Transcriptomic and functional pathways analysis of ascorbate-induced cytotoxicity and resistance of Burkitt lymphoma

Ascorbate is a pro-oxidant that generates hydrogen peroxide–dependent cytotoxity in cancer cells without adversely affecting normal cells. To determine the mechanistic basis for this phenotype, we selected Burkitt lymphoma cells resistant to ascorbate (JLPR cells) and their ascorbate-sensitive parental cells (JLPS cells). Compared with JLPS cells, the increased glucose uptake in JLPR cells (with upregulated glucose transporters, increased antioxidant enzyme activity, and altered cell cycling) conferred ascorbate–induced cytotoxicity and resistance. Transcriptomic profiles and function pathway analysis identified differentially expressed gene signatures for JLPR cells and JLPS cells, which differential expression levels of five genes (ATF5, CD79B, MHC, Myosin, and SAP18) in ascorbate-resistant cells were related to phosphoinositide 3 kinase, cdc42, DNA methylation and transcriptional repression, polyamine regulation, and integrin-linked kinase signaling pathways. These results suggested that coordinated changes occurred in JLPR cells to enable their survival when exposed to the cytotoxic pro-oxidant stress elicited by pharmacologic ascorbate treatment.

The 3-(4, 5 dimethylthiazol-2-yl)-2,5 tetrazolium bromide (MTT) assay was used to assess cytotoxicity. JLPS and JLPR cells were plated in 96-well plates at 10 4 cells/well and grown overnight in a 5% CO 2 incubator at 37°C. Next, the cells were treated with various concentrations of ascorbate or H 2 O 2 for 1 h. The cells were then collected, washed with PBS, and incubated in RPMI 1640 culture medium for an additional 24 h. The cells were then washed again with PBS three times and incubated in RPMI 1640 medium, then 10 μl of MTT (5 mg/ml) was added. After 4 h, 100 μl of DMSO was added to each well and measured at 570 nm.

Cell cycle analysis
Flow cytometry was used to determine cell cycle distribution. JLPS and JLPR cells (2.5 × 10 5 /ml) were treated with ascorbate or H 2 O 2 for 1 h. Then, the cells were washed with PBS, fixed in 95% ethanol, washed with 1% BSA in PBS, and resuspended in 1.0 g/ml of RNase. The cells were then stained with 50 μg/ml propidium iodide and analyzed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). Flow cytometry data were analyzed using the ModFit DNA analysis software program (Verity Software House, USA).

RNA purification
RNA was puried from the JLPS and JLPR cells using Trizol LS reagent (Invitrogen, Carlsbad, CA, USA) and the RNeasy mini kit (Qiagen, Valencia, CA, USA). The quality of the purified RNA was assessed using an Agilent 2100 bioanalyzer (Agilent Technologies, Waldbronn, DE, USA).

Quantitative real-time polymerase chain reaction analysis
Total RNA (1 μg) was reverse transcribed to cDNA using the First Strand cDNA synthesis kit (Invitrogen). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using the ABI Prism 7900 HT sequence detection system and the Taqman Universal PCR master mix (both from Applied Biosystems, Foster City, CA, USA). qPCR results were analyzed using the Sequence Detector software program version 2.0 (Applied Biosystems, Grand Island, NY) and normalized using 18S ribosomal RNA.

Microarray data analysis
The microarray images were analyzed using the GenePix software program version 5.1 (Molecular Devices, Sunnyvale, CA). The resultant gene lists and associated expression values were loaded into the NCI Microarray Facility mAdb database. We calculated the mean log 2 -transformed ratio of genes expressed in JLPR cells to that in JLPS cells for the results from experiments performed in triplicate. The mean values were calculated by taking the antilog as the ratio of the gene expression measures of resistant cells to those of their parental cells.

Statistical analysis
Statistical values are presented as means ± standard deviations. The Student t-test was used to assess differences between groups. Results were considered significant at P < 0.05.