Rho family proteins modulate rapid apoptosis induced by cytotoxic T lymphocytes and Fas

Little is known about the role of Rho proteins in apoptosis produced by stimuli evolved specifically to produce apoptosis, such as granzymes from cytotoxic T lymphocytes (CTLs) and Fas. Here we demonstrate that all three Rho family members are involved in CTL- and Fas-induced killing. Dominant-negative mutants of each Rho family member and Clostridium difficile toxin B, an inhibitor of all family members, strongly inhibited the susceptibility of cells to CTL- and Fas-induced apoptosis. Fas-induced caspase-3 activation was inhibited by C. difficile toxin. Activated mutants of each GTPase increased susceptibility to apoptosis, and activation of Cdc42 increased within 5 min of Fas stimulation. In contrast, during the time required for CTL and Fas killing, no apoptosis was produced by dominant-negative or activated mutants or by C. difficile toxin alone. Inhibition of actin polymerization using latrunculin A reduced the ability of constitutively active GTPase mutants to stimulate apoptosis and blocked Fas-induced activation of caspase-3. Furthermore, the ability of Rac to enhance apoptosis was decreased by point mutations reported to block Rac induction of actin polymerization. Rho family proteins may regulate apoptosis through their effects on the actin cytoskeleton.     

Genetic and antigenic characterization of recombinant nucleocapsid proteins derived from canine coronavirus and canine respiratory coronavirus in China

To characterize the antigenicity of nucleocapsid proteins (NP) derived from canine coronavirus (CCoV) and canine respiratory coronavirus (CRCoV) in China, the N genes of CCoV (CCoV-BJ70) and CRCoV (CRCoV-BJ202) were cloned from swabs obtained from diseased pet dogs in Beijing and then sequenced. The recombinant NPs (rNPs) were expressed in Escherichia coli and purified by nickel-affinity column and size exclusion chromatography. Sequencing data indicated that the N genes of CCoV-BJ70 and CRCoV-BJ202 belonging to two distinctly different groups were relatively conserved within each subgroup. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that rNPs of CCoV and CRCoV were expressed efficiently and isolated with a final purity of over 95%. Western blot analysis revealed the rNP from CRCoV could cross-react with mice antisera against human coronavirus (HCoV-229E, NL63, OC43, HKU1), while rNP of CCoV had cross-reactivity with only anti-sera against viruses belonging to the same group (HCoV-229E and NL63). In summary, CCoV and CRCoV rNPs were successfully expressed in E. coli and showed antigenic cross-reactivity with antisera raised against human coronaviruses. These findings indicate that further serologic studies on coronavirus infections at the animal-human interface are needed.     

Simple computational models of type I/type II cells in Fas signaling-induced apoptosis

Distinct apoptotic response of the type I/type II cells against Fas-ligand stimulation is considered to arise from the difference in dominant signaling pathways involved. In the type I cells, apoptotic signaling predominantly takes place via the direct activation of caspase-3 by activated caspase-8 (D channel) while mitochondrial pathway (M channel) plays a major role in the type II cells. To elucidate the selection mechanism of dominant pathway, we carried out systematic model analysis of the Fas signaling-induced apoptosis network. An increase in the expression level of caspase-8 induced a switch of dominant pathway from M- to D-channel (M–D transition), showing a phenotypic change from type II to type I cells. With the aid of sensitivity analysis and kinetic considerations, we succeeded in constructing a minimal network model relevant for the M–D transition, which revealed that mechanistic origin of the transition lies in the competition between the activated forms of caspase-8 and caspase-9 for their common substrate caspase-3. The pathway dominance was found to be primarily controlled by the balance between the activation rate of caspase-8 and the initial level of caspase-9. In the full network model, we showed that differential formation ability of the death-inducing signaling complex (DISC) can also induce M–D transition, in accordance with the experimental observations.     

New functions for the Snail family of transcription factors: Two-faced proteins

Comment on: Yang D, et al. Cell Cycle 2010; 9:2789-802.     

Inhibition of drug-induced Fas ligand transcription and apoptosis by Bcl-XL.

Fas/Fas ligand system triggers apoptosis in many cell types. Bcl-XL overexpresion antagonizes Fas/Fas ligand-mediated cell death. The mechanism by which Bcl-XL influences Fas-mediated cell death is unclear. We have found that microtubule-damaging drugs (e.g. Paclitaxel) induce apoptosis in a Fas/FasL-dependent manner. Inhibition of Fas/FasL pathway by anti-FasL antibody, mutant Fas or a dominant negative FADD blocks paclitaxel-induced apoptosis. Paclitaxel induced apoptosis through activation of both caspase-8 and caspase-3. Overexpression of Bcl-XL leads to inhibition of paclitaxel-induced FasL expression and apoptosis. Bcl-XL prevents the nuclear translocation of NFAT (nuclear factor of activated T lymphocytes) by inhibiting the activation of calcineurin, a calcium-dependent phosphatase that must dephosphorylate NFAT for it to move to the nucleus. The loop domain in Bcl-XL can suppress the anti-apoptotic function of Bcl-XL and may be a target for regulatory post-translational modifications. Upon phosphorylation, Bcl-XL loses its ability to bind with calcineurin. Without NFAT nuclear translocation, the FasL gene is not transcribed. Thus, paclitaxel and other drugs that disturb microtubule function kill cells, at least in part, through the induction of FasL, and Bcl-XL-mediated resistance to these agents is related to failure to induce FasL expression.     

Involvement of miltefosine-mediated ERK activation in glioma cell apoptosis through Fas regulation

The anti-neoplastic property of alkyl phospholipids has been tested for the treatment of several malignancies. In this study, we evaluated the efficacy of miltefosine (Hexadecylphosphocholine--an alkyl phospholipids analogue) on glioblastoma multiforme. In this study, we demonstrate that miltefosine-induced apoptosis is accompanied by elevated Fas, Fas-associated death domain (FADD) expression, caspase-8 activity and the increased distribution of Fas and FADD towards lipid raft microdomain to form death inducing signaling complex. Treatment with miltefosine resulted in increase in Ras, extracellular signal-regulated kinase (ERK) and p38MAPK activity. Expression of dominant-negative Ras (Ras N17) attenuated miltefosine-mediated apoptosis. Although inhibition of both ERK and p38MAPK decreased the pro-apoptotic effects of miltefosine, it was the inhibition of ERK and not p38MAPK activation that decreased Fas and FADD expression. An ERK-dependent increase in the expression of gammaH2AX-involved in response to DNA double-stranded breaks was also observed. Taken together, our findings suggest the involvement of ERK activation in miltefosine-induced glioma cell apoptosis.     

M and N proteins of SARS coronavirus induce apoptosis in HPF cells

Background: SARS-associated coronavirus (SARS-CoV) induced cell apoptosis and its structural proteins may play a role in this process. Objectives: To determine whether the structural proteins M and N of SARS-CoV induce apoptosis. Study design: We investigated human pulmonary fibroblast (HPF) cells, were transfected with plasmids containing the M or N gene, by TdT-mediated dUTP nick end labeling (TUNEL), Hoechst 33342 staining for nuclei, and observation of morphology. Results: We found that in the absence of serum about 16.34% of cells transfected by pcDNA3.1-M and 21.72% of N-transfected cells showed typical apoptotic characteristics, significantly different from mock-transfected cells (only 6.23%, p<0.01). Furthermore, the cells that were co-transfected with M and N proteins showed more obvious phenomena of cell death (about 36.03%). There was a statistical significance between M-transfected cells and co-transfected cells (p<0.01), and a remarkable difference between N-transfected cells and co-transfected cells (p<0.01). Conclusions: The results show that M and N proteins of SARS-CoV can induce apoptosis of HPF cells. Co-transfection of M and N enhances the induction of apoptosis by M or N alone, which also suggests that the structural proteins of SARS-CoV may play an important role not only in the process of invasion but also in the pathogenetic process in cells.     

Differential susceptibility to CD95 (Apo-1/Fas) and MHC class II-induced apoptosis during murine dendritic cell development

Disappearance of antigen presenting cells (APC) from the lymph node occurs following antigen specific interactions with T cells. We have investigated the regulation of CD95 (Apo-1/Fas) induced apoptosis during murine dendritic cell (DC) development. Consistent with the moderate levels of CD95 surface expression and low, or absent, MHC class II expression, immature DC in bone marrow cultures were highly sensitive to CD95 induced apoptosis, but insensitive to class II mediated apoptosis. In contrast, mature splenic, epidermal and bone marrow derived DC were fully resistant to CD95 induced cell death, but sensitive to class II induced apoptosis. Although caspase 3 and 8 activation was detected in immature DC undergoing CD95L-induced apoptosis, the pan-caspase inhibitor zVAD-fmk did not inhibit the early events of CD95-induced mitochondrial depolarisation or phosphatidyl serine exposure and only partially inhibited the killing of immature DC. In contrast, zVAD-fmk was completely effective in preventing CD95L mediated death of murine thymocytes. Collectively, these data do not support a major role of CD95: CD95L ligation in apoptosis of mature DC, but rather emphasise the existence of distinct pathways for the elimination of DC at different stages of maturation.