Increase in γ-globin mRNA content in human erythroid cells treated with angelicin analogs

Abstract

The aim of the present study was to identify molecular analogs of angelicin (ANG) able to increase erythroid differentiation of K562 cells and expression of gamma-globin genes in human erythroid precursor cells, with low effects on apoptosis. ANG-like molecules are well-known photosensitizers largely used for their antiproliferative activity in the treatment of different skin diseases (i.e., psoriasis, vitiligo, eczema, and mycosis fungoides). To verify the activity of these derivatives, we employed three experimental cell systems: (1) the human leukemic K562 cell line, (2) K562 cell clones stably transfected with a pCCL construct carrying green-EGFP under the gamma-globin gene promoter, and (3) the two-phase liquid culture of human erythroid progenitors isolated from normal donors and beta-thalassemia patients. The results of our study suggest that trimethyl ANG is a powerful inducer of erythroid differentiation, compared with known inducers, such as ANG, cytosine arabinoside, mithramycin, and cisplatin. These data could have practical relevance, because pharmacologically mediated regulation of human gamma-globin gene expression, with the consequent induction of fetal hemoglobin, is considered a potential therapeutic approach in hematological disorders including beta-thalassemia and sickle cell anemia.

A combined approach for β-thalassemia based on gene therapy-mediated adult hemoglobin (HbA) production and fetal hemoglobin (HbF) induction

“…To this aim, we used K562 cellular clones described in Guerrini et al [43] and Lampronti et al [44] and transduced the cells with the recombinant pCCL.Promβ.HcRed1.Promγ.EGFP vector (see Fig. 1a), carrying a green fluorescent protein (EGFP) and a red fluorescent protein (RFP) genes under the control of γ-globin and β-globin promoters, respectively [43, 44].…”

“…To this aim, we used K562 cellular clones described in Guerrini et al [43] and Lampronti et al [44] and transduced the cells with the recombinant pCCL.Promβ.HcRed1.Promγ.EGFP vector (see Fig. 1a), carrying a green fluorescent protein (EGFP) and a red fluorescent protein (RFP) genes under the control of γ-globin and β-globin promoters, respectively [43, 44]. Using this experimental cellular system, the increase of green (eGFP) signal is consistent with a γ-globin promoter-driven transcriptional activity, while the increase of the far red (RFP) signal is associated with β-globin promoter activity [43].…”

“…Human K562 cells [47] were used to obtain stable transfectants. In this system, increase of green eGFP signal will be consistent with a γ-globin gene promoter driven activity; on the contrary, increase of the far-red FP signal will be associated with β-globin promoter activity [43, 44]. For determining the promoter activity, cells were seeded at 12500 cells/ml and treated with MTH.…”

“…For the generation of stable K562 clones integrating human β-globin gene, the pCCL.β-globin.PGW vector was used [43, 44]. Transduction was carried out by plating 10 6  K562 cells in 9.5-cm 2 dishes with 45% RPMI and 45% I-MDM (Iscove's Modified Dulbecco's Medium, CAMBREX—Biowhittaker Europe), 10% FBS, 2 mM l -glutamine (CAMBREX—Biowhittaker Europe, Milan, Italy), 100 U/ml penicillin, and 100 mg/ml streptomycin in humified atmosphere of 5% CO 2 /air and adding the decided volume of the viral supernatant.…”

“…We analyzed the effect of MTH on K562 cell clones carrying the enhanced green fluorescent protein (eGFP) and the red fluorescent protein (RFP) driven by the γ-globin and β-globin promoter, respectively [43, 44]. We analyzed the effect of MTH on several transgenic K562 cell lines harboring different copies of the human β-globin gene to verify the possible co-expression of β-globin and γ-globin mRNAs, and the possible production of HbA and HbF under stimulation with MTH.…”