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Drp1 mediates caspase-independent type III cell death in normal and leukemic cells - PubMed

. 2007 Oct;27(20):7073-88.

doi: 10.1128/MCB.02116-06. Epub 2007 Aug 6.

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Drp1 mediates caspase-independent type III cell death in normal and leukemic cells

Marlène Bras et al. Mol Cell Biol. 2007 Oct.

Abstract

Ligation of CD47 triggers caspase-independent programmed cell death (PCD) in normal and leukemic cells. Here, we characterize the morphological and biochemical features of this type of death and show that it displays the hallmarks of type III PCD. A molecular and biochemical approach has led us to identify a key mediator of this type of death, dynamin-related protein 1 (Drp1). CD47 ligation induces Drp1 translocation from cytosol to mitochondria, a process controlled by chymotrypsin-like serine proteases. Once in mitochondria, Drp1 provokes an impairment of the mitochondrial electron transport chain, which results in dissipation of mitochondrial transmembrane potential, reactive oxygen species generation, and a drop in ATP levels. Surprisingly, neither the activation of the most representative proapoptotic members of the Bcl-2 family, such as Bax or Bak, nor the release of apoptogenic proteins AIF (apoptosis-inducing factor), cytochrome c, endonuclease G (EndoG), Omi/HtrA2, or Smac/DIABLO from mitochondria to cytosol is observed. Responsiveness of cells to CD47 ligation increases following Drp1 overexpression, while Drp1 downregulation confers resistance to CD47-mediated death. Importantly, in B-cell chronic lymphocytic leukemia cells, mRNA levels of Drp1 strongly correlate with death sensitivity. Thus, this previously unknown mechanism controlling caspase-independent type III PCD may provide the basis for novel therapeutic approaches to overcome apoptotic avoidance in malignant cells.

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Figures

FIG. 1.
FIG. 1.

CD47 ligation induces caspase-independent type III PCD. (A) Normal cells (B cells) or CLL cells left untreated (control) or incubated with HC, TSP, or CD47 MAb for 1 h were stained with MGG to assess cellular morphology (left panels). Representative micrographs of each treatment are shown. Alternatively, B lymphocytes from a representative CLL donor were left untreated (control) or incubated with HC or CD47 MAb for 6 h before being stained with green fluorescent CellTracker and Hoechst 33342 to evaluate cellular viability and chromatin condensation. HMC microscopy was used to visualize cells (right panels). Representative micrographs of each stain are shown. The frequencies of green cells and nuclear chromatin condensation were determined by microscopic observation at the indicated times. Each point indicates the mean ± standard deviation from four independent experiments. (B) Growth rate assessment in CD47 MAb treated Jurkat T cells. Cells were cultured for 1 h in the presence of 1 to 5 μg/ml CD47 MAb, followed by quantification of cell number with a Quantos cell proliferation assay kit. Cells were seeded in 96-well plates, and the cell number was analyzed at the times indicated in a plate reader fluorometer. One unit refers to the fluorescence emitted by 25,000 cells. Values are means (± standard deviations) from four independent experiments (left panel). Representative results obtained from cells treated with 5 μg/ml CD47 MAb at different times are shown in the right panel. Cell numbers were quantified as described above. Data represent the means ± standard deviations (n = 4). CD47 ligation induces loss of viability, and consequently the number of cells measured after CD47 ligation was significantly lower than that for control cells. (C) Electron micrographs of CLL cells left untreated (panels a and e) or incubated with CD47 MAb for either 1 h (panels b and f to h), 6 h (panel c), or 16 h (panel d). Panel e demonstrates a typical example of the mitochondrial (MT), ER, and Golgi apparatus (G) normal morphology. Organelles are marked with arrowheads. Panels f, g, and h, respectively, show the mitochondrial morphology, ER dilation and redistribution, and Golgi swelling observed in CD47 MAb-treated cells. Organelles are marked with arrows. Bars: a to d, 1 μm; e to h, 100 nm. (D) In situ evidence of ER redistribution and Golgi apparatus dilation observed after CD47 MAb treatment. CLL cells were fixed and stained for immunodetection of the ER transmembrane protein calnexin and the cis-Golgi KDEL receptor. Representative micrographs are shown. The redistribution of each protein marker was quantified by microscopic observation at the indicated times. Each histogram indicates the mean ± standard deviation for three fields of at least 100 cells within a representative experiment. (E) Kinetic analysis of the PS exposure, cell viability, ΔΨm dissipation, ROS production, and lysosomal permeabilization induced by TSP, HC, or CD47 MAb. After the indicated times, cells were labeled with DiOC6(3) and annexin V-APC, hydroethidine (HE), or LysoTracker Red. The data (means of a triplicate) obtained from a healthy donor or a representative CLL patient, after accounting for spontaneous apoptosis, are shown. This experiment was done four times, yielding low interexperiment variability (<5%). (F) Assessment of oligonucleosomal DNA fragmentation of CLL cells that were left untreated (lanes 1 and 2) or incubated with HC (lanes 3 and 4) or CD47 MAb (6 h) (lanes 5 and 6) in the absence (lanes 1, 3, and 5) or presence (lanes 2, 4, and 6) of the pan-caspase inhibitor z-VAD.fmk. (G) Quantification intracellular ATP levels in CLL cells treated at different times with CD47 MAb. HC and H2O2 were used as apoptotic and necrotic inducers, respectively. (H) Assessment of ΔΨm loss and PS exposure in CLL cells preincubated with pan-caspase inhibitor QVD.OPh or z-VAD.fmk or with specific inhibitors of caspases 2 (z-VDVAD.fmk), 3 and 7 (z-DEVD.fmk), 9 (z-LEHD.fmk), or 8 and 10 (z-IETD.fmk) before induction of apoptosis by CD47 MAb (1 h) or HC. Results are the means ± standard deviations from five experiments. (I) Fluorometric measurement of caspase 3/7 activity observed in cytosolic extracts obtained from CLL cells untreated (Co) or treated with CD47 MAb (6 h) or HC in the absence or presence of the pan-caspase inhibitor z-VAD.fmk. Results are the means ± standard deviations from three experiments. (J) Total cell lysates from CLL untreated (Co) or treated with HC or CD47 MAb (6 h) were probed for the detection of activated caspase 9 or caspase 3. Arrows indicate the cleaved form of caspase 9 or 3, which is revealed only after HC treatment. Equal loading was confirmed by tubulin assessment.

FIG. 2.
FIG. 2.

Implication of chymotrypsin-like serine proteases in CD47-mediated cell death. (A) ΔΨm loss induced by 1 h of CD47 ligation in CLL cells left untreated (time zero) or pretreated with inhibitors of chymotrypsin-like serine proteases (TPCK, at different concentrations), trypsin-like serine proteases (TLCK), type I or II calpains (MG101 and LLM), cathepsins B/D/L (z-FA.fmk and leupeptin), or proteasome (MG132, lactacystin, and NLVS). DEX, thapsigargin (Thaps.), hydroxychloroquine (HCQ), and brefeldin A/cycloheximide (BFA/CHX) were used as positive controls for trypsin-like, calpain, cathepsin, and proteasome activities, respectively. Data are means ± standard deviations from four independent experiments. (B) Effect of TPCK pretreatment on the inhibition of cell growth induced by CD47 ligation. Cells were cultured for 1 h in the absence (control) or presence of 5 μg/ml CD47 MAb, followed by quantification of cell number by a Quantos cell proliferation assay kit as for Fig. 1B. Alternatively, cells were pretreated with the inhibitor of chymotrypsin-like serine proteases TPCK at different concentrations before CD47 MAb treatment and proliferation was assessed as above. Note that TPCK reverses the effect of the CD47 MAb treatment. One unit refers to the fluorescence emitted by 25000 cells. Values are means (± standard deviations) from six independent experiments. (C) Fluorimetric measurement of chymotrypsin-like (Suc-AAPK.amc), trypsin-like (Boc-VLK.amc), and cathepsin (Z-RR.amc) protease activities observed in cytosolic extracts obtained from CLL cells incubated for 1 h with CD47 MAb in the absence or presence of the protease inhibitor TPCK or TLCK. One unit refers to the basal enzymatic activity measured in untreated cells. DEX and the lysosomotropic agent HCQ were used as positive controls for trypsin-like and cathepsin activities, respectively. Data are means ± standard deviations from four independent experiments. (D) B lymphocytes from CLL donors were left untreated (control), or incubated with CD47 MAb (1 h), CD47 MAb plus TPCK (20 μM), or CD47 MAb plus TLCK (20 μM) before assessment of chymotrypsin-like serine protease activity with a green fluorescent SerPase kit (FFCK). Representative micrographs of each treatment are shown. HMC microscopy was used to visualize cells. Note that TPCK significantly inhibits chymotrypsin-like serine protease labeling. (E) Normal B lymphocytes (B cells) or B lymphocytes from a CLL donor were left untreated (control) or incubated with CD47 MAb (1 h) or CD47 MAb plus TPCK (20 μM) before assessment of chymotrypsin-like serine protease activity as for panel D. Numbers indicate the percentage of cells positively stained. The experiment was repeated four times with a low variability (<5%).

FIG. 3.
FIG. 3.

CD47-mediated PCD functions independently of the most representative members of the Bcl-2 family of proteins. (A) Real-time reverse transcription-PCR quantification of Bcl-2, Bcl-XL, Mcl-1, Bax, Bak, and Bim mRNA transcripts obtained from healthy B lymphocytes (B cell) (n = 5) or CLL B lymphocytes (n = 15) that were left untreated (CLL) or incubated 1 h with CD47 MAb (CLL + CD47). Results for CLL cells are expressed as the mean ± standard error of the mean. (B) Neither overexpression of Bcl-2, Bcl-XL, or Mcl-1 nor downregulation of Bax, Bak, or Bim elicits a change in the mitochondrial damage induced by CD47 ligation. Jurkat cell lines were stably transfected with vector only (Jk-Neo) or cDNAs coding for human Bcl-2 (Jk-Bcl-2), human Bcl-XL (Jk-Bcl-XL), human Bcl-2 specifically targeted to the ER (Jk-Bcl-2-ER), or human Mcl-1 (Jk-Mcl-1). Bcl-2, Bcl-XL, and Mcl-1 expression levels were analyzed by Western blotting. After cells were treated with either CD47 MAb (1 h) or the inductor of apoptosis etoposide or thapsigargin, the frequency of cells with a low ΔΨm was determined. Data are means ± standard deviations from four independent experiments. The effect of Bax, Bak, and Bim downregulation on the ΔΨm loss provoked by the treatment of Jurkat cells with CD47 MAb (1 h), etoposide, or the microtubule-damaging agent paclitaxel (Taxol) is also shown. The frequency of ΔΨm dissipation was assessed by DiOC6(3) labeling. Data are shown as mean values ± standard deviations for three independent experiments. Downregulation of Bax, Bak, or Bim was confirmed by Western blotting. (C) Immunofluorescent staining of activated Bax or activated Bak in CLL cells left untreated (control), or treated with HC or CD47 MAb for 1 h. Representative micrographs of each stain are shown. Enlargements are shown for detailing Bax or Bak activation. Note that only in HC-treated cells did Bax and Bax become stained (activated). As a control of cell death in this particular experiment, cells were stained with annexin V-APC and the frequency of positive labeling was recorded and illustrated as a plot. Data are means of a quadruplicate ± standard deviations. (D) Bax or Bak activation, quantified by flow cytometry, in CLL cells treated as for panel C. Numbers indicate the percentage of cells positively stained. The experiment was repeated three times with low variability (<5%). (E) CD47 ligation, unlike HC, does not induce release of apoptogenic proteins from mitochondria. Immunofluorescent detection of Hsp60 (used as a structural unreleased mitochondrial protein, green fluorescence), AIF, cytochrome c (Cyt c), Smac/DIABLO (Smac), EndoG, and Omi/HtrA2 (Omi) in CLL cells left untreated (control) or incubated with HC or CD47 MAb for 6 h is shown. Individual cells are representative of the dominant phenotype. This experiment was repeated eight times, yielding comparable results. (F) CLL cells were treated as for panel E. Mitochondrial and cytosolic extracts were analyzed by Western blotting for the presence of AIF, cytochrome c (Cyt c), Smac/DIABLO (Smac), EndoG, and Omi/HtrA2 (Omi). Cox IV and tubulin were used to control fractionation quality and protein loading.

FIG. 4.
FIG. 4.

Drp1 redistributes from cytosol to mitochondria in CD47-mediated PCD. (A) Real-time reverse transcription-PCR quantification of Opa1, mitofusin 1 (Mnf1), mitofusin 2 (Mnf2), and Drp1 mRNA transcripts purified from B lymphocytes from five control donors (B cell) or from 30 CLL patients (described in Table S1 in the supplemental material). Results for CLL patients 1 to 28 are expressed as the mean ± standard error of the mean. Total cell lysates from control B cells (B cell) along with B cells from three representative CLL patients (patients 1, 29, and 30) were prepared, and the Drp1 protein expression level was analyzed by Western blotting. Tubulin level was assessed in the same membrane as a loading control. Note that B lymphocytes from CLL patients 29 and 30, like normal B cells, showed lower Drp1 mRNA and protein expression. (B) B lymphocytes from a representative control donor (B cell) or B cells isolated from the 30 CLL patients used for panel A were cultured for 20 h in presence of CD47 MAb (black bars) or HC (yellow bars). The percentage of cells with ΔΨm loss, after accounting for spontaneous apoptosis, is shown. Values are the means of a triplicate. Patients were divided into three groups according to their relative sensitivity to HC and CD47 MAb. In a similar set of experiments, B lymphocytes from control donors or CLL patients were treated with TSP and the percentage of cells with ΔΨm loss was assessed. Results are expressed as the mean ± standard deviation (n = 4). *, P < 0.001 (unpaired Student t test). (C) Normal B lymphocytes were left untreated (control) or treated with CD47 MAb for 1 h and then stained for the detection of Drp1 and cytochrome c (Cytoch. c) (used as mitochondrial marker) before being examined by fluorescence microscopy. Representative cells show that Drp1 has a cytosolic distribution in control cells, whereas it colocalizes with cytochrome c in a mitochondrial staining pattern in CD47-treated cells. The numbers of cells showing low ΔΨm (measured by flow cytometry), mitochondrial Drp1, or diffuse cytochrome c were quantified and plotted as a percentage of total cells. Data are the means ± standard deviations from five independent experiments. (D) CLL cells were left untreated (control) or treated with TSP (6 h) or CD47 MAb (1 h) and then stained for the detection of Drp1 or cytochrome c. The number of cells showing low ΔΨm, mitochondrial Drp1, or diffuse cytochrome c were quantified as for panel C. Kinetics of the Drp1 mitochondrial redeployment were detected by immunoblotting. After CD47 MAb treatment, CLL cells were subjected to subcellular fractionation, and mitochondrial and cytosolic fractions were blotted for Drp1 immunodetection. Fractionation quality and protein loading were verified by distribution of the specific subcellular markers Cox IV for mitochondria and tubulin for cytosol. (E) Immunoblot detection of Drp1 in mitochondrial and cytosolic fractions of control cells and cells stimulated with CD47 MAb for 1 h or treated with HC for 20 h. Cells were purified from a representative patient from each group identified in panel B. Cox IV and tubulin were used to control fractionation quality and protein loading. This experiment was repeated with cells purified from other patients from each group, yielding comparable results.

FIG. 5.
FIG. 5.

Drp1 regulates CD47-mediated PCD. (A) Effect of Drp1 silencing on CD47-mediated death. Jurkat cells were transfected with a scramble or a lamin A siRNA double-stranded oligonucleotide (control siRNA) or with three different siRNA double-stranded oligonucleotides designed against human Drp1 (siRNA Drp1a, siRNA Drp1b, and siRNA Drp1c). Total cell lysates from control and Drp1 siRNA cells were prepared, and the expression level of Drp1 was analyzed by Western blotting. Analysis of hFis1 expression in these extracts confirms the specificity of Drp1 silencing. The tubulin level was also analyzed as a loading control. At 24 h after the indicated transfection, the ΔΨm collapse or the PS exposure induced by treatment with CD47 MAb (1 to 6 h), HC, or the tyrosine kinase inhibitor STP was quantified by flow cytometry. Values are means ± standard deviations from five independent experiments. (B) Effect of Drp1 silencing on the CD47-mediated loss of viability. Jurkat cells were transfected with a control siRNA double-stranded oligonucleotide or with an siRNA double-stranded oligonucleotide designed against Drp1 as defined in panel A (Drp1a). At 24 h after the indicated transfection, cells were seeded in 96-well plates and the cell number was quantified as for Fig. 1B. Drp1 silencing, which precludes CD47-mediated cell death, contributes to cell growth. Data represent the means ± standard deviations (n = 4). (C) Drp1 overexpression promotes CD47-mediated ΔΨm loss. Jurkat cells were transiently transfected with pcDNA3.1 vector only (Jk-Neo), human Drp1 cDNA (Jk-Drp1), and two cDNAs coding for human Drp1 mutated in its GTPase function, Drp1K38A (Jk-Drp1K38A) and Drp1K679A (Jk-Drp1K679A). The expression level of Drp1 was assessed by immunoblotting. Equal loading was controlled by tubulin detection. The ΔΨm collapse induced by treatment with CD47 MAb (1 to 6 h), HC, or STP was quantified by flow cytometry as for panel A. Data represent the means ± standard deviations (n = 6). (D) Drp1, Drp1 K38A, and Drp1 K679A redistribute from cytosol to mitochondria after CD47 triggering. Jurkat cells were transiently transfected with pcDNA3.1 vector only (lanes 1), human Drp1 (lanes 2), human Drp1K38A (lanes 3), and human Drp1K679A (lanes 4) as for panel C. Total cell lysates were prepared, and the expression level of Drp1 was analyzed by Western blotting. The tubulin level was also analyzed as loading control. After treatment with CD47 MAb, cells were subjected to subcellular fractionation, and mitochondrial and cytosolic fractions were blotted for the immunodetection of Drp1. Fractionation quality and protein loading were verified by the distribution of the specific subcellular markers Cox IV for mitochondria and tubulin for cytosol. Immunodetection showed that, like Drp1, Drp1K38A and Drp1K679A redistribute from cytosol to mitochondria after CD47 ligation. (E) Effect of hFis1 silencing on CD47-mediated PCD. Jurkat cells were transfected with a control siRNA double-stranded oligonucleotide or with two siRNA double-stranded oligonucleotides designed against hFis1 (siRNA hFis1a and siRNA hFis1b). Immunodetection showed the changes in hFis1 expression. Analysis of Drp1 expression in these extracts confirms the specificity of hFis1 silencing. Equal loading was confirmed by tubulin detection. After CD47 MAb (1 to 6 h), HC or STP treatment, cells were stained with DiOC6(3) or annexin V-APC to quantify ΔΨm dissipation or PS exposure, respectively. Data are shown as the means ± standard deviations (n = 5).

FIG. 6.
FIG. 6.

Effect of Drp1, Drp1K38A, and Drp1K679A recombinant proteins on purified mitochondria. (A) Freshly isolated mitochondria were incubated with recombinant Drp1, Drp1K38A, or Drp1K679A protein, and the ΔΨm was measured by flow cytometry as described in Materials and Methods. Each histogram represents the analysis of 50,000 events. The mitochondrial uncoupler mClCCP (1 μM) was used as a positive control, and the mutant protein Drp1(1-335) was used as a negative control. (B) Detection of ROS generated by Drp1, Drp1K38A, or Drp1K679A in mitochondria. Purified mitochondria (100 μg/ml) were diluted in buffer containing L-012 (100 μM), and ROS production was detected. Drp1(1-335) and bovine serum albumin (BSA) recombinant proteins were used as negative controls. Chemiluminescence was registered at intervals of 30 s over 5 min with a luminometer, and the signal was expressed as counts/min at 5 min. Data are means ± standard deviations from four independent experiments. (C) Experiments similar to those for panels A and B were performed to analyze mitochondrial swelling. A decrease in A520 is consistent with an increase in mitochondrial volume (31). As a control for mitochondrial swelling induction, atractyloside (5 mM) or CaCl2 (100 μM) was used. Arrow, addition of each treatment. (D) Immunoblot of cytochrome c release detected in supernatants from mitochondria treated as for panel A, B, or C. Atractyloside (5 mM) and recombinant Bax (100 nM) were used as positive controls. Cox IV and pellet fractions were used to control fractionation quality and cytochrome c release.

FIG. 7.
FIG. 7.

Drp1 impairs mitochondrial electron transport. (A) Drp1, Drp1K38A, or Drp1K679A mitochondrial electron transfer inhibition measured by in situ MRC complex I activity detection. MRC complex I was visualized by Coomassie blue staining (left) or by specific Western blot detection. Treatment with rotenone (20 μM), a complex I inhibitor which blocks the reduction of nitroblue tetrazolium and prevents the detection of the complex I band, confirms the specificity of the reaction. Atractyloside and mClCCP were used as positive controls, and Drp1(1-335) was used as a negative control. (B) Drp1, Drp1K38A, or Drp1K679A blocks mitochondrial respiration in response to complex I substrates. Mitochondria (400 μg) were left untreated (control) or incubated with Drp1 (500 nM), and oxygen consumption was measured in a Clarke-type electrode. Representative curves are shown. Malate and glutamate (M/G) were added at 5 mM and rotenone at 2 μM. In a similar set of experiments, mitochondria were treated with Drp1, Drp1K38A, Drp1K679A, or the negative control Drp1(1-335) or bovine serum albumin (BSA) and analyzed for oxygen uptake. Rates of O2 consumption were calculated and expressed as nmol of O2/min/mg of protein. Data are means ± standard deviations from four independent experiments. (C) Oxygen consumption in CD47-treated cells. CLL or Jurkat cells were left untreated or pretreated with TPCK before being treated with CD47 MAb at different times. Alternatively, cells were incubated with HC or STP. Cells were then permeabilized with digitonin, and 400 μg of protein was loaded into the respiratory chamber. Oxygen consumption in presence of the complex I substrate M/G (5 mM each) was measured as for panel B. Representative curves obtained for untreated (control) cells or cells treated with CD47 MAb (1 h) are shown. Rates of O2 consumption were calculated and expressed as nmol of O2/min/mg of protein as for panel B. Data are means ± standard deviations from five independent experiments.

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