Myoblast-derived neuronal cells form glutamatergic neurons in the mouse cerebellum.

Production of neurons from non-neural cells has far-reaching clinical significance. We previously found that myoblasts can be converted to a physiologically active neuronal phenotype by transferring a single recombinant transcription factor, REST-VP16, which directly activates target genes of the transcriptional repressor, REST. However, the neuronal subtype of M-RV cells and whether they can establish synaptic communication in the brain have remained unknown. M-RV cells engineered to express green fluorescent protein (M-RV-GFP) had functional ion channels but did not establish synaptic communication in vitro. However, when transplanted into newborn mice cerebella, a site of extensive postnatal neurogenesis, these cells expressed endogenous cerebellar granule precursors and neuron proteins, such as transient axonal glycoprotein-1, neurofilament, type-III ß-tubulin, superior cervical ganglia-clone 10, glutamate receptor-2, and glutamate decarboxylase. Importantly, they exhibited action potentials and were capable of receiving glutamatergic synaptic input, similar to the native cerebellar granule neurons. These results suggest that M-RV-GFP cells differentiate into glutamatergic neurons, an important neuronal subtype, in the postnatal cerebellar milieu. Our findings suggest that although activation of REST-target genes can reprogram myoblasts to assume a general neuronal phenotype, the subtype specificity may then be directed by the brain microenvironment.

Reactivation of death receptor 4 (DR4) expression sensitizes medulloblastoma cell lines to TRAIL.

OBJECT: Apoptosis, a key cellular response to therapeutic agents is often inactivated in tumor cells. In this study, we evaluated the expression of the tumor necrosis family of death receptors, DR4 and DR5, in medulloblastoma tumor samples and cell lines to determine if epigenetic modulation of gene expression could sensitize tumor cell lines to TRAIL-mediated apoptosis. METHODS: Human medulloblastoma samples and cell lines were analyzed for DR4 and DR5 expression by quantitative PCR and immunofluorescence assays. Cell lines with downregulated expression of one or both genes were treated with the histone deacetylase inhibitor, MS-275, and the expression of DR4 and DR5 measured by quantitative PCR, Western blotting, flow cytometry and chromatin immunoprecipitation assays. Induction of apoptosis in the presence of MS-275 was evaluated by TUNEL assay and its ability to augment TRAIL-mediated cytotoxicity was determined by MTT assays, Western blotting and flow cytometry. RESULTS: Compared to normal cerebellum, DR4, but not DR5 expression was consistently downregulated in medulloblastoma tumor samples and in Daoy and D283 cell lines. Interestingly, MS-275 decreased cell growth and induced apoptosis in Daoy and D283 cells. In Daoy cells, this coincided with increased histone H3 and H4 acetylation at the DR4 promoter and enhanced DR4 gene and protein expression as well as elevated Caspase-8 activity. The involvement of DR4 in the cellular response to MS-275 was further confirmed by the observation that knockdown of DR4 and FADD abrogated apoptosis. Further, addition of TRAIL to MS-275 treated cells resulted in an enhancement of apoptosis, suggesting that the upregulated death receptors were functional. CONCLUSION: Our study provides an understanding of the role of DR4 in apoptosis of medulloblastoma cell lines and suggests a potential contribution of aberrant histone deacetylation to the resistance of medulloblastoma cells to therapeutic death.

Heparanase expression and TrkC/p75NTR ratios in human medulloblastoma.

Medulloblastoma (MB), the most devastating and common brain tumor in children, is highly invasive and extremely difficult to treat. Identifying the properties of MB tumors that cause them to invade and metastasize is therefore imperative for the development of novel treatments. We performed investigations to elucidate prognostic implications of heparanase (HPSE-1) and TrkC/p75(NTR) expression in MB using formalin-fixed, paraffin-embedded (FFPE) MB clinical specimens from children aged 1-19 years. Expressions of p75(NTR) and HPSE-1 correlated with each other (Pearson's correlation R = 0.899; P < 0.0001; R (2) = 81%; n = 14). In addition, TrkC:p75(NTR) ratios correlated with MB meningeal spread (R = 0.608; P = 0.0212; R (2) = 37%; n = 14). Secondly, using antibodies specific to TrkC and HPSE-1, we carried out immunohistochemistry (IHC) on 22 human MB tissue samples. IHC reaction scores revealed a significant expression of HPSE-1 in 76% of MB tissues from children aged 3 years and older (P = 0.0490; n = 17) while TrkC immunoreactivity was detected in 71% of these patient samples. Of note, TrkC was significantly present in 100% of MB female patients (P = 0.0313; n = 6). These studies support the role of p75(NTR) and HPSE-1 as two novel molecular determinants involved in the biology and clinical progression of MB.

Heparanase, TrkC and p75NTR: their functional involvement in human medulloblastoma cell invasion.

Medulloblastoma is the most common malignant childhood brain tumor. Although some previous reports have shown up to a 70% 5-year survival for some of these patients, it is at the cost of significant long-term treatment-related morbidity. The cellular mechanisms leading to metastatic disease in medulloblastoma are mainly unknown. For the first time, we demonstrate the differential expression of heparanase in medulloblastomas and how these differences at the mRNA and protein levels affect the activity and invasive properties of three newly developed cell lines. Furthermore, heparanase expression was confirmed in 7 (88%) of 8 medulloblastoma clinical samples by immunohistochemical staining. Heparanase was found to be localized in the cytoplasm and nucleus. Quantitative polymerase chain reaction revealed a negative correlation between heparanase and TrkC (which is associated with a favorable clinical outcome). The activation of TrkC or TrkC/p75NTR by NT-3 affected heparanase activity and cell-invasive properties of medulloblastoma cells in vitro. Taken together, our data extend the body of evidence that invasion and expression/functionality of heparanase, in a context linked to TrkC and p75NTR, may play critical roles in the disease progression of medulloblastoma.

Brain metastases in melanoma: roles of neurotrophins.

Brain metastasis, which occurs in 20% to 40% of all cancer patients, is an important cause of neoplastic morbidity and mortality. Successful invasion into the brain by tumor cells must include attachment to microvessel endothelial cells, penetration through the blood-brain barrier, and, of relevance, a response to brain survival and growth factors. Neurotrophins (NTs) are important in brain-invasive steps. Human melanoma cell lines express low-affinity NT receptor p75NTR in relation to their brain-metastatic propensity with their invasive properties being regulated by NGF, or nerve growth factor, the prototypic NT. They also express functional TrkC, the putative receptor for the invasion-promoting NT-3. In brain-metastatic melanoma cells, NTs promote invasion by enhancing the production of extracellular matrix (ECM)-degradative enzymes such as heparanase, an enzyme capable of locally destroying both ECM and the basement membrane of the blood-brain barrier. Heparanase is an endo-beta-d-glucuronidase that cleaves heparan sulfate (HS) chains of ECM HS proteoglycans, and it is a unique metastatic determinant because it is the dominant mammalian HS degradative enzyme. Brain-metastatic melanoma cells also produce autocrine/paracrine factors that influence their growth, invasion, and survival in the brain. Synthesis of these factors may serve to regulate NT production by brain cells adjacent to the neoplastic invasion front, such as astrocytes. Increased NT levels have been observed in tumor-adjacent tissues at the invasion front of human brain melanoma. Additionally, astrocytes may contribute to the brain-metastatic specificity of melanoma cells by producing NT-regulated heparanase. Trophic, autocrine, and paracrine growth factors may therefore determine whether metastatic cells can successfully invade, colonize, and grow in the CNS.