Induction of fetal hemoglobin and ABCB1 gene expression in 9-β-D-arabinofuranosylguanine-resistant MOLT-4 cells.
OBJECTIVE
To characterize resistance mechanisms to the nucleoside analog 9-β-D-arabinofuranosylguanine (AraG) in the T-cell acute lymphoblastic leukemia cell line MOLT-4 and its AraG-resistant variant.


METHODS
A gene expression microarray analysis was performed, as well as gene expression and enzyme activity measurements of key enzymes in the activation of AraG. Cytotoxicity of AraG and cross-resistance to other compounds were evaluated using a standard cytotoxicity assay.


RESULTS
Gene expression microarray analysis revealed that fetal hemoglobin genes and the multidrug resistance ABCB1 gene, encoding the drug efflux pump P-gp, were the most highly upregulated genes in the resistant cells, while genes traditionally associated with nucleoside analog resistance were not. Fetal hemoglobin and ABCB1 induction can be due to global DNA hypomethylation. This phenomenon was studied using AraG during a period of 4 weeks in MOLT-4 cells and the lung adenocarcinoma cell line A549, leading to up-regulation of hemoglobin gamma and ABCB1 as well as DNA hypomethylation. Inhibiting P-gp in the AraG-resistant MOLT-4 cells led to decreased proliferation, reduced hemoglobin expression, and highly induced ABCB1 expression.


CONCLUSIONS
We show that AraG can cause hypomethylation of DNA and induce the expression of the fetal hemoglobin gamma gene and the ABCB1 gene. We speculate that the induction of ABCB1/P-gp may occur in order to help with excretion of hemoglobin degradation products that would otherwise be toxic to the cells, and we present data supporting our theory that P-gp may be linked to the induction of hemoglobin.
Department of Medicine and Health, Linköping University, Linköping, Sweden. anna.fyrberg@liu.se
Anna
A
Fyrberg
Curt
C
Peterson
Bertil
B
Kågedal
Kourosh
K
Lotfi
D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis* / Q000235:genetics*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001087:Arabinonucleosides / Q000494:pharmacology*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002470:Cell Survival; D019175:DNA Methylation; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D005319:Fetal Hemoglobin / Q000032:analysis*; D006801:Humans; D046228:Microarray Analysis; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics; D012334:RNA, Neoplasm / Q000096:biosynthesis / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013778:Tetrazolium Salts; D013844:Thiazoles
C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents; D001087:Arabinonucleosides; D009363:Neoplasm Proteins; D012334:RNA, Neoplasm; D013778:Tetrazolium Salts; D013844:Thiazoles; C028771:9-arabinofuranosylguanine; D005319:Fetal Hemoglobin; C022616:thiazolyl blue