1. To identify key differentiation genes and characterize their molecular mechanisms of action A. Identification, Cloning and Characterization of CASZ1, the human homolog of the drosophila neural fate determination gene castor (dCas). We have identified, cloned and characterized CASZ1, the human homolog of the drosophila neural fate determination gene, dCas. We determined it mapped to chromosome 1p36.22, an allele was lost in both N-myc (NMA) amplified and non-NMA neuroblastoma cell lines and CASZ1 expression increased during retinoid differentiation. In drosophila, dCas is expressed exclusively at a late stage of neuroblast development that precedes the cessation of proliferation and the beginning of neuronal differentiation. The stage at which castor is expressed during drosophila development is analogous to later stages of human embryonic neural organogenesis, which implies that the human homologue- may have the same role as dCas in controlling cell fates within neuroblast cell lineages. The fact that this gene is homologous to a drosophila neural fate determination gene, is regulated during retinoid induced differentiation of NB cells and localizes to a chromosomal region of interest in NB (1p36) provided the rational to clone and characterize HCASZ1. Using in silico and standard cloning techniques, we identified two isoforms. Hcasz5 encodes a protein with 5 zinc fingers and belongs to the TFIIIA family of transcription proteins. There is a highly conserved para-ZnF sequence located upstream of each classic zinc finger, a feature that is identical to the zinc fingers in dCas. Hcasz11 is identical to hcasz5 for the first 1166 AA but contains an additional 6 TFIIIA class C2H2 zinc finger motifs that also contain the highly conserved para-ZnF sequence. As anticipated, both isoforms localize to the nucleus consistent with their putative function as transcriptional regulators. Both isoforms are highly expressed in human heart, skeletal muscle and fetal but not adult brain. Cell lines from NB tumors and rhabdomyosarcomas, a tumor of muscle origin, express to varying levels of the CASZ1 gene. CASZ1 gene expression was significantly increased after induction of differentiation in neuroblasts and C2C12 myoblasts, indicating that CASZ1 may play a role in controlling cell fates not only within neuroblast cell lineages but in myoblast cell lineages as well. B. Immunohistochemical analysis of DPYSL3/hUlip during human neural development and in neural tumors In a screen to isolate neural differentiation genes (from a retinoid induced NB cDNA library) we had identified an anonymous gene that we later cloned and characterized as hUlip (TUC4/CRMP4/DRP-3/DPYSL3), a human homolog of unc-33, a neurite regulatory gene in C. elegans. In this follow-up study, we detail an immunohistochemical analysis of hUlip during neural development and in nervous system tumors. This is the first report of a member of this gene family in humans. Most regions of the brain in the embryo express ULIP4/CRMP3 during development, whereas its expression in adulthood remains restricted to the dentate gyrus, the cerebellar granular layer and the inferior olive. This is consistent with our findings in our Ulip KO, in which there is an increased number of neurons in the adult dentate gyrus and abberrant neuritogenesis. The persistent expression of the ULIP4/CRMP3 gene in these selected areas of the adult nervous system suggests that the ULIP signaling pathway is implicated in some specific aspects of neuronal plasticity and disorders. hUlip immunoreactivity was also found in various brain tumors showing neuronal differentiation: central neurocytomas (6/6), medulloblastomas (5/11), atypical teratoid rhabdoid tumor (1/1) and gangliogliomas (4/7). Some astrocytic tumors also showed weak positivity; astrocytomas (1/5), anaplastic astrocytomas (2/5), glioblastomas (3/11). Subependymal giant cell astrocytomas and subependymomas, which are of controversial histogenetic origin, showed strong hUlip immunoreactivity. Finally hUlip protein was detected in NB tumors undergoing differentiation such as ganglioneuroma and gandlioneuroblastomas but not in neuroblastomas. C. MYCN regulation of cell cycle gene expression during differentiation of neuroblastoma cells The MYCN gene is amplified and over-expressed in the tumors of neuroblastoma patients that have the worst prognosis. Our previous studies have indicated that down-regulation of this gene is required prior to arrest of cell cycle progression and induction of differentiation by the biologic response modifiers retinoic acid or NGF. To delineate which cell cycle progression genes were regulated by expression of the MYCN gene, we used siRNAs targeted to MYCN to both target MYCN expression and assess which genes were specifically regulated by MYCN. We identified several siRNAs that could decrease both MYCN mRNA and protein expression. We identified that cdk6 levels were selectively targeted when MYCN gene expression decreased. Associated with the decrease in MYCN and cdk6 epxression was a decrease in cells in the S phase of the cell cycle. These data point to cdk6 as a key cyclin dependent cell cycle regulator in NB cells
| Woo, Chan-Wook; Tan, Fei; Cassano, Hope et al. (2008) Use of RNA interference to elucidate the effect of MYCN on cell cycle in neuroblastoma. Pediatr Blood Cancer 50:208-12 |
| Wei, J S; Song, Y K; Durinck, S et al. (2008) The MYCN oncogene is a direct target of miR-34a. Oncogene 27:5204-13 |
