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The ataxia telangiectasia mutated (ATM) and the ATM- related (ATR) kinases play a central role in facilitating the resistance of cancer cells to genotoxic treatment regimens. The components of the ATM and ATR regulated signaling pathways thus provide attractive pharmacological targets, since their inhibition enhances cellular sensitivity to chemo- and radiotherapy. Caffeine as well as more specific inhibitors of ATM (KU55933) or ATM and ATR (CGK733) have recently been shown to induce cell death in drug-induced senescent tumor cells. Addition of these agents to cancer cells previously rendered senescent by exposure to genotoxins suppressed the ATM mediated p21 expression required for the survival of these cells. The precise molecular pharmacology of these agents however, is not well characterized. Herein, we report that caffeine, CGK733, and to a lesser extent KU55933, inhibit the proliferation of otherwise untreated human cancer and non- transformed mouse fibroblast cell lines. Exposure of human cancer cell lines to caffeine and CGK733 was associated with a rapid decline in cyclin D1 protein levels and a reduction in the levels of both phosphorylated and total retinoblastoma protein (RB). Our studies suggest that observations based on the effects of these compounds on cell proliferation and survival must be interpreted with caution. The differential effects of caffeine/CGK733 and KU55933 on cyclin D1 protein levels suggest that these agents will exhibit dissimilar molecular pharmacological profiles. Background genotoxic anti-cancer therapeutics by sensitizing cancer ATM and ATR cooperate to mediate cellular responses to cells to these agents (reviewed ). The relative non-spe- DNA damage, following exposure to diverse genotoxic cificity of caffeine has lead to the search for more specific agents. These include induction of cell cycle arrest, DNA inhibitors of ATM and ATR. The small molecule inhibitor repair, maintenance of genomic stability, induction of 2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one premature senescence and cell death [1-3]. The coordi- (KU55933) has been shown to specifically inhibit ATM in nated activation of these processes has been defined as the the low nanomolar range (IC :12.9 nM). In contrast, DNA damage response pathway (DDR). Initial studies KU55933 did not inhibit ATR at doses of up to 100 μM demonstrated that inhibition of ATM and ATR by caffeine . KU55933 has been shown to sensitize cancer cells to significantly enhanced cellular sensitivity to ionizing radi- both IR and chemotherapeutic agents [6,7]. CGK733, a ation (IR) . Inhibiting ATM, ATR or their downstream thiourea-containing compound, was originally identified targets thus serves to widen the therapeutic window of as an inhibitor of ATM and ATR with an IC of ~200 nM Page 1 of 7 (page number not for citation purposes) Radiation Oncology 2009, 4:51 http://www.ro-journal.com/content/4/1/51 towards both kinases . This study was subsequently with 10% (v/v) fetal calf serum, 2 mM L-glutamine, 100 retracted, leaving the precise molecular pharmacology of units/ml penicillin and 100 μg/ml streptomycin at 37°C this compound unclear. Additional studies suggest how- in humidified 5% CO ever, that CGK733 inhibits ATM and ATR [9-12]. More recently, caffeine, CGK733 and KU55933 have been Cell proliferation assay shown to induce cell death in prematurely senescent Cells were seeded in 96-well plates at a predetermined breast cancer cells . The induction of premature senes- optimal cell density to ensure exponential growth for cence by genotoxic agents contributes to drug sensitivity duration of the assay. After a 24 h preincubation, growth and is primarily (but not solely) dependent on p53- medium was replaced with experimental medium con- induced p21 expression [14,15]. Cancer cells that have taining the appropriate drug concentrations or 0.1% (v/v) undergone drug-induced premature senescence are less vehicle control. After a 48 h incubation, cell proliferation sensitive to pro-apoptotic signaling and can re-enter the was estimated using the sulforhodamine B colorimetric cell cycle [16-20]. The study by Crescenzi et al.  sug- assay  and expressed as the mean ± SE for six replicates gests that ATM is required for the maintenance of the pre- as a percentage of vehicle control (taken as 100%). Exper- mature senescent phenotype and hence the survival of iments were performed independently at least three times. cancer cells exposed to genotoxins. Combining ATM and/ Statistical analyses were performed using a two-tailed Stu- or ATR inhibitors with genotoxins may thus further dent's t test. P < 0.05 was considered to be statistically sig- enhance the cytotoxicity of these agents, by preventing nificant. drug induced senescence as a therapeutic outcome . The molecular pharmacology of inhibitors like CGK733 Immunoblotting and KU55933 will require further characterization. ATR Cells treated as indicated were harvested in 5 ml of unlike ATM regulates cell cycle progression in the absence medium, pelleted by centrifugation (1,600 rpm for 5 min of DNA damage and is required for the viability of prolif- at 4°C), washed twice with ice-cold phosphate buffered erating human and mouse cells . Inhibitors that target saline (PBS) and lysed in ice-cold HEPES buffer [50 mM both ATM and ATR are thus likely to exhibit pharmacolog- HEPES (pH 7.5), 10 mM NaCl, 5 mM MgCl , 1 mM EDTA, ical profiles that are distinct from ATM selective inhibi- 10% (v/v) glycerol, 1% (v/v) Triton X-100 and a cocktail tors. It is also likely, that the genetic make up of a of protease inhibitors (Roche Diagnostics Scandinavia AB, particular subset of cancer cells influences their relative Bromma, Sweden)] on ice for 30 min. Lysates were clari- sensitivity to ATM and/or ATR inhibitors [21,22]. fied by centrifugation (13,000 rpm for 15 min at 4°C) and the supernatants then either analyzed immediately or Methods stored at -80°C. Equivalent amounts of protein (20 - 50 Reagents μg) from total cell lysates were resolved by SDS-PAGE and Stock solutions of caffeine (100 mM in water) (Sigma transferred onto 'nitrocellulose membranes. Membranes Aldrich, Stockholm, Sweden), CGK733 (20 mM) and were blocked in blocking buffer [5% (w/v) nonfat dried KU55933 (10 mM) (Calbiochem, VWR International AB, milk, 150 mM NaCl, 10 mM Tris (pH 8.0) and 0.05% (v/ Stockholm, Sweden) dissolved in dimethyl sulphoxide v) Tween 20]. Proteins were detected by incubation with (DMSO) were stored at -20°C. Lithium Chloride (40 primary antibodies at appropriate dilutions in blocking mM) (Sigma-Aldrich) was dissolved in sterile distilled buffer overnight at 4°C. Blots were then incubated at water and stored at 4°C. The proteasome inhibitor room temperature with horseradish peroxidase-conju- MG132 (50 mM) (Sigma- Aldrich) was dissolved in gated secondary antibody. Bands were visualized by DMSO and stored at -20°C. Caspase inhibitor I (40 mM) enhanced chemiluminescence (Supersignal West Pico; (Z-VAD (OMe)-FMK (Calbiochem) was dissolved in Pierce, Nordic Biolabs AB, Täby, Sweden) followed by DMSO and stored at -20°C. Monoclonal antibodies exposure to autoradiography film (General Electric Bio- raised against cyclin D1 (DCS-6) (Santa Cruz Biotechnol- Sciences, Uppsala, Sweden). ogy, Santa Cruz, CA), RB (G3-245) (Becton Dickinson AB, Stockholm, Sweden), α- Tubulin (Sigma- Aldrich), and Immunofluorescence microscopy Hsp60 (Abcam, Cambridge, United Kingdom) were used. MCF-7 cells were grown on sterile glass coverslips in 6- well plates to 80% confluence in media before being Cell culture washed three times in PBS. Cells were fixed in 4% formal- LNCaP, MCF-7, MDA-MB436 and T47D cells were cul- dehyde/PBS at room temperature for 10 minutes. Cover- tured in RPMI 1640 supplemented with 10% (v/v) fetal slips were washed twice in PBS and permeabilized in 0.2% calf serum, 2 mM L-glutamine, 100 units/ml penicillin Triton X100/PBS for 15 minutes. Following another three and 100 μg/ml streptomycin at 37°C in humidified 5% washes in PBS, coverslips were blocked in 3% bovine CO . HCT116 and BALB/c 3T3 cells were cultured in Dul- serum albumen (BSA)/PBS at room temperature for 30 becco's modified eagle medium (DMEM) supplemented min. Antibodies to Cyclin D1 (DCS-6) (Santa Cruz) (1:50 Page 2 of 7 (page number not for citation purposes) Radiation Oncology 2009, 4:51 http://www.ro-journal.com/content/4/1/51 dilution) were applied in 3% BSA/PBS medium overnight. Cyclin D1 activity is required for G1- S phase progression Cells were washed then washed 3 times in PBS, and incu- and the regulation of its expression and/or stability is bated with a rhodamine (TRITC)- conjugated goat anti- often deregulated in cancer cells (reviewed in ). Previ- mouse secondary antibody (1:200) (Jackson Immunore- ously, we demonstrated that cyclin D1 is essential for the search, Fisher Scientific AB, Gothenburg, Sweden) at proliferation of MCF-7 cancer cells . We thus investi- room temperature for 1 h. After a final 3 washes, cover- gated the effect of caffeine, CGK733 and KU55933 on the slips were mounted on glass slides with Vectorshield con- proliferation of a panel of human cancer cell lines derived taining 4', 6'-diamidino-2-phenylindole(DAPI) (Vector from solid tumors. Various studies have used CGK733 at Laboratories Ltd., Peterborough, United Kingdom). concentrations ranging from 0.6- 40 μM (Table 1). In our Images were obtained with a Zeiss AxioCam on a Zeiss study, CGK733 inhibited proliferation of MCF-7 and Axioplan 2 microscope with a 100 × objective using the T47D estrogen receptor (ER) positive breast cancer cells, appropriate filter sets. MDA-MB436 ER negative breast cancer cells, LnCap pros- tate cancer cells and HCT116 colon cancer cells (Figure Results 2A). Furthermore, CGK733 also suppressed proliferation In this work, we observe that CGK733 induces the loss of of non- transformed mouse BALB/c 3T3 embryonic cyclin D1 via the ubiquitin- dependent proteasomal deg- fibroblast cells (Figure 2A). The CGK733-mediated inhi- radation pathway in MCF-7 and T47D breast cancer cell bition of proliferation was dose dependent and significant lines. Culture of MCF-7 breast cancer cells with 10 μM at doses as low as 2.5 μM. Culture of MCF-7 and T47D CGK733 induced a detectable decline of cyclin D1 levels cells with 5 mM caffeine inhibited cell proliferation to a within 2 h of exposure, and this effect was maximal similar degree as CGK733 (Figure 2B). KU55933 maxi- between 4 and 6 h after exposure (Figure 1A). CGK733 mally inhibits cellular ATM kinase activity at 10 μM and induced the loss of cyclin D1 expression at concentrations was originally reported not to inhibit cell proliferation at as low as 5 μM and this activity was maximal at 10 to 20 this concentration . KU55933 has been used at doses μM (Figure 1B). CGK733 similarly induced loss of cyclin ranging from 1 to 40 μM (Table 1), and later studies sug- D1 protein in T47D cells at these concentrations (Figure gested that some cell lines exhibit sensitivity to this inhib- 1C). The phosphorylation of cyclin D1 residue threonine itor at concentrations between 10 and 20 μM . We 286 (T286) by GSK3β greatly enhances its degradation by observed that KU55933 inhibited proliferation of MCF-7 the ubiquitin- 26S proteasome pathway. Several agents and T47D cells at concentrations ranging from 10 to 30 that induce cyclin D1 ablation have been shown to do so μM (Figure 2B and 2C). The ability of caffeine and in a GSK3β dependent manner . The CGK733 CGK733 to induce the loss of cyclin D1 expression is thus induced attenuation of cyclin D1 levels was inhibited by likely to enhance their anti-proliferative activity at high the 26S proteasome inhibitor MG132 but not the GSK3β concentrations. inhibitor lithium chloride (LiCl) in MCF-7 and T47D cells (Figure 1D and 1E). CGK733 thus induces the loss of cyc- Crescenzi et al  recently reported that caffeine (1- 5 lin D1 protein via the ubiquitin- 26S proteasome pathway mM), CGK733 (10 - 20 μM) and KU55933 (20 - 40 μM) independently of GSK3β mediated T286 phosphoryla- induced senescent cancer cells to undergo caspase- tion. GSK3β regulates cyclin D1 stability by facilitating its dependent apoptosis. Our findings demonstrate, how- CRM1-dependent nuclear export and subsequent degra- ever, that these compounds inhibit the proliferation of dation within the cytoplasm [25,26]. Accordingly, otherwise untreated cancer and non- transformed cell CGK733 did not affect the subcellular localization of cyc- lines (Figure 2). In contrast to that study however, pan- lin D1 (Figure 1F). CGK733 also induced ubiquitin- caspase inhibition did not suppress the anti-proliferative dependent loss of cyclin D1 in LNCaP prostate cancer cells effect of CGK733 on MCF-7 cells in our experiments (Fig- (Figure 1G), indicating that this effect is not breast cancer ure 3A). Although the nuclei of CGK733 treated cells specific. We also observed that caffeine induced the ubiq- appeared condensed, we did not detect the nuclear frag- uitin- dependent loss of cyclin D1 in MCF-7 cells (Figure mentation normally detected in apoptotic MCF-7 cells 1H). In contrast, KU55933 did not affect cyclin D1 stabil- (Figure 3B) . Caffeine, CGK733 and KU55933 may ity at the commonly used concentration of 10 μM for up thus suppress proliferation of senescent and non- senes- to 24 h after exposure (Figure 1I). Cyclin D1 forms active cent cancer cells via different mechanisms. kinase complexes with cyclin dependent kinase 4 (CDK4) or CDK6 that phosphorylate and hence inactivate the Discussion retinoblastoma tumor suppressor protein (RB) (reviewed Small molecule selective inhibitors of ATM and/or ATR in ). Caffeine and CGK733 but not KU55933 induced provide powerful tools for studies on the cellular func- a significant decline in the levels of both phosphorylated tions of these kinases. Furthermore, these molecules may and total RB protein levels in MCF-7 cells (Figure 1I and eventually be used alongside regular anti-cancer agents as 1J). chemo- and radiosensitizers (reviewed in ). In this Page 3 of 7 (page number not for citation purposes) Radiation Oncology 2009, 4:51 http://www.ro-journal.com/content/4/1/51 Figure 1 Effect of CGK733, caffeine and KU55933 on cyclin D1 stability. A. MCF-7 breast cancer cells were cultured with 10 μM CGK733 for the indicated times. Total lysates were resolved by SDS- PAGE and analyzed for cyclin D1 expression. Gel loading was monitored with an antibody raised against Hsp60. B. MCF-7 cells were cultured with the indicated concentrations of CGK733 for 6 h and analyzed as in A. C. T47D breast cancer cells were treated as in B. D. MCF-7 cells were incubated with 10 μM CGK733 alone or in the presence of 40 mM LiCl or 25 μM MG132 for 6 h and analyzed for cyclin D1 expression. Gel loading was monitored with an antibody directed against α- Tubulin. E. T47D cells were treated as in D. F. MCF-7 cells were grown on coverslips and treated with 10 μM CGK733 ± 25 μM MG132 for 6 h. Cyclin D1 expression was determined by indi- rect immunofluorescence microscopy as described in Materials and methods. Scale bar 10 μm. G. LnCap prostate cancer cells were treated and analyzed as in D. H. MCF-7 cells were cultured with 5 mM caffeine ± 25 μM MG132 for 6 h and analyzed as in D. I. MCF-7 cells were cultured in the presence of 5 mM caffeine, 10 μM CGK733 or 20 μM KU55933 for 24 h and analyzed for RB and cyclin D1 expression. J. MCF-7 cells were cultured with the indicated concentrations of CGK733 for 24 h and ana- lyzed as in I. Page 4 of 7 (page number not for citation purposes) Radiation Oncology 2009, 4:51 http://www.ro-journal.com/content/4/1/51 E Figure 2 ffect of CGK733, caffeine and KU55933 on cell proliferation Effect of CGK733, caffeine and KU55933 on cell proliferation. A. MCF-7, T47D and MDA-MB436 breast cancer cells, LNCaP prostate cancer cells, HCT116 colon cancer cells and BALB/c 3T3 fibroblasts were cultured with the indicated doses of CGK733 for 48 h. Cell proliferation was measured as described in Materials and methods. Results represent the mean ± S.E. from three independent experiments. *, P < 0.05, compared with control; **, P < 0.005, compared with control. B. MCF-7 cells were cultured with the indicated doses of caffeine, CGK733 and KU55933 for 48 h. Cell proliferation was monitored as in A. Results represent the mean ± S.E. from three independent experiments. **, P < 0.005, compared with control. C. T47D cells were cultured with the indicated doses of caffeine, CGK733 and KU55933 for 48 h. Cell proliferation was monitored as in A. Results represent the mean ± S.E. from three independent experiments. *, P < 0.05, compared with control; **, P < 0.005, compared with control. Table 1: Inhibitors of ATM and/or ATR Study Inhibitor/Concentration Reference Caffeine CGK733 KU55933 Hickson et al., 2004 -- 0.03- 10 μM Cowell et al., 2005 - - 1-20 μM Byrant and Helleday, 2006 - - 2-20 μM Nakai-Murakami et al., 2007 - - 1 mM  Al-Minawi et al., 2008 2 mM 10 μM-  Yamauchi et al., 2008 - - 10 μM Cruet-Hennequart et al., 2008 - 10 μM10 μM Goldstein et al., 2008 - 0.6 μM-  Crescenzi et al., 2008 1-5 mM 10- 20 μM 20- 40 μM Bhattacharya et al., 2008 - 20 μM-  Selected studies involving the exposure of mammalian cell lines to the ATM and/or ATR inhibitors caffeine, CGK733 and KU55933. Page 5 of 7 (page number not for citation purposes) Radiation Oncology 2009, 4:51 http://www.ro-journal.com/content/4/1/51 in turn impact on their chemo- and radiosensitizing prop- erties. It remains unclear if the decline in cyclin D1 levels results from ATR inhibition, the inhibition of both ATM and ATR, or from the inhibition of an unknown target. It should be noted however, that the siRNA-mediated knockdown of ATR induced cyclin D1 accumulation in NIH 3T3 cells . It is also conceivable, that the antipro- liferative effects of ATM/ATR inhibitors observed at high does may result from off- target effects. It remains to be determined, if these agents exert cytotoxic effects on senes- cent cancer cells at lower doses . Observations on can- cer cell proliferation and survival based on the use of ATM/ATR inhibitors should thus be interpreted with cau- tion. Competing interests The authors declare that they have no competing interests. Authors' contributions JPA and PS conceived and designed the study. JPA per- formed the experiments. JPA and PS analyzed and inter- preted the data. JPA and PS drafted and wrote the manuscript. Acknowledgements JPA is the recipient of an EMBO Long Term Fellowship. This work was sup- Ca erati Figure 3 spase independent on inhibition of CGK733 on MCF-7 prolif- ported by the Swedish Cancer Fund (07-0759), the Chemical Biology Plat- Caspase independent inhibition of CGK733 on MCF-7 form at the University of Gothenburg, Assar Gabrielsson Cancer Fund proliferation. A. Cells were cultured with the indicated (FB07-32) and MedCoast Scandinavia. We kindly thank Dr. Hayley doses of CGK733 ± pan- caspase inhibitor z-VAD-fmk (40 Whitaker for the LNCaP cells. We also thank Prof. Jeanette Nilsson, Prof. μM) for 48 h. Cell proliferation was measured as described in Peter Carlsson, Dr. Marie Kannius-Janson and Ali Moussavi for the Materials and methods. Results represent the mean ± S.E. HCT116, T47D and MDA-MB436 cells. We also thank Prof. Julie Grantham from three independent experiments. B. MCF-7 cells were and Karen Brackley for the BALB/c 3T3 cells and their technical support. grown on coverslips and treated with 10 μM CGK733 or 10 μM KU55933 for 48 h. 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Radiation Oncology – Springer Journals
Published: Nov 10, 2009
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