okadaic-acid and Lymphoma--T-Cell

Stimuli that are mitogenic for mature T-cells induce cell cycle arrest in some T-cell tumors and T-cell hybridomas. The molecular mechanism of this growth inhibition is poorly understood. In this report, we show that in EL4, a murine T-lymphoma cell line, stimulation with concanavalin A or treatment with phorbol 13-myristate 12-acetate (PMA) inhibit growth, due to cell cycle arrest at both the G1 and the G2/M phases. The block at the G1 phase is accompanied by the appearance of a hypophosphorylated form of the retinoblastoma protein (pRb), due to the inhibition of G1 cyclin-Cdk complexes. However, the molecular mechanisms leading to this G1 cell cycle arrest differ between concanavalin A and PMA: concanavalin A inhibits both cyclin E-Cdk2 and cyclin D-Cdk4 complexes, while PMA inhibits only cyclin E-Cdk2. We demonstrate that concanavalin A inhibits cyclin D-Cdk4 activity by decreasing the amount of cyclin D. The inhibition of cyclin E-Cdk2 by both concanavalin A and PMA is due to increased binding of the Cdk inhibitor p21 to this complex. However, while stimulation of the cells with concanavalin A did not result in an evident increase of the total level of p21, treatment of the cells with PMA increased p21 levels significantly. Our results indicate, furthermore, that the G2/M block results from the inhibition of cyclin A- and cyclin B1-associated kinase activities. As for cyclin E-Cdk2, the inhibition of the cyclin A-Cdk2 complex is due to increased binding of the p21 inhibitor.

Topics: Animals; Concanavalin A; Cyclin-Dependent Kinases; G1 Phase; G2 Phase; Humans; Lymphoma, T-Cell; Mice; Mitosis; Okadaic Acid; Phorbol Esters; Phosphorylation; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

In untransformed T lymphocytes, pp19/cofilin, a cytoplasmic actin-binding protein, undergoes dephosphorylation and nuclear translocation in response to costimulation through accessory receptors (e.g., CD2), but not following TCR/CD3 triggering. In malignant T lymphoma cells, dephosphorylation and nuclear translocation of pp19/cofilin occur spontaneously through constitutive activation of a serine phosphatase. Blockade of these processes by the serine phosphatase inhibitor okadaic acid leads to apoptosis. Moreover, lowering the intracellular pp19/cofilin concentrations by antisense-cofilin transfection results in reduced cloning efficiencies. These findings provide support for the view that pp19/cofilin plays a critical role in the growth and survival of both untransformed and malignant T lymphocytes.

Topics: Actin Depolymerizing Factors; Apoptosis; Cell Transformation, Neoplastic; DNA, Antisense; Enzyme Inhibitors; Ethers, Cyclic; Humans; In Vitro Techniques; Lymphocyte Activation; Lymphoma, T-Cell; Microfilament Proteins; Microscopy, Confocal; Nerve Tissue Proteins; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; T-Lymphocytes; Transfection; Tumor Cells, Cultured

Transforming growth factor beta 1 (TGF-beta 1) is a multifunctional cytokine whose potent immunomodulatory activity is well documented. To explore the mechanisms of this activity we examined the effect of TGF-beta 1 on the production of IFN-gamma measured at the mRNA and protein levels in the YAC-1 T cell lymphoma. In previous studies, this model proved useful to characterize the mode of action of the immunosuppressant rapamycin (RAP). Here, we found that when induced by IL-1 or IL-1 + PMA, the production of IFN-gamma is suppressed by both TGF-beta 1 (ED50 = 1.9 pM) and RAP (ED50 = 0.2 nM). In contrast, when induced by the calcium ionophore ionomycin, in the absence or in the presence of PMA, this production is enhanced up to 10-fold by TGF-beta 1 (ED50 = 1.8 pM) and 1.5-3-fold by RAP. Therefore, in YAC-1 cells, TGF-beta 1 exerts opposite effects on IFN-gamma production depending on the mode of activation, and these effects parallel those of RAP. To further analyze the mode of action of TGF-beta 1 in this system, we used okadaic acid (OA), an inhibitor of serine/threonine protein phosphatases. Treatment with OA rendered the expression of IFN-gamma mRNA induced by IL-1 insensitive to TGF-beta 1 or RAP, indicating that activation of a phosphatase may play a role in the suppressive effect of both agents. However, OA did not prevent the augmentation of ionomycin-mediated induction of IFN-gamma mRNA by either TGF-beta 1 or RAP. Hence, the up-regulation of IFN-gamma production by TGF-beta 1 and RAP may involve a different biochemical mechanism than that mediating their suppressive action. These observations also favor the hypothesis that the two agents act on the same regulatory pathways. This was further supported by the finding that TGF-beta 1 and RAP modulate IFN-gamma production in an additive rather than synergistic fashion. However, their effects could be dissociated in mutants of YAC-1 cells selected for resistance to the inhibition of IL-1-mediated IFN-gamma induction by RAP. Moreover, the IFN-gamma modulatory action of RAP in YAC-1 cells was accompanied by an antiproliferative effect, whereas TGF-beta 1 failed to alter the growth of these cells. Therefore, the immunomodulatory action of TGF-beta 1 may result from the disruption of biochemical processes related to, although distinct from, those affected by RAP.

Topics: Animals; Cell Division; Drug Interactions; Drug Synergism; Ethers, Cyclic; Immunosuppressive Agents; Interferon-gamma; Interleukin-1; Ionomycin; Lymphoma, T-Cell; Mice; Okadaic Acid; Phosphoprotein Phosphatases; Polyenes; RNA, Messenger; Sirolimus; Swine; Tetradecanoylphorbol Acetate; Transforming Growth Factor beta