Identification of γ-globin-inducing compounds and extracts
The HS2 enhancer element of the LCR and proximal promoter sequences from the γ-globin gene [30
] were fused to the luciferase ‘reporter’ gene from H. pyralis [31
]. The TK-neo gene, present in cis
on the reporter plasmid, was used as the selectable marker for mammalian cell transfection. Human K562 cells, which display early erythroid and myeloid progenitor markers, were stably electroporated to express HS2γ-luciferase. Luciferase-‘tagged’ γ-globin YAC (γYAC) was constructed as previously described [32
] where the luciferase gene was recombined into YAC DNA 3′ of the γA
promoter within the context of the full β-globin locus. γYAC DNA was introduced into K562 cells and murine erythroleukemia (MEL) cells by protoplast fusion and cells selected for puromycin resistance. Individual clonal lines were evaluated by (i) basal luciferase expression; (ii) faithful integration of linear DNA as determined by Southern hybridization; and (iii) inducible regulation of the integrated reporter construct by hemin and 5-azacytidine (4 – 6 fold for HS2γ and >3 fold for γYAC). Expression levels from multiple K562 lines expressing HS2γ or γYAC genes were suitable for high throughput screening (HTS), while expression levels of γYAC in MEL-derived clones were insufficient.
Chemical compounds were synthesized as discrete compounds, dissolved in neat dimethyl sulfoxide (DMSO) and were screened on an individual basis at a concentration of ~12 uM. Fungal extracts were prepared by freeze drying the fermentation material, broth and mycelia together, followed by extraction with methanol. Extracts were dried and subjected to solvent fractionation to provide three crude fractions (water-methanol, chloroform and hexane) which were tested for biological activity at a 1:4 dilution of the starting culture volume (N/4). Active fractions were further purified by reverse-phase HPLC. Full characterization of select compounds was achieved using mass spectrometry and NMR techniques.
HS2γ/K562 and γYAC/K562 cells were plated at 10,000 cells per well in phenol red free RPMI-1640 media, 2% FCS (Hyclone), 20 mM l-glutamine, penicillin (100 units/ml), streptomycin (100 ug/ml), 20 mM HEPES, pH 7.4 in 96-well microtiter plates. Cells were induced with compound (~12 uM) or fungal extract (N/4) for a period of 24 hours. The final compound solvent concentration was 0.2% DMSO, which was used as the negative control. The positive control was 1 uM azacytidine. Upon completion of induction period, luciferase buffer was added to each well to achieve a final concentration of 100 mM Tris-acetate, pH 7.9, 20 mM magnesium acetate, 2 mM EGTA, 1% Brij 58, 100 mM 2-mercaptoethanol, 36 mM ATP, 150 uM luciferin. Cells were allowed to lyse for 5 min at room temperature after which the chemiluminescent signal was measured (Dynatech Lab ML1000- medium gain setting). A preliminary screen of approximately 5,000 compounds was performed, incorporating internal controls on each microtiter plate. These studies defined criteria to accept or reject high-throughput screen data, requiring that: 1) penicillic acid or 5-azacytidine induced HS2γ reporter gene expression >4 fold or γYAC expression >2 fold; 2) that the baseline signal was >0.05 luminometer units and; 3) that the coefficient of variation of controls on each microtiter plate was <20%.
MTT- (3-[4,5 Dimethylthiazol-2-yl-]-2,5 diphenyl tetrazolium bromide), a commonly used colorimetric substrate, was used to measure mitochondrial respiration in order to assess cell viability. Cells were plated as described for the luciferase reporter gene assays. After incubation with test compounds for 24 and 48 hrs, 20 ul of 3 mg/ml MTT was added and cells incubated for 2 hours at 37 °C, 5% CO2. One hundred microliters of 10% SDS was added to all wells for 4 hours at 37 °C to allow for lysis and solubilization of MTT. Mitochondrial conversion of MTT was assessed by measurement of light absorbance at 540 nM. Background (from the phosphate buffered saline control) was subtracted and the data expressed as % inhibition of cell growth.
HbF e.l.i.s.a.. of K562 hypotonic lysates
K562 cells were exposed in duplicate to compound for a period of 4 days. Cells were collected by centrifugation (1000 x g; 5 minutes), media aspirated, cells washed with phosphate buffered saline (PBS) and the cell pellet lyzed in ice cold H2O by vortexing for ~40s. Nuclei were removed by centrifugation (13,000 x g; 4 °C for 4 minutes). Cleared lysate was removed, protein concentration determined and extracts stored at − 70 °C. Horseradish peroxidase (HRP) conjugated monoclonal antibody to HbF (Isolabs/Wallace) was used for e.l.i.s.a. determination of HbF concentration. Anti HbF is approximately 100 times more reactive to HbF than HbA. Standard curves were generated using HbF standard (~10% HbF, 90% HbA) serially diluted 1:2 in 25 ug/ml BSA from 1 ug/ml HbF to 0.015 ug/ml HbF. 25 ug/ml BSA served as the assay background control. Hypotonic lysates were diluted 25 ug/ml. Protein samples were added in triplicate to Immulon-4 microtiter plates for colorimetric detection or Microlite II microtiter plates for chemiluminescent detection (Dynex Technologies), using an excess of 0.1M Na2HCO3 , pH 9.0 at room temperature for 2 hours. Plates were washed 4 times with 50 mM Tris pH 8.0, 150 mM NaCl and 0.05% Tween-20 (TST). Anti-HbF was diluted 1:1000– 1:2000 in TST containing 1 % BSA (Fraction V) and 100 ul was added to each well, incubated for 2 hrs at room temperature, washed 4 times with TST and HRP substrate added. For colorimetric detection, 100 ul of TMB substrate (Kirkegaard & Perry Laboratories) was added and incubated for 30 minutes, quenched by the addition of 20ul 4N H2SO4 and read at 450nm/630nm. For chemiluminescent detection 100 ul of complete LBA (Pierce Chemical Co.) was added per well and measured 2 hrs post substrate addition. Correlation coefficients of standard curves were 0.95 – 1.0 and not accepted if below 0.9.
Human primary blood progenitor culture
A two phase liquid culture system was used [33
] with modification. Human blood partially enriched for mononuclear cells (buffy coat) was diluted 1:1 with sterile PBS layed over 1.077 g/ml Ficoll (Invitrogen Corporation) and centrifuged at ~300 x g. Mononuclear cells at the Ficoll-aqueous interface were removed, diluted with PBS, collected by centrifugation (300 x g) and washed twice with PBS. Mononuclear cells containing erythroid progenitors from 2 units of blood were cultured in 80 mls ‘Phase I’ medium comprising: α modified Eagles media (αMEM; Invitrogen Corporation), 10% FCS (Stem Cell Technologies), 20 mM l-glutamine, penicillin (100 units/ml), streptomycin (100 ug/ml), human cytokines (R&D Systems Inc.) 10 ng/ml IL-3, 10 ng/ml IL-6, 10 ng/ml granulocyte colony stimulating factor (G-CSF), 10 ng/ml granulocyte-macrophage colony stimulating factor (GM-CSF), 20 ng/ml stem cell factor (SCF) and 1 ug/ml cyclosporin A (Cayman Chemical Corp.). Phase I culture was expanded for 5 – 7 days, collected by centrifugation, washed twice in αMEM and transferred to ‘Phase II’ medium comprising: αMEM, 10 – 30% FCS, 20 mM l-glutamine, penicillin (100 units/ml), streptomycin (100 ug/ml), 1U/ml erythropoietin (EPO), 10 ng/ml SCF, 1 uM dexamethasone, 10 uM β-mercaptoethanol. The effect of compound on HbF in primary cultures was assessed by exposing cells plated in Phase II media at 3 x 106
cells/ml, 5% CO2, 37degC, in a humidied incubator, to a range of compound concentrations for 4 days (corresponding to days 4 to 8 in Phase II).
K562 extract preparation and protein blotting
To measure histone acetylation, K562 cells (1 x 107 cells) were exposed to compounds for 24 hours. Cells were pelleted by centrifugation (500 x g, 5′), washed once with PBS followed by resuspension in 10 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT and 1.5 mM PMSF. HCl was added to 0.2 M for 30’ at 4degC. The extract was subjected to centrifugation at 11,000 x g, for 10’ at 4degC. The supernatant fraction was dialyzed against 0.1M acetic acid (once for 1 hour), followed by dialysis against water (three times). Cell extracts were subjected to SDS-PAGE, transferred to nitrocellulose membrane and incubated with anti-acetyl histone antibody (Upstate Biotechnology) for 2 hours at room temperature. Primary antibody incubation was followed by incubation with the appropriate HRP-conjugated secondary antibody for 1 hour at room temperature and chemiluminescent detection (ECL; Amersham Bioscience).
Stress gene profiling
HepG2 cells were stably transfected with individual reporter gene constructs employing the luciferase gene under the control of the stress-responsive promoters for jun, growth-arrest-and-DNA-damage 153 (GADD153), glutathione S-transferase Ya (GST Ya), heme mono-oxygenase (HMO), heat shock protein 70 (HSP70) or the hypoxia inducible factor 1 (HIF-1 RE), nuclear factor AT (NF-AT RE), nuclear factor kappa-B (NF-kB RE) or xenobiotic (XRE) response elements. Stably transfected clonal cell lines were isolated and selected for appropriate response to 2 or more known modulators of the particular promoter element. Assay conditions for each cell line were optimized for exposure time, serum concentration and cell number.
Cells were plated onto white 96-well microtiter plates precoated with collagen at either 10,000 (GADD153, HIF-1 RE) or 20,000 cells / well (JUN, GST Ya, HSP70, HMO, NF-AT RE, NF-kB RE, XRE) in 150 ml DMEM medium supplemented with L-glutamine, non-essential amino acids and either 1 % (GADD153, GST Ya, HIF1RE, HMO, NF-ATRE, NF-kBRE, XRE) or 10 % (JUN, HSP70) fetal calf serum. After 24 hr incubation (37 °C, 5% CO2) the cells were transferred to a fully robotic liquid and plate handling station where compounds were added in triplicate at 6 concentrations in a 5-fold dilution range. The plates were further incubated for either 6 hr (HMO), 8 hr (JUN, GADD153, GST Ya, NF-AT RE, NF-κB RE) 16 hr (HSP70, XRE) or 24 hr (GST Ya, assayed at two different time points) and luciferase assays performed.