Article Figures & Data
Figures
- Figure 1.
NF-κB is constitutively activated in CLL cells. EMSA studies of PB low-density cell nuclear extracts from 15 different patients with CLL are presented. Lane 1 of each sample depicts binding of CLL cell nuclear extract to a biotin-labeled (hot) NF-κB DNA probe, whereas lane 2 depicts a reduction in binding when an excess of unlabeled probe was added to the biotin-labeled (hot) κB DNA probe (hot + cold). In all 15 samples, NF-κB-DNA (p50/p65) complexes were detected and NF-κB-DNA binding was almost completely abolished by the cold probe.
- Figure 2.
U-STAT-3 binds NF-κB p50/p65 dimers. A, PB low-density cell-protein extracts from 4 different patients with CLL were immunoprecipitated with anti-STAT-3 antibodies. As shown, STAT-3 and the NF-κB p65 and p50 proteins were detected in the immunoprecipitates of all 3 PB samples. B, PB low-density cell-protein extracts from the same patients with CLL were immunoprecipitated with anti-p65 antibodies. Hela cell protein extract was used as a positive control. As shown, p65, p50, STAT-3, but not serine pSTAT-3 proteins were detected in all immunopreciptates. IP, immunoprecipitate; B, beads (control).
- Figure 3.
U-STAT-3-NF-κB complexes are detected in CLL cells but not in normal CD19+ cells. A, confocal microscopy studies of normal PB CD19+ cells detected a scattered signal of the NF-κB p65 protein in the cytoplasm and nucleus (left; green), faint signals of STAT-3 (middle; red) but no signal of STAT-3-p65 complexes (right; yellow). B, confocal microscopy studies of PB CLL cells detected a dense signal of p65 (green), STAT-3 (red), and STAT-3-p65 complexes (yellow) in the cytoplasm and mainly in the nucleus. One field of CLL cells (top) and a single CLL cell (bottom) are shown. Depicted are representative photomicrographs (×400) obtained from 2 different samples of normal CD19+ lymphocytes and CLL cells of patients 19 and 20.
- Figure 4.
The U-STAT-3/NF-κB complex binds to DNA. A, CLL cell nuclear proteins p65 and STAT-3 bind to a biotinylated κB-DNA probe. EMSA of CLL cell nuclear extract is depicted. As shown, the addition of anti-p65 or anti-STAT-3 antibodies to the κB DNA biotinylated probe induced a supershift, suggesting that p65 and STAT-3 bind to DNA. B, STAT-3-shRNA attenuates the binding of CLL cell nuclear extract to a biotin-labeled DNA probe. CLL cells were infected either with lentiviral STAT-3-shRNA or empty virus. Nuclear protein was extracted and EMSA was performed. As shown, NF-κB-DNA complexes were detected, and excess unlabeled (cold) DNA probe abolished the binding. Furthermore, infections with STAT-3-shRNA, but not with empty virus, significantly attenuated NF-κB-DNA binding.
- Figure 5.
U-STAT-3/NF-κB complexes activate NF-κB-target genes. A, STAT-3-shRNA downregulated mRNA levels of specific NF-κB-regulated genes. As shown, infection with lentiviral STAT-3-shRNA downregulated mRNA levels of VEGF C, CCL5, and CXCR5. As expected, lentiviral STAT-3-shRNA downregulated the level of STAT-3 mRNA as well. In this experiment we used PB cells from patient 21. B, STAT-3 coimmunoprecipitates with DNA of NF-κB-regulated genes. CLL cell-derived chromatin was immunoprecipitated by anti-STAT-3 antibodies or rabbit serum (IgG control). The coimmunoprecipitated DNA was amplified by PCR using promoter constructs of STAT-3, VEGF C, CCL5, CXCR5, or the control gene RPL30. As shown, DNA of STAT-3, VEGF C, CCL5, and CXCR5 but not the control RPL30 gene, coimmunoprecipitated with STAT-3 protein. STAT-3, VEGF C, CCL5, or RPL30 DNA was detected in whole cell chromatin-extracted DNA (Input).
Tables
- Table 1.
Patient characteristics
CLL Pt. no. Sex/age (y) WBC (109/L) Lymph. % Hb. (g/dL) Plts. (109/L) Rai stage CD38+/ CD19+% β2M (mg/dL) VH mutation ZAP- 70% Status Cytogenetics Previous treatment 1 M/58 73.7 90 13.1 128 1 NA 4.1 Y 80.9 Alive t 12 2 F/74 102.2 88 12.3 236 1 NA 1.9 N NA Alive NA 3 F/88 49.1 85 12.2 186 0 NA 3 ND NA Alive D13 4 M/61 52.9 84 14.8 129 1 0.9 2.7 N Pos. Alive del 11q FCR 5 M/63 143 82 13 164 1 97.5 3 Y 33.3 Alive del 11q FCR 6 F/70 118 94 12.5 178 1 62.5 3.2 Y 0 Alive t 12 Revlimid 7 M/60 133.5 96 13.1 113 0 NA 2.8 N 0 Alive del 13q 8 F/56 95.5 97 13 334 0 2.1 2.4 Y 0 Alive Dip. 9 F/74 66.7 94 12.5 231 1 NA 1.8 ND NA Alive NA 10 F/75 160.7 90 12.1 146 1 1 4 Y 44.6 Alive del 13q FCR 11 F/72 136.6 91 11.8 244 2 2 3.6 Y Pos. Alive t 12 Revlimid 12 M/63 96 92 14.7 155 0 0.3 3.5 Y 0 Alive del 13q 13 M/60 90.6 91 14.7 167 2 NA 4.8 Y 10.8 Alive del 13q FCR 14 F/70 26.4 93 10.1 101 3 0.2 3.2 Y 3.4 Alive del 13q Revlimid 15 F/58 172.6 98 13.3 212 1 NA 3.3 Y 0 Alive NA 16 F/72 46.9 79 9.8 272 3 2 4.7 Y Pos. Alive t 12 Revlimid 17 F/60 111.8 92 12.7 219 0 NA 3.5 Y 1.8 Alive NA Ritux. GM 18 F/75 60.9 89 12.5 179 0 NA 3.5 Y NA Alive NA 19 M/47 130.9 88 13 258 1 98.8 3.3 Y 46.2 Alive NA FCR 20 M/48 8.7 20 15.2 173 0 NA NA N Pos. Alive NA CFAR 21 F/58 184.4 88 13.6 216 1 NA 2.7 N Neg. Alive NA 22 F/75 258.9 94 11.8 140 1 1.1 4.2 Y 44.6 Alive del 13q Abbreviations: WBC, white blood cells; lymph., lymphocytes; Hb., hemoglobin; Plts., platelets; β2M, β2 microglobulin; M, male; F, female; NA, not available; VH mutation, hypermutation of the immunoglobulin heavy chain gene presented as N (negative; if % derivation from the germline sequence is ≤2%) or Y (positive, if % derivation from germline sequence is >2%); Ritux., Rituxan; GM, granulocyte-macrophage colony-stimulating factor; FCR, cyclophosphamide, fludarabine, and rituxan; CFAR, cyclophosphamide, fludarabine, alemtuzumab, and rituxan; Pos., positive (analyzed by immunohistochemistry only); Neg., negative; t, translocation; del, deletion.
Volume 9, Issue 4
