Izumi Nakashima

Modulation by glycyrrhizin of the cell-surface expression of H-2 class I antigens on murine tumour cell lines and normal cell populations

Glycyrrhizin (GL), a saponin fraction of licorice with defined chemical structure, was shown to display a definite action in vitro augmenting the cell-surface expression of H-2 class I antigens as well as class I gene transcription on various tumour cell lines. The magnitude of augmentation was varied among eight different cell lines tested, but reached more than three times. It was also found that GL enhanced the expression of H-2Dd antigens in some normal cell populations in vivo. The augmentation of H-2 class I antigens on tumour cell lines in vitro was probably not mediated by the interferon, which might have been produced by the cultured cells. These findings may suggest a new immunopharmacological action of GL.

Bid truncation mediated by caspases-3 and -9 in vinorelbine-induced apoptosis

Vinorelbine is a chemotherapeutic vinca alkaloid clinically prescribed for non-small cell lung cancer and breast cancer. Here we studied the mechanism for vinorelbine-induced apoptosis in a human T-cell lymphoma. Although vinorelbine induces DNA fragmentation that is inhibited by specific peptide inhibitors for caspases-9 and -3 in Jurkat cells, caspase-8 deficiency retards vinorelbine-induced apoptosis. Activation of caspase-8 is also observed in vinorelbine-treated cells, and the activity is diminished when the caspase-3 activity is blocked by a specific peptide inhibitor, Ac-DNLC-CHO. Blocking of the Fas receptor with an antagonistic anti-Fas antibody does not affect vinorelbine-induced DNA fragmentation. These results suggest that vinorelbine-induced apoptosis is enhanced by the activation of caspase-8 via caspase-9-mediated activation of caspase-3, but not through a Fas-triggered signal. Western blotting suggests that vinorelbine cleaves caspase-3, -9 and -8 and reduces the amount of mitochondrial cytochrome c. Caspase-8 deficiency suppresses all of these events. A downstream substrate for caspase-8, Bid, is also cleaved in vinorelbine-treated cells, but the Bid truncation is also observed in caspase-8-deficient Jurkat cells. Importantly, recombinant caspases-3 and -9, as well as caspase-8, directly cleaves recombinant Bid inxa0vitro. These results suggest that caspases-3 and -9 participate in Bid truncation, indicating a new mechanism for vinorelbine-induces apoptosis.

Control of Genetically Prescribed Protein Tyrosine Kinase Activities by Environment-Linked Redox Reactions

Recent observations on environment-linked control of genetically prescribed signaling systems for either cell activation or cell death have been reviewed with a focus on the regulation of activities of protein tyrosine kinases (PTKs). The environment-linked redox reactions seem to primarily affect cell surface receptors and cell membrane lipid rafts, and they induce generation of reactive oxygen species (ROS) in cells. ROS thus generated might upregulate the catalytic activities of PTKs through inactivating protein tyrosine phosphatases that dephosphorylate and inactivate autophosphorylated PTKs. Recent evidence has, however, demonstrated that ROS could also directly oxidize SH groups of genetically conserved specific cysteines on PTKs, sometimes producing disulfide-bonded dimers of PTK proteins, either for upregulation or downregulation of their catalytic activities. The basic role of the redox reaction/covalent bond-mediated modification of protein tertiary structure-linked noncovalent bond-oriented signaling systems in living organisms is discussed.

T Cell maturation stage-linked heterogeneity of the glycosylphosphatidylinositol membrane anchor of Thy-1

We showed that some of Thy-1 molecules on murine thymocytes are resistant to phosphatidylinositol-specific phospholipase C (PI-PLC) derived from Bacillus thuringiensis. Both immature thymocytes with low CD3 expression and mature thymic T lymphocytes with high CD3 expression carried the PI-PLC-resistant Thy-1, and the PI-PLC-sensitivity of Thy-1 extensively varied among thymocyte subpopulations. In contrast, the same PI-PLC fully hydrolysed the anchor of Thy-1 on peripheral T lymphocytes. When the latter cells were activated with mitogen in vitro, however, some Thy-1 on them became resistant to PI-PLC. We then found that virtually all Thy-1 molecules on thymocytes became sensitive to PI-PLC when they were treated with hydroxylamine that should cleave ester-linked lipids. The result ruled out the possibility that the PI-PLC-resistant Thy-1 had a transmembranous peptide sequence, and suggested the presence of an additional fatty acyl group on the inositol ring of the Thy-1 anchor. In addition, the molecular size of the PI-PLC-resistant membrane-bound Thy-1 was only marginally larger than that of the PI-PLC-sensitive solubilized Thy-1 in detergent-partitioning SDS-PAGE analysis.

1,4-butanediyl-bismethanethiosulfonate (BMTS) induces apoptosis through reactive oxygen species-mediated mechanism

Although methane sulfonate compounds are widely used for the protein modification for their selectivity of thiol groups in proteins, their intracellular signaling events have not yet been clearly documented. This study demonstrated the methane sulfonate chemical 1,4‐butanediyl‐bismethanethiosulfonate (BMTS)‐induced cascades of signals that ultimately led to apoptosis of Jurkat cells. BMTS induced apoptosis through fragmentation of DNA, activation of caspase‐9 and caspase‐3, and downregulation of Bcl‐2 protein with reduction of mitochondrial membrane potential. Moreover, BMTS intensely and transiently induced intracellular reactive oxygen species (ROS) production and ROS produced by BMTS was mediated through mitochondria. We also found that a reducing agent dithiothreitol (DTT) and an anti‐oxidant N‐acetyl cysteine (NAC) inhibited BMTS‐mediated caspase‐9 and ‐3 activation, ROS production and induction of Annexin V/propidium iodide double positive cells, suggesting the involvement of ROS in the apoptosis process. Therefore, this study further extends our understanding on the basic mechanism of redox‐linked apoptosis induced by sulfhydryl‐reactive chemicals. J. Cell. Biochem. 108: 1059–1065, 2009.