[CANCER RESEARCH 55, 1982-1988, May 1, 1995]
`
`Rapamycin Enhances Apoptosis and Increases Sensitivity to Cisplatin in Vitro1
`
`Yufang Shi, Andrea Frankel, Laszlo G. Radvanyi, Linda Z. Penn, Richard G. Miller, and Gordon B. Mills2
`
`Oncology Research, The Toronto Hospital, Toronto M5G 2C4, Canada ¡Y.S., A. F., G. B. M.], Ontario Cancer institute. Princess Margaret Hospital, Toronto M4X 1K9, Canada
`¡L.G. R., R. G. M.]. and Department of Microbiology,
`Immunology and Cancer, The Hospital
`for Sick Children, Toronto M5G 1X8, Canada [L Z. P.]
`
`ABSTRACT
`
`systems by the
`can be regulated in a number of different
`Apoptosis
`actions of cytokines. Rapamycin
`has been shown to exert
`its effects on
`growth factor-induced
`cell proliferation,
`at least in part, by blocking the
`activation
`of
`the p70 S6 kinase and thus preventing
`the downstream
`signaling process, such as the activation of the members of the cdk family.
`To determine whether
`this pathway plays a role in the regulation of
`apoptosis, we assessed the effect of rapamycin on apoptosis
`induced by
`interleukin 2 deprivation in murine T-cell
`lines, by T-cell receptor ligation
`in a murine T-cell hybridoma, by enforced i--//m- expression in murine
`fibroblasts,
`and by corticosteroids
`in murine T-lymphoma
`cell
`lines. Al
`though rapamycin did not induce apoptosis on its own, rapamycin aug
`mented apoptosis
`in each of the cell
`lines used as indicated by increased
`genomic DNA fragmentation,
`decreased cell viability, and characteristic
`apoptotic
`changes
`in morphology. These
`results
`suggest
`that a signal
`transduction pathway(s)
`inhibited by rapamycin plays an important
`role
`in the susceptibility
`of cells to apoptosis. Many chemotherapeutic
`agents
`kill cancer cells through the induction of apoptosis. Strikingly,
`rapamycin
`increased the ability of the alkylating agent, cisplatin,
`to induce apoptosis
`in the human promyelocytic
`leukemia
`cell
`line HI,-60 and the human
`ovarian cancer cell
`line SKOV3. These data suggest
`that a signal
`trans
`duction pathway,
`likely related to p70 S6 kinase,
`inhibited by rapamycin
`may be an important component of the pathway which prevents cell death
`in many cell lineages and also indicate that rapamycin has the potential
`to
`augment
`the efficacy of selected anticancer
`therapies.
`
`INTRODUCTION
`The discovery of the immunosuppressive
`
`drugs CsA,3 FK506, and
`
`rapamycin has revolutionized organ transplantation and the treatment
`of autoimmune
`diseases
`(1). These immunosuppressants
`exert
`their
`effects by binding to a class of intracellular proteins called immu-
`nophilins, specifically interfering with the signaling pathways leading
`to cytokine production or proliferation of T lymphocytes upon acti
`vation (2). CsA binds to an immunophilin called cyclophilin A, while
`FK-506 and rapamycin bind to FKBP (2). Both cyclophilin and FKBP
`are peptidyl-prolyl
`cis-trans
`isomerases,
`the enzymatic
`activity of
`which is inhibited by the immunosuppressants. However,
`inhibition of
`the peptidyl-prolyl
`cis-trans
`isomerase does not account for the dem
`onstrated immunosuppressive
`activity (3). The complex of cyclophilin
`A and cyclosporin A, as well as the complex of FK506 and FKBP,
`binds to and inactivates calcineurin, an intracellular calcium/calmod-
`ulin-activated protein phosphatase (2); while the complex of rapamy
`cin and FKBP exerts its effect, at least
`in part, on the p70 S6 kinase
`pathway (4). Studies of the mechanisms by which these immunosup-
`
`Received 11/4/94; accepted 3/1/95.
`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.
`' This project was supported by grants from the Medical Research Council of Canada
`and the National Cancer Institute of Canada (to G. B. M.). G. B. M. is a Medical Research
`Council of Canada Scientist, Y. S.
`is a recipient of a postdoctoral
`fellowship of the
`National Cancer
`Institute of Canada, and L. G. R. was supported in part by an Ontario
`Graduate Studentship.
`2 To whom requests for reprints should be addressed, at Section of Molecular Thera
`peutics, M. D. Anderson Cancer Center, University of Texas, C5.001, 1515 Holcombe
`Boulevard, Houston, TX 77030.
`binding
`3 The abbreviations used are: CsA, cyclosporin A; FKBP, FK-506/rapamycin
`protein;
`IL-2, interleukin 2; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl
`tetrazolium
`bromide; 125IUDR, 125I-labeled deoxyuridine.
`
`pressive drugs act have not only demonstrated how they function in
`cells but also have proved that
`they are useful
`tools to dissect cell
`signaling pathways
`(5).
`Unlike cyclosporin A and FK506, which suppress a calcium-de
`pendent pathway in the early stages of T-cell activation,
`rapamycin
`does not alter
`the early events
`following the activation of T cells
`through the T-cell antigen receptor, although it binds to and competes
`with FK506 for
`the same protein, FKBP (6).
`Instead,
`rapamycin
`inhibits signal
`transduction from the IL-2, epidermal growth factor,
`and other cytokine receptors,
`thus blocking the G¡to S phase transi
`tion required for cell cycle progression (6). In addition,
`rapamycin
`also inhibits the proliferation of 3T3 cells (7) and the hepatoma cell
`line H4 (8), presumably by blocking the effects of the growth factor-
`like activity of serum. The signal
`transduction pathway inhibited by
`the rapamycin-FKBP complex is not completely understood. Regard
`less of the mechanism,
`rapamycin blocks the activation of p70 S6
`kinase by diverse agents.
`It has also been shown that
`rapamycin
`blocks the activation of p34cdc2 kinase in T cells and in the myogenic
`cell
`line BC3H1 (9), presumably because p34cdc2 is a downstream
`target of p70 S6 kinase. This, combined with the evidence
`that
`p34cdc2 deregulation is an obligatory component of the induction of
`apoptosis by natural killers in target cells (10), suggests that rapamy
`cin could potentially alter the induction of apoptosis.
`is an active sui
`Apoptosis,
`a physiologically
`programmed event,
`cide process
`requiring energy-dependent
`participation
`of the dying
`cells (11). Apoptosis can be induced in T cells by activation, or in
`growth factor- and hormone-dependent
`cells by deprivation
`of the
`dependent
`factors, or in malignant cells by chemotherapeutic
`agents.
`Cyclosporin A and FK-506 have been shown to block activation-
`induced apoptosis in T-cell
`lines (12), while rapamycin does not (13).
`Herein, we report
`that rapamycin augments apoptosis in a number of
`different
`systems. This effect can be demonstrated
`in the murine
`T-cell
`line CTLL-2 induced by IL-2 withdrawal,
`in T-cell hybridomas
`induced by activation through the T-cell antigen receptor,
`in murine
`S49 cells treated with steroids, and in wye-transformed RAT-1 fibro
`blasts induced by culturing under
`low serum conditions. Strikingly,
`rapamycin also promotes apoptosis
`in the human promyelocytic
`line
`HL-60 cells and the human ovarian cancer SKOV3 cells induced
`by the chemotherapeutic
`drug, cisplatin,
`indicating potential clinical
`applications.
`
`MATERIALS AND METHODS
`
`Reagents. MTT and dexamethasone were purchased from Sigma Chemical
`Co. (St. Louis, MO). Rapamycin was provided by the National Cancer
`Insti
`tute, NIH (Bethesda, MD). Recombinant
`human IL-2 was obtained from the
`former Cetus Corp. (La Jolla, CA). Cisplatin was from David Bull Laboratories
`Pty Ltd. (Mulgrave, Victoria, Australia). FK520 was a gift of Dr. N. H. Sigal
`(Merck, Sharp and Dohme Research Laboratories, Rahway, NJ). Unless oth
`erwise indicated, all other chemicals were the purest grade available and were
`obtained from Sigma.
`Cell Lines. The T-cell hybridoma ALI was a gift of Dr. B. Singh (Uni
`versity of Western Ontario, London, Ontario, Canada; Ref. 14). They were
`recloned
`and selected
`for
`responsiveness
`to activation
`stimuli
`and T-cell
`receptor expression. A hamster anti-murine CD3 B cell hybridoma, 145-2cll
`(15), was used as sources of antibodies
`to the T-cell
`receptor complex. The
`IL-2-dependent mouse T cell
`line, CTLL-2, human promyelocytic
`leukemia
`
`1982
`
`Breckenridge Exhibit 1033
`Shi
`Page 001
`
`

`

`RAPAMYCIN ENHANCES APOPTOSIS
`
`line SKOV3 were obtained
`line HL-60, and human ovarian cancer cell
`cell
`from American Type Culture Collection (Rockville, MD). The mouse lym-
`phoma, S49, was kindly provided
`by Dr. G. T. Williams
`(University
`of
`Birmingham, Birmingham, United Kingdom). Rat-1 cells constitutively
`ex
`pressing c-myc under
`the control of the muLV retroviral promoter have been
`described
`previously
`(16). All cells were cultured
`at 37°C in humidified
`
`containing 5% CO2 in RPMI 1640 (GIBCO Laboratories, Grand
`atmosphere
`Island, NY) supplemented with 2 HIM L-glutamine,
`10 mM HEPES, 50 p,M
`2-mercaptoethanol,
`5-10% heat-inactivated
`fetal bovine serum (Sigma), and
`10 (AMgentamicin (GIBCO).
`Apoptosis
`Induction. The T-cell hybridoma Al.l was activated by anti-
`CD3 coated on tissue culture plastic by incubating with 0.05 M Tris-HCl
`(pH
`9.0) overnight
`at 4°Cor for
`l h at 37°C.Plates were washed with PBS to
`
`cells were added. Cells were harvested
`remove unbound antibody before Al.l
`for DNA fragmentation
`analysis after a 12-h incubation at 37°Cin humidified
`5% CO2. Apoptosis
`in CTLL-2 cells was induced by IL-2 starvation,
`in S49
`cells by dexamethasone
`treatment,
`in Rat-1-m^c cells by low serum, and in
`HL-60 cells and SKOV3 cells by cisplatin treatment as indicated.
`Genomic DNA Fragmentation Assay. Cells (4-6 X IO5) were harvested
`
`tube in 30 p.1 PBS and lysed with 30 fil of
`in an Eppendorf
`and resuspended
`lysis buffer
`[80 mM EDTA, 200 mM Tris (pH 8.0), 1.6% (w/v) sodium lauryl
`sarcosinate,
`and 5 mg proteinase K/ml]. The lysate was mixed and then
`incubated in a 50°Cwater bath for 1.5 h. After adding 0.2 mg/ml RNase A, the
`mixture was incubated in a 37°Cwater bath for an additional 30 min. The
`
`resulting DNA solution was analyzed on 1% agarose gels
`(10 mM Tris and 1 mM EDTA).
`by labeling
`Alternatively,
`genomic DNA fragmentation was quantitated
`actively dividing cells with 125IUDR at the concentration
`of IO6 cells/ml with
`1 fiCi/ml of 125IUDR at 37°Cfor 8-10 h. Labeled cells were harvested and
`
`in TAE buffer
`
`300000-1
`
`200000-
`
`100000-
`
`O ^^
`Q. ÃœJ
`
`5»
`
`O -H
`
`2 _-il>0
`
`[Rapamycln]
`
`ng ml
`
`cell proliferation. CTLL-2 cells were
`Fig. 1. Effect of rapamycin on IL-2-induced
`plated in 96-well plates at 1 X 10" cells/well with different concentrations
`of rapamycin
`and recombinant human IL-2. After 20 h, l ^tCi of [3H]thymidine was added to each well
`for 3 h. Cell proliferation was determined by measuring [3H]thymidine
`incorporation by
`scintillation counting. Results represent
`the mean of six wells; bars, SE.
`
`washed with cold media at least three times. Treatments were then carried out
`in 200 fj.1media in 96-well
`tissue culture plates; genomic DNA fragmentation
`was then assayed as follows. Cells were harvested in 1.5-ml Eppendorf
`tubes
`and lysed by adding 900 ju.1lysis buffer [5 mM Tris (pH 7.4), 2 mM EDTA, and
`0.5% Triton X-100 (nonionic detergent);
`total volume, 1.1 ml]. The Eppendorf
`tubes were then vortexed vigorously to ensure complete lysis of cells. After
`incubating on ice for 20 min, the tubes were centrifuged at 14,000 cpm for 20
`min in a microfuge. One ml of supernatant
`(containing fragmented DNA) was
`transferred to a new Eppendorf
`tube, leaving 100 ß\supernatant with the pellet
`to ensure that
`the pellets were not cotransferred.
`The radioactivity
`of
`the
`supernatant
`and the pellet were measured with a gamma counter. The percent
`age of fragmentation was calculated by:
`
`% DNA fragmentation =
`
`Supernatant cpm X 1.1
`Supernatant cpm + pellet cpm
`
`x 100
`
`MTT Staining. Cell viability was assessed essentially as described by
`Mosmann (17). Briefly, cells were incubated in 100 fil media in 96-well plates
`with additions
`as indicated. Following
`incubation,
`10 /nl of MTT solution
`(5 mg MTT/ml
`in H2O) were added and incubated at 37°Cfor 4 h. One
`hundred (¿1of acid-isopropanol
`(0.04 N HC1 in isopropanol) were added to each
`culture and mixed by pipetting or shaking on a plate shaker
`to dissolve the
`reduced MTT crystals;
`the relative cell viability was obtained by scanning with
`an ELISA reader with a 570-nm filter.
`Cell Proliferation Assay. Cells were incubated in 96-well plates with
`appropriate treatments. The proliferative
`response was determined by [3H]thy-
`midine incorporation in which 1 ^iCi of [3H]thymidine was added to each well
`and incubated for 3 h. Cells were then harvested onto glass-fiber
`filter paper,
`and the rate of [3H]thymidine
`uptake was quantitated
`by liquid scintillation
`
`counting.
`
`RESULTS
`
`reciprocal effect; the higher the concentration of IL-2 in the culture
`medium, the higher the concentration of rapamycin required to sup
`press the proliferation of CTLL-2 cells to background levels. (The
`effect of rapamycin on IL-2-induced proliferation was analyzed by
`two-way ANOVA, P < 0.0001.) This suggests that the concentration
`of IL-2 determines the quantity or quality of the transmembrane
`signals and that rapamycin is able to completely block IL-2-induced
`proliferation only in the presence of relatively low concentrations of
`IL-2.
`Since IL-2 induces proliferation in responsive cells, IL-2 must
`provide both mitogenic and cell survival signals upon the interaction
`with its receptor. It is conceivable that the effect of rapamycin on
`IL-2-induced proliferation of CTLL-2 cells might be due to the
`inhibition of cell survival signals rather than on the mitogenic signal
`induced by IL-2. We tested this hypothesis by using a model in which
`IL-2 deprivation induces apoptosis in CTLL-2 cells. As shown pre
`viously (19), 24 to 48 h after IL-2 withdrawal, the majority of CTLL-2
`cells undergo apoptosis. When rapamycin was added, there was a
`significant increase of apoptosis in CTLL-2 cells as indicated by
`genomic DNA fragmentation assessed on agarose gels (Fig. 2) and by
`release of label from 125IUDR-labeled cells (data not presented).
`However, this effect was only apparent when limiting concentrations
`of IL-2 were present. Indeed, in the presence of IL-2 (10 units/ml),
`rapamycin did not induce apoptosis at any concentration tested (Fig.
`2 and data not shown). This is apparently discordant to Fig. 1, in
`which cell proliferation induced by as much as 20 units/ml of IL-2 can
`still be decreased by high concentrations of rapamycin. The most
`likely explanation for this discrepancy is that apoptosis only occurs
`when IL-2 signals are decreased below a critical
`threshold. The
`immunosuppressant FK-520, an analogue of FK506 which competes
`with rapamycin for FKBP, did not promote apoptosis, but rather
`reversed the effect of rapamycin when FK520 was present at a 20-fold
`excess (Fig. 3),
`indicating that the apoptosis-promoting effect of
`rapamycin depends on the binding of rapamycin to FKBP.
`The distinction between cell survival and cell proliferation signals
`has been well established by genetic complementation of bcl-2 and
`The immunosuppressive effect of rapamycin is not through its
`myc (20, 21). The proto-oncogene c-myc, which plays an important
`effect on the signals from the T-cell antigen receptor but rather
`through the effects on the signals induced by IL-2 produced after
`role in cell proliferation and transformation, is also required for the
`T-cell activation (18). We tested the effect of rapamycin on IL-2-
`induction of apoptosis in some cell lineages (22, 23). When cells
`induced proliferation of a murine T-cell line, CTLL-2, by varying the
`constitutively expressing myc are cultured under reduced serum con
`concentrations of both IL-2 and rapamycin. As indicated by [3H]thy-
`ditions, they undergo apoptosis (23). In this case, apoptosis can be
`midine incorporation shown in Fig. 1, IL-2 and rapamycin showed a
`suppressed by cell survival signals provided by the constitutive
`1983
`
`Breckenridge Exhibit 1033
`Shi
`Page 002
`
`

`

`[rapamycin] ng/ml
`= 8 _
`08
`_
`
`RAPAMYCIN ENHANCES APOPTOSIS
`
`cally promoted anti-CD3-induced apoptosis in Al.l cells as indicated
`by genomic DNA fragmentation detected by agarose gel electrophore
`sis (Fig. 5, a and b) and by the release of 125IUDRfrom labeled cells
`(Fig. 5c). Rapamycin alone did not induce apoptosis in the T-cell
`hybridoma cells (Fig. 5).
`
`D Cl M 9
`a am a
`o «us rf
`u oÃ(cid:173)».ai
`
`O
`
`0.5
`[IL-2] lU/ml
`
`10
`
`I
`
`0.30
`
`[Rapa]
`
`ng/ml
`
`U ml
`
`genomic DNA fragmentation.
`IL-2 deprivation-induced
`Fig. 2. Rapamycin increases
`CTLL-2 cells (1 X Id6) were cultured without or with IL-2 at 0.5 or 10 units/ml, and
`rapamycin at (I, 1, 10, or 100 ng/ml. After 20 h, cells were harvested and lysed, and
`genomic DNA was extracted and analyzed by agarose gel electrophoresis
`(a). Densito-
`metric analysis of the fragmented DNA of the second hands in the agarose gel
`is also
`presented (h).
`
`expression of bcl-2 or the presence of high concentrations of serum
`(20, 23). In accordance with previous studies, when Rat-1 cells
`constitutively expressing c-mvc were cultured in media containing
`0.5% serum, there was a dramatic decrease in cell viability as indi
`cated by MTT reduction. This decrease in cell viability under low
`serum conditions was augmented by the addition of 10 ng/ml rapa
`mycin (Fig. 4). Thus, rapamycin increases programmed cell death
`induced by the constitutive expression of c-myc, likely by interfering
`with cell survival signals mediated by the low concentration of serum
`present in the assays (one-way ANOVA, P < 0.001). As with CTLL-2
`cells incubated with high concentrations of IL-2 (Figs. 1 and 2),
`rapamycin exerted little effect on apoptosis in RAT-1 cells in the
`presence of high concentrations of serum (P < 0.05).
`Activation-induced apoptosis in T-cell hybridoma cells has been
`used as a model system to explore the mechanism regulating negative
`selection during T-cell development in the thymus (24). Cyclosporin
`A and FK-506 can completely block activation-induced apoptosis in
`T-cell hybridoma A1.1 cells (12, 13) and other cells (data not shown).
`However, when the effect of rapamycin was tested, it was found that,
`unlike CsA or FK506, rapamycin does not block activation-induced
`apoptosis (13). We sought to determine whether rapamycin would
`promote apoptosis in this system following activation of ALI cells
`with varying concentrations of anti-CD3. At an optimal dose of
`anti-CD3 (2 ju.g/ml),rapamycin did not alter activation-induced ap
`optosis in A 1.1 cells, likely because the majority of the cells were
`committed to undergo apoptosis by anti-CD3 alone (25). However, at
`lower doses of anti-CD3 (0.5 /xg/ml or lower), rapamycin dramati
`
`1984
`
`«api . FK520
`
`on genomic DNA fragmentation
`rapamycin
`the effect of
`Fig. 3. FK520 reverses
`induced by IL-2 deprivation. CTLL-2 cells (1 x IO6) were cultured with or without
`rapamycin al 1 ng/ml
`in the presence or absence of FK520 at 20 ng/ml. Cells were
`harvested and assessed for genomic DNA fragmentation
`on an agarose gel following a
`20-h incubation (a). Densitometric analysis of the second bands in the gel is presented in b.
`
`rapamycin
`
`trapamycin
`
`0.5 i
`
`0.4-
`
`0.3-
`
`0.2-
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`"*
`= 9
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`z oit"
`s E
`at
`te
`
`0.5
`
`Serum Concentration
`
`(%)
`
`fibroblasts
`cell death in myr-transformed
`Fig. 4. Rapamycin enhances programmed
`induced by serum deprivation, mvc-transformed Rat-1 cells were cultured in RPMI
`supplemented with 5 or 0.5% fetal bovine serum for 50 h with or without rapamycin at 10
`ng/ml. The relative number of viable cells was determined by the reduction of MTT.
`
`Breckenridge Exhibit 1033
`Shi
`Page 003
`
`

`

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`
`RAPAMYCIN ENHANCES APOPTOSIS
`
`cells were stimulated with different
`cells. T-cell hybridoma Al.l
`in mouse T-cell hybridoma
`genomic DNA fragmentation
`activation-induced
`Fig. 5. Rapamycin augments
`concentrations
`of anti-CD3 (145-2cl 1) coated on tissue culture plastic with or without
`rapamycin at 10 ng/ml. Cells were harvested 12 h after culture and assessed for genomic DNA
`fragmentation by agarose gel electrophoresis
`(a). Densitometric analysis of the fragmented genomic DNA is presented in b. Al.l
`cells were also labeled with l25IUDRat 1 /xCi/1 X IO6
`cells/ml. After 10 h, cells were washed and cultured with 0.5 (J.g/ml anti-CD3 for 12 h. The percentage of genomic DNA fragmentation was assessed by the release of 12iIUDR-labeled
`fragmented DNA (c); bars, SE.
`
`*
`
`Rapa
`
`[Anli-CD3]
`
`Rapa
`
`Anti-COS
`
`AHII-CD3
`
`t Rapa
`
`is the
`in T cells and thymocytes
`apoptosis
`Corticosteroid-induced
`best characterized model system for studying programmed cell death
`(26). The corticosteroid-sensitive
`mouse lymphoma
`cell
`line S49
`undergoes
`characteristic
`apoptosis upon treatment with dexametha-
`sone. When 125IUDR-labeled S49 cells were incubated with a subop
`timal concentration
`of dexamethasone
`(10~7 M), rapamycin signifi
`
`(Fig. 6; one-way
`plus
`rapamycin;
`
`genomic DNA fragmentation
`increased
`cantly
`ANOVA;
`dexamethasone
`versus dexamethasone
`P < 0.001).
`reagents can induce
`It has been demonstrated that chemotherapeutic
`apoptosis
`in target cells (27). Rapamycin has already been tested
`clinically as an immunosuppressant
`in patients and is relatively non-
`toxic in short-term administration
`(28).
`If rapamycin can augment
`apoptosis
`induced by chemotherapeutic
`reagents,
`then the addition of
`rapamycin to chemotherapy
`protocols could potentially increase the
`efficacy of chemotherapy. Cisplatin, an effective chemotherapy agent,
`has been shown to induce apoptosis in a number of cell lineages (29).
`A 24-h incubation with cisplatin at 30 JAMor more induced DNA
`fragmentation in the human promyelocytic
`leukemia cell line HL-60,
`and as predicted,
`rapamycin augmented cisplatin-induced DNA frag
`mentation (Fig. la). The densitometric analysis of the gel is presented
`in Fig. Ib. However, as before, rapamycin did not augment
`the effect
`of cisplatin at doses which already induced maximal apoptosis
`(i.e.,
`100 fiM cisplatin). When HL-60 cells were incubated with cisplatin
`for 96 h, there was a dose-dependent
`reduction of cell viability, as
`determined
`by MTT reduction, which was detectable
`at 1 /XMand
`reached maximal effects at 5 to 10 /XM(Fig.
`lc; R2 = 0.995 from
`
`0.625 fiM to 5 JAM;note: the different concentrations of cisplatin in the
`experiments
`in Fig. 7, a and b, reflect
`the different periods of incu
`bation). We selected 2.5 /AM cisplatin as a suboptimal
`dose and
`determined
`the effect of
`rapamycin.
`In this
`system,
`rapamycin
`induced
`a dose-dependent
`augmentation
`of cisplatin-induced
`re
`duction of cell viability (Fig. Id;
`two-way ANOVA, P < 0.001 and
`R2 = 0.982 for
`rapamycin
`from 0.01 nM to 10 nM). In contrast,
`rapamycin
`alone did not
`significantly
`alter HL-60 viability
`at
`several concentrations
`tested (Fig. 7). The effect of rapamycin on
`1985
`
`of cisplatin was also
`doses
`by suboptimal
`induced
`cell death
`readily observed
`by changes
`in morphology
`at
`the microscopic
`level (Fig.
`le). Cisplatin at 2.5 LIMinduced morphological
`changes
`consistent with apoptosis
`in only a small proportion
`of cells;
`rapamycin alone had no effect. However, when cells were coincu-
`
`2.0-
`
`111
`
`COI*
`o cs s
`
`O s
`
`fi n
`0) â„¢
`Ü
`«I
`o I'
`
`Rapa
`
`Dex
`
`Dex + Rapa
`
`cells.
`in steroid-sensitive
`Fig. 6. Rapamycin increases genomic DNA fragmentation
`S49 cells were labeled with 125IUDR at 1 nCi/1 x 10" cells/ml. After 10 h, cells were
`washed and cultured with dexamethasone
`at 10~7 M for 36 h. Percentage
`of genomic
`DNA fragmentation
`was assessed
`by the release
`of
`'25IUDR-labeled
`fragmented
`DNA. Bars. SE.
`
`Breckenridge Exhibit 1033
`Shi
`Page 004
`
`

`

`RAPAMYCIN ENHANCES APOPTOSIS
`
`3
`
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`Breckenridge Exhibit 1033
`Shi
`Page 005
`
`

`

`RAPAMYCIN ENHANCES APOPTOSIS
`
`rapamycin,
`
`nearly all of
`
`bated with 2.5 JU.Mcisplatin and 1 ng/ml
`the cells underwent
`apoptosis.
`Cisplatin is currently the drug of choice for the treatment of ovarian
`cancer. Therefore, we examined the effect of rapamycin on the in
`duction of cell death in a relatively cisplatin-resistant
`ovarian cancer
`cell line, SKOV3. Since ovarian cancer cells frequently do not dem
`onstrate cisplatin-induced DNA ladders despite clearly undergoing
`apoptosis (30), we used cell viability as assessed by MTT reduction as
`a measure of apoptosis
`in these cells. As shown in Fig. If, cisplatin
`induced a dose-dependent
`reduction in cell viability of SKOV3 cells
`as determined by MTT reduction. The effect of cisplatin was detect
`able at 3 /xM and reached maximal effects at 25 to 50 /AM(R2 = 0.967
`
`from 1.56 JIM to 25 JU.M).We selected 5 /J.Mcisplatin as a suboptimal
`dose and studied the effect of rapamycin on cisplatin-induced
`cell
`death. We found that rapamycin induced a concentration-dependent
`enhancement
`of cisplatin-induced
`cell death (Fig. 7g;
`two-way
`ANO VA, P < 0.01 and R2 = 0.954 for rapamycin from 0.1 nM to 100
`
`could also enhance
`rapamycin
`HM). Thus,
`cisplatin in ovarian cancer cells.
`
`cell death induced by
`
`DISCUSSION
`
`and FK506, which inhibit activation-induced
`Unlike cyclosporin
`apoptosis
`in T cells (12, 13), rapamycin enhances
`the induction of
`apoptosis
`in a number of cell
`lineages mediated by a number of
`different mechanisms. Many cancer chemotherapeutic
`agents have
`been shown to exert their effects by inducing apoptosis in cancer cells.
`Our observation
`that
`low doses of
`rapamycin
`increase
`apoptosis
`induced by suboptimal
`doses of cisplatin,
`at
`least
`in vitro,
`in the
`human promyelocytic
`leukemia cell line HL-60 and the human ovar
`ian cancer cell line SKOV3 suggests a potential clinical application, as
`both drugs are currently in clinical use.
`transformation have led to
`Studies of the mechanisms of malignant
`the identification of genes that regulate cell proliferation,
`cell viabil
`ity, or both. Genes with the capacity to regulate cell viability include
`bcl-2, bcl-x, box, p53, retinoblastoma gene, raf, ras, ahi, Fas/APO-l,
`and c-myc (reviewed in Refs. 31 and 32). The hypothesis
`that cells
`require both proliferative
`and survival
`signals
`is best supported by
`genetic complementation
`experiments
`in which myc overexpression-
`induced cell death is suppressed by bcl-2 expression (20, 21). Our
`results demonstrating
`that
`rapamycin
`promotes
`serum deprivation-
`induced cell death in c-myc transfected RAT-1 cells suggest
`that
`rapamycin promotes cell death through the inhibition of cell survival
`signal(s).
`In IL-2-dependent
`cell lines, IL-2 provides both survival and
`proliferative signals. It has been shown that p70 S6 kinase is activated
`in CTLL-2 cells after
`IL-2 stimulation,
`and rapamycin specifically
`inhibits p70 S6 kinase activation (6). Therefore, p70 S6 kinase might
`be important
`in providing survival signals. Recent reports show that
`one of the downstream targets of p70 S6 kinase is p34cdc2 kinase,
`which has been shown to be important
`in the regulation of apoptosis
`in target cells by cytotoxic killing (10).
`It is currently used in the
`Cisplatin is a potent antitumor
`agent.
`treatment of many malignancies,
`including small cell lung, testicular,
`ovarian, head and neck, bladder, and esophageal
`cancers
`(33). Cis
`platin induces the formation of DNA adducts,
`including cross-links
`between DNA and protein or inter- and intrastrand cross-links in DNA
`(29). However,
`there is no correlation between the amount of cisplatin
`required to induce cell death and to inhibit DNA synthesis
`in DNA
`repair-deficient
`and DNA repair-proficient
`cells. Thus,
`the cytotoxic
`effect of cisplatin is not solely due to the inhibition of DNA synthesis
`or DNA damage.
`Instead,
`the ability of cisplatin to decrease cell
`viability was inhibited by cyclohexamide
`and was accompanied
`by
`genomic DNA fragmentation,
`characteristic of cells dying by apop
`
`the
`rapamycin enhances
`that
`results demonstrating
`tosis (33). Our
`cytotoxic effects of cisplatin suggests that intracellular
`signaling mol
`ecules inhibited by rapamycin,
`such as p70 S6 kinase, may play a
`generalized role in regulating apoptosis induced by chemotherapeutic
`agents.
`Although cisplatin has been widely used as an antitumor agent, high
`doses lead to severe multiorgan toxicities
`including kidney and bone
`marrow failure,
`intractable vomiting, peripheral neuropathy, deafness,
`seizures, and blindness, preventing dose intensification
`(34). Poten
`tially,
`rapamycin could reduce the dosage and augment
`antitumor
`activity of cisplatin without
`increasing toxic side effects. Furthermore,
`the amount of rapamycin required for this effect
`in vitro is only 1
`ng/ml, which is readily achieved in patients. For example,
`in renal
`transplant
`recipients,
`serum rapamycin concentrations
`as high as 10
`ng/ml have been achieved 72 h after administration without
`toxicity
`(35). Thus,
`if rapamycin potentiates
`the effects of cisplatin or other
`drugs on tumor cells without
`increasing multiorgan toxicity of the
`chemotherapeutic
`agents,
`the combination of rapamycin with conven
`tional chemotherapeutic
`agents may result
`in functional
`"dose inten
`sity," perhaps increasing survival
`rates.
`
`ACKNOWLEDGMENTS
`
`We are indebted to Mary Hill, Feng Wang, and Ajay Sharma for technical
`support.
`
`REFERENCES
`
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`
`Breckenridge Exhibit 1033
`Shi
`Page 006
`
`

`

`RAPAMYCIN ENH