がん遺伝子の単離とヒトがんにおける異常の解明

• Project/Area Number: 06283207
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas (A)
• Allocation Type: Single-year Grants
• Research Institution: The University of Tokyo
• Principal Investigator: 渋谷 正史 東京大学, 医科学研究所, 教授 (10107427)
• Co-Investigator(Kenkyū-buntansha): 矢花 直幸 東京大学, 医科学研究所, 助手 (90292846) ; 丸 義朗 東京大学, 医科学研究所, 助教授 (00251447) ; 後藤 典子 東京大学, 医科学研究所, 助手 (10251448) ; 松七五三 仁 東京大学, 医科学研究所, 助教授 (50251442)
• Project Period (FY): 1999
• Project Status: Completed (Fiscal Year 1999)
• Budget Amount: ¥186,000,000 (Direct Cost: ¥186,000,000); Fiscal Year 1999: ¥41,000,000 (Direct Cost: ¥41,000,000); Fiscal Year 1998: ¥40,000,000 (Direct Cost: ¥40,000,000); Fiscal Year 1997: ¥39,000,000 (Direct Cost: ¥39,000,000); Fiscal Year 1996: ¥30,000,000 (Direct Cost: ¥30,000,000); Fiscal Year 1995: ¥18,000,000 (Direct Cost: ¥18,000,000); Fiscal Year 1994: ¥18,000,000 (Direct Cost: ¥18,000,000)
• Keywords: 腫瘍血管 / 血管透過性 / VEGF / 腫瘍プロモーター / チロシンキナーゼ / シグナル伝達 / C-キナーゼ / KDR / がん関連遺伝子 / 受容体型チロシンキナーゼ / Flt / EGFR

Research Abstract

腫瘍血管形成や血管透過性亢進に関与すると考えられるVEGF(血管内皮増殖因子)およびその受容体(Flt-1,KDRキナーゼ)に関して分子生物学的、細胞生物学的解析を行い、本年度は以下の結果を得た。
(1)VEGFの遺伝子発現誘導物質のひとつとして、フォルボールエステル(TPA)が知られているが、他の腫瘍プロモーターが同様の活性をもつかは明らかでない。そこでnon-TPAタイプの腫瘍プロモーターによるVEGFの遺伝子発現調節作用を調べ、プロテインフォスファターゼ阻害剤Okadaic acidがVEGF mRNAレベルの上昇を引き起こすことをはじめて見出した。この作用はOkadaic acidのもつ2つの活性、プロテインフォスファターゼ1および2Aの阻害活性のうち後者に良い相関が見られたPKC阻害剤、TNF-alpha中和抗体で抑制できなかったことから、TPAとは異なるシグナル伝達によるものと考えられた。
(2)受容体KDRのシグナル伝達を初代培養血管内皮細胞およびKDR発現NIH3T3細胞をもちいて詳しく解析した。KDRはPDGF受容体と構造類似性を示すが、キナーゼ挿入配列の自己リン酸化モチーフが異なりシグナル伝達にかなりの違いが予想されていた。事実、Rasの活性化はほとんど認められず、PLC-gammaからPKCの活性化、Raf-MEK-MAPキナーゼの活性化が主なDNA合成経路であった。すなわち、ドミナントネガティブRasではVEGFによるDNA合成は抑制できず、PKC阻害剤で有効に抑制できた。これにより内皮細胞増殖の特徴のひとつが明らかとなった。

Research Products

• [Publications] Takakhashi, T: "VEGF activates Protein kinase C-dependent, but Ras-independent Raf-MEK-MAP kinase pathway for DNA synt"Oncogene. 18. 2221-2230 (1999)
• [Publications] Shibuya, M: "Structure and function of VEGF Receptor-1 and -2. in Curr."Topics Microbiol. Immunol.. 237. 59-83 (1999)
• [Publications] Takeda, N: "The BCR-ABL oncoprotein potentially interacts with the xeroderma pigmentosum group B protein"Proc. Natl. Acad. Sci.. 96. 203-207 (1999)
• [Publications] Maru, Y: "BCR binds to the Xeroderma Pigmentosum group B protein"Biochem. Biophys. Res Commun.. 260. 309-312 (1999)
• [Publications] Yoshiji, H: "Protein kinase C lies on the signaling pathwy for vascular endothelia growth factor-mediated tumor"Cancer Res.. 59. 4413-4418 (1999)
• [Publications] Wakiya, K: "Okadaic acid stimulates the expression of vascular endothelial growth gene"Biochem. Biophys. Res Commun.. 265. 584-588 (1999)
• [Publications] Maru,Y.: "v-Ras cooperates with integrin to induce tubulogenesis in sinusoidal endothelial cell line." J.Cell.Physiol.176. 223-234 (1998)
• [Publications] Luo,J.-C.: "Significant expression of vascular endthelial growth factor/vascular permeability factor in mouse ascites." Cancer Res.58. 2652-2660 (1998)
• [Publications] Hiratsuka,S.: "Flt-1 lacking the tyrosine kinase domain is sufficient for normal development and angiogenesis in mice." Proc.Natl.Acad.Sci.USA. 95. 9349-9354 (1998)
• [Publications] Olofsson,B.: "VEGF-B binds to VEGFR-1 and regulates plasminogen activator activity in endothelial cells." Proc.Natl.Acad.Sci.USA. 95. 11709-11714 (1998)
• [Publications] Toyoda,M.: "Involvement of MAP kinase-independent protein kinase C signaling pathway in the EGF-induced p21(WAF1/Cip1)" Biochem.Biophys.Res.Commun.250. 430-435 (1998)
• [Publications] Ogawa,S.: "A novel type of vascular endothelial growth factor ; VEGF-E (NZ-7 VEGF) preferentially utilizes KDR/Flk-1" J.Biol.Chem.273. 31273-31282 (1998)
• [Publications] Gotoh,N.: "Tyrosine phosphorylation sites of Shc,239/240 involved in EGF-induced mitotic signaling distinct..." Mol.Cell.Biol.17. 1824-1831 (1997)
• [Publications] Takahashi,T.: "The 230kDa mature form of KDR/Flk-1(VEGF receptor-2)activates the RLCg pathway and partially induceds..." Oncogene. 14. 2079-2089 (1997)
• [Publications] Tanaka,K.: "Characterization of the extracellular domain in the Vascular Endothelial Growth Factor Receptor-1..." Jpn.J.Cancer Res.88. 867-876 (1997)
• [Publications] Yoshiji,H.: "Enhancement of tumor growth and vascular endothelial growth factor expression in rat mammary carcinoma..." Cancer Res.(in press). (1997)
• [Publications] Sawano,A.: "The phosphorylated 1169-tyrosine containing region of the Flt-1 kinase(VEGFR-1)is a mojor binding..." Biochem.Biophys.Res.Commun.238. 487-491 (1997)
• [Publications] Maru,Y.: "Flt-1,a receptor for vascular endothelial growth factor,has transforming and morphogenic potentials..." Oncogene. (in press). (1997)
• [Publications] Sawano,A.: "Flt-1 but not KDR/Flk-1 tyrosine kinase is a receptor for Placeta Growth Factor (PIGF),which is related…" Cell Growth and Differentiation. 7. 213-221 (1996)
• [Publications] Ikeda,T.: "Characterization of the promoter region for flt-1 tyrosine kinase gene,a receptor for Vascular…" Growth Factors. 13. 151-162 (1996)
• [Publications] Yoshiji,H.: "Expression of Vascular Endothelial Growth Factor,its receptor and other angiogenic factors in human…" Cancer Res.56. 2013-2016 (1996)
• [Publications] Maru,Y.: "The dimerization property of glutathione S-transferase partially reactivates BCR-ABL lacking the…" J.Biol.Chem.271. 15353-15357 (1996)
• [Publications] Gotoh,N.: "A novel pathway from phosphorylation of tyrosine residues 239/240 of Shc,contributing to…" EMBO J.15. 6197-6204 (1996)
• [Publications] Wakiya,K.: "A cyclic AMP response element and an ETS motif are involved in the transcriptional regulation of…" J.Biol.Chem.271. 30823-30828 (1996)
• [Publications] Seetharam,L.: "A unique signal transduction from FLT tyrosine kinase,a receptor for vascular endothelial growth factor VEGF." Oncogene. 10. 135-147 (1995)
• [Publications] Shibuya,M: "Role of VEGF-Flt receptor system in normal and tumor angiogenesis." Adv.Cancer Res.67. 281-316 (1995)
• [Publications] Maru,Y.: "Tyrosine phosphorylation of BCR by FPS/FES protein-tyrosine kinases induces association on BCR with GRB2/SOS" Mol.Cell.Biol.15. 835-842 (1995)
• [Publications] Cohen,T.: "VEGF_<121>,a VEGF isoform lacking heparin binding ability,requires cell surface heparin-sulfates for effecient・・・" J.Biol.Chem.270. 11322-11326 (1995)
• [Publications] Sato,K.: "Stimulation by TSH and Graves′IgG of vascular endothelial growth factor mRNA expression in human thyroid・・・" J.Clin.Invest.96. 1295-1302 (1995)
• [Publications] Sawano,A.: "Flt-1 but not KDR/Flk-1 tyrosine kinase is a receptor for Placenta Grtowth Factor(PIGF),which is related・・・21GC06:Cell Growth and Differentiation" (in press). (1995)
• [Publications] Gotoh,N.et al.: "Epidermal growth factor receptor-mutant lacking---" Proc.Natl.Acad.USA. 91. 167-171 (1994)
• [Publications] Tanaka,S.et al.: "C3G,a guanine nucleotide-releasing protein expressed" Proc.Natl.Acad.USA. 91. 3443-3447 (1994)
• [Publications] Yamane,A.et al.: "A new communication system between hepatocytes and" Oncogene. 9. 2683-2690 (1994)
• [Publications] Kido,M.et al.: "Allel loss on chromosome 17 in ovarian cystic terato." Jpn.J.Clin.Oncol.24. 181-185 (1994)
• [Publications] Waltenberger,J.: "Different signal transduction properties of KDR and" J.Biol.Chem.269. 26988-26995 (1994)
• [Publications] Seetharam,L.et al.: "A unique signal transduction from FLT tyrosine" Oncogene. (in press).