Hallek M, Danhauser-Riedl S, Herbst R, Warmuth M, Winkler A, Kolb HJ, Druker B, Griffin JD, Emmerich B, Ullrich A

The chimaeric bcr/abl oncogene is detected in virtually all cases of chronic myelogenous leukaemia (CML). It encodes a constitutively active tyrosine kinase of 210 kDalton, p210bcr/abl, which stimulates a variety of cytosolic signalling intermediates. The effects of bcr/abl on the activity of growth factor receptors are less well known. In ...

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order to investigate interaction of p210bcr/abl with the receptor tyrosine kinase p145c-kit, we used two myeloid, factor-dependent cell lines, MO7 and 32D, to generate bcr/abl positive sublines, MO7p210 and 32Dp210, by transfection with the bcr/abl gene. Since 32D and 32Dp210 cells did not express p145c-kit, a c-kit retrovirus was used to generate c-kit positive cell lines (32Dkit, 32Dp210kit). In contrast to MO7 and 32Dkit cells, MO7p210 and 32Dp210kit cells were factor independent and did not respond to the growth-promoting effects of recombinant human Steel factor (rhSF). Preincubation with a monoclonal antibody (MAb) neutralizing the binding of SF to p145c-kit did not affect the growth of MO7p210 cells, thus eliminating the possibility of an autocrine SF secretion. 32Dkit cells transfected with bcr/abl containing an inactivating point mutation (Lys-->Arg271) in the Abl kinase domain (32Dp210(Arg271)kit) retained their responsiveness to the effects of rhSF. Immune complex kinase assays showed that the kinase activity of p145c-kit was several-fold higher in MO7p210 and 32Dp210kit cells than in MO7, 32Dkit and 32Dp210(Arg271)kit cells, suggesting that Abl kinase activity was necessary to activate p145c-kit. Co-immunoprecipitation experiments with anti-Kit and anti-Abl MAbs demonstrated that p145c-kit and p210bcr/abl were associated in an intracellular complex in human bcr/abl positive, c-kit positive cell lines (MO7p210; GM/SO). Finally, colony assays with bone marrow from bcr/abl positive CML patients showed that the haemopoietic progenitors of three of four patients did not respond to rhSF. Taken together, the results suggest that p145c-kit can be activated by p210bcr/abl via an Abl-kinase dependent mechanism involving the complex formation of both proteins. These findings could explain some clinical features (basophilia, increase of immature myeloid cells) of chronic-phase CML.

Date: Jul. 01, 1996

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