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Keywords:

  • PCDH10;
  • methylation;
  • lymphoma;
  • biomarker;
  • tumour suppressor gene

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussions
  6. Acknowledgements
  7. References
  8. Supporting Information

Epigenetic silencing of tumour suppressor genes (TSG) inactivates TSG functions. Previously, we identified PCDH10 as a methylated TSG in carcinomas. Here, we detected its frequent silencing and methylation in lymphoma cell lines including 100% Burkitt, 100% diffuse large B cell, 86% Hodgkin, 100% nasal natural killer/T-cell lymphoma and 1/3 of leukaemia cell lines, and in primary tumours but not in normal peripheral blood mononuclear cells or lymph nodes. PCDH10 silencing could be reversed by demethylation with 5-aza-2′-deoxycytidine. Methylation was further detected in 14% of Hodgkin lymphoma sera. Thus, PCDH10 methylation is frequently involved in lymphomagenesis and could serve as a tumour-specific biomarker.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussions
  6. Acknowledgements
  7. References
  8. Supporting Information

Aberrant methylation of tumour suppressor gene (TSG)-associated CpG islands leads to epigenetic loss of TSG functions, and is frequently involved in tumour development including haematological malignancies (Claus & Lubbert, 2003). It also acts as an epigenetic tumour marker for early cancer diagnosis, risk and prognosis assessment.

Epigenetic downregulation of cadherins and protocadherins as candidate TSGs (such as CDH1, CDH4, CDH13, FAT and protocadherin LKC) has been frequently observed in epithelial and haematologic tumours (Melki et al, 2000; Okazaki et al, 2002). Recently, we reported that PCDH10 (protocadherin 10/OL-PCDH/KIAA1400) was epigenetically silenced in carcinomas and further showed that PCDH10 is a functional TSG for carcinoma cells (Ying et al, 2006). Studies on mouse models of acute lymphocytic/myeloid leukaemia also demonstrated that PCDH10 is a frequently methylated gene involved in leukaemia transformation (Yu et al, 2005; Rosenbauer et al, 2006). Thus, we examined whether PCDH10 is frequently silenced in lymphomas and whether its methylation could serve as a biomarker for haematological tumours.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussions
  6. Acknowledgements
  7. References
  8. Supporting Information

Cell lines, tumour and serum samples

Twenty-seven cell lines were used: 13 non-Hodgkin lymphoma (non-HL) of B-cell type including seven Burkitt and six diffuse large B-cell lymphoma (DLBCL); four nasal natural killer (NK)/T-cell lymphoma; seven HL and three leukaemia. Ten normal peripheral blood mononuclear cells (PBMC) and lymph node samples, which were either of normal histology or with mild benign reactive hyperplasia from individuals without any malignancy, were collected. Cell lines were treated with 5 μM of 5-aza-2′-deoxycytidine (Aza) (Sigma, St. Louis, MO, USA) for 3 d (Ying et al, 2005, 2006). Serum samples from HL patients have been described (Murray et al, 2004).

Semi-quantitative reverse transcription polymerase chain reaction

Reverse transcription polymerase chain reaction was performed as previously described, using primers PCDH10F: 5′-ACTGCTATCAGGTATGCCTG and PCDH10R: 5′-GTCTGTCAACTAGATAGCTG, and GAPDH as a control (Ying et al, 2006).

Bisulphite treatment and promoter methylation analysis

Bisulphite modification of DNA, methylation-specific PCR (MSP) and bisulphite genomic sequencing (BGS) were performed as described (Tao et al, 1999; Ying et al, 2006). MSP primers were: methylation-specific, PCDH10bm1: 5′-TCGTTAAATAGATACGTTACGC, PCDH10bm2: 5′-TAAAAACTAAAAACTTTCCGCG, or unmethylation-specific, PCDH10bu1: 5′-GTTGTTAAATAGATATGTTATGT, PCDH10bu2: 5′-CTAAAAACTAAAAACTTTCCACA. Some MSP products were confirmed by direct sequencing. Semi-nested MSP was performed to detect PCDH10 methylation in serum DNA, using primers PCDH10BGSb1: 5′-TCACATTTACTAATACAAACAAA and PCDH10bm1 for the first round PCR, and primers PCDH10bm1 and PCDH10bm2 for the second round PCR. For BGS, bisulphite-treated DNA was amplified using primers BGS1: 5′-GTTGATGTAAATAGGGGAATT and BGS2: 5′-CTTCAACCTCTAAACCTATAA, cloned and sequenced (Tao et al, 1999; Ying et al, 2006).

Results and discussions

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussions
  6. Acknowledgements
  7. References
  8. Supporting Information

We firstly examined PCDH10 expression in 20 lymphoma cell lines. PCDH10 was silenced or reduced in 7/7 Burkitt, 6/6 DLBCL and 6/7 HL cell lines (Fig 1A). In contrast, PCDH10 expression was readily detected in all normal lymph node and PBMC samples (Fig 1C). We also detected PCDH10 methylation by MSP in all the cell lines with reduced or silenced expression (Fig 1A), as well as four nasal NK/T-cell lymphoma cell lines (Fig 1B). In most cell lines, methylation completely silenced PCDH10 expression. Among HL cell lines, PCDH10 was only expressed and unmethylated in HD-MY-Z, which is CD30-negative and may be unrepresentative of HL in general. Only partial methylation of PCDH10 was detected in 1/3 leukaemia cell lines (Fig 1B).

image

Figure 1.  (A) Representative analyses of PCDH10 expression levels by reverse transcription polymerase chain reaction (RT-PCR) (top two panels) and its methylation status by methylation-specific PCR (MSP) (bottom two panels) in lymphoma cell lines. GAPDH was used as a control. BL, Burkitt lymphoma; DLBCL, diffuse large B-cell lymphoma; HL, Hodgkin lymphoma; M, methylated; U, unmethylated. (B) Methylation status of PCDH10 promoter in leukaemia and nasal natural killer (NK)/T-cell lymphoma cell lines by MSP. (C) Expression of PCDH10 in normal lymph node (LN) and peripheral blood mononuclear cells (PBMC) samples. (D) Aza demethylation activated PCDH10 expression in silenced lymphoma cell lines. Representative results of PCDH10 methylation in primary lymphomas (E) and normal control tissues (F). FL, follicular lymphoma; PTLD, post-transplant lymphoma. (G) Detection of PCDH10 methylation in serum samples from HL patients. Right panel: PCR products were further confirmed by direct sequencing.

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To test whether methylation directly mediates PCDH10 silencing, we treated five cell lines with methylated and silenced PCDH10, using the demethylating agent Aza (Fig 1D). Aza restored PCDH10 expression in all of them, indicating that methylation directly mediates the transcriptional silencing of PCDH10 in lymphomas.

We further investigated PCDH10 methylation in multiple primary lymphomas and normal control tissues (lymph node, PBMC and purified CD3CD16+CD56+ normal NK cells), as well as serum samples from 14 HL patients. PCDH10 methylation was detected in various lymphomas including 90% (9/10) Burkitt, 80% (16/20) DLBCL, 63% (5/8) follicular, 2/4 mantle cell, 2/4 anaplastic large cell, 72% (31/43) nasal NK/T-cell, 20% (2/10) post-transplant and 28% (11/39) HL (Fig 1E and Supplementary Table I). Ten normal PBMC samples, normal NK cells and 10 normal lymph nodes showed no methylation at all (Fig 1F), indicating that PCDH10 methylation is common and tumour specific. Moreover, we detected PCDH10 methylation in the sera of two HL patients, confirmed by direct sequencing of MSP products (Fig 1G).

We further analysed nine cell lines, 10 primary tumours and two normal PBMC samples for PCDH10 methylation in more detail, using high-resolution BGS analysis (Fig 2). The results further supported our MSP data. Dense methylated CpG sites were detected in all silenced cell lines, but not in a PCDH10-expressing cell line (HD-MY-Z) and normal PBMCs (Fig 2A and B). In primary tumours, BGS results also revealed dense methylation in all tumours with methylation detected by MSP, while no methylated CpG site was detected in an unmethylated tumour (DLBCL13) (Fig 2C). As primary tumour tissues generally contain reactive infiltrating lymphocytes, unmethylated promoter alleles were detected in most tumours, except for two Burkitt lymphomas in which a high proportion of tissue cells were tumour cells.

image

Figure 2.  High-resolution mapping of the methylation status of individual CpG sites in the PCDH10 promoter by bisulphite genomic sequencing (BGS) in cell lines (A), normal peripheral blood mononuclear cells (PBMCs) (B) and primary tumours (C). Each row of circles (CpG sites) represents an individual allele of the promoter analysed. Arrow: transcription start site.

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Several human protocadherins, protocadherin LKC, PCDHGA11 and PCDH20, have been reported to be candidate TSGs with methylation in tumours (Okazaki et al, 2002; Waha et al, 2005; Imoto et al, 2006). We previously reported that PCDH10 is a functional TSG that is frequently inactivated epigenetically in carcinomas. Here, we further detected its frequent tumour-specific methylation in multiple lymphoma cell lines and primary tumours. We also detected PCDH10 methylation in the sera of HL patients. Aberrant TSG methylation in cancer patient sera has been suggested as a non-invasive molecular diagnostic biomarker (Hoque et al, 2004; Murray et al, 2004). Although we did not optimise our experimental system of serum collection, DNA extraction or bisulphite treatment specifically for the purpose of molecular diagnosis, our results still suggest that PCDH10 methylation in the sera of lymphoma patients could be further explored as a non-invasive biomarker for monitoring disease progression or relapse in patients with PCDH10 methylation. Our study, together with the two recent mouse leukaemia model studies (Yu et al, 2005; Rosenbauer et al, 2006), suggest that epigenetic inactivation of PCDH10 might be an important step in lymphomagenesis and leukaemia transformation.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussions
  6. Acknowledgements
  7. References
  8. Supporting Information

We thank Dr Riccardo Dalla-Favera for kindly providing DLBCL cell lines.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussions
  6. Acknowledgements
  7. References
  8. Supporting Information
  • Claus, R. & Lubbert, M. (2003) Epigenetic targets in hematopoietic malignancies. Oncogene, 22, 64896496.
  • Hoque, M.O., Begum, S., Topaloglu, O., Jeronimo, C., Mambo, E., Westra, W.H., Califano, J.A. & Sidransky, D. (2004) Quantitative detection of promoter hypermethylation of multiple genes in the tumor, urine, and serum DNA of patients with renal cancer. Cancer Research, 64, 55115517.
  • Imoto, I., Izumi, H., Yokoi, S., Hosoda, H., Shibata, T., Hosoda, F., Ohki, M., Hirohashi, S, & Inazawa, J. (2006) Frequent silencing of the candidate tumor suppressor PCDH20 by epigenetic mechanism in non-small-cell lung cancers. Cancer Research, 66, 46174626.
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  • Okazaki, N., Takahashi, N., Kojima, S., Masuho, Y. & Koga, H. (2002) Protocadherin LKC, a new candidate for a tumor suppressor of colon and liver cancers, its association with contact inhibition of cell proliferation. Carcinogenesis, 23, 11391148.
  • Rosenbauer, F., Owens, B.M., Yu, L., Tumang, J.R., Steidl, U., Kutok, J.L., Clayton, L.K., Wagner, K., Scheller, M., Iwasaki, H., Liu, C., Hackanson, B., Akashi, K., Leutz, A., Rothstein, T.L., Plass, C. & Tenen, D.G. (2006) Lymphoid cell growth and transformation are suppressed by a key regulatory element of the gene encoding PU.1. Nature Genetics, 38, 2737.
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  • Waha, A., Guntner, S., Huang, T.H., Yan, P.S., Arslan, B., Pietsch, T., Wiestler, O.D. & Waha, A. (2005) Epigenetic silencing of the protocadherin family member PCDH-gamma-A11 in astrocytomas. Neoplasia, 7, 193199.
  • Ying, J., Srivastava, G., Hsieh, W.S., Gao, Z., Murray, P., Liao, S.K., Ambinder, R., & Tao, Q. (2005) The stress-responsive gene GADD45G is a functional tumor suppressor, with its response to environmental stresses frequently disrupted epigenetically in multiple tumors. Clinical Cancer Research, 11, 64426449.
  • Ying, J., Li, H., Seng, T.J., Langford, C., Srivastava, G., Tsao, S.W., Putti, T., Murray, P., Chan, A.T., & Tao, Q. (2006) Functional epigenetics identifies a protocadherin PCDH10 as a candidate tumor suppressor for nasopharyngeal, esophageal and multiple other carcinomas with frequent methylation. Oncogene, 25, 10701080.
  • Yu, L., Liu, C., Vandeusen, J., Becknell, B., Dai, Z., Wu, Y.Z., Raval, A., Liu, T.H., Ding, W., Mao, C., Liu, S., Smith, L.T., Lee, S., Rassenti, L., Marcucci, G., Byrd, J., Caligiuri, M.A. & Plass, C. (2005) Global assessment of promoter methylation in a mouse model of cancer identifies ID4 as a putative tumor-suppressor gene in human leukemia. Nature Genetics, 37, 265274.

Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussions
  6. Acknowledgements
  7. References
  8. Supporting Information

Table SI. Summary of PCDH10 methylation in various lymphoma cell lines and primary lymphomas

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BJH+6512+Supplementary_Table_1.pdf90KSupporting info item

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