[CANCER RESEARCH 61, 5106 –5115, July 1, 2001]
`
`Pharmacological Inhibitors of the Mitogen-activated Protein Kinase (MAPK)
`Kinase/MAPK Cascade Interact Synergistically with UCN-01 to Induce
`Mitochondrial Dysfunction and Apoptosis in Human Leukemia Cells1
`Yun Dai, Chunrong Yu, Victor Singh, Lin Tang, Zhiliang Wang, Robert McInistry, Paul Dent, and Steven Grant2
`Division of Hematology/Oncology [Y. D., C. Y., V. S., Z. W., S. G.], Departments of Pharmacology [S. G.], Biochemistry [S. G.], Microbiology [L. T., S. G.], and Radiation
`Oncology [R. M., P. D.], Medical College of Virginia, Richmond, Virginia 23298
`
`ABSTRACT
`
`Interactions between the checkpoint abrogator UCN-01 and several
`pharmacological
`inhibitors of
`the mitogen-activated protein kinase
`(MAPK) kinase (MEK)/MAPK pathway have been examined in a variety
`of human leukemia cell lines. Exposure of U937 monocytic leukemia cells
`to a marginally toxic concentration of UCN-01 (e.g., 150 nM) for 18 h
`resulted in phosphorylation/activation of p42/44 MAPK. Coadministra-
`tion of the MEK inhibitor PD184352 (10 mM) blocked UCN-01-induced
`MAPK activation and was accompanied by marked mitochondrial dam-
`age (e.g., cytochrome c release and loss of DCm), caspase activation, DNA
`fragmentation, and apoptosis. Similar interactions were noted in the case
`of other MEK inhibitors (e.g., PD98059; U0126) as well as in multiple
`other leukemia cell types (e.g., HL-60, Jurkat, CCRF-CEM, and Raji).
`Coadministration of PD184352 and UCN-01 resulted in reduced binding
`of the cdc25C phosphatase to 14-3-3 proteins, enhanced dephosphoryl-
`ation/activation of p34cdc2, and diminished phosphorylation of cyclic
`AMP-responsive element binding protein. The ability of UCN-01, when
`combined with PD184352, to antagonize cdc25C/14-3-3 protein binding,
`promote dephosphorylation of p34cdc2, and potentiate apoptosis was mim-
`icked by the ataxia telangectasia mutation inhibitor caffeine. In contrast,
`cotreatment of cells with UCN-01 and PD184352 did not substantially
`increase c-Jun-NH2-terminal kinase activation nor did it alter expression
`of Bcl-2, Bcl-xL, Bax, or X-inhibitor of apoptosis. However, coexposure of
`U937 cells to UCN-01 and PD184352 induced a marked increase in p38
`MAPK activation. Moreover, SB203580, which inhibits multiple kinases
`including p38 MAPK, partially antagonized cell death. Lastly, although
`UCN-01 6 PD184352 did not induce p21CIP1, stable expression of a
`p21CIP1 antisense construct significantly increased susceptibility to this
`drug combination. Together, these findings indicate that exposure of
`leukemic cells to UCN-01 leads to activation of the MAPK cascade and
`that interruption of this process by MEK inhibition triggers perturbations
`in several signaling and cell cycle regulatory pathways that culminate in
`mitochondrial injury, caspase activation, and apoptosis. They also raise
`the possibility that disrupting multiple signaling pathways, e.g., by com-
`bining UCN-01 with MEK inhibitors, may represent a novel antileukemic
`strategy.
`
`INTRODUCTION
`
`UCN-01 (7-hydroxystaurosporine) is a derivative of staurosporine
`that was originally developed as an inhibitor of PKC3 (1). However,
`UCN-01 has since been shown to inhibit other kinases, including
`
`Chk1, which is responsible for phosphorylation, binding to 14-3-3
`proteins, and subsequent degradation of the cdc25c phosphatase (2).
`Degradation of cdc25c results in phosphorylation and inactivation of
`CDKs such as CDK1 (p34cdc2), which are critically involved in cell
`cycle arrest after DNA damage and other insults (3). In this way,
`UCN-01 acts as a checkpoint abrogator, an action that may account
`for its ability to enhance the lethal actions of various cytotoxic agents,
`including cisplatin (4), mitomycin C (5), camptothecin (6), fludara-
`bine (7), gemcitabine (8), and 1-b-D-arabinofuranosylcytosine (9, 10),
`among others. When administered alone, UCN-01 induces arrest in
`G2M or G0G1, depending upon cell type, or, alternatively, the p53 or
`pRb status of the cell (11, 12). UCN-01 is also a potent inducer of
`apoptosis, particularly in hematopoietic cells, a phenomenon that
`appears to be more closely related to dephosphorylation of CDKs than
`to inhibition of PKC (13).
`Phase I and pharmacokinetic studies of UCN-01 have been initiated
`in humans and have shown that this compound exhibits a very long
`plasma half-life, presumably a consequence of extensive binding to a
`1
`acidic glycoprotein (14). Nevertheless, free plasma levels of UCN-01
`capable of inhibiting Chk 1 and abrogating checkpoint control events
`appear to be achievable (15, 16). In a preliminary study (16), combi-
`nation of UCN-01 with established cytotoxic agents was associated
`with evidence of clinical activity in a patient with advanced non-
`Hodgkin’s lymphoma, raising the possibility that UCN-01 may en-
`hance the in vivo activity of conventional chemotherapeutic drugs.
`Despite the intense interest in UCN-01 as an antineoplastic agent,
`the mechanism(s) by which it induces cell death remain(s) incom-
`pletely understood. Recently, considerable attention has focused on
`the role of signal transduction pathways in the regulation of cell
`survival, particularly those related to three parallel MAPK modules.
`Of these, the SAPK/JNK and p38 kinase are primarily induced by
`environmental insults (e.g., DNA damage or osmotic stress) and are
`generally associated with pro-apoptotic actions (17, 18). In contrast,
`p42/44 MAPKs (ERKs) are induced by mitogenic or differentiation-
`related stimuli and are most frequently (although not invariably)
`associated with pro-survival activity (19, 20). In fact, there is evidence
`that the relative outputs of the JNK and p42/44 MAPK cascades
`determine whether a cell lives or dies in response to a noxious
`stimulus (e.g., growth factor deprivation; Ref. 21). p42/44 MAPK lies
`downstream of a signaling pathway consisting of PKC, Raf-1, and
`MEK1 (22). Investigation of the functional role of p42/44 MAPK in
`cell death decisions, as well as other biological processes, has been
`greatly facilitated by the development of pharmacological MEK in-
`hibitors, including PD98059 (23), U0126 (24), and SL327 (25). Re-
`cently, Seybolt-Leopold et al. (26) described a novel MEK inhibitor,
`PD184352, which is able to block MAPK activation and to inhibit the
`in vivo growth of colon tumor cells in mice. Aside from their intrinsic
`antitumor activity, MEK inhibitors may also have a role as potentia-
`tors of established chemotherapeutic drug action (27).
`The relationship between UCN-01 actions and activity of the
`
`Received 1/10/01; accepted 4/20/01.
`The costs of publication of this article were defrayed in part by the payment of page
`charges. This article must therefore be hereby marked advertisement in accordance with
`18 U.S.C. Section 1734 solely to indicate this fact.
`1 Supported by Awards CA 63753, CA 77141, and DK 52825 from the NIH, and by
`Awards 6630-01 from the Leukemia and Lymphoma Society of America and BC980148
`from the Department of Defense.
`2 To whom requests for reprints should be addressed, at Division of Hematology/
`Oncology, Medical College of Virginia, MCV Station Box 230, Richmond, VA 23298.
`Phone: (804) 828-5211; Fax: (804) 828-8079; E-mail: stgrant@hsc.vcu.edu.
`3 The abbreviations used are: PKC, protein kinase C; CDK, cyclin-dependent
`kinase; MAPK, mitogen-activated protein kinase; MEK, MAPK kinase; SAPK, stress-
`activated protein kinase; JNK, c-Jun NH2-terminal kinase; ERK, extracellular regu-
`lated kinase; TUNEL,
`terminal deoxynucleotidyl
`transferase-mediated nick end
`merase; RIPA, radioimmunoprecipitation assay; CHX, cycloheximide; GFX, bisindo-
`labeling; DiOC6, 3,3-dihexyloxacarbocynine; BrdUrd, bromodeoxyuridine; CREB,
`lylmaleimide; PMA, phorbol 12-myristate 13-acetate.
`cyclic AMP-responsive element binding protein; PARP, poly(ADP-ribose) poly-
`5106
`
`
`
`Downloaded from on April 18, 2016. © 2001 American Association for Cancercancerres.aacrjournals.org
`
`
`Research.
`
`Page 1 of 11
`
`Fluidigm
`Exhibit 1008
`
`

`

`INDUCTION OF APOPTOSIS BY UCN-01 AND MEK INHIBITORS
`
`MATERIALS AND METHODS
`
`MAPK pathway is poorly understood. Given the fact that UCN-01 can
`function as a PKC inhibitor (1) and that it has been shown to mimic
`some of the actions of the PKC down-regulator bryostatin 1 as well as
`the kinase inhibitor staurosporine (25), the possibility that UCN-01
`might block the downstream PKC targets MEK1/2 and MAPK ap-
`peared plausible. To address this issue, we have examined the apo-
`ptotic actions of UCN-01 in relation to its effects on the MEK/MAPK
`cascade. Contrary to expectations, exposure of multiple myeloid and
`lymphoid cell
`lines to submicromolar concentrations of UCN-01
`potentiated, rather than reduced, MAPK phosphorylation/activation.
`Moreover, interference with this process by several pharmacological
`MEK inhibitors, including PD98059, U0126, and PD184352, resulted
`in a highly synergistic enhancement of mitochondrial damage, caspase
`activation, and apoptosis in these cells. Together,
`these findings
`suggest that exposure of human leukemia cells to UCN-01 elicits a
`cytoprotective MAPK response and raise the possibility that combin-
`ing this agent with pharmacological MEK inhibitors may effectively
`lower the apoptotic threshold.
`
`tained and fixed with 4% formaldehyde. The slides were treated with acetic
`acid/ethanol (1:2), stained with terminal transferase reaction mixture contain-
`ing 1 3 terminal transferase reaction buffer, 0.25 units/ml terminal transferase,
`2.5 mM CoCl2, and 2 pmol fluorescein-12-dUTP (Boehringer Mannheim,
`Indianapolis, IN), and visualized using fluorescence microscopy.
`Analysis of Mitochondrial Membrane Potential (DCm). Cells (2 3 105)
`were incubated with 40 nM DiOC6 (Molecular Probes Inc., Eugene, OR) in
`PBS at 37°C for 20 min and then analyzed by flow cytometry as described
`previously (29). The percentage of cells exhibiting a low level of DiOC6
`uptake, which reflects loss of mitochondrial membrane potential, was deter-
`mined using a Becton Dickinson FACScan (Becton Dickinson, San Jose, CA).
`Cell Cycle Analysis and S-phase Content. Cells (2 3 106) were pelleted
`at 4°C, resuspended, fixed at 4°C with 67% ethanol overnight, and treated on
`ice with a propidium iodide solution containing 3.8 mM Na citrate, 0.5 mg/ml
`RNase A (Sigma Chemical Co.), and 0.01 mg/ml propidium iodide (Sigma
`Chemical Co.) for 3 h. Cell cycle analysis was performed by flow cytometry
`using Verity Winlist software (Topsham, ME).
`Incorporation of BrdUrd was monitored to evaluate S-phase content. For
`each condition, 2 3 106 cells (cell density 5 5 3 105/ml) were incubated with
`10 mM BrdUrd for 30 min at 37°C. After washing twice with 1% BSA/PBS, the
`cells were resuspended in 70% ethanol and fixed for 30 min on ice. The
`BrdUrd-labeled cells were denatured and nuclei released by incubation with 2
`N HCl/0.5% Triton X-100 for 30 min at room temperature. After centrifuga-
`tion, the pellet was resuspended in 0.1 M Na2B4O4 (pH 8.5) to neutralize the
`Cells. U937, HL-60, Jurkat, CCRF-CEM, and Raji cells are human histi-
`acid. Cells (1 3 106)/100 ml in 0.5% Tween 20/1% BSA/PBS were incubated
`ocytic lymphoma, acute promyelocytic leukemia, acute T-cell leukemia, acute
`with FITC-conjugated anti-BrdUrd (1:10; mouse monoclonal; DAKO, Carpin-
`lymphoblastic leukemia, and Burkitt lymphoma cell lines, respectively. All of
`teria, CA) for 30 min at 4°C. After washing once with 0.5% Tween 20/1%
`the cells were derived by the American Type Culture Collection and main-
`BSA/PBS, the cells were resuspended in PBS containing 5 mg/ml propidium
`tained in RPMI 1640 medium containing 10% FBS, 200 units/ml penicillin,
`iodide and analyzed by flow cytometry. The percentage of S-phase cells was
`200 mg/ml streptomycin, minimal essential vitamins, sodium pyruvate, and
`determined by measuring BrdUrd FITC-positive part in a dot plot of FL-3 (red
`glutamine, as reported previously (28). U937/p21AS and U937/pREP4 cells
`fluorescence) against FL-1 (green fluorescence).
`were obtained by stable transfection of cells with plasmids containing anti-
`Immunoblot and Immunoprecipitation Analysis. Whole-cell pellets
`sense-oriented p21 cDNA or an empty vector (pREP4), and clones were
`were lysed by sonication in 1 3 sample buffer [62.5 mM Tris base (pH6.8), 2%
`selected with hygromycin (29).
`SDS, 50 mM DTT, 10% glycerol, 0.1% bromphenol blue, and 5 mg/ml each
`Drugs and Reagents. Selective MEK inhibitors (PD98059 and UO126),
`chymostatin, leupeptin, aprotinin, pepstatin, and soybean trypsin inhibitor] and
`selective PKC inhibitors (GF 109203X or GFX I and safingol), and specific
`boiled for 5 min. For analysis of phospho-proteins, 1 mM each Na vanadate and
`inhibitors of p38 MAPK (SB203580) were supplied by Calbiochem (San
`Na PPi was added to the sample buffer. Protein samples were collected from
`Diego, CA) as powder. The MEK inhibitor PD184352 was kindly provided by
`the supernatant after centrifugation of the samples at 12,800 3 g for 5 min, and
`Dr. Judith Sebolt-Leopold (Warner Lambert/Parke-Davis Co., Ann Arbor, MI).
`protein was quantified using Coomassie Protein Assay Reagent (Pierce, Rock-
`Materials were dissolved in sterile DMSO and stored frozen under light-
`ford, IL). Equal amounts of protein (30 mg) were separated by SDS-PAGE and
`protected conditions at 220°C. UCN-01 was kindly provided by Dr. Edward
`electrotransferred onto a nitrocellulose membrane. For blotting phospho-
`Sausville (Developmental Therapeutics Program/Cancer Treatment and Eval-
`proteins, no SDS was included in the transfer buffer. The blots were blocked
`uation Program (CTEP), National Cancer Institute). It was dissolved in DMSO
`at a stock concentration of 1 mM, stored at 220°C, and subsequently diluted
`with 5% milk in PBS-Tween 20 (0.1%) at room temperature for 1 h and probed
`with the appropriate dilution of primary antibody overnight at 4°C. The blots
`with serum-free RPMI medium before use. Caffeine (Alexis Co., San Diego,
`CA) was dissolved in chloroform and stored at 220°C. In all of the experi-
`were washed twice in PBS-Tween 20 for 15 min and then incubated with a
`1:2000 dilution of horseradish peroxidase-conjugated secondary antibody
`ments, the final concentration of DMSO or chloroform did not exceed 0.1%.
`(Kirkegaard & Perry, Gaithersburg, MD) in 5% milk/PBS-Tween 20 at room
`Caspase inhibitor (Z-VAD-fmk) and caspase 8 inhibitor (Z-IETD-fmk) were
`temperature for 1 h. After washing twice in PBS-Tween 20 for 15 min, the
`purchased from Enzyme System Products (Livermore, CA), dissolved in
`proteins were visualized by Western Blot Chemiluminescence Reagent (NEN
`DMSO, and stored at 4°C. Cycloheximide was purchased from Sigma Chem-
`Life Science Products, Boston, MA). For analysis of phospho-proteins, Tris-
`ical Co. (St. Louis, MO), stored frozen in DMSO, and diluted in RPMI 1640
`buffered saline was used instead of PBS throughout. Where indicated, the blots
`medium before use.
`were reprobed with antibodies against actin (Signal Transduction Laboratories)
`Experimental Format. All of the experiments were performed using log-
`arithmically growing cells (3–5 3 105 cells/ml). Cell suspensions were placed
`or tubulin (Calbiochem) to ensure equal loading and transfer of proteins. The
`in sterile 25 cm2 T-flasks (Corning, Corning, NY) and incubated with MEK or
`following antibodies were used as primary antibodies: phospho-p44/42 MAPK
`(Thr202/Tyr204) antibody (1:1000; rabbit polyclonal; NEB, Beverly, MA);
`PKC inhibitors for 30 min at 37°C. At the end of this period, UCN-01 (or in
`p44/42 MAPK antibody (1:1000; rabbit polyclonal; NEB); phospho-p38
`some cases, caffeine) was added to the suspension, and the flasks were placed
`MAPK (Thr180/Tyr182) antibody (1:1000; rabbit polyclonal; NEB); phospho-
`in 37°C/5% CO2 incubator at various intervals, generally 18 h. In some studies,
`SAPK/JNK (Thr183/Tyr185) antibody (1:1000; rabbit polyclonal; Cell Sig-
`the p38 MAP kinase inhibitor SB203580 was added concurrently with MEK
`naling Technology, Beverly, MA); SAPK/JNK antibody (1:1000; rabbit poly-
`inhibitors. After drug treatment, cells were harvested and subjected to further
`clonal; Cell Signaling Technology); anti-phospho-CREB (1:1000;
`rabbit
`analysis as described below.
`Analysis of Apoptosis. The extent of apoptosis was evaluated by assess-
`polyclonal; Upstate Biotechnology, Lake Placid, NY); phospho-cdc2 (Tyr15)
`antibody (1:1000; rabbit polyclonal; Cell Signaling Technology); anti-p21Cip/
`ment of Wright-Giemsa-stained preparation under light microscopy and scor-
`WAF1 (1:500; mouse monoclonal; Transduction Laboratories, Lexington,
`ing the number of cells exhibiting classic morphological features of apoptosis.
`KY); anti-p27kip1 (1:500; mouse monoclonal; PharMingen, San Diego, CA);
`For each condition, 5 to 10 randomly selected fields/condition were evaluated,
`MAP kinase phosphatase-1 (M-18; 1:200; rabbit polyclonal; Santa Cruz Bio-
`encompassing at least 500 cells (28). To confirm the results of morphological
`technology Inc., Santa Cruz, CA); MAP kinase phosphatase-3 (C-20; 1:100;
`analysis, in some cases cells were also evaluated by TUNEL staining (30) and
`goat polyclonal; Santa Cruz Biotechnology Inc.); antihuman Bcl-2 oncoprotein
`assessment of oligonucleosomal DNA fragmentation of total DNA. DNA
`fragmentation was analyzed by 1.8% agarose gel electrophoresis as described
`(1:2000; mouse monoclonal; DAKO, Carpinteria, CA); Bax (N-20; 1:2000;
`rabbit polyclonal; Santa Cruz Biotechnology Inc.); Bcl-xS/L (S-18; 1:500;
`previously (31). For TUNEL staining, cytocentrifuge preparations were ob-
`5107
`
`
`
`Downloaded from on April 18, 2016. © 2001 American Association for Cancercancerres.aacrjournals.org
`
`
`Research.
`
`Page 2 of 11
`
`Fluidigm
`Exhibit 1008
`
`

`

`INDUCTION OF APOPTOSIS BY UCN-01 AND MEK INHIBITORS
`
`RESULTS
`
`to the flasks, after which they were placed in the incubator for 24 h. At the end
`of this period, cytospin preparations were obtained and stained with Wright-
`Giemsa, and the cells were scored under light microscopy for the typical
`morphological features of apoptosis.
`Statistical Analysis. For morphological assessment of apoptotic cells, cell
`cycle analysis, S-phase content, cdk1/cdc2 kinase assay, analysis of DCm, and
`clonogenic and cell proliferation assays, experiments were repeated at least
`three times. Values represent the means 6 SD for at least three separate
`experiments performed in triplicate. The significance of differences between
`experimental variables was determined using the Student t test.
`
`The effects of combined exposure of human monocytic leukemia
`cells (U937) to UCN-01 and the MEK inhibitor PD184352 were first
`examined in relation to MAPK activation and apoptosis (Fig. 1).
`Unexpectedly, incubation with UCN-01 (150 nM) induced phospho-
`rylation (activation) of MAPK by 2 h, and this effect persisted over
`the ensuing 18 h (Fig. 1A). Coincubation of U937 cells with
`PD184532 (10 mM) attenuated induction of phospho-MAPK at 2 h,
`and inhibition of MAPK activation was essentially complete after
`18 h. To determine what impact this phenomenon had on cell fate, the
`extent of apoptosis was monitored in cells exposed to each agent
`individually and in combination. Whereas exposure to PD184352 or
`
`rabbit polyclonal; Santa Cruz Biotechnology Inc.); antihuman/mouse XIAP
`(1:500; rabbit polyclonal; R&D System, Minneapolis, MN); anti-caspase-3
`(1:1000; rabbit polyclonal; PharMingen); cleaved-caspase-3 (Mr 17,000) anti-
`body (1:1000; rabbit polyclonal; Cell Signaling Technology); anti-caspase-9
`(1:1000; rabbit polyclonal; PharMingen); anti-PARP (1:2500; mouse mono-
`clonal; Calbiochem); and cleaved PARP (Mr 89,000) antibody (1:1000; rabbit
`polyclonal; Cell Signaling Technology).
`Immunoprecipitation was performed to determine the extent of cdc25C
`activation (32). Briefly, 2 3 107 cells were lysed in RIPA buffer (1% NP40,
`0.5% Na deoxycholate, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na vana-
`date, 5 mg/ml chymostatin, leupeptin, aprotinin, pepstatin, and soybean trypsin
`inhibitor, and 0.1% SDS in PBS) by syringing approximately 20 times with a
`23-gauge needle. Protein samples were centrifuged at 12,800 3 g for 30 min
`and quantified. Two-hundred mg of protein/condition were incubated under
`continuous shaking with 1 mg of anti-cdc25C (mouse monoclonal; PharMin-
`gen) overnight at 4°C. Twenty ml/condition of Dynabeads (goat antimouse
`IgG; Dynal, Oslo, Norway) were added and incubated for an additional 4 h.
`After washing three times with RIPA buffer, the bead-bound protein was
`eluted by vortexing and boiling in 20 ml of 13 sample buffer. The samples
`were separated by 12% SDS-PAGE and subjected to immunoblot analysis as
`described above. Anti-14-3-3b (rabbit polyclonal; Santa Cruz Biotechnology
`Inc.) was used as primary antibody at a dilution of 1:200.
`Analysis of Cytosolic Cytochrome c. Cells (2 3 106) were washed in PBS
`and lysed by incubating for 30 s in lysis buffer (75 mM NaCl, 8 mM Na2HPO4,
`1 mM NaH2PO4, 1 mM EDTA, and 350 mg/ml digitonin). The lysates were
`centrifuged at 12,000 3 g for 1 min, and the supernatant was collected and
`added to an equal volume of 2 3 sample buffer. The protein samples were
`quantified, separated by 15% SDS-PAGE, and subjected to immunoblot anal-
`ysis as described above. Anticytochrome c (mouse monoclonal; PharMingen)
`was used as primary antibody at a dilution of 1:500.
`Cdk1/cdc2 Kinase Assay. Cdk1/cdc2 Kinase Assay Kit (Upstate Biotech-
`nology) was used to determine the activity of cdk1/cdc2 kinase according to
`the manufacturer’s instructions. Briefly, 2 3 107 cells were lysed in RIPA
`buffer by sonication. Protein samples were centrifuged at 12,800 3 g for 30
`min and quantified. Fifty mg of protein/condition were incubated with 400
`mg/ml histone H1, 2 mCi of [g-32P]ATP, and 1:5 inhibitor cocktail in assay
`dilution buffer (total volume, 50 ml) at 30°C for 20 min. A 25-ml aliquot of
`reaction mixture was transferred onto P81 paper. After washing three times
`with 0.75% phosphoric acid and once with acetone, cpm of [g-32P] incorpo-
`rated into histone H1 was monitored using TRI-CARB 2100TR Liquid Scin-
`tillation Analyzer (Packard Instrument Co., Downers Grove, IL). In some
`cases, 10 ml of 23 sample buffer was added to 10 ml of the reaction mixture
`and boiled for 5 min. [g-32P]histone H1 was separated by 12% SDS-PAGE and
`visualized by exposure of the dried gels to X-ray film (KODAK) at 280°C
`for 1 h.
`Clonogenic Assay and Cell Proliferation Assays. Colony formation after
`drug treatment was evaluated using a soft agar cloning assay as described
`previously (33). Briefly, cells were washed three times with serum-free RPMI
`medium. Subsequently, 500 cells/well were mixed with RPMI medium con-
`taining 20% FBS and 0.3% agar and plated on 12-well plates (three wells/
`condition). The plates were then transferred to a 37°C/5% CO2, fully humid-
`ified incubator. After 10 days of incubation, colonies, consisting of groups of
`.50 cells, were scored using an Olympus Model CK inverted microscope, and
`Fig. 1. A, logarithmically growing U937 cells were incubated for the designated
`colony formation for each condition was calculated in relation to values
`intervals in the presence of 150 nM UCN-01 6 10 mM PD184352, after which cells were
`obtained for untreated control cells. For cell viability assays, CellTiter 96
`lysed, and proteins were separated by SDS-PAGE and probed with antibodies directed
`AQueous One Solution (Promega, Madison, WI) was used according to the
`against phospho-ERK, as described in “Materials and Methods.” Blots were subsequently
`manufacturer’s instructions, and the absorbance at 490 nm was recorded using
`stripped and reprobed with antibodies directed against total ERK. Two additional studies
`yielded equivalent results. B, cells were treated with PD184352 (PD) and/or UCN-01
`a 96-well plate reader (Molecular Devices, Sunnyvale, CA).
`(UCN; 61 mM CHX) as above for 18 h, after which Wright Giemsa-stained cytospin
`Normal Peripheral Blood Mononuclear Cells. Peripheral blood was ob-
`preparations were evaluated by light microscopy, and the percentage of cells exhibiting
`tained with informed consent from normal volunteers, diluted 1:3 with RPMI
`classic apoptotic features was determined by examining 5–10 randomly selected fields
`encompassing $500 cells. Values represent the means 6 SD for three separate experi-
`1640 medium, and layered over a cushion of 10 ml of Ficoll-Hypaque (specific
`ments performed in triplicate. C, cells were treated as in A, and the proteins were separated
`gravity, 1.077; Sigma Chemical Co.) in sterile 50-ml plastic centrifuge tubes.
`by SDS-PAGE and probed with antibodies directed against caspase-3, caspase-9, or
`These studies have been approved by the Human Investigations Committee of
`PARP. CF, cleavage fragment. Alternatively, cytosolic fractions were obtained as de-
`Virginia Commonwealth University. After centrifugation for 40 min at
`scribed in “Materials and Methods,” and expression of cytochrome c was monitored as
`400 3 g at room temperature, the interface layer, consisting of mononuclear
`above. Each lane was loaded with 30 mg of protein. Blots were stripped and reprobed with
`antibodies to actin or tubulin to ensure equal loading and transfer. D, cells were treated
`cells, was extracted with a sterile Pasteur pipette and diluted in fresh RPMI
`with UCN-01 6 PD184352 (6 1 mM cycloheximide) as above, after which the percentage
`medium. The cells were washed 32 in medium and resuspended in RPMI 1640
`of cells exhibiting reduced mitochondrial membrane potential (DCm) was determined by
`medium containing 10% FCS in 25-cm2 tissue culture flasks at a cell density
`monitoring DiOC6 uptake as described in “Materials and Methods.” Results represent the
`of 106 cells/ml. Various concentrations of UCN-01 6 PD 184352 were added
`means 6 SD for three separate experiments performed in triplicate.
`5108
`
`
`
`Downloaded from on April 18, 2016. © 2001 American Association for Cancercancerres.aacrjournals.org
`
`
`Research.
`
`Page 3 of 11
`
`Fluidigm
`Exhibit 1008
`
`

`

`INDUCTION OF APOPTOSIS BY UCN-01 AND MEK INHIBITORS
`
`investigated (23), and U0126 (20 mM), the affinity of which for the
`CDK ATP-binding site is significantly greater than that of PD98059
`(24). As noted in the case of PD184352, coadministration of mini-
`mally toxic concentrations of PD98059 or U0126 with 200 nM
`UCN-01 resulted in a marked potentiation of cell death, manifested by
`an increase in the morphological features of apoptosis (Fig. 3, A and
`C), PARP degradation, and release of cytochrome c into the cyto-
`plasm (Fig. 3, B and D). These findings demonstrated that multiple
`pharmacological MEK inhibitors are capable of substantially increas-
`ing the lethal actions of UCN-01 toward U937 cells.
`To establish whether the enhanced lethality of MEK inhibitors and
`UCN-01 was restricted to U937 cells or, instead, might be generalized
`to include other leukemia cell types, the effects of combined exposure
`to UCN-01 and PD184352 were examined in several additional leu-
`kemia cell lines (Fig. 4). Because the sensitivity of these cells to
`UCN-01 differed somewhat from that of U937 cells, slightly higher
`UCN-01 concentrations (e.g., 150 –300 nM) were used in some cases.
`On the basis of standard morphological criteria as well as evidence of
`PARP degradation, it can be seen that combined treatment with
`UCN-01 and PD184352, administered at concentrations that were
`marginally toxic by themselves, resulted in a dramatic increase in cell
`death in HL-60 promyelocytic leukemia cells, T-lymphoblastic
`CCRF-CEM and Jurkat cells, and B-lymphoblastic lymphoma Raji
`cells (Fig. 4, A and B). Qualitatively similar results were obtained
`when PD98059 and U0126 were used (data not shown). As in the case
`of U937 cells, UCN-01 treatment resulted in a substantial increase in
`MAPK activation in HL-60, CCRF-CEM, and in Jurkat cells (Fig.
`4C); moreover, this effect was blocked by PD184352 (5 mM). Thus,
`combined treatment with UCN-01 and MEK inhibitors prevented
`MAPK activation and produced a dramatic increase in apoptosis in a
`variety of myeloid and lymphoid cell lines.
`To investigate the hierarchy of events accompanying apoptosis
`induced by these agents, U937 cells were exposed to the combination
`of UCN-01 (150 nM) in conjunction with 10 mM PD184352 in the
`
`Fig. 2. Top panels, cells were exposed to UCN-01 (150 nM) 6 PD184352 (10 mM) as
`above for 18 h, after which cytospin preparations were obtained and TUNEL staining was
`performed as described in “Materials and Methods.” Cells were then viewed under
`fluorescence microscopy at 350 magnification. A, control; B, PD1843252; C, UCN-01; D,
`UCN-01 1 PD184352. Bottom panel, cells were treated as above after which cells were
`lysed, and DNA was extracted, separated by agarose gel electrophoresis, and stained with
`ethidium bromide as described in “Materials and Methods.” Lanes (20 mg of DNA each):
`M, molecular weight marker; A, control; B, PD184352; C, UCN-01; D, UCN-
`01 1 PD184352.
`
`150 nM UCN-01 alone was minimally toxic to these cells (,10%
`apoptosis in each case), combined treatment resulted in a dramatic
`increase in cell death (i.e., ;60%; Fig. 1B). Furthermore, this effect
`was not attenuated by coadministration of the protein synthesis inhib-
`itor CHX (1 mM). Consistent with these findings, combined treatment
`with UCN-01 and PD184352, but not individual exposure, induced
`marked cleavage of procaspases-3 and -9, PARP degradation, and
`cytochrome c release into the cytoplasmic S-100 fraction (Fig. 1C).
`Cotreatment of cells with UCN-01 and PD184352 also resulted in a
`marked increase in the number of cells exhibiting loss of the mito-
`chondrial membrane potential (e.g., Dc
`m; Fig. 1D), an action that was
`also not attenuated by CHX. TUNEL assays confirmed that a small
`number of cells exposed to UCN-01 or PD184352 alone for 18 h (Fig.
`2, B and C) displayed DNA breaks containing overhanging 39-OH
`ends, whereas coexposure resulted in a high percentage of TUNEL-
`positive cells. Similarly, agarose gel electrophoresis demonstrated a
`marked increase in oligonucleosomal DNA fragmentation in cells
`Fig. 3. A, U937 cells were exposed to UCN-01 (200 nM; UCN) 6 PD98059 (50 mM;
`PD98) for 24 h, after which the percentage of apoptotic cells was scored as described
`exposed to both agents (Fig. 2; bottom panel). Together, these find-
`above (Fig. 1). B, cells were exposed to UCN-01 (200 nM) 6 PD98059 (50 mM) for 24 h,
`ings indicate that coadministration of the MEK inhibitor PD184352
`after which cells were lysed, and proteins were separated by SDS-PAGE and probed for
`blocks MAPK activation and dramatically increases apoptosis in
`expression of PARP and cytosolic cytochrome c as described in Fig. 1. CF, cleavage
`fragment. C, cells were exposed to UCN-01 (200 nM) 6 20 mM U0126 (UO1) for 24 h,
`U937 cells exposed to a marginally toxic concentration of UCN-01.
`after which apoptosis was determined as in A. D, cells were exposed to UCN-01 6 U0126
`To determine whether these findings could be extended to other
`as above, after which expression of PARP and cytosolic cytochrome c were determined
`as above. For A and C, values represent the means 6 SD for three separate experiments
`known MEK inhibitors, U937 cells were incubated for 24 h with 200
`performed in triplicate. For B and D, each lane was loaded with 30 mg of protein. Blots
`nM UCN-01 either alone or in combination with PD98059 (50 mM), an
`were stripped and reprobed with antibodies directed against actin or tubulin to ensure
`aminoflavone that was among the earliest of the MEK inhibitors to be
`equal loading and transfer. Two additional studies yielded equivalent results.
`5109
`
`
`
`Downloaded from on April 18, 2016. © 2001 American Association for Cancercancerres.aacrjournals.org
`
`
`Research.
`
`Page 4 of 11
`
`Fluidigm
`Exhibit 1008
`
`

`

`INDUCTION OF APOPTOSIS BY UCN-01 AND MEK INHIBITORS
`
`Fig. 4. A, HL-60 promyelocytic leukemia cells, Jurkat and CCRF-
`CEM lymphoblastic leukemia cells, and Raji B-lymphoblastic leukemia
`cells were exposed to PD184352 (PD; 5 mM) 6 UCN-01 (UCN; 300 nM
`HL-60; 150 nM Jurkat; 200 nM CCRF; 200 nM Raji) for 24 h, after which
`the percentage of apoptotic cells was determined as described above.
`Values represent the means 6 SD for three separate experiments per-
`formed in triplicate. B, cells were treated as above, after which cells were
`lysed, and the lysates were separated by SDS-PAGE and probed with
`antibodies directed against PARP. Each lane was loaded with 30 mg of
`protein. Blots were subsequently stripped and reprobed with antibodies
`to tubulin or actin to ensure equivalent loading and transfer. CF, cleav-
`age fragment. C, cells were treated as above, after which Western
`analysis was performed to assess expression of phospho-ERK as de-
`scribed in “Materials and Methods.” For B and C, the results of a
`represen