Identification of fetal hemoglobin-inducing agents using the human leukemia KU812 cell line.
Fetal hemoglobin (HbF) ameliorates the clinical severity of sickle cell disease; therefore continued research to identify efficacious HbF-inducing agents is desirable. In this study, we investigated KU812 leukemia cells that express the fetal γ-globin and adult β-globin genes, as a system for screening and discovery of novel HbF inducers. KU812 cells were analyzed in the presence or absence of fetal bovine serum and then expression levels of the globin genes, cell surface markers and transcription factors were quantified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). For comparison, primary erythroid cells were grown in a two-phase liquid culture system. After drug inductions for 48-72 h, globin mRNA and HbF levels were quantified by RT-qPCR and enzyme-linked immunosorbent assay, respectively. Erythroid markers and transcription factors expression levels in KU812 cells were comparable to days 7-14 erythroid cells. We also tested several drugs including butyrate, trichostatin A, scriptaid, suberoylanilide hydroxamic acid and hydroxyurea, which induced γ-globin in KU812 cells; however, some agents also induced β-globin. A novel agent STI-571 was studied in the system, which non-selectively induced the globin genes. Additional studies showed comparable globin gene response patterns in KU812 and primary erythroid cells after treatments with the various drug inducers. Mechanisms of drug-mediated γ-globin induction in KU812 cells require signaling through the p38 mitogen-activated protein kinase pathway similar to that previously demonstrated in primary erythroid cells. These data suggest that KU812 cells serve as a good screening system to identify potential HbF inducers for the treatment of β-hemoglobinopathies.
Department of Neurology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
Sima
S
Zein
Wei
W
Li
Valya
V
Ramakrishnan
Tzu-Fang
TF
Lou
Sharanya
S
Sivanand
Ashley
A
Mackie
Betty
B
Pace
D000954:Antigens, Surface / Q000378:metabolism; D001549:Benzamides; D002087:Butyrates / Q000494:pharmacology; D002454:Cell Differentiation / Q000235:genetics; D045744:Cell Line, Tumor; D015672:Erythroid Precursor Cells / Q000187:drug effects / Q000378:metabolism; D005319:Fetal Hemoglobin / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D006898:Hydroxylamines / Q000494:pharmacology; D006918:Hydroxyurea / Q000494:pharmacology; D000068877:Imatinib Mesylate; D020014:K562 Cells; D010879:Piperazines / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D011804:Quinolines / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014157:Transcription Factors / Q000378:metabolism; D000077337:Vorinostat; D055544:beta-Globins / Q000235:genetics / Q000378:metabolism; D055545:gamma-Globins / Q000235:genetics / Q000378:metabolism
D000954:Antigens, Surface; D001549:Benzamides; D002087:Butyrates; D006877:Hydroxamic Acids; D006898:Hydroxylamines; D010879:Piperazines; D011743:Pyrimidines; D011804:Quinolines; D012333:RNA, Messenger; D014157:Transcription Factors; D055544:beta-Globins; D055545:gamma-Globins; C410733:scriptaid; C012589:trichostatin A; D000077337:Vorinostat; D000068877:Imatinib Mesylate; D005319:Fetal Hemoglobin; D006918:Hydroxyurea