[Show abstract][Hide abstract] ABSTRACT: For a progenitor cell to become a neuron, three activities must occur: neuronal differentiation program must be activated, elements repressing neuronal differentiation must be deactivated and competing differentiation programs must be silenced. It is known that NeuroD2 and related bHLH transcription factors induce neuronal differentiation, REST represses neuronal differentiation, and Zfhx1a prevents myogenic gene expression. We demonstrate that NeuroD2 suppresses REST during differentiation in culture. In the hippocampus of NeuroD2 knockout mice, higher level of REST is detected. Functional significance of NeuroD2-REST interplay is uncovered by showing that forced expression of REST interferes with neuronal differentiation in culture. NeuroD2 inhibits REST indirectly by involving the inhibitor of myogenic genes, Zfhx1a, which binds response elements in REST 5'-UTR. Our study supports a model wherein NeuroD2 induces transcription of neuronal genes and Zfhx1a, which in turn de-represses neuronal differentiation by down-regulating REST, and suppresses competing myogenic fate.
[Show abstract][Hide abstract] ABSTRACT: Loss-of-function studies have revealed the role of many basic helix-loop-helix (bHLH) transcription factors at specific points during development; however, the role of E proteins in the development of the nervous system has not been experimentally addressed. E proteins have been speculated to interact selectively with class II bHLH factors to form different neurogenic complexes. In this study, using coimmunoprecipitation in a culture model of neurogenesis (P19 cells), we show that E proteins E12, HEB, and E2-2 interact with neuroD2. Using electrophoretic mobility shift assay and P19 cell culture, we show that these heterodimers bind a neuroD2 preferred E box and induce neurogenesis equally well. We examine the mRNA levels of the three E proteins at 10 time points during brain development and show that E protein gene expression is regulated such that at certain times during development selective interaction between neuroD2 and a single E protein (HEB) is a possibility. This led us to study the brains of HEB and E2A knockout mice, which manifest no gross neuroanatomical, cellular, or behavioral deficits. These findings, together with homology in the primary peptide sequence of E proteins, suggest functional compensation among E proteins during development of the nervous system.
Journal of Neuroscience Research 05/2008; 86(7):1472-81. DOI:10.1002/jnr.21615 · 2.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Toward the goal of developing an optical imaging contrast agent that will enable surgeons to intraoperatively distinguish cancer foci from adjacent normal tissue, we developed a chlorotoxin:Cy5.5 (CTX:Cy5.5) bioconjugate that emits near-IR fluorescent signal. The probe delineates malignant glioma, medulloblastoma, prostate cancer, intestinal cancer, and sarcoma from adjacent non-neoplastic tissue in mouse models. Metastatic cancer foci as small as a few hundred cells were detected in lymph channels. Specific binding to cancer cells is facilitated by matrix metalloproteinase-2 (MMP-2) as evidenced by reduction of CTX:Cy5.5 binding in vitro and in vivo by a pharmacologic blocker of MMP-2 and induction of CTX:Cy5.5 binding in MCF-7 cells following transfection with a plasmid encoding MMP-2. Mouse studies revealed that CTX:Cy5.5 has favorable biodistribution and toxicity profiles. These studies show that CTX:Cy5.5 has the potential to fundamentally improve intraoperative detection and resection of malignancies.
Cancer Research 08/2007; 67(14):6882-8. DOI:10.1158/0008-5472.CAN-06-3948 · 9.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Suberoylanilide hydroxamic acid (SAHA) has been studied in adult solid and hematologic malignancies. However, little information has been reported on the effects of SAHA on central nervous system (CNS) tumors including medulloblastoma, the most common malignant brain tumor in children. We investigated SAHA in preclinical medulloblastoma models to determine its anti-cancer efficacy as well as its ability to affect intracranial lesions when administered systemically.
Tissue culture studies were performed treating primary human fibroblasts, established medulloblastoma cell lines, and primary human medulloblastoma tumors with SAHA. At 10 microM concentration, SAHA had little effect on normal fibroblasts but caused >90% apoptosis in cultured medulloblastoma cells. Primary medulloblastomas from patients were sensitive to SAHA compared to vehicle alone in ex vivo studies. In athymic mice with medulloblastoma xenograft tumors, oral SAHA resulted in apoptosis of tumor tissue and significantly slowed tumor growth. In the ND2:Smo transgenic mouse medulloblastoma model, SAHA treatment caused significant apoptosis in these cerebellar tumors.
SAHA effectively induces cell death in established medulloblastoma cell lines, human patient primary tumor cultures, medulloblastoma xenografts and intracranial spontaneous medulloblastomas. Fibroblasts in culture and mice treated with SAHA did not reveal prohibitive toxicity profiles. These findings support the advancement of SAHA to pediatric clinical trials.
Journal of Neuro-Oncology 10/2006; 79(3):259-70. DOI:10.1007/s11060-006-9142-0 · 3.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The basic helix-loop-helix (bHLH) transcription factor, neuroD2, induces neuronal differentiation and promotes neuronal survival. Reduced levels of neuroD2 were previously shown to cause motor deficits, ataxia, and seizure propensity. Because neuroD2 levels may be critical for brain function, we studied the regulation of neuroD2 gene in cell culture and transgenic mouse models. In transgenic mice, a 10-kb fragment of the neuroD2 promoter fully recapitulated the endogenous neuroD2 staining pattern. A 1-kb fragment of the neuroD2 promoter drove reporter gene expression in most, but not all neuroD2-positive neuronal populations. Mutation of two critical E-boxes, E4 and E5 (E4 and E5 situated 149 and 305 bp upstream of the transcriptional start site) eliminated gene expression. NeuroD2 expression was diminished in mice lacking neurogenin1 demonstrating that neurogenin1 regulates neuroD2 during murine brain development. These studies demonstrate that neuroD2 expression is highly dependent on bHLH-responsive E-boxes in the proximal promoter region, that additional distal regulatory elements are important for neuroD2 expression in a subset of cortical neurons, and that neurogenin1 regulates neuroD2 expression during mouse brain development.