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

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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