5-Azacytidine modulates the response of sensitive and multidrug-resistant K562 leukemic cells to cytostatic drugs.
In an endeavor to improve responsiveness of tumor cells to drug combination treatments, we analyzed the effect of 5-azacytidine (5AC) as a model compound for a new class of drugs, DNA-demethylating agents. We used parental K562/WT chronic myelogenous leukemia cells and a multidrug-resistant subline thereof, K562/ADM. Multidrug-resistant cells were more resistant to daunorubicin, but more sensitive to cisplatin than parental K562 cells as measured by growth inhibition and apoptosis assays. Resistance to daunorubicin can be explained by amplification of the MDR1 drug transporter gene. Cisplatin induced more DNA damage in specific genes and in the entire genome of K562/ADM cells compared to K562/WT cells using PCR stop assays and atomic absorption spectroscopy. Pretreatment with 5AC modulated the response of K562/ADM cells toward MDR-type drugs (daunorubicin, vincristine, etoposide) and reduced function and expression of MDR1 as analyzed by flow cytometry and RT-PCR. Analysis of CpG island methylation in the promotor region of the MDR1 gene by bisulfite sequencing and a methylation-sensitive HpaII-digestion/PCR approach revealed that methylation of the MDR1 promotor of K562/ADM cells was greater than in K562/WT cells. 5AC treatment completely abolished MDR1 promotor methylation. The unexpected observation that DNA demethylation by 5AC rather decreases than increases MDR1 expression in K5612/ADM cells points to still unexplored sequences in the MDR1 promotor whose transcriptional activity may be affected by the methylation status. 5AC pretreatment also modulated K562/WT and K562/ADM cells to non-MDR-type drugs such as cisplatin and increased cisplatin-induced DNA damage.
Efferth
T
T
Medizinische Klinik IV, Aachen Technical University (RWTH Aachen), Aachen, Germany. efferth@vcrp.de
Futscher
B W
BW
Osieka
R
R
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis / Q000187:drug effects; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001374:Azacitidine / Q000494:pharmacology*; D002455:Cell Division / Q000187:drug effects; D002945:Cisplatin / Q000494:pharmacology; D004249:DNA Damage / Q000187:drug effects; D019175:DNA Methylation / Q000187:drug effects; D003630:Daunorubicin / Q000494:pharmacology; D004347:Drug Interactions; D018432:Drug Resistance, Multiple; D005784:Gene Amplification / Q000187:drug effects; D019450:Genes, MDR / Q000187:drug effects / Q000235:genetics; D006801:Humans; D020014:K562 Cells / Q000187:drug effects*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000964:Antimetabolites, Antineoplastic; D000970:Antineoplastic Agents; D001374:Azacitidine; D002945:Cisplatin; D003630:Daunorubicin