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MINERVA BIOTEC 2007;19:123-32 Cellular biosensors for the identification
of fetal hemoglobin inducers
G. BREVEGLIERI 2, F. SALVATORI 1, A. FINOTTI 2, I. BERTUZZI 2, F. DESTRO 2, S. FALZONI 3, N. BIANCHI 1 M. BORGATTI 1, C. ZUCCATO 1, G. FERIOTTO 1, L. BREDA 4, S. RIVELLA 4, R. GAMBARI 1, 2 The development of cellular systems for the screening
1Laboratory for the Development of Pharmacological of molecules able to induce the production of fetal or
and Pharmacogenomic Therapy of Thalassemia adult hemoglobin in erythroid cells isolated from β-tha-
Biotechnology Centre, Ferrara, Italy lassemia patients is very important for the identifica-
2ER-GenTech, Department of Biochemistry tion of molecules of interest for pharmacological ther-
and Molecular Biology apy of thalassemia. This article reports the development
University of Ferrara, Ferrara, Italy of a cellular system for the identification of inducers of
3Department of Experimental and Diagnostic Medicine fetal hemoglobin preferentially acting on the human γ-
Section of General Pathology globin gene promoter. To achieve this aim, green and red
University of Ferrara, Ferrara, Italy fluorescence protein genes were cloned under the con-
eill Medical College of Cornell University, New York, NY, USA trol of γ-globin and β-globin promoters, respectively.
The developed K562 clones were tested for the increase
of fluorescence after treatment with sodium butyrate
and hydroxyurea, two well-known inducers of fetal
hemoglobin.

cin,4 tallimustine,5 cisplatin analogs.6 For instance, KEY WORDS: Fetal hemoglobin - beta-thalassemia - Histone
angelicin,7 an angular psoralen found in several med- deacetylases - Hydroxyurea - Green fluorescent proteins.
icinal plant extracts, was found to induce erythroid dif-ferentiation of the human leukemic K562 cell line andincreased HbF production in primary human ery- Pharmacologically mediated stimulation of human throid precursor cells. With respect to the screening β-globin gene expression and increase of fetal of HbF inducers, high-throughput approaches are haemoglobin (HbF) levels is considered a potential highly needed. To this aim, the use of K562 cells is therapeutic modality in hematological disorders, commonly considered for a first screening. However, including β-thalassemia and sickle cell anemia.1 the employment of this system gives few indications Based on this assumption, several HbF inducers on the mechanism of action of the inducer molecules.
have been recently studied, including hydroxyurea,1 Accordingly, several groups described the use of histone deacetylase-inhibitors,2 mithramycin,3 rapamy- reporter genes under the transcriptional control ofthe γ-globin gene promoter, in order to identify HbF .—R.G. is granted by Fondazione Cariparo (Cassa di inducers acting on the transcription efficiency of the mio di Padova e Rovigo), AIRC, Cofin-2005, by STAMINA Project (University of Ferrara), by UE ITHANET Project (eInfrastructure for the γ-globin gene. For instance, Skarpidi et al.8 developed Thalassaemia Research Network) and by Telethon (contract GGP07257).
a rapid and efficient method for detecting HbF induc- This research is also supported by Regione Emilia-Romagna (SpinnerProject) and by Associazione Veneta per la Lotta alla Talassemia (AVLT), ers, based on a recombinant DNA construct in which the coding sequences of two different luciferasereporter genes, firefly and renilla, are substituted for Address reprint requests to: R. Gambari, ER-GenTech, Section of those of human Aγ-globin and β-globin genes, respec- Molecular Biology, Department of Biochemistry and Molecular Biology,University of Ferrara, Ferrara, Italy. E-mail: gam@unife.it tively. The activity of these genes can be distinguished MINERVA BIOTECNOLOGICA CELLULAR SENSORS FOR β-THALASSEMIA THERAPY Figure 1.—Experimental strategy for the production of the pCCL.Promβ.HcRed1.Promγ.EGFP vector.
by a simple, highly sensitive enzymatic assay in cell gene locus. This approach has been recently described lysates. When potential HbF inducers were added to by Vadolas et al.,9 who developed a stable cellular cultures of cells stably transfected with the constructs, genomic reporter assay based on the green fluores- their effects could be determined by measuring the cent protein (GFP) gene under the Gγ-globin promot- changes in activity of the two luciferase genes. Specific er control in the intact human β-globin locus. They γ-globin gene inducers are recognized by their abili- demonstrated that stably transfected human ery- ty to increase the γ-firefly luciferase gene activity sig- throleukemic cell lines with a γ-GFP β-globin locusnificantly more than the β-renilla luciferase gene activ- construct can maintain a uniform basal level of GFPity, identified by an increased ratio of γ-firefly luciferase expression over long periods of continuous cultureactivity over total luciferase activity. and that the induction of GFP expression parallels the These results suggest that the use of the two induction of the endogenous γ-globin genes.
luciferase reporter genes provides a simple, highly Furthermore, they compared the GFP-induction poten- sensitive, and reproducible system for the detection of cy of a number of erythroid differentiating agents compounds that selectively increase γ-globin gene demonstrating that this system might help in identify-expression. ing novel HbF inducers.9, 10 Cellular genomic reporter assays for screening and Accordingly, the design and production of recom- evaluation of HbF inducers were also developed by binant clones for the screening of high numbers of introducing reporter genes within an intact β-globin potential HbF inducers was commenced preferen- MINERVA BIOTECNOLOGICA CELLULAR SENSORS FOR β-THALASSEMIA THERAPY Figure 2.—Strategy for the production of the experimental model system. The lentiviral vector pCCL.Promβ.HcRed1.Promγ.EGFP was inser- ted into K562 cells by transfection with cationic liposomes. Cells were then cloned by limited dilution, and after selecting stably transfectedclones, they were treated with potential inducers of fetal hemoglobin. The γ-globin promoter activation should cause an increase of green fluorescence, whereas the induction of β-globin promoter should lead to an increase of the red one. The fluorescence analysis can be final- ly performed both qualitatively by using a fluorescence microscope, and quantitatively by Wallac 1420 Victor3TM (Perkin Elmer) and FACScan(Becton Dickinson) instruments.
tially acting on the human γ-globin gene promoter. To Park Memorial Institute (RPMI) 1 640 medium (Sigmathis aim, the pCCL.PGK.GFP.WPRE vector was used to Aldrich) supplemented with 10% fetal bovine serum clone green and red fluorescent protein (FP) genes (FBS, Biowest), 100 units/mL penicillin and 100 under the control of γ-globin and β-globin promoters, µg/mL streptomycin (Pen-Strep, Cambrex-respectively. Human K562 cells were used to obtain Biowhittaker Europe). Cell growth was studied by determining the cell number per mL with a H5MCounter (SEAC). Materials and methods
Cell transfection with the reporter construct ector design and construction Human K562 cells were used to obtain stable For the production of vectors suitable for high- transfectants: 12 500 cells, seeded the day before throughput screening of HbF inducers, the pCCL.β-glo- the transfection in medium without Pen-Strep, were .WPRE vector was used to clone green transfected by using cationic liposomes and red FP genes under the control of γ-globin and β- (LipofectamineTM 2000 Reagent, Invitrogen-Life globin promoters, respectively (Figure 1).
Technologies): 1 µg of pCCL.Promβ.HcRed1.Promγ.
Cell lines and culture conditions EGFP vector was diluted in Opti-MEM® I (Gibco,Invitrogen-Life Technologies), in a final volume of The human leukemia K562 cells 11 were cultured 50 µL; then 2 µL of liposomes (1 µg/µL) were mixed in humified atmosphere of 5% CO2/air in the Roswell with 48 µL of Opti-MEM® I and incubated at room MINERVA BIOTECNOLOGICA CELLULAR SENSORS FOR β-THALASSEMIA THERAPY Median fluorescence Mean fluorescence value Figure 3.—Characterization of K562 cell clones stably transfected with the pCCL.Promβ.HcRed1.Promγ.EGFP vector. A, B) Fluorescent pro- tein expression analysis. Examples of qualitative (A) and quantitative (B) analysis of green fluorescence expressed by clone 4, by using a fluo-rescence microscope (DMIL, Leica), the Wallac 1420 Victor3TM (Perkin Elmer) and the FACScan (Becton Dickinson) instruments, respectively;C) PCR amplification, using primer hybridizing specifically on the vector backbone, of the genomic DNA of the eight clones selected, con-firming the transgene insertion.
temperature for 5 minutes, before being added to the continuing expressing green fluorescence over time vector; after a 20 minutes incubation at room tem- were selected as stable transfectants.
perature to allow DNA binding to liposomes, theobtained complex was added to the well containing HbF inducer compounds Inducer molecules were stored at -20 °C: sodium Cell cloning butyrate (Sigma Aldrich) was dissolved in ultrapurewater; hydroxyurea (Sigma Aldrich) was dissolved in After transfection, K562 cells were cloned in order DMSO/ethanol/ultrapure water 1:1:2. to obtain clones stably containing the reporter con-struct.
Treatment with inducer molecules Transfected cells were diluted with RPMI-20% FBS to a final concentration of 4 cells/mL, then 100 µL Treatment with chemical inducers was carried out were placed in wells of a 96-well plate; finally wells by adding the appropriate drug concentrations to were checked for the presence of single cells. By cells, usually seeded at 8 000 cells/mL. The medium using a fluorescence microscope (DMIL, Leica), clones was not changed during the induction period. After MINERVA BIOTECNOLOGICA CELLULAR SENSORS FOR β-THALASSEMIA THERAPY FACS analysis Figure 4.—Analysis of fluorescence levels of K562 cell clones, con-taining the reporter construct pCCL.Promβ.HcRed1.Promγ.EGFP, after treatment with 1.5 mM sodium butyrate for five days. A) Example of qualitative fluorescence analysis performed by using a fluorescence microscope (DMIL, Leica); B) example of histograms obtained byFACS analysis of treated and untreated cells; C) comparison between FACS analysis: median fold over control median and mean folds over control obtained by analyses with the Victor analysis: mean fold over control FACScan (Becton Dickinson) and the Wallac 1 420 Victor3TM (PerkinElmer), respectively.
five days of culture, cells were assayed for fluorescent with PBS 0.1% Triton X-100 and centrifuged to remove proteins expression. cell debris. Finally 100 µL of cell lysate were analyzedusing Victor3TM Multilabel Counter and Wallac 1420 Workstation software: the detection of the green flu-orescence was carried out in OptiPlate 96 (Perkin LUORESCENCE MICROSCOPE Elmer), by using a 485 nm excitation filter and a 535 A qualitative analysis of fluorescent proteins expres- nm emission filter; the lamp energy was 3 000. The sion was carried out by a fluorescence inverted micro- results were expressed as mean fold, the ratio between scope (DMIL, Leica), using filters suitable for both the mean relative fluorescence intensity values green and red FPs.
obtained by cells in the presence and in the absenceof treatment, respectively.
WALLAC 1 420 VICTOR3™ ANALYSIS For the determination of fluorescence intensity by using a Wallac 1 420 Victor3™ Multilabel Counter For the determination of fluorescence intensity by (Perkin Elmer), cells were harvested, washed and using a FACScan (Becton Dickinson), cells were har- counted; the same amounts of cells were then lysed vested and washed; then 10 000 cells were analyzed MINERVA BIOTECNOLOGICA CELLULAR SENSORS FOR β-THALASSEMIA THERAPY using the fl1 channel to detect green fluorescence TABLE I.—Comparison between fluorescence analyses performed and the Cell Quest Pro software. The results were by using FACScan (Becton Dickinson) and Wallac 1420Victor3TM (Perkin Elmer) instruments, after treatment of K562 expressed as median fold, the ratio between the medi- cell clones containing the reporter construct with 1.5 mM an of fluorescence intensity values obtained by cells sodium butyrate for 5 days. The values obtained by FACS analy- in the presence and in the absence of treatment, sis of 10000 cells by using the Cell Quest Pro software, inclu- respectively; also the median peak fluorescence (MPF) de: median fold over control, the ratio between the median offluorescence intensity values obtained by cells in the presence %, expressing the increase % of the fluorescence val- and in the absence of treatment, respectively; MPF% (Median ue caused by the inducer molecule, could be used. A Peak Fluorescence %), expressing the increase % of the fluo- graphic presentation of data was finally obtained by rescence value caused by the inducer molecule. The valuesobtained by Victor analysis after lysis of 0.7×106 cells by using histograms, showing the number of cells versus the the Wallac 1420 Workstation software, are expressed as mean expressed fluorescence intensity.
fold over control, the ratio between the mean relative fluore-scence intensity values obtained by cells in the presence and inthe absence of treatment, respectively. Median fold over control Mean fold over control Development of the pCCL.Promβ.HcRed1.Promγ.EGFP Clone 1 In order to develop the cellular model for HbF inducers screening the pCCL.Promβ.HcRed1.Promγ. Clone 5 EGFP vector was produced (Figure 1E). To reach this aim the pCCL.β-globin.PGK.GFP.WPRE vector was Clone 7 used (Figure 1A), kindly provided by Prof. Stefano Rivella (Weill Medical College of Cornell University,New York), who studied it for the β-globin gene trans-fer in gene therapy experiments for β-thalassemia.
the correct regulation and expression of globin genes, The third generation, self-inactivating lentiviral vector as in lentiviral vectors efficiently employed for gene contains all the necessary features for a safe and effi- therapy in murine models of β-thalassemia.12, 13 cient transgene integration into the target genome, A green FP and red (HcRed1) FP genes were cloned including the two modified viral LTRs (Long Terminal under the transcriptional control of human γ-globin Repeats) (essential for reverse transcription, integra- and β-globin promoters, respectively, downstream tion and gene expression), the packaging sequence the minimal LCR sequence still present in the pCCL.β- (necessary for the packaging of the RNA to be trans- globin.PGK.GFP.WPRE vector, according to the strat- ferred), the Rev Responsive Element (required for egy described in Figure 1. After removing from the RNA splicing and carriage out of the nuclear com- pCCL.β-globin.PGK.GFP.WPRE vector a 1 847 bp partment) and insulator elements, useful for isolation sequence containing PGK, GFP and WPRE, two dif- of the integrated construct from the chromatin near- ferent constructs were inserted, each consisting of a by, and for prevention of possible adverse effects.
reporter gene coding a fluorescent protein, under the The vector backbone also contains a GFP reporter control of a human globin gene promoter: gene, under the control of the strong phosphoglycerate Promβ.HcRed1 and Promγ.EGFP. kinase (PGK) promoter, together with the Woodchuck The two different fluorescent proteins, coded by Post-transcriptional Regulatory Element (WPRE) to genes contained in commercially available plasmids, ficient GFP expression. Finally, another can be discriminated thanks to their ability to gener- feature of the pCCL.β-globin.PGK.GFP.WPRE vector is ate fluorescence at two different wavelengths.
the human β-globin gene, under the control of both As for the nucleotide sequences of the globin gene its own promoter, and a minimal locus control region promoters, they were chosen in order to contain the (LCR) of the β-globin gene cluster. The LCR sequence major transcription regulatory elements, as describedspans about 3.2 kb and contains the functionally most elsewhere.8 For the β-globin promoter a 315 bp region, important sequences of the Hypersensitive Sites HS- immediately upstream the transcription initiation site, 2, HS-3, HS-4, which demonstrated to be necessary for was considered sufficient; on the contrary, the γ-glo- MINERVA BIOTECNOLOGICA CELLULAR SENSORS FOR β-THALASSEMIA THERAPY FACS analysis Figure 5.—Analysis of fluorescence levels of K562 cell clones, contai-ning the reporter construct pCCL.Promβ.HcRed1.Promγ.EGFP, after treatment with 150 µM hydroxyurea for five days. A) Example of qua- litative fluorescence analysis performed by using a fluorescence micro- scope (DMIL, Leica); B) example of histograms obtained by FACSanalysis of treated and untreated cells; C) comparison between median FACS analysis: median fold over control and mean folds over control obtained by analyses with the FACScan Victor analysis: mean fold over control (Becton Dickinson) and the Wallac 1 420 Victor3TM (Perkin Elmer),respectively.
bin promoter required a 1.4 kb region, in order to After these preliminary steps, the two reporter con- include a distal butyrate responsive-element.14 structs were sequentially inserted into the lentiviral Moreover, as proximity to LCR demonstrated to vector backbone. First, the Promβ.HcRed1 construct increase gene expression,15, 16 the β-globin promoter was cloned into the pCCL.β-globin vector, after diges- was placed between the LCR and the β-globulin pro- tion with the restriction endonucleases ClaI and XbaI moter, in order to obtain a lower γ promoter activity.8 (MBI Fermentas), to replace the normal β-globin gene The Promβ.HcRed1 construct was obtained from a (Figure 1B); then the Promγ.EGFP construct was pGL3.Basic (Promega) vector, after cloning the human cloned downstream the previous one by employing β-globin promoter, obtained from the pCCL.β-globin the restriction endonucleases BsiWI and MluI (MBI vector, and the HcRed1 gene, obtained from the com- Fermentas) (Figure 1E).
mercial plasmid pHcRed1-C1 (BD BiosciencesClontech) (Figure 1C). On the contrary, the Promγ. Production of the cellular model system EGFP construct was obtained from the pEGFP-C3plasmid (BD Biosciences Clontech), still containing the The cellular model system was produced according EGFP gene, after cloning the human γ-globin pro- to the strategy depicted in Figure 2. Human ery-moter obtained by PCR using normal human genom- throleukemic K562 cells were transfected with the ic DNA as template (Figure 1D).
pCCL.Promβ.HcRed1.Promγ.EGFP vector, by employ- MINERVA BIOTECNOLOGICA CELLULAR SENSORS FOR β-THALASSEMIA THERAPY ing cationic liposomes. Then, cells were cloned by TABLE II.—Comparison between fluorescence analyses performed limiting dilutions, in order to isolate cell clones stably by using FACScan (Becton Dickinson) and Wallac 1420Victor3TM (Perkin Elmer) instruments, after treatment of K562 containing the reporter construct, which should work cell clones containing the reporter construct with 150 µM as a cellular biosensor for HbF inducers screening. hydroxyurea for 5 days. The values obtained by FACS analy- In this system, increase of green EGFP signal would sis of 10000 cells by using the Cell Quest Pro software, inclu-de: median fold over control, the ratio between the median of be consistent with a γ-globin gene promoter driven fluorescence intensity values obtained by cells in the presence transcription activity; on the contrary, increase of the and in the absence of treatment, respectively; MPF% (Median far red FP signal would be associated with β-globin Peak Fluorescence %), expressing the increase % of the fluo- promoter activity. The fluorescence analysis can be rescence value caused by the inducer molecule. The valuesobtained by Victor analysis after lysis of 0.7×106 cells by using both qualitative, through the use of a fluorescence the Wallac 1420 Workstation software, are expressed as mean inverted microscope (DMIL, Leica), and quantitative, fold over control, the ratio between the mean relative fluore- by using instruments like Wallac 1420 Victor3TM (Perkin scence intensity values obtained by cells in the presence and inthe absence of treatment, respectively. Elmer) and FACScan (Becton Dickinson).
Following the described protocol, eight stably trans- fected clones were isolated (Figure 3). They were first Median fold over control Mean fold over control characterized by analysis of the basic fluorescencelevels. When examined under a fluorescence micro- scope, they all showed green fluorescence, even though at different levels, associated to low, but detectable, expression of the γ-globin promoter (Figure Clone 5 3A). This was confirmed by FACS and Victor analyses, giving median and mean fluorescence values, respec- tively, significantly different from those obtained bycontrol cells. Moreover, also the green fluorescencepeak of histograms obtained by FACS analysis wasalways shifted towards greater fluorescence values in able I). The values, even though differing depend- comparison with the control sample (Figure 3B).
ing on the clones and the analytical instrument Finally, the construct insertion was confirmed by employed, are however quite similar. For example, PCR amplification of genomic DNA extracted from FACS analysis gave a median fold over the control the eight selected clones, using primers specifically ranging from 2.62 to 4.41, also confirmed by MPF% hybridizing to the vector backbone: in all cases the values, expressing the percentage increase of fluo- expected 142 bp PCR band was obtained (Figure 3C).
rescence levels determined by the inducer molecule.
Moreover, also the FACS histograms showed an evi- Validation of the cellular biosensor dent shift of peaks of treated cells towards greaterfluorescence values, in comparison with the control The cellular biosensor was validated by treatment with sodium butyrate and hydroxyurea, two com- In conclusion, it is possible to state that all the eight pounds already known for their HbF inducing effects clones were significantly induced by sodium butyrate, on thalassemic patients. without evidence for growth or morphology alter- Sodium butyrate is a histone deacetylase inhibitor, able to stimulate γ-globin gene expression both in Similar results were obtained after the administra- vitro and in vivo.17 When administered to K562 clones, tion of hydroxyurea (HU), a ribonuclease reductase containing the reporter construct, at the concentra- inhibitor which blocks DNA synthesis and causes cell tion of 1.5 mM, it was found to stimulate a quantifiable death. HU is used as chemotherapeutic agent, as well increase of GFP signal, revealing a γ-globin promot- as for the treatment of sickle cell disease and β-tha- er induction. This could be noticed not only by pic- lassemia. HU was administered to K562 clones con- tures obtained using a fluorescence microscope taining the reporter construct, at the concentration of (Figure 4A), but also quantitatively by analyzing the 150 µM, and, after 5 days of treatment, the fluores- median and mean folds over control obtained by cence microscope analysis showed an evident increase Victor and FACS analyses, respectively (Figure 4C, of green fluorescence over the control. As found dur- MINERVA BIOTECNOLOGICA CELLULAR SENSORS FOR β-THALASSEMIA THERAPY ing experiments with butyrate, the level of induced In order to validate the experimental model, sodi- green fluorescence was variable in the different clones um butyrate and hydroxyurea were employed, two employed; no evidence for toxicity was found (Figure molecules well-known for their HbF induction activ- 5A). These conclusions were confirmed by quantita- ity, both in vitro and in vivo. Treatment of K562 cell tive analysis using FACS and Victor instruments: all clones carrying EGFP under the control of the γ-glo- clones were induced, with the FACS fluorescence bin gene promoter caused an evident, highly repro- peaks shifting towards higher values when compared ducible and quantifiable increase of green fluores- to controls (Figure 5B); it should be noted that medi- cence levels in all the treated cell clones, suggesting an folds and MPF% values were greater than those an inducer effect on the human γ-globin gene pro- obtained after the administration of sodium butyrate moter. Even though the different responses, all clones (Figure 5C, Table II). showed the inducing effect, demonstrating the biosen-sor ability in finding molecules able to stimulate γ-globin genes expression. As for the two different flu- orescence quantification methods employed, FACS There is general agreement that pharmacological- analysis is proposed as the method of choice, because ly mediated stimulation of human γ-globin gene of its ability to analyze single cellular events, giving aexpression and increase of production of fetal hemo- complete view of the fluorescence levels of cell pop- globin is a potential therapeutic approach in the exper- ulation analyzed. imental therapy of β-thalassemia and sickle cell ane-mia.1, 18, 19 For instance, hydroxyurea is used in the experimental therapy of these disorders, obtaining inabout 50% of the cases the conversion of patients 1. Fibach E, Burke LP, Schechter AN, Noguchi CT, Rodgers GP.
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novel HbF inducers appears to be relevant.19 4. Mischiati C, Sereni A, Lampronti I, Bianchi N, Borgatti M, Prus E et In order to screen potential HbF inducers several al. Rapamycin-mediated induction of gamma-globin mRNA accu-mulation in human erythroid cells. Br J Haematol 2004;126:612-21.
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ls with tallimustine. Br J Haematol 2001;113:951-61.
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Accumulation of gamma-globin mRNA in human erythroid cells these experimental systems are not suitable for high- treated with angelicin. Eur J Haematol 2003;71:189-95.
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From the Department of Orthopaedics, Clinical Sciences Lund, Lund University, Sweden The infected knee arthroplasty Anna Stefánsdóttir Anna Stefánsdóttir List of papers, 2 Results / Summary of papers, 20 Definitions and abbreviations, 3 Introduction, 4 Historical background, 4 The Swedish Knee Arthroplasty Register, 4 Definition of infected knee arthroplasty, 4