For constructing the tetracycline (Tet)-inducible enhanced green fluorescent protein (EGFP)-tagged Syk (Syk-EGFP) lentiviral vectors, cDNAs for Syk-EGFP, Syk-EGFP(K396R), Syk-EGFP(Y317F), Syk-EGFP(Y342F), Syk-EGFP(Y346F), Syk-EGFP(Y342F/Y346F), and Syk-EGFP(Y317F/Y342F/Y346F) were amplified by PCR from the corresponding EGFP-N2 (Clontech) constructs described previously (26). (12, 13). The formation of a Tel-Syk fusion protein that results from a chromosomal translocation results in a myeloid proliferative disorder, while Itk-Syk fusion proteins are found in some T-cell lymphomas (14, 15). The aberrant expression of Syk itself is also found in a variety of peripheral T-cell lymphomas (16). Even in nonhematological malignancies, Syk can play an important prosurvival function. Lung and pancreatic carcinomas that are dependent on activated K-Ras for viability are distinguished from those Rabbit Polyclonal to SLC9A6 not dependent on K-Ras by the expression of Syk (17). These K-Ras-dependent cells undergo apoptosis in response to the inhibition of Syk activity or knockdown of Syk expression. Retinoblastoma cells in which the expression of Syk is induced by changes in gene methylation also undergo apoptosis in response to reductions in the activity or level of the kinase (18). The survival of breast and ovarian cancer cells is promoted by the alternative splicing of Pregnenolone transcripts in response to epidermal growth factor, which enhances expression of the long form of the kinase (19). While the mechanisms by which Syk promotes cancer cell survival are incompletely understood, these observations have led to the exploration of Syk inhibitors as antitumor agents (e.g., see references 18 and 20 to 22). The ability to evade cell death is one of the fundamental hallmarks of a cancer cell (23). Programmed cell death in eukaryotic cells is regulated through the intrinsic pathway by members of the Bcl-2 family of proteins (24). These proteins function to modulate outer mitochondrial membrane channel opening and the release of cytochrome necessary for the formation of apoptosomes. The Bcl-2 family includes both pro- and antiapoptotic members. Among these are Bcl-xL and Bcl-xS, which are products of alternatively spliced transcripts of the gene (25). The product of the longer transcript, Bcl-xL, protects cells from apoptosis, while the smaller Bcl-xS protein promotes apoptosis by negatively regulating Bcl-xL and Bcl-2. The relative level of Bcl-xL and Bcl-xS in a cell is an important determinant of susceptibility to stress-induced cell death. In this study, we explored the mechanism by which Syk enhances cell survival by examining its effect on the responses of cancer cells to induced stress. We found that the presence of Syk increases the resistance of several cancer cell types to H2O2-induced apoptosis by protecting Bcl-xL mRNA from degradation by a Pregnenolone mechanism that involves the interaction of both Syk and the Bcl-xL mRNA with nucleolin (NCL). Reductions in the level of nucleolin destabilize the Bcl-xL message and inhibit the ability of Syk to protect cells from apoptosis induced by both oxidative and genotoxic stress. MATERIALS AND METHODS Plasmids and DNA constructs. For constructing the tetracycline (Tet)-inducible enhanced green fluorescent protein (EGFP)-tagged Syk (Syk-EGFP) lentiviral vectors, cDNAs for Syk-EGFP, Syk-EGFP(K396R), Syk-EGFP(Y317F), Syk-EGFP(Y342F), Syk-EGFP(Y346F), Syk-EGFP(Y342F/Y346F), and Syk-EGFP(Y317F/Y342F/Y346F) were amplified by PCR from the corresponding EGFP-N2 (Clontech) constructs described previously (26). These were then cloned into the Tet-inducible lentiviral vector pLVX-Tight-Puro (Clontech) between the MluI and EcoRI restriction sites. Lentiviral pGIPZ short hairpin RNA (shRNA) sets for the knockdown of nucleolin and Syk Pregnenolone were purchased from Thermo Scientific. The Bcl-xL expression plasmid pSFFV-neo Bcl-xL (27) was obtained from Addgene (plasmid 8749). Cell lines. A line of MCF7 cells lacking endogenous Syk (MCF7-BD) was described previously, as were MCF7-BD cells stably expressing exogenous Syk-EGFP (MCF7-Syk) (28). Syk-deficient MCF7-BD cells with tetracycline-regulated Syk-EGFP expression (MCF7-TRS) were constructed previously using a T-REx system (Invitrogen) (26). These cells were treated with 1 g/ml doxycycline to.