Structure analysis and antiviral activity of CW-33 analogues against Japanese encephalitis virus.

Japanese encephalitis virus (JEV) is a member of neurotropic flaviviruses transmitted by mosquito bites, causing severe central nervous system disorders. Current JEV genotype III vaccines have a low protection against genotype I isolates in the risk zone. The lead compound CW-33, ethyl 2-(3',5'-dimethylanilino)-4-oxo-4,5-dihydrofuran-3-carboxylate, demonstrates the antiviral activity against JEV with an IC50 values of 38.5 µM for virus yield reduction (Int J Mol Sci 2016,17: E1386). This study synthesized fourteen CW-33 analogues containing a fluoro atom or one methoxy group at the C-2, C-3, or C-4 of anilino ring, and then evaluated for their antiviral activity and mechanism. Among 6 amalogues, CW-33A (ethyl 2-(2-fluoroanilino)-4-oxo- 4,5-dihydrofuran-3-carboxylate), and CW-33D (ethyl 2-(3-methoxyanilino)-4-oxo- 4,5-dihydrofuran-3-carboxylate exhibited antiviral potentials in viral cytopathic effect (CPE) inhibition. CW-33A significantly suppressed the viral protein expression, genome synthesis and intracellular JEV particle production, showing a higher inhibitory effect on JEV yield than CW-33 and CW-33D. The study demonstrated that a mono-fluoro substitution on at the C-2 anilino ring of CW-33 improved the antiviral activity JEV, revealing the structure-activity relationship for developing novel agents against JEV infection.

An unusual presentation of cerebellar lymphoma.

We report a case of a 40 year old female who presented with a three month history of headaches with a background of Myasthenia Gravis (MG), treated with azathioprine. MRI brain demonstrated a rim-enhancing lesion in the left posterior fossa. CT scan of the chest abdomen and pelvis revealed no other lesion. The patient was taken for resection of the lesion via left retrosigmoid approach. Histologically the neoplasm was a large B-cell lymphoma, Epstein-Barr virus (EBV) positive, with clonality confirmed by IGH gene rearrangement studies. To the best of our knowledge this is the first case of primary central nervous system (CNS) EBV-positive diffuse large B-cell lymphoma of the posterior fossa in a patient with MG treated with azathioprine.

An oncolytic measles virus-sensitive Group 3 medulloblastoma model in immune-competent mice.

Background: Oncolytic measles virus (MV) is effective in xenograft models of many tumor types in immune-compromised mice. However, no murine cell line exists that is tumorigenic, grows in immune-competent mice, and is killed by MV. The lack of such a model prevents an examination of the effect of the immune system on MV oncotherapy. Methods: Cerebellar stem cells from human CD46-transgenic immunocompetent mice were transduced to express Sendai virus C-protein, murine C-Myc, and Gfi1b proteins. The resultant cells were injected into the brain of NSG mice, and a cell line, called CSCG, was prepared from the resulting tumor. Results: CSCG cells are highly proliferative, and express stem cell markers. These cells are permissive for replication of MV and are killed by the virus in a dose- and time-dependent manner. CSCG cells form aggressive tumors that morphologically resemble medulloblastoma when injected into the brains of immune-competent mice. On the molecular level, CSCG tumors overexpress natriuretic peptide receptor 3 and gamma-aminobutyric acid type A receptor alpha 5, markers of Group 3 medulloblastoma. A single intratumoral injection of MV‒green fluorescent protein resulted in complete tumor regression and prolonged survival of animals compared with treatments with phosphate buffered saline (P = 0.0018) or heat-inactivated MV (P = 0.0027). Conclusions: This immune-competent model provides the first platform to test therapeutic regimens of oncolytic MV for Group 3 medulloblastoma in the presence of anti-measles immunity. The strategy presented here can be used to make MV-sensitive murine models of any human tumor for which the driving mutations are known.

Increased cytomegalovirus replication by 5-Azacytidine and viral-induced cytoplasmic expression of DNMT­1 in medulloblastoma and endothelial cells.

Among all brain tumors diagnosed in children, medulloblastomas (MBs) are associated with a poor prognosis. The etiology of MB is not fully understood, yet the impact of epigenetic alterations of oncogenes has previously been established. During the past decade, the human cytomegalovirus (HCMV) has been detected in several types of cancer, including MB. Since DNA methylation occurs in the cell nucleus and this is considered a host defence response, we studied the impact of HCMV infection on DNA methyltransferase (DNMT­1) in MB (D324) cells, human umbilical vein endothelial cells (HUVECs) as well as in MB tissue sections. We hypothesized that infection and DNMT­1 intracellular localization are linked. Uninfected and HCMV­infected D324 cells and HUVECs were analyzed for HCMV immediate early (HCMV­IE) protein, HCMV­glycoprotein B (HCMV­gB) and DNMT­1 using immunofluorescence staining and quantitative ELISA. DNMT­1 localized to the nucleus of uninfected and HCMV­IE- expressing D324 cells and HUVECs, but accumulated in the extra nuclear space in all HCMV­gB-positive cells. Inhibition of HCMV late protein expression by Cymevene® (ganciclovir) prevented the cytoplasmic localization of DNMT­1. Treatment of HCMV­ infected D324 cells and HUVECs with the methylation inhibitor 5-Azacytidine (5AZA), significantly increased HCMV­IE and HCMV­gB gene transcription and protein expression. Immunohistochemical staining of DNMT­1 and HCMV proteins in MB cancer tissue sections revealed both nuclear and cytoplasmic DNMT­1 localization. In conclusion, DNMT­1 resides in the cytoplasm of HCMV­gB-expressing HUVECs and D324 cells. Increased viral protein synthesis in 5AZA-treated cells suggests that HCMV replication may benefit from a DNA methyltransferase-free cellular environment. Our findings emphasize the importance of assessing potential viral activation in the treatment of MB patients with epigenetic drugs.

Oncogenic role of cytomegalovirus in medulloblastoma?

Medulloblastoma is the most common solid tumor among children. Current therapeutic strategies for this malignancy include surgical resection, radiation therapy and chemotherapy. However, these treatments are accompanied with serious side effects such as neurological complications and psychosocial problems, due to the severity of treatment on the developing nervous system. To solve this problem, novel therapeutic approaches are currently being investigated. One of them is targeting human cytomegalovirus in medulloblastoma cancer cells. However, this approach is still under debate, since the presence of cytomegalovirus in medulloblastomas remains controversial. In this review, we discuss the current controversies on the role of cytomegalovirus in medulloblastoma oncogenesis and the potential of cytomegalovirus as a novel (immuno)therapeutic target.

Primary central nervous system extranodal NK/T cell lymphoma, nasal type, with antecedent hemophagocytic syndrome in a child.

Primary central nervous system (CNS) extranodal natural killer (NK)/T-cell lymphoma, nasal type (NKTCL), is an exceedingly uncommon entity. Here, we present a case of CNS NKTCL that manifested initially as hemophagocytic syndrome 4 months earlier in a 13-year-old girl. Histological examination revealed the cerebellum mass was composed of large-sized and atypical tumor cells, with an angiocentric and angiodestructive growth pattern and prominent necrosis. The tumor cells exhibited marked pleomorphism with conspicuous nucleoli and prominent mitotic activity. Immunohistochemical staining showed the tumor cells were positive for CD45, CD2, CD3ε, CD30, CD43, CD56, and granzyme B. Epstein-Barr virus--encoded ribonucleic acid was expressed in almost all of the nuclei of the lymphoma cells. The T-cell receptor γ chain gene rearrangement study showed no evidence of a clonal rearrangement. The patient was treated with etoposide and dexamethasone and died a few days after the operation. As far as we know, this case is the 1st pediatric and female patient of primary CNS NKTCL with antecedent hemophagocytic syndrome, which highlights the clinical data and is helpful for the diagnosis of this tumor.

Treatment of medulloblastoma with a modified measles virus.

Although treatment of medulloblastoma has improved, at least 30% of patients with this tumor die of progressive disease. Unfortunately, many of the children who survive suffer long-term treatment-related morbidity. Previous studies have demonstrated the efficacy of using oncolytic viruses to eradicate brain tumors. The objective of this study was to test the efficacy of measles virus in treating medulloblastoma. To determine whether medulloblastoma cells are susceptible, 5 different human medulloblastoma cell lines were analyzed for the expression of the measles virus receptor CD46. Fluorescence-activated cell-sorting analysis confirmed expression of CD46 on all cell lines tested, with UW288-1 having the most prominent expression and D283med displaying the lowest expression. CD46 expression was also demonstrated, using immunohistochemistry, in 13 of 13 medulloblastoma tissue specimens. All 5 medulloblastoma cell lines were examined for their susceptibility to measles virus killing in vitro. A measles virus containing the green fluorescent protein (GFP) gene as a marker for infection (MV-GFP) was used. All cell lines exhibited significant killing when infected with MV-GFP, all formed syncytia with infection, all showed fluorescence, and all allowed viral replicaton after infection. In an intracerebral murine xenograft model, a statistically significant increase in survival was seen in animals treated with the active measles virus compared with those treated with inactivated virus. These data demonstrate that medulloblastoma is susceptible to measles virus killing and that the virus may have a role in treating this tumor in the clinical setting.

Detection and quantitative analysis of human herpesvirus in pilocytic astrocytoma.

We investigated the hypothetical role of human herpesviruses (HHVs) in tumour formation of the cerebellum. Thirty-five samples of pilocytic astrocytoma and 10 control samples of cerebellum from patients who died of unrelated diseases were examined. Presence of the 8 known HHVs was first studied using specific real-time quantitative Polymerase Chain Reaction (qPCR) targeting viral DNA polymerase. HHV's DNA polymerase was found present in 20 samples (7 controls, 13 astrocytomas) and was absent in 25 samples (3 controls, 22 astrocytomas). DNA polymerase of Epstein-Barr Virus (EBV) was present in 16 samples, 7/10 controls (70%) and 9/35 astrocytomas (26%). HHV-1 and Varicella-Zoster virus were detected only twice and HHV-2, Cytomegalovirus, HHV-7 and HHV-8, only once. HHV-6 was not detected. In all cases, the gene copy numbers of DNA polymerase were low (<100/100 ng DNA). A second approach was to search for novel HHVs, using consensus-degenerated hybrid oligonucleotide primers (CODEHOP) PCR: no sequence indicative of a new HHV was detected. In summary, EBV was the most frequent HHV detected in pilocytic astrocytoma, but at very low levels. According to the actually accepted threshold the results suggest that EBV cannot be considered responsible for tumorigenesis of pilocytic astrocytoma.

Epstein-Barr virus involvement is a predictive factor for the resistance to chemoradiotherapy of gastric diffuse large B-cell lymphoma.

Primary gastric diffuse large B-cell lymphomas are generally well controlled by non-surgical treatment with combination chemotherapy followed by radiotherapy. We have previously reported that over 90% of patients achieved complete response (CR) with this therapeutic strategy: three cycles of cyclophosphamide, adriamycin, vincristine and prednisone followed by radiotherapy (40.5 Gy). Although the CR rate was very high, some patients still showed resistance to this combination therapy. In order to clarify the factors related to therapy resistance, we examined the relationship between Epstein-Barr virus (EBV), which was examined using an in situ hybridization technique, and the patients' clinical courses. Out of the 50 patients, four were EBV positive; over half of lymphoma cells were positive for EBV by in situ hybridization. Of the three EBV-positive patients, two showed progressive disease and one achieved partial response (PR). Two of the patients died of disease progression. The other patient achieved CR, but the lymphoma recurred with distant metastasis in the cerebellum 3 months after remission. In the present study, eight patients did not achieve CR or they relapsed, four patients showed progressive disease, one patient achieved PR, and three patients achieved CR with recurrence. Therefore, half of these unfavorable patients were EBV positive. This finding strongly indicated that EBV-associated gastric diffuse large B-cell lymphomas frequently show resistance to standard chemoradiotherapy, although some other adverse factors remain unclear.

Detection of JC virus DNA sequences in brain tumors in pediatric patients.

OBJECT: The JC virus is a human neurotropic polyomavirus that causes progressive multifocal leukoencephalopathy and is closely related to simian virus 40. Several recent reports have indicated a possible association between the JC virus and the development of various human brain tumors. The authors examined the presence of JC virus DNA sequences in primary brain tumors in pediatric patients to evaluate the hypothesis that particular brain tumors can arise in the pediatric population as a consequence of infection with the JC virus. METHODS: Genomic DNA sequences were isolated from 62 brain tumors (32 medulloblastomas, 18 ependymomas, five choroid plexus papillomas, and seven pilocytic astrocytomas) and analyzed for the presence of JC virus DNA by Southern blot hybridization and direct sequencing. The JC virus DNA sequence was detected in five ependymomas and one choroid plexus papilloma. Immunohistochemical studies revealed nuclear expression of the large T-antigen in a choroid plexus papilloma. None of the medulloblastomas or pilocytic astrocytomas contained JC virus DNA. CONCLUSIONS: The results of this study provide molecular evidence of the association between JC virus and the development of certain ependymomas and choroid plexus papillomas.

Increased p53 immunopositivity in anaplastic medulloblastoma and supratentorial PNET is not caused by JC virus.

BACKGROUND: p53 mutations are relatively uncommon in medulloblastoma, but abnormalities in this cell cycle pathway have been associated with anaplasia and worse clinical outcomes. We correlated p53 protein expression with pathological subtype and clinical outcome in 75 embryonal brain tumors. The presence of JC virus, which results in p53 protein accumulation, was also examined. METHODS: p53 protein levels were evaluated semi-quantitatively in 64 medulloblastomas, 3 atypical teratoid rhabdoid tumors (ATRT), and 8 supratentorial primitive neuroectodermal tumors (sPNET) using immunohistochemistry. JC viral sequences were analyzed in DNA extracted from 33 frozen medulloblastoma and PNET samples using quantitative polymerase chain reaction. RESULTS: p53 expression was detected in 18% of non-anaplastic medulloblastomas, 45% of anaplastic medulloblastomas, 67% of ATRT, and 88% of sPNET. The increased p53 immunoreactivity in anaplastic medulloblastoma, ATRT, and sPNET was statistically significant. Log rank analysis of clinical outcome revealed significantly shorter survival in patients with p53 immunopositive embryonal tumors. No JC virus was identified in the embryonal brain tumor samples, while an endogenous human retrovirus (ERV-3) was readily detected. CONCLUSION: Immunoreactivity for p53 protein is more common in anaplastic medulloblastomas, ATRT and sPNET than in non-anaplastic tumors, and is associated with worse clinical outcomes. However, JC virus infection is not responsible for increased levels of p53 protein.

T-antigen of human polyomavirus JC cooperates withIGF-IR signaling system in cerebellar tumors of the childhood-medulloblastomas.

Polyomaviruses are implicated in a number of cancers, and the transforming activity of their early protein, large T-antigen, has been documented in a variety of cell types and in experimental animals (1). Although the pathways by which T-antigen induces uncontrolled cell growth are not fully defined, T-antigen mediated inactivation of tumor suppressors, p53 and pRB, is well-documented in some malignancies (2). Here we postulate that functional interaction between the insulin-like growth factor (IGF-IR) and the T-antigen of human polyomavirus JC (JCV T-antigen) may contribute to the process of malignant transformation in medulloblastomas: (i) the IGF-IR signaling system is strongly activated in medulloblastoma cell lines and medulloblastoma biopsies; (ii) the cytoplasmic protein, insulin receptor substrate 1 (IRS-1), is translocated to the nucleus in the presence of JCV T-antigen; (iii) molecular characterization of the interaction between IRS-1 and JCV T-antigen indicates that the binding involves the N-terminal portion of IRS-1 (PH/PTB domain) and the C-terminal region of JCV T-antigen (aa 411-628); and finally (iv) competition for the IRS-1-JCV T-antigen binding attenuates anchorage-independent growth of T-antigen positive medulloblastoma cells in culture. Based on these findings, we propose a novel role for IRS-1 in JCV T-antigen-mediated deregulation of cellular equilibrium, which may involve uncoupling of IRS-1 from the surface receptor and translocation of its function to the nuclear compartment of the cell.

Insulin-like growth factor I receptor signaling system in JC virus T antigen-induced primitive neuroectodermal tumors--medulloblastomas.

Medulloblastomas represent about 25% of all pediatric intracranial neoplasms. These highly malignant tumors arise from the cerebellum, affecting mainly children between ages 5 and 15. Although the etiology of medulloblastomas has not yet been elucidated, several reports suggest that both the cellular protein insulin-like growth factor I (IGF-I) and the early protein of the human polyomavirus JC (JCV T antigen) may contribute to the development of these tumors. The results of this study show a potential functional cooperation between these two proteins in the process of malignant transformation. Both medulloblastoma cell lines and medulloblastoma biopsies are characterized by the abundant presence of the IGF-I receptor (IGF-IR) and its major signaling molecule, insulin receptor substrate 1 (IRS-1). Importantly, IRS-1 is translocated to the nucleus in the presence of the JCV T antigen. Nuclear IRS-1 was detected in T antigen-positive cell lines and in T antigen-positive biopsies from patients diagnosed with medulloblastoma. The IRS-1 domain responsible for a direct JCV T antigen binding was localized within the N-terminal portion of IRS-1 molecule and the competition for IRS-1 T antigen binding by a dominant-negative mutant of IRS-1 inhibited growth and survival of JCV T antigen-transformed cells in anchorage-independent culture condition.

Insulin-like growth factor-I receptor - a potential therapeutic target in medulloblastomas.

Medulloblastomas represent nearly 25% of all paediatric intracranial neoplasms. These highly malignant tumours arise from the cerebellum and affect predominantly children between the ages of 5 and 15. Although the aetiology of medulloblastomas has not been elucidated, several reports show that the insulin-like growth factor-I (IGF-I) signalling system is highly activated in medulloblastoma cell lines, medulloblastoma animal models and medulloblastoma biopsies, suggesting its contribution to the development and/or progression of these tumours. In addition, reports from multiple laboratories confirm a critical role for the IGF-I receptor (IGF-IR) in the process of cellular transformation. Taken together, these observations prompt the investigation of different strategies to impair the function of IGF-IR as a potential therapeutic tool, which by compromising growth and survival of medulloblastoma cells could supplement conventional therapeutic regiments against these malignant neoplasms of childhood.

Expression of a human polyomavirus oncoprotein and tumour suppressor proteins in medulloblastomas.

AIMS: Although the aetiology of medulloblastoma remains elusive, several lines of evidence suggest an association with the human neurotropic polyomavirus JC and its oncoprotein T antigen. The tumour forming properties of JC virus T antigen are the result, at least in part, of its ability to bind and inactivate tumour suppressor/cell cycle regulatory proteins, such as p53 and the retinoblastoma family of proteins. METHODS: To examine potential relations between these factors, immunohistochemistry was used to determine associations between the T antigen and the expression of p53 and the retinoblastoma proteins pRb, p107, and Rb2/p130 in eight medulloblastomas. RESULTS: Only the three medulloblastomas with T antigen expression also showed nuclear positivity with antibodies to p53. Although immunohistochemistry detected nuclear labelling for pRb in five of the cases, the three that were positive for T antigen showed the highest pRb labelling. The retinoblastoma related proteins p107 and Rb2/p130 were also immunopositive in most T antigen positive medulloblastomas. Double label immunohistochemistry also demonstrated p53 and pRb positivity in the same cells that were T antigen positive. CONCLUSIONS: These correlations suggest that associations between T antigen and p53 and/or T antigen and pRb occur in some of these tumours. These data provide indirect evidence that JC virus, acting through T antigen, might be involved in the formation and progression of medulloblastoma.

Detection of simian virus 40 DNA sequence in human primary glioblastomas multiforme.

OBJECT: Deoxyribonucleic acid oncoviruses can induce neoplastic transformation of cells because their viral proteins interfere with antiproliferative cellular proteins. Simian virus 40 (SV40) is a DNA virus that induces the emergence of ependymomas, choroid plexus tumors, mesotheliomas, osteosarcomas, sarcomas, and various tumors when injected into newborn hamsters. Recently, approximately 60% of human ependymomas, choroid plexus tumors, and mesotheliomas were reported to contain and express SV40 DNA sequences. In this study the presence of SV40 DNA sequences was investigated in human brain tumors. METHODS: Three of 32 glioblastomas mutiforme (GBMs), but none of two ependymomas and five medulloblastomas, were found to possess SV40 DNA sequences when examined using polymerase chain reaction (PCR). The DNA sequence analysis of PCR-amplified fragments disclosed that the samples were identical to the regulatory region of SV40. All three GBMs, which arose in elderly patients with wild-type p53, were considered to be primary (de novo) tumors. Although each of the three tumors was immunohistochemically negative for SV40 T antigen, in situ hybridization successfully demonstrated the messenger RNA for SV40 T antigen. CONCLUSIONS: The results of this study indicate that latent infection of SV40 in elderly people may be implicated in the tumorigenesis of certain primary GBMs.

Identification of a novel p53 mutation in JCV-induced mouse medulloblastoma.

Medulloblastoma, a malignant invasive tumor of the cerebellum, is one of the most common neoplasms of the nervous system in children. Utilization of the human neurotropic virus JC virus (JCV) early gene T-antigen allowed the development of a transgenic animal that models human medulloblastoma. Here we describe the characterization of two distinct populations of cells derived from the JCV-induced mouse medulloblastoma. Results from immunohistochemical and biochemical studies revealed the expression of T-antigen in some but not all tumor cells. In T-antigen-producing cells, T-antigen was found in association with wild-type p53 and pRb, two tumor suppressors that control cell growth and differentiation. In cells that lack expression of T-antigen, a novel mutant p53 with a deletion between residues 35 and 123 was detected. Morphological differences were observed between the two populations of cells, though there was no significant difference in their growth rates. However, subcutaneous transplantation of the T-antigen-positive, but not T-antigen-negative, cells resulted in the development of massive tumors in experimental animals. In light of earlier reports on the association of JCV with human medulloblastoma, the mouse cell lines described in this study may provide a valuable tool for deciphering the pathways involved in the formation and progression of medulloblastoma.