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(-)-Epigallocatechin-3-gallate (EGCG), a major constituent of green tea polyphenols, has been shown to suppress cancer cell proliferation and induce apoptosis. In this study we investigated its efficacy and the mechanism underlying its effect using human B lymphoblastoid cell line Ramos, and effect of co-treatment with EGCG and a chemotherapeutic agent on apoptotic cell death. EGCG induced dose- and time-dependent apoptotic cell death accompanied by loss of mitochondrial transmembrane potential, release of cytochrome c into the cytosol, and cleavage of pro-caspase-9 to its active form. EGCG also enhanced production of intracellular reactive oxygen species (ROS). Pretreatment with diphenylene iodonium chloride, an inhibitor of NAD(P)H oxidase and an antioxidant, partially suppressed both EGCG-induced apoptosis and production of ROS, implying that oxidative stress is involved in the apoptotic response. Furthermore, we showed that combined-treatment with EGCG and a chemotherapeutic agent, etoposide, synergistically induced apoptosis in Ramos cells.

The effect of adhesion via CD43 (leukosialin, sialophorin) on cell proliferation and phosphorylation signaling were examined in a growth factor-dependent hematopoietic progenitor cell line, TF-1. TF-1 cells promptly resulted in death after withdrawal of growth factors. However, the viable cell number increased when TF-1 cells were cultured on anti-CD43 monoclonal antibody-coated plates. In this case, sustained activation of protein tyrosine kinase Syk and extracellular signal-regulated kinase (Erk) 1/2 were detected. Overexpression of exogenous Syk on TF-1 cells by the adenovirus vector system induced enhancement of the cell proliferation accompanied with enhancement of the Erk activation by a dominant-positive effect. The signal transducer and activator of transcription (STAT) 5 seemed not to be associated with the CD43-mediated cell proliferation. These results indicated that adhesion via CD43 induces the proliferation of TF-1 cells in the absence of growth factors in part by Syk-dependent Erk 1/2 signaling.

Cell polarization and migration in response to CXCL12 is essential for hematopoiesis. To investigate the role of Syk in CXCL12/CXCR4-induced signaling, wild-type Syk or its dominant-negative form (DN-Syk) was introduced in mouse pro-B cells, BAF3. With CXCL12 stimulation, BAF3 cells became polarized with the formation of a leading edge and contractile uropod at the rear end with increased motility. Overexpression of wild-type Syk caused enhanced polarization, whereas DN-Syk inhibited cell polarity due to the loss of contractile structure at the rear end, and the altered phenotype was enhanced after CXCL12 stimulation. Motility of mutant BAF3 containing DN-Syk increased independent of CXCL12 stimulation. As beta1 integrin-mediated cell adhesion was inhibited, decreased adhesion might promote motility. CXCL12 stimulation led to prompt activation of RhoA, but expression of DN-Syk suppressed RhoA activation. These results demonstrate that Syk participates in CXCL12-induced cell polarization, which occurs in concert with cell adhesion mediated by beta1 integrin.

A human megakaryoblastic cell line, CMK, was treated with 12-o-tetradecanoylphorbol-13-acetate (TPA) for differentiation–induction. We examined TPA-induced activation of the MEK1-ERK1/2 pathway in the 100,000g Triton X-insoluble fraction of CMK cells as the membrane skeleton and researched the relation of the MEK1-ERK1/2 activation with integrin expression. We found that this activation was divided into two phases: the first activation occurred transiently in the membrane skeleton fraction of the suspended cell status and diminished after 1 h; and the second sustained activation was maintained by cell adhesion. TPA-treated CMK cells revealed increased expression of integrins αIIb and β3 only when the cell adhesion persisted, regardless of the difference of culture substratum. Sustained activation of the MEK1-ERK1/2 pathway is generated in the membrane skeleton by continuous cell adhesion and seems to be essential to TPA-induced megakaryocytic differentiation of CMK cells.