Choline Kinase Down-Regulation Increases the Effect of 5-Fluorouracil in Breast Cancer Cells

Research Article

Choline Kinase Down-regulation Increases the Effect of 5-Fluorouracil in Breast Cancer Cells

Noriko Mori, Kristine Glunde, Tomoyo Takagi, Venu Raman, and Zaver M. Bhujwalla

The John Hopkins University In vivo Cellular and Molecular Imaging Center Program, The Russell H. Morgan Department ofRadiology and Radiological Science, The Johns Hopkins University School ofMedicine, Baltimore, Maryland

Abstract An increase ofcellular phosphocholine (PC) and total choline- Identifying strategies to increase cancer cell kill while sparing containing compounds (tCho) has consistently been observed in normal tissue is critically important in cancer chemotherapy. cancer cells and tissue (3–5) and is closely related to malignant Choline kinase (Chk), the enzyme that converts choline to transformation, invasion, and metastasis (3, 6). Choline kinase (Chk), phosphocholine (PC), is elevated in cancer cells and presents a cytosolic enzyme that catalyzes the phosphorylation ofcholine to a novel target for increasing cell kill. Here, we have examined form PC by ATP in the presence of magnesium (7), is one of three the effects of transiently down-regulating Chk by small enzymes that, along with phosphatidylcholine-specific phospholi- interfering RNA against Chk (siRNA-chk) on PC and total pase C and CTP:phosphocholine cytidylyltransferase, can lead to choline-containing compound (tCho) levels and on the increased PC levels (8, 9). Increased expression ofChk has been observed in human lung, colon, prostate, and breast cancer as well as viability/proliferation of estrogen receptor–negative and estrogen receptor–positive breast cancer cell lines and a derived epithelial and hemopoietic cell lines (10–18). Carcinogens nonmalignant mammary epithelial cell line. We investigated such as polycyclic aromatic hydrocarbons and 1,2-dimethylhydra- the effects of combination treatment with transient siRNA- zine induce liver and colon cancers with increased Chk activity (17). chk transfection and the anticancer drug 5-fluorouracil Chk can also be induced by activating agents including hormones, (5-FU) in those cell lines. Microarray analysis of the invasive growth factors, oncogenes, and carcinogens (19–21). Because of its estrogen receptor–negative MDA-MB-231 cell line was done increased expression and activity in cancer, Chk has been proposed to characterize molecular changes associated with Chk down- as a target for antitumor therapy (16, 22, 23). Chk inhibition using the regulation. Chk down-regulation decreased PC and tCho pharmacologic inhibitor MN58b was recently shown to reduce pro- levels in the malignant cell lines, whereas the cell viability/ liferation of cancer cells in vitro and xenografts in vivo (22, 24–27). To avoid the side effects associated with pharmacologic inhibition, proliferation assays detected a decrease in proliferation in these cells. In contrast, Chk down-regulation had an almost we used small interfering RNA (siRNA-chk) to target Chk and negligible effect on PC and tCho levels as well as cell showed that both transient transfection and stable expression of viability/proliferation in the nonmalignant cell line. A siRNA against Chk induced differentiation and reduced proliferation combination of siRNA-chk with 5-FU treatment resulted in a in breast cancer cells (15). larger reduction of cell viability/proliferation in the breast Our purpose in this study was twofold. One was to examine the cancer cell lines; this reduction was evident to a much lesser effect of transiently down-regulating Chk in normal and malignant degree in the nonmalignant cells. Microarray analysis showed human mammary epithelial cells (HMEC) on cell viability and that Chk down-regulation affected 33 proliferation-related proliferation, and the second was to determine if combining Chk genes and 9 DNA repair–related genes. Chk down-regulation down-regulation with 5-fluorouracil (5-FU) treatment increased with siRNA-chk may provide a novel alternative to enhance cell kill in cancer cells. 5-FU is commonly used to treat a variety the effect of anticancer drugs in malignant cells. [Cancer Res ofcancers including breast, head and neck, and colon (28, 29). One 2007;67(23):11284–90] ofthe main mechanisms ofaction of5-FU is the inhibition of thymidylate synthase by the 5-FU metabolite 5-fluoro-2¶-deoxyur- idine-5¶-monophosphate, which leads to cell death (28). This effect Introduction is not specific to tumor cells, making 5-FU a highly toxic drug, with Although there is no dearth ofcytotoxic therapies available to a narrow margin ofsafety, which can cause severe hematologic kill cancer cells, most ofthese damage normal cells as well as toxicity and gastrointestinal hemorrhage (30). Several other cancer cells, resulting in unfortunate side effects such as diarrhea, chemotherapeutic agents such as mitomycin C have similar nausea, granulocytopenia, and cardiotoxicity, to name a few (1, 2). toxicity, and therefore combination therapies with agents that The ideal therapy would target cancer cells while sparing normal increase cell kill in cancer cells but not in normal cells would be tissue. Targeting pathways or molecules that are up-regulated in attractive to reduce toxicity and maintain or increase efficacy. cancer cells but not in normal cells can provide new strategies for In this study, we examined the effects of transient down- selective therapy to use either singly or in combination with regulation ofChk, using siRNA-chk transfection,on PC and tCho conventional chemotherapeutic agents. levels and on the viability/proliferation of two human breast cancer cell lines, MDA-MB-231 (estrogen receptor/progesterone receptor negative) and MCF-7 (estrogen receptor/progesterone receptor

Requests for reprints: Zaver M. Bhujwalla, The Russell H. Morgan Department of positive), and a nonmalignant HMEC line, MCF-12A. We investi- Radiology and Radiological Science, The Johns Hopkins University School ofMedicine, gated the effect of combining transient siRNA-chk transfection Room 208C, Traylor Building, 720 Rutland Avenue, Baltimore, MD 21205. Phone: 410- with the anticancer drug 5-FU on cell viability/proliferation in 955-9698; Fax: 410-614-1948; E-mail: [email protected]. I2007 American Association for Cancer Research. these cell lines as well. Because MDA-MB-231 cells were the more doi:10.1158/0008-5472.CAN-07-2728 aggressive, microarray analysis ofthese cells was done to

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comparable dose of5-FU, a greater reduction in cell viability/ proliferation was achieved with transient siRNA-chk transfection in breast cancer cells compared with nonmalignant HMECs.

Materials and Methods Cell culture. MDA-MB-231, an estrogen receptor–negative metastatic human breast cancer cell line, was grown in RPMI 1640 supplemented with 9% fetal bovine serum (FBS) and antibiotics (90 unit/mL penicillin and 90 Ag/mL streptomycin). MCF-7, an estrogen receptor–positive poorly metastatic human breast cancer cell line, was cultured in Eagle’s MEM supplemented with 9% FBS and antibiotics. MCF-12A, a spontaneously immortalized nonmalignant human mammary epithelial cell line, was Figure 1. Chk protein levels determined by immunoblot assay in MCF-12A, cultured in DMEM-Ham’s F12 medium supplemented as described MCF-7, and MDA-MB-231 control and siRNA-chk–treated cells. Fifty micrograms of protein from each cell line were loaded on a 10% reducing SDS-PAGE gel. previously (3). All cell lines were obtained from American Type Culture h-Actin protein levels were used for equal loading assessment. Collection (ATCC) and were maintained in a humidified atmosphere with 5% CO2 in air at 37jC. characterize molecular changes associated with Chk down- RNA interference experiments. siRNA-chk was designed as previously regulation. We chose to examine transient down-regulation described (15) and purchased from Dharmacon. Approximately 98 nmol/L of siRNA-chk were transfected for 48 h using Oligofectamine and Opti-MEM because this would be the most likely option in the clinical setting (both from Invitrogen) based on the observed decrease of Chk message using liposomal or viral mediated delivery ofsiRNA or short using reverse transcription-PCR analysis within this time (15). hairpin RNA. Dual-phase cell extraction and magnetic resonance spectroscopy Results from magnetic resonance spectroscopy of cell extracts study. Cells were transfected with siRNA-chk for 48 h and water-soluble and Chk expression levels following transient transfection of as well as lipid extracts were obtained from f2 107 control and siRNA- siRNA-chk showed that Chk plays an important role in generating chk–treated cells using the dual-phase extraction method (15). Control the high PC and tCho levels observed in MCF-7 and MDA-MB-231 cell extracts were obtained from cells without siRNA-chk treatment. Cell breast cancer cells but not in the nonmalignant HMEC line MCF- extracts were resuspended in 0.6-mL deuterated water for magnetic 12A. The cell viability/proliferation assay showed that Chk down- resonance spectroscopy analysis. Five microliters of0.75% (w/w) 3- (trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt (TMSP) in deuterated regulation reduced cell viability/proliferation of both estrogen 1 receptor/progesterone receptor–negative and estrogen receptor/ water were used as an internal standard. Fully relaxed H nuclear magnetic resonance spectra ofthe water-soluble extracts were acquired progesterone receptor–positive breast cancer cells but not on a Bruker Avance 500 spectrometer (Bruker BioSpin Corp.) as nonmalignant MCF-12A cells. Microarray analysis ofMDA-MB- previously described (15). Signal integrals ofN-(CH 3)3 offree choline 231 cells showed that Chk down-regulation affected 33 prolifera- (Cho), PC, and glycerophosphocholine (GPC) from within the 3.20 to 3.24 tion-related genes and 9 DNA repair–related genes in these cells. ppm region were determined and normalized to cell numbers and Combined treatment with siRNA-chk transfection and 5-FU compared with the standard. To determine concentrations, peak 1 reduced the cell viability/proliferation to levels that were integration (In) from H spectra for PC, GPC, Cho, and tCho (PC + significantly lower than either treatment alone in both breast GPC + Cho) was compared with that ofthe internal standard TMSP cancer cell lines. In contrast, although the nonmalignant HMECs according to the following equation: were sensitive to 5-FU treatment, the combination treatment resulted in a smaller reduction ofcell viability/proliferation ½ ¼ Imetabolite metabolite ATMSP compared with the malignant cells. These data show that for a ITMSP Ncell

Figure 2. Representative 1H magnetic resonance spectra obtained from water-soluble cell extracts of MCF-12A, MCF-7, and MDA-MB-231 control and siRNA-chk–treated cells. Spectra were acquired on a Bruker Avance 500 spectrometer and are expanded to display signals from the PC, GPC, and Cho regions only. www.aacrjournals.org 11285 Cancer Res 2007;67: (23). December 1, 2007

Figure 3. PC (white columns), GPC (striped columns), and tCho (PC + GPC + Cho; dotted columns) levels in water-soluble cell extracts obtained from 1H spectra of MCF-12A, MCF-7, and MDA-MB-231 control and siRNA-chk–treated cells. Percent change is normalized to values for control cells. Columns, mean of three or more cell extracts for each cell line; bars, SE. *, P V 0.03; **, P < 0.01, compared with control.

In this equation, [metabolite] is the molar concentration per cell ofthe described above using the RNeasy Mini Kit (Qiagen, Inc.) and QIAshredder metabolite expressed as mol/cell, ATMSP is the number ofmoles ofTMSP in homogenizer spin columns (Qiagen) as previously described (14, 31). The the sample, and Ncell is the cell number. Because the number ofprotons microarray hybridization was done at the JHMI Microarray Core Facility contributing to the signal ofall the choline (Cho) metabolites at 3.20 to 3.24 (Dr. Francisco Martinez Murillo, Johns Hopkins University School of ppm and to the TMSP peak at 0 ppm is the same, correction for differences Medicine) using the Human Genome U133 Plus 2.0 Array (Affymetrix, Inc.), in the number ofprotons was not required. which contains >47,000 transcripts, and the Affymetrix GeneChip platform. Immunoblot analysis. Approximately 3 106 cells were grown in GeneChips were analyzed by fluorescence detection using the Agilent culture medium overnight and transfected with siRNA-chk for 48 h. Cells GeneArray Scanner as previously described (14, 31). Data acquisition was were scraped into radioimmunoprecipitation assay buffer [50 mmol/L Tris done using the Micro Array Suite 5.0 software by Affymetrix as previously (pH 7.4), 150 mmol/L NaCl, 1 mmol/L EDTA, 1% Triton-100, 1% sodium described (14, 31). Experiments were done in duplicate. To estimate the deoxycholate, 1 mmol/L phenylmethylsulfonyl fluoride, 0.1% SDS, and gene expression signals, data analysis was conducted on the chips’ cell complete EDTA-free protease inhibitor cocktail (Roche)]. Cell lysates were intensity (CEL) file probe signal values at the Affymetrix probe pair [perfect incubated on ice for 15 min and spun down at 16,000 g (Refrigerated match (PM) probe and mismatch (MM) probe] level using statistical Centrifuge 5415 R, Eppendorf). Protein samples from whole-cell extracts techniques and package Robust Multiarray Analysis (14, 31). The criterion of were resolved on one-dimensional 10% SDS-PAGE gels and transferred onto the posterior probability >0.5, which means that the posterior probability is immunoblot polyvinylidene fluoride membranes (Bio-Rad) using a semidry larger than chance, was used to produce differentially expressed gene lists. transfer unit (Bio-Rad). The membranes were incubated overnight in All computations were done under the R package EBarrays environment as blocking solution [1% dry milk in PBS with 0.1% Tween 20] with custom- previously described (32–34). made polyclonal Chk antibody (Proteintech Group, Inc.; ref. 15) with an Statistical analysis. Data were expressed as mean F SE. The statistical appropriate dilution. Anti–h-actin antibody (Molecular Probes) was used significance of differences in cell metabolite levels and MTT assay result was for equal loading assessment. Secondary antibody was horseradish determined using an unpaired t test (two-tailed). P V 0.03 for cell metabolite peroxidase–conjugated antimouse immunoglobulin G (Vector Laborato- levels and P V 0.01 for MTT assay were considered to be significant. ries). Reactions were revealed using SuperSignal West Pico Substrate (Pierce Biotech.) recorded on Blue Bio film (Denville Scientific). Chk was detected Results at f48 kDa. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Chk protein level was down-regulated by transient siRNA- Cells were grown in culture medium without phenol red. Approximately chk transfection. The immunoblot assay results with Chk 4,000 cells were seeded in each well ofa 96-well plate and cultured antibody showed significant reduction of Chk protein expression overnight. Twenty-four hours later, siRNA-chk was transfected transiently levels in MCF-12A, MCF-7, and MDA-MB-231 cells after transient for 48 h, as previously described (15). No treatment or Oligofectamine siRNA-chk transfection (Fig. 1). Chk levels in nonmalignant MCF- without siRNA was used as negative control. For combination experiments, 12A cells were much lower than the two breast cancer cell lines. A 5-FU (final concentration, 5 g/mL) was added 24 h after transfection This assay was repeated thrice and the results were reproducible. without changing medium. Forty-eight hours after transfection and/or Significantly lower Chk protein levels after transient siRNA-chk 24 h after 5-FU treatment, cells were cultured for another 3 days in fresh transfection confirmed successful transfection and down-regula- culture medium. Cell viability/proliferation was evaluated with the 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) Cell Prolifer- tion ofChk levels. ation Assay (ATCC) and values were compared with those obtained from Magnetic resonance spectroscopy of a nonmalignant HMEC untreated cells. line and two human breast cancer cell lines after transient GeneChip microarray gene expression assay. Total cellular RNA was siRNA-chk transfection. Magnetic resonance spectra showed that isolated from f107 siRNA-chk–treated or control MDA-MB-231 cells as basal PC levels in the nonmalignant MCF-12A cells were much

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2007 American Association for Cancer Research. Breast Cancer Treatment and Choline Kinase Inhibition lower than in breast cancer cells (Fig. 2). A comparison ofPC, GPC, GeneChip microarray gene expression assay for prolifera- and tCho levels ofcells treated with siRNA-chk versus control cells tion-and DNA repair–related genes. Table 1 shows that 16 (Fig. 3) revealed a significant reduction of PC and tCho in the proliferation-related genes were significantly overexpressed and 17 malignant MDA-MB-231 breast cancer cells following transient proliferation-related genes significantly underexpressed following transfection with siRNA-chk compared with control cells. A siRNA-chk transfection in MDA-MB-231 cells. Nine DNA repair– significant reduction of PC in the estrogen receptor–positive breast related genes were significantly differentially expressed following cancer cell line MCF-7 was also observed after siRNA-chk transient siRNA-chk transfection in MDA-MB-231 cells as shown in transfection. There were no statistically significant changes in PC, Table 2. However, none ofthe genes directly involved in 5-FU tCho, or GPC in nonmalignant MCF-12A cells following transient metabolism, such as dihydropyrimidine dehydrogenase, thymidine siRNA-chk transfection (Fig. 3). We carried out control studies with phosphorylase, thymidylate synthase, human concentrative nucle- glyceraldehyde-3-phosphate dehydrogenase siRNA to rule out the oside transporter 1, or uridine phosphorylase, among others (28), possibility ofpoor transfectionofthese cells with siRNA (data not were differentially expressed in MDA-MB-231 cells transiently shown). transfected with siRNA-chk compared with control cells (data not Cell viability/proliferation assay of a nonmalignant HMEC shown). line and two human breast cancer cell lines after transient siRNA-chk transfection. Both breast cancer cell lines MDA-MB- Discussion 231 and MCF-7 showed significant reduction of viability/prolifer- We have previously shown that siRNA-chk induced differentia- ation following transient transfection using siRNA-chk alone tion and reduced cell proliferation markers in breast cancer cells compared with untreated cells (Fig. 4). In contrast, the reduction (15). There are at least three isoforms of Chk (chk-a1, chk-a2, and ofviability/proliferationwas negligible in nonmalignant MCF-12A chk-h) in mammalian cells (7). Our siRNA-chk was designed using cells (Fig. 4). the mRNA sequences transcribed from the chk-a and chk-b genes Cell viability/proliferation assay of a nonmalignant HMEC to allow down-regulation ofboth ofthese Chk mRNAs. Our line and two human breast cancer cell lines after treatment immunoblot assay confirmed successful transient transfection and with 5-FU alone or in combination with siRNA-chk transfection. down-regulation ofChk protein expression levels in all three cell Treatment with 5-FU alone resulted in a reduction ofviability/ lines used. Magnetic resonance spectroscopy studies confirmed the proliferation of all cell lines (Fig. 4). MCF-12A, which has the lowest successful down-regulation of siRNA-chk and showed the signifi- expression level ofChk protein, showed the highest reduction in cant dependence ofthe elevated PC levels in breast cancer cells on cell viability/proliferation by 5-FU alone. The combination Chk as evident from the low PC following transient siRNA-chk treatment of5-FU and siRNA-chk down-regulation resulted in a transfection. Because MCF-12A cells have low basal Chk protein 2-fold reduction of cell viability/proliferation in breast cancer cell and PC levels, the effect of Chk down-regulation by siRNA-chk was lines compared with 5-FU treatment alone (Fig. 4), but only a 10% not significant for PC levels in nonmalignant MCF-12A cells. additional reduction in the nonmalignant cell line. As a result, for Another possibility may be that PC in MCF-12A cells may not be a comparable dose of5-FU, the reduction in cell viability/ primarily dependent on Chk and may be derived through other proliferation was as much or higher for the breast cancer cells enzymes. The differences in Chk expression levels between compared with the nonmalignant cells, although the nonmalignant malignant and nonmalignant cells obtained here are consistent cells were more sensitive to 5-FU alone. with data from our previous study (15).

Figure 4. Results from the MTT cell proliferation assay of MCF-12A (white columns), MCF-7 (dotted columns), and MDA-MB-231 (striped columns) cells treated with siRNA-chk, Oligofectamine alone (oligo), 5-FU alone, or 5-FU combined with siRNA-chk. Percent change is normalized to untreated cells (no treat). Columns, mean of three or more assays for each cell line; bars, SE. *, P V 0.01; **, P < 0.0001, compared with no treatment.

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Cell viability/proliferation was significantly lower in both more resistant to 5-FU treatment. Combined treatment ofboth estrogen receptor/progesterone receptor–positive and estrogen breast cancer cell lines with siRNA-chk transfection and 5-FU receptor/progesterone receptor–negative breast cancer cells fol- reduced viability/proliferation to levels that were similar for both lowing transient siRNA-chk transfection. Importantly, transient estrogen receptor/progesterone receptor–positive and estrogen transfection of nonmalignant MCF-12A cells with siRNA-chk had a receptor/progesterone receptor–negative cells and f2-fold lower negligible effect on cell viability/proliferation. These data suggest than either treatment alone. Although the nonmalignant MCF-12A that transient Chk silencing may reduce cell viability/proliferation cells had the most profound reduction of cell viability/proliferation in cells with high Chk such as malignant cells but may not affect with 5-FU treatment, combining 5-FU treatment with siRNA-chk nonmalignant cells with low Chk. These data support exploring transfection resulted in a reduction of cell viability/proliferation in transient Chk silencing as a broad-spectrum chemotherapeutic the breast cancer cells that was as much or even more than that in agent for cancer cells. the nonmalignant cells. The increase ofcell kill was most likely The reduction of cell viability/proliferation following a single additive rather than synergistic because the microarray data dose of5-FU was highest in the nonmalignant cells with the lowest showed no effect with Chk down-regulation on 5-FU–related genes Chk expression. Malignant cells with higher Chk expression were such as thymidylate synthase.

Table 1. Genes that are involved in proliferation and contained on the Affymetrix Human Genome U133 Plus 2.0 Array, which are differentially expressed following siRNA-chk–mediated Chk down-regulation in MDA-MB-231 breast cancer cells

Gene title Gene symbol Representative public ID Fold change Probability

Cyclin-dependent kinase inhibitor 1B (p27, Kip1) CDKN1B BC001971 2.447 1.000 Cyclin-dependent kinase 5, regulatory subunit 1 (p35) CDK5R1 AL567411 2.121 0.999 Chemokine (C-X-C motif) ligand 1 CXCL1 NM_001511 2.089 0.979 (melanoma growth stimulating activity, a) Endothelin 1 EDN1 J05008 2.062 0.995 Neurofibromin 1 (neurofibromatosis, NF1 AW293356 1.997 0.993 von Recklinghausen disease, Watson disease) Dedicator ofcytokinesis 2 DOCK2 AI991459 1.958 0.987 Endothelin 1 EDN1 NM_001955 1.924 0.972 Lysosomal-associated membrane protein 3 LAMP3 NM_014398 1.909 0.973 Cyclin-dependent kinase inhibitor 1C (p57, Kip2) CDKN1C NM_000076 1.882 0.961 Signal-induced proliferation–associated 1–like 2 SIPA1L2 AB037810 1.872 0.949 Melanoma cell adhesion molecule MCAM BG105365 1.866 0.784 S-phase kinase-associated protein 2 (p45) SKP2 BC001441 1.823 0.833 EP300 interacting inhibitor ofdifferentiation 2 EID2 BE747815 1.747 0.543 FGFR1 oncogene partner; FGFR1OP; C9orf4 NM_007045 1.744 0.593 chromosome 9 open reading frame 4 SMAD family member 1 SMAD1 AU146891 1.713 0.622 Cell division cycle 25 homologue A (S. cerevisiae) CDC25A AY137580 1.707 0.585 NADPH oxidase, EF-hand calcium binding domain 5 NOX5 NM_024505 1.725 0.651 Centromere protein F, 350/400 ka (mitosin) CENPF NM_016343 1.732 0.558 Pre-B-cell colony enhancing factor 1 PBEF1 AA873350 1.751 0.691 DnaJ (Hsp40) homologue, subfamily A, member 2 DNAJA2 AW057513 1.846 0.919 Artemin ARTN AF115765 1.880 0.950 E74-like factor 4 (ets domain transcription factor) ELF4 U32645 1.945 0.975 Artemin ARTN NM_003976 1.957 0.985 Amphiregulin (schwannoma-derived growth factor); AREG; LOC727738 NM_001657 2.036 0.993 similar to amphiregulin precursor (colorectum cell–derived growth factor) Vesicle transport through interaction with VTI1B AI307763 2.043 0.996 t-SNAREs homologue 1B (yeast) Vesicle transport through interaction with VTI1B AI984620 2.081 0.998 t-SNAREs homologue 1B (yeast) Platelet-activating factor acetylhydrolase, PAFAH1B1 AA502643 2.200 0.957 soform Ib, 7a subunit 45 kDa Oncostatin M receptor OSMR NM_003999 2.457 1.000 Signal sequence receptor, a (translocon-associated protein a) SSR1 BF679286 2.497 1.000 Nuclear autoantigenic sperm protein (histone-binding) NASP AU144734 2.512 1.000 NADPH oxidase, EF-hand calcium binding domain 5 NOX5 NM_024505 2.965 1.000 Transforming growth factor, h1 (Camurati-Engelmann disease) TGFB1 BC000125 3.016 1.000 MAX interactor 1 MXI1 NM_005962 3.797 1.000

Abbreviation: t-SNARE, target soluble N-ethylmaleimide–sensitive factor adaptor protein receptor.

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Table 2. Genes that are involved in DNA repair and contained on the Affymetrix Human Genome U133 Plus 2.0 Array, which are differentially expressed following siRNA-chk–mediated Chk down-regulation in MDA-MB-231 breast cancer cells

Gene title Gene symbol Representative public ID Fold change Probability

Chromosome 11 open reading frame 30 C11orf30 BG272041 1.917 0.965 Fanconi anemia, complementation group M FANCM AK001672 1.822 0.907 Thymine-DNA glycosylase TDG NM_003211 1.769 0.587 HUS1 checkpoint homologue (S. pombe) HUS1 AI968626 1.740 0.589 Polymerase (DNA directed), E POLL W38444 1.714 0.560 Nei endonuclease VIII-like 3 (E. coli) NEIL3 NM_018248 1.901 0.957 Non-POU domain containing, octamer-binding NONO NM_007363 2.430 1.000 RAD23 homologue B (S. cerevisiae) RAD23B NM_002874 3.003 1.000 RAD23 homologue B (S. cerevisiae) RAD23B AF262027 3.217 1.000

The microarray analysis detected changes in the expression of33 In conclusion, the down-regulation ofChk by transient proliferation-related genes with Chk down-regulation. One gene transfection of siRNA-chk resulted in a significant reduction of cell significantly influenced by Chk down-regulation was MXI1 (3.8- proliferation in both estrogen receptor/progesterone receptor– fold), which is a c-Myc antagonist and is induced by hypoxia- positive and estrogen receptor/progesterone receptor–negative inducible factor 1. Whereas MXI1 is considered a potential tumor malignant human breast cancer cell lines but not in nonmalignant suppressor, it also protects cells from c-Myc-induced apoptosis HMECs. A combination ofsiRNA-chk transfection and 5-FU (35, 36). Overexpression ofboth negative (CDKN1B, CDKN1C, NF1, treatment resulted in increased cell kill in both cancer cell lines and SMAD1) and positive (EDN1 and FGFR1OP) regulators ofcell compared with each ofthese treatments given alone. Although the proliferation and underexpression of positive (PBEF1, DNAJA2, and nonmalignant cells were the most sensitive to 5-FU treatment, the SSR1) regulators ofcell proliferation were also observed. EDN1 is a combination treatment resulted in comparable or even higher potent vasoconstrictor peptide, which is also involved in differen- reduction ofcell viability/proliferation in the cancer cells compared tiation, apoptosis, and matrix metalloprotease expression (37). with the nonmalignant cells. Microarray analysis showed that Chk In a previous study, indomethacin-mediated down-regulation of down-regulation affected 33 proliferation-related genes and 9 DNA PC levels in breast cancer cells was also associated with repair–related genes. Our study supports the possibility that Chk overexpression ofEDN1 as detected by microarray analysis (31). silencing may provide a safe and effective therapy against cancer Nine DNA repair genes were affected by Chk down-regulation. One and be a novel alternative to enhance the effect of anticancer drugs. of the most significant effects was the 3-fold underexpression of The development oftherapy using RNA interference targeting Chk RAD23B (3.2-fold). The protein encoded by this gene contains an may be a useful and selective treatment strategy for breast cancer. NH2-terminal ubiquitin-like domain that can bind the proteosome (38, 39). Rad23/proteosome binding is necessary for efficient Acknowledgments nucleotide excision repair, which is the primary mechanism for Received 7/20/2007; accepted 9/24/2007. removing UV-induced DNA lesions (40). It has been shown that Grant support: NIH grant P50 CA103175. Rad23 has additional functions that are required for cell survival The costs ofpublication ofthis article were defrayed in part by the payment ofpage (40). We are currently exploring the role ofChk in multidrug charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. resistance and DNA repair to determine ifincreased expression of We thank Y. Mironchik for laboratory support and Drs. V.P. Chacko and J. Zhang Chk provides protection to cells during chemotherapy. for technical support for magnetic resonance spectroscopy experiments.

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