Journal of Cancer Prevention 2014; 19(2): 125-136
Published online June 30, 2014
© Korean Society of Cancer Prevention
J?zefa W?sierska-G?dek, and Sarah Heinzl
Cell Cycle Regulation Group, Department of Medicine I, Division: Institute of Cancer Research, Comprehensive Cancer Center, Medical University of Vienna, Austria
Correspondence to :
J?zefa W?sierska-G?dek, Cell Cycle Regulation Group, Department of Medicine I, Division: Institute of Cancer Research, Comprehensive Cancer Center, Medical University of Vienna, Borschkegasse 8 a, A-1090 Vienna, Austria, Tel: +43-1-40160-57592, Fax: +43-1-40160-957592, E-mail: Jozefa.Gadek-Wesierski@meduniwien.ac.at
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Cells harboring We determined drug cytotoxicity in human MCF-7 and SKBr-3 breast cancer cells using the CellTiterGLO Luminescent cell viability assay and a Tecan multi-label, multitask plate counter to measure generated luminescence. Changes in cell cycle progression were monitored by flow cytometric measurement of DNA content in cells stained with propidium iodide. Unlike NU1025, AZD2461, a new PARP-1 inhibitor, markedly reduced the numbers of living MCF-7 and SKBr-3 cells. ATM kinase inhibition (CP466722) was also cytotoxic for both MCF-7 and SKBr-3 cells. Furthermore, AZD2461 enhanced the cytotoxicity of CP466722 in both cell lines by inducing apoptosis, and concurrent inhibition of ATM and PARP-1 reduced cell proliferation more strongly than either single treatment. Our data show that inhibition of PARP-1 by AZD2461 is synthetically lethal for NU1025-resistant MCF-7 and SKBr-3 breast cancer cells. They also indicate that DNA damage signaling is essential for survival of both SKBr-3 and MCF-7 cells, especially after inactivation of PARP-1.
We determined drug cytotoxicity in human MCF-7 and SKBr-3 breast cancer cells using the CellTiterGLO Luminescent cell viability assay and a Tecan multi-label, multitask plate counter to measure generated luminescence. Changes in cell cycle progression were monitored by flow cytometric measurement of DNA content in cells stained with propidium iodide.
Unlike NU1025, AZD2461, a new PARP-1 inhibitor, markedly reduced the numbers of living MCF-7 and SKBr-3 cells. ATM kinase inhibition (CP466722) was also cytotoxic for both MCF-7 and SKBr-3 cells. Furthermore, AZD2461 enhanced the cytotoxicity of CP466722 in both cell lines by inducing apoptosis, and concurrent inhibition of ATM and PARP-1 reduced cell proliferation more strongly than either single treatment.
Our data show that inhibition of PARP-1 by AZD2461 is synthetically lethal for NU1025-resistant MCF-7 and SKBr-3 breast cancer cells. They also indicate that DNA damage signaling is essential for survival of both SKBr-3 and MCF-7 cells, especially after inactivation of PARP-1.
Keywords: Apoptosis, Caspase-3, Cell cycle, DNA repair, DNA damage, Poly(ADP-ribose) polymerases
DNA is error-prone in all organisms. Fortunately, mammalian cells have evolved mechanisms that detect and signal DNA injury, collectively called the DNA damage response (DDR).1 Ataxia telangiectasia mutated (ATM) protein kinase is an apical factor in DDR. Various chemicals and physical agents (e.g., ionizing radiation) that generate double-strand breaks (DSBs) strongly activate ATM kinase and induce downstream pathways that prevent doubling of the genetic information and cell division.2 However, ATM kinase, a Ser/Thr protein kinase, is mutated in the human genetic instability syndrome ataxia telangiectasia. Thus, patients bearing mutations in the
To survive DNA strand breaks, cells must rapidly sense and respond to them. Thus, mammalian cells have evolved several repair mechanisms. DNA single-strand breaks (SSBs) are repaired by base excision repair (BER),3 whereas DNA regions containing chemical adducts are corrected by nucleotide excision repair (NER).4 Poly(ADP-ribose) polymerase-1 (PARP-1), an extremely sensitive nuclear sensor of SSBs, mediates their signaling and is also involved in BER.5,6
Efficient repair of DNA DSBs is particularly important, as even a few unrepaired DSBs are thought to be harmful for cells.7 Thus, 3 distinct DSB repair processes have also evolved - homologous recombination (HR), non-homologous end-joining (NHEJ), and single strand annealing (SSA) - which differ in several aspects, particularly regarding the kinetics and fidelity. The most reliable and error-free is HR.8
BRCA1, encoded by the
We recently showed that inhibition of PARP-1 activity by NU1025 is strongly cytotoxic for
AZD2461, an inhibitor of PARP-124 and 2 ATM kinase inhibitors (KU55933 and CP466722)25,26 were obtained from AXON Medchem BV (Groningen, Netherlands), prepared as stock solutions in DMSO and stored at ?20°C until use. AZD2461 and KU55933 have been developed by AstraZeneca (London, UK) and CP466722 by Pfizer Inc. (New York, NY, USA).
Cells and treatment
Human MCF-7 and SKBr-3 primary breast carcinoma cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).20 MCF-7 cells were grown as a monolayer in phenol red-free Dulbecco’s medium supplemented with 10 % fetal calf serum at 37°C under an atmosphere containing 8% CO2 SKBr-3 cells were cultivated in DMEM medium with 10% fetal calf serum.27 Twenty-four hours after plating (at 60?70% confluence), the cells were treated with the ATM kinase inhibitors CP466722 (at concentrations ranging from 5 μM to 50 μM), KU55933 at a final concentration of 10 μM, and AZD2461 at concentrations ranging from 5 μM to 50 μM. The durations of the treatments are indicated in Figures 1?5.
Detection of chromatin changes in individual cells by fluorescence microscopy
Cells grown in 35 mm Petri dishes were treated with CP466722, AZD2461 or a combination of the 2 for the indicated durations then washed 3 times in phosphate buffered saline (PBS). The washed cells were immediately fixed in 3.7% paraformaldehyde in PBS, then washed four times in PBS and stained with Hoechst 33258 dissolved in PBS at a final concentration of 1.5 μg/mL.28 The stained cells were inspected under an Eclipse TE300 inverted fluorescence microscope (Nikon Corporation, Tokyo, Japan).
Determination of numbers of living cells
Numbers of viable human breast cancer cells and their sensitivities to the tested drugs at various concentrations were determined by measuring luminescent signals correlated with cellular ATP levels, generated using a CellTiter-Glo kit (Promega Corporation, Madison, WI, USA), as previously described.29 The assays were performed at least in quadruplicate, and the cells’ luminescence was measured using an Infinite M200PRO multi-label plate counter (Tecan Group Ltd., M?nnersdorf, Switzerland). Each data point presented in Figures 1?2; and Figure 4 represents the mean ± SD (bars) of replicates from at least 3 independent experiments. Effects of the combined CP66722 or KU55933 and AZD2461 treatments are shown in Figure 4.
Measurement of DNA concentration in single cells by flow cytometry
DNA contents of single cellswere measured by flow cytometry following Vindelov et al.30 with slight modifications as described elsewhere.31 Briefly, the adherent cells were detached from the substratum by limited trypsinization then all cells were harvested by centrifugation and washed in PBS. Aliquots of 1 × 106 cells were stained with propidium iodide as previously described and their fluorescence was measured using a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) after at least 2 hours incubation at 4°C in the dark. The DNA concentration in the harvested cells was evaluated using ModFITLT cell cycle analysis software (Verity Software House, Topsham, ME, USA) and DNA histograms were generated using the CellQuest software package (Becton Dickinson).
Electrophoretic separation of proteins and immunoblotting
Total cellular proteins dissolved in SDS sample buffer were separated on 8% or 10% SDS slab gels, transferred electrophoretically to polyvinylidenedifluoride membrane (PVDF); (GE Healthcare UK Ltd, Little Chalfont, Buckinghamshire, UK; formerly Amersham Biosciences) and immunoblotted as previously described.16 Equal protein loading was confirmed by Ponceau S staining. To determine the phosphorylation status of selected proteins, antibodies recognizing site-specific phosphorylated forms were diluted to a final concentration of 1:1000 in 1% BSA in Tris-saline-Tween-20 buffer.16 In some cases, blots were used for sequential incubations. Immune complexes were detected after incubation with appropriate horseradish peroxidase-coupled secondary antibodies using chemiluminescent ECL Plus western blotting reagents from GE Healthcare, followed by exposure of the blots to film or analysis using ChemiSmart5100 apparatus (PEQLAB, Biotechnologie GmbH, Erlangen, Germany).Analysis of interactions using the CalcuSyn method
The first was the combination index (CI) method of Chou and Talalay.32 The CalcuSyn software package (Version 2.0, Biosoft, Cambridge, UK), which is based on this method and takes into account both potency (median dose [Dm] or IC50) and the shape of the dose-effect curve (the
Statistical analyses were performed using GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA, USA) and significance levels were evaluated using Bonferroni’s and Dunnett’s multiple comparison tests. Differences between treatments were deemed to be extremely significant, very significant, significant and not significant if their P values (according to Bonferroni’s comparison test) were < 0.001, < 0.01, 0.01 < P < 0.05, and > 0.05, respectively. In the figures such differences are indicated by 3 asterisks (***), 2 asterisks (**), 1 asterisk (*), and no asterisks, respectively.
Previous reports (summarized in the Introduction) suggest that cells deficient in
Continuous treatment for 48 hours caused very significant reductions in numbers of viable SKBr-3 cells at a final concentration of 50 μM (Fig. 1B). After longer treatment (72 hours) very significant reductions in SKBr-3 cell numbers were also observed at a lower (CE = 10 μM) CP466722 dose. Exposure for 48 hours to the lowest CP466722 doses (CE = 5 μM and 10 μM) was less cytotoxic for MCF-7 cells (Fig. 1B). Nevertheless, at the highest dose their viability was reduced by 80% after 48 hours and 90% after 72 hours. In contrast, proliferation of SKBr-3 and MCF-7 cells was almost completely unchanged after 48 hours or 72 hours exposure to KU55933, another selective ATM inhibitor. Further, the anti-proliferative efficiencies of the 2 ATM inhibitors towards SKBr-3 cells at the same dose (CE = 10 μM) significantly differed (Fig. 1B).
Inhibition of Poly(ADP-ribose) polymerase-1 induces synthetic lethality in MCF-7 and SKBr-3 cells
Interference with PARP-1 activity using AZD2461 was cytotoxic to both the SKBR-3 line and (less strongly) MCF-7 line, reducing numbers of viable cells in a concentration- and time-dependent manner (Fig. 2). The finding that AZD2461 induced synthetic lethality in both cell lines, previously shown to be NU1025-resistant, indicates that even
Poly(ADP-ribose) polymerase-1 inhibition by AZD2461 induces accumulation of MCF-7 cells and weaker accumulation of SKBr-3 cells in the G2 phase
To determine effects of the tested drugs on cell cycle progression in human MCF-7 and SKBr-3 cells, exponentially growing cells were exposed to them both separately and in combination at a final concentration of 10 μM for 48 hours. Cells were then harvested and the DNA concentration in single cells was measured by flow cytometry. PARP-1 inhibition by AZD2461 increased proportions of MCF-7 cells in the G2 phase at the expense of proportions in the S-phase, and had similar (but weaker) effects on SKBr-3 cells (Fig. 3). In contrast, after exposure of MCF-7 and SKBr-3 cells to CP466722 proportions of cells in the G1 phase slightly increased. However, following concurrent inhibition of ATM kinase and PARP-1, proportions of MCF-7 cells in the G2 phase were 3-fold higher than in controls, while the changes in SKBr-3 cells were less pronounced (Fig. 3).
These results indicate that pharmacological inactivation of PARP-1 alone has different effects on the cell cycle progression in the 2 human breast cancer cell lines considered in this study.
Interference with Poly(ADP-ribose) polymerase-1 activity enhances cytotoxic action of ataxia telangiectasia mutated kinase inhibitors in both tested breast cancer cell lines by inducing apoptosis
In the second phase of the investigations we determined the sensitivity of the breast cancer cells to the combination of both types of drugs. Simultaneous inhibition of ATM kinase and PARP-1 activity was much more cytotoxic than inactivation of only one target (Fig. 4). Interference with PARP-1 activity strongly enhanced the action of KU55933; after treatment for 48 hours, the number of living MCF-7 cells was reduced by 50%, and after 72 hours by 45% (Fig. 4A and 4B). Concurrent administration of the PARP-1 inhibitor and CP466722 was much more effective at the higher dose (CE =10 μM) of the ATM inhibitor. Notably, after longer treatment (72 hours) with this drug combination no further increase in cytotoxicity was observed.
Moreover, analyses of DNA profiles revealed that the abundance of hypoploid cells increased after concurrent PARP-1 and ATM kinase inhibition for 48 hours, indicating that the drug combination induced programmed cell death. This effect was much more pronounced in SKBr-3 cells than in MCF-7 cells, possibly due to the apoptosis-resistance of MCF-7 cells caused by disruption of the
Chromatin changes after inhibition of poly(ADP-ribose) polymerase-1 in human SKBr-3 breast cancer cells
We used in situ monitoring techniques to monitor the changes in cell density and chromatin structure following the inhibition of ATM kinase and PARP-1 in examined breast cancer cells. MCF-7 and SKBr-3 cells were strongly affected by the inhibition of ATM kinase and PARP-1 (Fig. 5) and their density decreased substantially. Both apoptotic, mitotic and G2-arrested SKBr-3 cells were detected in the samples after treatment with AZD2461 (Fig. 5). Concurrent inhibition of ATM kinase and PARP-1 caused much more pronounced reduction in the cell density.
Poly(ADP-ribose) polymerase-1 inhibition by AZD2461 induces DNA damage response in breast cancer cells
Finally, we determined the changes in the functional status and expression of some key proteins after single treatment of SKBr-3 cells with the inhibitors and their combinations. An increase in the site-specific phosphorylation of 53BP1 and MRE11 proteins was observed after treatment for 24 hours with AZD2461 but not after exposure to CP466722 (Fig. 6). After combined treatment the phosphorylation of 53BP1 protein decreased. In contrast, the phosphorylation of MRE11 protein was enhanced after concurrent inhibition of PARP-1 and ATM in SKBr-3 cells. These data show that the functional status of key DDR regulators mediated by phosphorylation of their specific sites was differentially modulated after separate and combined exposure to ATM and PARP inhibitors.
Ataxia telangiectasia mutated inhibitors and AZD2461 synergistically interact
Our observation that treating MCF-7 and SKBr-3 cells with a PARP-1 inhibitor enhances the cytotoxic action of both examined ATM inhibitors prompted us to investigate the interactions between the 2 types of inhibitors using the CalcuSyn software package. The calculated CI was less than 1 for all combinations of compounds tested in MCF-7 cells, indicating that AZD2461 at CE = 10 μM synergistically interacts with CPP466722 at CE = 10 after 48 hours (Table).
Tumor suppressor genes play key regulatory roles in blocking cell division when DNA is damaged, broken or mutated, and initiating repair of DNA lesions. In heavily damaged cells some tumor suppressor proteins, e.g., p53, also trigger programmed cell death, thereby destroying them. Thus, tumor suppressor genes are crucial components of the machinery that maintains genomic stability and avoids the multiplication of cells harboring mutations or other defects.
As mentioned above, of 3 known DSB repair pathways HR is the most important due to its high fidelity.8
In this study 2 human breast cancer cell lines (human estrogen receptor-responsive MCF-7 and SKBr-3) differing in the functional status of important tumor suppressors were used. MCF-7 cells express
Our results show that both MCF-7 and SKBr-3 cells are sensitive to CP466722, an inhibitor of ATM kinase, and AZD2461, a new PARP-1 inhibitor that is under clinical investigation. AZD2461 induced synthetic lethality in both tested breast cancer cell lines that are insensitive to the PARP-1 inhibitor NU1025 even at higher doses (up to CE = 200 μM).16 Moreover, CP466722 was synthetically lethal for MCF-7 and SKBr-3 cells that are resistant to KU55933, another specific inhibitor of ATM kinase.
A key question to address is why BRCA1-proficient MCF-7 cells are sensitive to pharmacological interference with PARP-1 activity. As discussed above, deficiencies in not only BRCA1, but also in DNA damage signaling38,39 and components of the DSB repair machinery40?42 generally render cancer cells sensitive to PARP-1 inhibitors. During cellular processes such as DNA replication,7 transcription or recombination SSBs are generated that are usually repaired by BER. However, exposure to a PARP-1 inhibitor blocks BER, and SSBs are converted into DSBs. In HR-competent cells accumulating DSBs are repaired by HR and cells survive. However, in HR-deficient cells increasing levels of DSBs are lethal. In principle, they might be repaired by NHEJ, which is active throughout the cell cycle and accurately repairs “clean” DSBs, i.e., broken strands with compatible ends and undamaged terminal nucleotides. DNA termini harboring damaged nucleotides or mismatched termini may also be joined by NHEJ but this is associated with loss of nucleotides and increases in genomic instability.43 However a novel role of PARP in NHEJ regulation through retention of Ku 70 at DSBs was very recently discovered.40,44 Thus, in HR-deficient cells PARP inhibition leads to accumulation of DSBs and is synthetically lethal.
Furthermore, defects or deficiency in BER components (e.g., DNA polymerase β and XRCC1) confer hypersensitivity to PARP inhibitors.45 Germline mutations in several genes encoding other proteins involved in DSB repair (
Both cell lines examined in our study display deficits in DNA repair machinery. SKBr-3 cells are
Remarkably, cancer cells can develop resistance to PARP-1 inhibitors and BRCA-targeted therapies
Recently, several genetic modulators of PARP-inhibitor responses have been identified such as
Synergistic interaction between CP499722 and AZD2461 in MCF-7 cells.
|Drug combination||48 hr||72 hr||48 hr||72 hr|
|CP466722 (5 μM) + AZD2461 (10 μM)||0.896||1.468||1.423||2.151|
|CP466722 (10 μM) + AZD2461 (10 μM)||0.643||1.142||1.261||1.243|
CI, combination index..
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