Guoping Zhao

An Interaction between Bcl-xL and the Voltage-dependent Anion Channel (VDAC) Promotes Mitochondrial Ca2+ Uptake

Background: Ca2+ moves across the outer mitochondrial membrane (OMM) through the voltage-dependent anion channel (VDAC). Results: Disrupting the interaction between VDAC and the antiapoptotic protein Bcl-xL reduces mitochondrial Ca2+ uptake. Conclusion: Bcl-xL/VDAC interactions promote Ca2+ uptake by increasing transfer across the OMM. Significance: Mitochondrial matrix Ca2+ is tightly regulated at the OMM by the modulation of VDAC. The role of the antiapoptotic protein Bcl-xL in regulating mitochondrial Ca2+ ([Ca2+]mito) handling was examined in wild-type (WT) and Bcl-xL knock-out (Bcl-xL-KO) mouse embryonic fibroblast cells. Inositol 1,4,5-trisphosphate-generating agonist evoked cytosolic Ca2+ transients that produced a larger [Ca2+]mito uptake in WT cells compared with Bcl-xL-KO. In permeabilized cells, stepping external [Ca2+] from 0 to 3 μm also produced a larger [Ca2+]mito uptake in WT; moreover, the [Ca2+]mito uptake capacity of Bcl-xL-KO cells was restored by re-expression of mitochondrially targeted Bcl-xL. Bcl-xL enhancement of [Ca2+]mito uptake persisted after dissipation of the mitochondrial membrane potential but was absent in mitoplasts lacking an outer mitochondrial membrane. The outer membrane-localized voltage-dependent anion channel (VDAC) is a known Ca2+ permeability pathway that directly interacts with Bcl-xL. Bcl-xL interacted with VDAC1 and -3 isoforms, and peptides based on the VDAC sequence disrupted Bcl-xL binding. Peptides reduced [Ca2+]mito uptake in WT but were without effect in Bcl-xL-KO cells. In addition, peptides reduced [Ca2+]mito uptake in VDAC1 and VDAC3 knock-out but not VDAC1 and -3 double knock-out mouse embryonic fibroblast cells, confirming that Bcl-xL interacts functionally with VDAC1 and -3 but not VDAC2. Thus, an interaction between Bcl-xL and VDAC promotes matrix Ca2+ accumulation by increasing Ca2+ transfer across the outer mitochondrial membrane.

Up-regulation of ROS by mitochondria-dependent bystander signaling contributes to genotoxicity of bystander effects

Genomic instability can be observed in bystander cells. However, the underlying mechanism(s) is still relatively unclear. In a previous study, we found that irradiated cells released mitochondria-dependent intracellular factor(s) which could lead to bystander gamma-H2AX induction. In this paper, we used normal (rho(+)) and mtDNA-depleted (rho(0)) human-hamster hybrid cells to investigate mitochondrial effects on the genotoxicity in bystander effect through medium transfer experiments. Through the detection of DNA double-strand breaks with gamma-H2AX, we found that the fraction of gamma-H2AX positive cells changed with time when irradiation conditioned cell medium (ICCM) were harvested. ICCM harvested from irradiated rho(+) cells at 10 min post-irradiation (rho(+) ICCM(10 min)) caused larger increases of bystander gamma-H2AX induction comparing to rho(0) ICCM(10 min), which only caused a slight increase of bystander gamma-H2AX induction. The rho(+) ICCM(10 min) could also result in the up-regulation of ROS production (increased by 35% at 10 min), while there was no significant increase in cells treated with rho(0) ICCM(10 min). We treated cells with dimethyl sulfoxide (DMSO), the scavenger of ROS, and quenched gamma-H2AX induction by rho(+) ICCM. Furthermore, after the medium had been transferred and the cells were continuously cultured for 7 days, we found significantly increased CD59(-) gene loci mutation (increased by 45.9%) and delayed cell death in the progeny of rho(+) ICCM-treated bystander cells. In conclusion, the work presented here suggested that up-regulation of the mitochondria-dependent ROS might be very important in mediating genotoxicity of bystander effects.

ER stress modulates cellular metabolism.

Changes in metabolic processes play a critical role in the survival or death of cells subjected to various stresses. In the present study, we have investigated the effects of ER (endoplasmic reticulum) stress on cellular metabolism. A major difficulty in studying metabolic responses to ER stress is that ER stress normally leads to apoptosis and metabolic changes observed in dying cells may be misleading. Therefore we have used IL-3 (interleukin 3)-dependent Bak-/-Bax-/- haemopoietic cells which do not die in the presence of the ER-stress-inducing drug tunicamycin. Tunicamycin-treated Bak-/-Bax-/- cells remain viable, but cease growth, arresting in G1-phase and undergoing autophagy in the absence of apoptosis. In these cells, we used NMR-based SIRM (stable isotope-resolved metabolomics) to determine the metabolic effects of tunicamycin. Glucose was found to be the major carbon source for energy production and anabolic metabolism. Following tunicamycin exposure, glucose uptake and lactate production are greatly reduced. Decreased 13C labelling in several cellular metabolites suggests that mitochondrial function in cells undergoing ER stress is compromised. Consistent with this, mitochondrial membrane potential, oxygen consumption and cellular ATP levels are much lower compared with untreated cells. Importantly, the effects of tunicamycin on cellular metabolic processes may be related to a reduction in cell-surface GLUT1 (glucose transporter 1) levels which, in turn, may reflect decreased Akt signalling. These results suggest that ER stress exerts profound effects on several central metabolic processes which may help to explain cell death arising from ER stress in normal cells.

Left hemisphere lateralization for lexical and acoustic pitch processing in Cantonese speakers as revealed by mismatch negativity

For nontonal language speakers, speech processing is lateralized to the left hemisphere and musical processing is lateralized to the right hemisphere (i.e., function-dependent brain asymmetry). On the other hand, acoustic temporal processing is lateralized to the left hemisphere and spectral/pitch processing is lateralized to the right hemisphere (i.e., acoustic-dependent brain asymmetry). In this study, we examine whether the hemispheric lateralization of lexical pitch and acoustic pitch processing in tonal language speakers is consistent with the patterns of function- and acoustic-dependent brain asymmetry in nontonal language speakers. Pitch contrast in both speech stimuli (syllable /ji/ in Experiment 1) and nonspeech stimuli (harmonic tone in Experiment 1; pure tone in Experiment 2) was presented to native Cantonese speakers in passive oddball paradigms. We found that the mismatch negativity (MMN) elicited by lexical pitch contrast was lateralized to the left hemisphere, which is consistent with the pattern of function-dependent brain asymmetry (i.e., left hemisphere lateralization for speech processing) in nontonal language speakers. However, the MMN elicited by acoustic pitch contrast was also left hemisphere lateralized (harmonic tone in Experiment 1) or showed a tendency for left hemisphere lateralization (pure tone in Experiment 2), which is inconsistent with the pattern of acoustic-dependent brain asymmetry (i.e., right hemisphere lateralization for acoustic pitch processing) in nontonal language speakers. The consistent pattern of function-dependent brain asymmetry and the inconsistent pattern of acoustic-dependent brain asymmetry between tonal and nontonal language speakers can be explained by the hypothesis that the acoustic-dependent brain asymmetry is the consequence of a carryover effect from function-dependent brain asymmetry. Potential evolutionary implication of this hypothesis is discussed.

Activation of the Proapoptotic Bcl-2 Protein Bax by a Small Molecule Induces Tumor Cell Apoptosis

ABSTRACT The proapoptotic Bcl-2 protein Bax by itself is sufficient to initiate apoptosis in almost all apoptotic paradigms. Thus, compounds that can facilitate disruptive Bax insertion into mitochondrial membranes have potential as cancer therapeutics. In our study, we have identified small-molecule compounds predicted to associate with the Bax hydrophobic groove by a virtual-screen approach. Among these, one lead compound (compound 106) promotes Bax-dependent but not Bak-dependent apoptosis. Importantly, this compound alters Bax protein stability in vitro and promotes the insertion of Bax into mitochondria, leading to Bax-dependent permeabilization of the mitochondrial outer membrane. Furthermore, as a single agent, compound 106 inhibits the growth of transplanted tumors, probably by inducing apoptosis in tumors. Our study has revealed a compound that activates Bax and induces Bax-dependent apoptosis, which may lead to the development of new therapeutic agents for cancer.

Distinct roles of mitochondria- and ER-localized Bcl-xL in apoptosis resistance and Ca2+ homeostasis.

Bcl-xL localized to mitochondria is necessary and sufficient for apoptotic protection but is unable to restore Ca2+ homeostasis in Bcl-x-KO cells. ER-localized Bcl-xL is required for ER Ca2+ homeostasis but does not affect apoptosis unless Bcl-xL is present in additional cellular compartments.

Cellular ATP content was decreased by a homogeneous 8.5 T static magnetic field exposure: Role of reactive oxygen species

The literature on the impact of strong static magnetic fields (SMF) on human health is vast and contradictory. The present study focused on the cellular effects of strong homogeneous SMF in human-hamster hybrid (A(L) ) cells, mitochondria-deficient (ρ(0) A(L) ) cells, and double-strand break (DSB) repair-deficient (XRS-5) cells. Adenosine triphosphate (ATP) content was significantly decreased in A(L) cells exposed to 8.5u2009Tesla (T) but not 1 or 4u2009T SMF for either 3 or 5u2009h. In addition, ATP content significantly decreased in the two deficient cell lines exposed to 8.5u2009T SMF for 3u2009h. With further incubation of 12 or 24u2009h without SMF exposure, ATP content could retrieve to the control level in the A(L) cells but not ρ(0) A(L) and XRS-5 cells. Under a fluorescence reader, the levels of reactive oxygen species (ROS) in the three cell lines were significantly increased by exposure to 8.5u2009T SMF for 3u2009h. Concurrent treatment with ROS inhibitor, DMSO, dramatically suppressed the ATP content in exposed A(L) cells. However, the CD59 mutation frequency and the cell cycle distribution were not significantly affected by exposure to 8.5u2009T SMF for 3u2009h. Our results indicated that the cellular ATP content was reduced by 8.5u2009T SMF for 3u2009h exposure, which was partially mediated by mitochondria and the DNA DSB repair process. Moreover, ROS were involved in the process of the cellular perturbations from the SMF.

Mitochondrial dysfunction resulting from loss of cytochrome c impairs radiation-induced bystander effect

Cytochrome c is a pivotal protein that resides in mitochondria as component of mitochondria respiration and apoptosis initiator. Using murine cells lacking cytochrome c, we showed here that cytochrome c-deficient cells had attenuated reactive oxygen species/nitric oxide and micronuclei induction to radiation-induced bystander signals, indicating cytochrome c is essential for the bystander effect.

PFOS-induced apoptosis through mitochondrion-dependent pathway in human–hamster hybrid cells

Perfluorooctane sulfonate (PFOS) was listed as one of the persistent organic pollutants (POPs) in Stockholm Convention in 2009. Recent evidence showed that PFOS could induce apoptosis both in vivo and in vitro. However, the apoptotic mechanisms induced by PFOS as well as the possible relationship between apoptosis and other PFOS-induced endpoints, remain unclear. In the present study, normal human-hamster hybrid (AL) cells and mtDNA-depleted (ρ(0) AL) cells were exposed to PFOS, and assayed for cytotoxicity, mutagenicity, and apoptosis (caspase-3/7, caspase-9 activities). Our results showed that PFOS decreased cell viability in a time- and concentration-dependent manner in AL cells, but not in ρ(0) AL cells. However, long-term exposure to PFOS failed to induce the mutagenic effects at the CD59 locus in AL cells. Exposure to 200 μM PFOS significantly increased the activities of caspase-3/7 and caspase-9 in AL cells, but the activities of these caspases were not affected in ρ(0) AL cells. In addition, PFOS increased the levels of reactive oxygen species (ROS), superoxide anion (O2(-)), as well as nitric oxide (NO), and decreased mitochondrial membrane potential (MMP) at the concentrations of 100 and 200μM in AL cells. On the other hand, exposure to PFOS had no effect on intracellular ROS, O2(-), and NO production in ρ(0) AL cells. Caspase-3/7 activity, which was increased by 200 μM PFOS, could be suppressed by ROS/O2(-) scavengers and nitric oxide synthases (NOSs) inhibitors in AL cells. These results implicate that PFOS-induced apoptosis and oxidative stress is mediated by a mitochondrion-dependent pathway and that the induction of apoptosis might be a protective function against mutagenesis in AL cells exposed to PFOS.

Mutagenicity of PFOA in Mammalian Cells: Role of Mitochondria-Dependent Reactive Oxygen Species

Mutagenicity is often a prerequisite to the development of malignancy. Evidences have shown that exposure to perfluorooctanoic acid (PFOA) results in various cancer inductions. However, whether any mutagenic base exists is still puzzling. In the present study, we exposed exponentially growing AL cells to PFOA and assayed the cells for survival, mutation induction, and caspase-3/7, -9 activities. Mitochondrial-DNA deficient human-hamster hybrid (ρ(0) AL) cells and reactive oxygen species (ROS) inhibitor were used to elucidate the possible mechanism. Our results showed that treatment of AL cells with PFOA for 16 days induced significant mutagenic effects together with the increment of ROS, superoxide anions (O2(.-)), and nitrogen oxide (NO) levels, while treatment of ρ(0) AL cells did not have much change. Concurrent treatment of AL cells with ROS inhibitor significantly decreased the mutagenic potential of PFOA. In addition, caspase activities in AL cells were increased by PFOA exposure and suppressed by ROS/RNS (reactive oxygen/nitrogen species) inhibitors. Our results suggest that exposure to PFOA lead to mutagenicity induction in AL cells, and mitochondria-dependent ROS plays an important role in this process. This provides a direct base for PFOA mediated cancer induction.