Production of beta-globin and adult hemoglobin following G418 treatment of erythroid precursor cells from homozygous beta(0)39 thalassemia patients

Abstract

In several types of thalassemia (including beta(0)39-thalassemia), stop codon mutations lead to premature translation termination and to mRNA destabilization through nonsense-mediated decay. Drugs (for instance aminoglycosides) can be designed to suppress premature termination, inducing a ribosomal readthrough. These findings have introduced new hopes for the development of a pharmacologic approach to the cure of this disease. However, the effects of aminoglycosides on globin mRNA carrying beta-thalassemia stop mutations have not yet been investigated. In this study, we have used a lentiviral construct containing the beta(0)39-thalassemia globin gene under control of the beta-globin promoter and a LCR cassette. We demonstrated by fluorescence-activated cell sorting (FACS) analysis the production of beta-globin by K562 cell clones expressing the beta(0)39-thalassemia globin gene and treated with G418. More importantly, after FACS and high-performance liquid chromatography (HPLC) analyses, erythroid precursor cells from beta(0)39-thalassemia patients were demonstrated to be able to produce beta-globin and adult hemoglobin after treatment with G418. This study strongly suggests that ribosomal readthrough should be considered a strategy for developing experimental strategies for the treatment of beta(0)-thalassemia caused by stop codon mutations. Am. J. Hematol., 2009. (c) 2009 Wiley-Liss, Inc.

Figure 1

A: Map of the vector pCCL.βwt. PGW used to generate the K562 cellular clones carrying the wild-type and the β⁰39-thal mutated globin mRNA. βp, beta-globin promoter. The three exons, the two introns and the genomic region including the 3′ enhancer are indicated. B: Effects of 400 μg/ml G418 on the production of β-globin in K562-wt3 (D–G) and K562-m5 (H–M). As a reference control, the immunohistochemistry analysis of original wild-type K562 cells (not expressing β-globin mRNA) is shown in panels B and C; analysis performed on untreated (D, E, H, I) versus G418-treated (F, G, L, M) K562-wt3 and K562-m5 cells is shown. Staining of the cells with the β-globin-PE (PE, phycoerythrin) (Santa Cruz Biotechnology, Santa Cruz, CA) is shown in panels C, E, G, I, and M. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 2

A–N: Effects of G418 on the production of β-globin by K562-wt3 and K562-m5 cells. The FACS analysis is shown of untreated K562-wt3 (A, B) and K562-m5 (C, D) cells versus cells treated with 200 μg/ml (E–H) and 400 μg/ml (I–N) G418. A, B, E, F, I, L = K562-wt3 cells; C, D, G, H, M, N = K562-m5 cells. The arrows in panels D, H, and N are positioned on the intensity of the β-globin-PE peak of untreated cells (D), to help the reader to follow the shift of the right in G418-treated cells (H and N). O, P: Quantitative analysis of the FACS obtained in three independent experiments. GFP (closed symbols) and β-globin-PE (open symbols) fluorescence in K562-wt3 (O) and K562-m5 (P) cells treated with 100–400 μg/ml of G418 is reported. Data represent the average ± SD of fluorescence intensity. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 3

Proteomic analysis of untreated (A) and G418-treated (B) K562-m5 cells. In panels C–G, examples are reported, relative to two downmodulated spots (panels C and D) and three upmodulated spots (panels E–G). The quantitative data of four independent proteomic analysis are shown in the bottom of panels C–G. To obtain these data, the 2DE gels were scanned by a GS-800 Calibrated Densitometer (Bio-Rad, Hercules, CA), using the Quantity One (1D Analysis Software), version 4.6.1

Figure 4

A, B: Effect of 400 μg/ml G418 on the production of β-globin and HbA in erythroid precursor cells isolated from the peripheral blood of homozygous β⁰39-thalassemia patients. (A) FACS analysis; (B) HPLC analysis of lysates from untreated (upper panel) and G418-treated (lower panel) cells. C: Summary of the data on the increase of the percentage of HbA accumulation in erythroid precursor cells from β⁰39-thalassemic patients after treatment with G418; the data represent the mean ± SD from 10 different independent experiments using erythroid precursor cells from six homozygous β⁰39-thalassemic patients. which is available at www.interscience.wiley.com.]

Figure 5

A: RT-PCR quantitative analysis performed on RNA isolated from K562-wt3 (A, black symbols), K562-m5 (A, open symbols) and from erythroid cells from β⁰39-thalassemia patients (B, C), using primers amplifying β-globin mRNA sequences. In panels A and C, cells were treated with the indicated amounts of G418. In panels A and C, results are presented as fold induction of β-globin (panel A) and β⁰-globin mRNA (panel C) of G418-treated cells with respect to untreated controls (mean ± SD from three different determinations).