The IL-12Rbeta2 gene functions as a tumor suppressor in human B cell malignancies

Abstract

The IL-12Rbeta2 gene is expressed in human mature B cell subsets but not in transformed B cell lines. Silencing of this gene may be advantageous to neoplastic B cells. Our objective was to investigate the mechanism(s) and the functional consequence(s) of IL-12Rbeta2 gene silencing in primary B cell tumors and transformed B cell lines. Purified tumor cells from 41 patients with different chronic B cell lymphoproliferative disorders, representing the counterparts of the major mature human B cell subsets, tested negative for IL-12Rbeta2 gene expression. Hypermethylation of a CpG island in the noncoding exon 1 was associated with silencing of this gene in malignant B cells. Treatment with the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine restored IL-12Rbeta2 mRNA expression in primary neoplastic B cells that underwent apoptosis following exposure to human recombinant IL-12 (hrIL-12). hrIL-12 inhibited proliferation and increased the apoptotic rate of IL-12Rbeta2-transfected B cell lines in vitro. Finally, hrIL-12 strongly reduced the tumorigenicity of IL-12Rbeta2-transfected Burkitt lymphoma RAJI cells in SCID-NOD mice through antiproliferative and proapoptotic effects, coupled with neoangiogenesis inhibition related to human IFN-gamma-independent induction of hMig/CXCL9. The IL-12Rbeta2 gene acts as tumor suppressor in chronic B cell malignancies, and IL-12 exerts direct antitumor effects on IL-12Rbeta2-expressing neoplastic B cells.

Figure 1

Expression of IL-12R in human tonsil B cells. (A) Expression of IL-12Rβ1 and IL-12Rβ2 mRNA in tonsil B cells and their subsets. From left to right: molecular weight markers (MW); negative control (NC, represented by a Th2 cell clone for CD56, CD19, and IL-12Rβ2 primers; purified tonsil B cells for CD3γ primers; or water in the place of cDNA for IL12Rβ1 and GAPDH primers); positive control (PC, represented by a Th1 cell clone for CD3γ, IL12Rβ1, and IL-12Rβ2 primers; tonsil non_T cells for CD56 primers; or the RPMI 8866 B cell line for CD19 primers). The remaining lanes represent total tonsil B lymphocytes and naive, GC, and memory B cells, respectively, isolated from the same tonsil. On the right, the expected molecular weight of the amplified bands are shown. (B_N) Expression of IL-12Rβ2 protein in frozen tonsil tissue sections. (B) FM, GC, and SE, are boxed (magnification, ∞20). FM staining with CD19 mAb (C, green), anti_IL-12Rβ2 mAb (D, red), and both mAb's in combination with DAPI (E); in E, overlap of green and red colors gives rise to yellow staining, indicating that most cells coexpress CD19 and IL-12Rβ2; control FM staining with DAPI and with isotype- and fluorochrome-matched mAb's of irrelevant specificity (F). Staining of SEs (G_J) and GC areas (K_N) are shown in the same order as FM. Magnification (C_N), ∞100 for all panels.

Figure 2

IL-12R expression in human neoplastic B cells. Left panel (left to right): molecular weight markers; negative control, represented by a Th2 clone; positive control (tonsil B cells); two representative cases (patients 1 and 2 [Pt 1 and Pt 2]) each of MCL, MZL, and FL are shown. Right panel (left to right): molecular weight markers; negative control, represented by a Th2 clone; positive control (tonsil B cells). Fifteen B-CLL cases (Pt 1 to Pt 15) are shown.

Figure 3

Methylation analysis of nepolastic B cells versus their normal counterparts. (A) Top panel: Plot of the expected versus observed CpG dinucleotides frequency surrounding exon 1 of the IL-12Rβ2 gene. The CpG island and exon 1 are indicated. The broken line indicates the cut-off value of 0.60. Bottom panel: MSP analysis of cells expressing (Th1 clone, unfractionated tonsil B cells, and purified naive, GC, and memory B cells) and not expressing (RPMI 8866 LCL and Raji, BRGM, and Daudi BL cells) the IL12Rβ2 gene. Cells expressing the gene show only the amplification band corresponding to the unmethylated sequence (UM), whereas in nonexpressing cells, this band is present only in trace amounts, and the major product of amplification corresponds to the methylated (M) target sequence. (B) Induction of IL-12Rβ2 gene expression in Raji and RPMI 8866 cell lines treated with 5-Aza-2′-deoxycytidine. One experiment representative of the five performed is shown. From left to right: molecular weight markers; B cells cultured with medium alone (medium) or in the presence of 5ΒM 5-Aza-2′-deoxycytidine for 24 to 96 hours.

Figure 4

Induction of IL-12Rβ2 by treatment with 5-Aza-2′-deoxycytidine in primary tumors. (A) Left panel: Freshly isolated B-CLL cells (T0), B-CLL cells cultured with medium alone (medium) or in the presence of 5 ∝M 5-Aza-2′-deoxycytidine (Aza) for 48 to 96 hours. One sample representative of the four tested is shown. Right panel. Purified FL cells cultured with medium alone (medium) or in the presence of 5-Aza-2′-deoxycytidine for 72h. One sample representative of the two tested is shown. (B) Apoptosis of primary neoplastic B cells incubated with 5-Aza-2′-deoxycytidine for 72 hours and subsequently cultured with hrIL-12 for an additional 48 hours, as assessed by the TUNEL assay. Histogram in the left panel shows the percentages of apoptotic cells detected following culture with medium alone (white bar); with 5-Aza-2′-deoxycytidine for 72 hours and subsequently with medium for additional 48 hours (gray bar); or with 5-Aza-2′-deoxycytidine for 72 hours and subsequently with hrIL-12 for an additional 48 hours (black bar, Aza + IL-12). Two B-CLL and 2 FL samples are shown. In the right panel, one representative experiment performed with B-CLL1 sample is shown. TUNEL positive nuclei stained with FITC are green, whereas interphase nuclei stained with DAPI are blue (magnification, ∞20).

Figure 5

Characterization of IL-12Rβ2_transfected cells. (A) Expression of IL-12Rβ2 mRNA in IL-12Rβ2 and empty-vector_transfected Raji and RPMI 8866 cells. From left to right: molecular weight markers; negative control, represented by a Th2 clone; positive control (tonsil B cells); Raji cells transfected with the empty vector (vector) or with the IL-12Rβ2_containing vector (IL-12Rβ2); RPMI 8866 cells transfected with the empty vector or with the IL-12Rβ2_containing vector. (B) Induction of IFN-γ mRNA in Raji cells transfected with the empty vector or with the IL-12Rβ2_containing vector after treatment with IL-12 for 36 hours.

Figure 6

Assessment of proliferation and apoptosis of IL-12Rβ2_transfected Raji (A) and RPMI 8866 (B) cells following incubation with hrIL-12. (A and B, left panel) cells were cultured with hrIL-12 for 24 to 96 hours and counted by an electronic counter. One representative experiment out of the five performed is shown. (A and B, middle panel) [H3]-thymidine incorporation by cells incubated with hrIL-12 for 72 hour. The means ± SD from five independent experiments are shown. Results are expressed as cpm (A and B, right panel). Cells were cultured with hrIL-12 for 5 to 96 hours and tested for apoptosis by the TUNEL assay. The means ± SD from three independent experiments are shown. Results are expressed as percent apoptotic cells.

Figure 7

Tumorigenicity of IL-12Rβ2_transfected Raji cells in SCID-NOD mice. (A) The mean size ± SD of tumors formed by IL-12Rβ2_transfected Raji cells in SCID-NOD mice treated with hrIL-12 (black bar) or PBS (gray bar) is shown. The difference between mean size of tumors from hrIL-12 treated and PBS treated mice was statistically significant (P < 0.0001). (B) Two representative tumors grown in hrIL-12_treated and PBS-treated mice are shown. (C) Histological and immunohistochemical features of tumors from PBS treated (C, G, E, and I) and hrIL-12 treated (D, F, H, and J) SCID/NOD mice. IL-12Rβ2 gene_transfected Raji tumors from PBS-treated mice are formed by medium- to large-sized neoplastic cells with round to oval nuclei showing two or more nucleoli. The “starry sky” pattern determined by macrophages that have ingested apoptotic tumor cells is also present (arrows) (C). The tumor is well vascularized (E) and does not express detectable level of Mig/CXCL9 (G). An extremely high proliferation rate is evidenced by Ki-67 immunostaining (I). In tumors from hrIL-12 treated mice the above described architectural features are altered by frequent and extensive areas of ischemic-hemorrhagic necrosis (N) (D), which are associated with defective intratumoral microvessel network (F), and expression of Mig/CXCL9 in the cytoplasm and in the proximity of intact tumor cells (H). In addition, the number of proliferating cells in the viable neoplastic tissue surrounding necrotic areas, as assessed by Ki-67 staining, is strongly reduced (J). Magnification, ∞400, for all pictures shown.