THE ROLE OF ADENOSINE DEAMINASE AND DNA METHYLATION IN ACUTE MYELOID LEUKEMIA
(Abstract release date: 05/19/16)
EHA Library. Tekin-Bubenheim N. 06/09/16; 132458; E909
Disclosure(s): R.A. Padua receives research support from Abbvie and Genetech.
Dr. Nilgun Tekin-Bubenheim
Contributions
Contributions
Abstract
Abstract: E909
Type: Eposter Presentation
Background
Genes coding for epigenetic regulators are frequently mutated in acute myelogenous leukemia (AML). As epigenetic marks are reversible, drugs that target these events may have an important impact on AML survival rates. The objective of this study is to investigate the importance of a metabolic enzyme, Adenosine Deaminase(ADA), whose gene is upregulated by abnormal DNA methylation during AML induction. Analysis of the mouse microarray gene expression data showed that the ADA gene is highly upregulated in NRASD12/BCL2 MDS mouse models, which was validated by Q-PCR (Tekin and Padua, unpublished data). If the expression of different subtypes of AML samples is assessed, ADA levels are highest in the subgroup carrying inv(16) or t(8;21) chromosomal translocations. Notably, these subgroups of patients also have a conserved DNA methylation pattern (Figueroa et al. 2010).
Aims
Based on the observations, the following hypothesis has been formulated and was tested using human AML cells: Is ADA a key enzyme regulating the hypermethylation status in AML and contributing to disease progression?
Methods
To address the hypotheses, two approaches were taken: 1) To silence the ADA gene using shRNAs against ADA and to investigate its impact on DNA methylation 2) To overexpress ADA using a lentiviral vector expressing the ADA cDNA, and similarly investigate altered DNA methylation patterns. In initial experiments, three human cell lines were chosen: ME-1 AML cells carrying the inv(16), Kasumi-1 AML cells with t(8;21) and K562 t(9;22) cells, which was used as a control group. The cell lines were manipulated to overexpress or repress ADA by transduction with lentiviral vectors. As a control and comparison, azacitidine (AZA), a powerful demethylation agent, was also used. After transduction with lentiviral vectors and treating with AZA, RNA and DNA were extracted and proteins were purified. To investigate if inhibiting ADA mimics the effect of AZA, DNA methylation levels of Kasumi cells were measured by gene expression of the specific genes (CDKN1A, ITGA2B, LY86). Bisulphite conversions of the DNA samples were carried on for the evaluation of the methylation status for the future studies.
Results
ADA protein levels were determined via western blot to ensure the success of the ADA gene manipulation by lentiviral transduction. Expression of ADA and two related genes encoding downstream enzymes from the methylation pathway DNA methyltransferase 1 (DNMT1) and S-adenosylhomocysteine hydrolase (SAHH) were investigated by Q-PCR. ADA gene expression levels do not impact on SAHH and DNMT1 gene transcription, but we concluded that it does impact at the protein level. Interestingly, an inverse correlation between ADA and DNMT1 protein levels were determined between the cell lines. While ME-1 cell line has the highest ADA level and lowest DNMT1 level, K562 showed the highest DNMT1 levels with lowest ADA protein levels. In Kasumi cells a correlation between the expressions of methylated genes and ADA silencing was observed, which was similar to that observed upon AZA treatment. While AZA treatment resulted in >20 fold increase in the expression of methylated genes, silencing ADA increased the expression of methylated genes >2,5-10 fold.
Conclusion
The analysis of this study showed that silencing ADA can partly mimic the effect of AZA. To confirm the impact of ADA levels on DNA methylation, global gene analysis using bisulfite converted DNA samples, followed by promotor array or sequencing will be performed.
Session topic: E-poster
Keyword(s): DNA methylation, Lentiviral transduction, Overexpression, Silencing
Type: Eposter Presentation
Background
Genes coding for epigenetic regulators are frequently mutated in acute myelogenous leukemia (AML). As epigenetic marks are reversible, drugs that target these events may have an important impact on AML survival rates. The objective of this study is to investigate the importance of a metabolic enzyme, Adenosine Deaminase(ADA), whose gene is upregulated by abnormal DNA methylation during AML induction. Analysis of the mouse microarray gene expression data showed that the ADA gene is highly upregulated in NRASD12/BCL2 MDS mouse models, which was validated by Q-PCR (Tekin and Padua, unpublished data). If the expression of different subtypes of AML samples is assessed, ADA levels are highest in the subgroup carrying inv(16) or t(8;21) chromosomal translocations. Notably, these subgroups of patients also have a conserved DNA methylation pattern (Figueroa et al. 2010).
Aims
Based on the observations, the following hypothesis has been formulated and was tested using human AML cells: Is ADA a key enzyme regulating the hypermethylation status in AML and contributing to disease progression?
Methods
To address the hypotheses, two approaches were taken: 1) To silence the ADA gene using shRNAs against ADA and to investigate its impact on DNA methylation 2) To overexpress ADA using a lentiviral vector expressing the ADA cDNA, and similarly investigate altered DNA methylation patterns. In initial experiments, three human cell lines were chosen: ME-1 AML cells carrying the inv(16), Kasumi-1 AML cells with t(8;21) and K562 t(9;22) cells, which was used as a control group. The cell lines were manipulated to overexpress or repress ADA by transduction with lentiviral vectors. As a control and comparison, azacitidine (AZA), a powerful demethylation agent, was also used. After transduction with lentiviral vectors and treating with AZA, RNA and DNA were extracted and proteins were purified. To investigate if inhibiting ADA mimics the effect of AZA, DNA methylation levels of Kasumi cells were measured by gene expression of the specific genes (CDKN1A, ITGA2B, LY86). Bisulphite conversions of the DNA samples were carried on for the evaluation of the methylation status for the future studies.
Results
ADA protein levels were determined via western blot to ensure the success of the ADA gene manipulation by lentiviral transduction. Expression of ADA and two related genes encoding downstream enzymes from the methylation pathway DNA methyltransferase 1 (DNMT1) and S-adenosylhomocysteine hydrolase (SAHH) were investigated by Q-PCR. ADA gene expression levels do not impact on SAHH and DNMT1 gene transcription, but we concluded that it does impact at the protein level. Interestingly, an inverse correlation between ADA and DNMT1 protein levels were determined between the cell lines. While ME-1 cell line has the highest ADA level and lowest DNMT1 level, K562 showed the highest DNMT1 levels with lowest ADA protein levels. In Kasumi cells a correlation between the expressions of methylated genes and ADA silencing was observed, which was similar to that observed upon AZA treatment. While AZA treatment resulted in >20 fold increase in the expression of methylated genes, silencing ADA increased the expression of methylated genes >2,5-10 fold.
Conclusion
The analysis of this study showed that silencing ADA can partly mimic the effect of AZA. To confirm the impact of ADA levels on DNA methylation, global gene analysis using bisulfite converted DNA samples, followed by promotor array or sequencing will be performed.
Session topic: E-poster
Keyword(s): DNA methylation, Lentiviral transduction, Overexpression, Silencing
Abstract: E909
Type: Eposter Presentation
Background
Genes coding for epigenetic regulators are frequently mutated in acute myelogenous leukemia (AML). As epigenetic marks are reversible, drugs that target these events may have an important impact on AML survival rates. The objective of this study is to investigate the importance of a metabolic enzyme, Adenosine Deaminase(ADA), whose gene is upregulated by abnormal DNA methylation during AML induction. Analysis of the mouse microarray gene expression data showed that the ADA gene is highly upregulated in NRASD12/BCL2 MDS mouse models, which was validated by Q-PCR (Tekin and Padua, unpublished data). If the expression of different subtypes of AML samples is assessed, ADA levels are highest in the subgroup carrying inv(16) or t(8;21) chromosomal translocations. Notably, these subgroups of patients also have a conserved DNA methylation pattern (Figueroa et al. 2010).
Aims
Based on the observations, the following hypothesis has been formulated and was tested using human AML cells: Is ADA a key enzyme regulating the hypermethylation status in AML and contributing to disease progression?
Methods
To address the hypotheses, two approaches were taken: 1) To silence the ADA gene using shRNAs against ADA and to investigate its impact on DNA methylation 2) To overexpress ADA using a lentiviral vector expressing the ADA cDNA, and similarly investigate altered DNA methylation patterns. In initial experiments, three human cell lines were chosen: ME-1 AML cells carrying the inv(16), Kasumi-1 AML cells with t(8;21) and K562 t(9;22) cells, which was used as a control group. The cell lines were manipulated to overexpress or repress ADA by transduction with lentiviral vectors. As a control and comparison, azacitidine (AZA), a powerful demethylation agent, was also used. After transduction with lentiviral vectors and treating with AZA, RNA and DNA were extracted and proteins were purified. To investigate if inhibiting ADA mimics the effect of AZA, DNA methylation levels of Kasumi cells were measured by gene expression of the specific genes (CDKN1A, ITGA2B, LY86). Bisulphite conversions of the DNA samples were carried on for the evaluation of the methylation status for the future studies.
Results
ADA protein levels were determined via western blot to ensure the success of the ADA gene manipulation by lentiviral transduction. Expression of ADA and two related genes encoding downstream enzymes from the methylation pathway DNA methyltransferase 1 (DNMT1) and S-adenosylhomocysteine hydrolase (SAHH) were investigated by Q-PCR. ADA gene expression levels do not impact on SAHH and DNMT1 gene transcription, but we concluded that it does impact at the protein level. Interestingly, an inverse correlation between ADA and DNMT1 protein levels were determined between the cell lines. While ME-1 cell line has the highest ADA level and lowest DNMT1 level, K562 showed the highest DNMT1 levels with lowest ADA protein levels. In Kasumi cells a correlation between the expressions of methylated genes and ADA silencing was observed, which was similar to that observed upon AZA treatment. While AZA treatment resulted in >20 fold increase in the expression of methylated genes, silencing ADA increased the expression of methylated genes >2,5-10 fold.
Conclusion
The analysis of this study showed that silencing ADA can partly mimic the effect of AZA. To confirm the impact of ADA levels on DNA methylation, global gene analysis using bisulfite converted DNA samples, followed by promotor array or sequencing will be performed.
Session topic: E-poster
Keyword(s): DNA methylation, Lentiviral transduction, Overexpression, Silencing
Type: Eposter Presentation
Background
Genes coding for epigenetic regulators are frequently mutated in acute myelogenous leukemia (AML). As epigenetic marks are reversible, drugs that target these events may have an important impact on AML survival rates. The objective of this study is to investigate the importance of a metabolic enzyme, Adenosine Deaminase(ADA), whose gene is upregulated by abnormal DNA methylation during AML induction. Analysis of the mouse microarray gene expression data showed that the ADA gene is highly upregulated in NRASD12/BCL2 MDS mouse models, which was validated by Q-PCR (Tekin and Padua, unpublished data). If the expression of different subtypes of AML samples is assessed, ADA levels are highest in the subgroup carrying inv(16) or t(8;21) chromosomal translocations. Notably, these subgroups of patients also have a conserved DNA methylation pattern (Figueroa et al. 2010).
Aims
Based on the observations, the following hypothesis has been formulated and was tested using human AML cells: Is ADA a key enzyme regulating the hypermethylation status in AML and contributing to disease progression?
Methods
To address the hypotheses, two approaches were taken: 1) To silence the ADA gene using shRNAs against ADA and to investigate its impact on DNA methylation 2) To overexpress ADA using a lentiviral vector expressing the ADA cDNA, and similarly investigate altered DNA methylation patterns. In initial experiments, three human cell lines were chosen: ME-1 AML cells carrying the inv(16), Kasumi-1 AML cells with t(8;21) and K562 t(9;22) cells, which was used as a control group. The cell lines were manipulated to overexpress or repress ADA by transduction with lentiviral vectors. As a control and comparison, azacitidine (AZA), a powerful demethylation agent, was also used. After transduction with lentiviral vectors and treating with AZA, RNA and DNA were extracted and proteins were purified. To investigate if inhibiting ADA mimics the effect of AZA, DNA methylation levels of Kasumi cells were measured by gene expression of the specific genes (CDKN1A, ITGA2B, LY86). Bisulphite conversions of the DNA samples were carried on for the evaluation of the methylation status for the future studies.
Results
ADA protein levels were determined via western blot to ensure the success of the ADA gene manipulation by lentiviral transduction. Expression of ADA and two related genes encoding downstream enzymes from the methylation pathway DNA methyltransferase 1 (DNMT1) and S-adenosylhomocysteine hydrolase (SAHH) were investigated by Q-PCR. ADA gene expression levels do not impact on SAHH and DNMT1 gene transcription, but we concluded that it does impact at the protein level. Interestingly, an inverse correlation between ADA and DNMT1 protein levels were determined between the cell lines. While ME-1 cell line has the highest ADA level and lowest DNMT1 level, K562 showed the highest DNMT1 levels with lowest ADA protein levels. In Kasumi cells a correlation between the expressions of methylated genes and ADA silencing was observed, which was similar to that observed upon AZA treatment. While AZA treatment resulted in >20 fold increase in the expression of methylated genes, silencing ADA increased the expression of methylated genes >2,5-10 fold.
Conclusion
The analysis of this study showed that silencing ADA can partly mimic the effect of AZA. To confirm the impact of ADA levels on DNA methylation, global gene analysis using bisulfite converted DNA samples, followed by promotor array or sequencing will be performed.
Session topic: E-poster
Keyword(s): DNA methylation, Lentiviral transduction, Overexpression, Silencing
{{ help_message }}
{{filter}}