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Departments of Molecular and Biomedical Pharmacology (J.C.M., G.C., A.O., A.S., R.J.C.) and Statistics (C.S., A.S.), Markey Cancer Center, University of Kentucky, Lexington, Kentucky; and Department of Biology (A.O.), St. Mary's College, Notre Dame, Indiana
Received July 8, 2005; accepted September 8, 2005
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Abstract |
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). Because tumors replicate at a high rate, the resulting DNA damage reduces the tumor mass and suppresses spread of the tumor. However, DNA replication inhibitors generally fail to kill all of the cells within a tumor, and the surviving cells frequently develop drug resistance. One of the primary goals in cancer research is to develop new ways of inhibiting cancer cell growth, in part by improving the effectiveness of existing cancer treatment regimens.
Doxorubicin (Adriamycin) is an anthracycline antibiotic that is a component of many treatment regimens for solid tumors. Doxorubicin blocks the activity of topoisomerase II, a DNA unwinding protein, causing arrest of the cell cycle or apoptosis (Chabner et al., 2001). Doxorubicin resistance can emerge through altered availability of the drug or through its inactivation, through changes in topoisomerase II, or through changes in pathways mediating DNA repair and apoptosis (Longley and Johnston, 2005). In principle, identification of genes that are induced by doxorubicin could lead to new targets for improving the effectiveness of doxorubicin-based therapies.
Doxorubicin is a mainstay in the treatment of breast cancer, and several groups have used microarrays to screen for doxorubicin-regulated genes in breast cancer cell lines. Kudoh et al. (2000) identified 14 genes that are up-regulated and three genes that are down-regulated by doxorubicin in MCF-7 breast cancer cells. MCF-7 cells are a p53/estrogen receptor/progesterone receptor-positive cell line that is nontumorigenic in the absence of estradiol (Soule and McGrath, 1980). In general, genes that were up-regulated by doxorubicin, including cyclin D2 and Cdk6, direct cell cycle progression, whereas genes that were down-regulated, such as Bcl-2, inhibit apoptosis. The study by Kudoh et al. (2000) was performed with a microarray filter containing 5180 genes, and the authors anticipated further analyses when sequencing of the human genome was complete.
More recently, Troester et al. (2004) analyzed global gene expression patterns in two different p53-positive breast cancer cell lines (one line was the same MCF-7 cell line) treated with doxorubicin or 5-fluorouracil. These expression patterns were compared with those of mammary epithelial cells that were immortalized with telomerase (Troester et al., 2004). For MCF-7 cells, the results of Troester et al. (2004) differed from those of Kudoh et al. (2000) in that genes associated with proliferation, such as CDC2, cyclin A2, Ki67, and ribonucleotide reductase, were repressed, whereas cell cycle inhibitors such as p21WAF1 were induced. Similar results were found more recently by Elmore et al. (2005). All three studies analyzed expression patterns in MCF-7 cells treated with similar doxorubicin doses (1 µM and 1.7 µM, respectively) and similar time points, and two studies detected the induction of epoxide hydrolase by doxorubicin (Kudoh et al., 2000; Troester et al., 2004).
Other groups have used microarray-based screens to search for doxorubicin-regulated genes in hepatoma cells (Moriyama et al., 2003), lung cancer cells (Niiya et al., 2003), lymphoblasts (Hussain et al., 2004), and in breast cancer cells treated with hepatocyte growth factor/scatter factor (Yuan et al., 2001). Because these studies spanned a range of cell types and conditions, doxorubicin-regulated genes varied widely. Additional studies have used microarrays to identify expression patterns associated with doxorubicin-resistant cells. These studies identified midkine (Kang et al., 2004) and eukaryotic translation initiation factor 1A (Kang et al., 2004), among others (Kudoh et al., 2000; Ichikawa et al., 2004), as important in acquired doxorubicin resistance. Finally, other groups have compared doxorubicin-treated cells that display a senescent morphology with cells that have continued to proliferate. Chang et al. (2002) compared arrested and proliferating cells 10 days after a 24-h dose of doxorubicin and identified numerous genes affecting proliferation and arrest. The regulation of many of these genes was p53-dependent (Chang et al., 2002).
Unlike previous studies in MCF-7 cells, we have determined a doxorubicin-specific expression pattern in the highly tumorigenic MDA-MB-231 cell line (Zhang et al., 1991), which is p53/estrogen receptor/progesterone receptor-negative. In MDA-MB-231 cells, doxorubicin induced genes that regulate numerous pathways, including cellular proliferation and survival, deacidification, and membrane signaling. Using our microarray data set as candidate genes, we then inhibited three of the genes using RNAi and tested their roles in antineoplastic drug susceptibility. Our results support a model in which tumorigenic breast cancer cells undergo a doxorubicin-induced change in gene expression that is distinct from less malignant cells, and we have identified multiple genes that regulate antineoplastic drug susceptibility.
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Materials and Methods |
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For drug treatments, cells were split to a density of 500,000 cells per 100-mm dish and allowed to attach overnight. Cells were then treated with various drug concentrations and incubated for 24 h. Cells were harvested by scraping from the dish with a rubber policeman, centrifuging, and washing once with phosphate-buffered saline (PBS). Doses of various drugs were chosen because they cause toxicity after a prolonged incubation (data not shown). However, after 24 h, none of these agents caused pronounced cellular rounding, and the fluorescence-activated cell sorting profiles indicated that the cells were viable and largely nonapoptotic (Fig. 1).
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Reverse Transcription-Polymerase Chain Reaction. RNA was purified as described above and reverse transcribed (RT-PCR) using SuperScript II reverse transcriptase and random hexamers (both from Invitrogen, Carlsbad, CA). PCR reactions were performed with Taq polymerase (GenScript Corp., Scotch Plains, NJ) in an Eppendorf Mastercycler (Eppendorf North America, Westbury, NY) using 30 to 38 cycles of a program consisting of 94°C for 1 min., 55°C for 1 min., and 72°C for 1 min. PCR reactions contained primers to doxorubicin-induced genes and to actin, which was an internal control for the amount of cDNA template. DNA was then visualized by electrophoresis in 2.5% agarose 1000 (Invitrogen). The primer sequences for the various analyses are available on the web (http://www2.mc.uky.edu/Pharmacology/rjc_research.asp). Images of the agarose gels were captured as jpg files, and the intensities of the bands were quantitated using ImageQuant software (GE Healthcare, Little Chalfont, Buckinghamshire, UK). The ratios of doxorubicin signature genes to actin were calculated, and statistical tests were performed using Microsoft Excel (Microsoft, Redmond, WA).
Cell Cycle Analysis. MDA-MB-231 cells were treated with various drugs, harvested, washed with PBS, and fixed with 70% ethanol overnight. After fixing, the cells were washed again with PBS and resuspended at a density of 106 cells/ml in 20 µg/ml propidium iodide and 20 µg/ml DNase-free RNase. Samples were analyzed at the University of Kentucky Flow Cytometry Facility. The percentages of cells in the G1, S, and G2 phases of the cell cycle were calculated using ModFit Software (Verity Software House, Topsham, ME).
Western Blotting. Cell lysates were prepared by incubating cells in Nonidet P-40 buffer (1% Nonidet P-40, 20 mM Tris, 150 mM NaCl, 5 mM EDTA, 1 mM Na3VO4, pH 7.4, and 10 µg/ml of the protease inhibitors aprotinin and leupeptin) followed by centrifugation at maximum speed in a microcentrifuge. Lysates were then separated on 8 to 16% SDS-polyacrylamide gel electrophoresis gels. Proteins were then transferred to Immobilon P membranes (Millipore Corporation, Billerica, MA) and probed with various antibodies before visualization with the West Pico chemiluminescent substrate (Pierce Chemical, Rockford, IL). The antibodies to clusterin (H-330) and carbonic anhydrase II (H-70) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and the antibody to tubulin was from Fisher Scientific Co. (Pittsburgh, PA).
RNAi Transfections. MDA-MB-231 cells were plated at a density of 500,000 cells per 100-mm dish and left to attach overnight. After attachment, RNA oligonucleotide duplexes were diluted to 220 nM in 1 ml of Opti-MEM (Invitrogen) and left for 5 min. A 1:6 suspension of Oligofectamine (Invitrogen) in Opti-MEM was then added to the RNAi duplex solution, and the mixture was incubated at room temperature for 20 min. During the incubation, plated MDA-MB-231 cells were washed once with Opti-MEM and overlaid with 4.4 ml of Opti-MEM I. The RNAi duplex suspension was gently added to the cells, for a final concentration of 40 nM RNAi duplex. After a 4-h incubation at 37°C, 2.8 ml of culture medium containing 30% Serum Supreme (Fisher Scientific Co.) was added to the cells, and the cells were left overnight. The cells were then trypsinized from the dish and were plated at a density of 5000 cells per well in a 96-well dish or 500,000 cells per plate in a 100-mm dish.
Viability Assays. For measurements of cell growth, transfected cells were counted manually, plated in triplicate in 96-well dishes, and then treated with varying doses of doxorubicin, camptothecin, etoposide, or mechlorethamine for 96 h. Media were then removed and replaced with growth media containing 0.5 mg/ml 3-[4,5 dimethylthiazol-2-y]-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich) and incubated for 1 to 2 h. After incubation, MTT-containing media were removed, and 100 ml of dimethyl sulfoxide was added to each well. The cells were then incubated for 20 min on a rotating platform and the A595/A650 was determined using a Dynatech MR600 microplate reader (Dynex Technologies, Chantilly, VA). The percentage of viability was calculated as absorbance of cells treated with drug divided by the average absorbance of untreated cells. In each case, the results shown indicate representative results of at least three independent experiments. LD50 values were calculated from triplicate MTT assay measurements.
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Results |
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The doxorubicin-regulated genes are composed of multiple classes of genes, some of which are listed in Table 1. When sorted by annotation of biological function, there was a significant up- and down-regulation of metabolic genes and genes regulating cell cycle progression (see Supplemental data). Genes regulating DNA synthesis and chromatin assembly were significantly induced (Fisher's exact test, P = 0.03, respectively), whereas repressed genes included regulators of chemotaxis (P = 0.0005), ion transport (P = 0.03), and the inflammatory response (P = 0.04; see Supplemental data). Because of the global patterns of transcriptional changes, we have analyzed the expression of two transcription factors previously associated with proliferation, inhibition of differentiation/DNA binding 2 (Id2) and Atf3, and two metabolic genes regulating acid-base metabolism (carbonic anhydrase II; CAII) and phospholipid synthesis (phosphatidylinositol 3-kinase 55-kDa regulatory subunit p55PIK). We note that the microarray predicted that the most strongly up-regulated gene was Kreisler Maf-related leucine zipper homolog; Table 1), but we were unable to replicate this result in independent PCR analyses (data not shown).
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Doxorubicin-Regulated Genes Are Induced with Varying Kinetics and Drug Specificities. To examine the kinetics of gene induction, we analyzed RNA levels by RT-PCR using actin as an internal standard for cDNA loading. CAII and Id2 were induced quickly by doxorubicin, with a detectable increase 2 to 6 h after treatment (Fig. 2, A and B). In contrast, p55PIK and Atf3 were induced more slowly, reaching peak expression only after 24 h (Fig. 2, C and D, respectively). These results suggest that doxorubicin-regulated genes are induced at different rates.
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) caused arrest in S and G2/M (Fig. 1, B, D, and F, respectively), whereas the topoisomerase I inhibitor camptothecin (Chabner et al., 2001) arrested cells in G1 and G2/M (Fig. 1, C and F). The alkylating agent mechlorethamine, the active form of cyclophosphamide, which requires activation in the liver (Chabner et al., 2001), arrested cells primarily in G1 and S (Fig. 1, E and F).
Expression was analyzed after low or high doses of drugs (Fig. 3, A-D), and then the same assays were performed in triplicate for statistical analysis (Fig. 3, E-H). CAII was induced efficiently by multiple drugs (Fig. 3A). The induction of CAII by doxorubicin, camptothecin, etoposide, and mechlorethamine was significant compared with that of untreated cells (P 
0.01 for each drug), but not when CAII induction by any two drugs (i.e., doxorubicin and camptothecin) was compared (P > 0.05 for any pair). Id2 induction by doxorubicin was highly significant (Fig. 3B, lanes 1-3; P = 2 x 10-6 for untreated versus treated cells; Fig. 3F), and Id2 was induced to a lesser degree by camptothecin (Fig. 3B, lanes 4 and 5; 5-fold), etoposide (Fig. 3B, lanes 6 and 7; 8-fold), and mechlorethamine (Fig. 3B, lanes 8 and 9; 10-fold). In each case, the levels of induction by doxorubicin were significantly higher than for camptothecin, etoposide, and mechlorethamine (P < 0.0001 for each drug; Fig. 3F).
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The CAII and Clusterin Proteins Are Induced by Doxorubicin. As a consequence of its transcriptional regulation by doxorubicin, the CAII protein was induced by 1 µM doxorubicin (Fig. 4A) and by camptothecin (Fig. 4C, lane 3). CAII levels after treatment with etoposide or mechlorethamine were lower than levels after doxorubicin and camptothecin treatment (Fig. 4C, lanes 4 and 5), which is consistent with the RNA analysis for CAII (Fig. 3). Clusterin regulates chemotherapy resistance in osteosarcoma (Trougakos et al., 2004), and we found that clusterin was highly induced by 0.1 µM doxorubicin and was nearly saturated by a 0.5 µM dose (Fig. 4B). In addition, clusterin was highly induced by all four chemotherapeutic agents that we tested (Fig. 4D). We did not analyze Id2, Atf3, or p55PIK by Western blot because commercially available antibodies were not sufficiently specific.
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CAII, Id2, Atf3, and Clusterin Regulate Drug Susceptibility. To determine the roles of doxorubicin-induced genes, we attenuated the expression of several genes using RNAi oligonucleotide duplexes (Elbashir et al., 2001). CAII, Id2, and clusterin expression was inhibited by transiently transfecting MDA-MB-231 cells with RNAi duplexes targeting the genes (CAIIi, Id2i, or CLSi) or with a nonspecific control sequence (Fig. 6D, con). RNAi molecules targeting CAII, Id2, and clusterin inhibited the basal expression of Id2 (Fig. 6B, compare lanes 1 and 3) or attenuated the induction of each gene after doxorubicin treatment (Fig. 6, A-C, compare lanes 2 and 4). Furthermore, CAII protein levels were markedly decreased before (Fig. 6D, lanes 1 and 2) and after doxorubicin treatment (Fig. 6D, lanes 3 and 4) in CAIIi-transfected cells.
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0.007 for each dose). There was no change in viability in CLSi-transfected cells without drug treatment, consistent with the low levels of basal clusterin expression in this cell line (Figs. 4B, lane 1; 5B, lane 3; and 6C, lane 1). The loss of viability in CLSi-transfected cells resulted in a significant decrease in LD50 compared with control cells after treatment with doxorubicin, camptothecin, etoposide, and mechlorethamine (Fig. 7C; P 0.01 for each drug). We conclude that clusterin suppresses susceptibility to multiple chemotherapeutic drugs in MDA-MB-231 cells.
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0.005 for each drug). It is likely that this is a conservative estimate of the effect of CAII on drug susceptibility, because there was a low level of CAII expression in the CAIIi-transfected cells (Fig. 6D, lane 4). We conclude that CAII expression is associated with decreased susceptibility to multiple chemotherapeutic drugs.
Id2 inhibition decreased cell proliferation (Fig. 8A, black columns), even in the absence of doxorubicin. Growth inhibition was highly significant at 25 nM (P = 2 x 10-7) or 100 nM (P = 8 x 10-5) doses and was reflected in a 46% decrease in the viable cell count at 96 h after transfection. Id2 inhibition did not induce a significant increase in cell death, because we detected only a modest increase in the number of dead cells (5 ± 2 for control-transfected cells compared with 15 ± 5 for Id2i-transfected cells) by trypan blue exclusion assay. In contrast, the Id2i-transfected population contained a 12% increase in the number of cells arrested in S, G2, and M phase (data not shown) when measured by fluorescence-activated cell sorter analysis. Id2i-transfected cells exhibited decreased sensitivity to doxorubicin (Fig. 8B). At the 0.3 µM dose of doxorubicin, Id2i-transfected cells were significantly (P = 0.002) less sensitive than cells transfected with the control RNAi duplex (Fig. 8B). As a result, Id2i-transfected cells had LD50 values greater than control cells for doxorubicin (Fig. 7C) but not for camptothecin, etoposide, or mechlorethamine. We conclude that Id2 expression is associated with doxorubicin susceptibility in MDA-MB-231 cells.
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Discussion |
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Carbonic anhydrase II regulates the cellular acid/base balance by catalyzing the reaction 
. This reaction allows for efficient secretion of acid (Potter and Harris, 2003). Tumors have an acidic extracellular pH, which may contribute to drug resistance by reducing the uptake and cytotoxicity of weak bases such as doxorubicin (Raghunand et al., 1999). Our results are consistent with a model in which MDA-MB-231 cells induce CAII to improve survival, perhaps by limiting doxorubicin uptake. However, we note that CAII contributed to survival after treatment with camptothecin, etoposide, and mechlorethamine, suggesting a more universal mechanism for CAII. For example, CAII may influence water or ion exchange with the extracellular environment. Numerous genes regulating ion and water balance were altered after doxorubicin treatment, including Aqp3/aquaporin and multiple solute carrier proteins (Table 1), suggesting that changes in membrane permeability and ion balance contribute to chemotherapeutic responsiveness. Finally, CAII was expressed more strongly in MCF-7 cells than in MDA-MB-231 cells and was not induced by doxorubicin in the former cell line (Fig. 5A), suggesting that distinct pathways regulate CAII transcription in the two cell lines.
Several genes with reported antiapoptotic functions were induced by doxorubicin in MDA-MB-231 cells. Clusterin/apolipoprotein J/complement lysis inhibitor is a secreted protein that is induced by chemotherapy (Biroccio et al., 2003). Clusterin is overexpressed in tumors (Redondo et al., 2000; Chen et al., 2003) and has antiapoptotic functions in some cell types (Trougakos and Gonos, 2002; July et al., 2004). Although we did not detect a net effect on proliferation after clusterin inhibition (data not shown), clusterin inhibition had a pronounced effect on drug susceptibility in MDA-MB-231 cells (Fig. 7). Furthermore, clusterin was neither expressed constitutively nor induced in MCF-7 cells, suggesting that strategies targeting clusterin may be effective in highly tumorigenic cells. For all RNAi studies, we emphasize that changes in viability are conservative estimates, because transient transfections inhibit gene expression in less than 100% of the transfected cells.
Id2 is a basic helix-loop-helix protein that lacks a DNA binding domain and is capable of forming inactive heterodimers with other basic helix-loop-helix proteins (Sikder et al., 2003). Id2 is associated with proliferation, is up-regulated in cancers, and extends keratinocyte life span (Sikder et al., 2003). Id2 induction by doxorubicin was reported in previous studies of murine fibroblasts, where it interfered with activation of the MyoD muscle-specific transcription factor (Kurabayashi et al., 1994). In developing breast tissue, Id2 has been associated with proliferation (Mori et al., 2000) and differentiation (Parrinello et al., 2001; Miyoshi et al., 2002), but in human breast tumor samples, Id2 is frequently down-regulated (Itahana et al., 2003), and Id2 expression is associated with positive prognosis in breast cancer (Stighall et al., 2005). To clarify the role of Id2 in breast cell proliferation, Itahana et al. (2003) expressed Id2 in MDA-MB-231 cells under a constitutive promoter, and Id2 suppressed cell cycle progression and invasiveness. Our results using RNAi for Id2 are inconsistent with these overexpression studies, because Id2 inhibition suppressed proliferation. In some cases, constitutive overexpression of a gene phenotypically resembles the loss of the same gene, suggesting a sensitive regulation of the gene product. Indeed, Id2 expression is transcriptionally regulated during the cell cycle (Barone et al., 1994), and Id2 is phosphorylated by multiple kinases (Nagata et al., 1995; Hara et al., 1997), suggesting that constitutive expression of Id2 during G1 phase could delay cell cycle progression.
Id2 inhibition improved cell survival after doxorubicin treatment, suggesting that MDA-MB-231 cells induce Id2 as part of a pathway that leads to cell death. The mechanism through which Id2 regulated cell survival may include displacing other proteins from the Rb complex, which has been linked to chemotherapy susceptibility. However, doxorubicin-treated cells accumulate in S and G2/M (Fig. 1), a stage where Rb is hyperphosphorylated and has minimal protein interactions through its pocket domain. An alternate model is that Id2 up-regulates cell cycle progression through binding to helix-loop-helix proteins. As a result, loss of Id2 expression decreases proliferation and improves survival, perhaps by allowing cells with damaged DNA more time to complete DNA repair.
MCF-7 cells undergo a senescent-like change when treated with doxorubicin, whereas MDA-MB-231 cells do not (Elmore et al., 2002). A doxorubicin-resistant MCF-7 subclone overcame senescence by limiting the repression of positive cell cycle regulators (Elmore et al., 2005), including cdc2 and cyclin E2, which are normally suppressed by doxorubicin (Elmore et al., 2005). In contrast, Id2 (a gene associated with proliferation) caused decreased survival in MDA-MB-231 cells, the opposite result. We conclude that MCF-7 cells suppress chemotherapy susceptibility through a distinct mechanism from MDA-MB-231 cells.
Phosphatidylinositol 3-kinase (PI3K) has antiapoptotic functions in response to numerous stimuli (Fresno Vara et al., 2004), and we detected marked up-regulation of the PI3K regulatory subunit p55PIK/PI3K-p55
(Pons et al., 1995) by doxorubicin. Although the role of p55PIK in growth regulation is poorly understood, p55PIK binds to the Rb tumor suppressor protein (Xia et al., 2003), and overexpression of the amino-terminal Rb binding sequence of p55PIK causes growth arrest (Hu et al., 2005). p55PIK is also implicated in insulin and cytokine signaling (Mothe et al., 1997; Takahashi-Tezuka et al., 1997; Dey et al., 1998). In addition, we note that p55PIK was not induced by doxorubicin in MCF-7 cells, suggesting genetic factors that are altered in MDA-MB-231 cells regulate p55PIK expression.
Several other genes that are associated with proliferation were induced by doxorubicin. Atf3 is a member of the activating transcription factor/cAMP-responsive element binding protein family of bZip transcription factors (Hai and Hartman, 2001). Atf3 binds to the DNA sequence TGACGTCA and is associated with neoplastic transformation and the response to serum, stress, and damage (Yu et al., 1996; Amundson et al., 1999; Shtil et al., 1999; Hai and Hartman, 2001). Atf3 was up-regulated by doxorubicin in MDA-MB-231 and MCF-7 cells, suggesting that strategies targeting Atf3 would be unlikely to be specific for aggressive cancers.
Other prosurvival genes that were induced by doxorubicin include tumor necrosis factor-related apoptosis inducing ligand receptors (TRAIL-R3 and TRAIL-R4, which are "decoy receptors" for TRAIL (Kim and Seol, 2003). TRAIL-R3 and TRAIL-R4 bind to TRAIL, but they lack cytoplasmic death domains and do not induce apoptosis when engaged by TRAIL (Sheridan et al., 1997). Instead, TRAIL-R3 and TRAIL-R4 have antiapoptotic functions (Meng et al., 2000; Bernard et al., 2001). TRAIL-R3 has been reported previously as a doxorubicin-responsive gene in breast cancer (Ruiz de Almodovar et al., 2004), and our findings also implicate TRAIL-R4 in resistance to doxorubicin-mediated cell death. In addition, numerous genes that modulate cell death were down-regulated by doxorubicin, including multiple interleukins. Among these genes, interleukin-1
has been investigated previously for its ability to enhance the anticancer activity of doxorubicin (Nakamura et al., 1991; Monti et al., 1993).
Doxorubicin also induced the expression of proteins that metabolize xenobiotic compounds, including the cytochrome P450 proteins Cyp1A1 and Cyp1B1, the cytochrome P450 reductase, and the cytochrome b5-related P450 activator Iza1/Hpr6.6 (Table 1). Cyp1A1 and Cyp1B1 are induced in response to numerous aromatic compounds with similar structures to doxorubicin (Nebert and Russell, 2002). Hpr6.6 homologues activate and stabilize cytochrome P450 proteins (Min et al., 2004; Mallory et al., 2005), and Hpr6.6 regulates cell death after oxidative damage (Hand and Craven, 2003), which is generated by doxorubicin. As a group, these proteins probably modify doxorubicin or cellular metabolites induced by doxorubicin. Cytochrome P450 proteins probably act in concert with the drug transporter proteins GCN20/ABCF2 and MDRTAP, which were induced by doxorubicin (Table 1), in minimizing the effective concentrations of doxorubicin within the cell.
In summary, we have identified a group of genes that are induced by chemotherapeutic agents and that regulate drug susceptibility. The functions of individual genes could not be predicted based on their transcriptional pattern, because genes that were induced by doxorubicin had both positive and negative effects on doxorubicin susceptibility. In spite of this limitation, we detected two genes, CAII and clusterin, that were associated with cell survival after chemotherapeutic drug treatment. Because these genes were not transcriptionally altered in less tumorigenic cells, there may be a "therapeutic window" in which their activity can be inhibited in tumor cells without disrupting their function in nontumorigenic cells. A third gene, Id2, is associated with increased doxorubicin susceptibility, suggesting that Id2 might serve as positive prognostic indicator when it is induced by doxorubicin in clinical tumor samples.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS: RNAi, RNA inhibition; RT, reverse transcription; PCR, polymerase chain reaction; MTT, 3-[4,5 dimethylthiazol-2-y]-2,5-diphenyltetrazolium bromide; Id2, inhibitor of differentiation/DNA binding 2; Atf3, activating transcription factor 3; CAII, carbonic anhydrase II; p55PIK, phosphatidylinositol 3-kinase 55-kDa regulatory subunit; Rb, retinoblastoma tumor suppressor protein; PI3K, phosphatidylinositol 3-kinase; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand.
The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material. 
Address correspondence to: Dr. Rolf J. Craven, Department of Molecular and Biomedical Pharmacology, MS-305 University of Kentucky Medical Center, University of Kentucky, Lexington, KY 40536. E-mail: rolf.craven{at}uky.edu
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