Bioimaging analysis of nuclear factor-κB activity in Philadelphia chromosome-positive acute lymphoblastic leukemia cells reveals its synergistic upregulation by tumor necrosis factor-α-stimulated changes to the microenvironment

Abstract

To gain an insight into the microenvironmental regulation of nuclear factor (NF)-κB activity in the progression of leukemia, we established a bioluminescent imaging model of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) cells transduced with a NF-κB/luciferase (Luc) reporter and cocultured with murine stromal cells and cytokines. Stromal cells alone did not augment Luc activity, taken as an index of NF-κB, but Luc activity was synergistically upregulated by the combination of stromal cells and tumor necrosis factor (TNF)-α. Dehydroxymethylepoxyquinomicin (DHMEQ), a specific inhibitor of NF-κB DNA binding, rapidly induced the apoptosis of Ph+ALL cells, indicating that NF-κB is necessary for the growth and survival of these cells. However, the DHMEQ-induced suppression of NF-κB activity and the apoptosis of leukemia cells were attenuated by the presence of stromal cells and TNF-α. In NOD-SCID mice transplanted with NF-κB/Luc reporter-containing Ph+ALL cell lines and monitored periodically during the progression of the leukemia, murine TNF-α was significantly expressed in lesions in which the leukemia cells emitted a significant NF-κB signal. These results support the notion that TNF-α also triggers microenvironmental upregulation of NF-κB activity in vivo. Collectively, the results indicated that TNF-α-stimulated microenvironment may contribute to the survival and progression of Ph+ALL cells through the synergistic upregulation of NF-κB activity.

Figure 1

Nuclear factor (NF)‐κB is constitutive and inducible in Philadelphia chromosome‐positive acute lymphoblastic leukemia (Ph+ALL) cells. (A) Relative NF‐κB/luciferase (Luc) activity compared with basal levels. The IMS‐PhL1 cells were stimulated with the different cytokines (TRAIL 200 ng/mL; IL‐3 10 ng/mL; GM‐CSF 10 ng/mL; SCF 50 ng/mL; Flt3L 50 ng/mL; Fibronectin 100 μg/mL; IFN‐γ 1000 IU/mL) as indicated for 24 h and luc activity was measured using a multilabel counter. Data show the mean ± SD. TRAIL, TNF‐related apoptosis inducing ligand; IL‐3, interleukin‐3; GM‐CSF, granulocyte–macrophage colony‐stimulating factor; SCF, stem cell factor; Flt3L, Flt3‐ligand; IFN‐γ, interferon‐γ; TNF‐α, tumor necrosis factor‐α. (B) Bioluminescent images on the IVIS imaging system (Xenogen) showing background, basal and TNF‐α‐triggered NF‐κB/Luc activity. (C) Nuclear extracts prepared from Ph+ALL cell lines, treated with or without TNF‐α, were subjected to electrophoretic mobility shift assay (EMSA) or supershift assay using the antibodies indicated. Oct‐1 served as a loading control for the EMSA. Free indicates nuclear acid not bound with NF‐κB. (D) Results of the WST1 assay for the Ph+ALL cell lines (◆, IMS‐PhL1;, Sup‐B15; ▮, OM9;22) treated with TNF‐α for 48 h. Data show the mean ± SEM.

Figure 2

Nuclear factor (NF)‐κB is critical for the survival of Philadelphia chromosome‐positive acute lymphoblastic leukemia (Ph+ALL) cells. (A) Nuclear extracts prepared from IMS‐PhL1 cells treated with or without 10 μg/mL of dehydroxymethylepoxyquinomicin (DHMEQ) and/or 100 ng/mL of tumor necrosis factor (TNF)‐α for 6 and 12 h, as indicated, were subjected to an electrophoretic mobility shift assay (EMSA). Open arrowhead, DHMEQ‐sensitive complexes; closed arrowheads, p50, p65. (B) Relative NF‐κB/luciferase (Luc) activity of 100 ng/mL of TNF‐α‐stimulated IMS‐PhL1 cells treated with (+) or without (−) 10 μg/mL of DHMEQ for 48 h. (C) Results of the WST1 assay of Ph+ALL cell lines treated with 10 μg/mL of DHMEQ for 48 h. Data show the mean ± SEM. (◆), IMS‐PhL1; (), Sup‐B15; (▮), OM9;22; ), KOPN72; (○), KOPN30. (D) IMS‐PhL1 cells treated with 10 μg/mL DHMEQ for 48 h were analyzed by flow cytometry for annexin V/7‐AAD profiles. PE, phycoerythrin.

Figure 3

Tumor necrosis factor (TNF)‐α and stroma cells synergistically upregulate nuclear factor (NF)‐κB activity of Philadelphia chromosome‐positive acute lymphoblastic leukemia (Ph+ALL) cells. (A) Relative NF‐κB/luciferase (Luc) activity in IMS‐PhL1 cells that had been cultured for 24 h in the presence or absence of HESS5 cells, with or without 100 ng/mL of TNF‐α, and determined using a multilabel counter. Data show the mean ± SEM. (B) Bioluminescent images on the IVIS imaging system (Xenogen) showing NF‐κB/Luc activity of IMS‐PhL1 cells cultured under four different conditions, in the presence or absence of HESS5 cells, with or without TNF‐α, as indicated. (C) Relative NF‐κB/Luc activity of IMS‐PhL1 cells cultured with or without direct contact with HESS5 cells. The Luc activity of IMS‐PhL1 cells cultured without HESS5 cells was used as the basal level. The IMS‐PhL1 cells were treated overnight with TNF‐α. (D) Relative EF1α/Luc activity of IMS‐PhL1 cells cultured in the presence or absence of HESS5 cells and treated with graded concentrations of imatinib for 3 days. (□), 0 μM imatinib; (), 0.1 μM imatinib; (▪), 1 μM imatinib; (), 10 μM imatinib. These IMS‐PhL1 cells were treated with or without TNF‐α, as indicated, and the Luc activity of IMS‐PhL1 cells cultured in the absence of HESS5 and TNF‐α was used as the basal level. The EF1α/Luc activity in IMS‐PhL1 cells treated with 0.1 μM of imatinib and 100 ng/mL of TNF‐α, and in the absence and presence of HESS5, differed significantly (P < 0.05). The assays in (C,D) were conducted in triplicate and data were analyzed using Student’s t‐test. Data show the mean ± SEM.

Figure 4

Stromal cells attenuate dehydroxymethylepoxyquinomicin (DHMEQ) inhibition of nuclear factor (NF)‐κB activity in Philadelphia chromosome‐positive acute lymphoblastic leukemia (Ph+ALL) cells. (A) Bioluminescent images obtained using the IVIS imaging system (Xenogen) show the NF‐κB activity of IMS‐PhL1 cells treated with 10 μg/mL of DHMEQ and 100 ng/mL of tumor necrosis factor (TNF)‐α for periods indicated in the absence (upper) and presence (lower) of HESS5. (B) Nuclear extracts prepared from IMS‐PhL1 cells treated as indicated and subjected to the electrophoretic mobility shift assay. Oct‐1 served as a loading control. Free indicates nuclear acid not bound with NF‐κB.

Figure 5

In vivo imaging of nuclear factor (NF)‐κB activity during Philadelphia chromosome‐positive acute lymphoblastic leukemia (Ph+ALL) progression and its correlation with tumor necrosis factor (TNF)‐α expression. (A) NOD‐SCID mice were transplanted with 2 × 10⁶ IMS‐PhL1 reporter cells via the tail vein and in vivo bioluminescent activity was monitored with the IVIS imaging system (Xenogen) for the periods indicated. EF1α/Luc activity represents engraftment and propagation of IMS‐PhL1 cells (upper panels). (B) IMS‐PhL1‐NF‐kB/Luc leukemic mice were injected intraperitoneally with 500 ng TNF‐α on Day 23 after transplantation and NF‐κB/Luc activity was captured using the IVIS system 1 h before and then 6, 24 and 48 h after TNF‐α injection. (C) NOD‐SCID mice were transplanted with 2 × 10⁶ Sup‐B15‐NF‐kB/Luc cells and serial bioluminescent images were obtained using the IVIS system. (D) Pathological examination (H&E staining) of the liver and spleen from IMS‐PhL1‐NF‐kB/Luc leukemic mice (upper panels). Immunostaining with anti‐hCD45 MAb was also performed. Bone marrow infiltration was evaluated by flow cytometry with anti‐hCD45 MAb (lower panel). SSC, side scatter. Endogenous TNF‐α expression in leukemia‐infiltrated lesions was analyzed by quantitative reverse transcript (QR)‐PCR, and normalized against murine GAPDH mRNA copy numbers (SP: Sup‐B15, 1.4 ± 0.01 and IMS‐PhL1, 0.1 ± 0.001; BM: Sup‐B15, 2.3 ± 0.09 and IMS‐PhL1, 1.4 ± 0.07; Liver: Sup‐B15, 8.2 ± 0.89 and IMS‐PhL1, 7.8 ± 0.02; right lower panel). Mice not transplanted with leukemic cells was used as negative control. SP, spleen; BM, bone marrow; mTNF‐α, murine TNF‐α. (E) Immunostaining of murine TNF‐α in hepatic lesions from IMS‐PhL1 and Sup‐B15 leukemic mice, as well as control mice.