Research Article Mapping Genes that Contribute to Daunorubicin- Induced Cytotoxicity Shiwei Duan,1 Wasim K. Bleibel,1 Rong Stephanie Huang,1 Sunita J. Shukla,2 Xiaolin Wu,1 Judith A. Badner,3 and M. Eileen Dolan1 Departments of 1Medicine, 2Human Genetics, and 3Psychiatry, The University of Chicago, Chicago, Illinois Abstract for dGdC-rich regions flanked by A/T basepairs (2). Daunorubicin Daunorubicin is an anthracycline antibiotic agent used in has also been shown to inhibit DNA topoisomersase II by trapping the treatment of hematopoietic malignancies. Toxicities DNA strand passage intermediates, eventually resulting in DNA single-strand and double-strand breaks. Recent studies have shown associated with this agent include myelosuppression and cardiotoxicity; however, the genes or genetic determinants that the formation of DNA-anthracycline complexes can signifi- that contribute to these toxicities are unknown. We present cantly modify the ability of the helicases to separate DNA into an unbiased genome-wide approach that incorporates herita- single strands in an ATP-dependent fashion, thereby hindering bility, whole-genome linkage analysis, and linkage-directed the process of strand separation and limiting replication (3). There association to uncover genetic variants contributing to the are also reports that this drug can inhibit protein kinase C sensitivity to daunorubicin-induced cytotoxicity. Cell growth pathways (4). Patients often initially respond but then relapse due inhibition in 324 Centre d’ Etude du Polymorphisme Humain to resistance mechanisms, such as P-170 glycoprotein–mediated lymphoblastoid cell lines (24 pedigrees) was evaluated follow- drug efflux, altered topoisomerase II activity, or overexpression of ing treatment with daunorubicin for 72 h. Heritability analysis bcl-2 (5). showed a significant genetic component contributing to the The cumulative and dose-dependent toxicities that have limited cytotoxic phenotypes (h2 = 0.18–0.63at 0.0125, 0.025, 0.05, 0.1, the usage of daunorubicin are myelosuppression, mucositis, and 0.2, and 1.0 Mmol/L daunorubicin and at the IC , the dose cardiotoxicity (6–8). The risk of anthracycline-induced cardiotox- 50 icity is 10% to 26% and is dependent primarily on dose (9, 10). required to inhibit 50% cell growth). Whole-genome linkage Anthracycline-induced cardiotoxicity is thought to be mediated scans at all drug concentrations and IC50 uncovered 11 regions with moderate peak LOD scores (>1.5), including 4q28.2 to through reactive oxygen species production. Experiments con- 4q32.3 with a maximum LOD score of 3.18. The quantitative ducted on rat cardiomyocytes have shown that corticosterone can transmission disequilibrium tests were done using 31,312 high- inhibit apoptosis induced by doxorubicin, a structuralanalogueof frequency single-nucleotide polymorphisms (SNP) located in daunorubicin. This effect was mediated by the regulation of the 1 LOD confidence interval of these 11 regions. Thirty genes multiple genes, including antioxidant/detoxification enzymes, were identified as significantly associated with daunorubicin- receptors, signaling molecules, and amino acid and protein À induced cytotoxicity (P V 2.0  10 4, false discovery rate V 0.1). synthesis (11). Furthermore, Yi et al. have identified significant Pathway and functional gene ontology analysis showed that gene expression changes in mice after doxorubicin treatment, including a series of genes that encode oxidative stress-related these genes were overrepresented in the phosphatidylinositol signaling system, axon guidance pathway, and GPI-anchored proteins, signaltransduction, and apoptotic proteins (12). Matrix proteins family. Our findings suggest that a proportion of metalloproteinases 2 and 9 expression levels were enhanced in mice susceptibility to daunorubicin-induced cytotoxicity may be after acute doxorubicin treatment (13). In humans, tumor necrosis a y controlled by genetic determinants and that analysis using factor and phospholipase C- 1 have been shown to be criticalin linkage-directed association studies with dense SNP markers doxorubicin-induced cardiotoxicity (14). The genes important in can be used to identify the genetic variants contributing to daunorubicin-induced cardiotoxicity have not been well studied. In this report, we used classic and modern genetic approaches to cytotoxicity. [Cancer Res 2007;67(11):5425–33] identify genes that contribute to daunorubicin-induced cytotox- icity. To this end, lymphoblastoid cell lines (LCLs) derived from Introduction large Centre d’ Etude du Polymorphisme Humain (CEPH) reference Daunorubicin is a chemotherapeutic agent used in the treatment pedigrees of Northern and Western European descent were used to of hematopoietic malignancies, such as acute lymphocytic and identify the extent to which heritable factors contribute to drug acute myelogenous leukemia, as well as some lymphomas and cytotoxicity. There have been candidate gene approaches to study breast cancer (1). There are severalproposed mechanisms of the cellular sensitivity of daunorubicin in multiple tumor cell lines daunorubicin, most notably DNA intercalation, with a preference (15–19). However, our approach uses whole-genome linkage analysis and linkage-directed association studies to facilitate identifying regions within the genome that harbor genes contrib- Note: Supplementary data for this article are available at Cancer Research Online uting to daunorubicin-induced cytotoxicity. (http://cancerres.aacrjournals.org/). A Pharmacogenetics of Anticancer Agents Research Group study (http:// pharmacogenetics.org). Requests for reprints: M. Eileen Dolan, Department of Medicine, University of Materials and Methods Chicago, 5841 S. Maryland Avenue, Box MC2115, Chicago, IL 60637. Phone: 773-702- Cell lines. LCLs derived from 24 Caucasian Utah CEPH families (1331, 4441; Fax: 773-702-0963; E-mail: [email protected]. I2007 American Association for Cancer Research. 1333, 1334, 1340, 1341, 1344, 1345, 1346, 1347, 1349, 1350, 1358, 1362, 1375, doi:10.1158/0008-5472.CAN-06-4431 1408, 1413, 1416, 1420, 1423, 1444, 1447, 1454, 1459, and 1463) were www.aacrjournals.org 5425 Cancer Res 2007; 67: (11). June 1, 2007 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2007 American Association for Cancer Research. Cancer Research purchased from the Coriell Institute for Medical Research4 (Camden, NJ). heterozygous markers (heterozygosity: 1% at <0.7, 7% at 0.7–0.8, 28% at Cell lines were maintained in RPMI 1640 (Mediatech) supplemented with 0.8–0.9, 64% at 0.9–1) were used for linkage mapping studies. 15% fetalbovine serum (HyClone)and 1% L-glutamine (Invitrogen). Cell Linkage analysis. The genotypic data and map distances were lines were passaged thrice per week at a concentration of 350,000 cells/mL downloaded from the CEPH Version 9 database and the Marshfield map j 8 and kept at a temperature of 37 C, with 5% CO2 and 95% humidity. database using error-checked markers. MERLIN was used to perform Drug. Daunorubicin (NSC-82151) was kindly provided by the Drug nonparametric linkage analysis because it is robust to nonnormal Synthesis and Chemistry Branch, Division of Cancer Treatment, National distributions. For quantitative traits, MERLIN uses the following definitions: Cancer Institute (NCI), Bethesda, MD. Cell cytotoxicity assay. Cell growth inhibition was evaluated at 2 SðvÞ¼f founder alleles SalleleðvÞ ; concentrations of 0, 0.0125, 0.025, 0.05, 0.1, 0.2, and 1.0 Amol/L daunorubicin. These concentrations were selected through assay optimization over a large SalleleðvÞ¼f all carriers of allele ðyi À lÞ: range of daunorubicin treatment concentrations. We chose the daunorubicin concentrations within the limits of our assay that best characterized the The score for each inheritance vector S(v) is calculated by summing squared sigmoid shape of cell growth inhibition. Daunorubicin was prepared in PBS scores for each founder allele. The score for each founder allele is calculated by (pH 7.4; Invitrogen) immediately before use. The cytotoxic effect of summing the mean deviates (yi À l) for all individuals who carry the founder daunorubicin on these CEPH cell lines was determined using the nontoxic allele, in which yi is the phenotype for individual i, l is the population mean, colorimetric-based assay, alamarBlue (Biosource). Cell viability was assessed and v is the list of individuals who carry a particular founder allele. Inheritance on exponentially growing LCLs by trypan blue dye exclusion using the Vi-Cell vectors are used to construct a likelihood ratio test for linkage. XR viability analyzer (Beckman Coulter). Cells (100 AL) with viabilities of Single-nucleotide polymorphisms. From the online CEU dataset in the  5  4 >85% were plated at a density of 1 10 cells/mL (1 10 cells per well), in HapMap project (release 21)9, 31,312 high-frequency SNPs covering 1,278 triplicate, in 96-well round-bottomed plates (Corning). After 24 h incuba- genes within the 1 LOD confidence intervalof linkageregions with LOD tion, cells were treated with either vehicle (media contains 0.1% PBS) or scores of >1.5 were retrieved. To prevent possible genotyping errors, we increasing concentrations of daunorubicin for 72 h. At 72-h incubation excluded the SNPs with Mendelian transmission errors. Remaining SNPs time, untreated cells were in exponential growth. AlamarBlue was added used were those with three genotypes and two counts per genotype in the 24 h before absorbance reading at wavelengths