Introduction
Hemoglobin is an oxygen carrying transport protein found invariably in all the vertebrates including a few invertebrates. The major function of hemoglobin is in the transport of oxygen across tissues where oxygen ultimately aids in cellular respiration. Hemoglobin is a tetramer consisting of four protein subunits, in particular in humans they exist as HbA consisting of two alpha and 2 beta chains. Given its significance, it is important to understand the mechanisms and regulations underlying its production and the role of its variants at various stages of development in humans.
The chromosome 16 in humans, harbors both the alpha and the beta locus. The alpha locus encodes for the two alpha chains as well as the zeta chain whereas the beta locus encodes for the epsilon chain, the 2 gamma chains, delta chain and the beta chain. Regulation of alpha locus is controlled by the HS-40 enhance elements whereas regulation at the beta locus is by the locus control regions upstream of the beta gene and is complex in comparison given the switch that takes places at the three prominent stages of development. Embryonic hemoglobin(HbE) is made up of two alpha 2 and two zeta chains. There is a switch around 6 weeks of gestation from zeta to delta and the fetal hemoglobin(HbF) is made up of two alpha and 2 delta chains. There are two variants to the delta chain itself due to a mutation from G – A, hence the fetal hemoglobin is predominantly a heterogeneous tetramer. After birth there is a switch from delta to beta and around 10 months after birth the levels of fetal hemoglobin declines to <1%.
Often there are mutations associated with the beta globin gene either from A-T in both the alleles resulting the polymerization of BS resulting in sickle cell anemia or in one of the alleles or a mutation from GAG-AAG resulting in beta thalassemia.
Given the functional advantage of HbF having a higher oxygen affinity in comparison to HbA and also its role in the stability of HbS polymerization, the reactivation of gamma globin gene to increase HbF synthesis has opened doors for therapeutic options in that direction.
Research had first started with Hydroxyurea which has been the only drug till date to be approved for the treatment of sick cell disease but it wasn’t found to be effective in all SCD patients and also conferring to carcinogenicity upon long term treatments. Apart from hydroxyurea, studies was then done on cytotoxic agents, DNA methyltransferase inhibitors , histone deacetylase inhibitors, butyrate and short chain fatty acids.
Based on previous studies, our study questions the inducible properties of sodium butyrate , gemcitabine, aminolevulinic acid, succinylacetone and ALA in the induction of gamma globin in KU812 cell lines and hence identify the plausible mechanism based on their intrinsic properties. These studies may also answer the underlying mechanisms in the production of gamma globin and the regulation which thereby influences the reversible switch to the expression of gamma globin gene.
The major advantage over the use of KU812 cell lines is its property of spontaneous differentiation which have been previously studied to positively express markers for hematopoietic differentiation upon serum starvation. Although it would be useful to study the induction of gamma globin gene in primary erythroid cells, it is not possible to translate them into large scale drug screening. Given this barrier, it is more advantageous to use leukemia cell lines to study induction properties.
The 5 agents that have been used in this study are SA, ALA, SB and DT. Each of these agent has a significance behind the question to study their inducible properties. ALA is an important precursor of heme pathway and hence upon induction, gamma globin can be probably produced. Succinyl acetone(SA) is a major inhibitor of ALA and hence its inhibition can subsequently inhibit heme synthesis. Sodium butyrate(SB) falls under the class of histone deacetylase inhibitors and hence any kind of epigenetic regulation of this switch can be reversed resulting in the production of gamma globin.
Materials and methods
Culture of KU812 cell lines
Human leukemia KU812 cells were obtained from American Type Culture Collection and was cultured in IMDM media supplemented with 10% FBS along with Penicillin, Streptomycin and L-glutamine and incubated at 37C and 5% CO2. The cells were then seeded in a 6 well plate with a seeding density of 2 million cells/well. To study gamma globin induction, the cells were treated with 4 different agents ALA, SA, DT and SB at varying concentrations of 1.0mM ALA, 2.0mM ALA, 1.0mM SA, 1uM DT and 1.0mM SB along with a control well.
Protein extraction:
The cells were then collected and harvested at day 4 by centrifugation following the wash steps twice with phosphate buffered saline (10X) and were then subjected to lysis by the addition of lysis buffer (25mM HEPES pH 7.9, 0.3M NaCl, 1.5mM MgCl2, 0.5% Triton-X 100, 3mM DTT, Protease Inhibitors Cocktail [Roche, Cat# 05892970001] and Phosphate Inhibitors Cocktail [Roche, Cat# 04906845001]) and the whole cell extracts were isolated after incubation and centrifugation. The collected lysates were then stored at 4C until further use.
Total Protein quantification:
Using BSA as a protein standard, prepare several dilutions of the protein to be able to construct the protein standard curve. Also make 10X dilutions of the stored whole cell lysates
Western blot:
For fractionation by SDS-PAGE, the whole cell lysates were run on a 4–20% Mini-PROTEAN TGX Gel, 10-well(Bio-Rad) for around 30 minutes. The gel was then stained in 2, 2, 2-tricholorethanol and was incubated for 5 minutes. After incubation the gel was viewed under the Bio-Rad molecular imager gel doc system. The gel was then transferred to a PVDF (Polyvinylidene difluoride) membrane (Amber Biosciences) and was transferred using a blot transfer tank(Bio-Rad) for about 30 minutes. The transfer was confirmed by staining the membrane in Ponceau stain (0.1%(w/v) Ponceau S in 5%(v/v) acetic acid) and then washing it with TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20). For immunodetection, the membrane was blocked with 5% nonfat milk with TBST for around 30 minutes. After the 30-minute incubation, the membrane was further washed thrice for 10 minutes with TBST and then incubated with primary antibodies against gamma globin antibody (1:1000 dilution, Hemoglobin [51-7]: sc-21756) and actin antibody(1:1000 dilution, Anti-Actin, Clone 4, Cat. #MAB1501) for 1-2 hours or overnight. After the incubation, wash the membrane twice with TBST and follow it up with secondary incubation by incubating the membrane with goat-anti-mouse Horse radish peroxidase conjugated (1:1000 dilution, Cat. #31432, Thermo Scientific) for 1 hour. After incubation, wash the membrane 4 times with TBST. The bands were then detected by developing the membrane with the ECL system using the Amersham ECL Prime Western Blotting Detection Reagent kit and viewed using Bio-Rad molecular imager gel doc system.