Research Article Gemcitabine and Cytosine Arabinoside Cytotoxicity: Association with Lymphoblastoid Cell Expression Liang Li,1 Brooke Fridley,2 Krishna Kalari,2 Gregory Jenkins,2 Anthony Batzler,2 Stephanie Safgren,1 Michelle Hildebrandt,1 Matthew Ames,1 Daniel Schaid,2 and Liewei Wang1 Departments of 1Molecular Pharmacology and Experimental Therapeutics and 2Health Sciences Research, Mayo Clinic, Rochester, Minnesota Abstract test the hypothesis that variation in basal gene expression might affect sensitivity or resistance to chemotherapy, we have used a Two cytidine analogues, gemcitabine (dFdC) and 1-B-D- Human Variation Panel lymphoblastoid cell line model system arabinofuranosylcytosine (AraC), show significant therapeutic consisting of 197 cell lines for which we obtained gene expression effect in a variety of cancers.However, response to these drugs data using Affymetrix U133 Plus 2.0 GeneChips and drug-related varies widely.Evidence from tumor biopsy samples shows that cytotoxicity phenotypes. Although their expression profiles are expression levels for genes involved in the cytidine transport, not identical with those of tumor cells, these cell lines provide metabolism, and bioactivation pathway contribute to this an opportunity to query the effect on drug response of common variation in response.In the present study, we set out to test variation across the genome, providing data that cannot be the hypothesis that variation in gene expression both within obtained with other model systems such as the NCI-60 cell lines. and outside of this ‘‘pathway’’ might influence sensitivity to Therefore, these Human Variation Panel cell lines can serve as an gemcitabine and AraC.Specifically, Affymetrix U133 Plus 2.0 initial screen to identify candidate genes for which variation in GeneChip and cytotoxicity assays were performed to obtain expression might contribute to variation in drug response basal mRNA expression and IC values for both drugs in 197 50 phenotypes. ethnically defined Human Variation Panel lymphoblastoid cell We used two cytidine analogues, gemcitabine (dFdC) and 1-h-D- lines.Genes with a high degree of association with IC values 50 arabinofuranosylcytosine (AraC), to determine whether individual were involved mainly in cell death, cancer, cell cycle, and variation in basal gene expression might influence drug sensitivity. nucleic acid metabolism pathways.We validated selected These drugs have been used to treat many cancers, and they share significant genes by performing real-time quantitative reverse similar chemical structures, metabolic pathways, and mechanisms transcription-PCR and selected two representative candidates, of action (5–8). Both are prodrugs that must be transported into NT5C3 (within the pathway) and FKBP5 (outside of the cells, activated by kinases to form active diphosphorylated and pathway), for functional validation.Those studies showed triphosphorylated metabolites, and inactivated by dephosphoryla- that down-regulation of NT5C3 and FKBP5 altered tumor cell tion. Seventeen genes are involved in this cytidine analogue sensitivity to both drugs.Our results suggest that cell-based ‘‘pathway’’ (Supplementary Table S1). The triphosphates, AraCTP model system studies, when combined with complementary and dFdCTP, can be incorporated into DNA, terminating DNA functional characterization, may help to identify biomarkers synthesis (8, 9). dFdCDP can also inhibit ribonucleotide reductases, for response to chemotherapy with these cytidine analogues. enzymes that catalyze the conversion of ribonucleotides to [Cancer Res 2008;68(17):7050–8] deoxyribonucleotidase (10, 11). Gemcitabine is used to treat solid tumors (6, 12), whereas AraC Introduction is a major component of the therapy of acute myelogenous Studies of gene expression may make it possible to identify leukemia (AML; refs. 6, 7, 13). Clinical response to both drugs varies biomarkers that will help predict clinical response to antineoplastic widely (8, 14, 15). Most previous studies have focused on variation drug therapy. Many of these drugs have narrow therapeutic in the expression of genes within the known cytidine analogue indexes. Therefore, it is crucial to identify biomarkers that might metabolism and activation pathway (1, 16, 17). However, very little help to maximize efficacy and minimize drug-related toxicity. Most information is available with regard to genes outside of that previous studies have focused on the relationship of expression pathway. Therefore, we have used these lymphoblastoid cell lines to signatures in tumor tissue to therapeutic response (1–3). However, explore the possible contribution of individual variation in basal individual variation in expression patterns controlled by germline gene expression to gemcitabine and AraC sensitivity. DNA can also play an important role in response. Genes encoding Specifically, drug cytotoxicity and basal expression array data proteins involved in drug transport, metabolism, activation, were obtained for 197 lymphoblastoid cell lines. Selected genes deactivation, or drug targets and downstream signaling pathways significantly associated with cytotoxicity were then validated could all potentially influence drug response phenotypes (4). To functionally. A series of functional analyses were performed for two candidate genes, NT5C3, within the cytidine analogue pathway, and FKBP5, outside of that pathway. Specific siRNA ‘‘knockdown’’ confirmed the results of the association study, and the functional Note: Supplementary data for this article are available at Cancer Research Online effects of both genes on response to gemcitabine and AraC was (http://cancerres.aacrjournals.org/). Requests for reprints: Liewei Wang, Department of Molecular Pharmacology and explored. Therefore, the use of these cell lines to identify Experimental Therapeutics, Mayo Clinic, 200 First Street Southwest, Rochester, MN pharmacogenomic candidate genes for cytidine analogue cytotox- 55905. Phone: 507-284-5264; Fax: 507-284-4455; E-mail: [email protected]. I2008 American Association for Cancer Research. icity resulted in novel hypotheses that can now be tested in clinical doi:10.1158/0008-5472.CAN-08-0405 translational studies. Cancer Res 2008; 68: (17). September 1, 2008 7050 www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2008 American Association for Cancer Research. Cytidine Cytotoxicity and Genome-Wide Expression Materials and Methods siRNA studies. Cells were grown to 30% to 50% confluence in 6-well plates, and lipofectamine RNAMAX reagent (Invitrogen) was used to perform the Cell lines. Lymphoblastoid cell lines from 60 Caucasian-American, transfections. 54 African-American, 60 Han Chinese-American, and 23 Centre d’ Etude du Western blot analysis. Western blot analysis was performed with lysates Polymorphisme Humain (CEPH; Caucasian-American) unrelated subjects from cells transiently expressing siRNA 48 h after transfection with control, were purchased from the Coriell Institute. Human SU86 pancreatic cancer NT5C3, FKBP5, NT5C2, or FKBP1A siRNA. Specifically, 30 Ag of protein was cells were a gift from Dr. Daniel D. Billadeau, Mayo Clinic, Rochester, MN. subjected to electrophoresis on 12% SDS-PAGE gels. Proteins were Human breast cancer MDA-MB-231 cells were obtained from the American transferred to polyvinylidene difluoride membranes that were incubated Type Culture Collection. overnight at 4jC with primary antibodies, followed by the secondary Drugs and cell proliferation assays. AraC was purchased from Sigma- antibody. Bands were detected with enhanced chemiluminescence (Amer- Aldrich, and gemcitabine was provided by Eli Lilly. Drugs were dissolved sham). Antibodies were obtained from GenWay Biotech, Abcam, and Novus À j in DMSO and were frozen at 20 C. Assays were performed in triplicate Biologicals. at each drug concentration using the CellTiter 96 AQueous Non-Radio- Intracellular gemcitabine and AraC metabolites. High performance active Cell Proliferation Assay (Promega Corporation) in 96-well plates liquid chromatography (HPLC) was used to measure intracellular AraCDP, Â 4 (Corning) at a density of 5 10 cells per well. One hour after plating, AraCTP, dFdCDP, and dFdCTP concentrations. Nucleotide extracts were A 10 L of gemcitabine (0.1 nmol/L–1 mmol/L) or AraC (1 nmol/L– prepared with a modification of the method of Van Haperen and colleagues 10 mmol/L) was added to the wells, and plates were read in a Safire2 (18) after treatment for 8 h with the average IC50 concentration for each microplate reader (Tecan AG) after 72-h incubations. Twelve randomly drug. Cells (5 Â 106) were then centrifuged and washed with ice-cold PBS, selected lymphoblastoid cell lines were used to repeat the cytotoxicity followed by resuspension in 135 AL PBS with 15 AL of 100 Amol/L AraCTP studies 3 mo later. Human tumor cell line cytotoxicity was determined in or dFdCTP as internal standards. Subsequently, 50 AL of 40% TCA was a similar fashion except the cells were incubated overnight before the added, followed by vortexing and centrifugation. The supernatant was addition of drug. neutralized with 400 AL of trioctylamine/trichlorotrifluoroethane (1:4). Expression array data. Total RNA was extracted using Qiagen RNeasy After centrifugation, the aqueous phase was subjected to HPLC using a Mini kits (QIAGEN, Inc.). RNA quality was tested using an Agilent 2100 ZirChrom SAX HPLC column with photo-diode array detection and Bioanalyzer, followed by hybridization to Affymetrix U133