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Mol Cancer Ther. 2005;4:1617-1627
© 2005 American Association for Cancer Research

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

Teresa Kosakowska-Cholody1, W. Marek Cholody1, Anne Monks2, Barbara A. Woynarowska3 and Christopher J. Michejda1

1 Molecular Aspects of Drug Design, Structural Biophysics Laboratory, Center for Cancer Research; 2 Screening Technologies Branch, Laboratory of Functional Genomics, Science Applications International Corporation, National Cancer Institute at Frederick, Frederick, Maryland; and 3 Department of Radiation Oncology, University of Texas Health Science Center, San Antonio, Texas

Requests for reprints: Christopher J. Michejda, Molecular Aspects of Drug Design, Structural Biophysics Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD 21702. Phone: 301-846-1216; Fax: 301-846-6231. E-mail: michejda{at}ncifcrf.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
WMC-79 is a synthetic agent with potent activity against colon and hematopoietic tumors. In vitro, the agent is most potent against colon cancer cells that carry the wild-type p53 tumor suppressor gene (HCT-116 and RKO cells: GI50 <1 nmol/L, LC50 ~40 nmol/L). Growth arrest of HCT-116 and RKO cells occurs at the G1 and G2-M check points at sublethal concentrations (10 nmol/L) but the entire cell population was killed at 100 nmol/L. WMC-79 is localized to the nucleus where it binds to DNA. We hypothesized that WMC-79 binding to DNA is recognized as an unrepairable damage in the tumor cells, which results in p53 activation. This triggers transcriptional up-regulation of p53-dependent genes involved in replication, cell cycle progression, growth arrest, and apoptosis as evidenced by DNA microarrays. The change in the transcriptional profile of HCT-116 cells is followed by a change in the levels of cell cycle regulatory proteins and apoptosis. The recruitment of the p53-dependent apoptosis pathway was suggested by the up-regulation of p53, p21, Bax, DR-4, DR-5, and p53 phosphorylated on Ser15; down-regulation of Bcl-2; and activation of caspase-8, -9, -7, and -3 in cells treated with 100 nmol/L WMC-79. Apoptosis was also evident from the flow cytometric studies of drug-treated HCT-116 cells as well as from the appearance of nuclear fragmentation. However, whereas this pathway is important in wild-type p53 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 of WMC-79.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The bisimidazoacridones are bifunctional antitumor agents with strong selectivity against colon cancers (1, 2). Recent studies of the effect of bisimidazoacridones on sensitive colon tumors cells revealed that these compounds act as cytostatic agents that completely arrest cell growth at G1 and G2-M check points but do not trigger cell death even at high concentrations (10 µmol/L; ref. 3). The chemical structure of bisimidazoacridones is symmetrical in that it consists of two imidazoacridone moieties held together by linkers of various lengths and rigidities. We recently reported on the synthesis of unsymmetrical variants of the original bisimidazoacridones (4). WMC-79 (Fig. 1), a compound consisting of an imidazoacridone moiety linked to a 3-nitronaphthalimide moiety via 1,4-bispropenopiperazine linker, was found to be a potent but selective cytotoxic agent in a variety of tumor cell lines (4). However, it was more toxic against tumor cell lines that carry the wild-type p53 tumor suppressor gene.



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  		Figure 1. Chemical structure of WMC-79.

 
The p53 protein is a tightly regulated transcription factor that is elevated in response to DNA damage and has a critical function in maintaining the integrity of the genome. p53-driven cell cycle arrest prevents cells with altered DNA from proliferating and p53-controlled apoptosis selectively eliminates severely damaged cells (5–8). Whether the cell enters growth arrest or undergoes apoptosis depends on the final integration of incoming signals with antagonistic effects on cell growth. Many factors affect the cellular response to activated p53. These include cell type, oncogenic status of the cell, survival stimuli, intensity of stress signals, level of p53 expression, and interaction of p53 with specific proteins (9).

We hypothesized that WMC-79 binding to DNA is recognized as a damage that is not readily repaired in the tumor cells and which results in the activation of p53.

The aim of this study was to investigate the molecular mechanism by which WMC-79 induces growth arrest and apoptosis in the sensitive HCT-116 and RKO colon cancer cell lines and to determine the role of p53 in this mechanism.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
All mammalian cell culture reagents and trypan blue were purchased from Life Technologies, Invitrogen Corporation (Grand Island, NY). Other reagents were from Sigma-Aldrich (St. Louis, MO).

Cell Culture
The human colon cancer cell lines HCT-116 p53+/+, HCT-116 p53–/–, HCT-116 p21+/+, and HCT-116 p21–/– were a generous gift from Dr. Bert Vogelstein (Johns Hopkins University, Baltimore, MD) and were maintained in McCoy's 5A media. Human colon carcinoma HCT-116 (p53+/+), RKO (p53+/+), HCT-15 (p53+/–), COLO-205 (p53–/–), and HT-29 (p53–/–) cells were purchased from the American Type Culture Collection (Rockville, MD). HCT-116, RKO, and HT-29 cells were grown in DMEM; COLO-205 and HCT-15 cells were grown in RPMI 1640. All media were supplemented with 10% heat-inactivated fetal bovine serum, 2 mmol/L L-glutamine, 100 units/mL penicillin, and 100 µg/mL streptomycin. The cells were grown at 37°C in a humidified atmosphere consisting of 5% CO2 and 95% air.

Drugs and Drug Preparation Procedure
Stock solution of WMC-79 synthesized in our laboratory (4) was freshly prepared by dissolving the free base form of the compound in 2 equivalents of methanesulfonic acid (as 10 mmol/L water solution) and then diluted with water to a final concentration of 500 µmol/L. This solution was used to prepare 2 µmol/L working solution and its 10-fold serial dilutions in appropriate complete tissue culture media.

Cell Viability
3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide Assay. Cellular growth in the presence or absence of experimental agents was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)–based CellTiter96 Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI) according to the instruction of the manufacturer with small modifications as previously described (4, 10). Trypan blue exclusion assay was used to determine the number of live/dead cells in HCT-116 cultures exposed to WMC-79.

Fluorescence-Activated Cell Sorting. Tumor cells in exponential phase of growth were seeded at a density of 0.5 x 106 to 1 x 106 cells in 25 or 75 cm2 T flasks, allowed to attach for 24 hours, and then exposed to 10 or 100 nmol/L WMC-79. At appropriate intervals, drug-treated and control cells (attached and floating) were collected and washed twice in ice-cold PBS containing 1% fetal bovine serum. The cells were fixed in 70% ethanol and stored at –20°C until all time points had been collected. Fixed cells were rinsed twice in ice-cold PBS containing 10% fetal bovine serum, treated with RNase A (1 unit/106 cells) for 30 minutes at 37°C, and stained overnight with propidium iodide (50 µg/mL) at 4°C. Cell cycle analysis was done on a Beckman Coulter Epics XL-MCL flow cytometer (Fullerton, CA) with 10,000 events per collected sample.

Cellular Drug Localization by Confocal Microscopy
Cells in logarithmic growth phase were harvested by trypsinization and 50,000 to 100,000 cells were seeded in 35-mm glass-bottomed microwell dishes (MatTek Corporation, 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).

Western Blot Analysis
Immunoblot analysis of cell protein lysates was done according to the protocol of the manufacturer (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Briefly, cells were lysed on ice for 30 to 60 minutes in radioimmunoprecipitation assay buffer (1x PBS, 1% igepal, 0.5% sodium deoxycholate, 0.1% SDS) with freshly added inhibitors (10 µg/mL phenylmethylsulfonyl fluoride, 50 µg/mL aprotinin, and 1 mmol/L sodium orthovanadate). Cell lysate was passed through a 21-gauge needle followed by centrifugation at 10,000 x g for 10 minutes at 4°C. Protein concentration was determined using Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). Samples were mixed with 2x Laemmli buffer, denaturated at 100°C for 3 minutes, and proteins were separated by electrophoresis (NuPAGE 4-12% Bis-Tris Gel, Invitrogen, Life Technologies, Carlsbad, CA). Separated proteins were transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA) and subjected to immunoblotting with various primary antibodies. Positive antibody reactions were visualized with a horseradish peroxidase–conujugated secondary antibody and an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom) according to the protocol of the manufacturer. The membrane was then deprobed and reprobed with an anti-actin antibody to confirm that all samples contained similar amounts of proteins. TBS-0.05% Tween 20 was used as a wash buffer; 5% nonfat dry milk (Bio-Rad Laboratories) was dissolved in TBS-0.05% Tween 20 and was used as a blocking solution. The following antibodies were used in this study: mouse anti-Bax (Ab-3), mouse anti-Bcl-2 (Ab-1), mouse anti–cyclin D1 (Ab-3), mouse anti-E2F1 (Ab-1), mouse anti-Mdm2 (Ab-2), mouse anti-p21 (Ab-1), mouse anti-p53 (Ab-6), mouse anti-pRb (Ab-5), rabbit anti-caspase-7 (AB-1), rabbit anti-DR4 (AB-1), rabbit anti-DR5 (AB-2), rabbit anti-phospho-p53(Ser15) (Ab-3; Oncogene Research Products, Boston, MA), goat anti-actin (C-11), goat anti-caspase-8 (C-20), rabbit anti-caspase-9 (PharMingen, San Diego, CA), mouse anti–cyclin A, mouse anti–cyclin B1, rabbit anti–cyclin E (Biosource International, Camarillo, CA), and rabbit anti-phospho-Cdc2(Tyr15) (Cell Signaling Technology, Beverly, MA). All secondary antibodies (horseradish peroxidase conjugates) and Cruz Marker molecular weight standards were from Santa Cruz Biotechnology.

Gene Expression Profiling
Human HCT-116 colon adenocarcinoma cells at 60% confluency were exposed to 100 nmol/L WMC-79 for 3, 12, 24, and 72 hours. Total RNAs from tested and untreated cells were isolated using RNeasy Mini Kit (Qiagen, Valencia, CA) according to the instructions of the manufacturer. RNA was checked for purity and stability by gel electrophoresis. Drug-induced gene expression changes were evaluated by competitive hybridization of equal amounts of control versus drug-treated cDNA using 20K oligonucleotide microarrays (Hs-OperonV2-vB1.2p17-092903) purchased from the Advanced Technology Center, Center for Cancer Research, National Cancer Institute (Gaithersburg, MD) and data were analyzed through the Computer Information Technology Center mAdb website. Samples from two separate experiments were evaluated; experiment 1 involved 24 and 72 hours of exposure to the drug whereas experiment 2 involved 3, 12, and 24 hours of treatment. Each sample was run on a single microarray (duplicate 24-hour treatment). Gene expression changes common to all treatment conditions were selected based on those genes showing >3-fold change in expression in any two of the five arrays.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Activity of WMC-79 against Human Colon Tumor Cell Lines Depends on the Status of p53
The inhibition of cell proliferation and/or cell cytotoxicity induced by WMC-79 was measured by MTT assay, trypan blue exclusion, and cell cycle analysis. Initial experiments in colon cancer cell lines indicated that those carrying the wild-type p53 gene (HCT-116 and RKO) were the most sensitive, especially at the TGI and LC50 levels (Table 1). In follow-up experiments with isogenic HCT-116 cell lines engineered to express or be null for either p53 or p21 (HCT-116 p53+/+, HCT-116 p53–/–, HCT-116 p21+/+, and HCT-116 p21–/–), HCT p53–/– cells were ~10-fold resistant to WMC-79 as compared with HCT-116 p53+/+ whereas HCT-116 p21–/– cells were most sensitive to this treatment (Table 1). As evidenced by fluorescence-activated cell sorting (FACS) analysis, 24-hour treatment of HT-29, COLO-205, HCT-15, and HCT-116 colon cancer cells with 100 nmol/L WMC-79 caused p53-independent transient accumulation of cells in S phase (Fig. 2) but prolonged exposure to the drug led to G2-M growth arrest in cells with mutated p53 gene (Fig. 2A) and apoptosis in cells with wild-type p53 (Fig. 2B).


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  		Table 1. Activity of WMC-79 against selected human colon cancer cell lines

 


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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. Exponentially 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 correspond to the percentage values for the indicated stages of the cell cycle. Representative of three individual experiments.

 
The p53 wild-type cell lines, HCT-116 and RKO, were further used for a more extensive evaluation of the cytotoxic properties of WMC-79. A detailed study of the effect of WMC-79 treatment on dose and duration of treatment was carried out. WMC-79 at 10 nmol/L totally inhibited cell growth without evidence of cell death even after 120 hours of exposure (Figs. 3A and B and 2B). However, the cell growth arrest seemed to be irreversible as 6-hour exposure followed by 120-hour incubation in drug-free medium resulted in the same level of growth inhibition as 120-hour continuous drug exposure (Fig. 3A). Because the MTT viability assay cannot distinguish between total growth arrest and equilibrium between growth and death, we did additional experiments in which HCT-116 cultures exposed to 10 or 100 nmol/L WMC-79 were analyzed by counting of trypan blue–stained cells at various time points (Fig. 3B). At 10 nmol/L WMC-79, cell number was constant for the duration of the experiment (the number of dead cells was negligible). FACS analysis of HCT-116 cells exposed to 10 nmol/L WMC-79 (Fig. 2B) showed an apparent cell growth arrest at G1 and G2-M phases with complete depletion of S phase. This growth arrest persisted for the rest of the experiment with no distinct evidence of cell death at the 120-hour time point.



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  		Figure 3. The effect of different concentrations of WMC-79 and time of exposure on the growth of HCT-116 cells. A, cells grown in 96-well plates were exposed to various concentrations of the drug for different times and then incubated up to 120 h. The effect of the treatment was determined using MTT cell proliferation assay. Points, mean of three independent experiments; bars, SD. B, viable cell number counting. Cells (1 x 106) were plated in 75-cm2 flasks and, after attachment (24 h later), were exposed to 0, 10, and 100 nmol/L WMC-79. Following trypsin-mediated detachment, cells were counted at the indicated times by hemocytometry using trypan blue exclusion for cell viability. Each number represents the average of triplicate experiments.

 
In contrast, 100 nmol/L WMC-79 was clearly cytotoxic to these cells (Figs. 3A and B and 2B). As evidenced by FACS analysis, we observed initial accumulation of HCT-116 cells in S phase during the first 24 hours (from 31% for control to 44% for treated cells), a marked decreased in G1 phase (from 42.5% to 17.6%), and then massive cell death, which was evidenced by the appearance of sub-G1 cell population. This fraction increased steadily with time of exposure to the drug (Fig. 2B). Furthermore, a 6-hour exposure to 100 nmol/L WMC-79 followed by drug washout was sufficient to induce exactly the same cytotoxic result as that with continuous treatment with the drug (Fig. 3A). The cytotoxic effect of WMC-79 at 100 nmol/L was also confirmed by cell counting (Fig. 3B).

Cellular Drug Localization
To study WMC-79 localization in target cells, we took advantage of the intrinsic fluorescence of the drug that allowed direct visualization by confocal microscopy. Figure 4 presents the distribution of WMC-79 in HCT-116 cells. The drug easily crosses the cellular membrane and, within hours, accumulates in the nucleus where it stays until cell death, which for this cell line (HCT-116) is visible after 48 hours (fragmentation of nucleus).



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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 4',6-diamidino-2-phenylindole, a dye that localizes to the nucleus (data not shown).

 
Western Blot Analysis
We examined the effect of WMC-79 on expression of various cell cycle regulators and apoptosis-regulating proteins in RKO and HCT-116 cells. The time-dependent effects of exposure to 100 nmol/L WMC-79 on the protein levels in RKO cells are shown in Fig. 5A to D, and very similar results were seen for HCT-116 cells. Western blot analysis showed distinctly elevated levels of p53, p53 phosphorylated on Ser15, and MDM2 as early as 3 hours after the initiation of treatment with WMC-79. Prolonged exposure to WMC-79 enhanced the protein level of p21WAF1 (Fig. 5A). The level of Bcl-2 was slightly reduced as a result of the treatment but the level of Bax protein was significantly elevated. Likewise, the level of procaspase-9 was reduced (Fig. 5B). Activation of the death receptor pathway was indicated by the enhancement of DR4 and DR5 and the activation of caspase-8 and -7 (Fig. 5C). The effect of WMC-79 treatment on cell cycle proteins was also determined. Thus, cyclins A and D1 were up-regulated whereas cyclin E was slightly depressed. Cyclin B1 and cyclin-dependent kinase 1 phoshorylated on Tyr15, as well as pRb and E2F1 protein, were initially up-regulated but then showed a decrease at longer exposure times (Fig. 5D).



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  		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.

 
Regulation of mRNA Expression in HCT-116 Cells
WMC-79 was examined for its ability to alter gene expression in HCT-116 cells after treatment with 100 nmol/L for 3, 12, 24, and 72 hours to identify both early- and late-response genes. Data were examined from five arrays generated from two separate treatments with the 24-hour time point being common to both experiments. A selection of 901 drug-regulated genes (>1.8-fold change in expression) correlated at r = 0.61 between the two independent treatments at the 24-hour time point, signifying a similar response even with many of the minimally regulated genes. This subset of genes from within the same treatments but over different time points were often more highly correlated (0.8), indicating that most of the altered genes were down-regulated at an early time point (3 hours) and maintained for up to 72 hours.

More stringent selection identified 122 altered genes (>3-fold change in at least two arrays) of which ~45% were down-regulated and 55% were up-regulated, indicating no predominant transcriptional repression by WMC-79. Table 2 shows the down-regulated and up-regulated genes that meet the 3-fold change criterion. These data indicate that there is a clear bias towards down-regulation of genes (>30), as opposed to induction, as an early response to 100 nmol/L WMC-79 treatment (3- and 12-hour time points) in HCT-116 cells. Among the down-regulated genes, two well-defined groups can be easily distinguished. First are the genes involved in DNA replication such as MCM3, MCM4, MCM6, Pfs2, FEN1, RRM2, and RFC4 (11–15). The second identifiable group consists of KIF11, KIF22, MAD2L1, BUB1, HCAP-G, CCNB1, and SMC4L1, which are involved in mitosis (16–20).


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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

 
Unfortunately, at present, the role of the most down-regulated genes in colon cancer is not clear. However, it should be noted that the most down-regulated genes TAOK3, JUN, and IL6R (Table 2) are involved in signal transduction. Interestingly, PIK3C3, which encodes for the phosphatidylinositol-3-kinase, also belongs to the same functional group (21–23). In contrast, only four genes, all of which are direct transcriptional targets of p53, were up-regulated at 3 and 12 hours. Moreover, 18 of the most highly induced genes are known to be directly regulated by p53 [GDF15 (24), p21WAF1/CIP1, TP53I3, BTG2, FDXR, BAX, GADD45, TNFRSF10B, KAI1 (25), SFN, SAT, GPX1, NDRG1, DDIT4 (REDD1), PLK3, WIG1, LRDD, and 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.



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  		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).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
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 ~40 nmol/L in HCT-116 and RKO colon adenocarcinoma lines. The activity of the drug was time and dose dependent as shown by MTT assay, trypan blue exclusion, and cell cycle analysis. At 10 nmol/L, WMC-79 totally inhibited cell growth of HCT-116 cells whereas at >100 nmol/L, WMC-79 led to cell death in the entire cell population of cell lines bearing wild-type p53. WMC-79 is rapidly localized in the nucleus in HCT-116 cells where it binds to DNA. We hypothesize that WMC-79 binding to DNA is recognized as a damage that is not readily repaired in tumor cells, which results in the up-regulation of p53.

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

Dramatic up-regulation of p53 and p53 phosphorylated on Ser15 was observed as early as 3 hours in RKO and HCT-116 cells following treatment with 100 nmol/L WMC-79 and the levels increased with time (Fig. 5A). At the same time, up-regulation of MDM2 was detected, most likely as a result of the existing p53 autoregulatory loop (25, 36).

Following activation of p53, a dramatic increase in expression of p21WAF1/CIP1, both on gene transcription and protein levels, was detected (Table 2; Fig. 5A). This protein is an established direct transcriptional target of p53 that arrests cell growth in G1 and S phase in response to DNA damage (9). p21WAF1/CIP1 exerts a negative effect on G1 progression by inhibiting the activity of cyclin E/cyclin-dependent kinase 2 complexes that phosphorylate pRb and by inhibiting the function of proliferating cell nuclear antigen in S phase. In addition, as a transcription factor, p21 can directly down-regulate expression of many genes involved in DNA replication, repair, and mitosis (41).

Although G2 arrest can occur in the absence of p21 or p53, both of these proteins are essential for sustaining G2 arrest after DNA damage (42). Western blot analysis showed that WMC-79 markedly increased the expression of p21WAF1/CIP1 followed by elevated levels of cyclins A and D1 after 48 hours of exposure to the drug. Protein levels of E2F1, hyperphosphorylated Rb (ppRb), cyclin B1, and cyclin-dependent kinase 1 phoshorylated on Tyr15 (Cdc2-Tyr15) became elevated during the early time of exposure but decreased rapidly after 48 hours (Fig. 5D). Transcription of cyclin B1 can be directly repressed by p53 (43) and, indeed, we observed significant down-regulation of CCNB1 gene (Table 2). The time-dependent effect of WMC-79 on the expression of various cell cycle regulators is in complete accord with cell cycle analysis by FACS (Fig. 2B). These results are consistent with a mechanism in which WMC-79 exerts cytotoxicity by direct induction of apoptosis from S phase or arrest in G2-M phase and subsequent induction of apoptosis by depletion of cyclin B1, as was recently reported (44).

Numerous proapoptotic genes that are transcriptionally activated by p53 have been identified, suggesting that p53 apoptotic response is multifaceted (45). The BAX gene, a proapoptotic member of the Bcl-2 family, is an important target for p53. This protein together with an additional p53 target gene product (Noxa, PUMA, and p53AIPI) localizes to the mitochondria and induces the loss of the mitochondrial membrane potential and the release of cytochrome c. Cytochrome c interacts with Apaf-1, resulting in the activation of caspase-9, which in turn activates the effector caspase-3 (45, 46). Release of mitochondrial cytochrome c and activation of caspase-3 are blocked by antiapoptotic Bcl-2, another member of Bcl-2 family. It is clear that overexpression of Bcl-2 can block p53-mediated apoptosis. Bax binds to Bcl-2 and antagonizes its ability to block apoptosis so that a p53-dependent Bax synthesis could tip the scales toward apoptosis (9, 47). In summary, Bcl-2 protects cells from the induction of apoptosis whereas Bax promotes cell killing. In many studies, Bcl-2 gene expression was down-regulated and Bax expression was up-regulated in apoptosis (7, 47). Western blot analysis (Fig. 5B) showed a time-dependent elevation of Bax protein level and down-regulation of Bcl-2 and procaspase-9 proteins. This suggests that an increasing ratio of Bax/Bcl-2 proteins plays an important role in the induction of apoptosis by WMC-79 in HCT-116.

p53 has also been implicated in the membrane death receptor–induced pathway of apoptosis. It can up-regulate expression of TRAIL death receptors (DR4 and DR5; refs. 48, 49) and activate the CD95 (APO-1/Fas) receptor/ligand system (50). We found that incubation of RKO cells with 100 nmol/L WMC-79 induced elevation of DR4 and DR5 proteins and activation of caspase-8 (Fig. 5C). Together, our data suggest two mechanisms by which p53 may induce apoptosis: one involving activation of caspase-9 by up-regulation of Bax and down-regulation of Bcl-2 proteins, and the second through the activation of death receptors and caspase-8. Activated caspase-8 and caspase-9 can then activate the effectors caspase-3 and caspase-7. We reported earlier (4) that in response to 100 nmol/L WMC-79, the activity of caspase-3 was dramatically increased after 48 hours and reached an ~7-fold increase over the basal level in untreated cells.

In conclusion, our data show that WMC-79, a novel, very potent cytotoxic agent, is much more effective against colon cancer cells that carry wild-type p53 (~50% of all colon cancers) and, at concentration >100 nmol/L, kills cells by induction of apoptosis. The p53-dependent cell response and apoptotic pathway is evident by a change in expression of a large number of p53-regulated genes and overexpression of p53 itself, its Ser15-phoshorylated form, p21WAF1/CIP1, Bax, DR4, and DR5; down-regulation of Bcl-2; and finally activation of caspase-9, -8, -3 and -7. Apoptotic cell death was also confirmed by flow cytometry, cell morphology of WMC-79–treated colon cancer cells (Fig. 4), nuclear fragmentation, and formation of nucleosomal ladders in leukemia cell lines (4). Preliminary in vivo experiments on HCT-116 colon cancer xenografted in nude mice revealed good activity when the compound was administered i.v. (4). WMC-79 is somewhat related to the potent antitumor agent MLN944 (51), which also binds to DNA and apparently affects transcription. However, there are currently insufficient data to make a direct comparison between the two agents.

It should also be noted that WMC-79 is also cytotoxic to tumor cells in which the p53 gene is either mutated or not expressed. This suggests that other pathways leading to cell arrest and death are involved. Further experiments are needed to elucidate those mechanisms.


   Acknowledgments
 
We thank Dr. Bert Vogelstein for the HCT-116 p53+/+, p53–/– and HCT-116 p21+/+, p21–/– cells; Louise R. Finch and Refika B. Turnier for flow cytometric analysis; and John W. Connelly for assistance with the expression arrays.

Received 5/24/05; revised 7/11/05; accepted 7/25/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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