Changes in Apoptotic Pathways in MOLM-13 Cell Lines after Induction of Resistance to Hypomethylating Agents

Abstract

We established the following two variants of the MOLM-13 human acute myeloid leukemia (AML) cell line: (i) MOLM-13/DAC cells are resistant to 5-aza-2′-deoxycytidine (DAC), and (ii) MOLM-13/AZA are resistant to 5-azacytidine (AZA). Both cell variants were obtained through a six-month selection/adaptation procedure with a stepwise increase in the concentration of either DAC or AZA. MOLM-13/DAC cells are resistant to DAC, and MOLM-13/AZA cells are resistant to AZA (approximately 50-fold and 20-fold, respectively), but cross-resistance of MOLM-13/DAC to AZA and of MOLM-13/AZA to DAC was not detected. By measuring the cell retention of fluorescein-linked annexin V and propidium iodide, we showed an apoptotic mode of death for MOLM-13 cells after treatment with either DAC or AZA, for MOLM-13/DAC cells after treatment with AZA, and for MOLM-13/AZA cells after treatment with DAC. When cells progressed to apoptosis, via JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide) assay, we detected a reduction in the mitochondrial membrane potential. Furthermore, we characterized promoter methylation levels for some genes encoding proteins regulating apoptosis and the relation of this methylation to the expression of the respective genes. In addition, we focused on determining the expression levels and activity of intrinsic and extrinsic apoptosis pathway proteins.

Development of Multidrug Resistance in Acute Myeloid Leukemia Is Associated with Alterations of the LPHN1/GAL-9/TIM-3 Signaling Pathway

“…This approach is used in acute myeloid leukemia and myelodysplastic syndromes, as well as in other tumors. Although the use of hypomethylating agents is useful, it can lead to resistance against this treatment [ 10 , 50 ]. The ABCB1 drug transporter is considered to be a common cause of chemoresistance in many cancers, including AML [ 51 ].…”

“…The thermal cycling conditions were as follows: polymerase activation (95 °C, 10 min); PCR—40 cycles: denaturation (95 °C, 15 s) and annealing/extension (60 °C; 1 min). The relative expression of individual genes was evaluated by the Livak method (2 −ΔΔCT ) [ 10 ].…”

“…Intensive experimental and translational research has been conducted over the last 20 years on the use of immunomodulatory agents (e.g., lenalidomide [ 8 ]), demethylating agents (e.g., azacytidine and deoxyazacytidine [ 9 , 10 ]), proteasome inhibitors (e.g., bortezomib [ 11 , 12 ]) and histone deacetylase inhibitors (e.g., vorinostat [ 13 ]). Therapy with these and other promising drugs is applied in combination with standard intensive therapy, especially in patients with bone marrow malignancies, if the standard treatment alone fails.…”

“…We have shown in previous studies that the development of MDR in leukemic cells mediated by ABCB1 overexpression is associated with multiple and complex changes in the transcriptome, proteome, and cell surface glycome [ 10 , 18 , 19 , 20 , 21 , 22 , 23 ].…”

Analysis of 5-Azacytidine Resistance Models Reveals a Set of Targetable Pathways

“…Two other MDS/AML lines were also AZA-S: MOLM-13 (DSMZ, #ACC 554, derived from a 20-year-old male with MDS-EB in relapse to MDS/AML); SKM1 (DSMZ, #ACC 547, derived from a transition to MDS/AML). AZA resistant (AZA-R) subclones were obtained using a similar procedure as shown by a previous study [ 15 ]. Initially, cells were cultured for 48 h at a concentration of 10.000/well in triplicate 96-well plates in Iscove’s Modified Dulbecco’s medium (IMDM) + 20% fetal bovine serum (FBS) +1% Penicillin/Streptomycin (150 μL media/well).…”