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[Cancer Research 66, 3978-3980, April 15, 2006]
© 2006 American Association for Cancer Research

Priority Reports

Methylation of the IL-12Rß2 Gene as Novel Tumor Escape Mechanism for Pediatric B-Acute Lymphoblastic Leukemia Cells

Irma Airoldi1, Claudia Cocco1, Emma Di Carlo3, Silvia Disarò4, Emanuela Ognio2, Giuseppe Basso4 and Vito Pistoia1

1 Laboratory of Oncology, G. Gaslini Institute; 2 Animal Model Facility, Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy; 3 Department of Oncology and Neurosciences and Center of Excellence on Aging, G. D'Annunzio University, Chieti, Italy; and 4 Division of Hematology Oncology, Department of Pediatrics, University of Padova, Padova, Italy

Requests for reprints: Irma Airoldi, Laboratory of Oncology, G. Gaslini Institute, Largo G. Gaslini 5, 16148 Genova, Italy. Phone: 39-10-5636342; Fax: 39-10-3779820; E-mail: irmaairoldi{at}ospedale-gaslini.ge.it.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Previous studies have shown that the interleukin-12 receptor ß2 (IL-12Rß2) gene is expressed in normal naive, germinal center and memory B cells but not in their malignant counterparts. The aim of this study was to investigate (i) whether the IL-12Rß2 gene is silenced in B-cell acute lymphoblastic leukemia (B-ALL) cells, and (ii) what the functional implications of such silencing for tumor growth are. Here, we show that although mature B cells expressed both chains of the IL-12R, normal pro-B and pre-B cells failed to express the IL-12Rß2 chain. Similarly, primary tumor cells from pediatric pro-B, early pre-B, and pre-B ALL (30 cases) did not express the IL-12Rß2 chain. IL-12Rß2 gene silencing in B-ALL was found to depend on methylation of a CpG island in exon 1. Such methylation was not detected in normal early B cells that when differentiated into mature B cells expressed the IL-12Rß2 gene. Detection of IL-12Rß2 mRNA and protein in the tumorigenic 697 pre-B-ALL cell line allowed to perform functional experiments in severe combined immunodeficient/nonobese diabetic mice receiving 697 cells with or without human recombinant IL-12 (hrIL-12). hrIL-12 administration reduced tumor growth and metastasis through antiproliferative and proapoptotic rather than antiangiogenic, activities.

In conclusion, epigenetic silencing of the IL-12Rß2 gene represents a novel mechanism of tumor escape for B-ALL cells. (Cancer Res 2006; 66(8): 3978-80)


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Interleukin-12 (IL-12) is a cytokine bridging innate and adaptive immunity (1, 2). It is produced by antigen-presenting cells and regulates T-cell, natural killer, and B-cell functions (3–7). The IL-12 receptor (IL-12R) is a heterodimer composed of the ß1 and the ß2 chains, both necessary for high-affinity binding of and responsiveness to IL-12 (8, 9).

We have shown that the IL-12Rß2 gene, which is expressed in mature B cells, functions as a tumor suppressor in human chronic B-cell malignancies (10). Such methylation was not detected in normal bone marrow pro-B and pre-B cells, which, unlike mature B cells, failed to express the IL-12Rß2 gene. We have also shown that IL-12Rß2-deficient mice develop spontaneously a systemic disorder (11) characterized by multiorgan lymphoid infiltrates comprised predominantly of activated oligoclonal B cells and localized lymph node plasmacytoma. Taken together, these findings indicate that the IL-12Rß2 gene plays a broad role in the control of abnormal B-cell expansion and transformation.

In this study, we have asked the question of whether IL-12Rß2 gene silencing is a feature common to most B-cell lymphoproliferative disorders, including those derived from the early stages of B-cell differentiation [i.e., pro-B, early pre-B, and pre-B acute lymphoblastic leukemia (B-ALL)]. To investigate this issue, we tested tumor cells from 30 pediatric B-ALL patients and found that the IL-12Rß2 gene was consistently methylated. Such methylation was not detected in normal bone marrow pro-B and pre-B cells. Tumors formed in severe combined immunodeficient/nonobese diabetic (SCID/NOD) mice by an IL-12Rß2 expressing B-ALL cell line were significantly reduced in size upon human recombinant IL-12 (hrIL-12) administration, indicating that epigenetic silencing of the IL-12Rß2 gene is a novel mechanism of tumor escape in pediatric B-ALL.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Patient samples. Bone marrow aspirates from 10 pro-B, 10 early pre-B, and 10 pre-B pediatric ALL patients were done for diagnostic purpose after informed consent from children parents or their legal guardians. Bone marrow aspirates from four healthy donors were obtained following their informed consent. The study was approved by the local institutional review board. Mononuclear cell suspensions of leukemic and normal bone marrow cells were isolated on Ficoll-Hypaque density gradients and frozen in freezing solution until tested. Immunophenotypic characterization of B-ALL cases was carried out as described (12). Normal pro-B and pre-B cells were purified by fluorescence-activated cell sorting as CD19+CD10+CD20– or CD19+CD10+/–CD20+ cell fractions, respectively.

Cell culture, antibodies, and reagents. The RS4;11 pro-B ALL cell line and three pre-B ALL cell lines (NALM-6, REH, and 697) were cultured in RPMI 1640 with 10% FCS (Seromed-Biochrom KG, Berlin, Germany). REH and RS4;11 cells were cultured for 72 hours with 5-aza-2'-deoxycytidine (5 µmol/L; Sigma-Aldrich, St. Louis, MO). hrIL-12 was provided by Wyeth, Inc. (Cambridge, MA). Fluorochrome-conjugated anti-hIL-12Rß2, CD10, CD19, and CD20 for flow cytometric studies were from BD PharMingen (San Jose, CA). The CD10 (Novocastra, Newcastle upon Tyne, United Kingdom), anti-laminin (Biogenex, San Ramon, CA), CD31 (BD PharMingen), anti-proliferating cell nuclear antigen (PCNA), and CD19 (DakoCytomation, Glostrup, Denmark) monoclonal antibodies were used in immunohistochemistry, as described (11). Tissue samples were processed for histologic evaluation or for immunohistochemistry, as described (13). The rates of proliferating cells, as assessed by immunoreactivity for PCNA, and for apoptotic cells, as assessed by ApopTag Assay (Serologicals Corp., Norcross, GA), were determined as reported (10).

Reverse transcription-PCR, methylation assay, and flow cytometry. RNA was extracted from freshly isolated bone marrow cells and B-ALL cell lines using RNeasy Mini kit from Qiagen GmbH (Hilden, Germany). IL-12Rß1 and IL-12Rß2 expression was investigated by reverse transcription-PCR (RT-PCR; ref. 7).

DNA was extracted using GenElute DNA miniprep kit from Sigma (St. Louis, MO), and the methylation status of the target sequence was assessed by methylation-specific PCR (10, 14).

FACScan analysis (Becton Dickinson, San Jose, CA) was carried out as reported (7).

Mice studies. Fourteen- to 16-week-old female SCID/NOD mice were housed in sterile enclosure. All procedures involving mice were in accordance with institutional guidelines in compliance with national and international laws and policies (D.l.vo 27/01/1992, n.116, European Economic Community Council Directive 86/609, OJL 358, Dec. 1, 1987 and MIH Guide for the care and use of laboratory animals). Four groups of 10 animals each were injected i.v. with 5 x 106 697 cells. Two groups of mice were treated with three weekly doses of hrIL-12 (1 µg/mouse/dose) starting from 8 hours after injection of tumor cells. The other two groups of mice were injected with PBS following the same time schedule. One and 3 weeks after tumor cell inoculation, mice were sacrificed, and autopsies were carried out. Tumor masses were measured as described (10).

Statistical analysis. Differences in the number of PCNA-positive cells, apoptotic cells, and tumor microvessels were evaluated by Student's t test.


   Results and Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Mononuclear cells isolated from the bone marrow of B-ALL patients contained consistently higher than 98% CD19+ B cell blasts and were used as such for RT-PCR experiments. The IL-12Rß1 transcript was expressed in leukemic primary cells from all patients and cell lines (Fig. 1A ). In contrast, IL-12Rß2 mRNA was never detected in leukemic cells (Fig. 1A) with the exception of the 697 pre-B ALL cell line, which tested positive for IL-12Rß2 transcript (Fig. 1A) and protein (Fig. 1B).


Figure 1
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  		Figure 1. Expression of IL-12Rß1 and IL-12Rß2 mRNA in B-ALL cells. A, expression of IL-12Rß1 and IL-12Rß2 mRNA in neoplastic B cells from nine representative patients (Pt) with pro-B (n = 3), early pre-B (n = 3), and pre-B (n = 3) ALL and in four B-ALL cell lines by RT-PCR. MW, molecular weight; NC, negative control (Th2 clone); PC, positive control (tonsil B cells); lanes 4 to 12, B-ALL cases; lanes 13 to 16, B-ALL cell lines. B, constitutive IL-12Rß2 expression in 697 pre-B-ALL cells, as assessed by flow cytometry. Open profile, IL-12Rß2 staining; dark profile, isotype matched monoclonal antibody staining. C, methylation-specific PCR analysis of cells testing negative for IL12Rß2 gene expression. MW, molecular weight; NC, negative control (tonsil B lymphocytes); PC, positive controls (Raji B cells); lanes 4 to 12, same B-ALL cases as in (A); lanes 13 to 15, three B-ALL cell lines. D, IL-12Rß2 gene expression in RS4;11 and REH B-ALL cell lines treated for 72 hours with 5-aza-2'-deoxycytidine. MW, molecular weight markers; NC, negative control (Th2 clone); PC, positive controls (tonsil B cells); +Aza, RS4;11 and REH cells cultured with 5 µmol/L 5-aza-2'-deoxycytidine.

 
We next asked whether methylation of the CpG island previously identified in the noncoding exon 1 of the IL-12Rß2 gene (10) was involved in silencing of the latter in leukemic cells. Indeed, all primary B-ALL cells and cell lines but 697 (data not shown) displayed methylation of this CpG island (Fig. 1C). Additional evidence for methylation-dependent silencing of the IL-12Rß2 gene was provided by the de novo expression of IL-12Rß2 mRNA in RS4;11 and REH cell lines upon 72 hours of culture with the demethylating agent 5-aza-2'-deoxycytidine (Fig. 1D). Normal bone marrow pro-B and pre-B cells expressed IL-12Rß1 but not IL-12Rß2 mRNA in the absence of CpG island methylation (data not shown).

IL-12Rß2+ 697 cells injected i.v. in SCID/NOD mice gave rise after 3 weeks to bulky tumor masses that completely destroyed the ovary and invaded the pancreas, spleen, and liver. 697 cells injected i.p. or s.c. were not tumorigenic. hrIL-12-treated mice showed significantly smaller (P < 0.005) mesenteric and ovarian tumors than PBS-treated mice (Fig. 2A ) starting from 1 week after leukemic cell injection (Fig. 2B, a and b). The antitumor activity of hrIL-12 was related to antiproliferative (proliferation indexes: PBS-treated mice, 90.0 ± 7.2% versus hrIL-12-treated mice, 66.7 ± 6.5%) and proapoptotic (apoptotic indexes: PBS-treated mice, 8.3 ± 3.5% versus hrIL-12-treated mice, 18.6 ± 4.7%) rather than antiangiogenic (microvessel count: PBS-treated mice, 10.8 ± 3.1 versus hrIL-12-treated mice, 9.0 ± 2.8; Fig. 2B, c-h) effects.


Figure 2
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  		Figure 2. Tumor growth in hrIL-12 and PBS-treated SCID/NOD mice after i.v. injection of 697 cells. A, columns, mean size of tumors formed 3 weeks after injection of 697 cells in hrIL-12 (black) or PBS-treated mice (gray); bars, SD. The difference was statistically significant (P < 0.001). B, antihuman CD10 staining showed lower 697 cell implantation in the ovary of hrIL-12-treated animals (b) compared with PBS-treated mice (a) 7 days after cell inoculation. Twenty-one days after 697 cells inoculation, PBS-treated mice developed bulky masses (c) showing frequent mitotic figures (c, arrows) and high proliferative activity, as revealed by anti-PCNA immunostaining (e). In hrIL-12-treated mice, tumor masses showed more apoptotic features (d, inset) and reduced proliferative activity (f) compared with PBS-treated animals. The morphologic features of microvessel network were comparable in tumors from PBS-treated (g) and hrIL-12-treated (h) mice, as revealed by anti-laminin immunostaining. Magnification, x400 (a and b), x630 (c-h), x1,000 (inset).

 
This study shows for the first time that silencing of the IL-12Rß2 gene is a consistent feature in pro-B, early pre-B, and pre-B pediatric ALL and provides growth advantage to tumor cells. These results add to the previous demonstration that the IL-12Rß2 gene acts as tumor suppressor in a variety of chronic B-cell malignancies, whose normal counterparts express the functional heterodimeric IL-12R. Notably, the IL-12Rß2 transcript was not detected in pro-B or pre-B cells freshly isolated from normal bone marrow, but the CpG island in exon 1 was not methylated in the these cells that will express the IL-12Rß2 chain after maturation to B cells. This finding shows that expression of the IL-12R heterodimer is dispensable for successful early B-cell differentiation. Thus, the present results support a model whereby the IL-12R heterodimer is first expressed at late stages of human B-cell differentiation, as previously reported (7, 15), and retained until the plasmablast stage.5

Epigenetic silencing of the IL-12Rß2 gene in B-cell lymphoproliferative disorders, virtually recapitulating the whole spectrum of normal B cell differentiation, may play an important role in tumorigenesis. Whether or not IL-12Rß2 gene methylation is an early event in the pathogenesis of pediatric B-ALL, as already postulated for chronic B-cell malignancies (10), remains to be addressed in future studies.


   Acknowledgments
 
Grant support: Associazione Italiana per la Ricerca sul Cancro, Milano, Italy (V. Pistoia); Fondazione Cassa di Risparmio di Chieti, Italy (E. Di Carlo); Progetti di Ricerca di Interesse Nazionale (G. Basso); Associazione Italiana per la Ricerca sul Cancro (G. Basso); Consiglio Nazionale delle Ricerche Oncologia (G. Basso); Fondazione Città Della Speranza (G. Basso); Fondazione Gaslini, Genova, Italy (I. Airoldi); and FIRC fellowship, Milano, Italy (C. Cocco).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Chiara Bernardini for her excellent secretarial assistance.


   Footnotes
 
Note: I. Airoldi and C. Cocco contributed equally to this work.

5 I. Airoldi et al., in preparation. Back

Received 12/12/05. Revised 2/ 3/06. Accepted 2/17/06.


   References
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 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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  4. Wolf SF, Temple PA, Kobayashi M, et al. Cloning of cDNA for natural killer cell stimulatory factor, a heterodimeric cytokine with multiple biologic effects on T and natural killer cells. J Immunol 1991;146:3074–81.[Abstract]
  5. Manetti R, Gerosa F, Giudizi MG, et al. Interleukin 12 induces stable priming for interferon gamma (IFN-gamma) production during differentiation of human T helper (Th) cells and transient IFN-gamma production in established Th2 cell clones. J Exp Med 1994;179:1273–83.[Abstract/Free Full Text]
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