Contributions
Abstract: EP421
Type: E-Poster Presentation
Session title: Acute myeloid leukemia - Biology & Translational Research
Background
Genome integrity is maintained by the DNA damage response (DDR). DDR defects are described to contribute to leukemogenesis, namely to Acute myeloid leukemia (AML) development and progression. Micronutrients are DDR cofactors, critical for optimal DDR functioning. Zinc (Zn) is particularly relevant in apoptosis, cell cycle and DNA repair. AML patients commonly present decreased serum Zn levels, but its biological significance and impact in the DDR is not understood.
Aims
We explored the role of Zn in DDR modulation in AML cells and its therapeutic potential in combination with conventional chemotherapy and with a DDR inhibitor.
Methods
The AML cell line HEL was incubated for 15 days in three culture conditions: standard Zn concentration (Std), Zn depletion (ZnD) and supplementation with 40µM of ZnSO4 (ZnS). After 2, 7 and 15 days, cells were exposed to genotoxic stimuli: 10µM of H2O2 for 30 minutes or 1 minute of UV radiation (Ee=2.9841 W.cm-2). Chromosomal damage, cell death and division rates were studied by cytokinesis-block micronucleus assay. To monitor repair kinetics, levels of yH2AX were evaluated 0h, 1h and 24h after exposure. Expression of 14 DDR genes (PARP1, XRCC1, MSH2, MSH6, MLH1, XPA, ERCC1, RAD23B, RAD51, PRKDC, XRCC6, PALB2, FANCD2, and MGMT) was analysed by qRT-PCR. An immortalized cell line of normal lymphocytes (IMC) was submitted to the same experiments. Lastly, HEL cells were incubated with the DDR inhibitor olaparib and the chemotherapeutic compound cytarabine in monotherapy and combination with 40µM of ZnSO4. Cell viability was analysed by trypan blue assay and cell death by annexin V/7-AAD staining by flow cytometry. Data was analysed considering a confidence level of 95% (p<0.05).
Results
ZnS lymphocytes displayed lower basal damage than those in Std conditions. Lymphocytes in ZnD shown increased damage biomarkers through time (1.3-fold, p=0.0234). Cell death was lower in ZnS lymphocytes and division rates slightly increased, whereas in ZnD an increase in cell death and lowered division rates were observed. Oppositely, in AML cells, basal damage through time of Zn supplementation increased (1.2-fold, p=0.0470), cell death slightly increased and division rates decreased (1.1-fold, p=0.0117). After genotoxic stimuli, ZnS lymphocytes shown less damage levels, particularly after 7 and 15 days of supplementation. ZnD lymphocytes presented more damage biomarkers. AML cells cultured in ZnS had considerably higher damage levels after exposure than in Std conditions and the lowest damage scores were found in ZnD. yH2AX monitorization shown a repair response in ZnS lymphocytes that was not achieved in Zn absence. In AML cells, yH2AX activation persisted 24h after exposure, but ZnD cells shown a more evident repair kinetics. No significant differences were observed in DDR genes expression among all conditions. Olaparib and cytarabine reduced cell proliferation and viability in a dose and time-dependent manner, inducing cell death by apoptosis. Further, ZnSO4 improved cytotoxic effects as a marked decrease of the IC50 of each compound comparatively to monotherapy was observed (2.8-fold and 5.4-fold, respectively).
Conclusion
ZnS improved the DDR in normal cells while ZnD favoured genomic instability, confirming the importance of Zn in genome integrity. In AML cells, Zn increased the genotoxicity of H2O2 and UV radiation and the therapeutic effects of olaparib and cytarabine. These results suggest that Zn supplementation is a potential co-adjuvant strategy improving genotoxic or DDR-targeting therapies in AML.
Keyword(s): Acute myeloid leukemia, DNA damage, DNA repair, Zinc
Abstract: EP421
Type: E-Poster Presentation
Session title: Acute myeloid leukemia - Biology & Translational Research
Background
Genome integrity is maintained by the DNA damage response (DDR). DDR defects are described to contribute to leukemogenesis, namely to Acute myeloid leukemia (AML) development and progression. Micronutrients are DDR cofactors, critical for optimal DDR functioning. Zinc (Zn) is particularly relevant in apoptosis, cell cycle and DNA repair. AML patients commonly present decreased serum Zn levels, but its biological significance and impact in the DDR is not understood.
Aims
We explored the role of Zn in DDR modulation in AML cells and its therapeutic potential in combination with conventional chemotherapy and with a DDR inhibitor.
Methods
The AML cell line HEL was incubated for 15 days in three culture conditions: standard Zn concentration (Std), Zn depletion (ZnD) and supplementation with 40µM of ZnSO4 (ZnS). After 2, 7 and 15 days, cells were exposed to genotoxic stimuli: 10µM of H2O2 for 30 minutes or 1 minute of UV radiation (Ee=2.9841 W.cm-2). Chromosomal damage, cell death and division rates were studied by cytokinesis-block micronucleus assay. To monitor repair kinetics, levels of yH2AX were evaluated 0h, 1h and 24h after exposure. Expression of 14 DDR genes (PARP1, XRCC1, MSH2, MSH6, MLH1, XPA, ERCC1, RAD23B, RAD51, PRKDC, XRCC6, PALB2, FANCD2, and MGMT) was analysed by qRT-PCR. An immortalized cell line of normal lymphocytes (IMC) was submitted to the same experiments. Lastly, HEL cells were incubated with the DDR inhibitor olaparib and the chemotherapeutic compound cytarabine in monotherapy and combination with 40µM of ZnSO4. Cell viability was analysed by trypan blue assay and cell death by annexin V/7-AAD staining by flow cytometry. Data was analysed considering a confidence level of 95% (p<0.05).
Results
ZnS lymphocytes displayed lower basal damage than those in Std conditions. Lymphocytes in ZnD shown increased damage biomarkers through time (1.3-fold, p=0.0234). Cell death was lower in ZnS lymphocytes and division rates slightly increased, whereas in ZnD an increase in cell death and lowered division rates were observed. Oppositely, in AML cells, basal damage through time of Zn supplementation increased (1.2-fold, p=0.0470), cell death slightly increased and division rates decreased (1.1-fold, p=0.0117). After genotoxic stimuli, ZnS lymphocytes shown less damage levels, particularly after 7 and 15 days of supplementation. ZnD lymphocytes presented more damage biomarkers. AML cells cultured in ZnS had considerably higher damage levels after exposure than in Std conditions and the lowest damage scores were found in ZnD. yH2AX monitorization shown a repair response in ZnS lymphocytes that was not achieved in Zn absence. In AML cells, yH2AX activation persisted 24h after exposure, but ZnD cells shown a more evident repair kinetics. No significant differences were observed in DDR genes expression among all conditions. Olaparib and cytarabine reduced cell proliferation and viability in a dose and time-dependent manner, inducing cell death by apoptosis. Further, ZnSO4 improved cytotoxic effects as a marked decrease of the IC50 of each compound comparatively to monotherapy was observed (2.8-fold and 5.4-fold, respectively).
Conclusion
ZnS improved the DDR in normal cells while ZnD favoured genomic instability, confirming the importance of Zn in genome integrity. In AML cells, Zn increased the genotoxicity of H2O2 and UV radiation and the therapeutic effects of olaparib and cytarabine. These results suggest that Zn supplementation is a potential co-adjuvant strategy improving genotoxic or DDR-targeting therapies in AML.
Keyword(s): Acute myeloid leukemia, DNA damage, DNA repair, Zinc