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The mechanism of action of L-asparaginase in childhood acute lymfoblastic leukemia
Heřmanová, Ivana ; Starková, Júlia (advisor) ; Šálek, Cyril (referee) ; Trbušek, Martin (referee)
Acute lymphoblastic leukemia (ALL) is the most frequent type of childhood cancer. The key component in the therapy, L-asparaginase (ASNase), hydrolyzes plasma asparagine and glutamine. Leukemic cells are sensitive to the depletion due to low activity of asparagine synthetase. Although the treatment is very effective, resistance and side effects remain a serious problem in some cases and its mechanism of action is not well understood. In this study, we wanted to elucidate the effect of ASNS expression level on the sensitivity of ALL cells to ASNase treatment. Our aim was also to clarify the intracellular consequences of the amino acid depletion to define the reason of different patients' response. We used four ALL cell lines (NALM-6, RS4;11, REH, and UOCB-6) and 30 diagnostic bone marrow samples of ALL patients to study the relationship between ASNS expression and sensitivity to ASNase using MTS proliferation assay. RNA interference was used to study the effect of a range of ASNS levels on the response to ASNase treatment. Using a cell line model with a gradually knocked-down ASNS gene, we defined a cutoff level below which ASNS gene expression does not correlate with sensitivity to ASNase. Importantly, ASNS gene expression in patients' ALL blasts is below this level. We confirmed that there was no...
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Targeted therapy of AML1-ETO positive acute myeloid leukemia with histone deacetylase inhibitors
Zápotocký, Michal ; Trka, Jan (advisor) ; Stopka, Tomáš (referee) ; Trbušek, Martin (referee)
In t(8;21) acute myeloid leukaemia (AML), the leukemogenesis is supposed to be promoted by interference with expression of AML1 target genes. Repressor complex associated with AML1-ETO fusion protein recruits class I histone deacetylases (HDAC). Valproic acid (VPA) was found to have an extensive effect on AML blasts, via inhibition of class I HDAC. We aimed to characterize the differentiation effect of VPA on AML1-ETO-positive leukemic cells and to determine the expression pattern of AML1 target genes. Kasumi-1 (M2 AML1-ETO-positive), Kasumi-6 (M2 AML1-ETO- negative), MV4-11 (MLL-AF4-positive) and K562 cells were treated with VPA and 12-0-tetra- decanoylphorbol-13-acetate (TPA) and examined by flow cytometry and qRT-PCR. Two AML1-ETO- positive and two negative patients' bone marrow diagnostic samples were treated with VPA and TPA to confirm in vitro findings. Valproic acid induced apoptosis in AML1-ETO-positive and MLL- AF4-positive cells in dose dependent manner. But changes of immunophenotype proving the differentiation were observed purely in AML1-ETO-positive cell line (decreased CD33/34/117 and increased CD11a/11b expression). However, differentiated cells exhibited positivity of AnnexinV; hence the relationship between cell death and differentiation had to be evaluated. Apoptosis was blocked by...
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The mechanism of action of L-asparaginase in childhood acute lymfoblastic leukemia
Heřmanová, Ivana ; Starková, Júlia (advisor) ; Šálek, Cyril (referee) ; Trbušek, Martin (referee)
Acute lymphoblastic leukemia (ALL) is the most frequent type of childhood cancer. The key component in the therapy, L-asparaginase (ASNase), hydrolyzes plasma asparagine and glutamine. Leukemic cells are sensitive to the depletion due to low activity of asparagine synthetase. Although the treatment is very effective, resistance and side effects remain a serious problem in some cases and its mechanism of action is not well understood. In this study, we wanted to elucidate the effect of ASNS expression level on the sensitivity of ALL cells to ASNase treatment. Our aim was also to clarify the intracellular consequences of the amino acid depletion to define the reason of different patients' response. We used four ALL cell lines (NALM-6, RS4;11, REH, and UOCB-6) and 30 diagnostic bone marrow samples of ALL patients to study the relationship between ASNS expression and sensitivity to ASNase using MTS proliferation assay. RNA interference was used to study the effect of a range of ASNS levels on the response to ASNase treatment. Using a cell line model with a gradually knocked-down ASNS gene, we defined a cutoff level below which ASNS gene expression does not correlate with sensitivity to ASNase. Importantly, ASNS gene expression in patients' ALL blasts is below this level. We confirmed that there was no...
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Targeted therapy of AML1-ETO positive acute myeloid leukemia with histone deacetylase inhibitors
Zápotocký, Michal ; Trka, Jan (advisor) ; Stopka, Tomáš (referee) ; Trbušek, Martin (referee)
In t(8;21) acute myeloid leukaemia (AML), the leukemogenesis is supposed to be promoted by interference with expression of AML1 target genes. Repressor complex associated with AML1-ETO fusion protein recruits class I histone deacetylases (HDAC). Valproic acid (VPA) was found to have an extensive effect on AML blasts, via inhibition of class I HDAC. We aimed to characterize the differentiation effect of VPA on AML1-ETO-positive leukemic cells and to determine the expression pattern of AML1 target genes. Kasumi-1 (M2 AML1-ETO-positive), Kasumi-6 (M2 AML1-ETO- negative), MV4-11 (MLL-AF4-positive) and K562 cells were treated with VPA and 12-0-tetra- decanoylphorbol-13-acetate (TPA) and examined by flow cytometry and qRT-PCR. Two AML1-ETO- positive and two negative patients' bone marrow diagnostic samples were treated with VPA and TPA to confirm in vitro findings. Valproic acid induced apoptosis in AML1-ETO-positive and MLL- AF4-positive cells in dose dependent manner. But changes of immunophenotype proving the differentiation were observed purely in AML1-ETO-positive cell line (decreased CD33/34/117 and increased CD11a/11b expression). However, differentiated cells exhibited positivity of AnnexinV; hence the relationship between cell death and differentiation had to be evaluated. Apoptosis was blocked by...
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