WMC-79, a Potent Agent Against Colon Cancers, Induces Apoptosis Through a P53-Dependent Pathway

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WMC-79, a potent agent against colon cancers, induces apoptosis through a p53-dependent pathway

Teresa Kosakowska-Cholody,1 Introduction 1 2 W. Marek Cholody, Anne Monks, The bisimidazoacridones are bifunctional antitumor Barbara A. Woynarowska,3 agents with strong selectivity against colon cancers (1, 2). and Christopher J. Michejda1 Recent studies of the effect of bisimidazoacridones on sensitive colon tumors cells revealed that these com- 1 Molecular Aspects of Drug Design, Structural Biophysics pounds act as cytostatic agents that completely arrest cell Laboratory, Center for Cancer Research; 2Screening Technologies Branch, Laboratory of Functional Genomics, Science Applications growth at G1 and G2-M check points but do not trigger International Corporation, National Cancer Institute at Frederick, cell death even at high concentrations (10 Amol/L; ref. 3). Frederick, Maryland; and 3Department of Radiation Oncology, The chemical structure of bisimidazoacridones is symmet- University of Texas Health Science Center, San Antonio, Texas rical in that it consists of two imidazoacridone moieties held together by linkers of various lengths and rigidities. Abstract We recently reported on the synthesis of unsymmetrical variants of the original bisimidazoacridones (4). WMC-79 WMC-79 is a synthetic agent with potent activity (Fig. 1), a compound consisting of an imidazoacridone against colon and hematopoietic tumors. In vitro, the moiety linked to a 3-nitronaphthalimide moiety via agent is most potent against colon cancer cells that 1,4-bispropenopiperazine linker, was found to be a potent carry the wild-type p53 tumor suppressor gene (HCT- but selective cytotoxic agent in a variety of tumor cell f 116 and RKO cells: GI50 <1 nmol/L, LC50 40 nmol/L). lines (4). However, it was more toxic against tumor cell Growth arrest of HCT-116 and RKO cells occurs at the lines that carry the wild-type p53 tumor suppressor gene. G1 and G2-M check points at sublethal concentrations The p53 protein is a tightly regulated transcription factor (10 nmol/L) but the entire cell population was killed at that is elevated in response to DNA damage and has a 100 nmol/L. WMC-79 is localized to the nucleus where critical function in maintaining the integrity of the genome. it binds to DNA. We hypothesized that WMC-79 binding p53-driven cell cycle arrest prevents cells with altered DNA to DNA is recognized as an unrepairable damage in the from proliferating and p53-controlled apoptosis selectively tumor cells, which results in p53 activation. This eliminates severely damaged cells (5–8). Whether the cell triggers transcriptional up-regulation of p53-dependent enters growth arrest or undergoes apoptosis depends on genes involved in replication, cell cycle progression, the final integration of incoming signals with antagonistic growth arrest, and apoptosis as evidenced by DNA effects on cell growth. Many factors affect the cellular microarrays. The change in the transcriptional profile of response to activated p53. These include cell type, HCT-116 cells is followed by a change in the levels of oncogenic status of the cell, survival stimuli, intensity of cell cycle regulatory proteins and apoptosis. The stress signals, level of p53 expression, and interaction of recruitment of the p53-dependent apoptosis pathway p53 with specific proteins (9). was suggested by the up-regulation of p53, p21, Bax, We hypothesized that WMC-79 binding to DNA is DR-4, DR-5, and p53 phosphorylated on Ser15; down- recognized as a damage that is not readily repaired in the regulation of Bcl-2; and activation of caspase-8, -9, -7, tumor cells and which results in the activation of p53. and -3 in cells treated with 100 nmol/L WMC-79. The aim of this study was to investigate the molecular Apoptosis was also evident from the flow cytometric mechanism by which WMC-79 induces growth arrest and studies of drug-treated HCT-116 cells as well as from apoptosis in the sensitive HCT-116 and RKO colon cancer the appearance of nuclear fragmentation. However, cell lines and to determine the role of p53 in this whereas this pathway is important in wild-type p53 mechanism. colon tumors, other pathways are also in operation because colon cancer cell lines in which the p53 gene is mutated are also affected by higher concentrations Materials and Methods of WMC-79. [Mol Cancer Ther 2005;4(10):1617–27] Chemicals All mammalian cell culture reagents and trypan blue were purchased from Life Technologies, Invitrogen Cor- poration (Grand Island, NY). Other reagents were from Received 5/24/05; revised 7/11/05; accepted 7/25/05. Sigma-Aldrich (St. Louis, MO). Requests for reprints: Christopher J. Michejda, Molecular Aspects of Drug Cell Culture Design, Structural Biophysics Laboratory, Center for Cancer Research, +/+ National Cancer Institute at Frederick, Frederick, MD 21702. The human colon cancer cell lines HCT-116 p53 , HCT- Phone: 301-846-1216; Fax: 301-846-6231. E-mail: [email protected] 116 p53/, HCT-116 p21+/+, and HCT-116 p21/ were Copyright C 2005 American Association for Cancer Research. a generous gift from Dr. Bert Vogelstein (Johns Hopkins doi:10.1158/1535-7163.MCT-05-0170 University, Baltimore, MD) and were maintained in

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35-mm glass-bottomed microwell dishes (MatTek Corpo- ration, Ashland, MA). The following day, cells were washed and fresh medium containing 100 nmol/L WMC- 79 was added. Cells were examined at different time points under a Zeiss 410 laser scanning confocal microscope. Areas were imaged using appropriate laser lines for WMC- 79 excitation (488 nm). Figure 1. Chemical structure of WMC-79. Western Blot Analysis Immunoblot analysis of cell protein lysates was done McCoy’s 5A media. Human colon carcinoma HCT-116 according to the protocol of the manufacturer (Santa Cruz +/+ +/+ +/ (p53 ), RKO (p53 ), HCT-15 (p53 ), COLO-205 Biotechnology, Inc., Santa Cruz, CA). Briefly, cells were / / (p53 ), and HT-29 (p53 ) cells were purchased from lysed on ice for 30 to 60 minutes in radioimmunopreci- the American Type Culture Collection (Rockville, MD). pitation assay buffer (1 PBS, 1% igepal, 0.5% sodium HCT-116, RKO, and HT-29 cells were grown in DMEM; deoxycholate, 0.1% SDS) with freshly added inhibitors COLO-205 and HCT-15 cells were grown in RPMI 1640. All (10 Ag/mL phenylmethylsulfonyl fluoride, 50 Ag/mL media were supplemented with 10% heat-inactivated fetal aprotinin, and 1 mmol/L sodium orthovanadate). Cell bovine serum, 2 mmol/L L-glutamine, 100 units/mL lysate was passed through a 21-gauge needle followed by penicillin, and 100 Ag/mL streptomycin. The cells were centrifugation at 10,000 g for 10 minutes at 4jC. Protein grown at 37jC in a humidified atmosphere consisting of concentration was determined using Bio-Rad protein assay 5% CO2 and 95% air. (Bio-Rad Laboratories, Hercules, CA). Samples were Drugs and Drug Preparation Procedure mixed with 2 Laemmli buffer, denaturated at 100jC for Stock solution of WMC-79 synthesized in our laboratory 3 minutes, and proteins were separated by electrophoresis (4) was freshly prepared by dissolving the free base form (NuPAGE 4-12% Bis-Tris Gel, Invitrogen, Life Technologies, of the compound in 2 equivalents of methanesulfonic acid Carlsbad, CA). Separated proteins were transferred to (as 10 mmol/L water solution) and then diluted with water polyvinylidene difluoride membrane (Millipore, Bedford, to a final concentration of 500 Amol/L. This solution was MA) and subjected to immunoblotting with various primary used to prepare 2 Amol/L working solution and its 10-fold antibodies. Positive antibody reactions were visualized with serial dilutions in appropriate complete tissue culture a horseradish peroxidase–conujugated secondary antibody media. and an enhanced chemiluminescence detection system CellViability (Amersham Pharmacia Biotech, Little Chalfont, United 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Kingdom) according to the protocol of the manufacturer. Bromide Assay. Cellular growth in the presence or The membrane was then deprobed and reprobed with an absence of experimental agents was determined using anti-actin antibody to confirm that all samples contained the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium similar amounts of proteins. TBS-0.05% Tween 20 was used bromide (MTT)–based CellTiter96 Non-Radioactive Cell as a wash buffer; 5% nonfat dry milk (Bio-Rad Laboratories) Proliferation Assay (Promega, Madison, WI) according to was dissolved in TBS-0.05% Tween 20 and was used as a the instruction of the manufacturer with small modifica- blocking solution. The following antibodies were used in tions as previously described (4, 10). Trypan blue exclusion this study: mouse anti-Bax (Ab-3), mouse anti-Bcl-2 (Ab-1), assay was used to determine the number of live/dead cells mouse anti–cyclin D1 (Ab-3), mouse anti-E2F1 (Ab-1), in HCT-116 cultures exposed to WMC-79. mouse anti-Mdm2 (Ab-2), mouse anti-p21 (Ab-1), mouse Fluorescence-Activated Cell Sorting. Tumor cells in anti-p53 (Ab-6), mouse anti-pRb (Ab-5), rabbit anti-caspase- exponential phase of growth were seeded at a density of 7 (AB-1), rabbit anti-DR4 (AB-1), rabbit anti-DR5 (AB-2), 0.5 106 to 1 106 cells in 25 or 75 cm2 T flasks, allowed to rabbit anti-phospho-p53(Ser15) (Ab-3; Oncogene Research attach for 24 hours, and then exposed to 10 or 100 nmol/L Products, Boston, MA), goat anti-actin (C-11), goat anti- WMC-79. At appropriate intervals, drug-treated and caspase-8 (C-20), rabbit anti-caspase-9 (PharMingen, San control cells (attached and floating) were collected and Diego, CA), mouse anti–cyclin A, mouse anti–cyclin B1, washed twice in ice-cold PBS containing 1% fetal bovine rabbit anti–cyclin E (Biosource International, Camarillo, serum. The cells were fixed in 70% ethanol and stored at CA), and rabbit anti-phospho-Cdc2(Tyr15) (Cell Signaling 20jC until all time points had been collected. Fixed cells Technology, Beverly, MA). All secondary antibodies (horse- were rinsed twice in ice-cold PBS containing 10% fetal radish peroxidase conjugates) and Cruz Marker molecular bovine serum, treated with RNase A (1 unit/106 cells) for weight standards were from Santa Cruz Biotechnology. 30 minutes at 37jC, and stained overnight with propidium Gene Expression Profiling iodide (50 Ag/mL) at 4jC. Cell cycle analysis was done HumanHCT-116colonadenocarcinoma cells at 60% on a Beckman Coulter Epics XL-MCL flow cytometer confluency were exposed to 100 nmol/L WMC-79 for (Fullerton, CA) with 10,000 events per collected sample. 3, 12, 24, and 72 hours. Total RNAs from tested and Cellular Drug Localization by Confocal Microscopy untreated cells were isolated using RNeasy Mini Kit Cells in logarithmic growth phase were harvested by (Qiagen, Valencia, CA) according to the instructions of the trypsinization and 50,000 to 100,000 cells were seeded in manufacturer. RNA was checked for purity and stability

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by gel electrophoresis. Drug-induced gene expression accumulation of cells in S phase (Fig. 2) but prolonged changes were evaluated by competitive hybridization of exposure to the drug led to G2-M growth arrest in cells equal amounts of control versus drug-treated cDNA with mutated p53 gene (Fig. 2A) and apoptosis in cells with using 20K oligonucleotide microarrays (Hs-OperonV2- wild-type p53 (Fig. 2B). vB1.2p17-092903) purchased from the Advanced Tech- The p53 wild-type cell lines, HCT-116 and RKO, were nology Center, Center for Cancer Research, National further used for a more extensive evaluation of the Cancer Institute (Gaithersburg, MD) and data were cytotoxic properties of WMC-79. A detailed study of the analyzed through the Computer Information Technology effect of WMC-79 treatment on dose and duration of Center mAdb website. Samples from two separate treatment was carried out. WMC-79 at 10 nmol/L totally experiments were evaluated; experiment 1 involved 24 inhibited cell growth without evidence of cell death even and 72 hours of exposure to the drug whereas experi- after 120 hours of exposure (Figs. 3A and B and 2B). ment 2 involved 3, 12, and 24 hours of treatment. Each However, the cell growth arrest seemed to be irreversible sample was run on a single microarray (duplicate 24-hour as 6-hour exposure followed by 120-hour incubation in treatment). Gene expression changes common to all treat- drug-free medium resulted in the same level of growth ment conditions were selected based on those genes show- inhibition as 120-hour continuous drug exposure (Fig. 3A). ing >3-fold change in expression in any two of the five Because the MTT viability assay cannot distinguish arrays. between total growth arrest and equilibrium between growth and death, we did additional experiments in which Results HCT-116 cultures exposed to 10 or 100 nmol/L WMC-79 were analyzed by counting of trypan blue–stained cells at Activity of WMC-79 against Human ColonTumor Cell various time points (Fig. 3B). At 10 nmol/L WMC-79, cell Lines Depends on the Status of p53 number was constant for the duration of the experiment The inhibition of cell proliferation and/or cell cytotox- (the number of dead cells was negligible). FACS analysis of icity induced by WMC-79 was measured by MTT assay, HCT-116 cells exposed to 10 nmol/L WMC-79 (Fig. 2B) trypan blue exclusion, and cell cycle analysis. Initial showed an apparent cell growth arrest at G1 and G2-M experiments in colon cancer cell lines indicated that those phases with complete depletion of S phase. This growth carrying the wild-type p53 gene (HCT-116 and RKO) were arrest persisted for the rest of the experiment with no the most sensitive, especially at the TGI and LC50 levels distinct evidence of cell death at the 120-hour time point. (Table 1). In follow-up experiments with isogenic HCT-116 In contrast, 100 nmol/L WMC-79 was clearly cytotoxic cell lines engineered to express or be null for either p53 or +/+ / +/+ to these cells (Figs. 3A and B and 2B). As evidenced by p21 (HCT-116 p53 , HCT-116 p53 , HCT-116 p21 , FACS analysis, we observed initial accumulation of HCT- / / f and HCT-116 p21 ), HCT p53 cells were 10-fold 116 cells in S phase during the first 24 hours (from 31% for resistant to WMC-79 as compared with HCT-116 p53+/+ / control to 44% for treated cells), a marked decreased in G1 whereas HCT-116 p21 cells were most sensitive to this phase (from 42.5% to 17.6%), and then massive cell death, treatment (Table 1). As evidenced by fluorescence-activated which was evidenced by the appearance of sub-G1 cell cell sorting (FACS) analysis, 24-hour treatment of HT-29, population. This fraction increased steadily with time of COLO-205, HCT-15, and HCT-116 colon cancer cells with exposure to the drug (Fig. 2B). Furthermore, a 6-hour 100 nmol/L WMC-79 caused p53-independent transient exposure to 100 nmol/L WMC-79 followed by drug washout was sufficient to induce exactly the same cytotoxic Table 1. Activity of WMC-79 against selected human colon result as that with continuous treatment with the drug cancer cell lines (Fig. 3A). The cytotoxic effect of WMC-79 at 100 nmol/L was also confirmed by cell counting (Fig. 3B). c b Cell line p53 status GI50* TGI LC50 Cellular Drug Localization (nmol/L) (nmol/L) (nmol/L) To study WMC-79 localization in target cells, we took advantage of the intrinsic fluorescence of the drug that HCT-116 p53+/+ <1 10 F 140F 10 HCT-116 p53/ 4 F 0.4 90 F 11 400 F 50 allowed direct visualization by confocal microscopy. HCT-116 p53+/+ p21+/+ <1 10 F 1 100 F 15 Figure 4 presents the distribution of WMC-79 in HCT-116 HCT-116 p53+/+ p21/ <1 10 F 135F 5 cells. The drug easily crosses the cellular membrane and, RKO p53+/+ <1 10 F 135F 5 within hours, accumulates in the nucleus where it stays HCT-15 p53+/ 3.3 F 0.5 100 F 15 430 F 60 until cell death, which for this cell line (HCT-116) is visible COLO-205 p53/ 2.2 F 0.3 120 F 20 450 F 50 after 48 hours (fragmentation of nucleus). HT-29 p53/ 1.7 F 0.2 100 F 20 500 F 70 Western Blot Analysis We examined the effect of WMC-79 on expression of NOTE: The GI50, TGI, and LC50 values are the average of at least three independent determinations. various cell cycle regulators and apoptosis-regulating * GI50, concentration of the drug resulting in inhibition of cell growth to proteins in RKO and HCT-116 cells. The time-dependent 50% of controls. effects of exposure to 100 nmol/L WMC-79 on the protein cTGI, concentration of the drug resulting in total growth inhibition. b levels in RKO cells are shown in Fig. 5A to D, and very LC50, concentration of the drug required to reduce the initial cell number by 50%. similar results were seen for HCT-116 cells. Western blot

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Figure 2. Cell cycle analysis of human colon tumor cells treated with WMC-79. A, effects of 100 nmol/L WMC-79 on HT-29, COLO-205, and HCT-15 cells after 24 and 120 h of exposure. Cell cycle distribution of untreated HT-29 cells is shown for comparison. Other untreated cell lines had very similar profiles. B, dose and time dependence WMC- 79 on sensitive HCT-116 cells. Ex- ponentially growing cells were treated with a sublethal dose of 10 nmol/L and a toxic dose of 100 nmol/L and analyzed by FACS after 24, 72, and 120 h. The numerical data corre- spond to the percentage values for the indicated stages of the cell cycle. Representative of three individual experiments.

analysis showed distinctly elevated levels of p53, p53 pathway was indicated by the enhancement of DR4 and phosphorylated on Ser15, and MDM2 as early as 3 hours DR5 and the activation of caspase-8 and -7 (Fig. 5C). The after the initiation of treatment with WMC-79. Prolonged effect of WMC-79 treatment on cell cycle proteins was also exposure to WMC-79 enhanced the protein level of determined. Thus, cyclins A and D1 were up-regulated p21WAF1 (Fig. 5A). The level of Bcl-2 was slightly reduced whereas cyclin E was slightly depressed. Cyclin B1 and as a result of the treatment but the level of Bax protein was cyclin-dependent kinase 1 phoshorylated on Tyr15, as well significantly elevated. Likewise, the level of procaspase-9 as pRb and E2F1 protein, were initially up-regulated but was reduced (Fig. 5B). Activation of the death receptor then showed a decrease at longer exposure times (Fig. 5D).

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Regulation of mRNA Expression in HCT-116 Cells (>30), as opposed to induction, as an early response to WMC-79 was examined for its ability to alter gene ex- 100 nmol/L WMC-79 treatment (3- and 12-hour time pression in HCT-116 cells after treatment with 100 nmol/L points) in HCT-116 cells. Among the down-regulated for 3, 12, 24, and 72 hours to identify both early- and late- genes, two well-defined groups can be easily distinguished. response genes. Data were examined from five arrays First are the genes involved in DNA replication such generated from two separate treatments with the 24-hour as MCM3, MCM4, MCM6, Pfs2, FEN1, RRM2, and RFC4 time point being common to both experiments. A selection (11–15). The second identifiable group consists of KIF11, of 901 drug-regulated genes (>1.8-fold change in expres- KIF22, MAD2L1, BUB1, HCAP-G, CCNB1, and SMC4L1, sion) correlated at r = 0.61 between the two independent which are involved in mitosis (16–20). treatments at the 24-hour time point, signifying a similar Unfortunately, at present, the role of the most down- response even with many of the minimally regulated genes. regulated genes in colon cancer is not clear. However, This subset of genes from within the same treatments but it should be noted that the most down-regulated genes over different time points were often more highly corre- TAOK3, JUN, and IL6R (Table 2) are involved in signal lated (0.8), indicating that most of the altered genes were transduction. Interestingly, PIK3C3, which encodes for the down-regulated at an early time point (3 hours) and phosphatidylinositol-3-kinase, also belongs to the same maintained for up to 72 hours. functional group (21–23). In contrast, only four genes, all More stringent selection identified 122 altered genes of which are direct transcriptional targets of p53, were (>3-fold change in at least two arrays) of which f45% were up-regulated at 3 and 12 hours. Moreover, 18 of the most down-regulated and 55% were up-regulated, indicating highly induced genes are known to be directly regulated by no predominant transcriptional repression by WMC-79. p53 [GDF15 (24), p21WAF1/CIP1, TP53I3, BTG2, FDXR, BAX, Table 2 shows the down-regulated and up-regulated genes GADD45, TNFRSF10B, KAI1 (25), SFN, SAT, GPX1, that meet the 3-fold change criterion. These data indicate NDRG1, DDIT4 (REDD1), PLK3, WIG1, LRDD,and that there is a clear bias towards down-regulation of genes RRM2B (26–34)]. These results clearly point to the p53 pathway being affected by WMC-79 in HCT-116 cells. Analysis of the functional assignment of the 122 changed genes and of the pathways/networks involved in the response of HCT-116 cells to WMC-79 suggests that cell cycle control genes, particularly those that affect mitosis and DNA replication and repair, are important. Figure 6 shows the Gene Ontology categories selected through the online Expression Analysis Systematic Explorer program (35), which are significantly overrepresented in the selected gene set as compared with the Locus Link database. In keeping with the individual gene changes, categories such as DNA-dependent ATPase activity, M phase and mitotic cell cycle, DNA replication, and nuclear division are nonrandomly enriched and statistically different from Locus Link abundance.

Discussion We had previously reported that WMC-79 is a novel synthetic agent with potent but selective activity against colon cancer as well as some hematopoietic tumors (4). In the current study, we found that WMC-79 is especially active against colon cancer cells that carry the wild-type p53 tumor suppressor gene, with GI50 <1 nmol/L and LC50 f40 nmol/L in HCT-116 and RKO colon adenocarcinoma lines. The activity of the drug was time and dose dependent Figure 3. The effect of different concentrations of WMC-79 and time of as shown by MTT assay, trypan blue exclusion, and cell exposure on the growth of HCT-116 cells. A, cells grown in 96-well plates cycle analysis. At 10 nmol/L, WMC-79 totally inhibited cell were exposed to various concentrations of the drug for different times and growth of HCT-116 cells whereas at >100 nmol/L, WMC-79 then incubated up to 120 h. The effect of the treatment was determined using MTT cell proliferation assay. Points, mean of three independent led to cell death in the entire cell population of cell lines experiments; bars, SD. B, viable cell number counting. Cells (1 106) bearing wild-type p53. WMC-79 is rapidly localized in were plated in 75-cm2 flasks and, after attachment (24 h later), were the nucleus in HCT-116 cells where it binds to DNA. We exposed to 0, 10, and 100 nmol/L WMC-79. Following trypsin-mediated hypothesize that WMC-79 binding to DNA is recognized detachment, cells were counted at the indicated times by hemocytometry using trypan blue exclusion for cell viability. Each number represents the as a damage that is not readily repaired in tumor cells, average of triplicate experiments. which results in the up-regulation of p53.

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Figure 4. Cellular localization of WMC-79 in living HCT-116 cells. Attached cells were treated with 100 nmol/L WMC-79 and observed with a reverse optics confocal microscope at various times. Green color, native fluorescence of the drug induced by 488-nm excitation. A, 10 min after addition, the drug is localized in the cytoplasm; B, 3 h after addition, WMC-79 is exclusively accumulated in nuclei. C, after 48 h, fragmentation of nuclei is clearly visible. The more intense fluorescence in B and C corresponds to the longer exposure times to the drug. Nuclear staining was further substantiated by colocalization with 4V,6-diamidino-2-phenylindole, a dye that localizes to the nucleus (data not shown).

The p53 tumor suppressor is a critical mediator of including DNA-dependent protein kinase, ataxia telan- cellular responses, such as cell cycle arrest, senescence, giectasia mutated kinase, and Rad3-related kinase and apoptosis, caused by DNA damage and various (37, 38). The phosphorylation cascade involving residues stress signals. In normal, unstressed cells, p53 has a very S15, S20, and T18 impairs the binding of p53 to MDM2, short half-life (5-30 minutes) and is present at very low which prevents its degradation. The p53 stabilization cellular levels due to its continuous degradation medi- effect is additionally enhanced by phosphorylation of ated by MDM2. However, in response to DNA damage MDM2, which blocks its ability to associate with p53 and other stress signals, p53 rapidly stabilized and as well as inhibits its catalytic (ubiquitination) activity. accumulated in the cells due to a block of its degra- Thus, the p53 protein may be stabilized after DNA dation (36). It is generally accepted that stabilization damage even in tumor cells that overexpress MDM2 of p53 requires its phosphorylation by several kinases (36, 39, 40).

Figure 5. Western blot analysis of untreated and WMC-79 – treated RKO cells. Cells were grown in the absence or presence of 100 nmol/L WMC-79. After the indicated times, cells were lysed and total protein was extracted, separated by PAGE, electrotransferred to polyvinylidene difluoride membrane, and subjected to immunoblotting with indicated primary antibodies. See text for details. Actin was used as a loading control.

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Table 2. Gene expression changes in HCT-116 cells following 6, 12, 24, and 72 hours of exposure to 100 nmol/LWMC-79

Gene symbol Gene name Fold change*

3 h 12 h 24 h 72 h

A. Down-regulated genes TAOK3 TAO kinase 3 18.2 16.7 26.6 JUN v-jun sarcoma virus 17 oncogene homologue 11.6 8.5 11.8 IL6R interleukin 6 receptor 10.5 11.8 LOC90668 hypothetical protein BC008134 10.1 10.6 9.8 INSIG2 insulin-induced gene 2 9.4 13.3 8.11 PHF6 PHD finger protein 6 9.2 16.5 12.2 AGTR2 angiotensin II receptor, type 2 6.2 10.4 7.8 ZCCHC4 zinc finger, CCHC domain containing 4 5.8 4.4 4.8 CCNJ cyclin J 5.2 5.5 4.7 FBXW7 F-box and WD-40 domain protein 7 5.1 6.6 3.0 FLJ13273 hypothetical protein FLJ13273 4.9 4.4 4.3 CG018 hypothetical gene CG018 4.8 6.3 3.0 GPHN gephyrin 4.6 29.1 8.2 PEX5L peroxisomal biogenesis factor 5–like 4.5 4.3 4.3 MCSP mitochondrial capsule selenoprotein 4.1 3.8 4.3 LDB3 LIM domain binding 3 4.1 4.7 7.4 CYP7A1 cytochrome P450, family 7, subfamily A, polypeptide 1 4.0 4.0 SYNPO2 synaptopodin 2 3.7 3.9 3.0 TIE1 tyrosine kinase with immunoglobulin-like and 3.6 4.1 epidermal growth factor–like domains 1 TCF15 transcription factor 15 (basic helix-loop-helix) 3.6 3.5 JUB jub, ajuba homologue (Xenopuslaevis) 3.5 3.7 3.4 PIK3C3 phosphoinositide-3-kinase, class 3 3.3 3.7 PTPRZI protein tyrosine phosphatase, receptor-type, Z polypeptide 1 3.3 4.9 3.2 GPHA2 glycoprotein hormone a 2 3.2 3.2 3.3 STAR steroidogenic acute regulator 3.3 6.2 4.0 ATP5S ATP synthase, H-transporting, mitochondrial F0 complex 3.2 6.3 TFAP4 transcription factor AP-4 (activating enhancer binding protein 4) 4.7 3.5 RAB43 RAB43, member RAS oncogene family 4.2 3.0 PARP9 poly(ADP-ribose) polymerase family, member 9 4.1 5.7 SEC31L2 SEC31-like 2 (S. cerevisiae) 3.8 4.3 CCR2 chemokine (C-C motif) receptor 2 3.6 4.0 MITF microphthalmia-associated transcription factor 3.3 4.1 SLC19A1 solute carrier family 19 (folate transporter), member 1 3.3 3.4 SMC4L1 SMC4 structural maintenance of chromosomes 4–like 1 (yeast) 3.3 3.0 RRM2 ribonucleotide reductase M2 polypeptide 7.9 3.7 PPP2R3A protein phosphatase 2 (formerly 2A), regulatory subunit B00, a 7.8 4.4 Pfs2 DNA replication complex GINS protein PSF2 6.9 IFNAR1 IFN (a, h, and N) receptor 1 5.3 3.9 TUBB tubulin, h polypeptide 4.9 3.0 PLA2G2E phospholipase A2, group IIE 4.5 ZNF587 zinc finger protein 587 4.0 3.6 KIF22 kinesin family member 22 3.9 4.6 K-ALPHA-1 tubulin, a, ubiquitous 3.9 3.9 BUB1 BUB1 budding uninhabited by benzimidazoles 1 homologue (yeast) 3.9 MCM4 MCM4 minichromosome maintenance deficient 4 (S. cerevisiae) 3.9 CCNB1 cyclin B1 3.8 4.4 FEN1 flap structure–specific endonuclease 1 3.7 H2AFFP H2A histone family, member F, pseudogene 3.6 3.0 HIST1H2APS5 histone 1, H2a, pseudogene 5 3.6 3.0 HCAP-G chromosome condensation protein G 3.6 MCM3 MCM3 minichromosome maintenance deficient 3 (S. cerevisiae) 3.3 MAD2L1 MAD2 mitotic arrest deficient–like 1 (yeast) 3.3

*No entry indicates gene expression change <3. (Continued on the following page)

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Table 2. Gene expression changes in HCT-116 cells following 6, 12, 24, and 72 hours of exposure to 100 nmol/LWMC-79 (Cont’d)

Gene symbol Gene name Fold change*

3 h 12 h 24 h 72 h

KIF11 kinesin family member 11 3.3 MCM6 MCM6 minichromosome maintenance 3.2 deficient 6 (MIS5 homologue, S. pombe) RFC4 replication factor C (activator 1) 4, 37 kDa 3.2 SCRN3 secernin 3 3.0 3.0

B. Up-regulated genes GDF15 growth differentiation factor 15 3 5.5 19.1 23.9 CDKN1A cyclin-dependent kinase inhibitor 1A (p21, Cip1) 6.3 16.9 38.1 SAT spermidine/spermine N1-acetyl transferase 3.3 7.6 3.3 GPX1 glutathione peroxidase 1 3.2 4 10.8 TP5313 tumor protein p53 inducible protein 3 14.2 7.6 BTG2 BTG family, member 2 9.2 KRTAP2.1A keratin associated protein 2-1 6.0 11.6 TNFRSF10B tumor necrosis factor receptor superfamily, member 10b 5.9 5 WDR1 WD repeat domain 1 5.3 4.2 FLNA filamin A, a (actin binding protein 280) 5.6 6.0 RPS27L ribosomal protein S27–like 5 7.3 CSDA cold shock domain protein A 4.9 5.2 FHL2 four and a half LIM domains 2 4.9 4.5 NDRG1 N-myc downstream regulated gene 1 4.7 5.7 FBXO2 F-box protein 2 4.7 8.6 BLCAP bladder cancer associated protein 4.7 3.7 NGFRAP1 nerve growth factor receptor (TNFRSF16) associated protein 1 4.6 3.7 PSTPIP2 proline-serine-threonine phosphatase interacting protein 2 4.5 8.1 CYFIP2 cytoplasmic FMR1 interacting protein 2 4.5 5.5 PTGES prostaglandin E synthase 4.5 3.3 KAI1 CD82 antigen 4.5 ITGA3 integrin, a 3 (antigen CD49C) 4.4 18.3 ASS argininosuccinate synthetase 4.2 6.4 GADD45A growth arrest and DNA damage-inducible, a 4.3 3.8 SFN stratifin (14-3-3j) 4.2 6.3 PHLDA3 pleckstrin homology–like domain, family A, member 3 4.2 5 PLK3 polo-like kinase 3 (Drosophila) 4.2 3.6 KRT19 keratin 19 4.1 12.1 SDC4 syndecan 4 (amphiglycan, ryudocan) 4.1 11.9 TM7SF2 transmembrane 7 superfamily member 2 4.1 3.4 CLDN4 claudin 4 4.1 3.1 BAX BCL2-associated X protein 4.1 3 NINJ1 ninjurin 1 4.1 DDIT4 DNA damage-inducible transcript 4 4.1 4.1 CES2 carboxylesterase 2 (intestine, liver) 3.9 3.7 WIG1 p53 target zinc finger protein 3.9 3.5 HLA-C MHC, class I, C 3.8 6 C20orf108 chromosome 20 open reading frame 108 3.8 RAI3 retinoic acid induced 3 3.7 4.2 TETRAN tetracycline transporter–like protein 3.6 5.7 ACTN1 actinin, a 1 3.6 3.7 FDXR ferredoxin reductase 3.5 8 RGS16 regulator of G-protein signaling 16 3.5 4.7 LRDD leucine-rich repeats and death domain containing 3.5 3.9 CST3 cystatin C (amyloid angiopathy and cerebral hemorrhage) 3.4 9.9 TFF1 trefoil factor 1 (breast cancer, estrogen-inducible sequence expressed in) 3.4 4.7 S100A16 S100 calcium binding protein A16 3.3 5.4 CST6 cystatin E/M 3.3 3.4

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Mol Cancer Ther 2005;4(10). October 2005

Table 2. Gene expression changes in HCT-116 cells following 6, 12, 24, and 72 hours of exposure to 100 nmol/LWMC-79 (Cont’d)

Gene symbol Gene name Fold change*

3 h 12 h 24 h 72 h

MVP major vault protein (MVP), transcript variant 2 3.3 3.4 C9orf88 chromosome 9 open reading frame 88 3.3 3.3 PP1201 PP1201 protein 3.2 6.7 MKNK2 mitogen-activated protein kinase interacting serine/threonine kinase 2 3.2 3.4 TAF10 TAF10 RNA polymerase II 3.2 3.0 RAP2B RAP2B, member of RAS oncogene family 3.1 3.8 RRM2B ribonucleotide reductase M2 B (TP53 inducible) 3.1 4.4 TRIM8 tripartite motif–containing 8 3.1 3.5 MSF MLL septin–like fusion 3.0 4.2 PROCR protein C receptor, endothelial 3.0 4.2 RBP1 retinol binding protein 4 3.0 4.0

Dramatic up-regulation of p53 and p53 phosphorylated on and by inhibiting the function of proliferating cell nuclear Ser15 was observed as early as 3 hours in RKO and HCT- antigen in S phase. In addition, as a transcription factor, 116 cells following treatment with 100 nmol/L WMC-79 p21 can directly down-regulate expression of many genes and the levels increased with time (Fig. 5A). At the same involved in DNA replication, repair, and mitosis (41). time, up-regulation of MDM2 was detected, most likely Although G2 arrest can occur in the absence of p21 or as a result of the existing p53 autoregulatory loop (25, 36). p53, both of these proteins are essential for sustaining G2 Following activation of p53, a dramatic increase in arrest after DNA damage (42). Western blot analysis expression of p21WAF1/CIP1, both on gene transcription showed that WMC-79 markedly increased the expression and protein levels, was detected (Table 2; Fig. 5A). This of p21WAF1/CIP1 followed by elevated levels of cyclins A protein is an established direct transcriptional target of p53 and D1 after 48 hours of exposure to the drug. Protein that arrests cell growth in G1 and S phase in response to levels of E2F1, hyperphosphorylated Rb (ppRb), cyclin B1, DNA damage (9). p21WAF1/CIP1 exerts a negative effect on and cyclin-dependent kinase 1 phoshorylated on Tyr15 G1 progression by inhibiting the activity of cyclin E/cyclin- (Cdc2-Tyr15) became elevated during the early time of dependent kinase 2 complexes that phosphorylate pRb exposure but decreased rapidly after 48 hours (Fig. 5D).

Figure 6. Functional groups (Gene Ontology classification) that are significantly (Bonferroni adjusted P < 0.01) more enriched (% abundance) within the selected group of 128 genes than in the Locus Link database. The abundance of genes within a Gene Ontology classification is significantly higher (as percentage) in the selected gene group compared with the background abundance found in Locus Link. These groups represent nonrandom clustering of genes within functional groups (Gene Ontology classification).

Mol Cancer Ther 2005;4(10). October 2005

Transcription of cyclin B1 can be directly repressed by p53 dependent cell response and apoptotic pathway is evident (43) and, indeed, we observed significant down-regulation by a change in expression of a large number of p53- of CCNB1 gene (Table 2). The time-dependent effect of regulated genes and overexpression of p53 itself, its Ser15- WMC-79 on the expression of various cell cycle regulators phoshorylated form, p21WAF1/CIP1, Bax, DR4, and DR5; is in complete accord with cell cycle analysis by FACS down-regulation of Bcl-2; and finally activation of (Fig. 2B). These results are consistent with a mechanism in caspase-9, -8, -3 and -7. Apoptotic cell death was also which WMC-79 exerts cytotoxicity by direct induction of confirmed by flow cytometry, cell morphology of WMC- apoptosis from S phase or arrest in G2-M phase and 79–treated colon cancer cells (Fig. 4), nuclear fragmenta- subsequent induction of apoptosis by depletion of cyclin tion, and formation of nucleosomal ladders in leukemia B1, as was recently reported (44). cell lines (4). Preliminary in vivo experiments on HCT-116 Numerous proapoptotic genes that are transcriptionally colon cancer xenografted in nude mice revealed good activated by p53 have been identified, suggesting that activity when the compound was administered i.v. (4). p53 apoptotic response is multifaceted (45). The BAX gene, WMC-79 is somewhat related to the potent antitumor a proapoptotic member of the Bcl-2 family, is an important agent MLN944 (51), which also binds to DNA and target for p53. This protein together with an additional p53 apparently affects transcription. However, there are target gene product (Noxa, PUMA, and p53AIPI) localizes currently insufficient data to make a direct comparison to the mitochondria and induces the loss of the mitochon- between the two agents. drial membrane potential and the release of cytochrome c. It should also be noted that WMC-79 is also cytotoxic to Cytochrome c interacts with Apaf-1, resulting in the tumor cells in which the p53 gene is either mutated or not activation of caspase-9, which in turn activates the effector expressed. This suggests that other pathways leading to cell caspase-3 (45, 46). Release of mitochondrial cytochrome arrest and death are involved. Further experiments are c and activation of caspase-3 are blocked by antiapoptotic needed to elucidate those mechanisms. Bcl-2, another member of Bcl-2 family. It is clear that overexpression of Bcl-2 can block p53-mediated apoptosis. 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Teresa Kosakowska-Cholody, W. Marek Cholody, Anne Monks, et al.

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