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Abstract
By taking advantage of the established chicken B cell line, DT40 cells, which do not express tyrosine kinase Syk or Lyn, functional roles of Syk and Lyn in apoptotic response elicited by cellular stress were investigated. DT40 cells underwent apoptosis after hyperosmotic stress. In Syk-deficient DT40 cells, this apoptotic process was significantly enhanced. Ectopic expression of wild type, but not kinase-inactive, porcine Syk in Syk-deficient cells rescued cells from osmotic stress-induced apoptosis, demonstrating that the presence of functionally active Syk is necessary to protect cells from osmotic stress-induced apoptosis. In comparison, there was no effect on osmotic stress-induced apoptosis in Lyn-deficient DT40 cells. Interestingly, while Syk was not involved in ultraviolet C (UVC)-induced apoptosis, a deficiency of Lyn rendered cells resistant to UVC irradiation. These observations defined Syk and Lyn as important mediators of apoptosis in DT40 cells in response to osmotic stress and UVC irradiation, respectively. Furthermore, osmotic stress, but not UVC irradiation, could activate c-Jun N-terminal kinase (JNK) in DT40 cells. A deficiency in either Syk or Lyn did not affect the osmotic stress-induced activation of JNK. We, therefore, concluded that Syk and Lyn regulate the apoptotic responses to osmotic stress and UVC irradiation independently of the JNK pathway in DT40 cells.
Highlights
Apoptosis, which is widely observed in different cells of various organisms, is the unique morphological pattern of cell death characterized by chromatin condensation and membrane blebbing
These results demonstrate that in DT40 cells, Syk may function as a specific inhibitor of osmotic stress-induced apoptosis while Lyn acts as a positive mediator of ultraviolet C (UVC) irradiation-induced apoptosis
Cellular stress, including ionizing irradiation, hydrogen peroxide, sodium chloride, and low energy electromagnetic fields, activates several nonreceptor protein-tyrosine kinase (PTK) such as Btk, Syk, Lyn, and ZAP70 [7, 21, 22, 24, 25, 31, 32], which are predominantly expressed in lymphocytes
Summary
Materials—The generation of DT40/Lyn, DT40/Syk, DT40/Syk2/ Syk, and DT40/Syk2/Syk(K2) cells and antisera against Lyn or Syk was carried out as described previously [28]. Immunoblot Analysis—Cell extracts were immunoprecipitated with 0.3 mg of anti-Syk antibody, 1 mg of anti-JNK1 antibody, or 3 ml of anti-Lyn antisera with 40 ml of protein A-Sepharose 4B (50% slurry) for 1 h at 4 °C. Immunoprecipitates were washed three times with lysis buffer, once with 10 mM Hepes, pH 8.0, buffer containing 500 mM NaCl, and once with the same Hepes buffer without NaCl. The washed immunoprecipitates were boiled with SDS sample buffer for 3 min, resolved on a 10% SDS-polyacrylamide gel electrophoresis, transferred electrically to polyvinylidene difluoride membranes, and immunoprobed with 4G10 to detect tyrosine phosphorylation. 5 3 106 cells were lysed in 0.5 ml of lysis buffer (10 mM Tris-HCl, pH 7.5, 10 mM EDTA, 200 mM NaCl, 0.4% Triton X-100, and 0.1 mg/ml proteinase K) for 20 min at room temperature followed by a 30-min incubation with 0.1 mg/ml RNase A at 50 °C. DNA fragmentation was analyzed on a 2% agarose gel in the presence of 0.5 mg/ml ethidium bromide
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