Human KU812 Erythroleukemia Cells: A Model for Competitive γ-Globin Induction by Fetal Hemoglobin Inducing Drugs.

The control of human hemoglobin gene switching from fetal γ-globin to adult β-globin is important in erythroid maturation and treatment approaches for sickle cell disease. Drugs that reverse the γ to β-globin ratio (γ/β) have been used effectively in clinical settings. Interaction between globin genes and the locus control region is a widely accepted mechanism for competitive γ-gene silencing. Studies in K562 cells are limited and often not correlated with in vivo response in part due to a lack of β-globin expression. Therefore, we tested a KU812 erythroleukemia cell line containing active γ- and β-globin genes, as an in vitro model for screening HbF inducers. Cell viability was monitored by trypan blue exclusion and globin mRNA measured in K562 cells (γ-globin) and KU812 cells (γ and β globin) by quantitative-PCR (q-PCR). Treatments with the histone deacetylase (HDAC) inhibitors: sodium butyrate (NaB, 2mM), trichostatin A (TSA, 0.2–0.5μM), suberoylanilide hydroxamic acid (SAHA, 2.5–5μM) or STI571 (0.5μM) were completed. In addition, hydroxyurea (HU, 100μM) was tested as an HbF inducer control.

Results: KU812 cells grown in suspension in IMDM and 10% fetal bovine serum showed viability and proliferative capacity similar to K562 cells. Drug concentrations for SAHA and TSA were decreased to attain acceptable cell viability for 48hr inductions. Controls studies in K562 cells treated with NaB and TSA showed 60–80% viability and 3.2-fold increased γ-globin expression normalized by GAPD (Gγ/GAPD, See Table). For the new agents SAHA (0–5μM) and STI571 (0–2μM) dose response studies showed 62% and 66% viability in K562 cells at 50μM, and a concomitant increase in γ/GAPD mRNA from 1.7 to 40-fold respectively. For KU812 cells at steady-state we observed 40-fold higher γ vs. β globin mRNA. Inductions with NaB, TSA and SAHA produced increased γ/β ratios up to 4.5-fold along with β-gene repression; a scenario desirable in sickle cell patients. Although STI571 induced γ-globin 8-fold this effect was countered by 14-fold β-globin induction to produce a net reversal of the γ/β ratio (0.58). Control studies with HU showed on average an 1.4-fold increase in γ/β globin by 48 hrs suggesting that modest changes in this competitive ratio is sufficient to achieve significant clinical benefits. These data support KU812 cells as a good model for testing competitive γ-globin activation by drug inducers, which could not be ascertained in K562 cells. We plan to correlate:

1. γ/β ratios,

2. HbF protein (by ELISA; Bethyl Lab. INC., Montgomery, TX, and 3) histone H3 and H4 acetylation levels (by western blot) in K562 and KU812 cells. Preliminary ELISA data showed 2.4 to 6.4-fold increase in HbF protein by NaB and SAHA respectively in K562 cells. This data combined with the favorable γ/β globin ratio observed with SAHA suggests this agent might be an efficacious HbF inducer in vivo.

Summary: The γ/β globin mRNA ratio was determined in KU812 cells to establish a better measure of drug-mediated HbF induction in vivo. We will screen novel agents for therapeutic potential. KU812 cells might also serve as a good model to study the elusive mechanisms involved in the γ to β globin switch during erythroid maturation.