p190RhoGAP can act to inhibit PDGF-induced gliomas in mice: a putative tumor suppressor encoded on human chromosome 19q13.3

p190RhoGAP and Rho are key regulators of oligodendrocyte differentiation. The gene encoding p190RhoGAP is located at 19q13.3 of the human chromosome, a locus that is deleted in 50%-80% of oligodendrogliomas. Here we provide evidence that p190RhoGAP may suppress gliomagenesis by inducing a differentiated glial phenotype. Using a cell culture model of autocrine loop PDGF stimulation, we show that reduced Rho activity via p190RhoGAP overexpression or Rho kinase inhibition induced cellular process extension, a block in proliferation, and reduced expression of the neural precursor marker nestin. In vivo infection of mice with retrovirus expressing PDGF and the p190 GAP domain caused a decreased incidence of oligodendrogliomas compared with that observed with PDGF alone. Independent experiments revealed that the retroviral vector insertion site in 3 of 50 PDGF-induced gliomas was within the p190RhoGAP gene. This evidence strongly suggests that p190 regulates critical components of PDGF oncogenesis and can act as a tumor suppressor in PDGF-induced gliomas by down-regulating Rho activity.     

Astrocytes give rise to oligodendrogliomas and astrocytomas after gene transfer of polyoma virus middle T antigen in vivo

The cells of origin for oligodendrogliomas and astrocytomas are not known but are presumed to be oligodendrocyte and astrocyte precursors, respectively. In this paper we report the generation of mixed gliomas from in vivo transformation of glial fibrillary acidic protein (GFAP)-positive cells (differentiated astrocytes) with polyoma virus middle T antigen (MTA). MTA is a powerful oncogene that activates a number of signal transduction pathways, including those proposed to be involved in gliomagenesis, and has been shown to induce tumors in many cell types. We have achieved transfer of MTA expression specifically to GFAP(+) cells in vivo using somatic cell gene transfer, and find resultant formation of anaplastic gliomas with mixed astrocytoma and oligodendroglioma morphological features. We conclude that GFAP- expressing astrocytes, with appropriate signaling abnormalities, can serve as the cell of origin for oligodendrogliomas, astrocytomas, or mixed gliomas.     

A mouse model for glioma: biology, pathology, and therapeutic opportunities

The epidermal growth factor receptor (EGFR) gene is amplified or mutated in 30-50% of human glioblastoma multiforme. These mutations are usually associated with deletions of the INK4a-ARF locus, which encodes 2 gene products (p16INK4a and p19ARF) involved in cell cycle arrest and apoptosis. We have investigated the role of EGFR mutation in gliomagenesis using avian retroviral vectors to transfer a mutant EGFR gene to glial precursors and astrocytes in transgenic mice. These mice express tv-a, a gene encoding the retrovirus receptor TVA, which is under the control of brain cell type-specific promoters. We demonstrate that expression of a constitutively active, mutant form of EGFR in cells in the glial lineage can induce lesions with many similarities to human gliomas, including increased cell density, vascular proliferation, and immunohistochemical staining for glial fibrillary acidic protein (GFAP) and nestin. We also demonstrate that primary astrocytes cultured from transgenic mice expressing tv-a from the GFAP promoter are efficiently infected in culture, and such genetically modified cell cultures can be tumorigenic in nude mice. The combinations of genetic lesions (eg, mutated EGFR, INK4a-/-) leading to tumor formation in these 2 mouse systems are similar to those found in human gliomas. These genetically defined animal models for gliomas will allow for the testing of therapies that are targeted specifically at the gene products involved in the pathogenesis of gliomas.     

Recent Insights into PDGF‐Induced Gliomagenesis

Gliomas are aggressive and almost incurable glial brain tumors which frequently display abnormal platelet‐derived growth factor (PDGF) signaling. Evidence gained from studies on several in vivo animal models has firmly established a causal connection between aberrant PDGF signaling and the formation of some gliomas. However, only recently has significant knowledge been gained regarding crucial issues such as the glioma cell of origin and the relationship between the transforming stimulus and the cellular characteristics of the resulting tumor. Based on recent evidence, we propose that PDGF can bias cell‐fate decisions, driving the acquisition of cell type‐specific features by the progeny of multipotent neural progenitors, thus determining the shape and direction of the transformation path. Furthermore, recent data about the cellular mechanisms of PDGF‐driven glioma progression and maintenance indicate that PDGF may be required, unexpectedly, to override cell contact inhibition and promote glioma cell infiltration rather than to stimulate cell proliferation.     

Characterization of adult neural stem cells and their relation to brain tumors

The adult mammalian brain contains neural stem cells that are capable of generating new neurons and glia over the course of a lifetime. Neural stem cells reside in 2 germinal niches, the subventricular zone (SVZ) and the dentate gyrus subgranular zone. These primary progenitors have been identified in their niche in vivo; these cells have characteristics of astrocytes. Recent studies have shown that adult SVZ stem cells are derived from radial glia, the stem cells in the developing brain, which in turn are derived from the neuroepithelum, the earliest brain progenitors. Thus, SVZ stem cells are a continuum from neuroepithelium to radial glia to astrocytes, and are contained within what has been considered the lineage for astrocytes. However, it seems that only a small subset of the astrocytes present in the adult brain have stem cell properties. Recent findings have shown that SVZ stem cell astrocytes express a receptor for platelet-derived growth factor (PDGF), suggesting that the ability to respond to specific growth factor stimuli, such as PDGF, epidermal growth factor and others, may be unique to these stem cell astrocytes. Intriguingly, activation of these same signaling pathways is widely implicated in brain tumor formation. Since the adult brain has very few proliferating cells capable of accumulating the numerous mutations required for transformation, the adult neural stem and/or progenitor cells may be likely candidates for the brain tumor cell of origin. Indeed, activation of the PDGF or epidermal growth factor pathways in adult neural stem or progenitor cells confers tumor-like properties on these cells, lending support to this hypothesis.     

A constitutively active epidermal growth factor receptor cooperates with disruption of G1 cell-cycle arrest pathways to induce glioma-like lesions in mice

The epidermal growth factor receptor (EGFR) gene is amplified or mutated in 30%–50% of human gliobastoma multiforme (GBM). These mutations are associated usually with deletions of the INK4a–ARF locus, which encodes two gene products (p16^INK4a and p19^ARF) involved in cell-cycle arrest and apoptosis. We have investigated the role of EGFR mutation in gliomagenesis, using avian retroviral vectors to transfer a mutant EGFR gene to glial precursors and astrocytes in transgenic mice expressing tv-a, a gene encoding the retrovirus receptor. TVA, under control of brain cell type-specific promoters. We demonstrate that expression of a constitutively active, mutant form of EGFR in cells in the glial lineage can induce lesions with many similarities to human gliomas. These lesions occur more frequently with gene transfer to mice expressing tv-a from the progenitor-specific nestin promoter than to mice expressing tv-a from the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter, suggesting that tumors arise more efficiently from immature cells in the glial lineage. Furthermore, EGFR-induced gliomagenesis appears to require additional mutations in genes encoding proteins involved in cell-cycle arrest pathways. We have produced these combinations by simultaneously infecting tv-a transgenic mice with vectors carrying cdk4 and EGFR or by infecting tv-a transgenic mice bearing a disrupted INK4a–ARF locus with the EGFR-carrying vector alone. Moreover, EGFR-induced gliomagenesis does not occur in conjunction with p53 deficiency, unless the mice are also infected with a vector carrying cdk4. The gliomagenic combinations of genetic lesions required in mice are similar to those found in human gliomas.     

Tumor initiating cells in malignant gliomas: biology and implications for therapy

A rare subpopulation of cells within malignant gliomas, which shares canonical properties with neural stem cells (NSCs), may be integral to glial tumor development and perpetuation. These cells, also known as tumor initiating cells (TICs), have the ability to self-renew, develop into any cell in the overall tumor population (multipotency), and proliferate. A defining property of TICs is their ability to initiate new tumors in immunocompromised mice with high efficiency. Mounting evidence suggests that TICs originate from the transformation of NSCs and their progenitors. New findings show that TICs may be more resistant to chemotherapy and radiation than the bulk of tumor cells, thereby permitting recurrent tumor formation and accounting for the failure of conventional therapies. The development of new therapeutic strategies selectively targeting TICs while sparing NSCs may provide for more effective treatment of malignant gliomas.     

Regulation of platelet-derived growth factor-induced Ras signaling by poliovirus receptor Necl-5 and negative growth regulator Sprouty2

Necl-5, known as a poliovirus receptor and up-regulated in many cancer cells, enhances platelet-derived growth factor (PDGF)-induced activation of Ras-Raf-MEK-ERK signaling, but not PDGF-induced tyrosine phosphorylation of PDGF receptor, resulting in facilitation of cell proliferation. Here, we showed that Necl-5 interacted with Sprouty2, known to be a negative regulator of growth factor-induced signaling, and reduced the inhibitory effect of Sprouty2 on PDGF-induced Ras signaling. Necl-5 was reported to be down-regulated by its trans-interaction with nectin-3 upon cell-cell contact, initiating cooperative cell-cell adhesion with cadherin. This down-regulation of Necl-5 caused tyrosine phosphorylation of Sprouty2 by c-Src, which was activated by PDGF receptor in response to PDGF, and inhibited PDGF-induced Ras signaling. Thus, Necl-5 and Sprouty2 cooperatively regulate PDGF-induced Ras signaling. The roles of Necl-5 and Sprouty2 in contact inhibition for cell proliferation are also discussed.     

Platelet-derived growth factor-BB controls epithelial tumor phenotype by differential growth factor regulation in stromal cells

Platelet-derived growth factor (PDGF) stimulates tumor growth and progression by affecting tumor and stromal cells. In the HaCaT skin carcinogenesis model, transfection of immortal nontumorigenic and PDGF-receptor-negative HaCaT keratinocytes with PDGF-B induced formation of benign tumors. Here, we present potential mechanisms underlying this tumorigenic conversion. In vivo, persistent PDGF-B expression induced enhanced tumor cell proliferation but only transiently stimulated stromal cell proliferation and angiogenesis. In vitro and in vivo studies identified fibroblasts as PDGF target cells essential for mediating transient angiogenesis and persistent epithelial hyperproliferation. In fibroblast cultures, long-term PDGF-BB treatment caused an initial up-regulation of vascular endothelial growth factor (VEGF)-A, followed by a drastic VEGF down-regulation and myofibroblast differentiation. Accordingly, in HaCaT/PDGF-B transplants, initially enhanced VEGF expression by stromal fibroblasts was subsequently reduced, followed by down-regulation of angiogenesis, myofibroblast accumulation, and vessel maturation. The PDGF-induced, persistently increased expression of the hepatocyte growth factor by fibroblasts in vitro and in vivo was most probably responsible for enhanced epithelial cell proliferation and benign tumor formation. Thus, by paracrine stimulation of the stroma, PDGF-BB induced epithelial hyperproliferation, thereby promoting tumorigenicity, whereas the time-limited activation of the stroma followed by stromal maturation provides a possible explanation for the benign tumor phenotype.     

Platelet-derived growth factor-B enhances glioma angiogenesis by stimulating vascular endothelial growth factor expression in tumor endothelia and by promoting pericyte recruitment

Platelet-derived growth factor (PDGF)-B and its receptor (PDGF-R) beta are overexpressed in human gliomas and responsible for recruiting peri-endothelial cells to vessels. To establish the role of PDGF-B in glioma angiogenesis, we overexpressed PDGF-B in U87MG glioma cells. Although PDGF-B stimulated tyrosine phosphorylation of PDGF-Rbeta in U87MG cells, treatment with recombinant PDGF-B or overexpression of PDGF-B in U87MG cells had no effect on their proliferation. However, an increase of secreted PDGF-B in conditioned media of U87MG/PDGF-B cells promoted migration of endothelial cells expressing PDGF-R beta, whereas conditioned media from U87MG cells did not increase the cell migration. In mice, overexpression of PDGF-B in U87MG cells enhanced intracranial glioma formation by stimulating vascular endothelial growth factor (VEGF) expression in neovessels and by attracting vessel-associated pericytes. When PDGF-B and VEGF were overexpressed simultaneously by U87MG tumors, there was a marked increase of capillary-associated pericytes as seen in U87MG/VEGF(165)/PDGF-B gliomas. As a result of pericyte recruitment, vessels induced by VEGF in tumor vicinity migrated into the central regions of these tumors. These data suggest that PDGF-B is a paracrine factor in U87MG gliomas, and that PDGF-B enhances glioma angiogenesis, at least in part, by stimulating VEGF expression in tumor endothelia and by recruiting pericytes to neovessels.     

Platelet-derived growth factor gene delivery stimulates ex vivo gingival repair

Destruction of tooth support due to the chronic inflammatory disease periodontitis is a major cause of tooth loss. There are limitations with available treatment options to tissue engineer soft tissue periodontal defects. The exogenous application of growth factors (GFs) such as platelet-derived growth factor (PDGF) has shown promise to enhance oral and periodontal tissue regeneration. However, the topical administration of GFs has not led to clinically significant improvements in tissue regeneration because of problems in maintaining therapeutic protein levels at the defect site. The utilization of PDGF gene transfer may circumvent many of the limitations with protein delivery to soft tissue wounds. The objective of this study was to test the effect of PDGF-A and PDGF-B gene transfer to human gingival fibroblasts (HGFs) on ex vivo repair in three-dimensional collagen lattices. HGFs were transduced with adenovirus encoding PDGF-A and PDGF-B genes. Defect fill of bilayer collagen gels was measured by image analysis of cell repopulation into the gingival defects. The modulation of gene expression at the defect site and periphery was measured by RT-PCR during a 10-day time course after gene delivery. The results demonstrated that PDGF-B gene transfer stimulated potent (>4-fold) increases in cell repopulation and defect fill above that of PDGF-A and corresponding controls. PDGF-A and PDGF-B gene expression was maintained for at least 10 days. PDGF gene transfer upregulated the expression of phosphatidylinosital 3-kinase and integrin alpha5 subunit at 5 days after adenovirus transduction. These results suggest that PDGF gene transfer has potential for periodontal soft tissue-engineering applications.     

A recurrent 19q11-12 breakpoint suggested by cytogenetic and fluorescence in situ hybridization analysis of three glioblastoma cell lines

Loss of genetic material at chromosome 19 is a rather frequent finding in malignant gliomas. Loss of heterozygosity at region 19q13.3 is common in oligodendrogliomas and is also present, together with other genetic alterations on the same chomosome, in glioblastoma multiforme (GBM). Here we describe the results of cytogenetic and fluorescence in situ hybridization analysis on three GBM cell lines in which a series of complex chromosomal rearrangements affecting chromosome 19 were present. These genetic alterations suggest the presence of a common breakpoint at 19q11–12 which may point to the localization of a fragile site and/or to the presence of tumor suppressor gene(s) in the pericentromeric region of chromosome 19.     

Ink4a and Arf differentially affect cell proliferation and neural stem cell self-renewal in Bmi1-deficient mice

The Polycomb group (PcG) gene Bmi1 promotes cell proliferation and stem cell self-renewal by repressing the Ink4a/Arf locus. We used a genetic approach to investigate whether Ink4a or Arf is more critical for relaying Bmi1 function in lymphoid cells, neural progenitors, and neural stem cells. We show that Arf is a general target of Bmi1, however particularly in neural stem cells, derepression of Ink4a contributes to Bmi1(-/-) phenotypes. Additionally, we demonstrate haploinsufficient effects for the Ink4a/Arf locus downstream of Bmi1 in vivo. This suggests differential, cell type-specific roles for Ink4a versus Arf in PcG-mediated (stem) cell cycle control.     

100例中枢神经系统肿瘤的胶质纤维酸性蛋白免疫组化观察

对100例中枢神经系统肿瘤进行GFAP免疫组化观察。结果表明,GFAP主要分布于各种类型的星形细胞瘤,多形性胶质母细胞瘤,混合性胶质瘤及部分室管膜瘤。GFAP染色强度与瘤细胞分化程度有关。本文还探讨了室管膜瘤与少突胶质瘤存在GFAP问题。研究表明GFAP可作为诊断神经胶质瘤的一种有用标记物。     

Stratification of astrocytes in healthy and diseased brain

Astrocytes, a subtype of glial cells, come in variety of forms and functions. However, overarching role of these cell is in the homeostasis of the brain, be that regulation of ions, neurotransmitters, metabolism or neuronal synaptic networks. Loss of homeostasis represents the underlying cause of all brain disorders. Thus, astrocytes are likely involved in most if not all of the brain pathologies. We tabulate astroglial homeostatic functions along with pathological condition that arise from dysfunction of these glial cells. Classification of astrocytes is presented with the emphasis on evolutionary trails, morphological appearance and numerical preponderance. We note that, even though astrocytes from a variety of mammalian species share some common features, human astrocytes appear to be the largest and most complex of all astrocytes studied thus far. It is then an imperative to develop humanized models to study the role of astrocytes in brain pathologies, which is perhaps most abundantly clear in the case of glioblastoma multiforme.     

The role of astrocytes in the formation of cartilage in gliomas. An immunohistochemical study of four cases.

The occasional presence of focal cartilage in gliomas is generally attributed to metaplasia of the mesenchymal supportive elements. While this mechanism undoubtedly exists, the present report describes a different mode of development of cartilage in four gliomas occurring in young individuals. Two of the tumors were pontine astrocytomas, one was a mixed ependymoma and astrocytoma involving the fourth ventricle and the brainstem, and one was an extraspinal malignant astrocytoma in the lumbar region of a young boy who earlier had been diagnosed as having a pontine glioma for which he received radiation treatment. In all four tumors, transitions from astrocytic to cartilaginous elements were seen, characterized by an increasing deposition of chondroid ground substance between the astrocytes and a gradual morphologic changes of the glial cells to more rounded forms with a vacuolated cytoplasm, indistinguishable from chondrocytes of mesenchymal origin. Many of these cells retained positive staining for glial fibrillary acidic protein by the immunoperoxidase method, attesting to their astrocytic nature. The production of cartilage by neoplastic astrocytes may be related to their ability to secrete, in certain circumstances and occasionally in large amounts, basement membrane material and other forms of mucopolysaccharides, which may become condensed to form a chondroid ground substance. The process appears analogous to that of cartilage formation by epithelial cells in pleomorphic adenomas of the salivary glands.     

Reinduction of ErbB2 in astrocytes promotes radial glial progenitor identity in adult cerebral cortex

Radial glial cells play a critical role in the construction of mammalian brain by functioning as a source of new neurons and by providing a scaffold for radial migration of new neurons to their target locations. Radial glia transform into astrocytes at the end of embryonic development. Strategies to promote functional recovery in the injured adult brain depend on the generation of new neurons and the appropriate guidance of these neurons to where they are needed, two critical functions of radial glia. Thus, the competence to regain radial glial identity in the adult brain is of significance for the ability to promote functional repair via neurogenesis and targeted neuronal migration in the mature brain. Here we show that the in vivo induction of the tyrosine kinase receptor, ErbB2, in mature astrocytes enables a subset of them to regain radial glial identity in the mature cerebral cortex. These new radial glial progenitors are capable of giving rise to new neurons and can support neuronal migration. These studies indicate that ErbB2 signaling critically modulates the functional state of radial glia, and induction of ErbB2 in distinct adult astrocytes can promote radial glial identity in the mature cerebral cortex.     

The Potential Role of Platelet-Derived Growth Factor as an Autocrine or Paracrine Factor for Human Bone Cells

Platelet-derived growth factor, PDGF, is a potent mitogen for cells of mesenchymal origin such as fibroblasts, smooth muscle cells and glial cells. PDGF is thought to have the potential to act as both a paracrine and an autocrine factor. Studies described here extend these observations to human bone-derived cells. Exogenous PDGF induces biologic activity in two human osteogenic sarcoma cell lines and in one of these, the two PDGF genes, PDGF-1 and PDGF-2/c-sis are expressed. In addition, PDGF stimulates proliferation of normal osteoblastic cells derived from adult human cancellous bone. The expression of the PDGF-1 gene but not the PDGF-2/c-sis gene is demonstrated in normal human adult bone-derived cells by Northern blot analysis and synthesis of PDGF is shown by immunoprecipitation with PDGF antisera. These studies indicate that PDGF has the potential to act as a paracrine or autocrine regulator of bone cells.     

The potential role of platelet-derived growth factor as an autocrine or paracrine factor for human bone cells

Platelet-derived growth factor, PDGF, is a potent mitogen for cells of mesenchymal origin such as fibroblasts, smooth muscle cells and glial cells. PDGF is thought to have the potential to act as both a paracrine and an autocrine factor. Studies described here extend these observations to human bone-derived cells. Exogenous PDGF induces biologic activity in two human osteogenic sarcoma cell lines and in one of these, the two PDGF genes, PDGF-1 and PDGF-2/c-sis are expressed. In addition, PDGF stimulates proliferation of normal osteoblastic cells derived from adult human cancellous bone. The expression of the PDGF-1 gene but not the PDGF-2/c-sis gene is demonstrated in normal human adult bone-derived cells by Northern blot analysis and synthesis of PDGF is shown by immunoprecipitation with PDGF antisera. These studies indicate that PDGF has the potential to act as a paracrine or autocrine regulator of bone cells.