Isolation of hematopoietic-specific oncogenes by a novel expression screening method

通过新型表达筛选方法分离造血特异性癌基因

• 批准号: 10670964
• 负责人: MANO Hiroyuki
• 金额: $ 1.66万
• 依托单位: Jichi Medical School
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670964/
• 关键词: oncogene; leukemia; retrovirus; レトロウィルス; レトロウイルス

Identification of transforming genes has been largely relied on the transformation assay in mouse 3T3 fibroblast cells with the genomic DNA isolated from various cancer specimens. Although these studies have revealed the activation of ras, raf and myc oncogenes, they have been hampered by the fact that this kind of assay only allows the detection of genes, expression of which is active in 3T3 fibroblasts. Therefore, oncogenes in hematopoietic malignancies may not be transcriptionally active in 3T3 fibroblasts, and can not be identified in these types of assay.To overcome this limit, we have developed a novel screening method using mouse interleukin-3 (IL-3)-dependent BA/F3 cells and retrovirus. mRNAs were isolated from the bone marrow mononuclear cells in a patient with acute lymphoid leukemia (ALL), and converted into double-stranded cDNAs, which were subsequently ligated into pMX retrovirus vector in a uni-direction. With a help of BOSC23 packaging cell line, we could obtain a retroviral stock with a titer exceeding lx 10ィイD16ィエD1/mI. BAIF3 cells were infected with this retrovirus under the presence of engineered fibronectin (Takara Shuzo, Shiga, Japan) with an infection efficiency 【greater than or equal】90%.After the infection of retrovirus stock, BA/F3 cells were cultured in the absence of IL-3, generating a few IL-3-independent clones. Expression of leukemia-derived transforming genes were expected to render these cells factor-independency. Recovery of retroviral inserts from these transformed cells have identified a few cDNAs which can encode novel peptides. Studies are currently in progress to characterize the nature of these putative candidates of novel ALL oncogenes.

转化基因的鉴定在很大程度上依赖于小鼠3 T3成纤维细胞中的转化试验，该试验使用从各种癌症标本中分离的基因组DNA。尽管这些研究已经揭示了ras、raf和myc癌基因的激活，但是它们受到这样的事实的阻碍，即这种测定仅允许检测在3 T3成纤维细胞中表达有活性的基因。因此，在造血系统恶性肿瘤的癌基因可能不是转录活性在3 T3成纤维细胞，并不能在这些类型的assay.To克服这一限制，我们已经开发了一种新的筛选方法，使用小鼠白细胞介素-3（IL-3）依赖的BA/F3细胞和逆转录病毒。从1例急性淋巴细胞白血病（ALL）患者的骨髓单个核细胞中分离mRNA，转化为双链cDNA，然后将其单向连接到pMX逆转录病毒载体中。利用BOSC 23包装细胞系，我们可以获得滴度超过1 × 10 ~（-16）KD ~（-1）/ml的逆转录病毒储备液。在工程纤连蛋白（Takara Shuzo，滋贺，日本）存在下，用该逆转录病毒感染BAIF 3细胞，感染效率[大于或等于] 90%。感染逆转录病毒原液后，在不存在IL-3的情况下培养BA/F3细胞，产生一些IL-3非依赖性克隆。白血病来源的转化基因的表达预期使这些细胞因子独立性。从这些转化细胞中回收的逆转录病毒插入片段已经鉴定出一些可以编码新肽的cDNA。目前正在进行研究，以表征这些新ALL癌基因的推定候选者的性质。

项目成果

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Kitanaka,A.et al.：“人 B 细胞中非受体酪氨酸激酶 Tec 的表达和激活”血液。

Mano,J.et al.: "Tec/Bmx non-receptor tyrosine kinases are involved in regulation of Rho and serum response factor by Gα12/13" EMBO J.17. 5638-5646 (1998)

Mano, J. 等人：“Tec/Bmx 非受体酪氨酸激酶参与 Gα12/13 对 Rho 和血清反应因子的调节”EMBO J.17 (1998)。

Ohya, K. et al.: "Molecular cloning of a docking protein,BRDG1,that acts downstream of the Tec tyrosine kinase"Proc.Natl.Acad.Sci.USA. 96. 11976-11981 (1999)

Ohya, K. 等人：“作用于 Tec 酪氨酸激酶下游的对接蛋白 BRDG1 的分子克隆”Proc.Natl.Acad.Sci.USA。

KUME, A., et al.: "A G-CSF receptor-gyrase B fusion gene: A new type of molecular switch for expansion of genetically modified hematopoietic cells."Biochem Biophys Res Commun. 260. 9-12 (1999)

KUME, A. 等人：“G-CSF 受体-促旋酶 B 融合基因：用于扩增转基因造血细胞的新型分子开关。”Biochem Biophys Res Commun。

Yoshida,K., Yamashita,Y., Miyazato,A., Ohya,K., Kitanaka,A., Ikeda,U., Shimada,K., Yamanaka,T., Ozawa,K. and Mano,H.: "Mediation by the protein tyrosine kinase Tec of signaling between the B cell antigen receptor and Dok-1."J. Biol. Chem.. (in press).

吉田，K.，山下，Y.，宫里，A.，Ohya，K.，北中，A.，池田，U.，岛田，K.，山中，T.，小泽，K.