In vitro induction of fetal hemoglobin in human erythroid progenitor cells.
OBJECTIVE
Clinical heterogeneity among patients with sickle cell anemia (SCA) is influenced by the amount of fetal hemoglobin (HbF) within circulating erythrocytes. Current pharmacotherapy focuses on increasing HbF in order to reduce hemolysis and help prevent acute vaso-occlusive events. Hydroxyurea, a known S-phase-specific cytotoxic ribonucleotide reductase (RR) inhibitor, is an effective agent for HbF induction in patients with SCA, but the mechanisms by which hydroxyurea induces HbF in vivo have not been elucidated.


METHODS
We adapted an in vitro assay for HbF induction, growing burst-forming unit erythroid (BFU-E) colonies in methylcellulose from peripheral blood of children with SCA and extracting the hemoglobin for high-performance liquid chromatography analysis of HbF. Hydroxyurea and other known RR inhibitors, along with cytotoxic agents that are not RR inhibitors, were tested for the ability to induce HbF using this in vitro assay.


RESULTS
Hydroxyurea decreased the number of BFU-E colonies that grew in culture and significantly increased HbF from 13.6%+/-6.2% to 25.4%+/-8.0% at 50 microM HU (p=0.012). Three other known RR inhibitors also significantly induced HbF: 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone (p=0.025), guanazole (p=0.008), and gemcitabine (p=0.028). Cytarabine and alkylating agents BCNU and 4-hydroperoxycyclophosphamide, which are cytotoxic agents but not RR inhibitors, reduced BFU-E colony number but did not significantly induce HbF.


CONCLUSIONS
Hydroxyurea and other RR inhibitors significantly induce HbF in vitro in human erythroid progenitor cells. Inhibition of RR may be a critical mechanism by which hydroxyurea increases HbF in vivo in patients with SCA.
Ho
Janie A
JA
Division of Pediatric Hematology/Oncology, Duke University Medical Center, Durham, NC, USA.
Pickens
Chrisley V
CV
Gamcsik
Michael P
MP
Colvin
O Michael
OM
Ware
Russell E
RE
Gamscik
Michael P
MP
D000755:Anemia, Sickle Cell / Q000097:blood*; D002648:Child; D002851:Chromatography, High Pressure Liquid; D015672:Erythroid Precursor Cells / Q000187:drug effects / Q000378:metabolism*; D005319:Fetal Hemoglobin / Q000096:biosynthesis*; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D066298:In Vitro Techniques
D005319:Fetal Hemoglobin; D006918:Hydroxyurea