Computational protocol: Tumor-selective use of DNA base excision repair inhibition in pancreatic cancer using the NQO1 bioactivatable drug, β-lapachone

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Protocol publication

[…] For TUNEL analyses, cells were co-treated with ß-lap, MeOX, or ß-lap + MeOX for 2 h. Drug-containing media were removed and cells incubated in fresh complete media for 48 h. Cells were trypsinized, and both adherent and floating cells collected and washed in 1% BSA in PBS. After fixing cells in 70% ethanol, samples were washed and re-suspended in BSA/PBS buffer containing propidium iodine and saponin. Cells were analyzed on a FACSAria (BD Biosciences, San Jose, CA) and cell cycle distributions were modeled and calculated in FlowJo. [...] Gene expression data series were retrieved from the Gene Expression Omnibus (GEO) database subject to the following criteria: study included pancreatic cancer tumor or cell lines of pancreatic origin, more than 10 samples in the full study, processed using the GeneChip Human Genome U133 Plus 2.0 expression array. A total of 11 series met these criteria and were included in the cohort for analysis: GSE2109, GSE8332, GSE9599, GSE15471, GSE16515, GSE16648, GSE17891, GSE21654, GSE22337, GSE22780, GSE32676. The assembled cohort included 164 pancreatic tumor samples, 69 normal pancreas (n = 233) and 73 pancreas cell line specimens, for a total of 306 specimens. Within this assembled cohort were 59 matched-pair specimens from three independent studies, in which biopsies were taken from both tumor and adjacent normal lung for each patient studied. The specimen data files included in the cohort were downloaded as raw CEL files for post-processing as indicated, following the standard gene expression data preparation workflow. We used the R package aroma.affymetrix, which uses persistent memory to allow analyses of very large datasets. Data were processed using the linear model from RMA, then fit robustly using probe level models as described. Probe level models were fit to RMA-background corrected and quantile normalized data to obtain gene-level summaries. Gene-level summarization used the standard CDF provided by Affymetrix. The Welch’s t-test for unequal variance was used to compute p-values for the difference in the means. All analyses were performed in R. Statistical tests were performed using base R statistical functions, graphics were generated using the ggplot2 graphics package. […]

Pipeline specifications

Software tools FlowJo, Aroma.affymetrix, Ggplot2
Databases GEO
Applications Miscellaneous, Flow cytometry
Organisms Mus musculus, Homo sapiens/Mus musculus xenograft
Diseases Breast Neoplasms, Neoplasms, Pancreatic Neoplasms, Carcinoma, Pancreatic Ductal
Chemicals Dicumarol, Hydrogen Peroxide, NAD, NADP, Poly Adenosine Diphosphate Ribose