Heritable and non-genetic factors as variables of pharmacologic phenotypes in lymphoblastoid cell lines

Abstract

Publicly available genetic and expression data on lymphoblastoid cell lines (LCLs) make them a unique resource for understanding the genetic underpinnings of pharmacological outcomes and disease. LCLs have been used for pharmacogenomic discovery and validation of clinical findings associated with drug response. However, variation in cellular growth rate, baseline Epstein-Barr virus (EBV) copy number and ATP levels can all be confounders in such studies. Our objective is to better define confounding variables that affect pharmacological end points in LCLs. To this end, we evaluated the effect of these three variables on drug-induced cytotoxicity in LCLs. The drugs evaluated included daunorubicin, etoposide, carboplatin, cisplatin, cytarabine, pemetrexed, 5'-deoxyfluorouridine, vorinostat, methotrexate, 6-mercaptopurine, and 5-fluorouracil. Baseline ATP or EBV copy number were not significantly correlated with cellular growth rate or drug-induced cytotoxicity. In contrast, cellular growth rate and drug-induced cytotoxicity were significantly, directly related for all drugs except vorinostat. Importantly, cellular growth rate is under appreciable genetic influence (h²=0.30-0.39) with five suggestive linkage regions across the genome. Not surprisingly, a percentage of SNPs that significantly associate with drug-induced cytotoxicity also associate with cellular growth rate (P ≤ 0.0001). Studies using LCLs for pharmacologic outcomes should therefore consider that a portion of the genetic variation explaining drug-induced cytotoxicity is mediated via heritable effects on growth rate.

Figure 1

Growth rate between and within passages. Growth rate estimates from the same lines were evaluated for correlation across different passage times. (a) Plots of the estimates of growth rate at 72 h for the same cell lines within the same passage and treatment time using the Asian HapMap cells. The r² correlation is 0.89 with a slope of 0.80. (b) Plots of the estimates of growth rate at 72 h for the same cell lines across different passages, with years serving as a proxy for different passages using the CEU and YRI HapMap cells. The triangles represent the YRI lines whereas the squares represent the CEU lines. There was no difference across the two populations. The r² correlation is 0.274 with a slope of 0.48.

Figure 2

Correlation between growth rate and baseline EBV copy number. (a) Shows growth rate estimates calculated from alamarBlue growth inhibition assay correlated against baseline EBV copy number in 107 unrelated HapMap lymphoblastoid cell lines. The correlation (r²=0.00969) is not significant (P=0.30). (b) Uses the same growth rate estimates to show no significant relationship with baseline ATP levels as measured in Choy et al.

Figure 3

Genome-wide linkage scan for growth rate in lymphoblastoid cell lines. Growth rate was calculated from alamarBlue 72 h growth inhibition experiments using cell lines from the CEPH large pedigrees. Experiment 1 used 444 individuals from 34 families and experiments 2 and 3 used 328 individuals in 24 families. Five peaks reached a LOD score of 1.5, genome-wide suggestive significance.