5­Azacytidine treatment results in nuclear exclusion of DNA methyltransferase­1, as well as reduced proliferation and invasion in human cytomegalovirus­infected glioblastoma cells.

Glioblastoma (GBM) is the most aggressive form of brain tumor in adults, with a devastating outcome. Emerging evidence shows that human cytomegalovirus (HCMV) proteins and nucleic acids are present in GBM tissues. DNA methylation is important for the initiation and progression of cancer and is an established host response against invading nucleic acids. The expression and localization of DNA methyltransferase 1 (DNMT­1) was assessed, and the effects of DNA methylation inhibitor 5­azacytidine (5AZA) were analyzed in the context of the viral replication, proliferation and invasion capacities of HCMV­infected GBM U343MG cells. In addition, the expression of various HCMV proteins and DNMT­1 was examined in GBM tissue specimens obtained from five patients. DNMT­1 was localized in the nucleus of cells expressing HCMV­immediate early, whereas in cells expressing HCMV­glycoprotein gB (gB), extranuclear/cytoplasmic localization was observed. This was also observed in vitro in U343MG cells. In addition, DNMT­1 was localized to the extranuclear/cytoplasmic space of cells lining blood vessel walls within the GBM tumors. Treatment of infected U343MG cells with 5AZA did not affect viral replication, but reduced cell invasion and proliferation (P=0.05 and P<0.0001, respectively). However, 5AZA treatment of uninfected cells did not affect cell invasion (P=0.09), but proliferation was significantly reduced (P<0.0001). These findings may be of importance in further investigations aimed at using DNA methylation and viral inhibitors in GBM therapy.

Impact of Human Cytomegalovirus Infection and its Immune Response on Survival of Patients with Ovarian Cancer.

Human cytomegalovirus (HCMV) has been detected in various types of tumors. We studied the prevalence of HCMV in ovarian cancer and its relation to clinical outcome. Paraffin-embedded tissues obtained prospectively from 45 patients with ovarian cancer and 30 patients with benign ovarian cystadenoma were analyzed for expression of HCMV immediate-early protein (IE) and HCMV tegument protein (pp65) by immunohistochemistry. Plasma was analyzed for HCMV serology. HCMV-IgG levels were higher in patients with ovarian cancer or benign cystadenoma than in age-matched controls (P = .002, P < .0001, respectively). HCMV IgM was detected in 12% of ovarian cancer patients and 3% of patients with benign tumors but was absent in controls. In patients with ovarian cancer, higher IgG levels were associated with better outcomes (P = .04). Extensive HCMV-IE protein expression was detected in 75% of ovarian cancers and 26% of benign tumors; pp65 was detected in 67% of ovarian cancers and 14% of benign tumors. A higher grade of HCMV infection was associated with higher stage of disease. Extensive HCMV-pp65 expression was associated with shorter median overall survival than focal expression (39 versus 42.5 months, P = .03). At study closure, 58% of ovarian cancer patients with focal pp65 expression were alive versus 27% of patients with extensive pp65 expression (P = .03). Thus, HCMV proteins are detected at different levels in ovarian tumors and benign cystadenomas. Ovarian cancer patients with focal HCMV-pp65 expression in their tumors and high IgG levels against HCMV lived longer, highlighting a need for in-depth studies of the oncomodulatory role of HCMV in ovarian cancer.

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.

Establishment and characterization of an orthotopic patient-derived Group 3 medulloblastoma model for preclinical drug evaluation.

Medulloblastomas comprise a heterogeneous group of tumours and can be subdivided into four molecular subgroups (WNT, SHH, Group 3 and Group 4) with distinct prognosis, biological behaviour and implications for targeted therapies. Few experimental models exist of the aggressive and poorly characterized Group 3 tumours. In order to establish a reproducible transplantable Group 3 medulloblastoma model for preclinical therapeutic studies, we acquired a patient-derived tumour sphere culture and inoculated low-passage spheres into the cerebellums of NOD-scid mice. Mice developed symptoms of brain tumours with a latency of 17-18 weeks. Neurosphere cultures were re-established and serially transplanted for 3 generations, with a negative correlation between tumour latency and numbers of injected cells. Xenografts replicated the phenotype of the primary tumour, including high degree of clustering in DNA methylation analysis, high proliferation, expression of tumour markers, MYC amplification and elevated MYC expression, and sensitivity to the MYC inhibitor JQ1. Xenografts maintained maintained expression of tumour-derived VEGFA and stromal-derived COX-2. VEGFA, COX-2 and c-Myc are highly expressed in Group 3 compared to other medulloblastoma subgroups, suggesting that these molecules are relevant therapeutic targets in Group 3 medulloblastoma.

Human Cytomegalovirus Up-Regulates Endothelin Receptor Type B: Implication for Vasculopathies?

Background. Both endothelin receptor type B ([ETBR], a G protein-coupled receptor that mediates the vascular effects of the potent vasoconstrictor endothelin-1) and human cytomegalovirus ([HCMV], a ubiquitous herpesvirus) have been implicated in the pathogenesis of cardiovascular disease (CVD). The effects of HCMV infection on ETBR expression are unknown. We hypothesized that HCMV may contribute to the pathogenesis of CVD via ETBR modulation. Methods. Human CMV effects on ETBR were studied in vitro in endothelial cells (ECs) and smooth muscle cells (SMCs) and ex vivo in human carotid plaque tissue specimens. Expression of ETBR and viral immediate-early were quantified using quantitative polymerase chain reaction. Functional consequences after ETBR blockade in ECs were examined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide proliferation, wound healing, tube formation, and flow adhesion assays. Results. Human CMV is capable of upregulating both ETBR mRNA and protein expression in ECs and SMCs. The ETBR was also abundantly expressed in ECs, foam cells, and SMCs, and, more importantly, in HCMV-positive cells in human carotid plaques. Endothelin receptor type B blockade led to decreased proliferation and reduced tumor necrosis factor α-mediated leukocyte recruitment in both uninfected and HCMV-infected ECs. Direct HCMV infection was antimigratory and antiangiogenic in ECs. Conclusions. Human CMV may contribute to CVD via ETBR induction.