Bax-alpha promotes apoptosis induced by cancer chemotherapy and accelerates the activation of caspase 3-like cysteine proteases in p53 double mutant B lymphoma Namalwa cells

Stable transfected human p53 (mt/mt) B lymphoma Namalwa variant lines showing differential expression of the Bax-alpha protein were derived under hygromycin selection. Overexpression of Bax-alpha in these variant cells accelerates cell death induced by short or continuous treatments with various concentrations of camptothecin, etoposide, vinblastine and shows no accelerating cell death activity in cis-platinum and paclitaxel-treated cells. Activation of apoptosis and oligonucleosome-sized DNA fragmentation was observed in the variant lines with more pronounced effect in cells containing high level of Bax-alpha protein. These results suggest that increased cell death mediated by anticancer drugs correlates with Bax-alpha level of expression and that Bax-alpha sensitizes Namalwa cells treated at low drug concentrations. The extent of DNA synthesis inhibition following DNA topoisomerase inhibitor treatments was similar in control and all transfected Namalwa cells suggesting that Bax-alpha acts downstream of DNA topoisomerase-mediated DNA strand breaks. To define further the relation between Bax-alpha expression and apoptosis activation, kinetics of caspase activation was measured in drug-treated cells. Caspase activities were measured using specific fluorogenic peptide derivatives DABCYL-YVADAPV-EDANS and Ac-DEVD-AMC, substrates of the caspase 1-like and caspase 3-like families, respectively. In control and Bax-alpha transfected Namalwa cells no increase in caspase 1-like activity was detected following camptothecin and etoposide treatments. In contrast, a significant difference in Ac-DEVD-AMC hydrolysis activity was observed in Bax-alpha transfected Namalwa cells compared to that of control Namalwa cells after camptothecin and etoposide treatment. Increased caspase 3-like activity correlated also with poly(ADPribosyl) polymerase cleavage. Taken together, these results suggest that Bax-alpha sensitize B lymphoma cells to series of anticancer drugs and accelerates the activation of apoptotic protease cascade.     

Real time analysis of tumor necrosis factor-related apoptosis-inducing ligand/cycloheximide-induced caspase activities during apoptosis initiation

Employing fluorescence resonance energy transfer (FRET) imaging, we previously demonstrated that effector caspase activation is often an all-or-none response independent of drug choice or dose administered. We here investigated the signaling dynamics during apoptosis initiation via the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor pathway to investigate how variability in drug exposure can be translated into largely kinetically invariant cell death execution pathways. FRET-based microscopy demonstrated dose-dependent responses of caspase-8 activation and activity within individual living HeLa cells. Caspase-8 on average was activated 45-600 min after TRAIL/cycloheximide addition. Caspase-8-like activities persisted for 15-60 min before eventually inducing mitochondrial outer membrane permeabilization. Independent of the TRAIL concentrations used or the resulting caspase-8-like activities, mitochondrial outer membrane permeabilization was induced when 10% of the FRET substrate was cleaved. In contrast, in Bid-depleted cells, caspase-8-like activity persisted for hours without causing immediate cell death. Our findings provide detailed insight into the intracellular signaling kinetics during apoptosis initiation and describe a threshold mechanism controlling the induction of apoptosis execution.     

High throughput ratio imaging to profile caspase activity: potential application in multiparameter high content apoptosis analysis and drug screening

Recent advancement in the area of green fluorescent protein techniques coupled with microscopic imaging has significantly contributed in defining and dissecting subcellular changes of apoptosis with high spatio-temporal resolution. Although single cell based studies using EGFP and associated techniques have provided valuable information of initiation and hierarchical changes of apoptosis, they are yet to be exploited for multiparameter cell based real time analysis for possible drug screening or pathway defining in a high throughput manner. Here we have developed multiple cancer cell lines expressing FRET sensors for active caspases and adapted them for high throughput live cell ratio imaging, enabling high content image based multiparameter analysis. Sensitivity of the system to detect live cell caspase activation was substantiated by confocal acceptor bleaching as well as wide field FRET imaging. Multiple caspase-specific activities of DEVDase, IETDase and LEHDase were analysed simultaneously with other decisive events of cell death. Through simultaneous analysis of caspase activation by FRET ratio change coupled with detection of mitochondrial membrane potential loss or superoxide generation, we identified several antitumor agents that induced caspase activation with or without membrane potential loss or superoxide generation. Also, cells that escaped the initial drug-induced caspase activation could be easily followed up for defining long term fate. Employing such a revisit imaging strategy of the same area, we have tracked the caspase surviving fractions with multiple drugs and its subsequent response to retreatment, revealing drug-dependent diverging fate of surviving cells. This thereby indicates towards a complex control of drug induced tumor resistance. The technique described here has wider application in both screening of compound libraries as well as in defining apoptotic pathways by linking multiple signaling to identify non-classical apoptosis inducing agents, the greatest advantage being that the high content information obtained are from individual cells rather than being population based.     

TRAIL-induced apoptosis proceeding from caspase-3-dependent and -independent pathways in distinct HeLa cells

The apoptotic pathway in higher eukaryotes remains controversial with respect to the necessity of activation of caspase-3 in TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)-treated cells. In this study, a fluorescence resonance energy transfer (FRET) probe was developed to image the activation of caspase-3 and the related apoptotic pathway in TRAIL-treated cells in real time. Both kinds of apoptotic pathways were observed simultaneously in the same experiment proceeding from activation and non-activation of caspase-3. The total apoptotic rate was 56.08%, the apoptotic rates for activation and non-activation of caspase-3 pathways were 21.5% and 34.58%, respectively, which were examined later for Hoechst 33258 staining and morphological characteristics. The apoptotic rate due to the activation of caspase-3 pathways in TRAIL-treated cells has been independently measured to be around 25.11% by capillary electrophoresis (CE) analysis, which confirmed the apoptotic rate due to activation of caspase-3 pathways as found by FRET analysis. This result also suggests that rest apoptosis is preceded by caspase-3-independent pathways, as CE has the ability to quantitatively detect caspase-dependent apoptosis. The observation of the coexistence of caspase-3-dependent and caspase-3-independent apoptotic pathways in the TRAIL-treated cells was unusual in comparison with the previous reports.     

Single-cell fluorescence resonance energy transfer analysis demonstrates that caspase activation during apoptosis is a rapid process. Role of caspase-3

Activation of effector caspases is considered to be the final step in many apoptosis pathways. We transfected HeLa cells with a recombinant caspase substrate composed of cyan and yellow fluorescent protein and a linker peptide containing the caspase cleavage sequence DEVD, and we examined the cleavage kinetics at the single-cell level by fluorescence resonance energy transfer (FRET) analysis. Caspase activation in response to tumor necrosis factor-alpha, staurosporine, or etoposide resulted in cleavage of the linker peptide and subsequent disruption of the FRET signal. The time to caspase activation varied among individual cells, depending on the type of treatment and concentration used. However, once initiated, disruption of the FRET signal was always rapid (相似文献   

Role of death receptor and mitochondrial pathways in conventional chemotherapy drug induction of apoptosis

The molecular mechanism underlying chemotherapy-induced apoptosis is often debated because of contradicting reports of its signaling pathway. The focus of this ongoing debate is on the requirement of a death receptor and its role in subsequent activation of caspase-8. Understanding the precise mechanism responsible for apoptosis and identifying molecules targeted by chemotherapy will allow us to develop better therapeutic strategies that target the inherent abnormalities of cancer cells. To show conventional chemotherapy drugs can trigger the caspase cascade, including caspase-8, -9, -3 and DNA fragmentation factor, Jurkat T leukemia cells were treated with cisplatin or etoposide in a dose-dependent and a time-dependent manner. Cisplatin and etoposide all induced apoptosis in wild-type Jurkat T leukemia cells. On the other hand, when a pan-caspase inhibitor zVAD-FMK was pretreated, apoptosis did not occur, indicating that these chemotherapy drugs mediated caspase-dependent apoptosis. However, the chemotherapy drug induction of apoptosis was not inhibited by treatment of zIETD-FMK, a caspase-8 inhibitor. There was no difference in cell death between wild-type and caspase-8 or FADD-deficient Jurkat cells after treatment of chemotherapy drug. In addition, cisplatin-induced apoptosis is abrogated by the overexpression of either Bcl-2 or Bcl-x(L), which diminished changes of mitochondrial membrane potential and decreased the amount of cytochrome c released from mitochondria. Again, cisplatin-induced apoptosis was not diminished by c-FLIP-overexpression, whereas the c-FLIP-overexpressing cells were less sensitive to TRAIL-induced apoptosis than the wild type cells. Therefore, these results indicate that conventional chemotherapy drug-triggered apoptosis is indispensable, and its pathway is independent of the death receptor.     

利用荧光共振能量转移技术监测高通量低能量激光诱导细胞凋亡过程中caspase-3活性的动态变化

荧光共振能量转移可用于对生物大分子之间的距离进行定性、定量检测。应用荧光共振能量转移技术对高通量低能量激光诱导肺腺癌细胞凋亡过程中caspase-3的激活过程进行实时动态监测。实验结果表明:高通量低能量激光可以诱导肺腺癌细胞(human lung adenocarcinoma cell,ASTC-a-1)凋亡。同时荧光共振能量转移技术是一个有效的监测caspase-3在凋亡过程中活性动态变化的方法。     

Common regulation of apoptosis signaling induced by CD95 and the DNA-damaging stimuli etoposide and gamma-radiation downstream from caspase-8 activation.

The death receptor CD95 (APO-1/Fas), the anticancer drug etoposide, and gamma-radiation induce apoptosis in the human T cell line Jurkat. Variant clones selected for resistance to CD95-induced apoptosis proved cross-resistant to etoposide- and radiation-induced apoptosis, suggesting that the apoptosis pathways induced by these distinct stimuli have critical component(s) in common. The pathways do not converge at the level of CD95 ligation or caspase-8 signaling. Whereas caspase-8 function was required for CD95-mediated cytochrome c release, effector caspase activation, and apoptosis, these responses were unaffected in etoposide-treated and irradiated cells when caspase-8 was inhibited by FLIPL. Both effector caspase processing and cytochrome c release were inhibited in the resistant variant cells as well as in Bcl-2 transfectants, suggesting that, in Jurkat cells, the apoptosis signaling pathways activated by CD95, etoposide, and gamma-radiation are under common mitochondrial control. All three stimuli induced ceramide production in wild-type cells, but not in resistant variant cells. Exogenous ceramide bypassed apoptosis resistance in the variant cells, but not in Bcl-2-transfected cells, suggesting that apoptosis signaling induced by CD95, etoposide, and gamma-radiation is subject to common regulation at a level different from that targeted by Bcl-2.     

Human cytomegalovirus (HCMV) IE1 plays role in resistance to apoptosis with etoposide in cancer cell line by Cdk2 accumulation

Human cytomegalovirus (HCMV) has many strategies to survive the attack of the host. HCMV infection of host cells induces cellular activation and disturbance of the cell cycle. It is possible that HCMV modulates the behavior of certain cancer cells that are susceptible to HCMV infection. This study was performed to identify the possible mechanism of resistance to apoptotic stimuli in some cancer cell lines by HCMV infection. HCMV-infected cancer cells showed resistance to apoptosis induced by the topoisomerase II inhibitor etoposide. UMG1-2, which constitutively expresses HCMV immediate-early protein-1 (IE1), had resistance to apoptosis induced by etoposide as compared with the parental cell line U373MG. Measurement of caspases activity with fluorogenic substrates in etoposide-treated U373MG and UMG1-2 cells and the direct activation of caspase-3 with peptides containing arginine-glycine-aspartate in U373MG and UMG1-2 cells revealed that the inhibition level of apoptosis by HCMV IE1 would be upstream of caspase-3 in the caspase cascade pathway. Cellular expression of Cdk2 was increased in UMG1- 2 after etoposide treatment while the expression of E2F-1 in UMG1-2 was decreased as compared with that in U373MG. The Cdk2 inhibitor, roscovitine, decreased the resistance to apoptosis on etoposide-treated UMG1-2. These results suggest that aberrant HCMV infection confers resistance to anticancer drugs on some cancer cells and protects cells from apoptosis, possibly due to the deregulation of cyclin-dependent kinase by HCMV immediate-early protein.     

A high-throughput direct fluorescence resonance energy transfer-based assay for analyzing apoptotic proteases using flow cytometry and fluorescence lifetime measurements

Cytometry is a versatile and powerful method applicable to different fields, particularly pharmacology and biomedical studies. Based on the data obtained, cytometric studies are classified into high-throughput (HTP) or high-content screening (HCS) groups. However, assays combining the advantages of both are required to facilitate research. In this study, we developed a high-throughput system to profile cellular populations in terms of time- or dose-dependent responses to apoptotic stimulations because apoptotic inducers are potent anticancer drugs. We previously established assay systems involving protease to monitor live cells for apoptosis using tunable fluorescence resonance energy transfer (FRET)-based bioprobes. These assays can be used for microscopic analyses or fluorescence-activated cell sorting. In this study, we developed FRET-based bioprobes to detect the activity of the apoptotic markers caspase-3 and caspase-9 via changes in bioprobe fluorescence lifetimes using a flow cytometer for direct estimation of FRET efficiencies. Different patterns of changes in the fluorescence lifetimes of these markers during apoptosis were observed, indicating a relationship between discrete steps in the apoptosis process. The findings demonstrate the feasibility of evaluating collective cellular dynamics during apoptosis.     

Resistance to caspase-8 and -9 fragments in a malignant pleural mesothelioma cell line with acquired cisplatin-resistance

Apoptotic cysteine–aspartate proteases (caspases) are essential for the progression and execution of apoptosis, and detection of caspase fragmentation or activity is often used as markers of apoptosis. Cisplatin (cis-diamminedichloroplatinum (II)) is a chemotherapeutic drug that is clinically used for the treatment of solid tumours. We compared a cisplatin-resistant pleural malignant mesothelioma cell line (P31res1.2) with its parental cell line (P31) regarding the consequences of in vitro acquired cisplatin-resistance on basal and cisplatin-induced (equitoxic and equiapoptotic cisplatin concentrations) caspase-3, -8 and -9 fragmentation and proteolytic activity. Acquisition of cisplatin-resistance resulted in basal fragmentation of caspase-8 and -9 without a concomitant increase in proteolytic activity, and there was an increased basal caspase-3/7 activity. Similarly, cisplatin-resistant non-small-cell lung cancer cells, H1299res, had increased caspase-3 and -9 content compared with the parental H1299 cells. In P31 cells, cisplatin exposure resulted in caspase-9-mediated caspase-3/7 activation, but in P31res1.2 cells the cisplatin-induced caspase-3/7 activation occurred before caspase-8 or -9 activation. We therefore concluded that in vitro acquisition of cisplatin-resistance rendered P31res1.2 cells resistant to caspase-8 and caspase-9 fragments and that cisplatin-induced, initiator-caspase independent caspase-3/7 activation was necessary to overcome this resistance. Finally, the results demonstrated that detection of cleaved caspase fragments alone might be insufficient as a marker of caspase activity and ensuing apoptosis induction.     

In vivo imaging of early stage apoptosis by measuring real-time caspase-3/7 activation

In vivo imaging of apoptosis in a preclinical setting in anticancer drug development could provide remarkable advantages in terms of translational medicine. So far, several imaging technologies with different probes have been used to achieve this goal. Here we describe a bioluminescence imaging approach that uses a new formulation of Z-DEVD-aminoluciferin, a caspase 3/7 substrate, to monitor in vivo apoptosis in tumor cells engineered to express luciferase. Upon apoptosis induction, Z-DEVD-aminoluciferin is cleaved by caspase 3/7 releasing aminoluciferin that is now free to react with luciferase generating measurable light. Thus, the activation of caspase 3/7 can be measured by quantifying the bioluminescent signal. Using this approach, we have been able to monitor caspase-3 activation and subsequent apoptosis induction after camptothecin and temozolomide treatment on xenograft mouse models of colon cancer and glioblastoma, respectively. Treated mice showed more than 2-fold induction of Z-DEVD-aminoluciferin luminescent signal when compared to the untreated group. Combining D-luciferin that measures the total tumor burden, with Z-DEVD-aminoluciferin that assesses apoptosis induction via caspase activation, we confirmed that it is possible to follow non-invasively tumor growth inhibition and induction of apoptosis after treatment in the same animal over time. Moreover, here we have proved that following early apoptosis induction by caspase 3 activation is a good biomarker that accurately predicts tumor growth inhibition by anti-cancer drugs in engineered colon cancer and glioblastoma cell lines and in their respective mouse xenograft models.     

Involvement of protein kinase C-regulated ceramide generation in inostamycin-induced apoptosis

Activation of caspases is commonly involved in the apoptosis induced by various anticancer drugs. However, the upstream events leading to the activation of caspases seem to be specific to each anticancer drug. In the present study, we examined the possible involvement of protein kinase C (PKC) and ceramide generation in caspase-3(-like) protease activation induced by inostamycin, a phosphatidylinositol synthesis inhibitor. Treatment of cells with 12-O-tetradecanoyl phorbol-13-acetate (TPA), an activator of PKC, suppressed the release of cytochrome c from mitochondria and the activation of caspase-3(-like) proteases in inostamycin-treated cells, but not in other anticancer drug-treated cells. Inostamycin induced the elevation of intracellular ceramide levels, and fumonisin B1, an inhibitor of ceramide synthase, inhibited inostamycin-induced cytochrome c release, caspase-3(-like) protease activation, and apoptosis. Moreover, TPA also inhibited inostamycin-induced ceramide synthesis. Taken together, our results suggest that inostamycin-induced apoptosis is mediated by PKC-regulated ceramide generation, leading to the activation of a caspase cascade.     

Application of the fluorescence resonance energy transfer method for studying the dynamics of caspase-3 activation during UV-induced apoptosis in living HeLa cells

Activation of caspase-3 is a central event in apoptosis. We have developed a GFP-based FRET (fluorescence resonance energy transfer) probe that is highly sensitive to the activation of caspase-3 in intact living cells. This probe was constructed by fusing a CFP (cyan fluorescent protein) and a YFP (yellow fluorescent protein) with a specialized linker containing the caspase-3 cleavage sequence: DEVD. The linker design was optimized to produce a large FRET effect. Using purified protein, we observed a fivefold change in the fluorescence emission ratio when the probe was cleaved by caspase-3. To demonstrate the usefulness of this method, we introduced this FRET probe into HeLa cells by both transient and stable transfection. We observed that during UV-induced apoptosis, the activation of caspase-3 varied significantly between different cells; but once the caspase was activated, the enzyme within the cell became fully active within a few minutes. This technique will be highly useful for correlating the caspase-3 activation with other apoptotic events and for rapid-screening of potential drugs that may target the apoptotic process.     

Quantitative analysis of fluorescent caspase substrate cleavage in intact cells and identification of novel inhibitors of apoptosis

Caspase activation and proteolytic cleavage of specific target proteins represents an integral step in the pathway leading to the apoptotic death of cells. Analysis of caspase activity in intact cells, however, has been generally limited to the measurement of end-point biochemical and morphological markers of apoptosis. In an effort to develop a strategy with which to monitor caspase activity, early in the cell death cascade and in real-time, we have generated cell lines that overexpress recombinant GFP-based caspase substrates that display a quantifiable change in their spectral properties when cleaved by group II caspases. Specifically, tandem GFP substrates linked by a caspase-sensitive cleavage site show diminished fluorescence resonance energy transfer (FRET), as a consequence of cleavage, due to physical separation of the GFP moieties in apoptotic cells. We have evaluated the influence of different caspase-sensitive linkers on both FRET efficiency and cleavage by caspase-3. We also demonstrate that caspase activity as well as inhibition by pharmacological agents can be monitored, with minimal manipulation, in intact adherent cells seeded in a 96-well cell culture dish. Finally, we have adapted this technology to a high throughput screening platform to identify novel small molecule and cell permeable inhibitors of apoptosis. Based on a biochemical analysis of the compounds identified it is clear that this assay can be used to detect drugs which inhibit caspases directly as well as those which target upstream components of the caspase cascade.     

A high-throughput method for development of FRET-based indicators for proteolysis

SCAT3 is a fluorescence resonance energy transfer (FRET)-based indicator for activity of caspase-3, which is composed of an enhanced cyan fluorescent protein, a caspase-3-sensitive linker, and an enhanced yellow fluorescent protein with efficient maturation property (Venus). Despite its considerable promise, however, greater responsivity of fluorescence to the proteolysis has been desired for better understanding of spatio-temporal pattern of the activation of caspase-3 during apoptosis. In the present study, the length of linker regions of SCAT3 has been thoroughly optimized by use of a PCR technique. The bacterial colonies expressing the constructs were screened for high FRET efficiency using our home-made fluorescence image analyzer. The FRET signal of an improved SCAT3 changed by about tenfold during apoptotic events in mammalian cells, enabling visualization of caspase-3 activation with better spatial resolution than before. This new high-throughput method will be applicable to development and improvement of FRET-based indicators for proteolysis.     

Characterization of proteinase activation dynamics by capillary electrophoresis conjugating with fluorescent protein-based probe

In this paper, a novel strategy was reported to characterize dynamics of proteinase activation based on capillary electrophoresis (CE), using caspase-2 as the model system. A fusion protein conjugating an amino acid sequence VDVAD with two fluorescent proteins enhanced cyan fluorescence protein (ECFP) and red fluorescence protein (DsRed), ECFP-VDVAD-DsRed, was specially designed and expressed in HeLa cells as the substrate of proteinase caspase-2. In this substrate, the sequence VDVAD could be specifically recognized and cleaved by caspase-2 as soon as its activation was initiated with treatment of a certain dose of cisplatin to HeLa cells, which led to a break of the substrate into two fragments. Analyses of the cell lysates using CE in a time course of the apoptosis illustrated the dynamics of caspase-2 activation. It showed that the employment of fusion fluorescent protein greatly facilitated both CE separation and identification of the analytes. This result from cell colony by CE was compared with that from single cell achieved by optical imaging.     

Contrasting effects of α-tocopheryl succinate on cisplatin- and etoposide-induced apoptosis

Targeting mitochondria is a promising strategy in tumor cell elimination. d-α-tocopheryl succinate (α-TOS), a redox-silent analog of vitamin E, is a potentially powerful tool for fighting tumors by directly affecting mitochondria. However, when used at low concentrations it can suppress apoptosis induced by the conventionally used anticancer drug cisplatin. In cells treated with cisplatin, 30 μM α-TOS prominently attenuated the manifestation of characteristic features of apoptosis — release of cytochrome c from mitochondria, caspase-3-like activity, and cleavage of poly(ADP-ribose) polymerase. In contrast, cell death induced by etoposide was not inhibited but rather stimulated by α-TOS. Thus, co-treatment with α-TOS and conventional antitumor drugs should be carried out with caution.