Hematology and Oncology
Contributions
Type: Publication Only
Background
Leukemia initiating cells reside within specialized niches in the bone marrow where they undergo complex interactions with different stromal cell types. The bone marrow niche is characterized by a low oxygen tension resulting in high expression of hypoxia-inducible factor 1 alpha (HIF-1α). Expression of HIF-1α has been shown to represent a negative prognostic factor in acute myeloid leukemia (AML).
Aims
In the current study, we investigated the impact of hypoxic versus normoxic conditions on the sensitivity of AML cell lines and primary AML blasts to cytarabine.
Methods
AML cell lines HL60, Kasumi-1, MOLM-13, OCI-AML5, MV4-11 and KG-1 as well as primary blasts from AML patients were cultured under normoxic and hypoxic conditions (oxygen content of 20% vs. 6%, respectively). After an adaptation period of three days, cells were plated for proliferation and colony formation assays to investigate the susceptibility to cytarabine (Cell Pharm GmbH, Bad Vilbel, Germany). Proliferation and colony formation was determined after 3 and 7 days of cytarabine treatment, respectively. Using quantitative RT-PCR analysis, we investigated the mRNA expression of deoxycytidine kinase in AML cells cultured under normoxic vs. hypoxic conditions for 3 days. AML cells under hypoxic conditions were additionally treated with the HIF-1α inhibitor BAY87-2243 (Selleckchem, Houston, TX).
Results
AML cells cultured under 6% oxygen were significantly more resistant to cytarabine compared to cells cultured under normoxic conditions in proliferation assays. HL60 cells were the most susceptible cells to the drug-induced growth inhibition under hypoxic conditions for cytarabine concentrations between 10 nM and 1000 nM. Although less pronounced, Kasumi?1, MOLM-13, OCI-AML5 and MV4-11, also showed the hypoxia-induced resistance to cytarabine. Only in KG-1 cells, no significant differences in growth inhibition could be detected under normoxic vs. hypoxic conditions. Furthermore, increased sensitivity to cytarabine under normoxia could also be observed in primary AML samples (n=5).
Response to cytarabine under normoxic vs. hypoxic conditions was also analyzed by colony formation assays for several AML cell lines using cytarabine concentrations ranging from 25 nM to 100 nM. Colony numbers at day 7 of cytarabine treatment were significantly higher at 6% oxygen than at normoxia.
Interestingly upon cultivation under hypoxia, the expression of the cytarabine-activating enzyme deoxycytidine kinase was down-regulated in all analyzed AML cell lines and primary AML samples representing a possible mechanism for resistance to chemotherapy. Furthermore, the down-regulation of deoxycytidine kinase could directly be associated with HIF-1α as treatment with its inhibitor BAY87?2243 hampered the down-regulation of deoxycytidine kinase expression under hypoxic conditions.
Summary
In conclusion, our data reveal that the hypoxia-induced down-regulation of deoxycytidine kinase might represent one mechanism of drug resistance to cytarabine in acute myeloid leukemia.
Keyword(s): Drug resistance, Hypoxia-sensing, Microenvironment
Session topic: Publication Only
Type: Publication Only
Background
Leukemia initiating cells reside within specialized niches in the bone marrow where they undergo complex interactions with different stromal cell types. The bone marrow niche is characterized by a low oxygen tension resulting in high expression of hypoxia-inducible factor 1 alpha (HIF-1α). Expression of HIF-1α has been shown to represent a negative prognostic factor in acute myeloid leukemia (AML).
Aims
In the current study, we investigated the impact of hypoxic versus normoxic conditions on the sensitivity of AML cell lines and primary AML blasts to cytarabine.
Methods
AML cell lines HL60, Kasumi-1, MOLM-13, OCI-AML5, MV4-11 and KG-1 as well as primary blasts from AML patients were cultured under normoxic and hypoxic conditions (oxygen content of 20% vs. 6%, respectively). After an adaptation period of three days, cells were plated for proliferation and colony formation assays to investigate the susceptibility to cytarabine (Cell Pharm GmbH, Bad Vilbel, Germany). Proliferation and colony formation was determined after 3 and 7 days of cytarabine treatment, respectively. Using quantitative RT-PCR analysis, we investigated the mRNA expression of deoxycytidine kinase in AML cells cultured under normoxic vs. hypoxic conditions for 3 days. AML cells under hypoxic conditions were additionally treated with the HIF-1α inhibitor BAY87-2243 (Selleckchem, Houston, TX).
Results
AML cells cultured under 6% oxygen were significantly more resistant to cytarabine compared to cells cultured under normoxic conditions in proliferation assays. HL60 cells were the most susceptible cells to the drug-induced growth inhibition under hypoxic conditions for cytarabine concentrations between 10 nM and 1000 nM. Although less pronounced, Kasumi?1, MOLM-13, OCI-AML5 and MV4-11, also showed the hypoxia-induced resistance to cytarabine. Only in KG-1 cells, no significant differences in growth inhibition could be detected under normoxic vs. hypoxic conditions. Furthermore, increased sensitivity to cytarabine under normoxia could also be observed in primary AML samples (n=5).
Response to cytarabine under normoxic vs. hypoxic conditions was also analyzed by colony formation assays for several AML cell lines using cytarabine concentrations ranging from 25 nM to 100 nM. Colony numbers at day 7 of cytarabine treatment were significantly higher at 6% oxygen than at normoxia.
Interestingly upon cultivation under hypoxia, the expression of the cytarabine-activating enzyme deoxycytidine kinase was down-regulated in all analyzed AML cell lines and primary AML samples representing a possible mechanism for resistance to chemotherapy. Furthermore, the down-regulation of deoxycytidine kinase could directly be associated with HIF-1α as treatment with its inhibitor BAY87?2243 hampered the down-regulation of deoxycytidine kinase expression under hypoxic conditions.
Summary
In conclusion, our data reveal that the hypoxia-induced down-regulation of deoxycytidine kinase might represent one mechanism of drug resistance to cytarabine in acute myeloid leukemia.
Keyword(s): Drug resistance, Hypoxia-sensing, Microenvironment
Session topic: Publication Only