Apoptosis of NIH3T3 cells overexpressing the Src homology 2 domain protein Shb

To understand the role of the Src homology 2 (SH2) domain protein Shb in the signal transduction of tyrosine kinase receptor, NIH3T3 cells were transfected with a DNA construct expressing the Shb cDNA (NIHSHB cells). The NIHSHB cells expressed elevated levels of proteins with the estimated molecular weights of 77, 66 and 55 kDa as determined by immunoblotting. In contrast to the control cells, the NIHSHB cells failed to increase in cell number in the presence of 1% serum. This effect was largely due to apoptosis, since staining of pyknotic nuclei was observed using the terminal transferase labeling method. The NIHSHB cells displayed similar levels of c-myc mRNA and decreased contents of the p53 protein after culture in 1% serum compared with control cells. The addition of platelet-derived growth factor (PDGF-BB) restored the growth of the NIHSHB cells, whereas insulin-like growth factor-1 (IGF-1) failed to affect the proliferation of Shb overexpressing cells in 1% serum. We conclude that Shb overexpression is associated with cell degeneration under certain conditions, and that Shb could transduce apoptotic signals from tyrosine kinase receptors.