Biomedical subjects

David H Gutmann

Publications and source records attributed to David H Gutmann.

At least 73 records · Page 4Linked to original sources

PubMed2003

The neurofibromatosis 1 gene product neurofibromin regulates pituitary adenylate cyclase-activating polypeptide-mediated signaling in astrocytes.

Individuals with the neurofibromatosis 1 (NF1)-inherited tumor predisposition syndrome develop low-grade astrocytomas. The NF1 tumor suppressor gene product neurofibromin exhibits GTPase-activating activity (GAP) toward RAS, such that loss of neurofibromin expression leads to high levels of activated RAS and increased cell proliferation. Previous work has demonstrated that Nf1 inactivation in astrocytes leads to increased cell proliferation in vitro and in vivo, accompanied by increased RAS pathway activation. Studies on Nf1 mutant Drosophila have suggested that neurofibromin might also regulate cAMP signaling. Because intracellular cAMP levels have profound effects on astrocyte growth control, we sought to determine the contribution of neurofibromin to astrocyte cAMP regulation. In this report, we demonstrate that Nf1 inactivation in astrocytes results in reduced cAMP generation in response to PACAP and attenuated calcium influx and Rap1 activation. Based on the differential effects of forskolin and dibutyryl-cAMP on Nf1-/- astrocytes, neurofibromin likely functions at the level of adenylyl cyclase activation. Last, the reintroduction of a fragment of neurofibromin containing residues sufficient for restoring RAS-GAP function in Nf1-/- cells resulted in only partial restoration of neurofibromin-mediated cAMP regulation. These results demonstrate that neurofibromin positively influences cAMP generation and activation of cAMP growth regulatory targets in astrocytes and expands the role of the NF1 gene in astrocyte growth regulation.

Adenylyl Cyclases↗

PubMed2003

Identification of dominant negative mutants of Rheb GTPase and their use to implicate the involvement of human Rheb in the activation of p70S6K.

Rheb GTPases represent a unique family of the Ras superfamily of G-proteins. Studies on Rheb in Schizosaccharomyces pombe and Drosophila have shown that this small GTPase is essential and is involved in cell growth and cell cycle progression. The Drosophila studies also raised the possibility that Rheb is involved in the TOR/S6K signaling pathway. In this paper, we first report identification of dominant negative mutants of S. pombe Rheb (SpRheb). Screens of a randomly mutagenized SpRheb library yielded a mutant, SpRhebD60V, whose expression in S. pombe results in growth inhibition, G1 arrest, and induction of fnx1+, a gene whose expression is induced by the disruption of Rheb. Alteration of the Asp-60 residue to all possible amino acids by site-directed mutagenesis led to the identification of two particularly strong dominant negative mutants, D60I and D60K. Characterization of these dominant negative mutant proteins revealed that D60V and D60I exhibit preferential binding of GDP, while D60K lost the ability to bind both GTP and GDP. A possible use of the dominant negative mutants in the study of mammalian Rheb was explored by introducing dominant negative mutations into human Rheb. We show that transient expression of the wild type Rheb1 or Rheb2 causes activation of p70S6K, while expression of Rheb1D60K mutant results in inhibition of basal level activity of p70S6K. In addition, Rheb1D60K and Rheb1D60V mutants blocked nutrient- or serum-induced activation of p70S6K. This provides critical evidence that Rheb plays a role in the mTOR/S6K pathway in mammalian cells.

Amino Acid Sequence↗

PubMed2003

The 43000 growth-associated protein functions as a negative growth regulator in glioma.

Previous molecular analyses of human astrocytomas have identified many genetic changes associated with astrocytoma formation and progression. In an effort to identify novel gene expression changes associated with astrocytoma formation, which might reveal new potential targets for glioma therapeutic drug design, we used the B8-RAS-transgenic mouse astrocytoma model. Using multiplex gene expression profiling, we found that growth-associated protein 43 (GAP43) RNA and protein expression were lost in select human and mouse glioma cell lines. In this study, we demonstrate that re-expression of GAP43 in deficient C6 glioma cells results in growth suppression in clonogenic assays, as well as in multiple independently derived C6 glioma cell lines in vitro. GAP43-expressing C6 cells also exhibit reduced tumor growth as s.c. explants in immunocompromised mice in vivo. In addition, GAP43-expressing C6 clones demonstrate impaired cell motility and increased homophilic aggregation. GAP43 re-expression is also associated with reduced mitogen-activated protein kinase and AKT activation in C6 cells, suggesting that GAP43 functions as a novel glioma growth suppressor by modulating mitogenic signaling pathways.

Animals↗

PubMed2003

Mouse models of human cancer consortium symposium on nervous system tumors.

Nervous system tumors represent unique neoplasms that arise within the central and peripheral nervous system. Recent progress in generating genetically engineered mouse models of these tumors has advanced our understanding of the critical molecular and cellular events important for the development of these tumors. Recently, the National Cancer Institute-sponsored Mouse Models of Human Cancer Consortium convened a meeting on Nervous System Tumors to review recent advances and suggest directions for future research. Refined and novel approaches to modeling central nervous system tumors, including gliomas, meningiomas, medulloblastomas, and oligodendrogliomas, as well as peripheral nervous system tumors such as neurofibromas, schwannomas, and malignant peripheral nerve sheath tumors, were presented. In this review, we discuss the current status of mouse modeling of human nervous system cancers with a specific focus on unresolved scientific questions pertaining to the molecular genetics and cellular biology of these tumors.

Animals↗

PubMed2003

Oligodendrogliomas result from the expression of an activated mutant epidermal growth factor receptor in a RAS transgenic mouse astrocytoma model.

A significant proportion of human malignant gliomas exhibit amplification, overexpression, or mutations of the epidermal growth factor receptor (EGFR). To define the functional role(s) of the EGFR in the pathogenesis of gliomas, we established transgenic mice that express both wild-type (wt) and mutant (EGFRvIII) EGFR molecules using the human glial fibrillary acidic protein (GFAP) promoter. Both GFAP-EGFR(wt) and GFAP-EGFRvIII transgenic mice demonstrated increased numbers of astrocytes compared with control littermates, however, developed normally without formation of gliomas. To determine whether EGFR overexpression could modify the tumor phenotype in our previously reported GFAP-V(12)Ha-ras transgenic mouse astrocytoma model, mice expressing both activated RAS and EGFR were developed. GFAP-V(12)Ha-ras;GFAP-EGFRvIII, but not GFAP-V(12)Ha-ras;GFAP-EGFR(wt) double transgenic mice, had decreased survival with fifty percent of the mice dead at 2-4 weeks from gliomas, compared with 12-16 weeks for the GFAP-V(12)Ha-ras mice. Furthermore, GFAP-V(12)Ha-ras;GFAP-EGFRvIII mice developed oligodendrogliomas and mixed oligoastrocytoma tumors, instead of the fibrillary astrocytomas observed in GFAP-V(12)Ha-ras mice. In addition to yielding a spontaneous model of infiltrating oligodendroglioma, this study demonstrates that astrocyte-specific expression of EGFRvIII alone is insufficient for gliomagenesis but rather contributes to glioma progression in the context of existing predisposing genetic changes.

Animals↗

PubMed2003

Impaired glial glutamate transport in a mouse tuberous sclerosis epilepsy model.

Excessive astrocytosis in cortical tubers in tuberous sclerosis complex (TSC) suggests that astrocytes may be important for epileptogenesis in TSC. We previously demonstrated that astrocyte-specific Tsc1 gene inactivation in mice (Tsc1 cKO mice) results in progressive epilepsy. Here, we report that glutamate transporter expression and function is impaired in Tsc1 cKO astrocytes. Tsc1 cKO mice exhibit decreased GLT-1 and GLAST protein expression. Electrophysiological assays demonstrate a functional decrease in glutamate transport currents of Tsc1 cKO astrocytes in hippocampal slices and astrocyte cultures. These findings suggest that Tsc1 inactivation in astrocytes causes dysfunctional glutamate homeostasis, leading to seizure development in TSC.

Animals↗

PubMed2003

Role of the Rap1 GTPase in astrocyte growth regulation.

Tuberous sclerosis complex (TSC) is an autosomal dominant syndrome in which affected individuals develop nervous system abnormalities that might reflect astrocyte dysfunction. The TSC2 gene product, tuberin, encodes a GTPase-activating protein (GAP) domain, which regulates the activity of Rap1 in vitro. To determine whether dysregulated Rap1, resulting from TSC2 inactivation, leads to increased astrocyte proliferation in vivo, we generated transgenic mice expressing activated Rap1(G12V) specifically in astrocytes. We observed no statistically significant difference in the number of astrocytes between wild-type and GFAP-Rap1(G12V) littermates in vivo; however, during log-phase growth, we observed a 25% increase in GFAP-Rap1(G12V) astrocyte doubling times compared to wild-type controls. This decreased proliferation was associated with delayed MAP kinase, but not AKT, activation. Lastly, to determine whether constitutive Rap1 activation could reverse the increased astrocyte proliferation observed in transgenic mice expressing oncogenic Ras(G12V), we generated transgenic mice expressing both Ras(G12V) and Rap1(G12V) in astrocytes. These double transgenic mice showed a striking reversion of the Ras(G12V) astrocyte growth phenotype. Collectively, these results argue that the tumor suppressor properties of tuberin are unlikely to be related to Rap1 inactivation and that Rap1 inhibits mitogenic Ras pathway signaling in astrocytes.

Animals↗

PubMed2003

Neurofibromatosis 1: closing the GAP between mice and men.

Neurofibromatosis 1 (NF1) is a common genetic condition in which affected individuals are prone to the development of benign and malignant tumors. The NF1 tumor suppressor encodes a protein product, neurofibromin, which functions in part as a negative regulator of RAS. Loss of neurofibromin expression in NF1-associated tumors or Nf1-deficient mouse cells is associated with elevated RAS activity and increased cell proliferation. Despite this straightforward pathophysiologic association between neurofibromin, RAS, and tumorigenesis, recent insights from mouse and Drosophila modeling studies have suggested additional functions for neurofibromin and have implicated Nf1 heterozygosity in tumor formation. Lastly, Nf1 knockout mouse studies have also demonstrated important roles for cooperating genetic changes that accelerate tumorigenesis as well as modifier genes that impact on cancer susceptibility.

Animals↗

PubMed2003

Identification of a third Protein 4.1 tumor suppressor, Protein 4.1R, in meningioma pathogenesis.

Meningiomas are common central nervous system tumors; however, the mechanisms underlying their pathogenesis are largely undefined. In this report, we demonstrate that a third Protein 4.1 family member, Protein 4.1R, functions as a meningioma tumor suppressor. We observed loss of Protein 4.1R expression in two meningioma cell lines (IOMM-Lee, CH157-MN) by Western blotting as well as in 6 of 15 sporadic meningiomas by immunohistochemistry and fluorescence in situ hybridization. In support of a meningioma tumor suppressor function, Protein 4.1R overexpression resulted in reduced IOMM-Lee and CH157-MN cell proliferation. Similar to the Protein 4.1B and merlin tumor suppressors, Protein 4.1R membrane localization increased significantly under conditions of growth arrest in vitro. Lastly, we show that Protein 4.1R interacted with a subset of merlin/Protein 4.1B interactors including CD44 and betaII-spectrin. Collectively, these results suggest that Protein 4.1R functions as an important tumor suppressor in the molecular pathogenesis of meningioma.

Animals↗

PubMed2003

Expression of ICAM-1, TNF-alpha, NF kappa B, and MAP kinase in tubers of the tuberous sclerosis complex.

Individuals affected with tuberous sclerosis complex (TSC) develop cortical tubers characterized by disorganized cytoarchitecture and morphologically abnormal cell types, such as dysplastic neurons (DNs) and giant cells (GCs). As part of ongoing cDNA array analysis to study the molecular pathogenesis of tuber formation, we detected increased expression of intercellular adhesion molecule-1 (ICAM-1) mRNA, a cell adhesion molecule (CAM) that functions in cytokine signaling, in tubers. Western and immunohistochemical analyses revealed that ICAM-1 protein was selectively expressed in tubers, but was only minimally expressed in control cortex, adjacent nontuberal cortex, or in non-TSC focal cortical dysplasia. Increased expression of ICAM-1 was found in mice in which the Tsc1 gene was conditionally inactivated in astrocytes. Expression of molecules involved in ICAM-1 activation and cytokine signaling were increased in tubers, including tumor necrosis factor alpha (TNF-alpha), mitogen activated protein kinase (MAPK), and nuclear factor kappa B (NF-kappaB). Numerous CD68-immunoreactive macrophages were observed clustered around GCs further supporting an inflammatory response in tubers. Expression of caspase 8 and Fas support cytokine activation and detection of TUNEL reactivity suggests ongoing cell death in tubers. Specific alterations in ICAM-1, TNF-alpha, NF-kappaB1, and MAPK expression coupled with the detection of numerous CD68-immunoreactive macrophages suggests activation of proinflammatory cytokine signaling pathways in tubers that may culminate in cell death.

Adult↗

PubMed2003

Neurofibromatosis 2.

PURPOSE OF REVIEW: Recent clinical and molecular research on neurofibromatosis 2 (NF2) is reviewed, and the implications for clinical practice and research are discussed. RECENT FINDINGS: NF2 patients who are treated in specialty centers have a significantly lower risk of mortality than those who are treated in non-specialty centers. Vestibular schwannoma growth rates in NF2 are generally higher in younger people but are highly variable, even among multiple NF2 patients of similar ages in the same family. Radiation therapy is best reserved for NF2 patients who have particularly aggressive tumors, those who are poor surgical risks, those who refuse surgery, or those who are elderly. In-vivo studies have demonstrated that leptomeningeal cell activation of in mice results in leptomeningeal hyperplasia and meningioma formation. In-vitro studies have identified molecules that interact with the product (merlin or schwannomin), some of which (e.g., CD44 and paxillin) may play critical roles in merlin growth regulation. SUMMARY: NF2 patients should be referred to specialty treatment centers for optimal care. Clinical management of multiple patients in NF2 families cannot be based on the expectation of similar vestibular schwannoma growth rates, even when other clinical aspects of disease severity are similar. The availability of accurate mouse models of human NF2-associated tumors and the identification of molecules involved in merlin growth regulation now provide an opportunity to design targeted treatments for schwannomas and meningiomas.

Animals↗

PubMed2003

T-cadherin-mediated cell growth regulation involves G2 phase arrest and requires p21(CIP1/WAF1) expression.

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 G2 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 G2 arrest.

Aneuploidy↗

PubMed2003

Rap1 activity is elevated in malignant astrocytomas independent of tuberous sclerosis complex-2 gene expression.

Increased small GTPase protein mitogenic signaling is common in tumors. We have previously demonstrated that sporadic astrocytomas exhibit high levels of activated Ras important for tumor growth. Individuals with tuberous sclerosis complex (TSC) develop astrocytoma-like tumors resulting from mutations in the TSC2 protein, tuberin, which is hypothesized to function as a Rap1 GTPase activating protein (GAP). Since we have previously reported that high-grade astrocytomas frequently exhibit loss of tuberin expression or increased Rap1 levels, we sought to determine whether there is a correlation between decreased tuberin Rap1-GAP function or Rap1 overexpression and tumor Rap1 activity. In this study, we compared levels of Rap1-GTP, Rap1 and tuberin levels in normal brain tissue and 24 grade II-IV astrocytoma specimens. Whereas Rap1 overexpression was observed in astrocytomas of all malignancy grades, tuberin loss was seen most frequently in the higher-grade astrocytomas. In the grade IV glioblastoma multiforme tumors, Rap1 activity was 2-3-fold higher than in lower grade or non-neoplastic brain. However, there was no correlation between tuberin expression or Rap1 overexpression and the levels of Rap1 activity in the tumors studied, suggesting that the increased Rap1 activation is not the direct result of reduced tuberin Rap1-GAP function or elevated Rap1 protein expression.

Astrocytoma↗

PubMed2002

Functional analysis of the relationship between the neurofibromatosis 2 tumor suppressor and its binding partner, hepatocyte growth factor-regulated tyrosine kinase substrate.

Individuals with the neurofibromatosis 2 (NF2) inherited tumor predisposition syndrome are prone to the development of nervous system tumors, including schwannomas and meningiomas. The NF2 tumor suppressor protein, merlin or schwannomin, inhibits cell growth and motility as well as affects actin cytoskeleton-mediated processes. Merlin interacts with several proteins that might mediate merlin growth suppression, including hepatocyte growth factor-regulated tyrosine kinase substrate (HRS or HGS). Previously, we demonstrated that regulated overexpression of HRS in RT4 rat schwannoma cells had the same functional consequences as regulated overexpression of merlin. To determine the functional significance of this interaction, we generated a series of HRS truncation mutants and defined the regions of HRS required for merlin binding and HRS growth suppression. The HRS domain required for merlin binding was narrowed to a region (residues 470-497) containing the predicted coiled-coil domain whereas the major domain responsible for HRS growth suppression was distinct (residues 498-550). To determine whether merlin growth suppression required HRS, we demonstrated that merlin inhibited growth in HRS (+/+), but not HRS( -/-) mouse embryonic fibroblast cells. In contrast, HRS could suppress cell growth in the absence of Nf2 expression. These results suggest that merlin growth suppression requires HRS expression and that the binding of merlin to HRS may facilitate its ability to function as a tumor suppressor.

3T3 Cells↗

PubMed2002

Neurofibromatosis 2 (NF2) tumor suppressor schwannomin and its interacting protein HRS regulate STAT signaling.

Mutations in the neurofibromatosis 2 (NF2) gene with the resultant loss of expression of the NF2 tumor suppressor schwannomin are one of the most common causes of benign human brain tumors, including schwannomas and meningiomas. Previously we demonstrated that the hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) strongly interacts with schwannomin. HRS is a powerful regulator of receptor tyrosine kinase trafficking to the degradation pathway and HRS also binds STAM. Both of these actions for HRS potentially inhibit STAT activation. Therefore, we hypothesized that schwannomin inhibits STAT activation through interaction with HRS. We now show that both schwannomin and HRS inhibit Stat3 activation and that schwannomin suppresses Stat3 activation mediated by IGF-I treatment in the human schwannoma cell line STS26T. We also find that schwannomin inhibits Stat3 and Stat5 phosphorylation in the rat schwannoma cell line RT4. Schwannomin with the pathogenic missense mutation Q538P fails to bind HRS and does not inhibit Stat5 phosphorylation. These data are consistent with the hypothesis that schwannomin requires HRS interaction to be fully functionally active and to inhibit STAT activation.

Acute-Phase Proteins↗

PubMed2002

Protein 4.1 tumor suppressors: getting a FERM grip on growth regulation.

Members of the Protein 4.1 superfamily have highly conserved FERM domains that link cell surface glycoproteins to the actin cytoskeleton. Within this large and constantly expanding superfamily, at least five subgroups have been proposed. Two of these subgroups, the ERM and prototypic Protein 4.1 molecules, include proteins that function as tumor suppressors. The ERM subgroup member merlin/schwannomin is inactivated in the tumor-predisposition syndrome neurofibromatosis 2 (NF2), and the prototypic 4.1 subgroup member, Protein 4.1B, has been implicated in the molecular pathogenesis of breast, lung and brain cancers. This review focuses on what is known of mechanisms of action and critical protein interactions that may mediate the unique growth inhibitory signals of these two Protein 4.1 tumor suppressors. On the basis of insights derived from studying the NF2 tumor suppressor, we propose a model for merlin growth regulation in which CD44 links growth signals from plasma membrane to the nucleus by interacting with ERM proteins and merlin.

Animals↗

PubMed2002

The 4.1/ezrin/radixin/moesin domain of the DAL-1/Protein 4.1B tumour suppressor interacts with 14-3-3 proteins.

The Protein 4.1 family contains at least two members that function as tumour suppressors, the neurofibromatosis 2 gene product merlin and the recently identified differentially expressed in adenocarcinoma of the lung (DAL-1)/Protein 4.1B molecule. DAL-1/Protein 4.1B loss is observed in a variety of tumours, including breast and lung cancers as well as meningiomas. We have previously demonstrated that DAL-1/Protein 4.1B interacts with some but not all merlin-binding proteins, raising the possibility that DAL-1/Protein 4.1B associates with additional unique proteins specific to its function as a negative growth regulator. Using yeast two-hybrid interaction cloning, we identified three 14-3-3 isoforms, beta, gamma and eta, to be DAL-1/Protein 4.1B-binding proteins. These interactions were verified by using glutathione S-transferase affinity chromatography in vitro and co-immunoprecipitation in vivo. The interaction of 14-3-3 with DAL-1/Protein 4.1B was specific, as 14-3-3 did not bind to the related Protein 4.1 family members merlin, ezrin or radixin. The DAL-1/Protein 4.1B domain that mediates 14-3-3 binding was mapped to residues Pro(244) and Leu(280) within the 4.1/ezrin/radixin/moesin domain. The identification of this novel DAL-1/Protein 4.1B-interacting protein represents the first step towards elucidating its potentially unique mechanism of action.

14-3-3 Proteins↗

PubMed2002

Heterozygosity for the tuberous sclerosis complex (TSC) gene products results in increased astrocyte numbers and decreased p27-Kip1 expression in TSC2+/- cells.

Tuberous sclerosis complex (TSC) is an autosomal dominant tumor predisposition syndrome characterized by benign proliferations (hamartomas). In the brain, individuals with TSC develop autism, mental retardation and seizures associated with focal cortical dysplasias, subependymal nodules, and subependymal giant cell astrocytomas (SEGAs). We hypothesize that dysregulated astrocyte function due to mutations in the tumor suppressor genes, TSC1 and TSC2, may contribute to the pathogenesis of these brain abnormalities. In this report, we demonstrate that mice heterozygous for a targeted defect in either the Tsc1 or Tsc2 genes(Tsc1+/- and Tsc2+/- mice) exhibit a 1.5-fold increase in the number of astrocytes in vivo. Whereas increased astrocyte numbers in vivo were suggestive of a proliferative advantage, Tsc2+/- primary astrocyte cultures did not show a cell-autonomous growth advantage, anchorage-independent growth, increased saturation density, or increased fluid-phase endocytosis compared to wild type astrocytes. Tsc2 null mouse embryonic fibroblasts (MEFs) however, did exhibit increased saturation density compared to Tsc2 wild type controls. In both Tsc2+/- astrocytes and Tsc2 null mouse embryonic fibroblasts, p27-Kip1 expression was decreased compared to wild type cells, and was reversed by tuberin re-expression in Tsc2-/- MEFs. In contrast, no change in endocytosis was observed upon tuberin re-expression in Tsc2-/- MEFs. Collectively, these results suggest Tsc heterozygosity may provide a non-cell-autonomous growth advantage for astrocytes that may involve p27-Kip1 expression.

Animals↗