The tyrosine kinase src plays a crucial role in leukemic cell proliferation in AML

Abstract

10531 Background: Src-family tyrosine kinases are known to be involved in signal transduction pathways of growth factors and cytokines in hematopoetic cells. While the role of other src family members has been studied widely, only few data exist about c-src in leukemia. The actual study was performed to analyze src mutations in leukemic cells and to determine the role of pp60src in leukemic cell proliferation. Methods: AML cell lines and primary samples were analyzed for expression and activation of src by RT-PCR and Western blot analyses. Mutational analyses were performed by sequencing of C-terminal cDNA from 60 AML samples. The effects of src inhibition were studied by src-specific inhibitors PP1 and PP2 or by siRNA transfection. Effetcs of src inhibition were monitored in proliferation assays and analyzes of signalling through Erk1/2 and apoptosis by annexin V staining and DNA laddering. Results: In all 60 patients analyzed expression of c-src mRNA was detected by RT-PCR. Western blot analyses confirmed strong expression of src on the protein level and revealed a robust activation of the protein as determined by tyrosine phosphorylation. Incubation of leukemic cells with PP1 and PP2 caused significant inhibition of proliferation in a dose dependent manner. Similar results were observed after transfection with specific siRNAs. Src inhibition blocked phosphorylation of pp60src, Erk1/2 and induced apoptosis in leukemic cells. Mutational analyses as performed by SSCP/heteroduplex and bi-directionally sequencing revealed wildtype sequence in cell lines and 60 clinical samples. Conclusions: In summary, pp60src is highly expressed and activated in cell lines and clinical samples of human AML. Moreover, phosphorylation of src is essential for leukemic cell proliferation. Underlying mutations in the coding sequence of c-src causing constitutive activation could be excluded. These data suggest that pp60src plays a crucial role in AML and src inhibition by targeted therapy might offer a useful new approach in the treatment of AML. No significant financial relationships to disclose.