Spontaneous switch from Aγ- to β-globin promoter activity in a stable transfected dual reporter vector
Introduction
Thalassemia is a severe genetic disease caused by coinheritance of defective α- or β-globin alleles resulting in deficient globin expression. In the case of β-thalassemia, the disease is due to deletion or abnormal regulation of the β-globin gene. The resulting inefficient β-globin expression leads to accumulation of unpaired, insoluble α-globin chains in red cells. The consequent accelerated destruction of these cells induces ineffective erythropoiesis and severe anemia. The development of transfusion therapies coupled with administration of iron-chelating agents has greatly ameliorated the treatment and the quality of life of β-thalassemia patients. The overall treatment of this disorder, however, is still unsatisfactory. In fact, while the incidence of the disease is steadily decreasing in western countries, it remains high in economically challenged countries that do not have easy access to the technologies required to properly manage these complex therapies. The observation that some of the homozygote carriers of β-globin gene deletions are asymptomatic because of persistent expression in adult life of the fetal-specific β-like genes, the γ-globin genes (High Persistence of Fetal Hemoglobin Syndrome, HPFH), has inspired numerous studies aimed to identify chemical substances capable to pharmacologically re-activate γ-globin expression in patients with β-thalassemia. One of the pharmacological agents identified so far is hydroxyurea. Phase I–II clinical trials with hydroxyurea on β-thalassemia [1] and sickle cell [2], [3] patients have been successfully completed, and the drug is presently considered to be introduced in the current medical practice for these disorders. Hydroxyurea, however, is a compound not devoid of counter indications [4]. Therefore, the search for additional and less toxic agents is still in progress [5].
The ideal candidate for pharmaceutical treatment of β-thalassemia is a cheap chemical that could be administered orally, would be effective within reasonable doses, and would present no or little counter indications. The efficiency of the search for these agents is strictly dependent on the availability of a surrogate assay to measure the potentiality to reactivate γ-globin gene expression in vivo. Such surrogate assays should be rapid, cheap, reliable, and suitable to be adapted for automated screening. Assays of induction of γ-globin gene expression that use as target cells human erythroblasts and that determine the cellular globin content by HPLC represent the tests that would most reliably mimic the possible effects a chemical might induce in vivo [6]. These assays, however, as precise and indicative of clinical efficacy as they might be, are cumbersome and not easily adaptable for automatic screening. More suitable for this purpose would be surrogate assays based on murine erythroleukemia cell lines transfected with reporter constructs containing regions of the human γ-globin promoter linked to appropriate reporter genes. The accuracy of these assays depends on how closely the promoter regions that drive the expression of the reporter gene mimic the physiology of γ-globin gene activation in primary erythroid cells. Recently, it has been developed an assay based on the murine erythroid GM979 cell line stably transfected with a construct containing a human mini globin locus composed by a micro-LCR and by the promoters for Aγ- and β-globin, each one linked to a different Luciferase [7], [8]. The ratio between the expression of the two Luciferases reflects the relative activity of the two promoters and can be measured easily in to be automated assays. The suitability of this assay, however, for automated screening of chemical inducers of globin gene expression depends on how stable and consistent is the expression of the reporter in transfected cells over time.
Here it is characterized the expression over time of the mini locus dual reporter construct in stably transfected GM979 cells, from the first month up to 1–2 years from transfection. The results obtained indicate that GM979 μLCRβprRlucAγprFluc cells undergo an initial period of stabilization after which they express high levels of Luciferase activity driven both by the Aγ- and β-promoter. The Aγ-driven/(Aγ-driven + β-driven) reporter activity ratio, that was similar to the fetal-globin ratio at early bulk cultures, became similar to adult globin gene ratio at later time points. Butyric acid, a known fetal hemoglobin inducer, increased expression of the reporter from the Aγ-promoter both in early and in late bulk cultures but decreased that from the β-promoter only in late bulk cultures. As such, GM979 μLCRβprRlucAγprFluc cells from late bulk cultures represent a reliable assay for automated screening of chemical inducers of globin gene activation.
Section snippets
Establishment of the GM979 μLCRβprRlucAγprFluc cell line
The murine erythroleukemic GM979 cell line had been established from fetal liver cells [9] and was chosen for this study because its phenotype includes expression both of minor and major Hb. The GM979 μLCRβprRlucAγprFluc cell line was obtained by lipofecting GM979 cells with a μLCRβprRlucAγprFluc plasmid that contains a 3.1-kb μLCR cassette including the DNAse I hypersensitive core of the 5′ hypersensitive sites HS1, HS2, HS3, and HS4, linked to 315-bp of the human β-globin promoter and 1.4-kb
Increased Luciferase activity per cell, both from Aγ- and β-promoter, in GM979 μLCRβprRlucAγprFluc cells over time
The amount of Aγ-F and β-R Luciferase activity expressed over time by GM979 μLCRβprRlucAγprFluc cells is presented in Fig. 1. The activity of both the Aγ-driven Firefly and the β-driven Renilla expressed by the cells progressively increased during the culture (Fig. 1). The amount of the β-driven Renilla Luciferase, however, increased much more than that of the Aγ-driven Firefly Luciferase. In fact, the average activity of Aγ-F Luciferase per cell increased by 2-fold (from 0.05 ± 0.02 AFU at 2
Discussion
To provide for a fast and reliable assay for chemical inducers of human HbF, the murine GM979 cell line was transfected with a dual Luciferase construct (μLCRβprRlucAγprFluc) [8] that allows direct comparison of the reporter activity driven by the human Aγ- and β-globin promoters [7], [11]. Here we show that the resulting GM979 μLCRβprRlucAγprFluc cell line maintains the ability to produce both Firefly and Renilla Luciferases over a period of more than 1 year of continuous bulk culture (Fig. 1,
Acknowledgments
The authors gratefully acknowledge Prof. Francesco Antonio Manzoli for continuous support. This study was supported by, Progetti di ricerca di Interesse Nazionale 2001 and 2002 from the Ministry of Health, MIUR 60% Grant 2002, Grant E.1172 from Telethon Foundation, Progetti FIRB 2002 and 2003, National Project on Stem Cells, Institutional Funds from Istituto Superiore di Sanità, and grant NIH-HL20899.
References (18)
- et al.
Hydroxyurea can eliminate transfusion requirements in children with severe beta-thalassemia
Blood
(2003) - et al.
Predictors of fetal hemoglobin response in children with sickle cell anemia receiving hydroxyurea therapy
Blood
(2002) - et al.
Effect of hydroxyurea on growth in children with sickle cell anemia: results of the HUG-KIDS Study
J. Pediatr.
(2002) - et al.
In vitro induction of fetal hemoglobin in human erythroid progenitor cells
Exp. Hematol.
(2003) - et al.
Induction of human {gamma} globin gene expression by histone deacetylase inhibitors
Blood
(2004) - et al.
Novel in vitro assay for the detection of pharmacologic inducers of fetal hemoglobin
Blood
(2000) - et al.
Induction of fetal hemoglobin by propionic and butyric acid derivatives: correlations between chemical structure and potency of Hb F induction
Blood Cells Mol. Diseases
(2002) - et al.
Hydroxamide derivatives of short-chain fatty acids are potent inducers of human fetal globin gene expression
Exp. Hematol.
(2003) - et al.
Stable and unstable transgene integration sites in the human genome: extinction of the Green Fluorescent Protein transgene in K562 cells
Gene
(2000)
Cited by (5)
Pharmacological induction of human fetal globin gene in hydroxyurea-resistant primary adult erythroid cells
2015, Molecular and Cellular Biology