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Impact of P53 Knockout and Topotecan Treatment on Gene Expression Profiles in Human Colon Carcinoma Cells: a Pharmacogenomic Study

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Impact of P53 Knockout and Topotecan Treatment on Gene Expression Profiles in Human Colon Carcinoma Cells: a Pharmacogenomic Study [CANCER RESEARCH 63, 2782–2793, June 1, 2003] Impact of p53 Knockout and Topotecan Treatment on Gene Expression Profiles in Human Colon Carcinoma Cells: A Pharmacogenomic Study Sayed S. Daoud,1 Peter J. Munson, William Reinhold, Lynn Young, Vinay V. Prabhu, Qiang Yu, Jihyun LaRose, Kurt W. Kohn, John N. Weinstein, and Yves Pommier2 Department of Pharmaceutical Sciences, Washington State University, Pullman, Washington 99164-6510 [S. S. D.], and Mathematical and Statistical Computing Laboratory, Center for Information Technology [P. J. M., L. Y., V. V. P.] and Laboratory of Molecular Pharmacology, Center for Cancer Research [W. R., Q. Y., J. L., K. W. K., J. N. W., Y. P.], National Cancer Institute, NIH, Bethesda, Maryland 20892-4255 ABSTRACT through sequence-specific binding of its central domain to cis-acting elements within the promoters or introns of responsive genes. At To uncover transcriptional stress responses related to p53, we used present, more than 20 genes are known to be activated by p53, most cDNA microarrays (National Cancer Institute Oncochips comprising 6500 of them in growth arrest or apoptotic pathways (5, 6). Other promoters different genes) to characterize the gene expression profiles of wild-type p53 HCT-116 cells and an isogenic p53 knockout counterpart after treat- (many of which are viral or growth stimulatory) are repressed by p53 ment with topotecan, a specific topoisomerase I inhibitor. The use of the (7). Consequently, the downstream effects of activating p53 are com- p53 knockout cells had the advantage over p53-overexpressing systems in plex, and no single pathway mediates the full range of functions that p53 activation is mediated physiologically. RNA was extracted after of p53. low (0.1 ␮M)- and high (1 ␮M)-dose topotecan at multiple time points To analyze the p53 dependence of molecular events after DNA within the first6hoftreatment. To facilitate simultaneous study of the damage, we compared gene expression changes in a p53 wild-type p53 status and pharmacological effects on gene expression, we developed human colon carcinoma cell line, HCT-116 (p53ϩ/ϩ), with those in a novel “cross-referenced network” experimental design and used multi- an isogenic p53 knockout (p53Ϫ/Ϫ; Ref. 8) after treatment with the ple linear least squares fitting to optimize estimates of relative transcript topoisomerase I inhibitor topotecan. Topotecan (Hycamtin), a semi- levels in the network of experimental conditions. Approximately 10% of synthetic water-soluble derivative of camptothecin, is a clinically the transcripts were up- or down-regulated in response to topotecan in the p53؉/؉ cells, whereas only 1% of the transcripts changed in the p53؊/؊ useful agent. It is approved for first-line therapy of cisplatin-refractory cells, indicating that p53 has a broad effect on the transcriptional response ovarian cancer and second-line therapy of small cell lung cancer (9). to this stress. Individual transcripts and their relationships were analyzed Like other camptothecins, topotecan converts topoisomerase I into a using clustered image maps and by a novel two-dimensional analysis/ cellular poison by trapping topoisomerase I in a covalent complex visualization, gene expression map, in which each gene expression level is with DNA. The cytotoxic lesions result from breaks, generated by represented as a function of both the genotypic/phenotypic difference (i.e., collision of the complexes with DNA or RNA polymerase (10). We p53 status) and the treatment effect (i.e., of topotecan dose and time of chose to use the p53 knockout in this study, rather than an overex- exposure). Overall, drug-induced p53 activation was associated with a pressing p53 transfectant, so that the p53 expression would be phys- coherent genetic program leading to cell cycle arrest and apoptosis. We iological. identified novel p53-induced and DNA damage-induced genes (the pro- To simultaneously study the effects of p53 status and topotecan apoptotic SIVA gene and a set of transforming growth factor ␤-related genes). Genes induced independently of p53 included the antiapoptotic treatment at different concentrations and time points, we developed cFLIP gene and known stress genes related to the mitogen-activated (and present here) a new experimental design for microarray studies. protein kinase pathway and the Fos/Jun pathway. Genes that were neg- We term it a cross-referenced network (Fig. 1). The most frequently atively regulated by p53 included members of the antiapoptotic protein used design for two-color microarray experiments simply compares chaperone heat shock protein 70 family. Finally, among the p53-depend- each sample with a single internal reference sample by cohybridiza- ent genes whose expression was independent of drug treatment was tion. The network design uses internal reference samples, but it also ؉ S100A4, a small Ca2 -binding protein that has recently been implicated in provides the additional, global set of comparisons indicated in Fig. 1. p53 binding and regulation. The new experimental design and gene ex- Given this design, we were able to analyze the entire network of data pression map analysis introduced here are applicable to a wide range of by multiple regression in an appropriately weighted fashion to in- studies that encompass both treatment effects and genotypic or pheno- crease the statistical reliability of the results and conclusions in terms typic differences. of statistical consistency test. The data were then displayed using a novel visualization, GEM,3 to identify transcripts that differed in INTRODUCTION expression level in relation to p53 status and/or drug treatment. The p53 protein is a central transcription factor activated in re- sponse to a variety of cellular stresses, including DNA damage, MATERIALS AND METHODS mitotic spindle damage, heat shock, metabolic changes, hypoxia, viral infection, and oncogene activation (1, 2). p53 can induce growth arrest Cell Culture and Drug Treatment. Isogenic p53-null (p53Ϫ/Ϫ) and and apoptosis, events that prevent the survival of damaged cells. p53 wild-type (p53ϩ/ϩ) human HCT-116 colon carcinoma cells, kindly provided can also promote early senescence in response to unregulated mito- by Dr. Bert Vogelstein (8), were grown as monolayers in DMEM (Life genic signaling (3, 4). The transactivation function of p53 is mediated Technologies, Inc., Gaithersburg, MD) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT). Exponentially growing cultures at 80% conflu- ence were treated for 1 h with 0.1 ␮M (LD) or 1 ␮M (HD) topotecan obtained Received 8/30/02; accepted 3/31/03. The costs of publication of this article were defrayed in part by the payment of page from the Developmental Therapeutics Program, National Cancer Institute, NIH charges. This article must therefore be hereby marked advertisement in accordance with (Bethesda, MD). Cells were then washed twice with PBS (pH 7.4) resuspended 18 U.S.C. Section 1734 solely to indicate this fact. in drug-free medium and harvested 0, 1.5, 3, or 6 h later. Total RNA was 1 To whom requests for reprints may be addressed, at Department of Pharmaceutical Sciences Cancer Prevention & Research Center Washington State University, 259 Wegner Hall, Pullman, WA 99164-6510. E-mail: [email protected]. 3 The abbreviations used are: GEM, gene expression map; TGF, transforming growth 2 To whom requests for reprints may be addressed, at Laboratory of Molecular factor; LD, low dose; HD, high dose; CIM, clustered image map; RMS, root mean square; Pharmacology Center for Cancer Research National Cancer Institute/NIH Building 37, RT-PCR, reverse transcription-PCR; PCNA, proliferating cell nuclear antigen; HSP, heat Room 5068, Bethesda, MD 20892-4255. E-mail: [email protected]. shock protein. 2782 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2003 American Association for Cancer Research. PHARMACOGENOMIC OF p53 AND TOPOTECAN IN COLON CANCER Experimental Design. The 12 distinct cDNA preparations were as shown in Table 1. Each chip was hybridized using two cDNA preparations, each labeled with either Cy3 or Cy5. Every such comparison of preparation i with preparation j was also performed using reciprocal labeling, and the pair was replicated n (i, j) times. To compensate for any possible systematic bias caused by the chemical labeling difference between the two dyes, the experimental log-ratio, LЉ(i, j, k), and that from the reciprocal experiment, Ϫ LЉ(j, i, k), for each replicate k were averaged to give the final estimate of the log-expression ratio y(i, j) for that comparison. n͑i, j͒ Fig. 1. Experimental design of the cDNA hybridization experiments. Total RNA from ͸ ͑LЉ͑i, j, k͒ Ϫ LЉ͑ j, i, k͒͒ p53 wild-type (positive) cells [p, HCT-116 (p53ϩ/ϩ)] and p53 knockout (minus) cells [m, HCT-116 (p53Ϫ/Ϫ)] was collected at the beginning of treatment (t ϭ 0) or at the kϭ1 ϭ ϭ y͑i, j͒ ϭ (A) indicated time point (t 1.5, 3, or 6 h) after1hoftreatment with either a low (LD 0.1 2*n͑i, j͒ ␮M) or high concentration (HD ϭ 1 ␮M) of topotecan. As indicated by arrows, pairs of conditions were directly compared by labeling test samples with Cy3 or Cy5 and reference samples with Cy5 or Cy3 and by cohybridizing equal amounts of the product to a single When an observation fell below quality control cutoffs (based on the number array slide. The arrows are double-headed to indicate that hybridizations were done in of pixels in a spot and the intensity level above background), it was dropped pairs with reversal of the colors to eliminate any dye-related bias. Initially, all times and from the average. When the reciprocally labeled data were compared with doses were compared with the t ϭ 0 samples. Cell types (p, m) were then compared at t ϭ 0,3,and6hatHD.Thenetwork of comparisons was used mathematically (by replicate data for the p6LD versus p0 comparison, we noted that agreement weighted multiple linear least-squares regression) to calculate the relative expression between reciprocally labeled data were better, in general, than would be based differences between all pairs of conditions, not just those cohybridized on the same array.
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