Arsenic Trioxide Selectively Induces Early and Extensive Apoptosis ...

[Cell Cycle 2:4, 358-368, July/August 2003]; ©2003 Landes Bioscience

Report

ABSTRACT

*Correspondence to: Yair Gazitt, Ph.D.; Department of Medicine/Hematology; University of Texas Health Science Center; 7703 Floyd Curl Drive; San Antonio, Texas 78284 USA; Tel.: 210.567.4848; Fax: 210.567.1956; Email:[email protected]

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Received 04/15/03; Accepted 05/02/03

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Department of Medicine/Hematology; University of Texas Health Science Center; San Antonio, Texas USA

Arsenic trioxide (ATO) is effective in the treatment of acute promyelocytic leukemia (APL) and induces apoptosis in APL cells and in a great variety of other cancer cells. We have previously shown that ATO induces apoptosis in myeloma cells in two different modes depending on p53 status in the cells. In cells expressing mutated p53, ATO induced, G2/M arrest and activation caspase 8 and 3 and rapid and extensive apoptosis. Myeloma cells expressing w.t. p53, ATO induced G1 arrest and delayed apoptosis with activation of caspase 9 and 3. APO2/TRAIL receptor expression was induced in both cell types and APO2/TRAIL synergized with ATO in the induction of apoptosis. Here we tested the effect of ATO on mitochondrial membrane potential (MMP) in myeloma cells expressing mutated or w.t. p53. In myeloma cells expressing mutated p53, depolarization of MMP occurred early, concomitant with induction of APO2/TRAIL, activation of BID and release of AIF, preceding apoptosis. However, in cells expressing w.t. p53, APO2/TRAIL is not induced, BID is not cleaved and depolarization of MMP occurs concurrently with cytochrome c release and apoptosis. These results explain the greater sensitivity to ATO of cells with mutated p53 and suggest perhaps a general mechanism for ATO-induced apoptosis.

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Cagla Akay Yair Gazitt*

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Arsenic Trioxide Selectively Induces Early and Extensive Apoptosis via the APO2/Caspase-8 Pathway Engaging the Mitochondrial Pathway in Myeloma Cells with Mutant p53

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Arsenic trioxide, Apoptosis, Myeloma, TRAIL, BID, AIF

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KEY WORDS

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Previously published online as a Cell Cycle Paper in Press at: http://www.landesbioscience.com/journals/cc/toc.php?volume=2&issue=4

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INTRODUCTION

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Multiple myeloma (MM) is a clonal B-cell malignancy affecting both the immune system and bone destruction. It is the second most frequent hematological malignancy inflicting 40,000 people in the USA, with a 5-year survival of less than 20%.1-3 Therefore, new therapeutic modalities are needed for this disease. Arsenic trioxide (ATO), like all-trans retinoic acid (ATRA) is a potent drug in the treatment of acute promyelocytic leukemia (APL)4 and like ATRA has been shown to induce differentiation and apoptosis of APL cells, in vitro and in vivo in animal models.5-8 Most importantly, ATO is very effective in the treatment of APL patients with very little toxicity.9-10 In addition to its effect in APL, ATO is a potent inducer of apoptosis in a number of other cell types lacking the PML-RARα fusion protein.11-21 The exact mechanism of ATO-induced apoptosis in these cells is not yet clear. Several mechanisms were proposed to explain ATO-induced differentiation and apoptosis in cells lacking the PML-RARa protein. These mechanisms include mitochondrial damage, activation of histone deacetylase, blocking of cell cycle, activation of caspases and direct effect on mitochondrial membrane permeability pore.11-21 The pro-apoptotic Bcl-2 family member BID is a 'BH3-only' protein.22-25 BID is cleaved by active caspase-8 followed by post-translational myristoylation. The modified BID translocates to the mitochondria where the truncated p15tBID inserts into the membrane.26 tBID then activates the pro-apoptotic members BAX and BAK to inflict mitochondrial damage and apoptotic death.27-28 Hence, BID is a protein that connects activation of the extrinsic death receptor pathway to activation of the intrinsic pathways and thereby increases chemosensitivity.29 Mitochondria constitute the primary target for apoptosis through the release of proapoptotic proteins, in addition to caspases. Several apoptotic signals converge in mitochondria to induce depolarization of mitochondrial membrane potential (MMP) which can be blocked by the Bcl-2 family of proteins.30 Depolarization of MMP can occur as a result of primary caspase activation (e.g., caspase 8) or from caspase-independent pathway.31 In caspase dependent apoptosis, depolarization of MMP leads to the release of cytochrome

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ARSENIC TRIOXIDE-INDUCED APOPTOSIS IN MYELOMA

c which triggers the proteolytic maturation of caspases within the apoptosome, the complex of cytochrome c, Apaf-1, and caspase-9.32 In the caspase-independent pathway death effectors such as apoptosis inducing factors (AIFs) can be released from the mitochondria and induce apoptosis with or without caspase activation. AIF is capable of translocating to the nucleus and can bind directly to chromatin and induce chromatin condensation and partial DNA degradation.33 Hence, cytoplasmic extracts from dying cells can trigger the breakdown of isolated nuclei in a cell-free system, and this process is inhibited by a polyclonal antibody specific for AIF, suggesting that AIF is involved in DNA degradation during cell death.33 In addition to its apoptotic activity, AIF plays an important role in redox activity through its enzymatic NADH-like activity.34 p53 can induce apoptosis by activation the translocation of Bax from the cytosol to the mitochondria resulting in the release of cytochrome c and activation of caspase 9 through Apaf-1.35 However, p53 can regulate the synthesis and release of the proapoptotic protein SMAC/DIABLO to induce indirect activation of caspase-3 and apoptosis through its binding of anti-apoptotic proteins such as XIAP and cIAP-1, which are normally bound to caspase-3 and block its proteolytic activity.36,37 Also, p53 can regulate the BID expression and thereby p53 can actually link the extrinsic and intrinsic apoptotic pathways.29,38 TNF-related, apoptosis inducing ligand (APO2/TRAIL) belongs to the large family of TNF-like signal-inducing proteins, and their corresponding receptors, belong to the large family of TNF-like signal transduction receptor proteins.39-41 APO2/TRAIL induces a death signal following binding to the R1 or R2, APO2/TRAILreceptors.42-46 Normal cells escape APO2/TRAIL-induced apoptosis by virtue of coexpressing decoy receptor molecules such as R3 or R4, which are unable to transmit down-stream cell death signals and activation of caspase 8.42 Tumor cells generally do not express these decoy receptor molecules.42-46 Other mechanisms for APO2/TRAIL resistance include mutations in caspase-8.47-49 We have previously shown that APO2/TRAIL and Adenovirusmediated delivery of p53 are potent inducers of apoptosis in myeloma cells.50-52 More recently, we have shown that Ad-p53 synergizes with APO2/TRAIL in the induction of apoptosis in myeloma cells and other type of cells through upregulation of R1 and R2 APO2/ TRAIL receptors.53-54 Others have recently shown that treatment with various chemotherapeutic drugs55-57 or ionizing radiation58 also results in the induction of APO2/TRAIL receptors and APO2/TRAIL decoy receptors. In addition, APO2/TRAIL has been shown to exert an anti tumor effect in vivo in different xenograft models of cancer and exhibited very limited toxicity in monkeys.59 In an earlier study, we have shown that ATO induces apoptosis in myeloma cells in two different modes depending on the status of p53 in the cells.60 In cells expressing mutated p53, ATO induced rapid and extensive apoptosis concomitant with blocking of cells in G2/M and activation caspase 8 and caspase 3. In contrast, cell expressing w.t. p53 were relatively resistant to ATO, and ATO blocked cell cycling at G1 concomitant with activation of caspase-9 and caspase-3.60 We have also shown that ATO enhances the expression of R1 and R2 APO2/TRAIL receptors and hence synergizes with APO2/TRAIL in the induction of apoptosis in ATO-resistant cells.60 Here we tested the effect of ATO on mitochondrial membrane potential (MMP); generation of hydrogen peroxide (HP); and release of cytochrome c and AIF from mitochondria in myeloma cells expressing mutated or w.t. p53. We present evidence that in www.landesbioscience.com

cells expressing mutated p53, depolarization of MMP and generation of HP occur early on, before release of cytochrome c and apoptosis as measured by Annexin V. We also observed in these cells an early induction by ATO of APO2/TRAIL, in addition to APO2/TRAIL receptors and a concomitant cleavage of BID and early release of AIF and early apoptosis. In contrast, in cells expressing w.t. p53, APO2/TRAIL is not induced, BID is not cleaved and depolarization of MMP occurs concurrently with release of cytochrome c and apoptosis.

MATERIALS AND METHODS Cell Lines and Cell Culture Conditions. U266, RPMI 8226, IM9, HS-Sultan and ARH-77 cell lines were from ATCC. ARP-1 cells were isolated at Univ. of Arkansas.61 Bcl-2 and mock-transfected cells (neo) 8226 cells were generated and maintained in G418 as described before.61 Briefly, the Bcl-2 expression plasmid (obtained from Dr. S. Korsmeyer) contained the LTR-SV-NEO construct, driven by an SV-40 promoter and hu Bcl-2 cDNA and a neomycin selection gene in a partial Blue Script plasmid (Stratagene, La Jolla, CA).61 Cells were cultured in RPMI 1640 medium supplemented with 15% fetal calf serum) (FCS-medium, GIBCO, Grand Island, NY), in a 37˚C, CO2-incubator. Arsenic trioxide (ATO, Trisenox, Cell Therapeutics Inc., Seattle, WA) was added at concentrations of 0–10 µM. Staining with Annexin V and Detection of Apoptotic Cells. Apoptosis was determined by staining of exposed phosphatidylserine with Annexin V-FITC (BioVision, Palo Alto, CA) as recommended by the manufacturer. Stained cells were analyzed by flow cytometry (FACScalibur, BDIS, and San Jose, CA). Quantitation of apoptosis was done by the CellQuest program. Ten thousand cells were analyzed.52 Determination of Mitochondrial Membrane Depolarization (MMP) and Generation of Hydrogen Peroxide (HP). Tetramethylrhodamine ethyl ester (TMRE; Molecular probes, Eugene, OR) was used to determine depolarization of MMP. Hydroxy ethidine (HE; Molecular Probes) was used to determine generation of HP. HP catalyzes the conversion HE (non-fluorescent form) to ethidium bromide (EB; fluorescent at 520 nm). Briefly, cells (4 x 105/ml) were cultured in 1 ml of FCS-medium in a 24-well plate. At the time points indicated, ATO was added at concentration of 5 µM. For measurement of MMP depolarization or generation of HP, cells were washed and resuspended in FACS-buffer (PBS + 1%BSA) and TMRE (150 nM) or HE (20 µM) was added and cells were further cultured for 20 min. at 37˚C. Cells were then washed with FACS-buffer and TMRE uptake or generation of EB was determined by flow cytometry as recommended by the manufacturer. Quantitation of MMP depolarization was performed by measurement of the left shift in TMRE fluorescence of depolarized mitochondria and HE conversion to EB was determined by measurement of the right shift at 520 nm of newly formed EB. Analysis of the results was performed by the CellQuest program. Ten thousand cells were analyzed. Determination of Cytochrome c Release from Mitochondria by Flow Cytometry. Direct staining with FITC-anti cytochrome c antibody (clone 6H2; Santa Cruz Biotechnology, Inc.; Santa Cruz, CA) was used. Staining was performed as described before.60 Typically, cells were cultured with ATO as described above. Cultures were then harvested and cells were fixed for 30 min. at room temperature with 1 ml of 1% paraformaldehyde in PBS. Cells were then washed with PBS and permeated with 200 µl of permeation buffer (0.05% Triton X-100 in PBS and 2% BSA) containing 10 µl of FITC-anti cytochrome c antibody. Permeation/staining was carried out at 4˚C for 20 min. Unbound antibody was washed with PBS and cells analyzed by flow cytometry as described above for Annexin V. This staining method allows differential staining of cytosolic but not mitochondrial cytochrome c as indicated by confocal microscopy (Fig. 2). Staining for Cytochrome c and Confocal Microscopy. Cells were treated as above with ATO for 24 hours and were then stained as described above for flow cytometry. About 100,000 cells in 20 µl of anti-fade were layered on a slide and cells viewed using the Olympus FV500 Confocal Imaging System. Images were taken with x60 plan APO (1.4 aperture) lens and x40 UAPO 340 (1.53 aperture) lens.

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Figure 1. Bcl-2 does not block ATO-induced apoptosis in RPMI 8226 cells. RPMI 8226 cells and the Bcl-2 overexpressing RPMI8226 cells (8226/Bcl2) express mutated p53. Normal T-cells blasts were prepared from peripheral blood of a normal donor. Cells were cultured for up to 40 hours with 0–6 µM of ATO. Apoptosis was determined by the Annexin V method. For additional experimental details see Materials and Methods section. Ten thousand cells were analyzed. Bar represent ± SD derived from at least three experiments. Note that overexpression of Bcl-2 did not protect from ATO-induced apoptosis.

Determination by Flow Cytometry of AIF Release from Mitochondria and Translocation to the Nucleus. Cells were permeabilized with 1ml of hypotonic buffer consisting of 10 mM Tris buffer, pH 7.4 containing 5 mM MgCl2 and 4 µg of propidium iodide (PI) for 30 min. at 4˚C followed by centrifugation to remove the supernatant. Cell pellet was resuspended in 100 µl of same buffer and 10 µl of mouse monoclonal anti-AIF antibody was added and cells incubated at 4˚C for 25 min. followed by washing of Table 1

A LIST STUDY

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MYELOMA CELL LINES USED

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Cell Line

BCL-2 Status

p53 Status

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ARP-1

low*

null

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RPMI 8226

intermediate

mutated

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8226/bcl-2

high

mutated

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ARH-77

high

mutated

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U266

high

mutated

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HS-Sultan

very low

w.t

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IM9

low

w.t

54

The ARP-1/bcl-2 and 8226/bcl-2 are bcl-2 overexpressing ARP-1 and RPMI 8226 cells, following stable transfection with bcl-2 expressing vector.61 Cells transfected with empty vector were used as a control throughout this study. *The relative expression of Bcl-2 in all the cell lines used in this study was published recently.64,65

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unbound antibody. Secondary anti mouse-FITC (10 µl; Jackson Immunochemicals, Raritan, N.J.) was then added and protocol continued as described above for cytochrome c (Methods, Section 4). Mouse Ig matching control instead of anti-AIF was used for control. This PI/AIF staining combination allows differential staining of nuclear AIF as indicated by confocal microscopy (results not shown). Cell Fractionation and Preparation of the Cytosolic Cell Fraction. Cytosolic cell fraction was prepared by resuspending the cell pellets in mitochondria/ cytosol buffer (BioVision Palo Alto, CA) and passing 300 µl of cell suspension (1 x 107 cells/ml) through a syringe with a 27 gauge needle for several times at 4˚C. This was followed by centrifugation at x10,000 g for 20 min. at 4˚C. About 75% of cells were homogenized routinely by this procedure with 10%–15% loss of viability of mitochondria as determined by uptake of TMRE (Gazitt, 2003, manuscript, in preparation). Determination by Western Immunoblotting of Caspase 3 Activation, Cleavage of BID and Release of Cytochrome c, AIF and SMAC to the Cytosol. For determination of caspase 3 and BID activation, cells were treated with 5 µM of ATO for 0, 16, 24 and 48 hours and aliquots of 3–4 x 106 cells were washed (x2) with PBS and 50 µg of total cellular protein62 or 20 µg the cytosolic fraction protein fraction (see Methods Section 7) were loaded onto each lane and protein bands were resolved by SDS-PAGE. Loading controls were performed using the house keeping protein, α-actin (Sigma, St Louis MO). Gel electrophoresis, immunoblotting, detection of specific bands and quantitation of protein bands by densitometry were performed as previously described.62 Mouse monoclonal antibodies to AIF (clone E1), cytochrome c (clone 6H2) and caspase 3 (clone E8) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Goat polyclonal antibody to BID (N19) was also from Santa Cruz. Rabbit polyclonal anti SMAC antibody was from Oncogene Research Products (San Diego, CA). Biotin conjugated secondary antibodies were from Jackson Immunochemicals (Raritan, N.J.) A low molecular weight ladder of biotinylated protein markers (Biorad, Hercules, CA) was run for each gel. Proteins were tentatively identified according to their migration on the blot. Determination of APO2/TRAIL and APO2/TRAIL Receptors by Immunofluorescence Staining. To determine the effect of ATO on surface expression of APO2/TRAIL and APO2/TRAIL receptors cells were cultured for 24 hours with 3 µM of ATO at which time apoptosis was < 40%. Cells were then harvested for immunofluorescence staining and for determination of apoptosis. Indirect immunofluorescence staining for surface APO2/ TRAIL receptors and surface APO2/TRAIL was performed as described before for APO2/TRAIL receptor.54 Primary antibodies to APO2/TRAIL R1 and R2 were obtained from Immunex Corporation (Seattle, WA). Antibodies to APO2/TRAIL were from R&D Systems (Minneapolis, MN). Stained cells were analyzed by flow cytometry (FACScalibur, BDIS, San Jose, CA). Quantitation of apoptosis was done by the CellQuest program. Ten thousand cells were analyzed.

RESULTS ATO-Induced Apoptosis in Normal, Activated T-cells and in RPMI 8226 Cell Expressing Various Levels of Bcl-2. Table 1 outlines the myeloma cell lines used in this study. RPMI 8226 express mutated p53.63 RPMI 8226/Bcl-2 expresses ~5fold higher Bcl-2 compare to the parental 8226.65 In order to determine the role of p53 and Bcl-2 in ATO-induced apoptosis we used normal activated T-cells and RPMI 8226 myeloma cells (mutated p53) with varying levels of Bcl-2. We performed a time-dose titration of ATO to determine optimal conditions for maximal apoptosis in each cell type. The results obtained from RPMI 8226 and 8226/Bcl-2 myeloma cells and normal activated T-cells are depicted in Figure 1. We observed a time and dose dependent apoptosis between 1.5 to 6 µM of ATO with apoptosis of > 85% observed after 48 hours of treatment with 6 µM of ATO in RPMI 8226 cells. The kinetics and extent of apoptosis for RPMI 8226/Bcl-2 were similar to the kinetics and extent of apoptosis observed for the parental 8226 cells and therefore, overexpression of Bcl-2 does not confer resistance to ATO. In contrast, activated T-cells (w.t. p53) were markedly resistant to ATO at all time points tested and maximal apoptosis was only

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22%, following 48 hours exposure to 20 µM of ATO (Fig. 1). As reported before for RPMI 822660 G2/M block was observed also for the 8226/Bcl-2 cells, whereas no cell cycle effect was observed for activated T-cells (results not shown). Effect of ATO on MMP, Generation of HP, Release of Cytochrome c and Apoptosis in Myeloma Cells Expressing Mutated or w.t. p53. To study the effect of ATO on mitochondria we tested the effect of ATO on mitochondrial membrane potential, generation of hydrogen peroxide and release of cytochrome c from mitochondria to the cytosol. The results are presented in Figures 2–4. We first developed a fixation/permeation method to selectively stain cytosolic cytochrome c and an example is shown in a composite Confocal Imaging micrograph in Figure 2. U266 (mutated p53); IM9 and HS-Sultan cells (w.t. p53) were treated for 24 hours with 5 µM of Figure 2. ATO-induced cytochrome c release in HS-Sultan, IM9 and U266 cells. Cells were ATO and stained with FITC-anti cytochrome c anti- cultured for 24 hours with 5 µM of ATO. Staining for cytochrome c and confocal imaging were bodies. Control cells without ATO (CON) exhibited performed as described in the Materials and Methods section. Representative results from at only small background staining, whereas ATO-treated least 3 different experiments are shown. Micrographs are magnified x600. IM9 and HS-Sultan cells (30%–40% apoptosis) Cells were assayed at 0, 16, 24 and 48 hours post induction of apoptosis. exhibited strong diffuse cytoplasmic staining, in about 30% of cells, with The results from 1 representative experiment are shown in Figure 5. It is no nuclear staining. In contrast, ATO-treated U266 cells had dimmer clear that in cells with mutated p53, AIF is released from mitochondria to cytochrome c staining in about 60% of cells. This staining method was the cytosol as early as 16 hours post induction of apoptosis. At the same adapted for flow cytometry (see below). time, cytochrome c release is very small, if any. Hence, AIF is clearly associated Next, we compared the kinetics (0–24 hours) of the effect of 5 µM ATO with rapid depolarization of MMP and apoptosis in myeloma cells expressing on depolarization of MMP and generation of HP to the kinetics of mutated p53. In contrast, in cells expressing w.t. p53, the results are quite cytochrome c release and apoptosis by annexin V. Examples of the results the opposite, namely, cytochrome c was the main proapoptotic protein obtained for RPMI 8226 (mutated p53) and HS-Sultan (w.t. p53) are released from mitochondria and was released early, relative to AIF (Fig. 5). shown in Figures 3A and 3B, respectively. RPMI 8226 cells (mutated p53) On the other hand, SMAC expression was low relative to AIF and release of exhibited a rapid MMP depolarization with a net increase of 70% in cells SMAC from mitochondria, in both cell types was small relative to AIF (Fig. 5). with depolarized MMP and a net increase of 31% in cells with HP, as early Interestingly, addition of a wide rage of concentrations of permeable SMAC as 8–16 hours after treatment with ATO. However, the net increase in perpeptide (N7; Calbiochem; La Jolla, CA) did not result in blocking or stimcent cells with cytosolic cytochrome c and apoptosis by annexin V were only ulation of apoptosis in neither cell type (results not shown). Therefore, the 17% and 28%, respectively (Fig. 3A). role of SMAC in ATO-induced apoptosis is not clear at this point. In contrast to RPMI 8226 cells, treatment of HS-Sultan cells under Effect of ATO on the Expression of Surface APO2/TRAIL and similar conditions resulted in small (13% and 11%) increase in cells with Activation of BID in Myeloma Cells Expressing Mutated p53. We depolarized MMP and with cells positive to HP, respectively. Percent hypothesized that BID might be involved in the release of AIF from mitoincrease in cells with cytosolic cytochrome c and apoptosis were similar to chondria in cells expressing mutated p53. We therefore tested the effect of the percentage of cells with depolarized MMP and with HP (Fig. 3B). ATO on the activation of BID in cells expressing mutated vs. w.t. p53. The Similar trend was observed in cells treated with ATO for 24 hours, although results are summarized in Figure 6. Indeed, BID was cleaved in RPMI 8226, apoptosis and cytochrome c release were almost doubled (Fig. 3B). U266 and ARH-77 cells expressing mutated p53. In contrast, in cells Activated T-cells were practically resistant to doses of up to 20 µM of expressing w.t. p53, cleavage of BID was very little, if any. Caspase 3 cleavage ATO. Similar to the results obtained with SH-Sultan cells, activated normal followed the kinetics of BID cleavage (Fig. 6) and the extent of apoptosis by T-cells has relatively low levels of apoptosis and low percentage of cells with annexin V (results not shown) in both cell types. depolarized MMP and cytosolic cytochrome c. These cells, like HS-Sultan We have previously shown that ATO synergizes with APO2/TRAIL in cells (w.t. p53) we observed a small increase in the proportion of cells arrested the induction of apoptosis in myeloma cells and reported that this synergy in G1 (results not shown). The compiled data from 3 experiments are shown in Figures 4A and 4B is the result of ATO induction of surface APO2/TRAIL receptors.60 We also reported that in cells with mutated p53, caspase-8 is the major caspase for RPMI 8226, U266, ARH-77 and APR-1 cells (mutated p53); and activated in these cells. Since BID is specifically induced in cells with mutated HS-Sultan and IM9 cells (w.t. p53) treated with 5 µM of ATO for 0–40 p53, we hypothesized that ATO might induce APO2/TRAIL in these cells, hours. Clearly, cells with mutated p53 exhibited a rapid MMP depolarization in addition to APO2/TRAIL receptors, and thereby activating the casand generation of HP (8–16 hours) preceding cytochrome c release and pase-8/BID pathway of apoptosis.23-25 We therefore treated U266, ARH-77 apoptosis by annexin V, whereas, in cells with w.t. p53, MMP depolarization (mutated p53), HS-Sultan and IM9 cells (w.t. p53) with 5 µM of ATO for and generation of HP were concurrent or came after cytochrome c release 24 hours and tested the effect on surface expression of APO2/TRAIL as well and apoptosis (Fig. 4). These results further confirm a differential effect of as the expression of APO2/TRAIL receptors in cells undergoing apoptosis ATO on myeloma cells depending on p53 status and suggest a differential (30%–50%) by ATO. Examples from 1 representative experiment with effect also on mitochondria. U266 (mutated p53) and HS-Sultan (w.t. p53) is presented in Figure 7. Effect of ATO on the Release of AIF, Cytochrome c and SMAC from Indeed, in U266 cells, we observed a significant increase in percent cells Mitochondria in Myeloma Cells Expressing Mutated or w.t. p53. To further expressing R2 APO2/TRAIL receptor (solid thin line) from 36% to 89% and delineate the effect of p53 on the mitochondrial pathway of ATO-induced APO2/TRAIL (thick solid line) from 49%–92%, with a significant shift in apoptosis we compared the kinetics of the release from mitochondria of mean fluorescence intensity (MFI) for APO2/TRAIL from 10 units to 82 units three mitochondrial proapoptotic proteins: cytochrome c; apoptosis induc(Fig. 7). The expression of R1 APO2/TRAIL receptors did not significantly ing factor (AIF); and SMAC/DIABLO in cells expressing mutated p53 change in U266 cells (thin dashed line). The gray histograms represent (8226; U266) or w.t. p53 (IM9, HS-Sultan), by Western immunoblotting.

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Figure 3. (A) ATO induces a rapid depolarization of MMP preceding apoptosis and cytochrome c release in RPMI 8226 cells. RPMI 8226 cells (mutated p53) were cultured with 5 µM of ATO for up to 24 hours and aliquots were taken for determination of apoptosis; cytochrome c release; measurement of generation of hydrogen peroxide (HP) by HE and mitochondrial membrane depolarization (MMP) by TMRE. One representative experiment out of at least 3 experiments is shown. Note that MMP, as measured by TMRE uptake is the earliest apoptotic event in 8226 cells. For additional experimental details see Materials and Methods section. Ten thousand cells were analyzed. (B) Depolarization of MMP occurs late relative to cytochrome c release in ATO-induced apoptosis of HS-Sultan cells. HS-Sultan cells (w.t. p53) were cultured with 5 µM of ATO for up to 24 hours and aliquots were taken for determination of apoptosis; cytochrome c release; generation of hydrogen peroxide by HE and mitochondrial membrane depolarization (MMP) by TMRE. One representative experiment out of at least 3 experiments is shown. Note that cytochrome c release is the earliest apoptotic event in HS-Sultan cells. For additional experimental details see Materials and Methods section. Ten thousand cells were analyzed.

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staining with IgG1 control. Similar increases were observed in ARH-77 (mutated p53) cells (results not shown). In contrast to RPMI 8226 cells, only small increase in surface expression or APO2/TRAIL or R1 and R2 APO2/TRAIL receptors was observed in HS-Sultan cells (Fig. 7). Similarly, very small increase in the expression of APO2/TRAIL or APO2/ TRAIL receptors was observed in IM9 cells (w.t. p53) (results not shown). We further studied the effect of ATO on APO2/TRAIL induction in relation to the translocation of AIF from the mitochondria to the nucleus. We employed U266 cells (mutated p53) and HS-Sultan cells (w.t. p53) and treated these cells with 5 µM of ATO for 0, 8 and 16 hours and compared the effect on surface expression of APO2/TRAIL; depolarization of MMP; and AIF translocation to the nucleus. The results are depicted in Figures 8A and 8B. In U266, (Fig. 8A) APO2/TRAIL induction was significant (~40%) as early as 8 hours post induction, where MMP depolarization was evident in about 70% of the cells, at which time apoptosis by annexin V reached 31%. Similarly, AIF translocation to the nucleus occurred in 56% of the cells, 8 hours post induction of apoptosis, reaching 84% by 16 hours, at which time 94% of the cells exhibited depolarization of MMP. Therefore, in these cells, the APO2/ TRAIL/TRAIL receptor/BID/AIF pathway is engaged by ATO, preceding apoptosis by annexin V and coinciding with depolarization of MMP (Fig. 8A). U266 cells (mutated p53) were cultured with 5µM of ATO for up to 16 hours and Figure 4. (A) Effect of ATO on apoptosis, cytochrome c release, generation of hydrogen peroxide and aliquots were taken for determination of apopMMP depolarization in RPMI 8226, U266 and HS-Sultan cells. Cells were cultured with 5 µM of ATO tosis; surface expression of APO2/TRAIL; for up to 40 hours and aliquots were taken for determination of apoptosis; cytochrome c release; depolarization of mitochondrial membrane generation of hydrogen peroxide by HE and mitochondrial membrane depolarization (MMP) by TMRE. potential (MMP) by TMRE and translocation Note that depolarization of MMP, as measured by TMRE uptake is the earliest apoptotic event in of AIF to the nucleus. Note that induction of RPMI8226 and U266 cells (mutated p53) compared to HS-Sultan cells (w.t. p53). For additional APO2/TRAIL, depolarization of MMP and experimental details see Materials and Methods section. Ten thousand cells were analyzed. Bar represent translocation of AIF to the nucleus occur con± SD derived from at least 3 experiments. (B) Effect of ATO on apoptosis, cytochrome c release, generation of hydrogen peroxide and MMP depolarization in ARH-77, ARP-1 and IM9 cells. Cells were currently about 8 hours after addition of ATO, cultured with 5 µM of ATO for up to 40 hours and aliquots were taken for determination of apoptosis; preceding apoptosis by annexin V. For additional cytochrome c release; generation of hydrogen peroxide by HE and mitochondrial membrane depolarexperimental details see Methodology Section. ization (MMP) by TMRE. Note that depolarization of MMP, as measured by TMRE uptake is the earliest Ten thousand cells were analyzed. One repreapoptotic event in ARH-77 and ARP-1 cells (mutated p53) compared to IM9 cells (w.t. p53). For sentative experiment out of 3 experiments is additional experimental details see Materials and Methods section. Ten thousand cells were analyzed. shown. Bar represent ± SD derived from at least 3 experiments. Similar studies were performed with HS-Sultan cells (w.t. p53). The results are depicted in Figure 8B. In contrast to U266, there was practically no increase DISCUSSION in surface expression of APO2/TRAIL for up to 16 hours post induction ATO Induces Early Expression of Surface APO2/TRAIL and (compare to Fig. 7) and relatively small increase in nuclear AIF was observed APO2/TRAIL Receptors, BID Cleavage, Depolarization of MMP 8 and 16 hours post induction, similar to the increase in percent cells with and Release of AIF to the Cytosol in Myeloma Cells Expressing depolarized MMP and percent apoptotic cells. Therefore, in these cells the APO2/TRAIL and BID pathways are not engaged early on by ATO and Mutated p53. The mechanism of ATO-induced apoptosis is not yet translocation of AIF is modest, coinciding with apoptosis, measured by clear and different mechanisms were proposed for different type of annexin V and thus could be secondary to apoptosis (Fig. 8B). As was the cells and inconsistent results were reported by different groups for case for U266 cells about 20% of untreated HS-Sultan cells were positive for different type of cells. Previously, we performed detailed studies in nuclear AIF, suggesting perhaps a role for AIF in the regulation of a certain myeloma cells with varying p53 status in order to delineate the effect stage of the cell cycle. of p53 function on the sensitivity to apoptosis, G or G /M cell

A

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cycle arrest and on down stream caspases involved in ATO-induced apoptosis. We reported two distinct pathways for ATO-induced apoptosis depending on p53 status. In myeloma cells with mutated www.landesbioscience.com

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Figure 5. AIF is the major proapoptotic protein released from mitochondria in ATO-induced apoptosis of cells expressing mutated p53. Cells were cultured for 0, 16, 24 and 48 hours with 5 µM of ATO. Cell fractionation and isolation of the cytosolic fraction were carried out as described in the Methodology Section. SDS-PAGE, immunoblotting and detection of specific protein bands were performed as described in the Methodology Section. 20 µg of protein was loaded onto each lane. Representative results from at least 3 different experiments are shown. For loading controls, membranes were stripped and reprobed for cytosolic α-actin. Note that AIF is the major proapoptotic protein released from mitochondria in cells expressing mutated p53 whereas cytochrome c in the major proapoptotic protein released from mitochondria in cells expressing w.t. p53.

Figure 6. ATO induces BID activation in U266 and 8226 and ARH-77 cells expressing mutated p53. Cells were cultured for 0, 16, 24 and 48 hours with 5 µM of ATO. Extraction of total cellular protein, SDS-PAGE, immunoblotting and detection of specific protein bands were performed as described in the Materials and Methods section. 50 µg of protein was loaded onto each lane. Representative results from at least 3 different experiments are shown. For loading controls, membranes were stripped and reprobed for α-actin. For additional experimental details see Methodology Section. Note the differential effect of ATO on BID cleavage in cells with mutated (U266, 8226 and ARH-77) vs. w.t. p53 (IM9, HS-Sultan).

p53, ATO induced rapid apoptosis reaching > 50% in 8–16 hours concomitant with blocking of cells in G2/M and activation of caspase 8 and 3. In contrast, in myeloma cells expressing w.t. p53, partial or full resistance to ATO was observed concomitant with activation of p21 and activation of caspase 9.60 In this study we expanded our studies to include the effect of ATO on MMP, generation of HP and release of AIF, cytochrome c and SMAC from the mitochondria to the cytosol, in cells expressing w.t. or mutated p53. Striking differences were observed between the two types of cells. Cells expression mutated p53 exhibited substantial early (8–16 hours) depolarization of MMP and generation of HP, preceding cytochrome c release and apoptosis measured by annexin V. Most importantly, release of AIF in these cells was also early and was concurrent with depolarization of MMP and generation of HP and preceded apoptosis. Others have reported ATO-induced depolarization of MMP in APL cells.5,12,21 Most importantly, early on, APO2/ TRAIL and APO2/TRAIL receptors are induced concomitant with BID cleavage, in the absence of p53, although overexpression of p53 can induce the expression of APO2/TRAIL receptor, in myeloma cells.54 Together, these results and our previous observation that caspase-8 is the primary caspase involved in ATO-induced apoptosis in cells expressing mutated p53 strongly suggest that ATO engages the extrinsic apoptotic pathway in these cells resulting with a rapid release of AIF which leads to nuclear apoptosis. Support for this hypothesis comes from the fact that AIF is localized to the nucleus as early as 8–16 hours after induction of apoptosis and that cytochrome c release to the cytosol is relatively small in these cells compared to the rapid and substantial release of AIF. However, eventually, both pathways are engaged and caspase-3 is also cleaved and contributed to apoptosis. Direct effect of AIF on chromatin condensation, partial DNA degradation and “nuclear apoptosis” were previously reported for other cell types.30,33-34,66-69 Interestingly, the p53 regulated proapoptotic protein; SMAC70 was present in relatively small amounts and was released to the cytosol to some extent, both, in cell expressing w.t. as well as in cells expressing mutated p53. Since the TRAIL/TRAIL receptor pathway is engaged in myeloma cells expressing mutated p53, it is not surprising that overexpression of Bcl-2 in RPMI 8226 Figure 7. APO2/TRAIL and TRAIL receptors are induced in myeloma cells with mutated p53. U266 cells (mutated p53) and HS-Sultan cells (wt. p53) were cultured for 24 hours with or without 5 µM of ATO. Results from the viable cell fraction gated by light scatter are shown. Cells were stained for surface expression of APO2/TRAIL and R1 R2 TRAIL receptors as described in the Methodology Section. C--filled histogram = Ig control; dashed line = R1; solid thin line = R2; solid thick line = TRAIL. For additional experimental details see Materials and Methods section. Ten thousand cells were analyzed. One representative experiment out of 3 experiments is shown.

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cells did not block ATO-induced apoptosis (Fig. 1 and ref. 50 and 51). Our finding regarding ATO-induced apoptosis in cells with mutated p53 are not restricted to myeloma cells, since the p53 mutated Jurkat, T-ALL cells71 were also sensitive to ATO-induced apoptosis engaging APO2/ TRAIL/TRAIL receptor pathway, cleavage of BID, early depolarization of MMP, release of AIF to the cytosol and arrest of cells in the G2/M (Gazitt et al., manuscript, in preparation). It seems that over expression of Bcl-2 does not protect against ATO-induced apoptosis and the partial resistance of HS-sultan and IM9 cells correlates with p53 status and not on the expression of bcl-2. Similarly, U266 (high Bcl-2, mutated p53) ARP-1 cells (p53 null, low Bcl-2) behave in a similar fashion in response to ATO (Fig. 1, ref. 60 and Gazitt et al. manuscript in preparation). It has been suggested that resistance to apoptosis does not necessarily involve over-expression of Bcl-2 when other components of the apoptosis machinery become the rate limiting factors, such as mutations in caspases or Apaf-1, etc.75 Furthermore, cells expressing mutated p53 can be very sensitive to apoptosis due to direct activation of pathways downstream of p53.75 This might explain the complex effect of ATO in the induction of apoptosis as was observed in this paper and in our previous publication.60 The nature of the G2/M arrest induced by ATO is not yet clear at this point, however, in the absence of functional p53 and G1 arrest, DNA damage can result in a G2 arrest independent of p53 but involving other DNAdamage sensing proteins, such as Atm and Atr through a down stream activation of Chk1 and Chk2 kinases which phosphorylates the Cdc25 phosphatase and thus blocking Cdc25-regulated dephosphorylation of Cdc2. This can lead to blocking of the formation of the mitotic cyclin B/Cdc2 complex, effectively blocking cells in G2/M.72 Studies in our laboratory are conducted to elucidate the mechanism by which ATO blocks cell cycle in these cells. In these studies we employ a temperature conditional mutant to independently confirm the role of p53 in ATOinduced apoptosis. Transfection of w.t. p53expression construct into cells with no p53 or with mutated p53 does not work since the cells

A

B

Figure 8. (A) Effect of ATO on MMP depolarization, apoptosis, expression of APO2/TRAIL and translocation of AIF to the nucleus in U266 cells. U266 cells (mutated p53) were cultured with 5 µM of ATO for up to 16 hours and aliquots were taken for determination of apoptosis; surface expression of APO2/TRAIL; depolarization of mitochondrial membrane potential (MMP) by TMRE and translocation of AIF to the nucleus. Note that induction of APO2/TRAIL, depolarization of MMP and translocation of AIF to the nucleus occur concurrently about 8 hours after addition of ATO, preceding apoptosis by annexin V. For additional experimental details see Methodology Section. Ten thousand cells were analyzed. One representative experiment out of 3 experiments is shown. (B) Effect of ATO on MMP depolarization, apoptosis, expression of APO2/TRAIL and translocation of AIF to the nucleus in HS-Sultan cells. HS-Sultan cells (w.t. p53) were cultured with 5 µM of ATO for up to 16 hours and aliquots were taken for determination of apoptosis; surface expression of APO2/TRAIL; depolarization of mitochondrial membrane potential (MMP) by TMRE and translocation of AIF to the nucleus. Note the small increase in surface expression of APO2/TRAIL and the small and relatively late depolarization of MMP and translocation of AIF to the nucleus in these cells and low level apoptosis, compared to U266 cells. For additional experimental details see Materials and Methods. Ten thousand cells were analyzed. One representative experiment out of 3 experiments is shown.


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A

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Figure 9. (A) A proposed model for ATO-induced apoptosis in cells expressing mutated p53. Based on the results reported by us before60 and the results presented in this paper, we propose that in the absence of functional p53, ATO induces the expression of APO2/TRAIL and TRAIL receptors and thereby engaging the caspase-8/BID/Bax pathway resulting in a rapid depolarization of MMP, release of AIF followed by activation of caspase-3 and nuclear/cell apoptosis. According to this model, release of cytochrome c, SMAC and activation of caspase-9 are secondary apoptotic events of dying cells. (B) A proposed model for ATO-induced apoptosis in cells expressing w.t. p53. Based on the results reported by us before60 and the results presented in this paper, we propose that in the presence of functional p53, ATO induces primarily expression p21 and arrest of cells in G1 of the cell cycle. In these cells, p53 can also activate Bax (with no BID cleavage) and Bax translocates to the mitochondria. These events lead to the release of cytochrome-c, SMAC and activation of the caspase-9-dependent apoptosome/mitochondrial pathway. In this case, APO2/TRAIL and TRAIL receptors and caspase-8/BID pathway are not engaged and the observed depolarization of MMP and release of small amounts of AIF are secondary apoptotic events of dying cells.

undergo rapid apoptosis before addition of ATO (see also refs. 52 and 53). Also, the fact that Jurkat cells (mutated p53) behave in a similar way as myeloma cells expressing mutated p53 suggests a generalized role for p53 in ATO-induced apoptosis (Gazitt et al., in preparation). Finally, although hydrogen peroxide was generated early on in 366

these cells in response to ATO, its role in ATO-induced apoptosis is still under investigation in our lab. Based on the current results and our previous publication,60 we propose a model for ATO-induced apoptosis in cells expressing mutated p53. The model is summarized in Figure 9A. ATO-Induced Early Cytochrome c and SMAC Release to the Cytosol in Myeloma Cells Expressing w.t. p53. In contrast to cells with mutated p53, in cells expressing w.t. p53 the primary apoptotic events included early release of cytochrome c to the cytosol and activation of caspase 3 and caspase-9.60 We did not observe in these cells induction of APO2/TRAIL, cleavage of BID or substantial release of AIF to the cytosol and its translocation to the nucleus. Hence the APO2/TRAIL pathway is not the primary apoptotic pathway in these cells and the observed partial and delayed depolarization of MMP and slight release of AIF could be secondary to apoptosis in these cells. Instead, in cells expressing w.t. p53 the primary apoptotic pathway engaged by ATO is the p53-mediated Bax translocation to the mitochondria, cytochrome c release to the cytosol and activation of caspase-9.60 SMAC, the p53 regulated proapoptotic regulatory protein36,70 could also be involved to some extent in ATO-induced apoptosis in these cells. In these cells, ATO activates p53-dependent cell cycle regulation through activation of p21 and arrest of the cells in G1 of the cell cycle as reported by us before.60 In the presence of functional p53, ATO acts like DNA damaging agent (e.g., UV and ionizing radiation), most likely inducing DNA breaks which trigger p53-dependent DNA repair apparatus involving upregulation of gadd45, p21 and blocking of G1 cyclins followed by G1 arrest and eventually leading to differentiation and/or apoptosis.5,73-74 The fact that activated normal T-cells were relatively resistant to ATO and that the pattern of apoptosis in these cells was similar to the one observed in myeloma cells expressing w.t. p53 suggest a general mechanism for ATO-induced apoptosis in cells with w.t. p53. Based on the current results and our previous publication,60 we propose a model for ATO-induced apoptosis in cells expressing w.t. p53. The model is summarized in Figure 9B. Our interpretation of the effect of ATO in vitro could explain the relative low hematological toxicity observed in patients undergoing treatment with ATO, since G1 block, unlike G2 block is less toxic to the cells and can be reversible. Finally, it is important to mention that ATO has been used in myeloma patients in phase I–II clinical trials as a single agent, or in combination with ascorbic acid, or in combination with thalidomide. Given the low toxicity of ATO and the activation of the Bcl-2-independent APO2/TRAIL/BID/AIF pathway in myeloma cells expressing mutated p53, prescreening for p53 mutations should increase the efficacy of ATO in multiple myeloma patients. Acknowledgements FACS analyses were performed in the Institutional Flow Cytometry (FC) Core Facility and the Institutional Confocal Imaging (CI) Core Facility. The authors would like to thank Mr. Charles Thomas (FC) and Dr. Victoria Frohlich (CI) for performing these analyses. We would also like to thank Immunex for supplying the anti APO2/TRAIL-receptor antibodies and Cell Therapeutics Inc for supplying ATO (Trisenox) References 1. Gregory W, Richards M, Malpas J. Combination chemotherapy versus melphalan and prednisone in the treatment of multiple myeloma: an overview of published trials. J Clin Oncol 1992; 10:336-42. 2. Alexanian R, Dimopoulos MA, Delasalle K, and Barlogie B. Primary dexamethasone treatment for multiple myeloma. Blood 1992; 80:887-92. 3. Attal M, Harousseau JL, Stoppa AM, Sotto JJ, Fuzibet JG, Rossi JE, Casassus P, Maisoneuve H, Facon T, Ifrah N, Payen C, and Bataille R. A prospective randomized trial of autologous transplantation and chemotherapy in multiple myeloma. N Eng J Med 1996; 335:91-7

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