Affymetrix GeneChip Human Genome HG-U133A [HG-U133A]

Influence of wild-type MLL on glucocorticoid sensitivity and response to DNA-damage in pediatric acute lymphoblastic leukemia.

Drug-Gene Modeling in Pediatric T-Cell Acute Lymphoblastic Leukemia Highlights Importance of 6-Mercaptopurine for Outcome

Glucocorticoid resistance in T-lineage acute lymphoblastic leukaemia is associated with a proliferative metabolism

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Baseline glucocorticoid resistance profiles were measured in a panel of 15 paediatric T-Cell acute lymphoblastic leukaemia cell lines and correlated with Affymetrix Gene expression to identify genes and pathways associated with drug resistance in T-ALL. The cell lines' resistance to Methylprednisolone (MPRED) and Dexamethasone (DEX) was then tested using the MTT assay and IC50 values (drug concentration lethal to 50% of cells) for each drug were calculated.

DEX IC50

MPRED IC50

Transcription profling by array of 15 untreated paediatric T-cell acute lymphoblastic leukaemia cell lines to identify genes associated with drug resistance

Glucocorticoid Resistance Gene Profiling in T-ALL Cell Lines

Baseline glucocorticoid resistance profiles were measured in a panel of 15 paediatric T-Cell acute lymphoblastic leukaemia cell lines and correlated with Affymetrix Gene expression to identify genes and pathways associated with drug resistance in T-ALL. The cell lines' resistance to Methylprednisolone (MPRED) and Dexamethasone (DEX) was then tested using the MTT assay and IC50 values (drug concentration lethal to 50% of cells) for each drug were calculated.


P-MTAB-27351

Cell lines were grown in RPMI-1640 supplemented with 2mM L-glutamine, 10nM 2-mercaptoethanol and 10-20% heat-inactivated fetal calf serum. All PER cell line media contained additional non-essential amino acids and pyruvate, whilst 300 units/ml interleukin-2 is required for growth of PER-427 and PER-487 (Kees et al., 2003). Cell lines were cultured in the absence of antibiotics; testing for mycoplasma was routinely performed by PCR, and immunophenotyping performed by indirect immunofluorescence and flow cytometry 

P-MTAB-27352

Expression assays were performed using untreated cells. Resistance was tested using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Alley et al., 1988). Cells in exponential growth phase were counted by trypan blue exclusion and seeded in fresh media at a density of 5x105 to 1.5x106 cells/ml in a 96-well plate in the presence or absence of each drug. Drugs were serially diluted in fresh media, with each drug concentration tested in triplicate. Culture plates were incubated for 4 days at 37¡C prior to the addition of 10_L of filter-sterilized MTT (5 mg/ml). Plates were re-incubated for 6 hours before addition of 100_L of acidified isopropyl alcohol solution to dissolve formazan crystals and measurement of absorbance at 590 nm. The IC50 (drug concentration lethal to 50% of cells) was used as the measure of drug resistance. Where 50% cytotoxicity was not achieved by even the highest dose, the IC50 was recorded as double the highest concentration tested.

P-MTAB-27353

Total RNA was extracted from untreated cells in exponential growth phase using a combination of TRIZOL Reagent (Invitrogen, Carlsbad, CA), RNeasy Mini kit (QIAGEN, Valencia, CA) and ethanol precipitation. The integrity of the RNA preparation was assessed using agarose gel electrophoresis and analysis on the Agilent 2100 Bioanalyzer

P-MTAB-27354

According to instructions for Affymetrix HG_U133A

P-MTAB-27355

P-MTAB-27356

Gene expression data were extracted and normalized using robust multi-array analysis (RMA) as referenced in E-TABM-125.

P-MTAB-27357

Normalised expression values were log 2 transformed

Transcription profling by array of 15 untreated paediatric T-cell acute lymphoblastic leukaemia cell lines to identify genes associated with drug resistance

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