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Adhesion-dependent growth of ATL cells ; significance of signaling pathway through adhesion molecules

ATL细胞的粘附依赖性生长；

基本信息

批准号：
12670992
负责人：
NAGAI Kazuhiro
金额：
$ 2.43万
依托单位：
University of Nagasaki
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670992/
关键词：
Adult T-cell leukemia leukemic progenitor cell adhesion molecule signal transduction

项目摘要

HTLV-I-transformed T cell lines and clinical samples of ATL cells were inoculated at various concentrations into the liquid culture which had a feeder layer of murine marrow stromal cell line, MS-5 cells (Kirin Brewery co. ltd., Japan). ATL cells grew in close contact wiih MS-5 layer and formed so-called "cobblestone areas (CA)" without any exogenous cytokines. Not only IL-2-independent cell lines (MT-1, MT-2, and HUT-102), but also some IL-2-dependent cell lines (ST-1, and KOB) showed similar growth pattern without IL-2. Contact-inhibition by insertion of a transwell membrane in the culture dishes resulted in no growth of these cell lines. In clinical samples, eight of ten (80.0 %) cases of acute ATL cells showed CA formation from day 10 to day 14, whereas two samples of chronic and one of smoldering ATL did not. Morphology and immunophenotyping indicated that CA-composing cells were originated from ATL cells. Analysis of prdviral integration pattern by southern blot hybridization sho … More wed that CA-composing cells were identical clone with primary ATL cells. Results of in situ hybridization using oligo-DNA probe complementary to the Tax mRNA revealed in two cases tested down-regulation of the expression of Tax gene of CA-composing ATL cells compared with the growing cells in liquid culture with IL-2. The production of p40tax and p19gag was decreased in CA cells by immunostaining in five cases. Semi-solid collagen gel culture was employed to assess clonal cell growth on MS-5 layer in three cases, and there was a linear relationship between inoculated cell number and CA counts. In seven cases, the frequency of clonogenic CA-forming ATL cells was ; 0.03-1.04 % (mean ; 0.22 %). Immunostaining of phospholyrated p44/p42 MAP kinase in CA-composing cells indicated that, in CA-composing ATL cells, MAP kinase signaling pathway might be activated constitutively. Although some adhesion molecules (I.e. VLA-4, LFA-1 etc.) were blocked by specific monoclonal antibodies, none of them could inhibit CA formation of ATL cells. Thus, for the present, one possibility is that there might be novel adhesion molecules which control this adhesion-dependent growth of ATL cells. Using our newly established co-culture system which provides the adhesion-dependent growth of primary acute ATL cells without HTLV-I-related proteins, as in vivo ATL cells, further investigation concerning significance of Ras/MAPK pathway and adhesion molecules is now in progress. Less

将HTLV-1转化的T细胞系和ATL细胞的临床样品以各种浓度接种到液体培养物中，所述液体培养物具有鼠骨髓基质细胞系MS-5细胞的饲养层（Kirin Brewery有限公司，日本）。ATL细胞与MS-5细胞层紧密接触生长，在无外源性细胞因子的情况下形成所谓的“鹅卵石区（cobblestone area，CA）”。不仅IL-2非依赖性细胞系（MT-1、MT-2和HUT-102），而且一些IL-2依赖性细胞系（ST-1和KOB）在没有IL-2的情况下显示出相似的生长模式。通过在培养皿中插入transwell膜的接触抑制导致这些细胞系不生长。在临床样本中，10例急性ATL细胞中有8例（80.0%）从第10天到第14天显示CA形成，而2例慢性ATL和1例阴燃ATL样本则没有。形态学和免疫表型分析表明CA合成细胞来源于ATL细胞。应用Southern印迹杂交技术分析prd病毒整合模式 ...更多信息证明CA合成细胞与原代ATL细胞为同一克隆。原位杂交的结果，使用寡核苷酸探针互补的Tax mRNA显示在两种情况下测试的下调的Tax基因的表达CA组成的ATL细胞相比，在液体培养中与IL-2的生长细胞。5例CA细胞p40 tax和p19 gag表达减少。采用半固体胶原凝胶培养法检测3例MS-5细胞上的克隆细胞生长情况，接种细胞数与CA计数呈线性关系。在7例病例中，克隆性CA形成ATL细胞的频率为0.03- 1.04%（平均0.22%）。磷酸化p44/p42 MAP激酶在CA合成细胞中的免疫染色表明，在CA合成ATL细胞中，MAP激酶信号通路可能被组成性激活。虽然一些粘附分子（即VLA-4、LFA-1等）但均不能抑制ATL细胞CA的形成。因此，就目前而言，一种可能性是可能存在控制ATL细胞的这种粘附依赖性生长的新型粘附分子。使用我们新建立的共培养系统，该系统提供了原代急性ATL细胞的粘附依赖性生长，而没有HTLV-I相关蛋白，作为体内ATL细胞，关于Ras/MAPK途径和粘附分子的意义的进一步研究正在进行中。少