Inactivation of the Lamin A/C Gene by CpG Island Promoter Hypermethylation in Hematologic Malignancies, and Its Association With Poor Surviv
http://www.100md.com
《临床肿瘤学》
the Cancer Epigenetics and Lymphoma Laboratories, Molecular Pathology Program, Spanish National Cancer Centre, Madrid, Spain
ABSTRACT
PURPOSE: Lamins support the nuclear envelope and provide anchorage sites for chromatin, but they are also involved in DNA synthesis, transcription, and apoptosis. Although the lack of expression of A-type lamins in lymphoma and leukemia has been reported, the mechanism was unknown. We investigated the possible role of CpG island hypermethylation in lamin A/C silencing and its prognostic relevance.
PATIENTS AND METHODS: The promoter CpG island methylation status of the lamin A/C gene, encoding the A-type lamins, was analyzed by bisulfite genomic sequencing and methylation-specific polymerase chain reaction in human cancer cell lines (n = 74; from 17 tumor types), and primary leukemias (n = 60) and lymphomas (n = 80). Lamin A/C expression was determined by reverse-transcription polymerase chain reaction, Western blot, immunohistochemistry, and immunofluorescence.
RESULTS: Lamin A/C promoter CpG island methylation was found in hematologic malignancies: seven (50%) of 14 leukemia- and five (42%) of 13 lymphoma cell lines. The presence of hypermethylation was associated with the loss of gene expression while a demethylating agent restored expression. In primary malignancies, lamin A/C hypermethylation was present in 18% (nine of 50) of acute lymphoblastic leukemias and 34% (14 of 41) of nodal diffuse large B-cell lymphomas. The presence of lamin A/C hypermethylation in nodal diffuse large B-cell lymphomas correlated strongly with a decrease in failure-free survival (Kaplan-Meier, P = .0001) and overall survival (Kaplan-Meier, P = .0005).
CONCLUSION: Epigenetic silencing of the lamin A/C gene by CpG island promoter hypermethylation is responsible for the loss of expression of A-type lamins in leukemias and lymphomas. The finding that lamin A/C hypermethylation is associated with poor outcome in diffuse large B-cell lymphomas suggests important clinical implications.
INTRODUCTION
The human genome is organized into chromosomes located together in the nucleus and separated from the cytoplasm by a double membrane studded with pores, which is known as the nuclear envelope. The lamins provide structural support for the nuclear envelope though a meshwork of filaments that are attached to the inner layer of the nuclear membrane, forming the lamina.1 While the nuclear lamina was initially considered to be a rigid structure, it has been recently shown that it is, in fact, highly dynamic in nature, with suggested roles in the nonrandom positioning of subchromosome domains, the overall organization of chromatin, and possibly also in the regulation of gene expression.2,3
The principal component of the nuclear lamina consists of lamins, members of the intermediate-filament family of proteins. Protein-protein interactions between the various lamins are responsible for the formation of the 10-nM diameter filaments that constitute the nuclear lamina. Lamins are also found in the nucleoplasm and in intranuclear and transnuclear channels.4,5 Lamins can be categorized into two subfamilies: A-type, which are expressed in most differentiated somatic cells, and B-type, which are expressed in nearly all cells and are essential for cell viability.6 The lamin A/C gene encodes the A-type lamins A and C, isoforms that arise as a result of alternative RNA splicing. Although A-type lamins are important in maintaining lamina stability, they also seem to have important additional roles in regulating transcription.6 With regard to the multiple functions of A-type lamins, mutations in the lamin A/C gene have been shown to cause several inherited diseases. Some, such as Emery-Dreifuss muscular dystrophy and Dunnigan-type familial partial lipodystrophy, are rather tissue-specific, but others such as the Hutchinson-Gilford progeria and atypical Werner syndromes, are more generalized.7 Importantly, it has been described that the expression of the A-type lamins is reduced or absent in subsets of cells with a low degree of differentiation and/or cells that are highly proliferating,8,9 including human malignancies,6 especially leukemias and lymphomas.10,11 However, the molecular defect underlying the loss of A-type lamins in human cancer remained unknown.
Transcriptional inactivation by CpG island promoter hypermethylation is a well-established mechanism for gene silencing in human tumors.12-14 Classical tumor-suppressor genes and genes involved in chemosensitivity, such as hMLH1, p16INK4a, and BRCA1, or the DNA repair gene MGMT, undergo epigenetic inactivation by hypermethylation of their regulatory regions.12-14 In this article, we demonstrate the presence of promoter CpG island hypermethylation in the lamin A/C gene and correlate this to loss of RNA and protein expression in leukemia and lynphoma malignancies. Furthermore, we also report that lamin A/C CpG island promoter hypermethylation is a significant predictor of poor outcome in nodal diffuse large B-cell lymphomas, a potentially relevant finding for the clinical management of these patients.
PATIENTS AND METHODS
Human Cancer Cell Lines and Study Subjects
The 74 human cancer cell lines from 17 tumor types examined in this study were obtained from the American Type Culture Collection (Rockland, MD), the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) and have been described elsewhere.15,16 The cancer cell lines included the following tumor types: leukemia (n = 14), lymphoma (n = 13), lung (n = 15), colon (n = 8), breast (n = 7), prostate (n = 3), cervix (n = 2), myeloma (n = 2), head and neck (n = 2), bladder (n = 1), kidney (n = 1), liver (n = 1), ovary (n = 1), liposarcoma (n = 1), rhabdomyosarcoma (n = 1), choriocarcinoma (n = 1), and glioma (n = 1). In this set, the 14 leukemia cell lines were MOLT-4, MOLT-16, MV4-11, REH, HEL-R, THP-1, KG1a, HL-60, NB4, K-562, Jurkat, SD-1, P12 Ichikawa, and ML1, and the 13 lymphoma cell lines were RAJI, KARPAS-422, U937, HT, NAMALWA, HUT78, DOHH2, L-428, HDLM2, KM-H2, HDMyZ, SU-DHL-1, and WSU-NHL. Cell lines were maintained in appropriate media and treated with 1 μmol/L 5-aza-2'-deoxycytidine (Sigma, St Louis, MO) for 3 days to achieve demethylation, as previously described.15,16
The collection of primary hematologic malignancies analyzed has been described elsewhere.16,17 The ethics committee approved the study protocol. All samples were frozen in liquid nitrogen immediately after resection and stored at –80°C until processing. DNA was extracted by standard methods. In brief, we studied specimens of acute lymphoblastic leukemia (n = 50), acute myeloid leukemia (n = 10), and non-Hodgkin lymphoma (NHL; n = 80). The histologic types for the NHL samples were nodal diffuse large B-cell lymphoma (n = 41), extranodal diffuse large B-cell lymphoma (n = 10), cutaneous T-lymphoma (n = 17), Burkitt's lymphoma (n = 6), and follicular lymphoma (n = 6). The nodal diffuse large B-cell lymphomas were diagnosed from 1990 to 1999, the stages being evaluated according to standard protocols. All had histologically verified malignancies. All patients were treated with the same standard chemotherapy regimen. The response to treatment was evaluated after the patients had completed therapy. A complete response was defined as the absence of any detectable disease. Partial remission was defined as 50% reduction in tumor volume. Failure was defined as anything less than a partial response, progressive disease, or treatment-related death.
Analysis of Lamin A/C Promoter-Associated CpG Island Methylation Status
We established the lamin A/C gene CpG island methylation status by polymerase chain reaction (PCR) analysis of bisulfite-modified genomic DNA, which induces chemical conversion of unmethylated, but not methylated, cytosine to uracil, using two procedures.
First, methylation status was analyzed by bisulfite genomic sequencing of both strands of the lamin A/C CpG islands. The primers used for lamin A/C were 5'-GAG GAG GAT TTA TTA GAG TTT TT-3'(sense) and 5'-ATCA TTA AAC TCC TAC AAA TCC-3' (antisense), encompassing the transcription start site (Fig 1A). The second analysis used methylation-specific PCR using primers specific for either the methylated or modified unmethylated DNA.18 Primer sequences of lamin A/C for the unmethylated reaction were 5'-AGG ATT TAT TAG AGT TTT TGT TTT GGT GTT -3' (sense) and 5'-CAA AAT ACA CCA ACC AAC TAA CTC TCA-3' (antisense) and for the methylated reaction, 5'- TTA TTA GAG TTT TTG TTT CGG CGT C-3' (sense) and 5'-CGC CGA CCG ACT AAC TCT CG-3' (antisense). Primers were also located encompassing the transcription start site (Fig 1A). The annealing temperature for both unmethylated and methylated reactions was 60°C. DNA from normal lymphocytes treated in vitro with SssI methyltransferase was used as a positive control for methylated alleles. DNA from normal lymphocytes was used as a positive control for unmethylated alleles. PCR products were loaded onto nondenaturing 3% agarose gels, stained with ethidium bromide and visualized under an ultraviolet transilluminator.
Lamin A/C RNA and Protein Analysis
RNA was isolated using TRIzol (Life Technologies, Gaithersburg, MD). RNA (2 μg) was reverse-transcribed using SuperScript II reverse transcriptase (RT; Gibco/BRL, Barcelona, Spain) and amplified using specific primers for lamin A (forward: 5'-ATG GAG ACC CCG TCC CAG -3'; reverse: 5'-TTA CAT GAT GCT GCA GTT CTG G-3') and lamin C (forward: 5'-GAG ATG ATC CCT TGC TGA-3', reverse: 5'-GTT CCA AAA CAT TCT TTA ATG-3'). PCR was performed for 30 cycles (94°C for 30 seconds, 62°C for 30 seconds, and 72°C for 1.5 minutes and (94°C for 30 seconds, 57°C for 30 seconds, and 72°C for 30 seconds) for lamin A and C, respectively, in a final volume of 25 μL containing 1 x PCR buffer (Gibco/BRL), 1.5 mmol/L MgCl2, 0.3 mmol/L of dNTP, 0.25 mmol/L of each primer, and 2 U of taq polymerase (Gibco/BRL). RT-PCR primers were designed between different exons and encompassing large introns to avoid any amplification of genomic DNA. GAPDH was used as an internal control to ensure cDNA quality and loading accuracy. The amplification products were resolved by 2% agarose gel electrophoresis and visualized by ethidium bromide staining.
Nuclear lysates for protein analysis were prepared as described elsewhere19 and analyzed by Western blotting using the antibody anti-lamin A+C.ab8984 (mouse monoclonal antibody immunoglobulin G; Abcam Limited, Cambridge, UK). Equal loading was tested by re-probing with a polyclonal antibody against human nucleolin C23 (mouse monoclonal immunoglobulin G1; Santa Cruz Biotechnology, Santa Cruz, CA). Gels were cast using the XCell SureLock Mini-Cell system (Invitrogen Corp/NOVEX, Carlsbad, CA) and developed using ECL immunodetection reagents (Amersham Pharmacia Biotech, Piscataway, NJ).
Immunohistochemistry and Immunofluorescence
For immunohistochemistry, cells were centrifuged, washed in phosphate-buffered saline, fixed in formaldehyde (4%) for 2 hours, subsequently in ethanol (70%) overnight, and included in agarose (2%). After being dehydrated with increasing ethanol concentrations, the cells were put in xilol and embedded in paraffin blocks. The blocks were sectioned at a thickness of 3 μm and dried for 16 hours at 56°C before being dewaxed in xylene and ethanol. Antigen retrieval was achieved by heat treatment in a pressure-cooker for 2 minutes in 10 mmol/L citrate buffer (pH 6.5). After incubation with antibody (lamin A/C monoclonal antibody 636; Novocastra, Newcastle Upon Tyne, UK), immunodetection was performed with EnVision-HRP (DakoCytomation, Copenhagen, Denmark) and peroxidase activity was developed using 3,3-diaminobenzydine chromogen as substrate. Sections were counterstained with hematoxylin. A positive control was included with each batch of staining to ensure consistency between consecutive runs.
For immunofluorescence, cells were grown on coverslips in P60 dishes, fixed in 4% formaldehyde and stained as previously described.19 Cells to be analyzed for 5mC were postfixed in cold methanol, treated with 1M HCl at 37°C for 30 minutes and stained. Confocal optical sections were obtained using a Leica TCS SP microscope (Leica Microsystems, Wetzlar, Germany) equipped with krypton and argon lasers. Images were acquired with Leica LCS Lite software and processed with Adobe Photoshop 5.0 (Adobe Systems Inc, San Jose, CA) and publicly available National Institutes of Health image software.
Statistical Analysis
Contingency tables were analyzed by Fisher's exact test. Disease-free and overall survival curves were estimated by the Kaplan-Meier method and were compared with the use of the log-rank test. All statistical analyses were performed using the SPSS version 10.1 program (SPSS Inc, Chicago, IL).
RESULTS
Lamin A/C CpG Island Hypermethylation in Leukemia and Lymphoma Cell Lines and Its Association With Transcriptional Gene Silencing
Lamin A/C is a candidate gene for hypermethylation-associated inactivation in human cancer since a 5'-CpG island is located around the transcription start site (Fig 1A). In order to assess the methylation status of the promoter-associated CpG island of lamin A/C, we screened 74 human cancer cell lines from 17 tumor types using bisulfite genomic sequencing and methylation-specific PCR targeted to the area surrounding its transcription start site, as described in Patients and Methods. All samples tested were found by both methods to be unmethylated at the lamin A/C CpG island, except hematologic malignancy cell lines (Fig 1). Lamin A/C CpG island-promoter hypermethylation was found in seven (50%) of 14 leukemia cell lines (MOLT-4, MOLT-16, REH, HEL-R, KG1a, Jurkat, and P12 Ichikawa) and five (42%) of 13 lymphoma cell lines (RAJI, KARPAS-422, HT, DOHH2, and L-428). The remaining leukemia and lymphoma cell lines were unmethylated at the lamin A/C CpG island. All normal tissues analyzed, including lymphocytes and bone marrow, were completely unmethylated at the lamin A/C CpG island-promoter. The results were confirmed by bisulfite genomic sequencing (Figs 1A and 1B) and methylation-specific PCR (Fig 1C).
Having noted lamin A/C promoter hypermethylation in hematologic malignant cell lines, we assessed the association between this epigenetic aberration and putative transcriptional inactivation of the lamin A/C gene at the RNA and protein levels. First, RT-PCR analysis with primers specific for both transcripts arising from the lamin A/C CpG island, lamin A and lamin C, was carried out. The leukemia- and lymphoma-cell lines, REH, Jurkat and RAJI, hypermethylated at the lamin A/C CpG island, did not show expression of the lamin A and lamin C RNA transcripts (Fig 2A). However, the leukemia SD-1 and breast MDA-MB-231 cancer-cell lines, which are unmethylated at the lamin A/C promoter, expressed both lamin A and C transcripts (Fig 2A). Protein expression was analyzed by Western blot, immunohistochemistry, and immunofluorescence and confirmed that the lamin A/C hypermethylated cell lines REH, Jurkat, and RAJI lack expression of both lamin A and C isoforms, while the lamin A/C unmethylated cell lines SD-1 and MDA-231 expressed both protein isoforms (Figs 2B to 2D).
We established a further link between lamin A/C CpG island hypermethylation and gene silencing by the treatment of the methylated leukemia and lymphoma cell lines with a DNA demethylating agent. Treatment of the REH, Jurkat, and RAJI cell lines with the demethylating drug, 5-aza-2'-deoxycytidine, restored expression of both lamin A and lamin C RNA transcripts and protein splice variants in the three cell lines (Fig 2A and 2B).
Lamin A/C Is Hypermethylated in Primary Leukemias and Lymphomas and Is a Predictor of Poor Outcome in Nodal Diffuse Large B-Cell Lymphoma
Once the functional consequences of lamin A/C CpG island hypermethylation had been determined, we considered the relevance of lamin A/C methylation in human primary hematologic malignancies by studying a large set of leukemias and lymphomas. In leukemia, lamin A/C CpG island hypermethylation was found in 18% (nine of 50) of acute lymphoblastic leukemias, but was absent in acute myeloid leukemia (AML; n = 10). These AML cases did not include any erythroleukemia, while the two AML cell lines found hypermethylated at lamin A/C were of this subtype. In lymphomas, lamin A/C methylation was observed in 34% (14 of 41) of nodal diffuse large B-cell lymphoma and in 17% (one of six) of Burkitt's lymphomas, but was not found in cutaneous T-cell lymphoma (n = 17), extranodal diffuse large B-cell lymphoma (n = 10), and follicular lymphoma (n = 6). Illustrative examples are shown in Figure 3A. Unlike the cell lines that were either completely methylated or completely unmethylated at lamin A/C CpG island (Fig 1B), in these clinical samples, there is always an amplification in the unmethylated PCR lane, indicating the presence of normal tissues. Consistent with the data derived from hematopoietic cell lines, semiquantitative RT-PCR analysis of 24 nodal diffuse large B-cell lymphomas correlated loss of lamin A/C expression with promoter hypermethylation. Seventeen lymphomas lacking lamin A/C methylation expressed high levels of lamin A/C mRNAs, whereas seven lymphomas with lamin A/C methylation expressed little or no detectable lamin A/C mRNA transcripts (Fig 3B).
We next determined whether there was a relationship between the hypermethylation status of the gene lamin A/C and the outcome of our set of 41 nodal diffuse large B-cell lymphoma patients (this clinical information was not available for the acute lymphoblastic leukemias patients). Lamin A/C hypermethylation was observed in 34% (14 of 41) of patients, as described above. Demographic and clinical characteristics versus lamin A/C methylation status is shown in Table 1. For these lymphomas, lamin A/C hypermethylation was independent of the age of onset (Fisher's exact two-tail test, P = .18) and sex of the patient (Fisher's exact two-tail test, P = .32) and the International Prognostic Indicator (IPI; Fisher's exact two-tail test, P = 1). Most importantly, we found that the presence of lamin A/C-promoter hypermethylation was associated with a statistically significant decrease in failure-free survival (Kaplan-Meier, P = .0001; Fig 4A) and overall survival rates (Kaplan-Meier, P = .0005; Fig 4B). Therefore, lamin A/C CpG island hypermethylation is a likely predictor of poor outcome in nodal diffuse large B-cell lymphoma patients.
DISCUSSION
A-type lamins are essential components of the nuclear lamina.2,6 Together with B-type lamins, they are the most prominent intermediate filaments forming this network of 10-nM diameter filaments located on the inner side of the nuclear membrane. A-type lamins are expressed in most differentiated somatic cells, and preliminary studies have suggested that lamin expression may be reduced in transformed cells,2,6 especially in hematologic malignancies.10,11 Our results demonstrate for the first time that in leukemias and lymphomas, an important mechanism for the loss of lamin A/C expression is aberrant transcriptional silencing by CpG island promoter hypermethylation.
The consequences of lamin A/C gene inactivation by CpG island hypermethylation in hematologic maligancies should be assessed from the standpoint of basic research and translational studies. With regard to basic research, it is important to understand the downstream biologic and cellular consequences for the methylation-mediated loss of lamin A and C in cancer cells. In the same way that the term "guardian of the genome" was proposed for p53 a decade ago, it may be now acceptable to describe A-type lamins as guardians of the soma6 for their dual role as "tensegrity elements" that protect chromatin from damage and also act as multifunctional regulators of gene transcription.2,6 It has been proposed that the loss of lamin A/C expression in malignant B- and T-lymphocytes reflects a block in the differentiation pathway of these cells.10 Our finding of the epigenetic silencing of the lamin A/C gene may aid understanding as to how lamins contribute to cellular transformation.
In this work, we have focused on translational studies, showing that the presence of lamin A/C hypermethylation in nodal diffuse large B-cell lymphoma patients is a significant predictor of shorter failure-free survival and overall survival. These lymphomas are the most common type of NHL, accounting for approximately 30% to 40% of adult NHLs. They represent a heterogeneous group of lymphoid neoplasms taking into account clinical, histologic, immunophenotypic, cytogenetic, and molecular genetic features,20,21 highlighting the need to determine parameters identifying patients at high risk of progression and death. Several molecular markers, such as BCL-6, BCL-2, p53, and specific microarray expression patterns17,22-24 or MGMT hypermethylation,25 have demonstrated potential as prognostic factors for the disease. However, the most commonly used indicator of prognosis is the International Prognostic Index (IPI). The IPI takes into account factors that are mostly linked to patient characteristics (age, performance status) and to disease extension and growth (disease stage, lactate dehydrogenase levels, and extent of extranodal involvement). The presence of lamin A/C hypermethylation was independent of the IPI value, confirming its potential role as a clinical marker. A second translational point relates to treatment. Current chemotherapy regimens have improved the outcome for patients with hematologic malignancies, but many patients do not respond to and/or benefit from the use of the standard anticancer drugs. Recently, the US Food and Drug Administration has approved the use of the demethylating agent 5-azacytidine at low doses for the treatment of myelodysplastic syndrome based on the capacity of the drug to reactivate CpG island hypermethylated genes.26 In this new scenario, lamin A/C is an excellent target gene for the use of this type of drugs in order to achieve its demethylation and reactivation in hematologic malignancies.
In summary, our study provides a mechanistic explanation for the observed loss of A-type lamins in hematologic malignancies, demonstrating that the lamin A/C gene encoding these central components of the nuclear lamin undergoes transcriptional silencing by promoter CpG island hypermethylation in leukemias and lymphomas. Although, the ultimate consequences of lamin A/C epigenetic inactivation for the molecular biology of the tumoral cell remain unknown, hypermethylation emerges as a strong indicator of poor prognosis for lymphoma patients. These data should encourage further research into the role of lamins in the development of human neoplasia.
Authors' Disclosures of Potential Conflicts of Interest
The authors indicated no potential conflicts of interest.
NOTES
Supported by the Health (FIS) and Science (I+D) Departments, Spanish Government; and Fundacion Telefonica.
Terms in blue are defined in the glossary, found at the end of this issue and online at www.jco.org.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
REFERENCES
Gruenbaum Y, Wilson KL, Harel A, et al: Review: Nuclear lamins–structural proteins with fundamental functions. J Struct Biol 129:313-323, 2000
Broers JL, Machiels BM, van Eys GJ, et al: Dynamics of the nuclear lamina as monitored by GFP-tagged A-type lamins. J Cell Sci 112:3463-3475, 1999
Taddei A, Hediger F, Neumann FR, et al: The function of nuclear architecture: A genetic approach. Annu Rev Genet 38:305-345, 2004
Aebi U, Cohn J, Buhle L, et al: The nuclear lamina is a meshwork of intermediate-type filaments. Nature 323:560-564, 1986
Fricker M, Hollinshead M, White N, et al: Interphase nuclei of many mammalian cell types contain deep, dynamic, tubular membrane-bound invaginations of the nuclear envelope. J Cell Biol 136:531-544, 1997
Hutchison CJ, Worman HJ: A-type lamins: Guardians of the soma Nat Cell Biol 6:1062-1067, 2004
Broers JL, Hutchison CJ, Ramaekers FC: Laminopathies. J Pathol 204:478-488, 2004
Rober RA, Weber K, Osborn M: Differential timing of nuclear lamin A/C expression in the various organs of the mouse embryo and the young animal: A developmental study. Development 105:365-378, 1989
Broers JL, Machiels BM, Kuijpers HJ, et al: A- and B-type lamins are differentially expressed in normal human tissues. Histochem Cell Biol 107:505-517, 1997
Stadelmann B, Khandjian E, Hirt A, et al: Repression of nuclear lamin A and C gene expression in human acute lymphoblastic leukemia and non-Hodgkin's lymphoma cells. Leuk Res 14:815-821, 1990
Lin F, Worman HJ: Expression of nuclear lamins in human tissues and cancer cell lines and transcription from the promoters of the lamin A/C and B1 genes. Exp Cell Res 236:378-384, 1997
Jones PA, Laird PW: Cancer epigenetics comes of age. Nat Genet 21:163-167, 1999
Esteller M: CpG island hypermethylation and tumor suppressor genes: A booming present, a brighter future. Oncogene 21:5427-5440, 2002
Herman JG, Baylin SB: Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med 349:2042-2054, 2003
Paz MF, Fraga MF, Avila S, et al: A systematic profile of DNA methylation in human cancer cell lines. Cancer Res 63:1114-1121, 2003
Ropero S, Setien F, Espada J, et al: Epigenetic loss of the familial tumor-suppressor gene exostosin-1 (EXT1) disrupts heparan sulfate synthesis in cancer cells. Hum Mol Genet 13:2753-2765, 2004
Saez AI, Saez AJ, Artiga MJ, et al: Building an outcome predictor model for diffuse large B-cell lymphoma. Am J Pathol 164:613-622, 2004
Herman JG, Graff JR, Myohanen S, et al: Methylation-specific PCR: A novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A 93:9821-9826, 1996
Espada J, Ballestar E, Fraga MF, et al: Human DNA methyltransferase 1 is required for maintenance of the histone H3 modification pattern. J Biol Chem 279:37175-37184, 2004
Harris NL, Jaffe ES, Stein H, et al: A revised European-American classification of lymphoid neoplasms: A proposal from the International Lymphoma Study Group. Blood 84:1361-1392, 1994
A clinical evaluation of the International Lymphoma Study Group classification of non-Hodgkin's lymphoma: The Non-Hodgkin's Lymphoma Classification Project. Blood 89:3909-3918, 1997
Shipp MA, Ross KN, Tamayo P, et al: Diffuse large B-cell lymphoma outcome prediction by gene-expression profiling and supervised machine learning. Nat Med 8:68-74, 2002
Rosenwald A, Wright G, Chan WC, et al: The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma. N Engl J Med 346:1937-1947, 2002
Gascoyne RD: Emerging prognostic factors in diffuse large B cell lymphoma. Curr Opin Oncol 16:436-441, 2004
Esteller M, Gaidano G, Goodman SN, et al: Hypermethylation of the DNA repair gene O(6)-methylguanine DNA methyltransferase and survival of patients with diffuse large B-cell lymphoma. J Natl Cancer Inst 94:26-32, 2002
Esteller M: DNA methylation and cancer therapy: New developments and expectations. Curr Opin Oncol 17:55-60, 2005(Ruben Agrelo, Fernando Se)
ABSTRACT
PURPOSE: Lamins support the nuclear envelope and provide anchorage sites for chromatin, but they are also involved in DNA synthesis, transcription, and apoptosis. Although the lack of expression of A-type lamins in lymphoma and leukemia has been reported, the mechanism was unknown. We investigated the possible role of CpG island hypermethylation in lamin A/C silencing and its prognostic relevance.
PATIENTS AND METHODS: The promoter CpG island methylation status of the lamin A/C gene, encoding the A-type lamins, was analyzed by bisulfite genomic sequencing and methylation-specific polymerase chain reaction in human cancer cell lines (n = 74; from 17 tumor types), and primary leukemias (n = 60) and lymphomas (n = 80). Lamin A/C expression was determined by reverse-transcription polymerase chain reaction, Western blot, immunohistochemistry, and immunofluorescence.
RESULTS: Lamin A/C promoter CpG island methylation was found in hematologic malignancies: seven (50%) of 14 leukemia- and five (42%) of 13 lymphoma cell lines. The presence of hypermethylation was associated with the loss of gene expression while a demethylating agent restored expression. In primary malignancies, lamin A/C hypermethylation was present in 18% (nine of 50) of acute lymphoblastic leukemias and 34% (14 of 41) of nodal diffuse large B-cell lymphomas. The presence of lamin A/C hypermethylation in nodal diffuse large B-cell lymphomas correlated strongly with a decrease in failure-free survival (Kaplan-Meier, P = .0001) and overall survival (Kaplan-Meier, P = .0005).
CONCLUSION: Epigenetic silencing of the lamin A/C gene by CpG island promoter hypermethylation is responsible for the loss of expression of A-type lamins in leukemias and lymphomas. The finding that lamin A/C hypermethylation is associated with poor outcome in diffuse large B-cell lymphomas suggests important clinical implications.
INTRODUCTION
The human genome is organized into chromosomes located together in the nucleus and separated from the cytoplasm by a double membrane studded with pores, which is known as the nuclear envelope. The lamins provide structural support for the nuclear envelope though a meshwork of filaments that are attached to the inner layer of the nuclear membrane, forming the lamina.1 While the nuclear lamina was initially considered to be a rigid structure, it has been recently shown that it is, in fact, highly dynamic in nature, with suggested roles in the nonrandom positioning of subchromosome domains, the overall organization of chromatin, and possibly also in the regulation of gene expression.2,3
The principal component of the nuclear lamina consists of lamins, members of the intermediate-filament family of proteins. Protein-protein interactions between the various lamins are responsible for the formation of the 10-nM diameter filaments that constitute the nuclear lamina. Lamins are also found in the nucleoplasm and in intranuclear and transnuclear channels.4,5 Lamins can be categorized into two subfamilies: A-type, which are expressed in most differentiated somatic cells, and B-type, which are expressed in nearly all cells and are essential for cell viability.6 The lamin A/C gene encodes the A-type lamins A and C, isoforms that arise as a result of alternative RNA splicing. Although A-type lamins are important in maintaining lamina stability, they also seem to have important additional roles in regulating transcription.6 With regard to the multiple functions of A-type lamins, mutations in the lamin A/C gene have been shown to cause several inherited diseases. Some, such as Emery-Dreifuss muscular dystrophy and Dunnigan-type familial partial lipodystrophy, are rather tissue-specific, but others such as the Hutchinson-Gilford progeria and atypical Werner syndromes, are more generalized.7 Importantly, it has been described that the expression of the A-type lamins is reduced or absent in subsets of cells with a low degree of differentiation and/or cells that are highly proliferating,8,9 including human malignancies,6 especially leukemias and lymphomas.10,11 However, the molecular defect underlying the loss of A-type lamins in human cancer remained unknown.
Transcriptional inactivation by CpG island promoter hypermethylation is a well-established mechanism for gene silencing in human tumors.12-14 Classical tumor-suppressor genes and genes involved in chemosensitivity, such as hMLH1, p16INK4a, and BRCA1, or the DNA repair gene MGMT, undergo epigenetic inactivation by hypermethylation of their regulatory regions.12-14 In this article, we demonstrate the presence of promoter CpG island hypermethylation in the lamin A/C gene and correlate this to loss of RNA and protein expression in leukemia and lynphoma malignancies. Furthermore, we also report that lamin A/C CpG island promoter hypermethylation is a significant predictor of poor outcome in nodal diffuse large B-cell lymphomas, a potentially relevant finding for the clinical management of these patients.
PATIENTS AND METHODS
Human Cancer Cell Lines and Study Subjects
The 74 human cancer cell lines from 17 tumor types examined in this study were obtained from the American Type Culture Collection (Rockland, MD), the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) and have been described elsewhere.15,16 The cancer cell lines included the following tumor types: leukemia (n = 14), lymphoma (n = 13), lung (n = 15), colon (n = 8), breast (n = 7), prostate (n = 3), cervix (n = 2), myeloma (n = 2), head and neck (n = 2), bladder (n = 1), kidney (n = 1), liver (n = 1), ovary (n = 1), liposarcoma (n = 1), rhabdomyosarcoma (n = 1), choriocarcinoma (n = 1), and glioma (n = 1). In this set, the 14 leukemia cell lines were MOLT-4, MOLT-16, MV4-11, REH, HEL-R, THP-1, KG1a, HL-60, NB4, K-562, Jurkat, SD-1, P12 Ichikawa, and ML1, and the 13 lymphoma cell lines were RAJI, KARPAS-422, U937, HT, NAMALWA, HUT78, DOHH2, L-428, HDLM2, KM-H2, HDMyZ, SU-DHL-1, and WSU-NHL. Cell lines were maintained in appropriate media and treated with 1 μmol/L 5-aza-2'-deoxycytidine (Sigma, St Louis, MO) for 3 days to achieve demethylation, as previously described.15,16
The collection of primary hematologic malignancies analyzed has been described elsewhere.16,17 The ethics committee approved the study protocol. All samples were frozen in liquid nitrogen immediately after resection and stored at –80°C until processing. DNA was extracted by standard methods. In brief, we studied specimens of acute lymphoblastic leukemia (n = 50), acute myeloid leukemia (n = 10), and non-Hodgkin lymphoma (NHL; n = 80). The histologic types for the NHL samples were nodal diffuse large B-cell lymphoma (n = 41), extranodal diffuse large B-cell lymphoma (n = 10), cutaneous T-lymphoma (n = 17), Burkitt's lymphoma (n = 6), and follicular lymphoma (n = 6). The nodal diffuse large B-cell lymphomas were diagnosed from 1990 to 1999, the stages being evaluated according to standard protocols. All had histologically verified malignancies. All patients were treated with the same standard chemotherapy regimen. The response to treatment was evaluated after the patients had completed therapy. A complete response was defined as the absence of any detectable disease. Partial remission was defined as 50% reduction in tumor volume. Failure was defined as anything less than a partial response, progressive disease, or treatment-related death.
Analysis of Lamin A/C Promoter-Associated CpG Island Methylation Status
We established the lamin A/C gene CpG island methylation status by polymerase chain reaction (PCR) analysis of bisulfite-modified genomic DNA, which induces chemical conversion of unmethylated, but not methylated, cytosine to uracil, using two procedures.
First, methylation status was analyzed by bisulfite genomic sequencing of both strands of the lamin A/C CpG islands. The primers used for lamin A/C were 5'-GAG GAG GAT TTA TTA GAG TTT TT-3'(sense) and 5'-ATCA TTA AAC TCC TAC AAA TCC-3' (antisense), encompassing the transcription start site (Fig 1A). The second analysis used methylation-specific PCR using primers specific for either the methylated or modified unmethylated DNA.18 Primer sequences of lamin A/C for the unmethylated reaction were 5'-AGG ATT TAT TAG AGT TTT TGT TTT GGT GTT -3' (sense) and 5'-CAA AAT ACA CCA ACC AAC TAA CTC TCA-3' (antisense) and for the methylated reaction, 5'- TTA TTA GAG TTT TTG TTT CGG CGT C-3' (sense) and 5'-CGC CGA CCG ACT AAC TCT CG-3' (antisense). Primers were also located encompassing the transcription start site (Fig 1A). The annealing temperature for both unmethylated and methylated reactions was 60°C. DNA from normal lymphocytes treated in vitro with SssI methyltransferase was used as a positive control for methylated alleles. DNA from normal lymphocytes was used as a positive control for unmethylated alleles. PCR products were loaded onto nondenaturing 3% agarose gels, stained with ethidium bromide and visualized under an ultraviolet transilluminator.
Lamin A/C RNA and Protein Analysis
RNA was isolated using TRIzol (Life Technologies, Gaithersburg, MD). RNA (2 μg) was reverse-transcribed using SuperScript II reverse transcriptase (RT; Gibco/BRL, Barcelona, Spain) and amplified using specific primers for lamin A (forward: 5'-ATG GAG ACC CCG TCC CAG -3'; reverse: 5'-TTA CAT GAT GCT GCA GTT CTG G-3') and lamin C (forward: 5'-GAG ATG ATC CCT TGC TGA-3', reverse: 5'-GTT CCA AAA CAT TCT TTA ATG-3'). PCR was performed for 30 cycles (94°C for 30 seconds, 62°C for 30 seconds, and 72°C for 1.5 minutes and (94°C for 30 seconds, 57°C for 30 seconds, and 72°C for 30 seconds) for lamin A and C, respectively, in a final volume of 25 μL containing 1 x PCR buffer (Gibco/BRL), 1.5 mmol/L MgCl2, 0.3 mmol/L of dNTP, 0.25 mmol/L of each primer, and 2 U of taq polymerase (Gibco/BRL). RT-PCR primers were designed between different exons and encompassing large introns to avoid any amplification of genomic DNA. GAPDH was used as an internal control to ensure cDNA quality and loading accuracy. The amplification products were resolved by 2% agarose gel electrophoresis and visualized by ethidium bromide staining.
Nuclear lysates for protein analysis were prepared as described elsewhere19 and analyzed by Western blotting using the antibody anti-lamin A+C.ab8984 (mouse monoclonal antibody immunoglobulin G; Abcam Limited, Cambridge, UK). Equal loading was tested by re-probing with a polyclonal antibody against human nucleolin C23 (mouse monoclonal immunoglobulin G1; Santa Cruz Biotechnology, Santa Cruz, CA). Gels were cast using the XCell SureLock Mini-Cell system (Invitrogen Corp/NOVEX, Carlsbad, CA) and developed using ECL immunodetection reagents (Amersham Pharmacia Biotech, Piscataway, NJ).
Immunohistochemistry and Immunofluorescence
For immunohistochemistry, cells were centrifuged, washed in phosphate-buffered saline, fixed in formaldehyde (4%) for 2 hours, subsequently in ethanol (70%) overnight, and included in agarose (2%). After being dehydrated with increasing ethanol concentrations, the cells were put in xilol and embedded in paraffin blocks. The blocks were sectioned at a thickness of 3 μm and dried for 16 hours at 56°C before being dewaxed in xylene and ethanol. Antigen retrieval was achieved by heat treatment in a pressure-cooker for 2 minutes in 10 mmol/L citrate buffer (pH 6.5). After incubation with antibody (lamin A/C monoclonal antibody 636; Novocastra, Newcastle Upon Tyne, UK), immunodetection was performed with EnVision-HRP (DakoCytomation, Copenhagen, Denmark) and peroxidase activity was developed using 3,3-diaminobenzydine chromogen as substrate. Sections were counterstained with hematoxylin. A positive control was included with each batch of staining to ensure consistency between consecutive runs.
For immunofluorescence, cells were grown on coverslips in P60 dishes, fixed in 4% formaldehyde and stained as previously described.19 Cells to be analyzed for 5mC were postfixed in cold methanol, treated with 1M HCl at 37°C for 30 minutes and stained. Confocal optical sections were obtained using a Leica TCS SP microscope (Leica Microsystems, Wetzlar, Germany) equipped with krypton and argon lasers. Images were acquired with Leica LCS Lite software and processed with Adobe Photoshop 5.0 (Adobe Systems Inc, San Jose, CA) and publicly available National Institutes of Health image software.
Statistical Analysis
Contingency tables were analyzed by Fisher's exact test. Disease-free and overall survival curves were estimated by the Kaplan-Meier method and were compared with the use of the log-rank test. All statistical analyses were performed using the SPSS version 10.1 program (SPSS Inc, Chicago, IL).
RESULTS
Lamin A/C CpG Island Hypermethylation in Leukemia and Lymphoma Cell Lines and Its Association With Transcriptional Gene Silencing
Lamin A/C is a candidate gene for hypermethylation-associated inactivation in human cancer since a 5'-CpG island is located around the transcription start site (Fig 1A). In order to assess the methylation status of the promoter-associated CpG island of lamin A/C, we screened 74 human cancer cell lines from 17 tumor types using bisulfite genomic sequencing and methylation-specific PCR targeted to the area surrounding its transcription start site, as described in Patients and Methods. All samples tested were found by both methods to be unmethylated at the lamin A/C CpG island, except hematologic malignancy cell lines (Fig 1). Lamin A/C CpG island-promoter hypermethylation was found in seven (50%) of 14 leukemia cell lines (MOLT-4, MOLT-16, REH, HEL-R, KG1a, Jurkat, and P12 Ichikawa) and five (42%) of 13 lymphoma cell lines (RAJI, KARPAS-422, HT, DOHH2, and L-428). The remaining leukemia and lymphoma cell lines were unmethylated at the lamin A/C CpG island. All normal tissues analyzed, including lymphocytes and bone marrow, were completely unmethylated at the lamin A/C CpG island-promoter. The results were confirmed by bisulfite genomic sequencing (Figs 1A and 1B) and methylation-specific PCR (Fig 1C).
Having noted lamin A/C promoter hypermethylation in hematologic malignant cell lines, we assessed the association between this epigenetic aberration and putative transcriptional inactivation of the lamin A/C gene at the RNA and protein levels. First, RT-PCR analysis with primers specific for both transcripts arising from the lamin A/C CpG island, lamin A and lamin C, was carried out. The leukemia- and lymphoma-cell lines, REH, Jurkat and RAJI, hypermethylated at the lamin A/C CpG island, did not show expression of the lamin A and lamin C RNA transcripts (Fig 2A). However, the leukemia SD-1 and breast MDA-MB-231 cancer-cell lines, which are unmethylated at the lamin A/C promoter, expressed both lamin A and C transcripts (Fig 2A). Protein expression was analyzed by Western blot, immunohistochemistry, and immunofluorescence and confirmed that the lamin A/C hypermethylated cell lines REH, Jurkat, and RAJI lack expression of both lamin A and C isoforms, while the lamin A/C unmethylated cell lines SD-1 and MDA-231 expressed both protein isoforms (Figs 2B to 2D).
We established a further link between lamin A/C CpG island hypermethylation and gene silencing by the treatment of the methylated leukemia and lymphoma cell lines with a DNA demethylating agent. Treatment of the REH, Jurkat, and RAJI cell lines with the demethylating drug, 5-aza-2'-deoxycytidine, restored expression of both lamin A and lamin C RNA transcripts and protein splice variants in the three cell lines (Fig 2A and 2B).
Lamin A/C Is Hypermethylated in Primary Leukemias and Lymphomas and Is a Predictor of Poor Outcome in Nodal Diffuse Large B-Cell Lymphoma
Once the functional consequences of lamin A/C CpG island hypermethylation had been determined, we considered the relevance of lamin A/C methylation in human primary hematologic malignancies by studying a large set of leukemias and lymphomas. In leukemia, lamin A/C CpG island hypermethylation was found in 18% (nine of 50) of acute lymphoblastic leukemias, but was absent in acute myeloid leukemia (AML; n = 10). These AML cases did not include any erythroleukemia, while the two AML cell lines found hypermethylated at lamin A/C were of this subtype. In lymphomas, lamin A/C methylation was observed in 34% (14 of 41) of nodal diffuse large B-cell lymphoma and in 17% (one of six) of Burkitt's lymphomas, but was not found in cutaneous T-cell lymphoma (n = 17), extranodal diffuse large B-cell lymphoma (n = 10), and follicular lymphoma (n = 6). Illustrative examples are shown in Figure 3A. Unlike the cell lines that were either completely methylated or completely unmethylated at lamin A/C CpG island (Fig 1B), in these clinical samples, there is always an amplification in the unmethylated PCR lane, indicating the presence of normal tissues. Consistent with the data derived from hematopoietic cell lines, semiquantitative RT-PCR analysis of 24 nodal diffuse large B-cell lymphomas correlated loss of lamin A/C expression with promoter hypermethylation. Seventeen lymphomas lacking lamin A/C methylation expressed high levels of lamin A/C mRNAs, whereas seven lymphomas with lamin A/C methylation expressed little or no detectable lamin A/C mRNA transcripts (Fig 3B).
We next determined whether there was a relationship between the hypermethylation status of the gene lamin A/C and the outcome of our set of 41 nodal diffuse large B-cell lymphoma patients (this clinical information was not available for the acute lymphoblastic leukemias patients). Lamin A/C hypermethylation was observed in 34% (14 of 41) of patients, as described above. Demographic and clinical characteristics versus lamin A/C methylation status is shown in Table 1. For these lymphomas, lamin A/C hypermethylation was independent of the age of onset (Fisher's exact two-tail test, P = .18) and sex of the patient (Fisher's exact two-tail test, P = .32) and the International Prognostic Indicator (IPI; Fisher's exact two-tail test, P = 1). Most importantly, we found that the presence of lamin A/C-promoter hypermethylation was associated with a statistically significant decrease in failure-free survival (Kaplan-Meier, P = .0001; Fig 4A) and overall survival rates (Kaplan-Meier, P = .0005; Fig 4B). Therefore, lamin A/C CpG island hypermethylation is a likely predictor of poor outcome in nodal diffuse large B-cell lymphoma patients.
DISCUSSION
A-type lamins are essential components of the nuclear lamina.2,6 Together with B-type lamins, they are the most prominent intermediate filaments forming this network of 10-nM diameter filaments located on the inner side of the nuclear membrane. A-type lamins are expressed in most differentiated somatic cells, and preliminary studies have suggested that lamin expression may be reduced in transformed cells,2,6 especially in hematologic malignancies.10,11 Our results demonstrate for the first time that in leukemias and lymphomas, an important mechanism for the loss of lamin A/C expression is aberrant transcriptional silencing by CpG island promoter hypermethylation.
The consequences of lamin A/C gene inactivation by CpG island hypermethylation in hematologic maligancies should be assessed from the standpoint of basic research and translational studies. With regard to basic research, it is important to understand the downstream biologic and cellular consequences for the methylation-mediated loss of lamin A and C in cancer cells. In the same way that the term "guardian of the genome" was proposed for p53 a decade ago, it may be now acceptable to describe A-type lamins as guardians of the soma6 for their dual role as "tensegrity elements" that protect chromatin from damage and also act as multifunctional regulators of gene transcription.2,6 It has been proposed that the loss of lamin A/C expression in malignant B- and T-lymphocytes reflects a block in the differentiation pathway of these cells.10 Our finding of the epigenetic silencing of the lamin A/C gene may aid understanding as to how lamins contribute to cellular transformation.
In this work, we have focused on translational studies, showing that the presence of lamin A/C hypermethylation in nodal diffuse large B-cell lymphoma patients is a significant predictor of shorter failure-free survival and overall survival. These lymphomas are the most common type of NHL, accounting for approximately 30% to 40% of adult NHLs. They represent a heterogeneous group of lymphoid neoplasms taking into account clinical, histologic, immunophenotypic, cytogenetic, and molecular genetic features,20,21 highlighting the need to determine parameters identifying patients at high risk of progression and death. Several molecular markers, such as BCL-6, BCL-2, p53, and specific microarray expression patterns17,22-24 or MGMT hypermethylation,25 have demonstrated potential as prognostic factors for the disease. However, the most commonly used indicator of prognosis is the International Prognostic Index (IPI). The IPI takes into account factors that are mostly linked to patient characteristics (age, performance status) and to disease extension and growth (disease stage, lactate dehydrogenase levels, and extent of extranodal involvement). The presence of lamin A/C hypermethylation was independent of the IPI value, confirming its potential role as a clinical marker. A second translational point relates to treatment. Current chemotherapy regimens have improved the outcome for patients with hematologic malignancies, but many patients do not respond to and/or benefit from the use of the standard anticancer drugs. Recently, the US Food and Drug Administration has approved the use of the demethylating agent 5-azacytidine at low doses for the treatment of myelodysplastic syndrome based on the capacity of the drug to reactivate CpG island hypermethylated genes.26 In this new scenario, lamin A/C is an excellent target gene for the use of this type of drugs in order to achieve its demethylation and reactivation in hematologic malignancies.
In summary, our study provides a mechanistic explanation for the observed loss of A-type lamins in hematologic malignancies, demonstrating that the lamin A/C gene encoding these central components of the nuclear lamin undergoes transcriptional silencing by promoter CpG island hypermethylation in leukemias and lymphomas. Although, the ultimate consequences of lamin A/C epigenetic inactivation for the molecular biology of the tumoral cell remain unknown, hypermethylation emerges as a strong indicator of poor prognosis for lymphoma patients. These data should encourage further research into the role of lamins in the development of human neoplasia.
Authors' Disclosures of Potential Conflicts of Interest
The authors indicated no potential conflicts of interest.
NOTES
Supported by the Health (FIS) and Science (I+D) Departments, Spanish Government; and Fundacion Telefonica.
Terms in blue are defined in the glossary, found at the end of this issue and online at www.jco.org.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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