The Tyrosine Kinase src Is Highly Activated in AML and Plays a Crucial Role in Leukemic Cell Proliferation.

Abstract

The tyrosine kinase pp60src is involved in several signal transduction pathways such as signalling of hematopoetic growth factors and cytokines. The viral form v-src was the first oncogene described and mutations of regulatory tyrosine residues in cellular src (c-src) have been linked to malignant transformation. However, no mutations in the gene of c-src have been described in leukemia so far, although some data of src mutations in solid tumors have been reported. The current study was undertaken to examine the role of src in acute myeloid leukemia (AML).Blood and bone marrow specimen of patients with newly diagnosed or recurrent AML treated at our institution were sampled. AML cell lines or CD34 positive cells of healthy donors served as positive and negative controls, respectively. RNA was isolated, and RT-PCR was performed using 4 different primer pairs spanning the coding region of c-src. Protein expression and phosphorylation was studied after protein extraction and Western blot analyses using src and phospho-src specific antibodies. The effect of src inhibitors PP1 and PP2 on leukemic cell proliferation was studied in human and murine cell lines. Mutational analyses of the coding region were performed using SSCP/heteroduplex and bi-directionally sequencing.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. Inhibition of src phosphorylation by src-specific inhibitors PP1 and PP2 was detected by Western blot using an antibody specific for phospho-src. Incubation of leukemic cells with PP1 and PP2 caused significant inhibition of proliferation in a dose dependent manner. Mutational analyses as performed by SSCP/heteroduplex and bi-directionally sequencing revealed wildtype sequence in all cell lines and 60 clinical samples.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. Mutations in the coding sequence of c-src causing constitutive activation could be excluded by mutational analyses of primary AML samples. 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.