as an effective inducing agent for fetal hemoglobin and as an effective therapy for the treatment of sickle cell disease
Abstract: Sickle cell disease (SCD) affects millions worldwide and it arises because of a defect in the beta-globin gene. . Lab scale analysis of the potential of sodium butyrate as an efficient inducer of fetal hemoglobin (HbF) levels as an effective therapy in patients with sickle cell disease was performed. Experiments were performed using K562 cells treated with sodium butyrate for 24, 48 and 72 hours using power tools such as quantitative PCR, ELISA and FACS. In our experiment, we performed qPCR for K562 cells treated with sodium butyrate for 72 hours and performed ELISA and FACS on K562 cell cultures treated for 24 hours. Here, we report the results of our experiment on 24 hour sodium butyrate treated samples. Results indicate that sodium butyrate induces fetal hemoglobin production in the cells. Hence, sodium butyrate is an effective therapeutic agent for SCD patients and can be studied further on a large scale to evaluate its potency.
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Sickle Cell Disease (SCD) was first coined in 1910 by a dental student based on the observation on the peculiar appearance of the red blood corpuscles. . Linus Pauling was the first to hypothesize that in 1945 that the disease originates from a defect in the hemoglobin molecule. . Sickle cell disease which affects millions worldwide arises because of a genetic defect in the beta-globin gene, a protein component of hemoglobin. . This defect results in the red blood cells being deformed, clumped and broken apart. . The resulting clogged blood vessels damage kidneys, liver, spleen and lungs. .
This has led to a search for alternate ways to treat patients and helping them to lead a normal life. Human α-Globin and β-Globin genes are present on chromosomes 16 and 11 respectively. . In fetal life, the predominant form of hemoglobin expressed is HbF called fetal hemoglobin (α2γ2). . During the post natal period, HbF is gradually replaced by HbA (α2β2). . The clinical symptoms of the patients with the disease start manifesting after the switch from HbF to HbA. . The permanent cure for the disease is a bone marrow transplant to give recipients blood forming cells that form normal beta globin. . Such transplants are rare due to donor compatibility issues. .
Pharmacologic induction of HbF has been observed to be an effective therapeutic strategy for lessening the severity of the condition. . Some of the inducing agents studied were 5-azacytidine, hydroxyurea, butyrate and decitabine. . In this study, we analysed the effects of sodium butyrate on K562 cells. Sodium Butyrate (NaC3H7COO), a short chain fatty acid, is a histone deacetylase inhibitor and is produced in the colonic lumen due to the microbial degradation of dietary fibres. . Arginine butyrate has been found to induce HbF production in majority of the SCD patients. . Though butyrate is efficient, the difficulty lies in administering large volumes of the drug through venous catheters which is a major challenge. .
K562 cell line is an erythroleukemia cell line derived from a chronic myeloid leukemia patient in blast crisis. . These cells are multipotential, hematopoetic malignant cells that spontaneously differentiate into progenitors of erythrocytes, granulocytes and monocytes. . These cells are used for hemoglobin synthesis, tumorigenicity, differentiation, cell biology studies. .
In our experiment, we measured the efficiency of sodium butyrate to induce fetal hemoglobin in K562 cells. K562 cells were treated with 0.5M, 2M and 10M sodium butyrate for 24 and 72 hours with the untreated cells being used as the control. The fetal hemoglobin gene expression was evaluated using the techniques Quantitative Real Time PCR (qPCR), ELISA for protein quantification and Flourescence Activated Cell Sorting (FACS) to sort cell based on markers. First, we isolated RNA from K562 cells which were treated with 0.5M, 2M and 10M sodium butyrate and the untreated cells. The purity of the extracted RNA was determined. The RNA samples were then subjected to qPCR.
Real-Time Quantitative PCR or in short qPCR is a wonderful technology for accurate, sensitive and fast quantification of nucleic acids. . In quantitation PCR, a specific detection method allows quantification of the product. . The risk for contamination is very low as tubes do not need to be opened. . In classical PCR, the products of amplification have to be run on a gel for the detection of the product. . This step can be avoided in qPCR since this technique combines both amplification of the products with detection in a single tube. . Detection methods are based on changes in the fluorescence of the product which is proportional to the target increase. . In our experiment, the starting material is RNA hence we performed the reverse transcription of RNA to obtain cDNA (complementary DNA). This was done in a two step qRT-PCR reaction where reverse transcription and qPCR reaction were performed using 2 different sets of tubes. .
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Enzyme Linked ImmunoSorbent Assay, known as ELISA is a technique used to identify the presence and the quantity of a protein in a sample. . The antibody is coated onto a well and the amount of antigen that binds to the antibody can be calculated. . In our experiment, we used ELISA to determine fetal hemoglobin induction in response to sodium butyrate treatment of K562 cells. In this the wells were coated with sheep anti-Human Hemoglobin affinity purified antibody which bound to the fetal hemoglobin in the sample. TMB substrate was added and the enzyme reaction produced a yellow colour with sulphuric acid which was read at 450 nm to determine the quantity of HbF in the sample.
The Coomassie brilliant blue protein assay, known as the Bradford assay is widely used because of ease of performance, rapidity, relative sensitivity, and speciï¬city for proteins. . Coomassie Blue dye binds the protein and the absorbance is measured at 595 nm. . This is based on the principle that the absorbance maximum for an acidic solution of Coomassie Brilliant Blue shifts from 465nm to 595 nm when the binding occurs. . Both hydrophobic and ionic interactions stabilize the dye, causing a visible colour change. .
Fluorescence Activated Cell Sorting (FACS) is a useful technique to sort the proteins of multicellular organisms that are phenotypically different. . It is one the types of flow cytometry which can measure the physical and chemical properties of cells. . The cells are scanned by a laser and the light scattered by the cells reveal information about the size and other properties of the cells. . In our experiment, this technique was used to measure how much of fetal hemoglobin was expressed by the cells. In this approach, cells were passed through a laser beam. The cells were fluorescently tagged which are excites when laser beam passes through them and emit light of a longer wavelength than the light source. The fluorescent emission was measured after excitation.
These methods are power tools that can help to improvise research strategies to treat human diseases.
MATERIALS AND METHODS
K562 CELLS, Stat-60, chloroform, isopropanol, Oligo (dT) primers, RNase free water, magnesium chloride, dNTP mix, RNasin Ribo Inhibitor, ImProm-II RT, ImProm-II (5X) buffer, Sybergreen, primers BP274, BP276, BP291, BP292, coating buffer (0.05 M carbonate-Bicarbonate,pH 9.6), wash solution (50mM Tris, 0.14 M NaCl, 0.05% Tween 20,pH 8.0), blocking solution (50mM Tris, 0.14 M NaCl, 1% BSA, pH 8.0), sample diluent (50mM Tris, 0.14 M NaCl, 1% BSA, 0.05% Tween 20, pH 8.0), 3,3',5,5' Tetramethylbenzidine (TMB), 2M sulfuric acid, ethanol, propidium iodide (PI), Phosphate Buffer Saline (PBS).
K562 Cell Culture Treatment
K562 (Chronic Myeloid Leukemia Cell Line) was maintained in IMDM medium with 10% fetal bovine serum and antibiotics such as penicillin and streptomycin. The cells were counted and viability was measured with 0.4% trypan blue dye. The K562 cell line was treated with 0.5mM, 2mM and 10mM, incubated at 37C , 5% carbon dioxide for 24, 48 and 72 hours. The cells were then harvested for RNA extraction.
Quantitative Polymerase Chain Reaction
RNA Isolation: RNA was isolated from both treated and untreated K562 cell samples at 72 hours to identify genes that were either activated or repressed by sodium butyrate treatment to accomplish γ-globin and fetal hemoglobin induction. The cells were centrifuged in a 15 ml tube at 300 rpm for 5 minutes and the supernatant was removed. 1 ml of Stat-60 was added and the tube was vortexed. The tube was let stand for 5 minutes at room temperature. 200 µl of chloroform was added and the tube was vortexed and centrifuged for 15 minutes at 4C. The upper aqueous phase was transferred to new labeled tubes, 1:1 ratio of isopropanol was added and vortexed. The tube was placed in -70C freezer overnight. The tube was then spun for 20 minutes at 4C, the isopropanol was aspirated. The pellet was washed with 200 µl 70% ethanol, vortexed and spun for 1 minute. The ethanol was aspirated, let stand at room temperature for 5 minutes and the pellet was resuspended in 50 µl RNase free water. The RNA isolated was subjected to reverse transcription to generate cDNA molecules.
Reverse Transcription PCR: 1 µl Oligo(dT) primer, RNA and water were added at different concentrations of the treated and untreated samples to a tube to make up the final volume to 5 µl. The tube was vortexed and spun. The samples were kept on ice at all times. 225 µl of the master mix containing magnesium chloride, dNTP mix, RNasin Ribo Inhibitor, ImProm-II RT, ImProm-II (5X) buffer and RNase free water was prepared. 15 µl of the master mix was added to 5 µl of the RNA/oligo (dT) mix to make up the final volume upto 20 µl. Reverse Transcription was then performed on the samples using the parameters:
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Incubation at 25C for 5 minutes (Annealing).
Incubation at 42C for 60 minutes (Extension).
Incubation at 70C for 15 minutes (Termination).
The DNA was stored at -20C until ready for qPCR which was done the following week.
A. Gamma Globin qPCR: The Sybergreen master mix (Sybergreen, Primer BP274, Primer BP276, water) was prepared. The negative controls (R1, R2) and standards (R3-R10) were prepared. The standards were prepared with different dilutions. 20 µl of the Sybergreen Master mix was added to 5 µl of the unknown cDNA sample (U11-46) to make the total volume of each sample upto 25 µl. The samples were loaded in the 96-well plate. The primers used were:
γ-Globin: Forward (BP274): 5′-GGCAACCTGTCCTCTGCCTC-3′
Reverse (BP276): 5′-GAAATGGATTGCCAAAACGG-3′
B. GAPD qPCR: The Sybergreen master mix (Sybergreen, Primer BP291, Primer BP292, water) was prepared. The negative controls (R1, R2) and standards (R3-R10) were prepared. The standards were prepared with different dilutions. 20 µl of the Sybergreen Master mix was added to 5 µl of the unknown cDNA sample (U11-46) to make the total volume of each sample upto 25 µl. The samples were loaded in the 96-well plate. Amplification was done by using iCycler qPCR program from Bio-Rad. The primers used were:
GAPD: Forward (BP291): 5′-GAAGGTGAAGGTCGGAGT-3′
Reverse (BP292): 5′-GAAGATGGTGATGGGATTTC-3′
Enzyme Linked ImmunoSorbent Assay (ELISA)
K562 cell culture treated with sodium butyrate for 24 hours was used for this procedure.
A 4x8 ELISA plate was coated with 100 µl of coating antibody. The coating antibody used was Sheep anti-Human Hemoglobin affinity purified). The plate was incubated at room temperature for 45 minutes. The plate was then washed thrice with the wash solution. 200 µl of blocking solution was added to the wells in the plate and incubated at room temperature for 30 minutes. The plates were then washed thrice. Standards and 1:5 and 1:10 diluted samples were prepared.
100 µl of the sample or the standard was added to assigned wells.
After incubation at room temperature for 45 minutes, the wells were washed five times with the wash buffer. The HRP Detection antibody was diluted to 1:10000 and 100 µl of the antibody solution was transferred to the wells. After incubation at room temperature for 45 minutes, the wells were washed with the wash buffer five times. 100 µl of the TMB substrate solution was added to the wells and incubated at room temperature for 5 minutes. 100 µl of 2M sulfuric acid was added to the wells and the absorbance of the samples were read at 450 nm.
Bradford Assay: 2 µl of the samples and 498 µl of Bradford reagent were mixed to a 1:250 dilution. The absorbance of all the samples was read at 595 nm in the spectrophotometer.
Fluorescence Activated Cell Sorting (FACS)
The propidium iodide treated and untreated 24 hour samples were arranged in a 24-well plate.
The cells were transferred from the 24-well plate to 1.5 ml tube taking care that all the cells were transferred. The cells were spun at 2000 rpm at room temperature for 5 minutes. The medium was removed and 1 ml 1X PBS was added to the pellet and resuspended by inverting the tube several times. The cells were then spun at room temperature for 5 minutes. The PBS was discarded and the cells were resuspended in 0.3 ml of fresh cold 1X PBS. The tubes were vortexed gently while adding 0.7 ml cold ethanol to the cell suspension. The cell mixture was incubated on ice for 45 minutes. The fixed cells were pelleted by centrifugation for 10 minutes. The solution was discarded, 1 ml of cold PBS was added to resuspend the cells. The cells were pelleted by centrifugation for 5 minutes. The cell pellet was resuspended in 500 µl PI solution and incubated in ice for 1 hour. The cells were transferred to 5 ml FACS tubes and the samples were analyzed using FACS Calibur. The data was recorded.
Our sample consisted of 0.6 million cells as observed under the microscope. 85.71% of the cells were found to be viable after the cell count. After RNA extraction, the RNA samples were run on an agarose gel electrophoresis to determine the purity and degradation of the sample as seen in In Fig. 1. In lane 4, 3 bands can be observed which are the 3 RNA subunits 28S, 18S and 5S RNA. The purity of the sample was checked by UV Spectrophotometry. A260/280 ratio was found to be 1.6079. RNA concentration was 365.9098 µg/ml.
Ladder 1 2 3 4 5
Fig. 1. Gel Electrophoresis of the RNA samples were performed as seen in lanes 1-5. 3 bands were observed in each lane. In lane 4, the RNA subunits 28S, 18S and 5S were seen. The 5S subunit appeared to be degraded. In the other lanes, the RNA was found to be degraded, contaminated or present as bands.
Quantitative Polymer Chain Reaction
Gamma-globin cDNA was obtained by reverse transcription PCR (RT-PCR) of 72 hour sodium butyrate treated K562 cells. qPCR reaction on the samples with GAPD as the control was conducted and the results were tabulated. Table 1 shows the raw data for Gamma-globin and GAPD genes. SQ represents the standard quantity obtained by real time PCR reaction. Ct value for gamma-globin PCR reaction was 0.991 and PCR efficiency was 82.5%. Ct value for GAPD PCR reaction was 0.997 and PCR efficiency was 89.2 %.
Table. 1. Raw Data
The Gamma-globin/GAPD expression was normalized and mean, standard deviation, SEM (Standard Error of Mean), and the Fold Change were calculated (Table 2) and were plotted and compared on a bar graph. The mean (Fig. 2(a).) and the fold change (Fig. 2(b).) was plotted against the treatment of the samples and were compared.
Table. 2. Data Analysis-Gamma-globin Normalization
Fig. 2(a). Graph of mean vs the treatment of the cells which shows the variation in the expression with treatment. (b). Graph of the fold change of treated compared to the untreated versus the treatment of the cells. Cells treated with 2mM NaB appeared to induce a higher amount of fetal hemoglobin in the cells when compared to the untreated, 0.5mM and 10mM NaB treated samples.
Enzyme Linked ImmunoSorbent Assay (ELISA)
ELISA was then performed to determine the amount of HbF (antigen) present in the sample which bound to the antibody. Bradford Assay was used to calculate the entire protein content of the sample and used for normalization of the HbF content in cells. The absorbance of the 24 hour sodium butyrate treated samples was tabulated and averaged to a single value. (Table 3).
Table. 3. Summary of Total Hemoglobin ELISA Data
Standard Dilutions (ng/ml)
Duplicate 1 (Absorbance)
Duplicate 2 (Abs)
Unknown Sample Dilutions
The protein concentrations calculated by the Bradford Assay protocol were tabulated in Table. 4. Fetal hemoglobin levels were normalized with the total protein content in the cells and were tabulated in Table. 5. We used the values of the undiluted samples to calculate the concentrations of the diluted ones as the presence of BSA (Bovine Serum Albumin) led to an increase in absorbance and hence improper reading. Finally, a bar graph of normalized fetal hemoglobin levels in cells versus the concentrations was generated in Fig. 3.
Table. 4. Summary of protein concentrations obtained by Bradford Assay for unknown experimental samples.
0.5mM NaB undiluted
2mM NaB undiluted
10mM NaB undiluted
Table. 5. Normalized fetal hemoglobin levels for unknown experimental samples.
Normalized HbF levels
Fig. 3. A bar graph of normalized HbF levels versus the sample dilutions.
Fluorescence Activated Cell Sorting (FACS)
FACS of the 24 hour sodium butyrate treated samples was carried out and the results were reported. The untreated samples and the 2mM sodium butyrate treated samples were reported here and the remainder of the results will be reported in my batchmates' report.
Fig. 4(a). is a histogram with the detection of the red fluorescence plotted against the cell count for the propidium iodide untreated, sodium butyrate untreated samples. Fig. 4(b). is a histogram with the detection of the red fluorescence plotted against the cell count for the propidium iodide treated, sodium butyrate untreated samples. Fig. 4(c). is a histogram and a part of 4(b) zoomed to show the detection range and also the four gate markers: M1, M2, M3 and M4.
(a) (b) (c)
Fig. 4 (a). Histogram of the untreated sample, no PI treatment with the Flt-2 (detection of the red fluorescence emitted by the propidium iodem) as the x axis and cell count as the y axis. The detecting range is marked by a gate marker>=102. (b). Histogram of the untreated sample, PI treated with the Flt-2 as the x axis and cell count as the y axis. The detecting range is marked by a gate marker M1>=102. (c). Histogram zoomed in from (b) to the detection region of propidium iodide staining. 4 gate markers M1, M2, M3 and M4 are shown in the plot.
Fig. 5(a). is a histogram with the detection of the red fluorescence plotted against the cell count for the duplicated, propidium iodide untreated, sodium butyrate untreated samples. Fig. 5(b). is a histogram with the detection of the red fluorescence plotted against the cell count for the propidium iodide treated, sodium butyrate untreated samples. Fig. 5(c). is a histogram and a part of 5(b) zoomed to show the detection range and also the four gate markers: M1, M2, M3 and M4.
Fig 5(a). Histogram of the untreated duplicated sample with the Flt-2 (detection of the red fluorescence emitted by the propidium iodem) as the x axis and cell count as the y axis. The detecting range is marked by a gate marker>=102. (b). Histogram of the untreated sample with the Flt-2 as the x axis and cell count as the y axis. The detecting range is marked by a gate marker M1>=102. (c). Histogram zoomed in from (b) to the detection region of propidium iodide staining. 4 gate markers M1, M2, M3 and M4 are shown in the plot.
Fig. 6(a). is a histogram with the detection of the red fluorescence plotted against the cell count for the propidium iodide untreated, 2mM sodium butyrate treated samples. Fig. 6(b). is a histogram with the detection of the red fluorescence plotted against the cell count for the propidium iodide treated, 2mM sodium butyrate treated samples. Fig. 6(c). is a histogram and a part of 6(b) zoomed to show the detection range and also the four gate markers: M1, M2, M3 and M4.
Fig 6(a). Histogram of the 2mM treated, no PI sample with the Flt-2 (detection of the red fluorescence emitted by the propidium iodem) as the x axis and cell count as the y axis. The detecting range is marked by a gate marker>=102. (b). Histogram of 2mM NaB treated sample, PI treated with the Flt-2 as the x axis and cell count as the y axis. The detecting range is marked by a gate marker M1>=102. (c). Histogram zoomed in from (b) to the detection region of propidium iodide staining. 4 gate markers M1, M2, M3 and M4 are shown in the plot.
The background fluorescence of the cells, percentage of the cells that were PI positive and in various stages of the cell cycle were reported in Table. 6. The number within the brackets represents the value for the duplicated samples.
Table. 6. Summary table from the histogram statistical tables. The number within the brackets represents the value for the duplicated samples.
% of PI positive cells in the sample
% of dead cells in the sample
% cells in G0/G1phase in the sample
% of cells in S phase in the sample
% of cells in G2/M phase in the sample
Sickle cell disease originates from an abnormality in the hemoglobin molecule. . It affects millions worldwide. . The discovery of the molecular basis of sickle cell disease was an important landmark in molecular medicine. . The modern molecular and cell biology tools have refined our understanding of its physiology and have led to the discovery of new therapies. . A large number of clinical, and laboratory observations support the notion that fetal hemoglobin (HbF) administration can ameliorate the clinical severity of SCD. . It was proposed that pharmacological agents that alter the epigenetic configuration of the γ-globin genes may provide a viable therapeutic approach to the induction of Hb F. . This report presents sodium butyrate as an effective therapy for fetal hemoglobin induction in K562 cells. We used techniques such as qPCR, ELISA and FACS to analyse the effect of 0.5 mM, 2 mM and 10 mM concentrations of sodium butyrate.
We first extracted RNA from K562 cells to be used in quantitative PCR reaction. Cell viability was 85.71% which was good. As seen in Fig. 1., lane 4, RNA subunits could be clearly seen. Subunit 5 appeared to be degraded probably due to keeping the sample outside the ice bucket for a longer time than was necessary. A 260/280 ratio of 1.6079 indicates that the sample was probably contaminated with protein during the extraction procedure which lowered the ratio. .
Quantitative PCR results indicated that the mean expression of Gamma-globin/GAPD normalization increased with increase in sodium butyrate concentration. The fold change in expression compared to the untreated control, the standard deviation value show a progressive increase in gene expression , reaching an optimum value at 2mM NaB concentration. This could mean that a 2 mM concentration of NaB is an optimum concentration for Gamma-globin gene expression. As can be observed from the graph, Fig. 2(a) and (b), there was an increase in gene expression with an increase in NaB concentration, reaching an optimum at 2 mM NaB concentration.
ELISA was then performed to determine the amount of HbF (antigen) present in the sample which bound to the antibody. Bradford Assay was used to calculate the entire protein content of the sample and used for normalization of the HbF content in cells. We exchanged our 72 hour samples with the other group with 24 hour samples. An increase in fetal hemoglobin production with an increase in NaB concentration was observed which seemed to be consistent with qPCR results. Furthermore, the results of the use of two dilutions: 1:5 and 1:10 seemed to indicate that a 1:10 dilution induces a higher amount of fetal hemoglobin in the cells. Hence, a higher dilution and higher NaB concentration are effective inducers of fetal hemoglobin in cells.
The background fluorescence in samples, Table. 6., increased with an increase in NaB concentration. Since in an FACS experiment, the cells containing the protein of interest are tagged and fluoresce, this can be interpreted as an increase in HbF concentration with an increase in NaB treatment. There was an increase in the percentage of cells in G0/G1 phase (Table. 6) which is the initiation phase of the cell cycle with an increase in NaB treatment. A larger amount of the cells were in the G2/Mitosis phase indicating that NaB induces HbF concentration in the cells.
Hence, taken together, these results seemed to indicate that sodium butyrate is an effective therapeutic agent for inducing fetal hemoglobin in K562 cells. These results need to be evaluated further in large scale to be used for sickle cell anaemia patients and to help in ameliorating the effects of the disease. An earlier study on fetal hemoglobin effect showed that patients with high HbF levels had an improved life expectancy. . Fetal hemoglobin level is known to be relatively stable throughout life. . Adults who had lower HbF levels as children were likely to die earlier than those who had higher levels. . Hence, fetal hemoglobin should be considered as an important factor in selecting patients for bone marrow transplantation and for long term treatment with agents. . In another study, it was found that fetal hemoglobin has an inhibitory effect on the extent and kinetics of sickle cell hemoglobin polymerization. . It predicts that even small increases of fetal hemoglobin levels reduces the pain rate in patients and may ultimately improve survival. .