Cancer cell lines

GCs-sensitive model of acute lymphoblastic leukemia (ALL), CEM-C7-14 and GC-resistant model of acute lymphoblastic leukemia (ALL), CEM-C1-15 cell lines were obtained from Brad E Thompson (The University of Texas, USA). These cell lines were derived from the parental line CCRF-CEM, grown in Dexamethasone to select for resistant and sensitive clones. Both cell lines were derived from 4 years Caucasian female suffering from acute lymphoblastic leukemia. CEM-C1-15 cells display a typical multiple drug resistance (MDR). CEM-C1-15 cell line is GCs -resistant clone obtained by growing CEM-C7-14 cells in dexamethasone (Medh et al., 1998). C7 cells are ALL cell line that undergoapoptosis when incubated with GCs while C1 are not sensitive to apoptosis initiated by GCs (Medh et al., 2003).

DT40 cells were kind gift from Professor Julian Sale (MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus). These DT40 cells are chicken lymphoblast cell line derived from chicken primary immune organ (bursa of fabricious) infected with

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avian lymphoid leukosis. Employing chicken DT40 B cell line is of importance due high replication efficiency and for easy monitoring of cell death, growth curve and gene expression (Winding and Berchtold, 2001), which serve as a prototype for experimental biology and molecular biology for the efficiency of homologous gene of interest (Molnar et al., 2014)

Epithelial cell lines; HACAT (Human immortalized keratinocytes primary adherent cell line), MCF-7(Human breast cancer adherent cell line) and BEAS2B (Human normal lung, bronchus epithelial cell line) were obtained from ATCC. Peripheral Blood Mononuclear Cells (PBMC), were kind gift from Dr Lucy Smyth, Lecturer in Human Physiology-

University of Salford. A limited number of the PBMCs was available due to experiments and the types of performed assays and ethics application details. Especially the choice of CEM-C7-14 and CEM-C1-15 cell line are good system for GR sensitivity/resistance. Disadvantage of using this system is that extrapolating from cell lines to patients is difficult. However, given thatno good mouse models of ALL are available and time consuming/cost prohibitive, this system is important tool to uncovering potential new and better therapeutic approaches.

2.2.1.1 Maintenance of the cells

Roswell park memorial institute (RPMI)-1640 growth medium (Sigma) supplemented with 10% heat-inactivated foetal calf serum (FCS) and 1% penicillin/streptomycin (Labtech), 2 mmol/L L-glutamine (Labtech) was used to maintain CEM-C7-14 and CEM- C1-15 cells.

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DT40 cells growth medium consisted of Dulbecco's Modified Eagle's Medium (DMEM) (ATCC® 30-2002™) supplemented with 7% heat-inactivated foetal calf serum (FCS) and 1% penicillin/streptomycin (Labtech), 2 mmol/L L-glutamine and chicken serum 3% (Sigma –Aldrich) supplemented with 50 µM of 2- mercaptoethanol from Sigma.

Epithelial cells HACAT and MCF-C7 were maintained in Dulbecco's Modified Eagle's Medium (DMEM) (ATCC® 30-2002™) supplemented with 10% FCS and 1% penicillin/streptomycin (Labtech). BEBM medium was used to grow BEAS-2B cells. Cells were transferred to media containing Dextran Coated Charcoal (DCC) treated FBS (from Cyclone) before treatment with compounds. PBMCs were defrosted from -80 freezers, fresh RPMI medium added, spun down using the centrifuge, then the medium replaced by RPMI medium with DCC then treatment added.

Cells were incubated at 37 oC in the incubator (Galaxy S -Wolf laboratories) in humidified atmosphere of 5% CO2.

2.2.1.2 Passaging of the cells

Passaging of the cells was carried out 3 times a week for CEM-C7-14, CEM-C1-15 and epithelial cells were spitted twice a week, while DT40 cells were sub cultured every 24- 48 h due to high proliferation rate. Cells were diluted in fresh medium to a density of about (0.3 – 1.0) × 106 cells/ml in either T 25 or T 75 flasks. Cells were inspected daily and counting was carried out using microscope Motif AE31 to ensure proper density.

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2.2.1.3 Counting of the cells

Cells were counted to determine the optimal density. 10 µL was taken directly from cell suspension or diluted with trypan blue (to count the viable cells) and placed on

disposable haemocytometer chamber (C-chips of dimensions 25 mm (W) x75 mm (L) x Thickness 1.6 mm from LabTech). Cells were examined under light microscope Motif AE 31 using 10X lens. The average of total number of cells was counted in the four large corner squares and cell concentration calculated according to this formula:

Total cells/ml=(Total cells counted/No. of squares) x dilution factor x 104 cells/ml The viable cells number was measured by Trypan blue exclusion staining based on the fact that dead cells only can take the dye and viable cells exclude it. 1:1 volume of cell suspension and 0.4% trypan blue dye (dilution factor is 2) were mixed in Eppendorf tube and vortexed then incubated for less than 3 min at room temperature, then the

indicated volume was taken and cells were counted following above mentioned protocol.

2.2.1.4 Freezing of the cells

To provide low passage numbers for the experiments, cells were cryopreserved by centrifugation at 1500 RPM for 5 mins, supernatant was removed, and then 2 ml sterile media consisting of 90% FCS and 10% DMSO, was added to the pelleted cells and mixed by pipetting. Afterwards the cells suspension was transferred into 2 sterile cryovials, 1ml each. Vials were immediately placed in a freezer box at -80 °C and for longer storage

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transferred into the Dewar containing liquid nitrogen.

2.2.1.5 Thawing of cells

Cells were defrosted by placing them in a warm water bath at 37 OC for less than one minute; the content was transferred to T25 sterile flask containing 4ml fresh media, mixed carefully and kept in incubator for 24 h. Next day, the cells were centrifuged and the media replaced to remove DMSO.