TO THE EDITOR
Mantle cell lymphoma (MCL) and follicular lymphoma (FL) are two forms of B-cell lymphoma derived from neoplastic transformation of their normal counterparts in the mantle zone and germinal center, respectively.1 MCL is characterized in virtually all cases by t(11;14), with dysregulation of the cell cycle by upregulation of cyclin D1 located at 11q13.1 On the other hand, FL is characterized by t(14;18) and upregulation of BCL-2 located at 18q21, which confers resistance to apoptosis on the malignant lymphoid cells.1
ATM (A–T mutated) gene (localized to chromosome 11q22.3) encodes a cell cycle checkpoint-specific, serine–threonine kinase. In response to double-stranded DNA breaks (DSBs), ATM phosphorylates a panel of proteins including p53, Chk1, BRCA1 and Nbs1, resulting in cell cycle arrest and DNA repair, and is thus important in maintaining the integrity of the genome. This ensures passage of an intact genome in dividing cells, and elimination of cells carrying nonrepairable DSBs.2 Mutation of ATM leads to ataxia telangiectasia, an autosomal recessive disorder, which is characterized by neurodegeneration, immunodeficiency and increased risk of cancer including lymphomas.
In addition to the upregulation of cylin D1 associated with t(11;14) in MCL, a commonly deleted region at 11q22–q23 that encompasses the ATM locus has been identified in MCL. Subsequent mutation analysis of ATM, in 12 sporadic cases of MCL, showed a deletion of one ATM gene copy in seven, all of which harbored an inactivating point mutation in the remaining allele. In addition, biallelic ATM mutations were identified in two MCL patients without 11q deletions.3 Using a microarray to screen lymphomas from 120 patients (including 28 MCL and 18 FL) for all possible ATM coding and splice junction mutations, ATM mutations were shown to be most frequent (12/28; 43%) in the mantle cell (MCL) subtype, but absent in FL.4
In addition to deletion and mutation, methylation of gene promoter sites has now been identified as a frequently encountered mechanism of gene silencing in tumors. Indeed, ATM is frequently methylated in breast cancer.5 Catalyzed by DNA methyltransferase, DNA methylation involves the addition of a methyl group to the carbon 5 position of the cytosine ring in the CpG dinucleotide, to form methylcytosine.6 In many cancers, the CpG islands of selected genes are aberrantly methylated (hypermethylated), resulting in the repression of transcription of these genes.6 Methylation, thus, can serve as an alternative mechanism of gene inactivation during tumorigenesis. There is only one paper on methylation of ATM in lymphoma,7 including FL but not MCL, and no methylation was identified. Here, we report on our studies to determine if hypermethylation of ATM contributes to lymphomagenesis by combining with upregulation of cyclin D1 in MCL, and BCL-2 in FL.
Diagnosis of MCL and FL was made according to the standard criteria. Patients were staged according to the Ann Arbor system. Immunophenotyping was performed on cryostat sections and paraffin sections with standard immunoperoxidase techniques. Paraffin sections of formalin- or B5-fixed tissue were stained with hematoxylin/eosin to confirm the diagnosis of lymphoma, and examined for the expression of B- and T-cell markers. The panel of antibodies used included CD3 (Leu4, Becton Dickinson, San Jose, CA, USA), CD3 (polyclonal, Dako, Carpinteria, CA, USA), CD5 (Dako), CD10 (J5, Coulter, Miami, FL, USA), CD19 (Leu12, Becton Dickinson), CD20 (L26, Dako), CD22 (Dako), CD23 (Novocastra, Newcastle Upon Tyne, England) and cyclin D1 (Zymed, San Francisco, CA, USA). All eight cases of MCL were classical variant with the phenotype of CD5+, CD10− and CD23−. The 21 patients of FL belonged to FL grade II according to the WHO classification.
DNA was extracted from B-cell lines developed at different stages of differentiation including precursor B-cell derived from common acute lymphoblastic leukemia (697), follicular center B-cell derived from Burkitt's lymphoma (Nalm-6, Raji, Daudi, Jijoye, BJAB and Ramos), mature B-cell derived from multiple myeloma (U-266 and SK-MM-2), one MCL cell line (Granta-519) and two FL cell lines (Su-DHL-6 and DHL-16), and frozen lymph node biopsy tissue from patients with MCL (n=8) and FL (n=21) by standard proteinase K digestion and phenol–chloroform extraction method.8 The methylation-specific polymerase chain reaction (MSP), to determine gene promoter methylation, was performed as described previously.4 Briefly, treatment of DNA with bisulfite to convert unmethylated cytosine (but not methylated cytosine) to uracil was performed with a commercially available kit (CpGenome DNA modification kit, Intergen, New York, USA) according to the manufacturer's instructions. Primers are designed to amplify either methylated (M-MSP) or unmethylated (U-MSP) sequences. For ATM gene methylation, primers were designed to amplify the GC-rich region (−288 to −68) within the ATM promoter. MSP analysis was carried out in 20 μl PCR reactions containing 2 μl of bisulfite-treated genomic DNA, dNTPs (200 μ M each), oligonucleotide primers (25 pmol each/reaction), 2.0 mM MgCl2 and 0.625 U of AmpliTaq-Gold polymerase (PE Biosystem, Foster City, CA, USA) in 1 × PCR buffer. MSP primers for analysis of the ATM gene promoter were: 5′-AGAGTTTTGGAGTTTGAGTTGA-3′ (sense) and 5′-ACCCTACATAACTACCCAACACC-3′ (antisense) for the amplification of the promoter existing in an unmethylated state, and 5′-AGTTTCGGAGTTCGAGTCGA-3′ (sense) and 5′-AACCCTACGTAACTACCCAACG-3′ (antisense) for the amplification of the promoter existing in a methylated state. Touchdown PCR was then carried out as follows. After an initial denaturation and hot start at 95°C for 10 min, the cycling protocol entailed five cycles of 95°C for 30 s, 61°C for 30 s and 72°C for 30 s. In each cycle after the first, the annealing temperature was decreased by one degree. The PCR was then completed with 35 cycles of 30 s at 95°C, 30 s at 57°C (for both methylated and unmethylated primers) and 30 s at 72°C, followed by 7 min at 72°C. MSP reactions were analyzed by electrophoresis on 2% agarose gels and were visualized by ethidium bromide staining. DNA from the peripheral blood of eight normal donors was used as negative control, while methylated control DNA (CpGenome Universal Methylated DNA, Intergen) was used as positive control in all the experiments. Bisulfite genomic sequencing was performed to confirm the methylation status. In brief, the strand-specific primers for the bisulfite-converted single-stranded DNA of the ATM promoter were: BGS-F, 5′-GTTGGTTATTGGTGGATATGG-3′ (sense); and BGS-R, 5′-ATCAAAAACCACTCTAAAAAAATACA-3′ (antisense) for amplification. Touchdown PCR was used as described above. The annealing temperature for BGS primers was 54°C. For direct sequencing, the PCR products were treated with ExoSAP-IT (USB, Cleveland, OH, USA), and sequenced with the BigDye Terminator 3.1 Cycle Sequencing Kit in the 3730 DNA Analyzer (PE Biosystem, Foster City, CA, USA).
All of the eight normal peripheral blood samples exhibited completely unmethylated ATM, as represented by the four samples shown in Figure 1. Universally methylated DNA as a positive control showed complete methylation by supporting amplification in M-MSP but not in U-MSP. Conversely, normal control DNA showed unmethylated gene status by the presence of amplification in U-MSP but not in M-MSP. All B-cell cell lines showed unmethylated ATM (Figure 1), as did all of the lymph node DNA samples from patients with MCL or FL (Figure 1). Sequencing of gel-purified positive control DNA confirmed ATM amplification (Figure 2).
In contrast to the frequent methylation of ATM in breast cancer,5 our study on a full range of B-cell lines with variable degrees of differentiation and primary tumor samples of both MCL and FL show that ATM methylation is far less frequent in lymphomagenesis than deletion and mutation.
Sanchez-Beato M, Sanchez-Aguilera A, Piris MA . Cell cycle deregulation in B-cell lymphomas. Blood 2003; 101: 1220–1235.
Shiloh Y . ATM and related protein kinases: safeguarding genome integrity. Nat Rev Cancer 2003; 3: 155–168.
Schaffner C, Idler I, Stilgenbauer S, Dohner H, Lichter P . Mantle cell lymphoma is characterized by inactivation of the ATM gene. Proc Natl Acad Sci USA 2000; 97: 2773–2778.
Fang NY, Greiner TC, Weisenburger DD, Chan WC, Vose JM, Smith LM et al. Oligonucleotide microarrays demonstrate the highest frequency of ATM mutations in the mantle cell subtype of lymphoma. Proc Natl Acad Sci USA 2003; 100: 5372–5377.
Vo QN, Kim WJ, Cvitanovic L, Boudreau DA, Ginzinger DG, Brown KD . The ATM gene is a target for epigenetic silencing in locally advanced breast cancer. Oncogene. 2004; 23: 9432–9437.
Chim CS, Liang R, Kwong YL . Gene promotor hypermethylation in hematological neoplasia. Hematol Oncol 2002; 20: 167–176.
Gronbaek K, Worm J, Ralfkiaer E, Ahrenkiel V, Hokland P, Guldberg P . ATM mutations are associated with inactivation of the ARF-TP53 tumor suppressor pathway in diffuse large B-cell lymphoma. Blood 2002; 100: 1430–1437.
Chim CS, Fung TK, Cheung J, Liang R, Kwong YL . SOCS1 and SHP1 hypermethylation in multiple myeloma: implications for epigenetic activation of the Jak/STAT pathway. Blood 2004; 103: 4630–4635.
We express our sincere thanks to all medical and nursing staff in the Department of Medicine, Queen Mary Hospital, for the provision of expert medical care. This project was supported by a CRCG grant of the University of Hong Kong.
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