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Isolation and characterization of daunorubicin-resistant AML-2 sublines
Abstract:
An attempt was made to isolate resistant sublines of acute myelogenous leukemia (OCI/ AML-2) cells by chronic exposure to gradually increasing concentrations of daunorubicin in order to determine the mechanism of its resistance to this drug. Four daunorubicin-resistant sublines, AML-2/D100, /D250, /D500, and /D1,000 were isolated. The values of relative resistance of each daunorubicin-resistant AML subline were about 3, 6, 18, and 23-fold, respectively, as compared to the AML-2 line with an IC50 of 5 nM. The daunorubicin-resistant AML-2 sublines also showed cross resistance to various anticancer drugs including another anthracycline doxorubicin, a Vinca alkaloid vincristine, and an epipodophyllotoxin etoposide. A functional assay using flow cytometry showed decreased accumulation of daunorubicin in these sublines as compared to that of AML-2, which was reversed by cyclosporin A or cyanide. The development of the ATP-dependent multidrug resistant phenotype was due to low to high levels of expression of P-glycoprotein (PGP). The major mechanisms of increased PGP appears to be associated with gene amplification. In addition, other mechanisms such as increased stability of protein or mRNA might be involved depending on the concentration of daunorubicin used for selection. However, a multidrug resistance-associated protein (MRP) was not involved in these resistant sublines. These daunorubicin-resistant AML-2 sublines could provide a useful model for the study of multidrug resistance mediated by PGP.
Insights
Researchers developed daunorubicin-resistant acute myelogenous leukemia (AML) cell lines to study drug resistance mechanisms. These resistant AML cells exhibit cross-resistance to other chemotherapy drugs, primarily due to increased P-glycoprotein (PGP) expression.
Area of Science:
- Oncology
- Pharmacology
- Molecular Biology
Background:
- Acute myelogenous leukemia (AML) remains a challenging cancer with limited treatment options.
- Understanding drug resistance mechanisms is crucial for improving AML therapy efficacy.
- Daunorubicin is a key chemotherapeutic agent used in AML treatment.
Purpose of the Study:
- To generate and characterize daunorubicin-resistant sublines of OCI/AML-2 cells.
- To investigate the underlying mechanisms of acquired resistance to daunorubicin in AML.
- To explore potential cross-resistance patterns with other anticancer drugs.
Main Methods:
- Chronic exposure of OCI/AML-2 cells to increasing concentrations of daunorubicin to isolate resistant sublines.
- Quantification of relative drug resistance using IC50 values.
- Flow cytometry analysis to assess intracellular daunorubicin accumulation.
- Evaluation of P-glycoprotein (PGP) and multidrug resistance-associated protein (MRP) involvement.
Main Results:
- Four daunorubicin-resistant AML sublines (AML-2/D100, /D250, /D500, /D1,000) were successfully isolated with 3 to 23-fold increased resistance.
- Resistant sublines displayed cross-resistance to doxorubicin, vincristine, and etoposide.
- Decreased daunorubicin accumulation in resistant cells was observed, reversible by cyclosporin A or cyanide.
- Resistance was attributed to ATP-dependent P-glycoprotein (PGP) upregulation, likely via gene amplification, with potential contributions from protein/mRNA stability.
Conclusions:
- Acquired daunorubicin resistance in AML cells is mediated by P-glycoprotein (PGP) overexpression.
- Gene amplification is a primary mechanism for increased PGP expression in these resistant sublines.
- The developed resistant AML-2 sublines serve as a valuable model for studying PGP-mediated multidrug resistance in cancer therapy.