Quantitative PCR analysis of HbF inducers in primary human adult erythroid cells

Abstract

The development and evaluation of drugs to elevate fetal hemoglobin in the treatment of the genetic diseases of hemoglobin would be facilitated by the availability of reliable cell assays. We have used real-time, quantitative polymerase chain reaction (PCR) analyses of globin messenger RNA (mRNA) levels in a biphasic, erythropoietin-dependent primary culture system for human adult erythroid cells in order to assay compounds for their ability to modulate levels of adult (beta) and fetal (gamma) globin mRNA. Complementary DNA synthesized from total RNA extracted at timed intervals from aliquots of cells were assayed throughout the period that the culture was studied. gamma-globin mRNA levels were found to be much lower (less than 1%) than beta-globin mRNA levels. At concentrations of agents chosen for minimal effect on cell division, we find that the 3 drugs studied, 5-azacytidine (5 micromol/L), hydroxyurea (40 micromol/L), and butyric acid (0.5 mmol/L), significantly increase gamma-globin mRNA levels. Interestingly, hydroxyurea also had a small stimulatory effect on beta-globin mRNA levels, while butyric acid caused a twofold inhibition of beta-globin mRNA levels, and 5-azacytidine had little effect on beta-globin mRNA levels. The net result of all 3 drugs was to increase the gamma/(gamma + beta) mRNA ratios by threefold to fivefold. These data suggest that the mechanism is distinct for each drug. The profile of butyric-acid-induced changes on globin gene expression is also quite distinct from changes produced by trichostatin A, a known histone deacetylase inhibitor. Quantitative PCR analyses of human erythroid cells should prove useful for studying the mechanism(s) of action of known inducers of gamma-globin and identifying new drug candidates.