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Department of Anatomical and Cellular Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong SAR, China
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. In B-cell malignancies, loss of DAP kinase expression is commonly associated with promoter hypermethylation. These characteristics of DAP kinase may be of particular relevance in multiple myeloma (MM), a B-lineage malignancy in which prolonged survival capacity of the malignant plasma cells may be critical in the induction and maintenance of tumor cells. Purpose: The involvement and potential role of DAP kinase in MM pathogenesis was examined.
Experimental Design: In this investigation, methylation-specific PCR was conducted on primary MM and MM cell lines. Methylation status findings were correlated with clinical parameters.
Results: We first demonstrated frequent DAP kinase hypermethylation in 24 of 36 primary MMs (20 of 26 at diagnosis and 4 of 10 with relapse/residual MM after treatment), 1 of 2 patients with monoclonal gammopathy of undetermined significance, and 1 of 3 MM cell lines studied. The high frequency of DAP kinase hypermethylation was similarly observed in MM of different stages, immunoglobulin isotypes, and histological grades, with or without plasmacytomas. Although not statistically significant, the overall survival of patients with DAP kinase methylation was notably shortened among 23 MM patients followed prospectively (P = 0.38 by Kaplan-Meier method and log-rank test). This preliminary finding suggests prognostic implications of DAP kinase in MM that may deserve further investigation.
Conclusions: Our data suggest an important role for DAP kinase in MM tumorigenesis.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A number of cytokines are involved in MM pathogenesis (5, 6, 7, 8)
. Interleukin 6 inhibits apoptosis and supports the growth of MM cells (5)
. IFNs mediate MM growth inhibition via modification of the cell cycle and the interleukin 6 signaling mechanism (6)
. IFN-
has been used in the maintenance therapy of some MM patients (7)
. Because occasional growth stimulation by IFN- was also observed in some MM cell lines (8)
, IFN-, which shows consistent antiproliferative activity, has been suggested as a potential alternative in MM management (6)
.
DAP kinase is a novel calcium/calmodulin-dependent and cytoskeletal-associated serine/threonine kinase with death-inducing functions (9)
. Overexpression of DAP kinase killed HeLa cells in the absence of any external stimuli (9)
. Mapped to chromosome 9q34.1, the DAP kinase gene was initially isolated as a positive mediator of apoptosis induced by IFN-, using a strategy of functional cloning (10
, 11)
. Loss of expression of DAP kinase was frequently found in B-cell lymphoma and some carcinoma cell lines, which highlights its potential role as a tumor suppressor (12)
. In B-cell malignancies, the loss of expression was commonly associated with hypermethylation of the DAP kinase CpG island (13)
. Recently, DAP kinase has also been found to mediate apoptosis induced by TNF- and Fas, whose expression is found in some MM cell lines and patient-derived primary cells (14, 15, 16, 17)
. However, a poor response to Fas-induced apoptosis was observed in a majority of the MM cases and a correlation between Fas antigen expression and susceptibility to Fas-mediated apoptosis in MM could not be established (15, 16, 17)
.
These findings have prompted us to examine the methylation status of DAP kinase in MM, which may have potential implications in our understanding of its pathogenesis and prognosis.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Normal Controls and Positive Control.
Thirty normal PB samples were obtained from healthy volunteer staff and students of the Chinese University of Hong Kong to serve as normal controls. Raji (ATCC), a Burkitt’s lymphoma cell line, was used as a methylated control (12
, 13)
.
DNA Extraction and MSP.
The buffy coat fraction was isolated from BM aspirate, and total genomic DNA was extracted using standard SDS-proteinase K treatment, followed by phenol/chloroform/isoamylalcohol extraction. DNA methylation patterns in the CpG island of DAP kinase were determined by chemical treatment with sodium bisulfite (CpGenome DNA Modification Kit; Intergen) and subsequent use of the previously described PCR procedure (13
, 21
, 22)
. Bisulfite treatment allows differentiation between methylated and unmethylated cytosine residues to be distinguished and detected by sequence-specific PCR primers (13
, 21
, 22)
. Bisulfite-treated buffy coat DNA (1 µg) was amplified using primers DAPUF (5'-GGA-GGA-TAG-TTG-GAT-TGA-GTT-AAT-GTT-3') and DAPUR (5'-CAA-ATC-CCT-CCC-AAA-CAC-CAA-3') for the unmethylated sequence and primers DAPMF (5'-GGA-TAG-TCG-GAT-CGA-GTT-AAC-GTC-3') and DAPMR (5'-CCC-TCC-CAA-ACG-CCG-A-3') for the methylated sequence (13
, 23)
. PCR was conducted using the GeneAmp DNA Amplification Kit and AmpliTaq Gold polymerase (Perkin-Elmer, Foster City, CA) according to the conditions described previously (13)
. In brief, 25 µl of PCR mixture contained 1x PCR buffer [10 mM Tris-HCl (pH 8.3) and 50 mM KCl], 2 mM MgCl2, deoxynucleotide triphosphates (each at 250 µM), primers (1 µM each per reaction), bisulfite-modified DNA (80 ng) or unmodified DNA (80 ng), and 1 unit of AmpliTaq Gold polymerase. Reactions were hot started at 95°C for 10 min, and the annealing temperature was 58°C. Amplification was carried out in a Thermal Cycler 480 (Perkin-Elmer) for 35 cycles. Ten µl of the PCR reaction were eletrophoresed onto 10% polyacrylamide gels, stained with ethidium bromide, and visualized under UV light. Methylated control (Raji), unmethylated control (normal blood sample), and negative control (water blank) were included in each experiment.
Culture of Cell Lines.
HS-Sultan, NCI-H929, and U266 human MM-derived cell lines and the Raji (Burkitt’s lymphoma) cell line were purchased from ATCC. These cell lines were cultured in RPMI 1640 supplemented with 10% or 15% heat-inactivated fetal bovine serum (Life Technologies, Inc., Gaithersburg, MD).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 9 G
, 5 A, 2 A, 1 D, 2 BJP, and 1 BJP) and 2 patients with MGUS (G and G), were analyzed for DAP kinase hypermethylation (Table 1)
. The male:female ratio was 1.7:1, with a median age of 66 years (range, 29–89 years). Five patients defaulted, 15 patients died (median survival, 7 months), and 18 were still alive after a median follow-up time of 23 months. In the MM group, 26 patients were analyzed at diagnosis, and 10 patients were analyzed at variable times after treatment (median treatment duration, 12 months). Twenty-eight patients had stage III disease, five patients had stage II disease, and three patients had stage I disease. Eight patients had plasmacytomas, as documented by histology. Six plasmacytomas involved bone, one plasmacytoma was found in chest wall muscle, and one plasmacytoma was found in pleura. Histologically, 10 MMs were classified as mature, 16 MMs were classified as intermediate, and 9 MMs were classified as blastic. One patient who fulfilled the other criteria of MM had less than 5% of mature plasma cells in the BM at both diagnosis and relapse. Excluding this patient, the mean percentage of plasma cell infiltration was 46 ± 31% (range, 10–100%). The complete blood count revealed a mean hemoglobin of 9.1 ± 2.2 g/dl (range, 4.5–15 g/dl), WBC count of 6.5 ± 2.6 x 109/liter (range, 2.7–15x 109/liter), and platelet count of 200.8 ± 102.9 x 109/liter (range, 9–467x 109/liter). Most MM patients were treated with melphalan and prednisone or dexamethasone with or without radiotherapy. A few patients were treated with vincristine, adriamycin, and dexamethasone or with cyclophosphamide, etoposide, vincristine, adriamycin, and dexamethasone. One patient with stable MM was given supportive blood transfusions only.
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. These data are consistent with previous studies, which showed no evidence of methylation of the gene in both normal controls and EBV-immortalized lymphoblastoid cell lines (12
, 13)
. In contrast, a high frequency of hypermethylation of DAP kinase was found in the primary MM (67%; Table 1
; Fig. 1B
). Among the three MM cell lines tested, HS-Sultan demonstrated DAP kinase methylation. In all of the DAP kinase-methylated primary MM cases, unmethylated DNA bands were also observed as a result of the presence of normal BM cellular elements in all these samples (Fig. 1B)
. On the contrary, the HS-Sultan MM cell line revealed a complete methylation pattern with absence of the unmethylated band (Fig. 1B)
. One patient was diagnosed with MM because of the presence of bone plasmacytomas and significant G paraproteinemia with immunoparesis. Despite repeated normal morphological findings on BM examination (<5% plasma cells) during diagnosis and relapse, DAP kinase hypermethylation was detected in this patient’s BM sample at relapse. It is also noteworthy that of the two patients with MGUS tested, one demonstrated DAP kinase hypermethylation.
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). Moreover, a generally poorer treatment response was observed in patients with methylated DAP kinase as compared with patients with unmethylated DAP kinase. In the methylated DAP kinase group, 2 patients were treated at the plateau phase, and the remaining 15 patients with active disease showed variable responses to chemotherapy that were considered good (rapidly decreasing paraprotein levels with improvement of symptoms), fair (slowly settling response), or poor (progressive disease) for 5 patients in each category. In contrast, among the six patients with unmethylated DAP kinase, one was in plateau phase, one demonstrated a fair response to chemotherapy, and the other four patients demonstrated good responses to chemotherapy, which were also more sustainable compared with the responses of patients with methylated DAP kinase who also had a good initial response.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(9
, 10)
, has been shown to be commonly inactivated by promoter hypermethylation in B-cell malignancies (12
, 13)
. In the present investigation, using MSP, we first demonstrated a high incidence of DAP kinasehypermethylation in primary MM and in MM cell lines. Our finding is consistent with the type of DAP kinase alteration found previously in B-cell malignancies and may extend its role in the pathogenesis of this spectrum of diseases. In the HS-Sultan MM cell line, it was also observed that DAP kinase methylation was associated with loss of transcription, which was restored after demethylation treatment with 5-aza-2'-deoxycytidine concomitant with the reappearance of the unmethylated alleles detected by MSP.4 It has been postulated that loss of DAP kinase may confer a selective advantage during the multiple stages of metastasis for tumor cells with resistance to various apoptotic stimuli encountered after detachment from the original tumor and transport in the circulation (24) . In lung carcinoma, loss of DAP kinase has been associated with a more aggressive highly metastatic phenotype. Thus, loss of DAP kinase expression provides a unique mechanism that links suppression of apoptosis to metastasis (24) . Although slowly proliferating MM cells are localized predominantly in the BM, the disease manifests itself in a disseminated form with the consistent presence of a circulatory pool of MM precursors (25) . Failure to eradicate and control this proliferative circulatory pool of MM cells may be the cause of the high fatality and poor treatment outcomes in this malignancy. It is possible that the selective advantage conferred by the inactivation of DAP kinase may play a role in the induction and maintenance of the circulatory MM tumor pool. The high frequency of DAP kinase hypermethylation in MM suggests that it may play a critical role in the etiology of MM. Furthermore, its frequent occurrence in MM of all stages, immunoglobulin isotypes, and histological grades and in MM with or without plasmacytomas may indicate that it is an early event in MM pathogenesis. This is further supported by the presence of DAP kinase hypermethylation in MGUS, which may represent a pre-MM condition.
There was apparently a shorter (3-fold) survival duration in the methylated DAP kinase group than in the unmethylated DAP kinase MM group (Fig. 2)
. In a recent study, a shorter 5-year survival rate has also been observed in patients with non-small cell lung cancer with DAP kinase hypermethylation (26)
. Whether this poor prognosis for patients with DAP kinase methylation in MM is related to enhanced survival of the circulatory MM precursor cells requires further study in the future.
The Fas antigen is a member of the TNF receptor family of proteins and is expressed in many neoplastic and normal cells including hematopoietic cell lines, lymphoma cells, and activated normal T and B lymphocytes (27
, 28)
. Although Fas expression was observed on some MM cell lines and patient-derived primary cells, a poor response to Fas-induced apoptosis was demonstrated in a majority of the MM cases (15, 16, 17)
. The high expression of Fas antigen and the low number of cells induced to apoptose may suggest a defect in the Fas signaling pathway. Consistent with these observations, it has been shown recently that expression of DAP kinase antisense RNA protected HeLa cells from killing by anti-Fas/APO-1 agonistic antibodies (14)
. Thus, DAP kinase not only mediates cell deaths induced by IFN- but also by TNF- and Fas activation (14)
. The high frequency of DAP kinase hypermethylation (thus downstream signaling defect) in primary MM found in this study may explain, in part, the poor response of apoptosis induced by Fas activation. However, it was also observed that pretreatment with IFN- augmented Fas-induced apoptosis in the MM cells (29)
. This suggests that a DAP kinase-independent pathway may operate in this latter death scenario.
We have demonstrated previously that there is a frequent hypermethylation of p16 and p15 genes in MM (20
, 30
, 31)
. Taken together, these data support the argument that multiple epigenetic events may be a common and important mode of gene inactivation that may coexist with other genetic alterations in the constellation of changes associated with MM transformation. The therapeutic potential of IFNs acts via their effects on growth inhibition and induction of apoptosis (6)
. In MM, IFN- has been used in maintenance therapy. Because occasional growth-stimulatory effects on MM cells by IFN- were also observed (8)
, it has been suggested that IFN- may be an alternative (6)
. However, the treatment potential of IFN- in MM may likely be limited in the presence of a high incidence of DAP kinase methylation, such as that found in this study. In contrast, clinical evaluation of the use of a demethylating agent, alone or in combination with other therapeutic agents, may be worthwhile in MM, where concurrent methylation of multiple genes is involved (20
, 30
, 31)
.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Research Grant 2040738 from the Chinese University of Hong Kong. 
2 To whom requests for reprints should be addressed, at Hematology Section, Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, China. Phone: 852-2632-2179; Fax: 852-2637-6274; E-mail: margaretng{at}cuhk.edu.hk
3 The abbreviations used are: MM, multiple myeloma; DAP, death-associated protein; MSP, methylation-specific PCR; MGUS, monoclonal gammopathy of undetermined significance; TNF, tumor necrosis factor; BM, bone marrow; ATCC, American Type Culture Collection; PB, peripheral blood.
Received 11/ 6/00; revised 2/17/01; accepted 3/ 1/01.
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  E. De Bruyne, T. J. Bos, K. Asosingh, I. Vande Broek, E. Menu, E. Van Valckenborgh, P. Atadja, V. Coiteux, X. Leleu, K. Thielemans, et al. Epigenetic Silencing of the Tetraspanin CD9 during Disease Progression in Multiple Myeloma Cells and Correlation with Survival Clin. Cancer Res., May 15, 2008; 14(10): 2918 - 2926. [Abstract] [Full Text] [PDF]
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T. Takahashi, N. Shivapurkar, J. Reddy, H. Shigematsu, K. Miyajima, M. Suzuki, S. Toyooka, S. Zochbauer-Muller, J. Drach, G. Parikh, et al. DNA Methylation Profiles of Lymphoid and Hematopoietic Malignancies Clin. Cancer Res., May 1, 2004; 10(9): 2928 - 2935. [Abstract] [Full Text] [PDF]
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D. Levy, G. Plu-Bureau, Y. Decroix, D. Hugol, W. Rostene, A. Kimchi, and A. Gompel Death-Associated Protein Kinase Loss of Expression Is a New Marker for Breast Cancer Prognosis Clin. Cancer Res., May 1, 2004; 10(9): 3124 - 3130. [Abstract] [Full Text] [PDF]
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 M. T. Voso, A. Scardocci, F. Guidi, G. Zini, A. Di Mario, L. Pagano, S. Hohaus, and G. Leone Aberrant methylation of DAP-kinase in therapy-related acute myeloid leukemia and myelodysplastic syndromes Blood, January 15, 2004; 103(2): 698 - 700. [Abstract] [Full Text] [PDF]
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A. N. Reddy, W. W. Jiang, M. Kim, N. Benoit, R. Taylor, J. Clinger, D. Sidransky, and J. A. Califano Death-Associated Protein Kinase Promoter Hypermethylation in Normal Human Lymphocytes Cancer Res., November 15, 2003; 63(22): 7694 - 7698. [Abstract] [Full Text] [PDF]
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O. Galm, H. Yoshikawa, M. Esteller, R. Osieka, and J. G. Herman SOCS-1, a negative regulator of cytokine signaling, is frequently silenced by methylation in multiple myeloma Blood, April 1, 2003; 101(7): 2784 - 2788. [Abstract] [Full Text] [PDF]
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X174 RF DNA/HaeIII fragments) is indicated on the left. A, the DAP kinase gene is unmethylated in all of the PB samples from healthy donors. B, HS-Sultan contains only methylated sequence, whereas U266 and NCI-H929 contain only unmethylated DAP kinase. In primary MM, P2, P3, P6, and P7 contain both unmethylated and methylated DAP kinase, whereas P1, P4, and P5 contain only unmethylated DAP kinase.