Article Figures & Data
Figures
- Fig. 1.
Modulation of DEK by Nutlin-3 in primary B-CLL samples. After exposure to Nutlin-3 (10 μmol/L), B-CLL samples were analyzed for DEK protein (A) and mRNA (B). A, DEK protein levels, analyzed by Western blot, are shown for representative B-CLL patient samples. Tubulin staining is shown as loading control. Protein bands were quantified by densitometry and level of DEK was calculated for each time point after normalization to tubulin in the same sample. Unstimulated basal expression was set as unity (hatched line). Columns, mean of determinations each done in triplicate; bars, SD. *, P < 0.05 with respect to nontreated cultures (time 0). B, levels of DEK and MDM2 mRNA were analyzed by quantitative RT-PCR. Results are expressed as fold of DEK and MDM2 modulation by Nutlin-3, after 48 h of treatment, with respect to the control nontreated cultures set to 1 (hatched line). Columns, mean of results from experiments each done in triplicate; bars, SD.
- Fig. 2.
Effect of Nutlin-3 on DEK expression in the p53wild-type SKW6.4 B lymphoblastoid cell line. SKW6.4 cell line was either left nontreated or exposed to Nutlin-3 (10 μmol/L). Levels of p53, MDM2, and DEK proteins were simultaneously assessed by Western blot analysis in cell lysates harvested at the indicated time points. Tubulin staining is shown as a loading control. Representative examples of Western blot results of four independent experiments are shown. Protein bands were quantified by densitometry and level of DEK was calculated for each time point after normalization to tubulin in the same sample. Results are expressed as DEK protein modulation by Nutlin-3 with respect to the control nontreated cultures set to 1 (hatched line). Columns, mean of three independent experiments; bars, SD. *, P < 0.05 with respect to nontreated cultures.
- Fig. 3.
Comparative analysis of the effects of Nutlin-3 and chlorambucil in p53wild-type and in p53mutated B lymphoblastoid cell lines. The p53wild-type SKW6.4 and the p53mutated BJA B-cell lines were either left nontreated (Ntr.) or exposed to Nutlin-3 (Nutl.) or chlorambucil (Chlor.), both used at 10 μmol/L. A, levels of p53, MDM2, and DEK proteins were simultaneously assessed by Western blot analysis in cell lysates harvested at the indicated time points. Tubulin staining is shown as a loading control. Representative examples of Western blot results of three independent experiments are shown. Protein bands were quantified by densitometry, and DEK levels were calculated for each time point after normalization to tubulin in the same sample. Results are expressed as DEK protein modulation by either Nutlin-3 or chlorambucil with respect to the control nontreated cultures. Columns, mean of three independent experiments; bars, SD. *, P < 0.05 with respect to nontreated cultures. B, levels of DEK and MDM2 mRNA were analyzed by quantitative RT-PCR. Results are expressed as fold of DEK and MDM2 modulation in Nutlin-3– or chlorambucil-treated cultures with respect to the control nontreated cultures set to 1 (hatched line). Columns, mean of results from experiments each done in triplicate; bars, SD. *, P < 0.05 with respect to nontreated cultures. C, data of cell culture viability upon the indicated treatments are presented; columns, mean of five independent experiments each done in duplicate. *, P < 0.05 with respect to nontreated cultures (set to 100).
- Fig. 4.
Effect of p53 silencing on the ability of Nutlin-3 to downregulate DEK in p53wild-type B lymphoblastoid cells. SKW6.4 cells were transfected with either control scrambled (scr.) siRNA or p53 siRNA before treatment with Nutlin-3 for 48 h. After transfection, levels of p53 (A) and DEK (B) mRNA and proteins were assessed by quantitative RT-PCR and Western blot analysis. RT-PCR results are expressed as fold of p53 and DEK modulation with respect to the scrambled control transfected cultures. Representative examples of Western blot results of three independent experiments are shown; tubulin staining is shown as loading control. Protein bands were quantified by densitometry and levels of p53 and DEK were calculated for each time point after normalization to tubulin in the same sample. Results are expressed as p53 or DEK protein modulation with respect to the control scrambled-transfected cultures set to 1 (hatched line). Columns, mean of three independent experiments; bars, SD. *, P < 0.05.
- Fig. 5.
Effects of DEK downmodulation on Nutlin-3–mediated cytotoxicity in p53wild-type lymphoblastoid cell line. SKW6.4 cells were transfected with either control scrambled (scr.) siRNA or DEK siRNA before treatment with Nutlin-3 for 48 h. After transfection, levels of DEK mRNA and protein were assessed by quantitative RT-PCR and Western blot followed by densitometric analyses (A). Results were expressed as fold of DEK modulation with respect to the control scrambled-transfected cultures set to 1 (hatched line). B and C, cultures transfected with either control scrambled siRNA or DEK siRNA were analyzed for apoptosis induction (B) and p53 accumulation (C) upon treatment with Nutlin-3. Columns, mean of results from three independent experiments; bars, SD. *, P < 0.05. C, representative examples of Western blot results of three independent experiments are shown; tubulin staining is shown as loading control.
- Fig. 6.
Lack of DEK down-modulation by Nutlin-3 in normal CD19+ B cells. After exposure to Nutlin-3, samples from normal CD19+ B cells were analyzed for DEK protein (A) and mRNA levels (B). A, Western blot analysis of DEK protein levels, as well as of p53 accumulation upon Nutlin-3 treatment, is shown for a representative CD19+ B-cell culture. Tubulin staining is shown as loading control. B, levels of DEK and MDM2 mRNA were analyzed by quantitative RT-PCR. After normalization to the level of glyceraldehyde-3-phosphate dehydrogenase mRNA, results were expressed as fold of DEK and MDM2 modulation in Nutlin-3–treated cultures with respect to the control nontreated cultures. C, normal CD19+ B-cell cultures were analyzed for apoptosis upon 48 h of treatment with Nutlin-3. Columns, mean of results from experiments each done in triplicate; bars, SD.
Tables
- Table 1.
Clinical and laboratory features of patients with CLL
Patient no. Sex Age, y Rai stage DT* FISH† ZAP-70‡ 1 F 80 1 28 +12 High 2 F 74 2 6 13q−, +12 Low 3 M 71 0 16 17p−, +12 High 4 M 67 4 >24 ND Low 5 F 55 0 10 11q− High 6 M 57 1 60 ND Low 7 M 62 0 >72 11q− Low 8 M 64 1 14 13q− High 9 M 51 0 37 Nor Low 10 M 59 2 28 13q− Low 11 M 61 2 3 17p− ND 12 M 62 4 18 ND Low 13 F 71 2 28 Nor High 14 M 69 2 19 13q−; 17p− Low 15 M 62 4 6 17p− High 16 F 79 3 16 12+ Low 17 F 60 1 28 Nor Low 18 M 73 1 16 Nor Low 19 M 86 3 19 13q− ND 20 F 64 0 ND Nor High 21 M 68 0 14 ND Low
Volume 16, Issue 6
