Gemcitabine and cytosine arabinoside cytotoxicity: association with lymphoblastoid cell expression

Abstract

Two cytidine analogues, gemcitabine (dFdC) and 1-beta-d-arabinofuranosylcytosine (AraC), show significant therapeutic effect in a variety of cancers. However, response to these drugs varies widely. Evidence from tumor biopsy samples shows that expression levels for genes involved in the cytidine transport, metabolism, and bioactivation pathway contribute to this variation in response. In the present study, we set out to test the hypothesis that variation in gene expression both within and outside of this "pathway" might influence sensitivity to gemcitabine and AraC. Specifically, Affymetrix U133 Plus 2.0 GeneChip and cytotoxicity assays were performed to obtain basal mRNA expression and IC(50) values for both drugs in 197 ethnically defined Human Variation Panel lymphoblastoid cell lines. Genes with a high degree of association with IC(50) values were involved mainly in cell death, cancer, cell cycle, and nucleic acid metabolism pathways. We validated selected significant genes by performing real-time quantitative reverse transcription-PCR and selected two representative candidates, NT5C3 (within the pathway) and FKBP5 (outside of the pathway), for functional validation. Those studies showed that down-regulation of NT5C3 and FKBP5 altered tumor cell sensitivity to both drugs. Our results suggest that cell-based model system studies, when combined with complementary functional characterization, may help to identify biomarkers for response to chemotherapy with these cytidine analogues.

Figure 1

Association between expression array data and IC₅₀ values for gemcitabine and AraC. Each dot on the y-axis represents the –log₁₀ (p-value) for the probe set with the lowest p-value for each gene. Probe sets are plotted on the x-axis with regard to the chromosomal location of their genes. Red dots represent probe sets for genes listed in Supplementary Table 1 that encode proteins within the cytidine analogue metabolism and target pathway.

Figure 2

NT5C3 functional validation in two tumor cell lines. (A) Western blot analyses showing significantly decreased levels of NT5C3 protein in SU86 pancreatic cancer and MDA-MB-231 breast cancer cells after treatment with specific siRNAs. The “insert” shows average levels of protein as a percentage of control. Error bars represent SEM values for 3 experiments. (B) SU86 and MDA-MB-231 cytotoxicity. Both cell lines were sensitized to gemcitabine and AraC as determined by MTS assay after the down regulation of NT5C3 gene expression. Error bars represent SEM values for 3 independent experiments. (C) Levels of intracellular phosphorylated gemcitabine and AraC metabolites were correlated with NT5C3 gene expression in 14 randomly selected sensitive and resistant lymphoblastoid cell lines. _[S2]Rp and p-values for metabolites, with expression adjusted for IC₅₀, were R_AraCDP = −0.50, p_AraCDP = 0.084; R_AraCTP = −0.65, p_AraCTP = 0.016; R_GemDP = −0.49, p_GemDP = 0.045; and R_GemTP = −0.52, p_GemTP = 0.033. _[S3]

Figure 3

FKBP5 functional validation in two tumor cell lines. (A) Western blot analyses showing significantly decreased levels of FKBP5 protein in SU86 pancreatic cancer and MDA-MB-231 breast cancer cells after treatment with FKBP5-specific siRNA. The “insert” shows average levels of expressed protein as a percentage of control. Error bars represent SEM values for 3 experiments. (B) SU86 and MDA-MB-231 cytotoxicity. Both cell lines became more resistant to gemcitabine and AraC as determined by MTS cytotoxicity assay after the down regulation of FKBP5. Error bars represent SEM values for 3 independent experiments.

Figure 4

Caspase activity in SU86 and MDA-MB-231 cells after FKBP5 siRNA treatment. Caspase activity was measured using the Caspase-Glo® 3/7 activity assay in (A, C) for MDA-MB-231 cells and (B, D) for SU86 cells treated with FKBP5 siRNA in the presence of (A, B) gemcitabine or (C, D) AraC. Error bars represent SEM values for 3 independent experiments.