Tsu-Chung Chang

Differential properties of D4/LyGDI versus RhoGDI: phosphorylation and rho GTPase selectivity

RhoA/B/C and CDC42/Rac, which form two subgroups of the rho guanosine triphosphatase (GTPase) family, regulate various aspects of actin cytoskeleton organisation. In cytosol, guanosine diphosphate (GDP) dissociation inhibitor (GDI) interacts with and maintains rho GTPases in their inactive GDP‐bound form. RhoGDI is a ubiquitously expressed GDI, whereas D4/LyGDI is hematopoietic cell‐specific and 10‐fold less potent than RhoGDI in binding to and regulating rho GTPases. We have combined microanalytical liquid chromatography with the use of specific antibodies in order to separate D4/LyGDI and RhoDGI‐complexes from the cytosol of U937 cells and to demonstrate that the two GDIs associate with different rho protein partners. RhoGDI can form a complex with CDC42Hs, RhoA, Rac1 and Rac2, while none of these GTPases was found to interact with D4/LyGDI. In addition, we found that stimulation of U937 cells with phorbol ester leads to phosphorylation of D4/LyGDI. Our results suggest that LyGDI forms complexes with specific rho GTPases expressed in hematopoietic cells where it may regulate specific pathways.

Isolation and characterization of a novel 98-kd Dermatophagoides farinae mite allergen

BACKGROUNDnExposure to allergens from house dust mites is a significant cause of immediate hypersensitivity. Thus far, the active mite allergens defined are low molecular weight (MW) proteins or glycoproteins. However, other important mite allergens remain to be investigated. In this study a high MW mite antigen with a high IgE-binding activity was characterized.nnnMETHODSnAn anti-Dermatophagoides farinae (Df) monoclonal antibody, mAb642, which recognized a 98-kd allergenic mite protein, was used for affinity chromatography. The purified Df642 was characterized biochemically and immunologically.nnnRESULTSnCompetitive ELISA demonstrated that mAb642 was inhibited by the interaction between serum IgE from allergic patients and Df642 antigen in a dose-dependent fashion. The IgE reactivity to both 98-kd and 92-kd components was removed or diminished by preincubation of asthmatic sera with Df642-coated CNBr-activated cellulose-4B gel. Two-dimensional immunoblot analysis revealed that there are at least 4 isoforms of Df642 that represent a minor component in the crude mite extract. The allergenicity of Df642 was assayed by IgE immunoassay with a large panel of 67 sera from asthmatic patients with positive skin reactions, and Df 642 showed positive IgE reactivity with more than 80% of the sera tested. Thus it should be classified as an important allergen. In addition, amino acid sequence analysis revealed that Df642 shares more than 50% homology with paramyosin from invertebrates.nnnCONCLUSIONnWe have identified and characterized a 98-kd house dust mite allergen that showed greater than 80% IgE reactivity with sera from patients allergic to mites. This is the first high MW allergen characterized to date, and it shares high sequence homology with paramyosins in invertebrates.

Dexamethasone suppresses apoptosis in a human gastric cancer cell line through modulation of bcl‐x gene expression

Treatment of human gastric cancer TMK‐1 cells with transcription and translation inhibitors rapidly triggered cell apoptosis. Along with cell apoptosis, the Bcl‐xS level was markedly upregulated suggesting a crucial role of this protein in promoting the apoptotic process. In the presence of dexamethasone, however, cell apoptosis was greatly attenuated as demonstrated by DNA histogram shift and DNA fragmentation. Studies using the glucocorticoid receptor antagonist RU486 indicated that attenuation of apoptosis was mediated through glucocorticoid receptors. Dexamethasone not only suppressed the apoptosis‐associated upregulation of Bcl‐xS but also enhanced the basal level of Bcl‐xL in the cells. In addition, bcl‐x mRNA stability was significantly extended in the presence of dexamethasone. These results indicate that dexamethasone exerted a protective effect and delayed apoptosis of TMK‐1 cells by modulating bcl‐x gene expression.

Effects of transcription and translation inhibitors on a human gastric carcinoma cell line: Potential role of Bcl-Xs in apoptosis triggered by these inhibitors☆

The effects of the macromolecular synthesis inhibitors 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB), actinomycin D, and cycloheximide on the human gastric cancer TMK-1 cell line were studied. These agents inhibited DNA, RNA, or protein synthesis efficiently and induced cell death rapidly in a wide range of concentrations. After 8 hr of exposure to these agents, the cells exhibited morphological features of apoptosis, including cell shrinkage, nuclear condensation, DNA fragmentation, and formation of apoptotic bodies. Western blot analysis revealed that these inhibitors altered the protein levels of apoptosis-related gene products such as c-Myc, Bcl-X(S), and the mutant p53 (mp53) in TMK-1 cells markedly. The c-myc mRNA and protein levels were decreased initially and were then induced markedly to a new level after 4 hr of exposure to DRB, a RNA polymerase II inhibitor. The Bcl-X(S) levels were increased rapidly after treatment with all of these agents, whereas the levels of Bcl-X(L) and Bax remained largely unchanged. Northern blot analysis indicated that the c-myc overexpression is concomitant to DRB-induced DNA fragmentation and that the increased mp53 protein level was mainly a posttranscriptional event. Our observations suggest that the up-regulation of Bcl-X(S) may serve as an important mechanism for the apoptosis triggered by these inhibitors. This study also provides evidence for the notion that interference with the cellular survival pathway may lead to apoptosis.

Caffeic acid phenethyl ester induces E2F‐1‐mediated growth inhibition and cell‐cycle arrest in human cervical cancer cells

Caffeic acid phenyl ester (CAPE) has been identified as an active component of propolis, a substance that confers diverse activities in cells of various origins. However, the molecular basis of CAPE‐mediated cellular activity remains to be clarified. Here, we show that CAPE preferentially induced S‐ and G2/M‐phase cell‐cycle arrests and initiated apoptosis in human cervical cancer lines. The effect was found to be associated with increased expression of E2F‐1, as there is no CAPE‐mediated induction of E2F‐1 in the pre‐cancerous cervical Z172 cells. CAPE also up‐regulated the E2F‐1 target genes cyclin A, cyclin E and apoptotic protease activating of factor 1 (Apaf‐1) but down‐regulated cyclin B and induced myeloid leukemia cell differentiation protein (Mcl‐1). These results suggest the involvement of E2F‐1 in CAPE‐mediated growth inhibition and cell‐cycle arrest. Transient transfection studies with luciferase reporters revealed that CAPE altered the transcriptional activity of the apaf‐1 and mcl‐1 promoters. Further studies using chromatin immunoprecipitation assays demonstrated that E2F‐1 binding to the apaf‐1 and cyclin B promoters was increased and decreased, respectively, in CAPE‐treated cells. Furthermore, E2F‐1 silencing abolished CAPE‐mediated effects on cell‐cycle arrest, apoptosis and related gene expression. Taken together, these results indicate a crucial role for E2F‐1 in CAPE‐mediated cellular activities in cervical cancer cells.