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ABSTRACT: We have shown previously that the multifunctional cytokine scatter factor/hepatocyte growth factor (SF/HGF) is elevated in human malignant gliomas. In this study we investigated how human SF/HGF expression affects the malignancy of the U373 human glioblastoma cell line in vivo and in vitro. Human SF/HGF gene transfer increased U373 glioblastoma tumorigenicity by > or = 20-fold and enhanced the growth rate of intracerebral U373 xenografts by 3- to 8-fold. SF/HGF expression had no effect on the proliferation of glioblastoma cell monolayers but increased their anchorage-independent colony formation in soft agar by 5- to 8-fold. These results are the first to show that SF/HGF expression by human glioblastoma cells enhances their growth dysregulation in vitro and malignancy in vivo.
Biochemical and Biophysical Research Communications 07/1997; 235(3):743-7. · 2.48 Impact Factor
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ABSTRACT: Scatter factor (SF), also known as hepatocyte growth factor, is angiogenic in systemic tissue, and SF titers correlate with the malignancy and metastatic phenotype of certain systemic cancers. Human gliomas express SF and its receptor c-met, but their role in the malignant progression of these tumors has not been defined. To examine this, 9L glioma cells that express c-met but not SF were transfected with human SF cDNA, and their behavior in vitro and in vivo was examined. SF gene expression was detected in conditioned medium of 9L-SF but not in control 9L-neo-transfected cell lines, by reverse transcriptase-PCR, immunoblot, ELISA, and scatter activity assays. Gliomas derived from 9L-SF and control 9L-neo cell lines implanted in the caudate/putamen of Fisher 344 rats (intracranially) and in the flanks of SCID/Beige mice (subcutaneously) were examined. Extracts from intracranial (i.c.) gliomas contained elevated levels of SF protein as determined by ELISA (1 to 5.5 ng SF/mg protein), whereas no SF was detected in control tumors. Reverse transcriptase-PCR of RNA from i.c. gliomas revealed that only 9L-SF gliomas expressed SF and both 9L-neo and 9L-SF gliomas expressed the c-met SF receptor. By postimplantation Day 14, 9L-SF i.c. gliomas were approximately 5-fold larger than 9L-neo control tumors (p < 0.001). Subcutaneous 9L-SF glioma growth was also greater than that in controls, although the differences were more variable. SF-producing i.c. gliomas contained elevated levels of 48-kd urokinase (3.5-fold) and 92-kd type IV collagenase (2.8-fold), both enzymes that correlate with the malignant progression of human gliomas (p < 0.001). SF-producing and control 9L cell lines did not differ in rates of proliferation, thymidine incorporation, or adhesion-independent growth in vitro. Conditioned medium from 9L-SF cells stimulated thymidine incorporation into microvessel brain endothelial cells 3- to 4-fold higher than did CM from 9L-neo controls (p < 0.001). Intracranial 9L-SF gliomas were more angiogenic than controls based on elevated peak (2.25-fold; p < 0.005) and mean (1.7-fold; p < 0.008) blood vessel densities. These results suggest that SF production by glioma cells enhances glioma malignancy in vivo, in part, by paracrine mechanisms involving glioma-associated angiogenesis.
Laboratory Investigation 04/1997; 76(4):565-77. · 3.64 Impact Factor
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ABSTRACT: Fibroblast growth factor (FGF) is an endothelial cell mitogen and serves as a mitogen and/or differentiating factor that can be neuroprotective for other cell types within the CNS. We established brain microvascular endothelial cell lines that secrete FGF-1 with the ultimate goal of examining their usefulness as a cellular platform for FGF gene delivery to brain. A chimeric gene consisting of the secretory sequence of FGF-4 linked at the 5' end of human FGF-1 (sp-hst/KS3:FGF-1) was transfected into rat microvascular endothelial cells previously altered to express the lacZ reporter gene (RBEZ), and numerous clones were found to secrete FGF-1 (RBEZ-FGF). Immunoblotting of conditioned medium demonstrated an 18-kDa protein corresponding to FGF-1. Conditioned medium from RBEZ-FGF cells enhanced [3H]thymidine incorporation in BALB/c3T3 fibroblasts by up to sevenfold when compared with conditioned medium of control cell lines, corresponding to as much as 110 ng of active FGF-1/mg of cell protein/24 h. RBEZ-FGF cell lines remained contact-inhibited and proliferated independent of exogenous endothelial mitogens, in contrast to control lines that are mitogen-dependent. Incubation of PC12 cells with RBEZ-FGF cells or their conditioned medium induced neurite outgrowth by PC12 cells. RBEZ-FGF cells survived following implantation to neonatal and adult rat caudate-putamen for at least 21 days based on 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-gal) histochemistry, and FGF-1 gene expression by these cells in vivo was demonstrated by in situ hybridization and reverse transcriptase-PCR. These findings suggest that endothelial cells may be useful for FGF gene delivery to the CNS.
Journal of Neurochemistry 11/1996; 67(4):1643-52. · 4.06 Impact Factor
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ABSTRACT: Malignant brain neoplasms present great therapeutic challenges due to their extremely aggressive behavior and relative isolation by the blood-brain and blood-tumor barriers. Endothelial cells may be versatile platforms for delivering genes to solid tumors by virtue of their location at blood-tissue interfaces and their proliferation in response to endothelial mitogens produced by tumors. Immortalized rat brain endothelial cells that express the E. coli lacZ reporter gene and the gene for murine interleukin-2 (RBEZ-IL2) were co-inoculated with 9L glioma cells to Fisher rats to examine the effects of endothelial cell-based cytokine delivery on glioma growth in vivo. 9L glioma growth was not affected by the implantation of control RBEZ cells. The growth of subcutaneous and intracranial 9L gliomas was significantly inhibited by RBEZ-IL2 cells (P < 0.005 and P < 0.01, respectively) when compared to control transfected RBEZ cells. Rats receiving intracranial 9L glioma cells with RBEZ-IL2 cells showed increased survival (P < 0.001). Histologic and immunohistologic analysis showed enhanced activation of microglia/macrophages and CD8-positive T lymphocytes and/or natural killer cells within brain at sites of 9L inoculation with RBEZ-IL2 cells. This report establishes that immortalized endothelial cells can be used for cytokine gene delivery and to activate anti-tumor host responses to experimental gliomas within the central nervous system.
Brain Research 09/1996; 731(1-2):161-70. · 2.73 Impact Factor
