Limits...
The anti-leukemic activity of sodium dichloroacetate in p53mutated/ cells is mediated by a p53-independent ILF3/p21 pathway.

Agnoletto C, Brunelli L, Melloni E, Pastorelli R, Casciano F, Rimondi E, Rigolin GM, Cuneo A, Secchiero P, Zauli G - Oncotarget (2015)

Bottom Line: By using a proteomic approach, we demonstrated that DCA up-regulated the ILF3 transcription factor, which is a known regulator of p21 expression.The role of the ILF3/p21 axis in mediating the DCA anti-leukemic activity was underscored by knocking-down experiments.Indeed, transfection with ILF3 and p21 siRNAs significantly decreased both the DCA-induced p21 expression and the DCA-mediated cytotoxicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Morphology, Surgery and Experimental Medicine and LTTA Centre, University of Ferrara, Ferrara, Italy.

ABSTRACT
B-chronic lymphocytic leukemia (B-CLL) patients harboring p53 mutations are invariably refractory to therapies based on purine analogues and have limited treatment options and poor survival. Having recently demonstrated that the mitochondria-targeting small molecule sodium dichloroacetate (DCA) exhibits anti-leukemic activity in p53wild-type B-CLL cells, the aim of this study was to evaluate the effect of DCA in p53mutated B-CLL cells and in p53mutated/ leukemic cell lines. DCA exhibited comparable cytotoxicity in p53wild-type and p53mutated B-CLL patient cell cultures, as well as in p53mutated B leukemic cell lines (MAVER, MEC-1, MEC-2). At the molecular level, DCA promoted the transcriptional induction of p21 in all leukemic cell types investigated, including p53 HL-60. By using a proteomic approach, we demonstrated that DCA up-regulated the ILF3 transcription factor, which is a known regulator of p21 expression. The role of the ILF3/p21 axis in mediating the DCA anti-leukemic activity was underscored by knocking-down experiments. Indeed, transfection with ILF3 and p21 siRNAs significantly decreased both the DCA-induced p21 expression and the DCA-mediated cytotoxicity. Taken together, our results emphasize that DCA is a small molecule that merits further evaluation as a therapeutic agent also for p53mutated leukemic cells, by acting through the induction of a p53-independent pathway.

Show MeSH

Related in: MedlinePlus

Induction of p21 by DCA in p53mutated/ leukemic cellsLevels of p21 were analyzed by quantitative RT-PCR in the p53wilde-type and p53mutated B-CLL patient samples, as well as in the p53mutated B leukemic cell lines (MAVER, MEC-1 and MEC-2) and in the p53 HL-60 cells. In A, levels of p21 modulation upon 24 hours of treatment with DCA (30 mM) are shown. Representative western blot results documenting induction of p21 protein by DCA in 2 B-CLL patient samples are shown in the insets. In B, the same cell cultures were exposed for 24 hours to Nutlin-3 (10 μM) before measurement of p21 levels. In parallel, as a positive control, p21 induction by Nutlin-3 was assessed in a p53wild-type B-CLL sample. In A and B, mRNA levels are expressed as fold of modulation with respect to the control untreated cultures set at 1. Results are reported as means±SD of at least three independent experiments, performed in duplicate, carried out on each leukemic sample.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4385858&req=5

Figure 3: Induction of p21 by DCA in p53mutated/ leukemic cellsLevels of p21 were analyzed by quantitative RT-PCR in the p53wilde-type and p53mutated B-CLL patient samples, as well as in the p53mutated B leukemic cell lines (MAVER, MEC-1 and MEC-2) and in the p53 HL-60 cells. In A, levels of p21 modulation upon 24 hours of treatment with DCA (30 mM) are shown. Representative western blot results documenting induction of p21 protein by DCA in 2 B-CLL patient samples are shown in the insets. In B, the same cell cultures were exposed for 24 hours to Nutlin-3 (10 μM) before measurement of p21 levels. In parallel, as a positive control, p21 induction by Nutlin-3 was assessed in a p53wild-type B-CLL sample. In A and B, mRNA levels are expressed as fold of modulation with respect to the control untreated cultures set at 1. Results are reported as means±SD of at least three independent experiments, performed in duplicate, carried out on each leukemic sample.

Mentions: In the next set of experiments, we have investigated the mRNA levels of p21, whose in vitro induction represents a positive prognostic marker of response to therapy [20]. Surprisingly, DCA was equally effective in increasing the transcription levels of p21 in both primary p53wild-type and p53mutated B-CLL leukemic cells (Figure 3A). The induction of p21 was confirmed at the protein level by Western blot analysis. Moreover, DCA significantly increased the steady-state mRNA levels in all the p53mutated B cell lines (MAVER, MEC-1 and MEC-2) as well as in p53 HL-60 (Figure 3A), thus confirming the ability of DCA to induce p21 expression independently of the presence of p53. In parallel, a confirmation of the lack of functional p53 transcriptional activity in both p53mutated primary B-CLL cells and p53mutated MAVER, MEC-1 and MEC-2 cell lines as well as in p53 HL-60 was provided by the use of Nutlin-3, a small molecule activator of p53 [21]. Indeed, while Nutlin-3 (10 μM) potently induced p21 transcription in primary p53wild-type B-CLL cells, it did not promote any modulation of p21 mRNA over the baseline in both primary p53mutated B-CLL patient samples and p53mutated/ leukemic cell lines (Figure 3B), thus confirming the p53-independent activation of p21 by DCA in these cell models.


The anti-leukemic activity of sodium dichloroacetate in p53mutated/ cells is mediated by a p53-independent ILF3/p21 pathway.

Agnoletto C, Brunelli L, Melloni E, Pastorelli R, Casciano F, Rimondi E, Rigolin GM, Cuneo A, Secchiero P, Zauli G - Oncotarget (2015)

Induction of p21 by DCA in p53mutated/ leukemic cellsLevels of p21 were analyzed by quantitative RT-PCR in the p53wilde-type and p53mutated B-CLL patient samples, as well as in the p53mutated B leukemic cell lines (MAVER, MEC-1 and MEC-2) and in the p53 HL-60 cells. In A, levels of p21 modulation upon 24 hours of treatment with DCA (30 mM) are shown. Representative western blot results documenting induction of p21 protein by DCA in 2 B-CLL patient samples are shown in the insets. In B, the same cell cultures were exposed for 24 hours to Nutlin-3 (10 μM) before measurement of p21 levels. In parallel, as a positive control, p21 induction by Nutlin-3 was assessed in a p53wild-type B-CLL sample. In A and B, mRNA levels are expressed as fold of modulation with respect to the control untreated cultures set at 1. Results are reported as means±SD of at least three independent experiments, performed in duplicate, carried out on each leukemic sample.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4385858&req=5

Figure 3: Induction of p21 by DCA in p53mutated/ leukemic cellsLevels of p21 were analyzed by quantitative RT-PCR in the p53wilde-type and p53mutated B-CLL patient samples, as well as in the p53mutated B leukemic cell lines (MAVER, MEC-1 and MEC-2) and in the p53 HL-60 cells. In A, levels of p21 modulation upon 24 hours of treatment with DCA (30 mM) are shown. Representative western blot results documenting induction of p21 protein by DCA in 2 B-CLL patient samples are shown in the insets. In B, the same cell cultures were exposed for 24 hours to Nutlin-3 (10 μM) before measurement of p21 levels. In parallel, as a positive control, p21 induction by Nutlin-3 was assessed in a p53wild-type B-CLL sample. In A and B, mRNA levels are expressed as fold of modulation with respect to the control untreated cultures set at 1. Results are reported as means±SD of at least three independent experiments, performed in duplicate, carried out on each leukemic sample.
Mentions: In the next set of experiments, we have investigated the mRNA levels of p21, whose in vitro induction represents a positive prognostic marker of response to therapy [20]. Surprisingly, DCA was equally effective in increasing the transcription levels of p21 in both primary p53wild-type and p53mutated B-CLL leukemic cells (Figure 3A). The induction of p21 was confirmed at the protein level by Western blot analysis. Moreover, DCA significantly increased the steady-state mRNA levels in all the p53mutated B cell lines (MAVER, MEC-1 and MEC-2) as well as in p53 HL-60 (Figure 3A), thus confirming the ability of DCA to induce p21 expression independently of the presence of p53. In parallel, a confirmation of the lack of functional p53 transcriptional activity in both p53mutated primary B-CLL cells and p53mutated MAVER, MEC-1 and MEC-2 cell lines as well as in p53 HL-60 was provided by the use of Nutlin-3, a small molecule activator of p53 [21]. Indeed, while Nutlin-3 (10 μM) potently induced p21 transcription in primary p53wild-type B-CLL cells, it did not promote any modulation of p21 mRNA over the baseline in both primary p53mutated B-CLL patient samples and p53mutated/ leukemic cell lines (Figure 3B), thus confirming the p53-independent activation of p21 by DCA in these cell models.

Bottom Line: By using a proteomic approach, we demonstrated that DCA up-regulated the ILF3 transcription factor, which is a known regulator of p21 expression.The role of the ILF3/p21 axis in mediating the DCA anti-leukemic activity was underscored by knocking-down experiments.Indeed, transfection with ILF3 and p21 siRNAs significantly decreased both the DCA-induced p21 expression and the DCA-mediated cytotoxicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Morphology, Surgery and Experimental Medicine and LTTA Centre, University of Ferrara, Ferrara, Italy.

ABSTRACT
B-chronic lymphocytic leukemia (B-CLL) patients harboring p53 mutations are invariably refractory to therapies based on purine analogues and have limited treatment options and poor survival. Having recently demonstrated that the mitochondria-targeting small molecule sodium dichloroacetate (DCA) exhibits anti-leukemic activity in p53wild-type B-CLL cells, the aim of this study was to evaluate the effect of DCA in p53mutated B-CLL cells and in p53mutated/ leukemic cell lines. DCA exhibited comparable cytotoxicity in p53wild-type and p53mutated B-CLL patient cell cultures, as well as in p53mutated B leukemic cell lines (MAVER, MEC-1, MEC-2). At the molecular level, DCA promoted the transcriptional induction of p21 in all leukemic cell types investigated, including p53 HL-60. By using a proteomic approach, we demonstrated that DCA up-regulated the ILF3 transcription factor, which is a known regulator of p21 expression. The role of the ILF3/p21 axis in mediating the DCA anti-leukemic activity was underscored by knocking-down experiments. Indeed, transfection with ILF3 and p21 siRNAs significantly decreased both the DCA-induced p21 expression and the DCA-mediated cytotoxicity. Taken together, our results emphasize that DCA is a small molecule that merits further evaluation as a therapeutic agent also for p53mutated leukemic cells, by acting through the induction of a p53-independent pathway.

Show MeSH
Related in: MedlinePlus