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Frequent and simultaneous epigenetic inactivation of TP53 pathway genes in acute lymphoblastic leukemia.

Vilas-Zornoza A, Agirre X, Martín-Palanco V, Martín-Subero JI, San José-Eneriz E, Garate L, Álvarez S, Miranda E, Rodríguez-Otero P, Rifón J, Torres A, Calasanz MJ, Cruz Cigudosa J, Román-Gómez J, Prósper F - PLoS ONE (2011)

Bottom Line: We found that 154 genes were methylated in more than 10% of ALL samples.The results obtained with the initial group of 48 patients was validated retrospectively in a second cohort of 200 newly diagnosed ALL patients.Methylation of at least 1 of the 13 genes implicated in the TP53 pathway was observed in 78% of the patients, which significantly correlated with a higher relapse (p = 0.001) and mortality (p<0.001) rate being an independent prognostic factor for disease-free survival (DFS) (p = 0.006) and overall survival (OS) (p = 0.005) in the multivariate analysis.

View Article: PubMed Central - PubMed

Affiliation: Hematology Service and Area of Cell Therapy, Clínica Universidad de Navarra, Foundation for Applied Medical Research, University of Navarra, Pamplona, Spain.

ABSTRACT
Aberrant DNA methylation is one of the most frequent alterations in patients with Acute Lymphoblastic Leukemia (ALL). Using methylation bead arrays we analyzed the methylation status of 807 genes implicated in cancer in a group of ALL samples at diagnosis (n = 48). We found that 154 genes were methylated in more than 10% of ALL samples. Interestingly, the expression of 13 genes implicated in the TP53 pathway was downregulated by hypermethylation. Direct or indirect activation of TP53 pathway with 5-aza-2'-deoxycitidine, Curcumin or Nutlin-3 induced an increase in apoptosis of ALL cells. The results obtained with the initial group of 48 patients was validated retrospectively in a second cohort of 200 newly diagnosed ALL patients. Methylation of at least 1 of the 13 genes implicated in the TP53 pathway was observed in 78% of the patients, which significantly correlated with a higher relapse (p = 0.001) and mortality (p<0.001) rate being an independent prognostic factor for disease-free survival (DFS) (p = 0.006) and overall survival (OS) (p = 0.005) in the multivariate analysis. All these findings indicate that TP53 pathway is altered by epigenetic mechanisms in the majority of ALL patients and correlates with prognosis. Treatments with compounds that may reverse the epigenetic abnormalities or activate directly the p53 pathway represent a new therapeutic alternative for patients with ALL.

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Activation of TP53 function induces cell apoptosis in ALL.TOM-1 and NALM-20 cells were treated with 5-aza-2-deoxycitidine, Curcumin and Nutlin-3. A. Apoptosis induced by 5-aza-2-deoxycitidine treatment: Annexin V analysis by flow citometry (grey: early apoptotic; black: late apoptotic/death cells), Caspase-3 analysis by flow citometry and Western blot analysis of the 85-kDa fragment of PARP. B–D. Curcumin treatment; Western blot analysis and quantification of Caspase 8. PRO-C8: full length caspase-8 fragment; p43/p41: cleaved intermediate p43/p41 fragment of caspase-8 and p18: caspase-8 active fragment p18. Numbers at the bottom of the figure represent the quantification of these three fragments of caspase-8 (B); Activation of apoptosis measure by Annexin V (grey: early apoptotic; black: late apoptotic/death cells) and Caspase-3 (FACS) and by the detection of the 85-kDa fragment of PARP (C). Upregulation in the amount of acetylated histone 3 by Western blot is shown. Total histone 3 and β-actin are loading controls (C); qRT-MSP and pyrosequencing of hypermethylated genes implicated in TP53 pathway before and after treatment with Curcumin (D). E. Western blot analysis of p21 and p53, levels of CDKN1A and TP53 mRNA by Q-RT-PCR and detection of apoptosis by Annexin V (grey: early apoptotic; black: late apoptotic/death cells), Caspase-3 and detection of the 85-kDa fragment of PARP by western blot after treatment with Nutlin-3. β-actin was used as a loading control in all cases. A,C and E, the mean ± SD of at least 3 different experiments is shown. A-E, a representative example of at least 3 different experiments is shown.
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pone-0017012-g005: Activation of TP53 function induces cell apoptosis in ALL.TOM-1 and NALM-20 cells were treated with 5-aza-2-deoxycitidine, Curcumin and Nutlin-3. A. Apoptosis induced by 5-aza-2-deoxycitidine treatment: Annexin V analysis by flow citometry (grey: early apoptotic; black: late apoptotic/death cells), Caspase-3 analysis by flow citometry and Western blot analysis of the 85-kDa fragment of PARP. B–D. Curcumin treatment; Western blot analysis and quantification of Caspase 8. PRO-C8: full length caspase-8 fragment; p43/p41: cleaved intermediate p43/p41 fragment of caspase-8 and p18: caspase-8 active fragment p18. Numbers at the bottom of the figure represent the quantification of these three fragments of caspase-8 (B); Activation of apoptosis measure by Annexin V (grey: early apoptotic; black: late apoptotic/death cells) and Caspase-3 (FACS) and by the detection of the 85-kDa fragment of PARP (C). Upregulation in the amount of acetylated histone 3 by Western blot is shown. Total histone 3 and β-actin are loading controls (C); qRT-MSP and pyrosequencing of hypermethylated genes implicated in TP53 pathway before and after treatment with Curcumin (D). E. Western blot analysis of p21 and p53, levels of CDKN1A and TP53 mRNA by Q-RT-PCR and detection of apoptosis by Annexin V (grey: early apoptotic; black: late apoptotic/death cells), Caspase-3 and detection of the 85-kDa fragment of PARP by western blot after treatment with Nutlin-3. β-actin was used as a loading control in all cases. A,C and E, the mean ± SD of at least 3 different experiments is shown. A-E, a representative example of at least 3 different experiments is shown.

Mentions: In order to prove that abnormal hypermethylation of genes implicated in the TP53 pathway could be responsible at least in part for the resistance to apoptosis in ALL, TOM-1 and NALM-20 cells were treated with either a demethylating agent (5-aza-2′-deoxycytidine), a caspase-8 activator (Curcumin) that induces apoptosis downstream and independently of p53 and Nutlin-3, an inhibitor of MDM2 and thus an activator of the p53 pathway. We have previously demonstrated that TOM-1 shows no TP53 mutation or deletion while NALM-20 shows a deletion of one TP53 allele [11]. Treatment of ALL cells with 5-aza-2′-deoxycytidine induced a non-specific demethylation and upregulation of expression of genes implicated in TP53 pathway (Figure S4), as well as an increase in apoptosis of TOM-1 and NALM-20 cells as measured by Annexin-V, caspase-3 or PARP expression (Figure 5A).


Frequent and simultaneous epigenetic inactivation of TP53 pathway genes in acute lymphoblastic leukemia.

Vilas-Zornoza A, Agirre X, Martín-Palanco V, Martín-Subero JI, San José-Eneriz E, Garate L, Álvarez S, Miranda E, Rodríguez-Otero P, Rifón J, Torres A, Calasanz MJ, Cruz Cigudosa J, Román-Gómez J, Prósper F - PLoS ONE (2011)

Activation of TP53 function induces cell apoptosis in ALL.TOM-1 and NALM-20 cells were treated with 5-aza-2-deoxycitidine, Curcumin and Nutlin-3. A. Apoptosis induced by 5-aza-2-deoxycitidine treatment: Annexin V analysis by flow citometry (grey: early apoptotic; black: late apoptotic/death cells), Caspase-3 analysis by flow citometry and Western blot analysis of the 85-kDa fragment of PARP. B–D. Curcumin treatment; Western blot analysis and quantification of Caspase 8. PRO-C8: full length caspase-8 fragment; p43/p41: cleaved intermediate p43/p41 fragment of caspase-8 and p18: caspase-8 active fragment p18. Numbers at the bottom of the figure represent the quantification of these three fragments of caspase-8 (B); Activation of apoptosis measure by Annexin V (grey: early apoptotic; black: late apoptotic/death cells) and Caspase-3 (FACS) and by the detection of the 85-kDa fragment of PARP (C). Upregulation in the amount of acetylated histone 3 by Western blot is shown. Total histone 3 and β-actin are loading controls (C); qRT-MSP and pyrosequencing of hypermethylated genes implicated in TP53 pathway before and after treatment with Curcumin (D). E. Western blot analysis of p21 and p53, levels of CDKN1A and TP53 mRNA by Q-RT-PCR and detection of apoptosis by Annexin V (grey: early apoptotic; black: late apoptotic/death cells), Caspase-3 and detection of the 85-kDa fragment of PARP by western blot after treatment with Nutlin-3. β-actin was used as a loading control in all cases. A,C and E, the mean ± SD of at least 3 different experiments is shown. A-E, a representative example of at least 3 different experiments is shown.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3046174&req=5

pone-0017012-g005: Activation of TP53 function induces cell apoptosis in ALL.TOM-1 and NALM-20 cells were treated with 5-aza-2-deoxycitidine, Curcumin and Nutlin-3. A. Apoptosis induced by 5-aza-2-deoxycitidine treatment: Annexin V analysis by flow citometry (grey: early apoptotic; black: late apoptotic/death cells), Caspase-3 analysis by flow citometry and Western blot analysis of the 85-kDa fragment of PARP. B–D. Curcumin treatment; Western blot analysis and quantification of Caspase 8. PRO-C8: full length caspase-8 fragment; p43/p41: cleaved intermediate p43/p41 fragment of caspase-8 and p18: caspase-8 active fragment p18. Numbers at the bottom of the figure represent the quantification of these three fragments of caspase-8 (B); Activation of apoptosis measure by Annexin V (grey: early apoptotic; black: late apoptotic/death cells) and Caspase-3 (FACS) and by the detection of the 85-kDa fragment of PARP (C). Upregulation in the amount of acetylated histone 3 by Western blot is shown. Total histone 3 and β-actin are loading controls (C); qRT-MSP and pyrosequencing of hypermethylated genes implicated in TP53 pathway before and after treatment with Curcumin (D). E. Western blot analysis of p21 and p53, levels of CDKN1A and TP53 mRNA by Q-RT-PCR and detection of apoptosis by Annexin V (grey: early apoptotic; black: late apoptotic/death cells), Caspase-3 and detection of the 85-kDa fragment of PARP by western blot after treatment with Nutlin-3. β-actin was used as a loading control in all cases. A,C and E, the mean ± SD of at least 3 different experiments is shown. A-E, a representative example of at least 3 different experiments is shown.
Mentions: In order to prove that abnormal hypermethylation of genes implicated in the TP53 pathway could be responsible at least in part for the resistance to apoptosis in ALL, TOM-1 and NALM-20 cells were treated with either a demethylating agent (5-aza-2′-deoxycytidine), a caspase-8 activator (Curcumin) that induces apoptosis downstream and independently of p53 and Nutlin-3, an inhibitor of MDM2 and thus an activator of the p53 pathway. We have previously demonstrated that TOM-1 shows no TP53 mutation or deletion while NALM-20 shows a deletion of one TP53 allele [11]. Treatment of ALL cells with 5-aza-2′-deoxycytidine induced a non-specific demethylation and upregulation of expression of genes implicated in TP53 pathway (Figure S4), as well as an increase in apoptosis of TOM-1 and NALM-20 cells as measured by Annexin-V, caspase-3 or PARP expression (Figure 5A).

Bottom Line: We found that 154 genes were methylated in more than 10% of ALL samples.The results obtained with the initial group of 48 patients was validated retrospectively in a second cohort of 200 newly diagnosed ALL patients.Methylation of at least 1 of the 13 genes implicated in the TP53 pathway was observed in 78% of the patients, which significantly correlated with a higher relapse (p = 0.001) and mortality (p<0.001) rate being an independent prognostic factor for disease-free survival (DFS) (p = 0.006) and overall survival (OS) (p = 0.005) in the multivariate analysis.

View Article: PubMed Central - PubMed

Affiliation: Hematology Service and Area of Cell Therapy, Clínica Universidad de Navarra, Foundation for Applied Medical Research, University of Navarra, Pamplona, Spain.

ABSTRACT
Aberrant DNA methylation is one of the most frequent alterations in patients with Acute Lymphoblastic Leukemia (ALL). Using methylation bead arrays we analyzed the methylation status of 807 genes implicated in cancer in a group of ALL samples at diagnosis (n = 48). We found that 154 genes were methylated in more than 10% of ALL samples. Interestingly, the expression of 13 genes implicated in the TP53 pathway was downregulated by hypermethylation. Direct or indirect activation of TP53 pathway with 5-aza-2'-deoxycitidine, Curcumin or Nutlin-3 induced an increase in apoptosis of ALL cells. The results obtained with the initial group of 48 patients was validated retrospectively in a second cohort of 200 newly diagnosed ALL patients. Methylation of at least 1 of the 13 genes implicated in the TP53 pathway was observed in 78% of the patients, which significantly correlated with a higher relapse (p = 0.001) and mortality (p<0.001) rate being an independent prognostic factor for disease-free survival (DFS) (p = 0.006) and overall survival (OS) (p = 0.005) in the multivariate analysis. All these findings indicate that TP53 pathway is altered by epigenetic mechanisms in the majority of ALL patients and correlates with prognosis. Treatments with compounds that may reverse the epigenetic abnormalities or activate directly the p53 pathway represent a new therapeutic alternative for patients with ALL.

Show MeSH
Related in: MedlinePlus