Individuals with the neurofibromatosis 1 (NF1) inherited tumor syndrome develop low-grade gliomas (astrocytomas) at an increased frequency, suggesting that the NF1 gene is a critical growth regulator for astrocytes. In an effort to determine the contribution of the NF1 gene product, neurofibromin, to astrocyte growth regulation and NF1-associated astrocytoma formation, we generated astrocyte-specific Nf1 conditional knockout mice (Nf1 GFAP CKO) by using Cre/LoxP technology. Transgenic mice were developed in which Cre recombinase was specifically expressed in astrocytes by embryonic day 14.5. Successive intercrossing with mice bearing a conditional Nf1 allele (Nf1flox) resulted in GFAP-Cre Nf1flox/flox (Nf1 GFAP CKO) animals. No astrocytoma formation or neurological impairment was observed in Nf1 GFAP CKO mice after 20 months, but increased numbers of proliferating astrocytes were observed in several brain regions. To determine the consequence of Nf1 inactivation at different developmental times, the growth properties of embryonic day 12.5 and postnatal day 2 Nf1 null astrocytes were analyzed. Nf1 null astrocytes exhibited increased proliferation but lacked tumorigenic properties in vitro and did not form tumors when injected into immunocompromised mouse brains in vivo. Collectively, our results suggest that loss of neurofibromin is not sufficient for astrocytoma formation in mice and that other genetic or environmental factors might influence NF1-associated glioma tumorigenesis.Neurofibromatosis 1 (NF1) is the most common cancer predisposition syndrome affecting the nervous system, with an incidence of 1 in 3,500 births worldwide (16). Early in life, individuals with NF1 develop café-au-lait spots, skinfold freckling, and iris hamartomas (Lisch nodules). In addition, approximately 15 to 20% of children with NF1 develop glial cell tumors (astrocytomas) involving the optic nerve, chiasm, hypothalamus, and brain stem (31). Although classified as grade I juvenile pilocytic astrocytomas, these tumors can be associated with significant morbidity as a result of visual loss or neurological compromise.Since individuals affected with NF1 develop tumors at an increased frequency, the NF1 gene is hypothesized to function as a tumor suppressor. Identification of the NF1 gene (10,42,43) and its protein product, the 220-to 250-kDa cytoplasmic neurofibromin protein, revealed that a small portion of the molecule has sequence similarity with the catalytic domain of a family of proteins termed GTPase-activating proteins (GAPs) (4,34,45). GAP molecules function as negative regulators of small mitogenic GTPase proteins, like p21 ras , and inactivate these signaling proteins by accelerating their conversion from active, GTP-bound conformations to inactive, GDP-bound forms (6). In NF1-associated tumors, loss of neurofibromin results in increased p21 ras mitogenic signaling and augments cell proliferation, leading to tumor formation (5,7,12).

Members of the cadherin family have been implicated as growth regulators in multiple tumor types. Based on recent studies from our laboratory implicating T-cadherin expression in mouse brain tumorigenesis, we examined the role of T-cadherin in astrocytoma growth regulation. In this report, we show that T-cadherin expression increased during primary astrocyte physiologic growth arrest in response to contact inhibition and serum starvation in vitro, suggesting a function for T-cadherin in astrocyte growth regulation. We further demonstrate that transient and stable reexpression of T-cadherin in deficient C6 glioma cell lines results in growth suppression. In addition, T-cadherin-expressing C6 cell lines demonstrated increased homophilic cell aggregation, increased cell attachment to fibronectin, and decreased cell motility. Cell cycle flow cytometry demonstrated that T-cadherin reexpression resulted in G 2 phase arrest, which was confirmed by mitotic index analysis. This growth arrest was p53 independent, as T-cadherin could still mediate growth suppression in p53 ؊/؊ mouse embryonic fibroblasts. T-cadherin-expressing C6 cell lines exhibited increased p21 CIP1/WAF1 , but not p27 Kip1 , expression. Lastly, T-cadherin-mediated growth arrest was dependent on p21 CIP1/WAF1 expression and was eliminated in p21 CIP1/WAF1 -deficient fibroblasts. Collectively, these observations suggest a novel mechanism of growth regulation for T-cadherin involving p21 CIP1/WAF1 expression and G 2 arrest.Astrocytomas are the most common primary malignant cancer affecting the nervous system, and despite aggressive therapy, the median survival of patients diagnosed with a highgrade astrocytoma (glioma) is only 9 to 12 months (35). These malignant glial tumors are hypothesized to develop as the result of the stepwise accumulation of specific genetic changes in astrocytes or astroglial precursors that promote astrocyte transformation and malignant progression (8). Genetic events important for astrocytoma formation and progression involve alterations in pathways involved in mitogenic signaling, cell cycle growth regulation, and environmental sensing. Previous studies have demonstrated that astrocytomas harbor changes in the epidermal and platelet-derived growth factor signaling pathways, involving amplification, mutation, or overexpression of these receptor tyrosine kinase molecules to result in increased mitogenic signaling. Similarly, astrocytomas harbor alterations in the p53 and retinoblastoma (Rb) cell cycle regulatory pathways. Inactivating mutations in the p53, p16, and Rb genes have been reported as well as overexpression of regulators of these pathways, including cyclin-dependent kinase 4 (cdk4) and MDM2.In contrast, alterations in molecules involved in environmental sensing have not been explored in great detail in astrocytomas. Gene expression profiling experiments from our laboratory on a mouse astrocytoma model have implicated the novel cadherin molecule, T-or H-cadherin, in astrocytoma progression. Based on these initial observ...

Individuals with the neurofibromatosis 1 (NF1) tumor predisposition syndrome develop low-grade pilocytic astrocytomas at an increased frequency. Previously, we demonstrated that astrocytes from mice heterozygous for a targeted mutation in the Nf1 gene (Nf1+/- astrocytes) exhibit a cell autonomous growth advantage associated with increased RAS pathway activation. In this report, we extend our initial characterization of the effect of reduced Nf1 gene expression on astrocyte function by demonstrating that Nf1+/- astrocytes exhibit decreased cell attachment, actin cytoskeletal abnormalities during the initial phases of cell spreading, and increased cell motility. Whereas these cytoskeletal abnormalities were also observed in Nf1-/- astrocytes, astrocytes expressing a constitutively active RAS molecule showed increased cell motility and abnormal actin cytoskeleton organization during cell spreading, but exhibited normal cell attachment. Based on ongoing gene expression profiling experiments on human astrocytoma tumors, we demonstrate increased expression of two proteins implicated in cell attachment, spreading and motility (GAP43 and T-cadherin) in Nf1+/- and Nf1-/- astrocytes. These results support the emerging notion that tumor suppressor gene heterozygosity results in abnormalities in cell function that may contribute to the pathogenesis of non-tumor phenotypes in NF1.

Mutations of the neurofibromatosis 2 (NF2) tumor suppressor gene cause the inherited disorder NF2 and are also common in malignant mesothelioma, which is not a characteristic feature of NF2. The authors report an asbestos-exposed person with NF2 and malignant mesothelioma. Immunohistochemical analysis of the mesothelioma confirmed loss of expression of the NF2 protein, and comparative genomic hybridization revealed losses of chromosomes 14, 15, and 22, and gain of 7. The authors propose that a person with a constitutional mutation of an NF2 allele is more susceptible to mesothelioma.

Based on previous studies demonstrating increased RAS activity in human astrocytomas, we developed a transgenic mouse model (B8) that targets an activated RAS molecule to astrocytes. Within 3 to 4 months after birth, these mice develop high-grade astrocytomas that are histologically identical to human astrocytomas. To characterize genetic events associated with B8 mouse astrocytoma formation, we employed comparative gene expression profiling of wild-type cultured mouse astrocytes, non-neoplastic B8 astrocytes, B8 astrocytoma cultures, and two other astrocytoma cultures from independently derived RAS transgenic mouse lines. We identified several classes of gene expression changes, including those associated with the non-neoplastic state in the B8 transgenic mouse, those associated with astrocytoma formation, and those specifically associated with only one of the three independently derived transgenic mouse astrocytomas. Differential expression of several unique genes was confirmed at the protein level in both the RAS transgenic mouse astrocytomas and two human glioblastoma multiforme cell lines. Furthermore, reexpression of one of these downregulated astrocytoma-associated proteins, GAP43, resulted in C6 glioma cell growth suppression. The use of this transgenic mouse model to identify novel genetic changes that might underlie the pathogenesis of human high-grade astrocytomas provides a unique opportunity to discover future targets for brain tumor therapy.