1 Department of Obstetrics and Gynaecology, Division of Gynaecology, University Hospital of Zurich, Switzerland; 2 Department of Medicine and the Cancer Centre, University of California at San Diego, La Jolla, CA, USA
Received 10 January 2003; revised 7 February 2003; accepted 17 February 2003
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ataxia-telangiectasia is a pleiotropic autosomal recessive disorder caused by mutations in the ATM gene. In addition to a profound cancer predisposition, another hallmark of ataxia-telangiectasia is radiosensitivity. Recently, p53-null mouse fibroblasts have been reported to be radiosensitised by the concurrent loss of ATM.
Materials and methods:
We compared the sensitivity of atm+/+/p53/ and atm//p53/ mouse embryonic fibroblasts to different classes of chemotherapeutic agents using the MTT assay, Trypan Blue exclusion and fluorescence-activated cell sorting for cell cycle and apoptosis analyses.
Results:
Loss of ATM function in p53-deficient cells resulted in a 2- to 4-fold increase in sensitivity to the topoisomerase I poisons camptothecin and topotecan, to the topoisomerase II poisons doxorubicin, epirubicin and etoposide, and to the antimetabolites 5-fluorouracil and gemcitabine, but not to the platinum compounds cisplatin, carboplatin and oxaliplatin, the taxanes docetaxel and paclitaxel, or to busulfan. Loss of ATM function did not result in increased apoptosis, but resulted in increased Trypan Blue staining in response to epirubicin, suggesting that processes other than apoptosis may mediate cytotoxicity. ATM deficiency did not alter the extent of G1/S or G2/M cell cycle phase accumulation produced by epirubicin, suggesting that enhanced sensitivity was not due to failure of checkpoint activation.
Conclusions:
We provide further evidence that ATM is involved in regulating cellular defences against some cytotoxic agents in the absence of p53. Tumour-targeted functional inhibition of ATM may be a valuable strategy for increasing the efficacy of anticancer agents in the treatment of p53-mutant cancers.
Key words: antimetabolites, ATM, cancer, drug sensitivity, p53, topoisomerase poisons
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ATM is mutated in patients with ataxia-telangiectasia, a pleiotropic autosomal recessive disorder characterised by progressive neurodegeneration, premature senescence, immunodeficiency, predisposition to cancer, chromosomal instability and hypersensitivity to -irradiation [4, 5]. ATM is a PI-3-like protein kinase operating upstream of p53 [6] and it has been shown to bind to free DNA ends produced by DNA double-strand breaks that occur during normal replication and recombination, or in response to exogenous genotoxic stress [7]. It is known to interact with a variety of other targets in addition to p53, including c-Abl, BRCAl, CHK2, MDM2 and DNA-PK [8, 9]. ATM plays a key role in sensing DNA damage and in propagating signals that modulate protective cellular responses to genotoxic agents. It serves a surveillance function that helps maintain genomic integrity by promoting cell cycle arrest and damage repair, and possibly by recruiting repair proteins to the site of damage to prevent double-strand break repair from entering an error-prone pathway [10]. Ataxia-telangiectasia cells display defective p53 induction, abrogation of G1/S and G2/M cell cycle checkpoints, and hypersensitivity to
-irradiation [5], suggesting that ATM and p53 interact in a common pathway in response to this type of DNA damage. However, some p53-null mouse tissues have been shown to be rendered radiosensitive by the concurrent loss of the ATM gene [11], suggesting the existence of an ATM effector pathway that is activated in response to ionising radiation but which does not depend on p53.
Because a very large fraction of human cancers is functionally p53 deficient, and hence may be resistant to chemotherapeutic agents, a means of sensitising p53-deficient tumours assumes great importance. Therefore, we were interested in determining whether p53-deficient cells could be sensitised to a panel of clinically important chemotherapeutic agents by the additional loss of ATM function. We report here that loss of ATM function in p53-null mouse embryonic fibroblasts results in hypersensitivity to a panel of chemotherapeutic agents, indicating that ATM plays a role in protective responses to DNA damage independently of p53. The increased drug sensitivity of ATM-deficient cells was not accompanied by increased apoptosis or by further alteration of cell cycle checkpoint activation. Our data support the concept that tumour-targeted functional inhibition of ATM may be a valuable approach to improving the effectiveness of chemotherapeutic agents in the treatment of p53-mutant cancers.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Drugs
Camptothecin and busulfan were purchased from Sigma (Buchs, Switzerland). The following drugs were generous gifts: epirubicin and doxorubicin (Pharmacia & Upjohn, Dubendorf, Switzerland), topotecan (SmithKline Beecham, Thorishaus, Switzerland), docetaxel (Aventis, Zurich, Switzerland), oxaliplatin (Sanofi-Synthelabo, Meyrin, Switzerland), etoposide, paclitaxel, cisplatin and carboplatin (Bristol-Myers Squibb, Baar, Switzerland), 5-fluorouracil (Roche, Reinach, Switzerland) and gemcitabine (Eli Lilly, Vernier, Switzerland).
Immunoblot analysis
To provoke p53 induction, exponentially growing cells were exposed to 25 nM epirubicin for 24 h and then collected as described. Cells were washed in cold phosphate-buffered saline (PBS) and lysed on ice in 150 mM NaCl containing 5 mM EDTA, 1% Triton X-100, 10 mM TrisHCl (pH 7.4), 5 mM dithiothreitol (DTT), 100 µg/ml phenylmethylsulfonyl fluoride and 1 µg/ml aprotinin, followed by centrifugation at 14 000 g for 20 min at 4°C. The protein amount was determined using the Bio-Rad protein assay dye (Bio-Rad, Glattbrugg, Switzerland). After centrifugation, 150 µg (for ATM) or 50 µg (for p53) of protein was boiled in an equal volume of 100 mM TrisHCl (pH 6.8) containing 20% glycerol, 200 mM DTT, 4% sodium dodecylsulphate (SDS) and 0.2% bromophenol blue. The proteins were separated using SDSPAGE on a 5% gel for ATM and on a 10% gel for p53 analysis, followed by blotting onto a polyvinylidene difluoride membrane (Amersham Pharmacia Biotech, Little Chalfont, UK). ATM protein was detected using a mouse monoclonal antibody directed against amino acids 25773056 (ATM-2CI; GeneTex, Inc., San Antonio, TX, USA), whereas p53 protein was detected using the mouse monoclonal antibody pAb 240 (Santa Cruz Biotechnology Inc., Basel, Switzerland). After washing the blots, horseradish peroxidase-conjugated antimouse antibody (BD Biosciences, Allschwil, Switzerland) was added and the complexes were visualised by enhanced chemiluminescence (Amersham Pharmacia Biotech).
Growth inhibition assay
Epirubicin, doxorubicin, topotecan, cisplatin, carboplatin, oxaliplatin, 5-fluorouracil and gemcitabine were diluted in 0.9% NaCl immediately before use. Stock solutions of etoposide, camptothecin, paclitaxel and busulfan were prepared in dimethyl sulphoxide (DMSO), whereas docetaxel was dissolved in methanol. The final concentration of DMSO or methanol in the cultures was <0.1% at all drug concentrations and in controls. Previous experiments (data not shown) have established that neither 0.1% DMSO nor 0.1% methanol affects the viability or growth of these cell lines. Growth inhibition in response to drug treatment was determined by the MTT assay [13]. Cells growing in the log phase were harvested by brief trypsinisation and washed once with medium containing 15% fetal calf serum. MTT assays were performed by seeding 500 (atm//p53/) or 1000 (atm+/+/p53/) cells into 96-well plates 24 h before incubation without or with the drug for 5 days. A volume of 20 µl MTT in PBS was added to a final concentration of 0.5 mg/ml, followed by incubation at 37°C for 4 h, aspiration of the medium and addition of DMSO 200 µl. Optical density was measured by the Emax microplate reader E9336 (Molecular Devices, Clearwater, MN, USA) at 540 nm, setting the value of the cell lines in medium to 1.0 (control) and the value of the no cells blank to zero. Differences in drug sensitivity of the respective cell lines were determined from at least four independent experiments and are reported as the concentration required to suppress growth by 50% (IC50).
TUNEL apoptosis assay
Cells were grown to 70% confluence in 60 mm dishes in triplicate cultures and then treated with 25 nM epirubicin. Adherent and floating cells were collected at the time points indicated and washed in PBS. Cells were then fixed in 4% paraformaldehyde on ice for 30 min, followed by dropwise addition of ice-cold 70% ethanol. Samples were stored at 4°C until further use. Samples were washed twice with PBS and resuspended in the TUNEL reaction mixture and incubated at 37°C for 2 h, according to the manufacturers protocol (Roche Molecular Biochemicals, Basel, Switzerland). Cells were analysed by flow cytometry (EPICS ELITE; Beckmann-Coulter, Hialeah, FL, USA).
Trypan Blue exclusion assay
Cell viability after drug treatment was determined by means of the Trypan Blue exclusion assay. Cells were grown to 60% confluence and incubated without (controls) or with 25 nM epirubicin for 24, 48 or 72 h. At the indicated time points, floating and adherent cells were collected. Cells were then incubated with Trypan Blue solution (0.1% final concentration) for 1 min, and the number of Trypan Blue-positive and -negative cells was determined using a haematocytometer.
Cell cycle analysis
Cells were grown to 50% confluence in 60 mm dishes. Cells were then incubated with or without (controls) epirubicin 10 nM. This dose inhibited growth of ATM-deficient cells by at least 70% in the MTT assay. Before collection by trypsinisation at the times indicated, cells were incubated with BrdU 10 µM (Serva, Heidelberg, Germany) at 37°C for 8 h. Samples were washed in ice-cold PBS, resuspended in PBS 100 µl, fixed by dropwise addition of ice-cold 70% ethanol and stored at 4°C until use. Then, ethanol was removed by centrifugation (3000 g) and cells were resuspended and incubated in 2 N HCl/0.5% Triton X-100 for 30 min at room temperature, followed by centrifugation (3000 g) and resuspension in 0.1 M Na2B4O7 to neutralise the acid. After collection of the pellet by centrifugation, cells were resuspended and incubated in PBS 500 µl /0.5% Tween-20/1% bovine serum albumin (BSA) and further incubated with FITC-conjugated anti-BrdU antibody (5 µg/ml; BD Biosciences) at room temperature for 30 min. Nuclei were then collected by centrifugation (3000 g) and incubated in 1 ml of propidium iodide staining solution (50 µg/ml propidium iodide and 100 U/ml RNase A in PBS) at room temperature for 1 h, followed by washing once in PBS containing 0.5% BSA. All light-sensitive steps were carried out in twilight. Samples were analysed for BrdU incorporation and DNA content by flow cytometry (EPICS ELITE; Beckmann-Coulter), and the percentage of cells in each phase was determined using the MultiCycle for Windows Software (Phoenix Flow Systems, San Diego, CA, USA).
Statistical analysis
Mean ± SD are indicated for all data sets. The two-sided paired t-test was performed to compare the effects of loss of ATM function on drug sensitivity. P <0.05 was considered to be statistically significant.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ATM may function as a sensor of DNA double-strand breaks arising from -irradiation [11] and, as reported here, from some types of anticancer agents including topoisomerase poisons or antimetabolites. However, it does not sense lesions produced by microtubule poisons or platinum compounds. This is perhaps not surprising for docetaxel and paclitaxel, since they are not known to interfere with DNA. Perhaps, as for UV radiation-introduced DNA damage [14], ATR, another member of the PIK family [9, 15], is responsible for sensing damage produced by platinum drugs. ATR and ATM are partially redundant, and they act in parallel but overlapping DNA damage signalling pathways, but respond primarily to different kinds of lesions [7, 15].
ATM regulates cell cycle checkpoint activation upon DNA damage causing cells to arrest in G1/S and/or G2/M, ensuring that cells delay entry into mitosis and putatively permitting time for damage repair prior to the onset of mitosis [16]. Although stress may cause overriding of the G1/S and the G2/M arrests in p53-mutant cells [2], damage-mediated activation of ATM may stop cells at the G2/M checkpoint in these cells [16]. However, loss of ATM does not substantially affect either G1/S or G2/M checkpoint activation in p53-deficient cells after -irradiation [17]. Our data show that the increased sensitivity of these cells to epirubicin cannot be explained by the lack of ATM-mediated cell cycle checkpoint control. Indeed, ATM, unlike ATR, does not seem to modulate the length and magnitude of the G2 arrest induction [18, 19].
The primary mechanism by which ATM exerts its protective effect may be through modulating damage repair and decreasing the threshold for cell kill [10]. ATM immunoprecipitates with DNA damage repair proteins including MLH1, MSH2, MSH6 and BRCA1, and may facilitate recruitment of repair proteins to the site of the lesion [20]. When such damage remains unrepaired as the cell enters mitosis, the increased rate of chromosome breaks and aberrant chromosome segregation results in increased cell killing [11]. Our results indicate that the increased chemosensitivity of the ATM-deficient cells may be a result of processes other than apoptosis.
This study supports the concept that tumour-targeted functional inhibition of ATM increases the efficacy of some anticancer agents in the treatment of p53-deficient cancers, which comprise a large proportion of human tumours [1].
![]() |
Acknowledgements |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Levine AJ. p53, the cellular gatekeeper for growth and division. Cell 1997; 88: 323331.[ISI][Medline]
3. Bunz F, Hwang PM, Torrance C et al. Disruption of p53 in human cancer cells alters the responses to therapeutic agents. J Clin Invest 1999; 104: 263269.
4. Savitsky K, Bar-Shira A, Gilad S et al. A single ataxia-telangiectasia gene with a product similar to PI-3 kinase. Science 1995; 268: 17491753.[ISI][Medline]
5. Lavin MF, Shiloh Y. The genetic defect in ataxia-telangiectasia. Annu Rev Immunol 1997; 15: 177202.[CrossRef][ISI][Medline]
6. Kastan MB, Zhan Q, El-Deiry WS et al. A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia. Cell 1992; 71: 587597.[ISI][Medline]
7. Smith GC, Cary RB, Lakin ND et al. Purification and DNA binding properties of the ataxia-telangiectasia gene product ATM. Proc Natl Acad Sci USA 1999; 96: 1113411139.
8. Rotman G, Shiloh Y. ATM: from gene to function. Hum Mol Genet 1998; 7: 15551563.
9. Khanna KK, Jackson SP. DNA double-strand breaks: signaling, repair and the cancer connection. Nature Genet 2001; 27: 247254.[CrossRef][ISI][Medline]
10. Morrison C, Sonoda E, Takao N et al. The controlling role of ATM in homologous recombinational repair of DNA damage. EMBO J 2000; 19: 463471.
11. Westphal CH, Hoyes KP, Canman CE et al. Loss of atm radiosensitizes multiple p53 null tissues. Cancer Res 1998; 58: 56375639.[Abstract]
12. Westphal CH, Rowan S, Schmaltz C et al. Atm and p53 cooperate in apoptosis and suppression of tumorigenesis, but not in resistance to acute radiation toxicity. Nature Genet 1997; 16: 397401.[ISI][Medline]
13. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1985; 65: 5563.[CrossRef][ISI]
14. Xu Y, Baltimore D. Dual roles of ATM in the cellular response to radiation and in cell growth control. Genes Dev 1996; 10: 24012410.[Abstract]
15. Gatei M, Zhou BB, Hobson K et al. Ataxia telangiectasia mutated (ATM) kinase and ATM and Rad3 related kinase mediate phosphorylation of Brca1 at distinct and overlapping sites: in vivo assessment using phospho-specific antibodies. J Biol Chem 2001; 276: 1727617280.
16. Falck J, Mailand N, Syljuasen RG et al. The ATM-Chk2-Cdc25A checkpoint pathway guards against radioresistant DNA synthesis. Genes Dev 2001; 15: 10671077.
17. Westphal CH, Schmaltz C, Rowan S et al. Genetic interactions between ATM and p53 influence cellular proliferation and irradiation-induced cell cycle checkpoints. Cancer Res 1997; 57: 16641667.[Abstract]
18. Pincheira J, Bravo M, Navarrete MH et al. Ataxia-telangiectasia: G(2) checkpoint and chromosomal damage in proliferating lymphocytes. Mutagenesis 2001; 16: 419422.
19. Cliby WA, Lewis KA, Lilly KK, Kaufmann SH. S phase and G2 arrests induced by topoisomerase I poisons are dependent on ATR kinase function. J Biol Chem 2002; 277: 15991606.
20. Wang Y, Cortez D, Yazdi P et al. BASC, a supercomplex of BRCA1-associated proteins involved in the recognition and repair of aberrant DNA structures. Genes Dev 2000; 4: 927939.