ABSTRACT
In order to clarify the role of HMW FGF-2 in glioma development and angiogenesis, we over-expressed different human FGF-2 isoforms in C6 rat glioma cell line using a tetracycline-regulated expression system. Phenotypic modifications were analyzed in vitro and compared to untransfected cells or to cells over-expressing 18 kDa FGF-2 or all FGF-2 isoforms. In particular, we demonstrate that HMW FGF-2 has unique features in inhibiting glioma cell proliferation. HMW FGF-2 expressing cells showed a cell-cycle arrest at the G2M, demonstrating a role of HMW FGF-2 in controlling the entry in mitosis. Moreover, hydroxyurea was ineffective in blocking cells at the G1S boundary when HMW FGF-2 was expressed. We also show that the HMW FGF-2 isoforms inhibit 4E-BP1 phosphorylation at critical sites restoring the translation inhibitory activity of 4E-BP1. In vivo, inhibition of tumor growth was observed when cells expressed HMW FGF-2. This indicates that HMW FGF-2 inhibits tumor growth in glioma cells by acting on cell-cycle progression and protein translation.
Subject(s)
Cell Cycle/genetics , Cell Proliferation , Fibroblast Growth Factor 2/genetics , Glioma/genetics , Protein Biosynthesis/genetics , Animals , Cell Cycle/physiology , Cell Line, Tumor , Down-Regulation , Glioma/metabolism , Glioma/pathology , Humans , Mice , Mice, SCID , Molecular Weight , Protein Isoforms/genetics , Rats , Transfection , Tumor Burden/genetics , Xenograft Model Antitumor AssaysABSTRACT
UNLABELLED: Using a proteomic analysis of human hepatocellular carcinoma (HCC), we identified the overexpression in 4 tumors of RuvB-like 2 (RUVBL2), an ATPase and putative DNA helicase known to interact with beta-catenin and cellular v-myc myelocytomatosis viral oncogene homolog (c-myc). RUVBL2 expression was further analyzed in tumors with quantitative reverse-transcription polymerase chain reaction analysis and immunohistochemistry; in addition, RUVBL2 expression in a HuH7 cell line was silenced by small interfering RNA or increased with a lentiviral vector. RUVBL2 messenger RNA overexpression was confirmed in 72 of 96 HCC cases, and it was associated with poorly differentiated tumors (P = 0.02) and a poor prognosis (P = 0.02) but not with beta-catenin mutations or c-myc levels. Although RUVBL2 was strictly nuclear in normal hepatocytes, tumoral hepatocytes exhibited additional cytoplasmic staining. There was no mutation in the coding sequence of RUVBL2 in 10 sequenced cases. Silencing RUVBL2 in HuH7 HCC cells reduced cell growth (P < 0.001) and increased apoptosis, as shown by DNA fragmentation (P < 0.001) and caspase 3 activity (P < 0.005). This was associated with an increased expression of several proapoptotic genes and with an increased conformational activation of Bak-1 and Bax. On the other hand, HuH7 cells with an overexpression of RUVBL2 grew better in soft agar (P < 0.03), had increased resistance to C2 ceramide-induced apoptosis (P < 0.001), and gave rise to significantly larger tumors when injected into immunodeficient Rag2/gammac mice (P = 0.016). CONCLUSION: RUVBL2 is overexpressed in a large majority of HCCs. RUVBL2 overexpression enhances tumorigenicity, and RUVBL2 is required for tumor cell viability. These results argue for a major role of RUVBL2 in liver carcinogenesis.
Subject(s)
Adenosine Triphosphatases/metabolism , Carcinoma, Hepatocellular/metabolism , Carrier Proteins/metabolism , DNA Helicases/metabolism , Liver Neoplasms/metabolism , ATPases Associated with Diverse Cellular Activities , Adenosine Triphosphatases/genetics , Aged , Aged, 80 and over , Animals , Apoptosis/genetics , Apoptosis/physiology , Carcinoma, Hepatocellular/etiology , Carcinoma, Hepatocellular/pathology , Carrier Proteins/genetics , Caspase 3/genetics , Caspase 3/metabolism , Cell Line, Tumor , Cell Proliferation , DNA Fragmentation , DNA Helicases/genetics , Female , Gene Expression Regulation, Neoplastic , Humans , Liver Neoplasms/etiology , Liver Neoplasms/pathology , Male , Mice , Mice, Inbred Strains , Middle Aged , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transplantation, HeterologousABSTRACT
Tumor angiogenesis is a fast growing sub-domain of angiogenesis research and tumor biology. Basic mechanisms have been unraveled and many key players identified. For many years, tumor vascularization was explained solely by the ingrowth of new vessels into the tumor from preexisting one's. However, in recent years, additional mechanisms have been recognized. These include angioblasts recruitment, cooption, vasculogenic mimicry and mosaic vessels. These different mechanisms may exist concomitantly in the same tumor or may be selectively involved in a specific tumor type or host environment. In this article, we will review, in depth, these different mechanisms and also discuss some aspects of anti-angiogenic tumor therapy.
Subject(s)
Neoplasms/blood supply , Neovascularization, Pathologic/physiopathology , Angiogenesis Inhibitors/therapeutic use , Animals , Antineoplastic Agents/therapeutic use , Humans , Neoplasms/drug therapy , Neovascularization, Pathologic/drug therapyABSTRACT
We have investigated growth and vascularization of malignant glioma in mice upon conditional inhibition of fibroblast growth factor (FGF) receptor activity. C6 rat glioma cells were transfected with a dominant-negative fibroblast growth factor receptor-2 (FGFR2-DN) cDNA under the control of a tetracycline-regulated expression promoter (tet off) and implanted in the brain of immunodeficient mice. Magnetic resonance imaging analysis showed a significant decrease in tumor growth 14 days after implantation when FGFR2-DN was expressed compared to control. This size difference disappeared after 20 days. However, after 20 days, tumor and endothelial cells apoptosis were higher in the FGFR2-DN group and consequently angiogenesis was decreased whereas tumor cells were similarly associated with blood vessels at the tumor periphery. Pericyte coverage was not different between the two groups but a higher amount of pericytes not associated with vessels was found in the FGFR2-DN expressing group. This demonstrates, that conditional expression of inhibitor of FGF receptor activity in gliomas implanted in the brain of immunodeficient mice can be achieved efficiently, and that FGFs are major players in glioma development and in glioma angiogenesis.
