virus (HCV) infection is a major cause of hepatocellular carcinoma (HCC) worldwide. The HCV non­structural protein 3 (NS3) protein is considered to affect normal cellular functions and to be involved in HCV carcinogenesis. The expression of microRNA (miRNA) is altered in human HCC, thus implicating its role in hepatocarcinogenesis. To investigate the mechanisms by which the HCV NS3 protein affects the expression of miRNA in malignant hepatocytes, if any, the present study constructed expression vectors encoding the HCV NS3 and NS3/4A proteins, which were stably transfected into HepG2 cells. The biological behaviors of the HepG2 transfectants and their differential expression levels of miRNA expression were investigated. Compared with the HepG2­vector cells, the HepG2­NS3 cells grew at a slower rate, were arrested in the G0/G1 cell cycle phase, formed more colonies and developed larger tumors at a faster rate. Co­expression of HCV NS4A resulted in the inhibition of HCV NS3­stimulated tumorigenicity. A total of 35 miRNAs were dysregulated, 26 of which were downregulated and nine of which were upregulated, in the HepG2­NS3 cells, and 75 miRNAs were altered in HepG2­NS3/4A cells, of which 20 were downregulated and 55 were upregulated). In addition, significant decreases in the mRNA levels of p53 and p21 were observed, which confirmed differential expression of miRNA. These results suggested that differential miRNA profiling in malignant hepatocytes may account for the variable pathophysiological manifestations associated with the HCV NS3 protein. These differentially expressed miRNAs may offer potential as candidates for the development of miRNA­based therapeutics.

Decreased Expression of Innate Immunity-Related Genes in Peripheral Blood Mononuclear Cells from Patients with IgG4-Related Disease.

BACKGROUND: IgG4-related disease (IgG4-RD) is a new clinical entity of unknown etiology characterized by elevated serum IgG4 and tissue infiltration by IgG4-positive plasma cells. Although aberrancies in acquired immune system functions, including increases in Th2 and Treg cytokines observed in patients with IgG4-RD, its true etiology remains unclear. To investigate the pathogenesis of IgG4-RD, this study compared the expression of genes related to innate immunity in patients with IgG4-RD and healthy controls. MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMCs) were obtained from patients with IgG4-RD before and after steroid therapy and from healthy controls. Total RNA was extracted and DNA microarray analysis was performed in two IgG4-RD patients to screen for genes showing changes in expression. Candidate genes were validated by real-time RT-PCR in 27 patients with IgG4-RD and 13 healthy controls. RESULTS: DNA microarray analysis identified 21 genes that showed a greater than 3-fold difference in expression between IgG4-RD patients and healthy controls and 30 genes that showed a greater than 3-fold change in IgG4-RD patients following steroid therapy. Candidate genes related to innate immunity, including those encoding Charcot-Leyden crystal protein (CLC), membrane-spanning 4-domain subfamily A member 3 (MS4A3), defensin alpha (DEFA) 3 and 4, and interleukin-8 receptors (IL8R), were validated by real-time RT-PCR. Expression of all genes was significantly lower in IgG4-RD patients than in healthy controls. Steroid therapy significantly increased the expression of DEFA3, DEFA4 and MS4A3, but had no effect on the expression of CLC, IL8RA and IL8RB. CONCLUSIONS: The expression of genes related to allergy or innate immunity, including CLC, MS4A3, DEFA3, DEFA4, IL8RA and IL8RB, was lower in PBMCs from patients with IgG4-RD than from healthy controls. Although there is the limitation in the number of patients applied in DNA microarray, impaired expression of genes related to innate immunity may be involved in the pathogenesis of IgG4-RD as well as in abnormalities of acquired immunity.

NAB2-STAT6 fusion gene analysis in two cases of meningeal solitary fibrous tumor/hemangiopericytoma with late distant metastases.

We present two cases of meningeal solitary fibrous tumor (SFT)/hemangiopericytoma (HPC) with immunohistochemistry of STAT6 and analysis of NAB2-STAT6 fusion genes. Case 1 was a 37-year-old male with a left middle fossa tumor; case 2 was a 68-year-old female with a cerebellar tumor. They showed late metastasis to the lung or bone 8 or 13 years, respectively, after the first surgery. Histology of both primary and metastatic tumors showed a cellular hemangiopericytomatous pattern with nuclear atypia. The primary tumors showed nuclear staining of STAT6, but both metastatic tumors showed nuclear and cytoplasmic STAT6. DNA sequencing revealed two kinds of NAB2-STAT6 fusion genes. One consisted of exon 6 of NAB2, intron 6 of NAB2, and the middle of exon 17 of STAT6 (observed in the primary and metastatic tumors of case 1); the other consisted of exon 6 of NAB2 and the beginning of exon 17 of STAT6 (observed in the metastatic tumor of case 2). The primary tumor of case 2 had both fusion genes. To the best of our knowledge, we are the first to report NAB2-STAT6 fusion gene analysis in primary and metastatic meningeal SFT/HPCs and a case showed different fusion gene status in the metastatic tumor.

Replication of IMR-32-adapted JC virus clones in human embryonic kidney cells.

It has been difficult to study JCV replication because of its restricted host range. In this study, JCV replication was examined using different clones in 293 cells. RT-PCR assay revealed that large T antigen expression in cells transfected with IMR-32-adapted JCVs was significantly greater than in those transfected with Mad-1 or CY. DNA replication assay and viral load verified that the IMR-32-adapted JCVs were replication-competent in 293 cells, but not Mad-1 or CY JCVs. These results suggest that a 293 culture system with IMR-32-adapted JCVs may be a useful tool for assessing replication of JCV in vitro.

TNF-α stimulates efficient JC virus replication in neuroblastoma cells.

JC polyomavirus (JCV) causes progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease of the central nervous system (CNS) in immunocompromised patients, and particularly in the severe immunosuppression associated with acquired immunodeficiency syndrome (AIDS). HIV-1 can lead to the production of tumor necrosis factor-alpha (TNF-α) in the CNS. Our aim was to examine the effects of TNF-α on JCV gene expression and replication using a human neuroblastoma cell line, IMR-32, transfected with JCV DNA, M1-IMRb. Quantitative RT-PCR analysis of JCV large T antigen and VP1 mRNA, the viral DNA replication assay, and the DNase protection assay were carried out. TNF-α treatment of IMR-32 cells transfected with JCV DNA induced large T antigen mRNA and JCV DNA replication, while other effects on VP1 mRNA expression and virus production were marginal. In addition, ELISA analysis of the nuclear p65 subunit of nuclear factor κB (NF-κB), which is a hallmark of NF-κB pathway activation, of IMR-32 cells upon TNF-α treatment showed that TNF-α treatment activated the NF-κB pathway in IMR-32 cells. Taken together, our results suggest that TNF-α stimulation could induce JCV replication associated with the induction of JCV large T antigen mRNA through the NF-κB pathway in IMR-32 cells transfected with JCV DNA. Our findings may contribute to further understanding of the pathogenesis of AIDS-related PML.

Proteasome inhibitors and knockdown of SMG1 cause accumulation of Upf1 and Upf2 in human cells.

The ubiquitin-proteasome system (UPS) is one of the most promising anticancer drug targets of the century. However, the involved molecular mechanisms are still unclear. The nonsense-mediated mRNA decay (NMD) pathway is a highly conserved pathway which degrades nonsense mutation­containing mRNA selectively and efficiently. In this pathway, the SMG1-Upf1-eRF (SURF) complex binds to Upf2 on the exon junction complex and finally causes degradation of nonsense-containing mRNA. To reveal the relationship between the UPS and NMD pathways, we analyzed the effects of proteasome inhibitors on Upf1 and Upf2. The data showed that treatment with proteasome inhibitors caused the accumulation of the Upf1 and Upf2 proteins in A549 cells. In addition, we found that knockdown of SMG1 also caused the upregulation of Upf1 and Upf2 proteins, which was confirmed by different target sequences of siRNA. SMG1 and UPS appear to participate in different pathways of the degradation of Upf1 and Upf2, since simultaneous treatment with both of them caused additive effects. This study demonstrated the quantitative regulation of Upf1 and Upf2 proteins by UPS and SMG1.

Chronic ingestion of ethanol induces hepatocellular carcinoma in mice without additional hepatic insult.

BACKGROUND: Chronic intake of alcohol increases the risk of gastrointestinal and hepatic carcinogenesis. The present study was focused to investigate the incidence and mechanism of pathogenesis of hepatocellular carcinoma (HCC) during chronic ingestion of alcohol without any additional hepatic injury. METHODS: Ethanol was administered to Institute for Cancer Research male mice through drinking water for 70 weeks at concentrations of 5 % (first week), 10 % (next 8 weeks), and 15 % thereafter. The animals were killed at 60 and 70 weeks, the livers were examined for hepatic tumors, and evaluated for foci of cellular alteration (FCA). Immunohistochemical staining was performed in the liver sections for cytochrome P4502E1 (CYP2E1), 4-hydroxy-nonenal (4-HNE), and proto-oncogene, c-Myc. RESULTS: At the 60th week, 40 % of the mice in the ethanol group had visible white nodules (5-10 mm) in the liver, but not in the control mice. At the 70th week, several larger nodules (5-22 mm) were present in the livers of 50 % mice in the ethanol group. In the control group, one mouse developed a single nodule. All nodules were histologically trabecular HCC composed of eosinophilic and vacuolated cells. In the livers of both control and ethanol group, several foci with cellular alteration were present, which were significantly higher in ethanol group. Staining for CYP2E1, 4-HNE and c-Myc depicted marked upregulation of all these molecules in the FCA. CONCLUSIONS: Our data demonstrated that upregulation of CYP2E1 and subsequent production of reactive oxygen species along with the persistent expression of c-Myc play a significant role in the pathogenesis of HCC during chronic ingestion of ethanol.

Suppressive effect of PARP-1 inhibitor on JC virus replication in vitro.

The incidence of progressive multifocal leukoencephalopathy (PML) has increased due to the AIDS pandemic, hematological malignancies, and immunosuppressive therapies. Recently, the number of cases of monoclonal antibody-associated PML has increased in patients treated with immunomodulatory drugs such as natalizumab. However, no common consensus regarding PML therapy has been reached in clinical studies. In order to examine the suppression of JC virus (JCV) replication by 3-aminobenzamide (3-AB), a representative PARP-1 inhibitor, a DNA replication assay was carried out using the neuroblastoma cell line IMR-32 and IMR-adapted JCV. The suppression of JCV propagation by 3-AB was also examined using JCI cells, which are a carrier culture producing continuously high JCV titers. The results indicated that PARP-1 inhibitors, such as 3-aminobenzamide (3-AB), suppress JCV replication and propagation significantly in vitro, as judged by DNA replication assay, hemagglutination, and real-time PCR analysis. It has been also shown that 3-AB reduced PARP-1 activity in IMR-32 cells. According to the results of the MTT assay, the enzyme activity of 3-AB-treated cells was slightly lower than that of DMSO-treated cells. However, the significant suppression of JCV propagation is not related to the slight decrease in cell growth. To our knowledge, this is the first report that PARP-1 inhibitor suppresses the replication of JCV significantly in neuroblastoma cell lines via the reduction of PARP-1 activity. Thus, PARP-1 inhibitors also may be a novel therapeutic drug for PML.

AT motif binding factor 1 (ATBF1) is highly phosphorylated in embryonic brain and protected from cleavage by calpain-1.

ATBF1 is a transcription factor that regulates genes responsible for repairing tissues and the protection of cells from oxidative stress. Therefore reduction of ATBF1 promotes susceptibility to varieties of human diseases including neurodegenerative diseases and malignant tumors. The instability of the protein was found to be an important background of diseases. Because ATBF1 is composed of a large 404-kDa protein, it can be easily targeted by proteinases. The protein instability should be a serious problem for the function in the cells and practically for our biochemical study of ATBF1. We have found that calpain-1 is a protease responsible for the degeneration of ATBF1. We observed distinct difference between embryo and adult brain derived ATBF1 regarding the sensitivity to calpain-1. The comparative study showed that eight phosphorylated serine residues (Ser1600, Ser2634, Ser2795, Ser2804, Ser2900, Ser3431, Ser3613, Ser3697) in embryonic brain, but only one site (Ser2634) in adult brain. As long as these amino acids were phosphorylated, ATBF1 derived from embryonic mouse brain showed resistance to cleavage; however, treatment with calf intestine alkaline phosphatase sensitized ATBF1 to be digested by calpain-1. An inhibitor (FK506) against calcineurin, which is a serine/threonine specific phosphatase enhanced the resistance of ATBF1 against the digestion by calpain-1. Taken together, these results demonstrate that these phosphorylation sites on ATBF1 function as a defensive shield to calpain-1.