Fate of nanoparticles in the central nervous system after intrathecal injection in healthy mice.

Cerebrospinal fluid (CSF) is produced in the cerebral ventricles and circulates within the subarachnoid space (SAS) of the brain and spinal cord, where it exchanges with interstitial fluid of the parenchyma. The access of CSF to the entire central nervous system (CNS) makes it an attractive medium for drug delivery. However, few intrathecal (IT) therapies have reached the clinic due, in part, to limited distribution and rapid clearance. Given the success of nanoparticle (NP) carriers in prolonging circulation and improving delivery of systemically administered agents, we sought to evaluate the distribution of IT injected NPs within the CNS. We administered fluorescent, 100 nm PEGylated-NPs into the cisterna magna of healthy mice and studied their distribution along the brain and spinal cord. Our data demonstrate that NPs are capable of distributing rapidly through the SAS along the entire neuraxis with reproducible, anatomically defined patterns of delivery. NPs were well retained within the leptomeninges for over 3 weeks, showing preference for ventral surfaces and minimal penetration into the CNS parenchyma. Clearance of NPs occurred across the cribriform plate into the nasal mucosa, with a small fraction of NPs localizing with nerve roots exiting the spinal column. Larger 10 µm particles were also capable of moving through the SAS but did not achieve as widespread distribution. These studies demonstrate the ability of NPs to achieve widespread delivery along the neuraxis and highlight IT administration as a potentially significant route of administration for delivery of nanomedicine to the subarachnoid space.

Survivin as a therapeutic target in Sonic hedgehog-driven medulloblastoma.

Medulloblastoma (MB) is a highly malignant brain tumor that occurs primarily in children. Although surgery, radiation and high-dose chemotherapy have led to increased survival, many MB patients still die from their disease, and patients who survive suffer severe long-term side effects as a consequence of treatment. Thus, more effective and less toxic therapies for MB are critically important. Development of such therapies depends in part on identification of genes that are necessary for growth and survival of tumor cells. Survivin is an inhibitor of apoptosis protein that regulates cell cycle progression and resistance to apoptosis, is frequently expressed in human MB and when expressed at high levels predicts poor clinical outcome. Therefore, we hypothesized that Survivin may have a critical role in growth and survival of MB cells and that targeting it may enhance MB therapy. Here we show that Survivin is overexpressed in tumors from patched (Ptch) mutant mice, a model of Sonic hedgehog (SHH)-driven MB. Genetic deletion of survivin in Ptch mutant tumor cells significantly inhibits proliferation and causes cell cycle arrest. Treatment with small-molecule antagonists of Survivin impairs proliferation and survival of both murine and human MB cells. Finally, Survivin antagonists impede growth of MB cells in vivo. These studies highlight the importance of Survivin in SHH-driven MB, and suggest that it may represent a novel therapeutic target in patients with this disease.

Distinct roles for fibroblast growth factor signaling in cerebellar development and medulloblastoma.

Cerebellar granule neurons are the most abundant neurons in the brain, and a critical element of the circuitry that controls motor coordination and learning. In addition, granule neuron precursors (GNPs) are thought to represent cells of origin for medulloblastoma, the most common malignant brain tumor in children. Thus, understanding the signals that control the growth and differentiation of these cells has important implications for neurobiology and neurooncology. Our previous studies have shown that proliferation of GNPs is regulated by Sonic hedgehog (Shh), and that aberrant activation of the Shh pathway can lead to medulloblastoma. Moreover, we have demonstrated that Shh-dependent proliferation of GNPs and medulloblastoma cells can be blocked by basic fibroblast growth factor (bFGF). But while the mitogenic effects of Shh signaling have been confirmed in vivo, the inhibitory effects of bFGF have primarily been studied in culture. Here, we demonstrate that mice lacking FGF signaling in GNPs exhibit no discernable changes in GNP proliferation or differentiation. In contrast, activation of FGF signaling has a potent effect on tumor growth: treatment of medulloblastoma cells with bFGF prevents them from forming tumors following transplantation, and inoculation of tumor-bearing mice with bFGF markedly inhibits tumor growth in vivo. These results suggest that activators of FGF signaling may be useful for targeting medulloblastoma and other Shh-dependent tumors.

Review: personalized mice: modelling the molecular heterogeneity of medulloblastoma.

Medulloblastoma, the most common malignant paediatric brain tumour, is thought to arise from mutations in progenitors or stem cells in the cerebellum. Recent molecular analyses have highlighted the heterogeneity of these tumours, and demonstrated that they can be classified into at least four major subtypes that differ in terms of gene expression, genomic gains and losses, epidemiology and patient outcome. Along with analysis of human tumours, a variety of animal models of medulloblastoma have been developed using transgenic and knockout technology as well as somatic gene delivery. These models have provided valuable insight into the origins of the disease and the signalling pathways that control tumour growth. But the degree to which current models recapitulate the heterogeneity of the human disease remains unclear. Here we review the recent literature on the genomics of medulloblastoma and discuss the relationship of mouse models to the subtypes of the disease. Judicious use of existing models, and generation of additional models for poorly studied subtypes of medulloblastoma, will increase our understanding of tumour biology and allow evaluation of novel approaches to treatment of the disease.

Caught in the matrix: how vitronectin controls neuronal differentiation.

Cerebellar granule cells are the most abundant neurons in the brain and are crucial to the circuitry that controls motor coordination. The proliferation of granule cell precursors (GCPs) is controlled by the secreted signaling molecule Sonic hedgehog (Shh), but the factors that regulate GCP differentiation remain a mystery. A recent study suggests that the extracellular matrix protein vitronectin might tell GCPs when to stop dividing and begin differentiation.

The developmental biology of brain tumors.

Tumors of the central nervous system (CNS) can be devastating because they often affect children, are difficult to treat, and frequently cause mental impairment or death. New insights into the causes and potential treatment of CNS tumors have come from discovering connections with genes that control cell growth, differentiation, and death during normal development. Links between tumorigenesis and normal development are illustrated by three common CNS tumors: retinoblastoma, glioblastoma, and medulloblastoma. For example, the retinoblastoma (Rb) tumor suppressor protein is crucial for control of normal neuronal differentiation and apoptosis. Excessive activity of the epidermal growth factor receptor and loss of the phosphatase PTEN are associated with glioblastoma, and both genes are required for normal growth and development. The membrane protein Patched1 (Ptc1), which controls cell fate in many tissues, regulates cell growth in the cerebellum, and reduced Ptc1 function contributes to medulloblastoma. Just as elucidating the mechanisms that control normal development can lead to the identification of new cancer-related genes and signaling pathways, studies of tumor biology can increase our understanding of normal development. Learning that Ptc1 is a medulloblastoma tumor suppressor led directly to the identification of the Ptc1 ligand, Sonic hedgehog, as a powerful mitogen for cerebellar granule cell precursors. Much remains to be learned about the genetic events that lead to brain tumors and how each event regulates cell cycle progression, apoptosis, and differentiation. The prospects for beneficial work at the boundary between oncology and developmental biology are great.

Evidence that haploinsufficiency of Ptch leads to medulloblastoma in mice.

The PTCH gene encodes a putative tumor suppressor protein; germline alterations in PTCH have been found in patients with the nevoid basal cell carcinoma syndrome (NBCCS). Medulloblastoma, a brain tumor, develops in about 3% of NBCCS patients, and mutations in PTCH have also been described in a subset of sporadic medulloblastomas. The search for the causes of medulloblastoma has been hindered by the lack of an appropriate model system for this tumor type. Recently, a transgenic mouse hemizygous for the Ptch gene was generated by homologous recombination. Medulloblastomas were found in about 19% of these mice within the first 25 weeks after birth. The status of the wild-type PTCH allele in these tumors has not been investigated. For clearer definition of the role of PTCH as a tumor suppressor in medulloblastoma, 13 cerebellar tumors from transgenic Ptch(+/-) mice were examined for alterations in the remaining Ptch allele. A single mutation was found in one tumor, a C-to-A substitution changing a tyrosine to a stop codon; all other tumors exhibited a wild-type sequence. Two tumors with normal Ptch cDNA were examined by in situ hybridization. Ptch cDNA was found in tumor cells but not in associated tumor stroma. We also examined the mRNA expression levels for the remaining Ptch allele, as well as for Gli1, a gene known to be transcriptionally activated by Ptch inactivation. Blot analysis of RNA from the 13 tumors shows that Ptch mRNA of appropriate size is expressed in all tumors at varying levels. Expression of Gli1 was increased in tumors compared to normal cerebellum. These results suggest that deletion of one copy of Ptch may be sufficient to promote medulloblastoma development in mice.

Losses of the tumor suppressor BIN1 in breast carcinoma are frequent and reflect deficits in programmed cell death capacity.

Oncogenic activation of MYC occurs often in breast carcinoma and is associated with poor prognosis. Loss or inactivation of mechanisms that restrain MYC may therefore be involved in tumor progression. In this study, we show that the MYC-interacting adaptor protein BIN1 is frequently missing in malignant breast cells and that this loss is functionally significant. BIN1 was expressed in normal and benign cells and tissues but was undetectable in 6/6 estrogen receptor-positive or estrogen receptor-negative carcinoma cell lines examined. Similarly, complete or partial losses of BIN1 were documented in 30/50 (60%) cases of malignant breast tissue analyzed by immuno-histochemistry or RT-PCR. Abnormalities in the organization of the BIN1 gene were apparent in only a minority of these cases, suggesting that most losses were due to epigenetic causes. Nevertheless, they were functionally significant because ectopic BIN1 induced programmed cell death in malignant cells lacking endogenous BIN1 but had no effect on the viability of benign cells. We propose that loss of BIN1 may contribute to breast cancer progression by eliminating a mechanism that restrains the ability of activated MYC to drive cell division inappropriately.

Control of neuronal precursor proliferation in the cerebellum by Sonic Hedgehog.

Cerebellar granule cells are the most abundant type of neuron in the brain, but the molecular mechanisms that control their generation are incompletely understood. We show that Sonic hedgehog (Shh), which is made by Purkinje cells, regulates the division of granule cell precursors (GCPs). Treatment of GCPs with Shh prevents differentiation and induces a potent, long-lasting proliferative response. This response can be inhibited by basic fibroblast growth factor or by activation of protein kinase A. Blocking Shh function in vivo dramatically reduces GCP proliferation. These findings provide insight into the mechanisms of normal growth and tumorigenesis in the cerebellum.

A role for the putative tumor suppressor Bin1 in muscle cell differentiation.

Bin1 is a Myc-interacting protein with features of a tumor suppressor. The high level of Bin1 expression in skeletal muscle prompted us to investigate its role in muscle differentiation. Significant levels of Bin1 were observed in undifferentiated C2C12 myoblasts, a murine in vitro model system. Induction of differentiation by growth factor withdrawal led to an upregulation of Bin1 mRNA and to the generation of higher-molecular-weight forms of Bin1 protein by alternate splicing. While Bin1 in undifferentiated cells was localized exclusively in the nucleus, differentiation-associated isoforms of Bin1 were found in the cytoplasm as well. To examine the function of Bin1 during differentiation, we generated stable cell lines that express exogenous human Bin1 cDNA in the sense or antisense orientation. Cells overexpressing Bin1 grew more slowly than control cells and differentiated more rapidly when deprived of growth factors. In contrast, C2C12 cells expressing antisense Bin1 showed an impaired ability to undergo differentiation. Taken together, the results indicated that Bin1 expression, structure, and localization are tightly regulated during muscle differentiation and suggested that Bin1 plays a functional role in the differentiation process.

The putative tumor suppressor BIN1 is a short-lived nuclear phosphoprotein, the localization of which is altered in malignant cells.

BIN1 is a putative tumor suppressor that was identified in a genetic screen for polypeptides that interact with the MYC oncoprotein. Using a set of six monoclonal antibodies, we identified and examined biochemical features and localization of cellular BIN1. Epitope mapping indicated that a putative nuclear localization motif and the MYC-binding domain were among the regions recognized by five antibodies. In immunoprecipitation and Western analyses, cellular BIN1 was identified in human and rodent cells as a monomeric phosphoprotein of M(r) approximately 70,000. Pulse-chase experiments showed that BIN1 was short-lived, with a half-life of approximately 2 h. Cell immunofluorescence experiments revealed overlapping but unique nuclear localization patterns distinguished by two different antibodies. In normal cells, BIN1 was predominantly nucleoplasmic but was also present in a subnuclear compartment. Conversely, in a panel of tumor cells that expressed BIN1, the predominant localization was the subnuclear compartment. Taken together, the results suggested that the antibodies recognized different isoforms or conformations of BIN1, the localization of which varied between normal and tumor cells. This study will facilitate further analysis of the structure and regulation of BIN1 in normal and malignant cells.

Retinal development: communication helps you see the light.

Recent studies suggest that interactions between neurons, glial cells and endothelial cells are critical in determining the structure of the retina and the optic nerve. Dysregulation of these interactions can lead to disruption of retinal architecture and impairment of vision.

Structural analysis of the human BIN1 gene. Evidence for tissue-specific transcriptional regulation and alternate RNA splicing.

BIN1 is a putative tumor suppressor that was identified through its interaction with the MYC oncoprotein. To begin to identify elements of BIN1 whose alteration may contribute to malignancy, we cloned and characterized the human BIN1 gene and promoter. Nineteen exons were identified in a region of >54 kilobases, six of which were alternately spliced in a cell type-specific manner. One alternately spliced exon encodes part of the MYC-binding domain, suggesting that splicing controls the MYC-binding capacity of BIN1 polypeptides. Four other alternately spliced exons encode amphiphysin-related sequences that were included in brain-specific BIN1 species, also termed amphiphysin isoforms or amphiphysin II. The 5'-flanking region of BIN1 is GC-rich and lacks a TATA box but directs transcriptional initiation from a single site. A approximately 0. 9-kilobase fragment from this region was sufficient for basal transcription and transactivation by MyoD, which may account for the high levels of BIN1 observed in skeletal muscle. This study lays the foundation for genetic and epigenetic investigations into the role of BIN1 in normal and neoplastic cell regulation.

Lipopolysaccharide prevents apoptosis and induces responsiveness to antigen receptor cross-linking in immature B cells.

Unlike mature B cells, immature B cells are not activated in response to antigen receptor cross-linking. To examine the mechanisms underlying this unresponsiveness, we have studied the effects of reagents that have been shown to alter the responses of immature B cells to antigen receptor stimulation. Bacterial lipopolysaccharide (LPS) is a polyclonal B-cell activator, and has been shown to interfere with B-cell tolerance induction in vivo and in vitro. Here we show that LPS can also overcome the unresponsiveness of immature B cells to stimulation with anti-receptor (anti-mu) antibodies. LPS synergizes with anti-mu to induce a proliferative response that exceeds the response of immature B cells to LPS alone. Moreover, pretreatment of immature cells with LPS allows them to proliferate in response to subsequent stimulation with anti-mu antibodies. This induction of responsiveness to anti-mu requires exposure to LPS for at least 8 hr. Although the mechanisms of induction are not fully understood, one component of the LPS effect appears to involve enhancement of immature B-cell survival in culture. Neonatal splenic B cells undergo spontaneous apoptosis at a much higher rate than mature B cells, but we have found that LPS causes a dramatic inhibition of apoptosis, even when it is present for only the first 8 hr of culture. The ability of LPS to promote survival of immature B cells and allow them to proliferate in response to antigen receptor stimulation provides a system for investigation of the biochemical mechanisms of unresponsiveness and tolerance susceptibility.

BIN1 is a novel MYC-interacting protein with features of a tumour suppressor.

BIN1 is a novel protein that interacts with the functionally critical Myc box regions at the N terminus of the MYC oncoprotein. BIN1 is structurally related to amphiphysin, a breast cancer-associated autoimmune antigen, and RVS167, a negative regulator of the yeast cell cycle, suggesting roles in malignancy and cell cycle control. Consistent with this likelihood, BIN1 inhibited malignant cell transformation by MYC. Although BIN1 is expressed in many normal cells, its levels were greatly reduced or undetectable in 14/27 carcinoma cell lines and 3/6 primary breast tumours. Deficits were functionally significant because ectopic expression of BIN1 inhibited the growth of tumour cells lacking endogenous message. We conclude that BIN1 is an MYC-interacting protein with features of a tumour suppressor.

Resistance of a variant ras-transformed cell line to phenotypic reversion by farnesyl transferase inhibitors.

Pharmacological inhibitors of the housekeeping enzyme farnesyl transferase (FT) inhibit the growth of ras-transformed cells in vitro and in vivo without antiproliferative effects on normal cells. In one direction to analyze the basis for this selectivity and to study modes of drug resistance that arise in animals, we characterized a variant ras-transformed cell line, 749r-1, which was resistant to phenotypic reversion with FT inhibitors. The transformed phenotype, growth potential, and actin cytoskeleton of 749r-1 cells were unaffected by treatment with the FT inhibitor 1-739,749 at concentrations up to 30-fold higher than those sufficient to revert ras-transformed cells. Resistance correlated with a reduced ability of L-739,749 to inhibit the farnesylation of Ras and lamin B and with a reduction in the susceptibility of endogenous FT to drug inhibition. These effects were not due to mutation of the FT subunits, changes in intracellular drug accumulation, or amplification of the multiple drug resistance gene (MDR). However, a similar reduction in the ability of L-739,749 to inhibit Ras farnesylation was also seen in ras-transformed cells rendered resistant by ectopic expression of farnesyl-independent RhoB, suggesting some mechanistic overlap. We concluded that 749r-1 cells sustained a stable alteration that conferred drug resistance by a novel mechanism.

Immature stage B cells enter but do not progress beyond the early G1 phase of the cell cycle in response to antigen receptor signaling.

In contrast to mature B cells, immature stage B cells do not proliferate following Ag receptor cross-linking with anti-Ig Abs. To determine where in the cell cycle immature B cells arrest, we have examined the expression of specific G, cell cycle regulators. Following surface IgM (sIgM) cross-linking on mature B cells, we observed increased expression of the early G1 kinase, cyclin-dependent kinase 4 (cdk4), and one of its regulatory subunits, cyclin D2. Mature B cells also showed increased expression of components required for G1/S transition, including cyclin E and cdk2. Whereas immature stage B cells increased expression of cyclin D2 and cdk4 after anti-IgM stimulation, unlike mature stage B cells they failed to express cyclin E and cdk2. Expression of cyclin D2 and cdk4 indicates that these cells can exit G0 and enter the initial G1 phase following sIgM ligation. Interestingly, IL-4, which by itself does not stimulate proliferation of immature B cells, induced expression of cyclin E and cdk2. These latter results suggest that IL-4 complements sIgM, signaling for proliferation by increasing the basal levels of late G1 cell cycle regulators. Consistent with this idea, IL-4 synergizes with anti-Ig Abs to promote cell cycle progression and proliferation of immature B cells. Finally, c-myc, a transcriptional regulator of some members of the cell cycle machinery, is not induced following sIgM cross-linking of immature cells. This lack of inducible expression contrasts with that seen in mature stage B cells, and in immature stage cells stimulated to proliferate with LPS. These results suggest that c-myc may be a component of the signaling pathway that induces cyclin E and cdk2 expression.