技术资料

We describe a novel form of tumor cell plasticity characterized by reversible adaptive plasticity in murine and human neuroblastoma. Two cellular phenotypes were defined by their ability to exhibit adhered,anchorage dependent (AD) or sphere forming,anchorage independent (AI) growth. The tumor cells could transition back and forth between the two phenotypes and the transition was dependent on the culture conditions. Both cell phenotypes exhibited stem-like features such as expression of nestin,self-renewal capacity,and mesenchymal differentiation potential. The AI tumorspheres were found to be more resistant to chemotherapy and proliferated slower in vitro compared to the AD cells. Identification of specific molecular markers like MAP2,β-catenin,and PDGFRβ enabled us to characterize and observe both phenotypes in established mouse tumors. Irrespective of the phenotype originally implanted in mice,tumors grown in vivo show phenotypic heterogeneity in molecular marker signatures and are indistinguishable in growth or histologic appearance. Similar molecular marker heterogeneity was demonstrated in primary human tumor specimens. Chemotherapy or growth factor receptor inhibition slowed tumor growth in mice and promoted initial loss of AD or AI heterogeneity,respectively. Simultaneous targeting of both phenotypes led to further tumor growth delay with emergence of new unique phenotypes. Our results demonstrate that neuroblastoma cells are plastic,dynamic,and may optimize their ability to survive by changing their phenotype. Phenotypic switching appears to be an adaptive mechanism to unfavorable selection pressure and could explain the phenotypic and functional heterogeneity of neuroblastoma. View Publication

Neuron-specific in vitro screening strategies have the potential to accelerate the evaluation of chemicals for neurotoxicity. We examined neurite outgrowth as a measure of neuronal response with a commercially available rat cortex progenitor cell model,where cells were exposed to a chemical during a period of cell differentiation. In control cultures,the fraction of beta-III-tubulin positive neurons and their neurite length increased significantly with time,indicating differentiation of the progenitor cells. Expression of glial fibrillary acidic protein,an astrocyte marker,also increased significantly with time. By seeding progenitor cells at varying densities,we demonstrated that neurite length was influenced by cell-cell spacing. After ten days,cultures seeded at densities of 1000 cells/mm(2) or lower had significantly shorter neurites than cultures seeded at densities of 1250 cells/mm(2) or higher. Progenitor cells were exposed to lithium,a neuroactive chemical with diverse modes of action. Cultures exposed to 30 mmol/L or 10 mmol/L lithium chloride (LiCl) had significantly lower metabolic activity than control cultures,as reported by adenosine triphosphate content,and no neurons were observed after ten days of exposure. Cultures exposed to 3 mmol/L,1 mmol/L,or 0.3 mmol/L LiCl,which encompass lithium's therapeutic range,had metabolic activity similar to control cultures. These cultures exhibited concentration-dependent decreases in neurite outgrowth after ten days of LiCl exposure. Neurite outgrowth results were relatively robust,regardless of the evaluation methodology. This work demonstrates that measurement of neurite outgrowth in differentiating progenitor cell cultures can be a sensitive endpoint for neuronal response under non-cytotoxic exposure conditions. View Publication

The WNT pathway plays multiple roles in neural development and is crucial for establishment of the embryonic cerebellum. In addition,WNT pathway mutations are associated with medulloblastoma,the most common malignant brain tumor in children. However,the cell types within the cerebellum that are responsive to WNT signaling remain unknown. Here we investigate the effects of canonical WNT signaling on two important classes of progenitors in the developing cerebellum: multipotent neural stem cells (NSCs) and granule neuron precursors (GNPs). We show that WNT pathway activation in vitro promotes proliferation of NSCs but not GNPs. Moreover,mice that express activated β-catenin in the cerebellar ventricular zone exhibit increased proliferation of NSCs in that region,whereas expression of the same protein in GNPs impairs proliferation. Although β-catenin-expressing NSCs proliferate they do not undergo prolonged expansion or neoplastic growth; rather,WNT signaling markedly interferes with their capacity for self-renewal and differentiation. At a molecular level,mutant NSCs exhibit increased expression of c-Myc,which might account for their transient proliferation,but also express high levels of bone morphogenetic proteins and the cyclin-dependent kinase inhibitor p21,which might contribute to their altered self-renewal and differentiation. These studies suggest that the WNT pathway is a potent regulator of cerebellar stem cell growth and differentiation. View Publication

Appropriate number of neurons and glial cells is generated from neural stem cells (NSCs) by the regulation of cell cycle exit and subsequent differentiation. Although the regulatory mechanism remains obscure,Id (inhibitor of differentiation) proteins are known to contribute critically to NSC proliferation by controlling cell cycle. Here,we report that a transcriptional factor,RP58,negatively regulates all four Id genes (Id1-Id4) in developing cerebral cortex. Consistently,Rp58 knockout (KO) mice demonstrated enhanced astrogenesis accompanied with an excess of NSCs. These phenotypes were mimicked by the overexpression of all Id genes in wild-type cortical progenitors. Furthermore,Rp58 KO phenotypes were rescued by the knockdown of all Id genes in mutant cortical progenitors but not by the knockdown of each single Id gene. Finally,we determined p57 as an effector gene of RP58-Id-mediated cell fate control. These findings establish RP58 as a novel key regulator that controls the self-renewal and differentiation of NSCs and restriction of astrogenesis by repressing all Id genes during corticogenesis. View Publication

BACKGROUND Medulloblastoma is a highly malignant pediatric brain tumor that requires surgery,whole brain and spine irradiation,and intense chemotherapy for treatment. A more sophisticated understanding of the pathophysiology of medulloblastoma is needed to successfully reduce the intensity of treatment and improve outcomes. Nuclear factor kappa-B (NFκB) is a signaling pathway that controls transcriptional activation of genes important for tight regulation of many cellular processes and is aberrantly expressed in many types of cancer. METHODS To test the importance of NFκB to medulloblastoma cell growth,the effects of multiple drugs that inhibit NFκB,pyrrolidine dithiocarbamate,diethyldithiocarbamate,sulfasalazine,curcumin and bortezomib,were studied in medulloblastoma cell lines compared to a malignant glioma cell line and normal neurons. Expression of endogenous NFκB was investigated in cultured cells,xenograft flank tumors,and primary human tumor samples. A dominant negative construct for the endogenous inhibitor of NFκB,IκB,was prepared from medulloblastoma cell lines and flank tumors were established to allow specific pathway inhibition. RESULTS We report high constitutive activity of the canonical NFκB pathway,as seen by Western analysis of the NFκB subunit p65,in medulloblastoma tumors compared to normal brain. The p65 subunit of NFκB is extremely highly expressed in xenograft tumors from human medulloblastoma cell lines; though,conversely,the same cells in culture have minimal expression without specific stimulation. We demonstrate that pharmacological inhibition of NFκB in cell lines halts proliferation and leads to apoptosis. We show by immunohistochemical stain that phosphorylated p65 is found in the majority of primary tumor cells examined. Finally,expression of a dominant negative form of the endogenous inhibitor of NFκB,dnIκB,resulted in poor xenograft tumor growth,with average tumor volumes 40% smaller than controls. CONCLUSIONS These data collectively demonstrate that NFκB signaling is important for medulloblastoma tumor growth,and that inhibition can reduce tumor size and viability in vivo. We discuss the implications of NFκB signaling on the approach to managing patients with medulloblastoma in order to improve clinical outcomes. View Publication

The aim of this investigation was to characterize the proliferative precursor cells in the adult mouse hippocampal region. Given that a very large number of new hippocampal cells are generated over the lifetime of an animal,it is predicted that a neural stem cell is ultimately responsible for maintaining this genesis. Although it is generally accepted that a proliferative precursor resides within the hippocampus,contradictory reports exist regarding the classification of this cell. Is it a true stem cell or a more limited progenitor? Using a strict functional definition of a neural stem cell and a number of in vitro assays,we report that the resident hippocampal precursor is a progenitor capable of proliferation and multipotential differentiation but is unable to self-renew and thus proliferate indefinitely. Furthermore,the mitogen FGF-2 stimulates proliferation of these cells to a greater extent than epidermal growth factor (EGF). In addition,we found that BDNF was essential for the production of neurons from the hippocampal progenitor cells,being required during proliferation to trigger neuronal fate. In contrast,a bona fide neural stem cell was identified in the lateral wall of the lateral ventricle surrounding the hippocampus. Interestingly,EGF proved to be the stronger mitogenic factor for this cell,which was clearly a different precursor from the resident hippocampal progenitor. These results suggest that the stem cell ultimately responsible for adult hippocampal neurogenesis resides outside the hippocampus,producing progenitor cells that migrate into the neurogenic zones and proliferate to produce new neurons and glia. View Publication

The mechanisms underlying the decision of a stem or progenitor cell to either self-renew or differentiate are incompletely understood. To address the role of Myc in this process,we expressed different forms of the proto-oncogene Myc in multipotent neural progenitor cells (NPCs) using retroviral transduction. Expression of Myc in neurospheres increased the proportion of self-renewing cells fivefold,and 1% of the Myc-overexpressing cells,but none of the control cells,retained self-renewal capacity even under differentiation-inducing conditions. A Myc mutant (MycV394D) deficient in binding to Miz-1,did not increase the percentage of self-renewing cells but was able to stimulate proliferation of NPCs as efficiently as wild-type Myc,indicating that these two cellular phenomena are regulated by at least partially different pathways. Our results suggest that Myc,through Miz-1,enhances self-renewal of NPCs and influences the way progenitor cells react to the environmental cues that normally dictate the cellular identity of tissues containing self-renewing cells. View Publication

Adult hippocampal neurogenesis is stimulated by chronic administration of antidepressants (ADs) and by voluntary exercise. Neural progenitor cells (NPCs) in the dentate gyrus (DG) that are capable of continuous proliferation and neuronal differentiation are the source of such structural plasticity. Here we report that mice lacking the receptor tyrosine kinase TrkB in hippocampal NPCs have impaired proliferation and neurogenesis. When exposed to chronic ADs or wheel-running,no increase in proliferation or neurogenesis is observed. Ablation of TrkB also renders these mice behaviorally insensitive to antidepressive treatment in depression- and anxiety-like paradigms. In contrast,mice lacking TrkB only in differentiated DG neurons display typical neurogenesis and respond normally to chronic ADs. Thus,our data establish an essential cell-autonomous role for TrkB in regulating hippocampal neurogenesis and behavioral sensitivity to antidepressive treatments,and support the notion that impairment of the neurogenic niche is an etiological factor for refractory responses to an antidepressive regimen. View Publication

Neurogenesis persists in the adult brain subventricular zone where neural stem/progenitor cells (NSPCs) lie close to brain endothelial cells (BECs). We show in mouse that BECs produce bone morphogenetic proteins (BMPs). Coculture of embryonic and adult NSPCs with BECs activated the canonical BMP/Smad pathway and reduced their proliferation. We demonstrate that coculture with BECs in the presence of EGF and FGF2 induced a reversible cell cycle exit of NSPCs (LeX+) and an increase in the amount of GFAP/LeX-expressing progenitors thought to be stem cells. Levels of the phosphatidylinositol phosphatase PTEN were upregulated in NSPCs after coculture with BECs,or treatment with recombinant BMP4,with a concomitant reduction in Akt phosphorylation. Silencing Smad5 with siRNA or treatment with Noggin,a BMP antagonist,demonstrated that upregulation of PTEN in NSPCs required BMP/Smad signaling and that this pathway regulated cell cycle exit of NSPCs. Therefore,BECs may provide a feedback mechanism to control the proliferation of NSPCs. View Publication

The regulated production of neurons in the hippocampus throughout life underpins important brain functions such as learning and memory. Surprisingly,however,studies have so far failed to identify a resident hippocampal stem cell capable of providing the renewable source of these neurons. Here,we report that depolarizing levels of KCl produce a threefold increase in the number of neurospheres generated from the adult mouse hippocampus. Most interestingly,however,depolarizing levels of KCl led to the emergence of a small subpopulation of precursors (approximately eight per hippocampus) with the capacity to generate very large neurospheres (textgreater 250 microm in diameter). Many of these contained cells that displayed the cardinal properties of stem cells: multipotentiality and self-renewal. In contrast,the same conditions led to the opposite effect in the other main neurogenic region of the brain,the subventricular zone,in which neurosphere numbers decreased by approximately 40% in response to depolarizing levels of KCl. Most importantly,we also show that the latent hippocampal progenitor population can be activated in vivo in response to prolonged neural activity found in status epilepticus. This work provides the first direct evidence of a latent precursor and stem cell population in the adult hippocampus,which is able to be activated by neural activity. Because the latent population is also demonstrated to reside in the aged animal,defining the precise mechanisms that underlie its activation may provide a means to combat the cognitive deficits associated with a decline in neurogenesis. View Publication