Rapid diagnosis of ataxia-telangiectasia by flow cytometric ... - Nature

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Jul 17, 2008 - monocytic leukemia with t(5;12) chromosomal translocation. Cell 1994; 77: 307–316. 7 Lee BH, Tothova Z, Levine RL, Anderson K, Buza-Vidas ...
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myelogenous leukemia, but are prevalent in chronic myelomonocytic leukemia. Blood 1990; 76: 1214–1219. Loh ML, Martinelli S, Cordeddu V, Reynolds MG, Vattikuti S, Lee CM et al. Acquired PTPN11 mutations occur rarely in adult patients with myelodysplastic syndromes and chronic myelomonocytic leukemia. Leuk Res 2005; 29: 459–462. Quesnel B, Preudhomme C, Vanrumbeke M, Vachee A, Lai JL, Fenaux P. Absence of rearrangement of the neurofibromatosis 1 (NF1) gene in myelodysplastic syndromes and acute myeloid leukemia. Leukemia 1994; 8: 878–880. Golub TR, Barker GF, Lovett M, Gilliland DG. Fusion of PDGF receptor beta to a novel ets-like gene, tel, in chronic myelomonocytic leukemia with t(5;12) chromosomal translocation. Cell 1994; 77: 307–316. Lee BH, Tothova Z, Levine RL, Anderson K, Buza-Vidas N, Cullen DE et al. FLT3 mutations confer enhanced proliferation and survival properties to multipotent progenitors in a murine model of chronic myelomonocytic leukemia. Cancer Cell 2007; 12: 367–380. Levine RL, Loriaux M, Huntly BJ, Loh ML, Beran M, Stoffregen E et al. The JAK2V617F activating mutation occurs in chronic myelomonocytic leukemia and acute myeloid leukemia, but not in acute lymphoblastic leukemia or chronic lymphocytic leukemia. Blood 2005; 106: 3377–3379.

9 Lorenzo F, Nishii K, Monma F, Kuwagata S, Usui E, Shiku H. Mutational analysis of the KIT gene in myelodysplastic syndrome (MDS) and MDS-derived leukemia. Leuk Res 2006; 30: 1235–1239. 10 Padua RA, Guinn BA, Al-Sabah AI, Smith M, Taylor C, Pettersson T et al. RAS, FMS and p53 mutations and poor clinical outcome in myelodysplasias: a 10-year follow-up. Leukemia 1998; 12: 887–892. 11 Reiter A, Sohal J, Kulkarni S, Chase A, Macdonald DH, Aguiar RC et al. Consistent fusion of ZNF198 to the fibroblast growth factor receptor-1 in the t(8;13)(p11;q12) myeloproliferative syndrome. Blood 1998; 92: 1735–1742. 12 Deguchi K, Gilliland DG. Cooperativity between mutations in tyrosine kinases and in hematopoietic transcription factors in AML. Leukemia 2002; 16: 740–744. 13 Loriaux MM, Levine RL, Tyner JW, Frohling S, Scholl C, Stoffregen EP et al. High-throughput sequence analysis of the tyrosine kinome in acute myeloid leukemia. Blood 2008; 111: 4788–4796. 14 Sjoblom T, Jones S, Wood LD, Parsons DW, Lin J, Barber TD et al. The consensus coding sequences of human breast and colorectal cancers. Science 2006; 314: 268–274. 15 Tomasson MH, Xiang Z, Walgren R, Zhao Y, Kasai Y, Miner T et al. Somatic mutations and germline sequence variants in the expressed tyrosine kinase genes of patients with de novo acute myeloid leukemia. Blood 2008; 111: 4797–4808.

Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)

Rapid diagnosis of ataxia-telangiectasia by flow cytometric monitoring of DNA damage-dependent ATM phosphorylation

Leukemia (2009) 23, 409–414; doi:10.1038/leu.2008.195; published online 17 July 2008

Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by cerebellar ataxia, telangiectasias, immune defects and a predisposition to malignancy. The birth frequency of A-T is estimated to be about 1 in 100 000–300 000. The A-T heterozygote frequency is estimated to be 0.5–1%. The patient shows progressive cerebellar ataxia since infancy. Telangiectasia typically develops after 3–5 years of age. Sixty to 80% of patients show immunodeficiency. Twenty to 30% of patients develop a malignancy, mainly leukemia or lymphoma. Laboratory finding shows decreased level of serum IgA, IgG2 and IgE. Increased level of serum a-fetoprotein is a specific feature. Peripheral circulating lymphocytes show characteristic intra-locus rearrangements involving T-cell receptor and/or immunoglobulin loci. Chromosomal breakage and hypersensitization to ionizing radiation (IR) are a hallmark cell biological feature of the A-T cell. A-T cells also show improper cell cycle regulation after IR, which has been known as radioresistant DNA synthesis. The responsible gene, ataxia-telangiectasia mutated (ATM), contains 66 exons spanning approximately 150 kb of genomic DNA at 11q22.3. cDNA contains 10 140 bp. The ATM protein contains 3056 amino acids coded by a 9168-bp open reading frame.1,2 The ATM gene is also known to be mutated secondarily in various hematological malignancies, and is speculated to work as a tumor suppressor gene.3 ATM protein is one of the members of phosphoinositide 3-kinase-related kinases. Exposure of cells to genotoxic stresses such as anticancer drugs or irradiation induces DNA double-strand breaks. The central player in such DNA damage response is ATM.4 ATM

is present as a dimer or higher-order multimer in unstressed cells with the kinase domain bound to the region surrounding serine (Ser) 1981. Cellular irradiation induces rapid intermolecular autophosphorylation of Ser 1981 and this phosphorylation event results in dimer dissociation and initiation of cellular ATM kinase activity.5 Diagnosis of A-T is based on clinical features combined with laboratory findings. Several cell biological and molecular approaches, such as protein truncation assay, yeast-based protein truncation assay, western blotting, RNA restriction finger printing assay, and others, have been used to reveal ATM mutation prior to straightforward genome sequencing.2 However, there is no standard method that could be applied in clinical diagnosis, genetic counseling or carrier prediction. Here, we describe a novel method for A-T diagnosis using measurement of phosphorylation status of ATM by flow cytometry. DNA damage response occurring in cells was monitored by measuring the phosphorylation level of ATM at Ser 1981 using the flow cytometry technique. Phosphorylation of ATM at Ser 1981 is the hallmark of ATM activation and can be detected by the anti-phospho-ATM-specific antibody 10H11.E12 (Cell Signaling, Danvers, MA, USA). Epstein–Barr virus (EBV)transformed wild-type lymphoblastoid cell line (LCL-WT) was irradiated or treated with hydrogen peroxide (H2O2) to induce DNA damage. Dose-dependent phosphorylation of ATM was detected 1 h after irradiation or H2O2 treatment by flow cytometry. An irradiation dose of 0.5 Gy was sufficient to induce phosphorylation of ATM at Ser 1981 and the level of phosphorylation increased in a dose-dependent manner (Figure 1a). Phosphorylation of ATM was seen in cells treated with 0.1 mM H2O2, and the level of phosphorylation increased in a dose-dependent manner up to 0.5 mM. Leukemia

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Figure 1 ATM phosphorylation status of LCL-WT detected by flow cytometry. (a) Upper panel: Irradiation induces ATM phosphorylation in a dose-dependent manner in LCL-WT. ATM phosphorylation was measured 1 h after irradiation. Radiation dosage is described above the dot blot gram. Right histogram panel corresponds to the results from dot blot gram. Black line: control, nonirradiated; red line: 0.5 Gy; green line: 1 Gy; pink line: 5 Gy; blue line: 10 Gy. Lower panel: H2O2-dependent ATM phosphorylation in LCL-WT. ATM phosphorylation was measured 1 h after H2O2 treatment. H2O2 concentration is described above the dot blot gram. Right histogram panels correspond to the results from dot blot gram. Black line: control, non-treatment; red line: 0.1 mM; green line: 0.5 mM; blue line: 1 mM. (b) Upper panel: Time kinetics of ATM phosphorylation after irradiation in LCL-WT. ATM phosphorylation was measured 10–60 min after 10 Gy irradiation. Time point when ATM phosphorylation was measured is described above the dot blot gram. Cont.: control non-treatment. Right histogram panels correspond to the results from dot blot gram. Black line: control, non-treatment; green line: 10 min; pink line: 30 min; blue line: 60 min. Lower panel: Time kinetics of ATM phosphorylation after H2O2 treatment in LCL-WT. ATM phosphorylation was measured at the indicated time points after H2O2 treatment. Right histogram panels correspond to the results from dot blot gram. Black line: control, non-treatment; blue line: 15 min; pink line: 30 min; green line: 60 min. (c) ATM phosphorylation status in ataxia-telangiectasia (A-T) LCL in comparison with LCL-WT. ATM phosphorylation was measured 1 h after 10 Gy irradiation, or 1 h after 1 mM H2O2 treatment in LCL-WT (upper panel) and AT52RM with ATM deficiency (lower panel). Right histogram panels correspond to the results from dot blot gram. Black line: control, non-treatment; red line: irradiated cell; green line: H2O2 treated cells.

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Treatment with H2O2 appeared to reach plateau at the doses of 0.5 and 1 mM H2O2 based on the equivalent level of phosphorylation seen by flow cytometry (Figure 1a). Next, the time kinetics of ATM phosphorylation status was investigated in 10 Gy IR- or 0.5 mM H2O2-treated cells. ATM phosphorylation was detected 10 min after IR and 15 min after H2O2 treatment, and reached its near-maximum level at 60 min after IR and 30 min after H2O2 treatment (Figure 1b). To validate the phosphorylation status demonstrated by flow cytometry in LCL-WT, the same assay was applied to AT52RM, an A-T LCL with null ATM expression by compound heterozygous truncation mutations 7626 C4T/8365 del A, and was

compared with that in LCL-WT. Phosphorylation after 10 Gy IR and 0.5 mM H2O2 treatment was detected in LCL-WT but not in AT52RM (Figure 1c). These results are compatible with the interpretation that the phosphorylation detected by flow cytometry corresponds to ATM phosphorylation. This observation prompted us to investigate whether the flow cytometric monitoring of DNA damage response is applicable for A-T diagnosis in the clinical setting. Peripheral blood mononuclear cells (PBMNCs) were cultured using media with 700 U/ml interleukin-2 (IL-2) and co-cultured with anti-CD3 antibody, which activates peripheral blood T cells (activated T cell)6 and provides enough numbers of Leukemia

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413 compared with LCL-WT (Figure 3a). The phosphorylation level in obligate A-T heterozygotes correlated with the modest level of ATM protein detected by the western blotting method (Figure 3b). Diagnosis of A-T is made on the basis of clinical symptoms, including ataxia and telangiectasia and the support of laboratory data. Although the demonstration of lack of ATM protein by western blotting method is a straightforward approach for the screening of A-T patients, ATM protein is not expressed enough to identify wild-type controls in PBMNC, thus making it difficult to distinguish normal, heterozygous and homozygous individuals. Establishment of cell lines or stimulation of PBMNC is required for western blotting analysis. Protein truncation assay is not practical for clinical use because of the complexity of the technique and requirement of several special equipment. Furthermore, these methods failed to identify the missense mutations with normal expression levels of mutant ATM. Sequence-based genetic diagnosis is not easy because of the large size of the ATM gene. The rapid screening methods that precisely predict the presence of ATM mutation will save effort, time and cost for the selection of patients who need whole ATM gene sequencing. Under the light of these previous laboratory experiences, we have eagerly awaited the development of a convenient screening procedure. Here we developed a method to detect the expression levels of phosphorylated ATM protein, which is based on the combinational analyses of ATM protein and its phosphorylation levels. This functional analysis is expected to enable screening of genomic condition of A-T gene before

lymphocytes to analyze ATM activation starting from a small amount of PBMNC. Then, flow cytometric analysis for ATM phosphorylation was employed in wild-type and A-T-derived activated T cells using the same protocol as that for EBVtransformed LCLs. As a result, activated T cells from wild-type control showed substantial increase of phosphorylated ATM after 10 Gy irradiation and 0.5 mM H2O2 treatment. On the other hand, A-T patient-derived activated T cells, which respond to IL-2 and CD3 as much as those of wild-type control (data not shown), showed no ATM phosphorylation (Figure 2a). Then, it was investigated whether this assay can be applied to PBMNC without stimulation. PBMNC from wild-type or A-T patient was isolated using Percoll gradient and irradiated soon after isolation in RPMI medium with 10% fetal calf serum. At 30 min after irradiation, PBMNC was assessed by flow cytometric analysis for ATM phosphorylation. Wild-type PBMNC showed increased phosphorylation of ATM after irradiation but no phosphorylation was detected in A-T-derived PBMNC (Figure 2b). These results suggested that flow cytometric analysis for ATM phosphorylation can be applied for the bed-side diagnosis of A-T patients even without precultivation with IL-2 and anti-CD3 antibody. Next, this assay was tested to see whether the obligate A-T heterozygous carrier is distinguishable from a wild-type individual. Two independent obligate A-T heterozygotederived EBV-LCLs (LCL-Hetero.), 227RM (7517 del 4/wt) and 373 RM (8283 del TC/wt), were enrolled in this study. Both of these LCLs showed increased ATM phosphorylation. However, the intensity of phosphorylation level was rather modest when

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actin Figure 3 ATM phosphorylation status in A-T heterozygous carrier derived LCLs in comparison with LCL-WT and AT52RM with ATM deficiency. (a) ATM phosphorylation was measured 1 h after 10 Gy irradiation. Right histogram panel corresponds to the results from dot blot gram. Black line: AT52RM; blue line: 227RM; green line: 373RM; red line: LCL-WT. The histogram illustrates ATM phosphorylation after irradiation of each cell line. The data from irradiated samples is shown. The histogram from un-irradiated sample of each LCL overlapping with that of irradiated AT52RM was omitted. (b) western blotting data of ATM protein expression from LCL-WT, AT52RM and LCL-Hetero. (227RM, 373RM). Leukemia

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414 genome sequencing. Flow cytometry is well equipped in most of the clinical laboratories, is relatively easy to manipulate and is easily accessible. Once the procedure is developed, it enables us to share this method between basic and clinical researchers. The results on EBV-LCLs further suggest that this method will facilitate the diagnosis of heterozygous carriers. Thus, an advantage of our flow cytometric analysis using ATM phosphorylation also exists in the diagnosis of carriers with missense mutations. It was shown that overexpression of dominantnegative form of misssense mutation at the kinase domain interferes with the ATM autophosphorylation after DNA damage. Further investigation is underway to find whether we can identify the individuals with obligatory heterozygous missense mutation by this assay without EBV transformation. Flow cytometric analysis enables us to screen simultaneously relatively large numbers of patient samples. We have previously reported that activation of DNA damage checkpoints plays an important role in the prevention of leukemic transformation in myelodysplastic syndrome. Phosphorylation of ATM is detected in refractory anemia with excess blast (RAEB)-II phase but not in RA phase.7 It is often difficult to morphologically classify these two conditions by microscopy. Measurement of ATM phosphorylation using flow cytometry might help to subclassify myelodysplastic syndrome. Furthermore, acquired mutation of ATM gene is frequently identified in hematological malignancies, such as T-prolymphocytic leukemia, mantle cell lymphoma, B-chronic lymphocytic leukemia. Loss of ATM function shows poorer clinical outcome in B-chronic lymphocytic leukemia.8 The classification of risk groups using ATM activation may add one of the options for selecting the therapy. As with the other clinical applications, measurement of ATM phosphorylation using flow cytometry may be useful for identification of individuals who may show abnormal response against genotoxic stress such as exposure to radiation or chemotherapeutic agents. If these individuals are screened at the bedside, we can avoid the adverse effect of anticancer drugs by reducing the administration doses. Additionally, this screening method will extend the opportunities for risk assessment of cancer development on the basis of population by identifying heterozygous carriers of the ATM gene in individuals with or without cancer.

Acknowledgements This work was supported by a Grant-in-Aid from the Ministry of Education, Science, and Culture, Japan, by a Grant-in-Aid for Cancer Research from the Ministry of Health, Labor and Welfare, Japan, and by a Grant-in-Aid for Child Health and Development from the Ministry of Health, Labor and Welfare, Japan.

M Honda1, M Takagi1, L Chessa2, T Morio1 and S Mizuatni1 1 Department of Pediatrics and Developmental Biology, Graduate School of Medicine, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan and 2 Department of Experimental Medicine Pathology, II Faculty of Medicine, University ‘La Sapienza’, Rome, Italy E-mails: [email protected] or [email protected] References 1 Savitsky K, Sfez S, Tagle DA, Ziv Y, Sartiel A, Collins FS et al. The complete sequence of the coding region of the ATM gene reveals similarity to cell cycle regulators in different species. Hum Mol Genet 1995; 4: 2025–2032. 2 Lavin MF, Gueven N, Bottle S, Gatti RA. Current and potential therapeutic strategies for the treatment of ataxia-telangiectasia. Br Med Bull 2007; 81–82: 129–147. 3 Stankovic T, Stewart GS, Byrd P, Fegan C, Moss PA, Taylor AM. ATM mutations in sporadic lymphoid tumours. Leuk Lymphoma 2002; 43: 1563–1571. 4 Shiloh Y. ATM and related protein kinases: safeguarding genome integrity. Nat Rev Cancer 2003; 3: 155–168. 5 Bakkenist CJ, Kastan MB. DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature 2003; 421: 499–506. 6 Sekine T, Shiraiwa H, Yamazaki T, Tobisu K, Kakizoe T. A feasible method for expansion of peripheral blood lymphocytes by culture with immobilized anti-CD3 monoclonal antibody and interleukin-2 for use in adoptive immunotherapy of cancer patients. Biomed Pharmacother 1993; 47: 73–78. 7 Horibe S, Takagi M, Unno J, Nagasawa M, Morio T, Arai A et al. DNA damage check points prevent leukemic transformation in myelodysplastic syndrome. Leukemia 2007; 21: 2195–2198. 8 Austen B, Skowronska A, Baker C, Powell JE, Gardiner A, Oscier D et al. Mutation status of the residual ATM allele is an important determinant of the cellular response to chemotherapy and survival in patients with chronic lymphocytic leukemia containing an 11q deletion. J Clin Oncol 2007; 25: 5448–5457.

Expression of cyclin A in childhood acute lymphoblastic leukemia cells reveals undisturbed G1–S phase transition and passage through the S phase

Leukemia (2009) 23, 414–417; doi:10.1038/leu.2008.200; published online 31 July 2008

Pediatric acute lymphoblastic leukemia (ALL) is characterized by arrested differentiation of the malignant cell clone with retention of proliferative ability. Earlier we had demonstrated that the majority of leukemic cells reside in the G1 phase of the cell cycle, with a minor proportion of cells in the S and G2 phases and only a few cells in the true G0 phase.1 Although the majority of ALL cells reside in the G1 phase of the cell cycle, attempts to induce differentiation in vivo or in vitro have not been successful. Leukemia

In view of this arrested differentiation, we became interested in the distribution of ALL cells within the G1 phase relative to the restriction (R) point. The R point determines whether a cell is committed to proliferation or retains the ability to differentiate.2 Progression through the R point is regulated by the functional inactivation of the retinoblastoma protein (pRb) through a complex stepwise process that includes a sequential phosphorylation by at least two different cyclin-dependent kinase (cdk) complexes, cyclin D/cdk4,6 and cyclin E/cdk2 inducing a loss of nuclear tethering and the activation of E2F transcriptional programs that permit G1–S phase transition.3 In pediatric ALL cell samples, both cyclin D/cdk4,6 and cyclin E/cdk2 kinase complexes were active; correspondingly,