Pharmacogenomic Identification of Targets for Adjuvant Therapy with the Topoisomerase Poison Camptothecin
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[CANCER RESEARCH 64, 2096–2104, March 15, 2004] Pharmacogenomic Identification of Targets for Adjuvant Therapy with the Topoisomerase Poison Camptothecin Jonathan P. Carson, Nianyi Zhang, Garrett M. Frampton, Norman P. Gerry, Marc E. Lenburg, and Michael F. Christman Department of Genetics and Genomics, Boston University School of Medicine, Boston, Massachusetts ABSTRACT treatment (7). Moreover, it remains unclear whether such information could be of therapeutic value. In the following study, we present data The response of tumor cells to the unusual form of DNA damage caused generated using the Affymetrix U133A chip (22,283 probe sets) from by topoisomerase poisons such as camptothecin (CPT) is poorly under- total RNA collected from HeLa cells treated with CPT at a dose (10 stood, and knowledge regarding which drugs can be effectively combined with CPT is lacking. To better understand the response of tumor cells to M) and time (8 h) sufficient to induce rapid apoptosis. We observed CPT and to identify potential targets for adjuvant therapy, we examined significant 3-fold changes in expression of 683 genes, including novel global changes in mRNA abundance in HeLa cells after CPT treatment transcripts and several putative p53-regulated genes. Consistent with using Affymetrix U133A GeneChips, which include all annotated human the induction of certain p53-responsive genes, we observed increased genes (22,283 probe sets). Statistical analysis of the data using a Bayesian/ protein levels of p53, phosphorylation on serine 15, and S-phase arrest Cyber t test and a modified Benjamini and Hochberg correction for after CPT treatment. Moreover, we detected striking evidence of multiple hypotheses testing identified 188 probe sets that are induced and nuclear factor (NF)-B transactivation and epidermal growth factor 495 that are repressed 8 h after CPT treatment at a False Discovery Rate receptor (EGFR) survival signaling (via HB-EGF). We reasoned that of <0.05 and a minimum 3-fold change. This pharmacogenomic approach changes in the gene expression pattern might represent regulation of led us to identify two pathways that are CPT induced: (a) the epidermal pathways important for CPT survival. The data were used to generate growth factor receptor; and (b) nuclear factor- B-regulated antiapoptotic factors. Experiments using HeLa cells in our lab and prior animal model the hypothesis that inhibition of the NF- B and EGFR pathways studies performed elsewhere confirm that inhibitors of these respective would increase the sensitivity of HeLa cells to CPT. Administration of pathways super-additively enhance CPT’s cytotoxicity, suggesting their an inhibitor of this pathway, AG1478, yielded a superadditive in- potential as targets for adjuvant therapy with CPT. crease in apoptosis when administered in combinations with CPT. Inhibition of NF-B signaling has been shown to dramatically in- crease tumor cell sensitivity to CPT-11 (8, 9). These results suggest INTRODUCTION both that CPT may be effective in mobilizing a p53 response in cervical carcinomas and that data from microarray experiments can The ability to induce apoptosis in tumor cells is critical to elicit a form the basis for rational hypotheses in the treatment of cancer. positive response to cytotoxic chemotherapy (1). In recent years, the characterization of the complete human transcriptome has enabled investigators to observe the changes in gene expression that accom- MATERIALS AND METHODS pany programmed cell death induced by diverse insults. This molec- ular profiling of gene expression from tumor biopsies has begun to RNA Isolation. Total RNA was extracted from HeLa cells plated 24 h previously at a density of ϳ40,000 cells/cm2 (ϳ75% confluence) using an yield diagnostic and prognostic information of unprecedented accu- RNAeasy mini-kit (Qiagen, Valencia, CA). racy (2). Chemicals. All chemicals and reagents, unless otherwise specified, were Camptothecin (CPT) is a plant alkaloid derived from the tree, purchased from Sigma-Aldrich (St. Louis, MO). Camptotheca acuminata. Its derivatives have shown promise in clin- Cell Culture. HeLa cells were purchased from the American Type Culture ical trials for use against small cell lung carcinoma (3) and other Collection (Manassas, VA) and maintained at 37°C in 5% CO2 in DMEM malignancies, including cervical cancer (4). The compound exhibits (Mediatech, Herndon, VA) containing 10% fetal bovine serum supplemented high specificity for topoisomerase I (topo I), which it inhibits by with penicillin/streptomycin (Hyclone, Logan, UT). Experiments listed here preventing the religation step of this enzyme’s DNA relaxing activity. were conducted on cells that had been passaged three to seven times following In the presence of CPT, topo I forms a covalent intermediate with the arrival from American Type Culture Collection. 3Ј-phosphate end of the broken DNA strand (5). This single-strand Apoptosis Response and p53 Activation. HeLa cells were plated at a density of 106 cells/6 cm-diameter tissue culture dish and incubated 16 h. nick lesion is believed to convert into an irreversible double-strand Culture medium was then removed and replaced with media containing vary- break after it encounters the replication fork machinery in S phase. ing doses of CPT and/or inhibitors. At times of data collection, both floating This unusual form of DNA damage induces a signaling cascade that (apoptotic and/or necrotic cells) and adherent cell material from each dish were is propagated by ATM-family kinases in human cells (6), which collected on ice using cell lifters and centrifugation (5 min ϫ 200 g). Next, cell phosphorylate numerous targets involved in transcription, DNA re- pellets were resuspended in ice-cold PBS, centrifuged again (5 min ϫ 200 g), pair, cell cycle arrest, and apoptosis. and lysed in 200 l of lysis buffer [1% Igepal, 0.5% deoxycholate, 2 mM DTT, A comprehensive survey of the entire transcriptome after CPT 150 mM NaCl, and 20 mM HEPES (pH 7.4); supplemented with phosphatase treatment via the Affymetrix U133A Chip has not been reported, and protease inhibitors]. Protein concentrations in lysates were determined by although one study examined a small number of genes after CPT the Bio-Rad assay, and 100 g/lane samples were immediately prepared for loading onto 12% acrylamide SDS-PAGE by boiling (5 min) in 1% SDS sample buffer. Completed gels were then electrotransferred to nitrocellulose Received 7/8/03; revised 1/14/04; accepted 1/15/04. membranes, which were stained with Ponceau S and cut in half across protein Grant support: J. P. Carson is supported by an Oncobiology postdoctoral training ϳ fellowship from the National Cancer Institute. M. F. Christman is supported by grants bands corresponding to Mr 35,000. Membrane halves corresponding to from the NIH. higher molecular weight proteins were Western blotted with primary antibod- The costs of publication of this article were defrayed in part by the payment of page ies directed against whole p53 protein (Ab-12; Oncogene Research Products, charges. This article must therefore be hereby marked advertisement in accordance with San Diego, CA) and serine-15-phosphorylated p53 (Cell Signaling Technol- 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Michael F. Christman. Phone: (617) 414-1636; Fax: (617) 414- ogy, Beverly, MA); membranes corresponding to the lower molecular weight 1646; E-mail: [email protected]; Internet address: http://www.gg.bu.edu. proteins were Western blotted with an antibody directed against the Mr 23,000 2096 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2004 American Association for Cancer Research. CAMPTOTHECIN WHOLE-GENOME MICROARRAYS caspase-specific cleavage product of poly(ADP-ribose) polymerase (R&D were generated and hybridized to U133A GeneChips as per a standard Systems, Minneapolis, MN). These Western blot procedures were conducted protocol (Affymetrix). This procedure was repeated four times on according to the directions of the manufacturer, and immunoreactive bands separate occasions; one CPT-treated sample failed to hybridize. Gene were visualized by chemiluminescent exposure to instant film such that sub- expression changes between the three CPT-treated samples and four ϳ saturation exposures ( 1 min) were obtained. These film images were then untreated samples were then tabulated and analyzed after normaliza- digitally scanned and converted to .tiff images by Adobe PhotoShop with the tion (“Materials and Methods”). “Autolevels” (ShftϩCtrlϩL) image function selected. Scanned Western blot images were then analyzed with the Scion Image Data Analysis. For each gene, fold-change (FC) and statistical software package freely available from ScionCorp.1 The immunoreactive significance of differential expression (p) were calculated. FC was bands were highlighted, and pixel counts of each band were obtained via the calculated using the average signal from the two groups. Statistical 3 Integrated Density analysis option. Integrated densities were linearly normal- significance was calculated using a t-Test implemented in Cyber-T. ized such that the gel band corresponding to the positive control in the Cyber-T uses a Bayesian estimate of the variance within treatment experiment (10 M CPT at 8 h.) was assigned an immunoreactivity value groups based on a prior distribution obtained from an estimate of the of 100. variance from genes at a similar expression level. This provides WST-1 Viability Assay. HeLa cells were plated