Altered expression of c-myc, p16 and p27 in rat colon tumors and its reversal by short-term treatment with chemopreventive agents

Lianhui Tao1,3, Paula M. Kramer1, Wei Wang1, Siming Yang1, Ronald A. Lubet2, Vernon E. Steele2 and Michael A. Pereira1

1 Department of Pathology, HEB, Rm 202, Medical College of Ohio, 3055 Arlington Ave., Toledo, OH 43614-5806, USA and
2 Chemopreventive Agent Development Research Group, Division of Cancer Prevention, National Cancer Institute, Bethesda, MD 20892, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Modulation of gene expression in tumors has the potential of being a surrogate end-point biomarker for chemoprevention. Thus, we determined the modulation by chemopreventive agents of the protein and mRNA expression of genes in rat colon tumors. Male F344 rats were administered three weekly injections of 15 mg/kg azoxymethane. Forty-seven weeks later, they received aspirin (600), calcium chloride (50 000), 2-(carboxyphenyl) retinamide (2-CPR, 315), {alpha}-difluoromethylornithine (DFMO, 3000), piroxicam (200), quercetin (33 600), 9-cis retinoic acid (9-cis RA, 30), rutin (3000), or sulindac (280) in their diet at the indicated mg/kg concentration for 7 days and were then killed. In colon tumors relative to the mucosa, the protein and mRNA levels of c-myc were increased, while the levels of p16 and p27 were decreased. Calcium chloride, DFMO, piroxicam and sulindac administered for 7 days decreased the mitotic index and reduced the protein and mRNA levels of c-myc in colon tumors. Calcium chloride, DFMO and piroxicam increased the protein and mRNA levels of p16 and along with sulindac increased the protein level of p27, but not its mRNA. The other agents failed to modulate both the mitotic index and the expression of the genes. The ability of the chemopreventive agents to prevent colon tumors was determined. Male F344 rats were administered three weekly injections of 15 mg/kg azoxymethane and 8 weeks later they were administered aspirin, 2-CPR, DFMO, piroxicam, 9-cis RA and rutin in their diet. The rats were killed 26 weeks after they started to receive the chemopreventive agents. The multiplicity of colon tumors was reduced by DFMO and piroxicam, increased by rutin and not affected by the other agents. Hence, agents that prevented colon cancer decreased the mitotic index and altered the expression of c-myc, p16 and p27 suggesting that modulation in the expression of these genes are potential biomarkers for chemopreventive activity.

Abbreviations: AOM, azoxymethane; CDKI, cyclin-dependent kinase inhibitors; 2-CPR, 2-(carboxyphenyl) retinamide; 9-cis RA, 9-cis retinoic acid; DFMO, {alpha}-difluoromethylornithine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase gene


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Colon cancer is a leading cause of cancer death in the world (13). In 2000 in the USA, colon cancer accounted for ~20% of all cancer deaths, with an estimated 98 200 new cases and 48 100 deaths (3). About 5–6% of the population in the USA has a lifetime risk of developing colon cancer (3). However, though the use of animal bioassays and in vitro studies, numerous potential chemopreventive agents for colon cancer have been identified. Non-steroidal anti-inflammatory drugs (NSAID, piroxicam and sulindac), polyamine inhibitors ({alpha}-difluoromethylornithine, DFMO) and dietary components (calcium salts) are among the agents that have demonstrated reproducible efficacy in preventing colon cancer in laboratory animals (2,46). These agents are efficacious in preventing colon cancer when administered after initiation by a carcinogen (azoxymethane, AOM) indicating that they prevented the promotion/progression of cancer (2,48). Sulindac has been shown to induce the regression of pre-existing polyps in individuals with familiar adenomatosis polyposis (912). Hence, tumorigenesis in the colon appears to have the potential of being prevented and reversed by chemopreventive agents.

Surrogate end-point biomarkers are being developed for potential clinical use in indicating the efficacy of chemopreventive agents in phase I and II trials prior to actually demonstrating prevention of cancer in phase III trials. These biomarkers are biological end-points that can be measured quantitatively and are modulated by chemopreventive agents in parallel with the efficacy of the agent to prevent cancer. Biomarkers have the advantage of requiring a shorter duration of treatment with a chemopreventive agent than a tumor end-point. Hence, biomarkers might be used to rank agents with greater or lesser promise prior to their evaluation in much larger and more expensive phase III trials that are likely to employ a tumor end-point.

Numerous molecular events are involved in the evolution of colon cancer, many of which correlate to enhance cell proliferation and to decrease apoptosis in lesions (2,5). These biological events are regulated in part by the expression of protooncogenes and tumor suppressor genes. The protein level of protooncogenes and tumor suppressor genes may be controlled by various mechanisms including transcription factors, DNA methylation and modification of their protein (phosphorylation, farnesylation, ubiquitination, etc.) that are examples of potentially reversible alterations controlling the synthesis and degradation of proteins (1317). These sites of epigenetic regulation of gene expression are potential biomarkers for cancer chemoprevention.

c-myc is a protooncogene associated with increased cell proliferation and apoptosis (18). The c-myc protein is a transcription factor that forms a heteromeric complex with the Myn or the Max protein to activate the transcription of growth-related genes including cdc25A and cyclins A, D1 and E. Increased expression of the c-myc gene has been identified as an early event of colon carcinogenesis in both human and rodent (16,17,1921). Expression of the c-myc gene can be controlled by alteration in transcription factors, modulation by the ß-catenin/TCF4 pathway and methylation of the gene (13,14,2226).

Expression of the tumor suppressor genes, p16INK4A (p16) and p27Kip1 (p27) is decreased in many human tumors including colon cancer (2730). The p16 and p27 proteins are cyclin-dependent kinase inhibitors (CDKI) that inhibit cyclin/cyclin kinase complexes and thereby inhibit progression of the cell cycle (27). CDKI are classified into two major families (i.e. the INK4 family that includes p16 and the Cip/Kip family that includes p27). The p16 protein binds to CDK4 and CDK6 to inhibit their interaction with cyclin D (28,29). This reduces the phosphorylation of the retinoblastoma protein (pRb), increasing its stability. The resulting increase in pRb inhibits the transit from the G1 to S phase of the cell cycle. The p27 protein inhibits transit from S to G2 phase by interacting with cyclins A, B and E and with CDK2 (30). A variety of mechanisms can control the expression of the p16 and p27 genes including genetic alterations such as mutations and deletions of the gene, and epigenetic alterations such as hypermethylation of the promoter of the gene or increased turnover of the protein due to phosphorylation (2730).

In the studies reported here, we determined the ability of short-term exposure to chemopreventive agents with varied levels of efficacy to modulate the expression of the mRNA and protein of the c-myc, p16 and p27 genes in colon tumors. The results demonstrate (i) that the expression of these proteins is altered in rat colon tumors and (ii) that efficacious chemopreventive agents can reverse the alterations in the expression of these genes. Therefore, modulation of the expression of one or more of these genes in colon tumors may be a potential surrogate end-point biomarker for chemoprevention.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals and reagents
2-(Carboxyphenyl)retinamide (2-CPR), 9-cis retinoic acid (9-cis RA) and DFMO were obtained from the National Cancer Institute, DCP Repository (Rockville, MD). AOM, calcium chloride, piroxicam, quercetin, rutin and sulindac were purchased from Sigma Chemical (St Louis, MO) and aspirin was from Acros organics (Pittsburgh, PA). AIN-76A diet was purchased from Dyets (Bethlehem, PA). Monoclonal mouse antibody for c-myc (Ab-2) was obtained from Oncogene Research Products (Cambridge, MA). Mouse monoclonal antibody for p27 (F-8), rabbit polyclonal antibody for p16 (M-156), anti-mouse or rabbit IgG-horseradish peroxidase (HRP) and protein molecular weight standards were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Enhanced-chemiluminescence western blotting detection reagents were obtained from Amersham (Arlington Heights, IL).

Animals
Male F344 rats were obtained at 6 weeks of age from Charles River Laboratories (Frederick, MD). They were housed in the AAALAC-accredited laboratory animal facility at the Medical College of Ohio and in accordance with the US DHHS Guide for the ‘Care and Use of Laboratory Animals’. The Medical College of Ohio, Institutional Animal Care and Use Committee approved the protocol for the study. Solid-bottomed polycarbonate cages with stainless steel wire-bar lids and Bed-o-Cob bedding (Andersons, Toledo, OH) were used to house 2 rats/cage. The light cycle consisted of 12 h each of dark and light. Yellow light was used in the animal room containing the rat treated with 2-CPR and 9-cis RA. The animal rooms were maintained at 64–76°F and 55 ± 15% relative humidity. Drinking water and AIN 76A diet were supplied to the animals ad libitum.

Experiment 1: effect of short-term exposure to chemopreventive agents
Starting at 7 weeks of age, the rats were injected i.p. with 15 mg/kg AOM weekly for 3 consecutive weeks. At 47 weeks after the initial dose of AOM, the rats were assigned using a stratified randomization procedure to the different treatment groups. Each treatment group contained six rats. They were then administered aspirin (600), calcium chloride (50 000), 2-CPR (315), DFMO (3000), piroxicam (200), quercetin (33 600), 9-cis RA (30), rutin (3000) or sulindac (280) in their diet at the indicated mg/kg concentration. We have administered previously these concentrations of the chemopreventive agents to rats without causing toxicity (7,3135). After 7 days of exposure to the chemopreventive agents (week 48), the rats were euthanized. At necropsy, the colon tumors were harvested. A piece of the tumor containing the area of attachment was fixed in 10% buffered formalin overnight, transferred to 70% alcohol and embedded in paraffin for histology. The rest of the tumor was rapidly frozen in liquid nitrogen and stored at –70°C for protein and mRNA determination. The area of attachment was used for histology to distinguish adenocarcinomas by invasion past the epithelial basement membrane. Furthermore, the use of tumor tissue for molecular biology that was away from the area of attachment minimized possible contamination with non-tumor tissue.

Paraffin sections (5 µm) were stained with hematoxylin and eosin for histopathologic evaluation. Colon tumors were classified as adenomas or adenocarcinomas, using the classification described by Elwell and McConnell (36). Tumors that did not exhibit invasion through the muscularis mucosa were classified as adenomas, while those with invasion were adenocarcinomas. The number of mitotic figures in at least 1000 epithelial cells/tumor was determined and the mitotic index was calculated as the percentage of cells with mitotic figures.

Experiment 2: prevention of colon tumors
Male F344 rats at 7 weeks of age were injected i.p. with 15 mg/kg AOM weekly for 3 consecutive weeks. Ten weeks after the first dose of AOM, rats started to receive aspirin, 2-CPR, DFMO, piroxicam, 9-cis RA and rutin in their diet at the same concentration used in Experiment 1. Each treatment group contained 36 rats. The rats were killed 36 weeks after the first dose of AOM. At necropsy, the colons were excised, flushed with 0.9% saline, slit open longitudinally and examined for tumors. A piece of each colon tumor that contained the area of attachment was fixed in 10% buffered formalin overnight, transferred to 70% alcohol and embedded in paraffin. The rest of the tumor was rapidly frozen in liquid nitrogen and stored at –70°C. The tumors were evaluated using the classification described above for Experiment 1.

Western blot analyses of the protein expression of c-myc, p16 and p27
Tissue was homogenized in a buffer containing 20 mM Tris–HCl (pH 7.5), 10 mM EGTA (pH 7.5), 1 mM EDTA (pH 8.0), 10 mM ß-mercaptoethanol, 1 mM phenylmethyl-sulfonyl fluoride, 0.02% leupeptin, 0.04% trypsin inhibitor, 0.25 M sucrose and 0.1% Triton X-100. After sonication, the homogenate was centrifuged at 12 000 g for 30 min. Protein concentration in the supernatant was determined using the Bio-Rad Protein Assay (Bio-Rad, Richmond, CA). The supernatant (30 mg protein) was electrophoresed on 15% SDS–PAGE mini gels under reducing conditions and blotted electrophoretically to Immobilon-P membranes. Molecular weight standards were included with each gel. The membranes were incubated in a 5% milk/Tris-buffered saline + Tween 20 (TBST, pH 7.6) blocking solution for 1 h and probed with mouse monoclonal antibodies for c-myc and p27 and rabbit polyclonal antibody for p16 at room temperature for 3 h. The membranes were then washed with TBST (pH 7.6) and incubated with 1:1000 dilution of HRP-conjugated secondary antibody IgG for 1 h. The membranes were washed again in TBST, treated with enhanced-chemiluminescence western blotting detection reagents and exposed to Kodak autoradiograph films. The membranes were then stained with Coomassie Brilliant Blue R-250 to demonstrate equal loading of the samples onto the gel.

Analysis of the mRNA expression of c-myc, p16 and p27 genes
Expression of the mRNA for the c-myc, p16 and p27 was evaluated by reverse transcription–polymerase chain reaction (RT–PCR). Total RNA was extracted using TRIzol Reagent and treated with DNase I at 37°C for 30 min. cDNA was synthesized from 1 µg of total RNA using 15 U AMV (avian myeloblastosis virus) reverse transcriptase in 20 µl of reaction mixture containing 0.5 µg oligo(dT)15 primer, 5 mM MgCl2, 1 µg/µl recombinant RNasin ribonuclease inhibitor, reverse transcription buffer [10 mM Tris–HCl (pH 9.0), 50 mM KCl and 0.1% Triton X-100] and 1 mM of each deoxynucleoside triphosphate. The reaction was at 42°C for 40 min, followed by 99°C for 5 min and 4°C for 5 min. The cDNA samples were diluted 5-fold with nuclease-free water and used for PCR amplification. c-myc, p16 and p27 genes were co-amplified with the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) in 50 µl of reverse transcription buffer containing 10 ng/µl first strand cDNA, 100 µM each deoxynucleoside triphosphate, 1 mM MgCl2, 1.25µ of Taq DNA polymerase and 25 pmol of upstream and downstream primers for c-myc, p16, p27 and GAPDH. Primer sequences for c-myc (GenBank accession number Z38066) were upstream: 5'-TGA CGA GAC CTT CGT GAA GA-3' (453–472 bp) and downstream: 5'-ATT GAT GTT ATT TAC ACT TAA GGG T-3' (821–845 bp). For p16 (GenBank accession number L81167) the upstream primer sequence was 5'-CAT CTC CGA GAG GAA GGC GAA CT-3' (1–23 bp) and the downstream primer sequence was 5'-CGC AGT TCG AAT CTG CAC CAT AG-3' (217–239 bp). For 27 (GenBank accession number D86924) the upstream primer sequence was 5'-GAG GGC AGA TAC GAG TGG CAG-3' (211–231 bp) and the downstream primer sequence was 5'-CTG GAC ACT GCT CCG CTA ACC-3' (428–448 bp). For GAPDH (GenBank accession number AF106860) the upstream primer sequence was 5'-ATG GTG AAG GTC GGT GTG AAC G-3' (850–871 bp) and the downstream primer sequence was 5'-GTT GTC ATG GAT GAC CTT GGC C-3' (1323–1344 bp). The incubation underwent 30 cycles of 94°C for 60 s, 57°C for 60 s and 72°C for 60 s. This was followed by incubation at 72°C for 10 min. The PCR products were electrophoresed in 1% agarose gel containing 0.1 µg/ml ethidium bromide in 0.5x TBE buffer. After electrophoresis, the gels were photographed under ultraviolet-irradiation and the optical density of the mRNA of genes measured with the Scion Image Analysis System (Scion, Frederick, MD). The optical density for the mRNA of c-myc, p16 and p27 genes was standardized using the density of GAPDH.

Statistical analysis
The data were analyzed unless otherwise noted by an analysis of variance followed by a Tukey test with a P-value <0.05 indicating statistical significance.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tumors used for molecular biology and mitotic index
The colon tumor multiplicity was 2.42 ± 0.17 (mean ± SE) in the rats of experiment 1: effect of short-term exposure to chemopreventive agents. Four adenocarcinomas, each from a different rat were randomly selected for the comparison of the protein levels of c-myc, p16 and p27 in tumors and normal mucosa. Four other adenocarcinomas, each from a different rat were then randomly selected from the control diet group and from each of the groups that received the chemopreventive agents for 7 days. These tumors were used for the determination of the effect of the chemopreventive agents on the protein and mRNA levels of c-myc, p16 and p27. Each tumor sample was analyzed separately for protein and mRNA expression.

The mitotic index in the tumors was determined after 7 days of treatment with the chemopreventive agents (Table IGo). DFMO and piroxicam caused the most striking decrease in the mitotic index, while calcium chloride and sulindac decreased it by a lesser, but significant extent. The other agents, aspirin, 2-CPR, 9-cis RA, quercetin and rutin did not significantly alter the mitotic index.


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Table I. Effect of 7 days of treatment with chemopreventive agents on the mitotic index in tumors
 
Protein levels of the c-myc, p16 and p27 genes in colon tumors
Preliminary to our studies of the effect of chemopreventive agents, we examined the expression of the c-myc, p16 and p27 genes in colon tumors (Figure 1Go). The c-myc protein migrated during electrophoresis as two bands of 65 and 49 kDa. The upper (major) band is probably the phosphorylated form of the c-myc protein and the lower band the unphosphorylated form of the protein (37). The expression level of both bands of the c-myc protein was significantly greater in colon tumors than in the colonic mucosa. In contrast, p16 and p27 migrated as single bands that were of greater intensity in the colonic mucosa than in the tumors. These studies demonstrated roughly a 3-fold increase in c-myc expression and a 60 and 80% decrease in the expression of p16 and p27, respectively, when comparing colon tumors to normal mucosae. Furthermore, the levels of these proteins were fairly consistent among the tumors as indicated by the small standard errors.




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Fig. 1. c-Myc, p16 and p27 protein levels in colon mucosa and tumors. (A) Representative SDS–PAGE gels probed for c-myc, p16 or p27. Lane 1 contained molecular weight standards, lanes 2–5 contained protein from ‘normal’ colonic mucosa of four different rats and lanes 6–9 contained protein from colon tumors of four different rats that did not receive a chemopreventive agent. For c-myc, two bands were detected with the upper band (65 kDa) probably the phosphorylated form of the c-myc protein and the lower band (49 kDa) the unphosphorylated form. For gels probed for p16 and p27, a single band was present of ~16 and 27 kDa, respectively. (B) Mean optical density of the protein bands for c-myc, p16 and p27. Results are mean ± SE of the optical density for bands from at least four mucosa or four tumors from different animals. The * indicates significant difference between the protein level in colonic mucosae and tumors with a P-value < 0.05.

 
Effect of chemopreventive agents on the protein levels of the c-myc, p16 and p27 genes
After 7 days of treatment with the chemopreventive agents, calcium chloride, DFMO, piroxicam and sulindac decreased the level of the c-myc protein in colon tumors (Figure 2Go). Both bands that stained for c-myc were decreased indicating that both the phosphorylated and unphosphorylated forms of c-myc were decreased. Thus, short-term treatment with certain chemopreventive agents decreased the protein level of the c-myc gene towards the level found in ‘normal’ colonic mucosa. Seven days of treatment with the other agents (aspirin, 2-CPR, 9-cis RA, quercetin and rutin) did not affect the expression of the c-myc protein in colon tumors.




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Fig. 2. Effect of 7 days of treatment with a chemopreventive agent on the protein level of c-Myc in colon tumors. (A) Representative SDS–PAGE gels probed for c-myc. Lane 1 contained molecular weight standards, lane 2 contained protein from a colon tumor of a rat that did not receive a chemopreventive agent and lanes 3–5 contained protein from tumors of three different rats that for 7 days prior to death were administered the chemopreventive agent, Asp, aspirin; Ca2+, calcium chloride; 9-cis, 9-cis RA; DFMO, {alpha}-difluoromethylornithine; Pirox, piroxicam; Quer, quercetin and Sulin, sulindac. As in Figure 1Go, the two bands are probably the phosphorylated (65 kDa) and unphosphorylated (49 kDa) forms of c-myc. (B) Mean optical density of the phosphorylated c-myc protein band. Results are mean ± SE of the optical density for bands from at least four tumors from different animals. The * indicates significant difference from colon tumors of rats that were not administered the chemopreventive agent with a P-value < 0.05.

 
The effect of the chemopreventive agents administered for 7 days on the level of the p16 and p27 proteins in colon tumors is presented in Figures 3 and 4GoGo. Calcium chloride, DFMO and piroxicam increased the relatively low protein level of p16 and p27 found in colon tumors. Sulindac increased the protein level of the p27 but not the p16 gene. The other agents did not affect the protein level of either the p16 or p27 gene. Interestingly, only treatment with piroxicam resulted in the appearance of the two bands that stained for p27. Three other tumors from rats that were administered piroxicam also contained two bands when probed for 27 (data not presented). The lower molecular weight p27 band that was present in tumors from rats administered piroxicam was absent in tumors from rats administered calcium chloride, DFMO or sulindac, even though the higher molecular weight band was strongly expressed.




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Fig. 3. Effect of 7 days of treatment with a chemopreventive agent on the protein level of p16 in colon tumors. (A) Representative gels probed for p16. Lane 1 contained molecular weight standards with the 23 kDa molecular weight standard being shown, lane 2 contained protein from a colon tumor of a rat that did not receive a chemopreventive agent and lanes 3–5 contained protein from tumors of three different rats that were administered the chemopreventive agent, as indicated in Figure 2Go. (B) Mean optical density of the p16 protein band. Results are mean ± SE of the optical density for bands from at least four tumors from different animals. The * indicates significant difference from colon tumors of rats that were not administered the chemopreventive agent with a P-value < 0.05.

 



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Fig. 4. Effect of 7 days of treatment with a chemopreventive agent on the protein level of p27 in colon tumors. (A) Representative gels probed for p27. Lane 1 contained Santa Cruz Marker molecular weight standards, lane 2 contained protein from a colon tumor of a rat that did not receive a chemopreventive agent and lanes 3–5 contained protein from tumors of three different rats that were administered the chemopreventive agent, as indicated in Figure 2Go. Except for the rats administered piroxicam, a single band of ~27 kDa was present. A second band of smaller molecular weight and of less intensity was present in tumors only from rats administered piroxicam. (B) Mean optical density of the p27 protein band. Results are mean ± SE of the optical density for bands from at least four tumors from different animals. The * indicates significant difference from colon tumors of rats that were not administered the chemopreventive agent with a P-value < 0.05.

 
Effect of chemopreventive agents on the mRNA levels of c-myc, p16 and p27 genes
To further investigate the mechanism(s) for the altered protein levels of the c-myc, p16 and p27 genes, we examined the expression of their mRNA in normal colonic epithelium, in colon tumors and in tumors after short-term exposure to the chemopreventive agents (Figure 5Go). The mRNA for c-myc was increased 3.3-fold in colon tumors relative to colonic mucosa (Figure 5BGo). Calcium chloride, DFMO, piroxicam and sulindac decreased the level of the mRNA for c-myc in colon tumors by about the same extent, 60–63% decrease, so that it was no longer different from the colonic mucosa (Figure 5BGo). None of the other agents altered the level of the mRNA for c-myc.






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Fig. 5. Effect of 7 days of treatment with a chemopreventive agent on the mRNA expression of c-myc, p16 and p27 in colon tumors. The c-myc, p16 and p27 genes were co-amplified with GAPDH and electrophoresed in 1% agarose gel containing 0.1 mg/ml ethidium bromide. (A) Representative gels of the RT–PCR products of the c-myc, p16 and p27 genes. The gels were photographed under ultraviolet-irradiation. The first lane contained size markers (bp, base pairs), the next two lanes labeled ‘control’ contained product resulting from colon tumors of rats not administered a chemopreventive agent, and the remaining lanes except for the last lane, contained product resulting from tumors from rats administered the indicated chemopreventive agent. The last lane contained product resulting from RNA isolated from colonic mucosa of untreated rats. The ratio of the density for the target gene product divided by the density of the GAPDH gene product is presented in (B) for c-Myc, (C) for p16 and (D) for p27. Results are means ± SE from four tumors, each from a different rat. The * indicates significant difference from colon tumors of rats that were not administered the chemopreventive agent with a P-value < 0.05.

 
In contrast to c-myc, the mRNA levels of the tumor suppressor genes, p16 and p27 were reduced in colon tumors relative to colonic mucosa. Thus, the mRNA expression of p16 was almost completely absent in colon tumors (Figure 5CGo), while expression of p27 was reduced by 70% relative to colonic mucosa (Figure 5DGo). DFMO and piroxicam caused the greatest increase in p16 mRNA, while calcium chloride and sulindac caused more modest, but significant increase in its expression (Figure 5CGo). The limited effect of sulindac on the mRNA level of p16 paralleled its insignificant increase in the protein level of p16. None of the other agents altered the level of the mRNA for the p16 gene. With respect to the mRNA level of p27, it was decreased in colon tumors relative to the colonic mucosa by ~70%. However, none of the chemopreventive agents increased the mRNA level of p27, despite the fact that some of the agents, calcium chloride, DFMO, piroxicam and sulindac increased the level of its protein (Figure 5DGo).

Prevention of colon tumors
The effect on colon tumor multiplicity of aspirin, 2-CPR, DFMO, piroxicam, 9-cis RA and rutin administered from 10 to 36 weeks after the first of 3 weekly doses of AOM is presented in Figure 6Go. No more than two of the 36 animals in any treatment group died during the study. Also, none of the chemopreventive agents significantly altered the body weight of the rats or produced clinical signs of toxicity. DFMO and piroxicam strongly reduced the yield of colon tumors per rat by ~100 and 75%, respectively (P < 0.01), while rutin significantly increased multiplicity of tumors by 55% (P < 0.01). The other agents (aspirin, 2-CPR and 9-cis RA) did not affect the multiplicity of colon tumors. DFMO and piroxicam also significantly decreased the incidence of rats with colon tumors from 21 of 29 rats (72.4%) on the control diet to 0 of 36 (0%) and 10 of 35 (28.6%), respectively. The incidence of rats with colon tumors in the treatment groups that received the other agents ranged from 55.6 to 88.6% and was not significantly different from the 72.4% incidence in rats administered the control diet.



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Fig. 6. Effect of the chemopreventive agents on the yield of AOM-induced colon tumors. The results are means ± SE of the number of colon tumors per rat. The * indicates statistical significance from the rats that were not administered a chemopreventive agent by an ANOVA followed by the Tukey test with a P-value < 0.05.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Besides potentially shortening the duration of clinical studies, surrogate end-point biomarkers particularly those related to the carcinogenic process can help determine the mechanism of the chemopreventive agents. One mechanism proposed for chemopreventive agents is to decrease the level of cell proliferation in colon tumors (38). Colon cancers in humans have been reported to have increased expression of the c-myc protooncogene and decreased expression of the p16 and p27 tumor suppressor genes (21,22,2730). Relative to the mucosa, AOM-induced colon tumors in rats were found to also have increased expression of the c-myc gene and decreased expression of the p16 and p27 genes. A wide variety of mechanisms including mutation, chromosomal deletion, gene amplification, altered methylation of the gene and its promoter, phosphorylation followed by degradation of the protein, etc., could be responsible for the altered protein and mRNA expression of the c-myc, p16 and p27 genes in colon tumors. Some of these mechanisms especially the epigenetic mechanisms could be susceptible to reversal by short-term treatment with chemopreventive agents. Thus, the protein and mRNA levels of the c-myc, p16 and p27 genes are altered similarly in human colon cancer and in AOM-induced colon cancer in rats and possibly by reversible mechanisms.

The agents used to evaluate modulation of the protein and mRNA levels of genes in colon tumors included aspirin, calcium chloride, 2-CPR, DFMO, piroxicam, quercetin, 9-cis RA, rutin and sulindac. DFMO and piroxicam have been shown previously to be strong preventive agents in the AOM-induced colon cancer model (5,7,31), which we confirmed. This study also confirmed our previous finding that aspirin at 600 mg/kg in the diet did not prevent AOM-induced colon cancer (31). In a prior study, a higher concentration of 9-cis RA than that used in the present study did prevent colon cancer (34). However, because of toxicity in the prior study, the present study used a lower concentration of 9-cis RA that was ineffective in preventing colon cancer. 2-CPR and quercetin although proposed as chemopreventive agents have in our previous studies actually increased the yield of AOM-induced colon cancer (34,35). In the study reported here, rutin, the parent compound of quercetin enhanced the yield of colon tumors induced by AOM. Thus, it appears that rutin and its aglycone, quercetin can enhance the yield of colon cancer induced by AOM in rats. We did not assay calcium chloride and sulindac for the ability to prevent colon cancer. However, both agents have been reported previously to prevent AOM-induced colon cancer, although with apparently less efficacy than DFMO or piroxicam (4,39,40). Hence, for the evaluation of the biomarkers, four agents (calcium chloride, DFMO, piroxicam and sulindac) that prevented, three agents (aspirin, 9-cis RA and 2-CPR) that did not affect and two agents (quercetin and rutin) that increased the yield of colon cancer in rats were employed.

Seven days of treatment with calcium chloride, DFMO, piroxicam or sulindac decreased the mRNA and protein level of the c-myc gene in colon tumors toward the lower level of expression found in normal colonic epithelium. The alteration in c-myc mRNA expression that was caused by these agents paralleled their effect on its protein expression implying that the altered expression of c-myc was mediated at transcription. The agents (aspirin, 2-CPR, 9-cis RA, quercetin and rutin) that did not prevent colon cancer in rats did not affect either the mRNA or protein level of c-myc. Thus, the increased expression of the c-myc gene found in colon tumors was reversible and only by agents that demonstrated the ability to prevent colon cancer in the model. c-myc expression is a particularly interesting biomarker as increased expression of this gene has been associated with alteration in the development of colon cancer in rats and in humans (1923).

Calcium chloride, DFMO and piroxicam increased the mRNA and protein level of the p16 gene. Interestingly, DFMO and piroxicam were the most potent agents both in preventing colon cancer and in increasing the expression of the mRNA and protein of the p16 gene. The agents that were ineffective in preventing colon cancer in rats, aspirin, 2-CPR, 9-cis RA, quercetin and rutin did not affect the mRNA or protein level of the gene in the tumors. Hence, the alteration in protein level of this gene was closely in line with its mRNA expression implying transcriptional control for its protein expression. Down regulation of the p16 gene is often achieved epigenetically by hypermethylation of its promoter (28,29,41). We are presently determining whether the chemopreventive agents that increased the expression of the mRNA and protein of the p16, do so by reversing the hypermethylation of the gene in AOM-induced colon tumors.

Calcium chloride, DFMO, piroxicam and sulindac increased the protein level of the p27 gene, but none of these agents altered the mRNA level of the gene. This would imply that the increased level of the p27 protein resulting from treatment with the chemopreventive agents was not mediated at the transcription level. Degradation of the p27 protein appears to involve phosphorylation and ubiquitination of the protein that leads to its degradation in protesomes (42). Thus, it is proposed that the chemopreventive agents increased the protein level of p27 by preventing its phosphorylation or ubiquitination so that it is not degraded. The agents that did not prevent colon cancer failed to alter the expression of p27.

The present results demonstrate that short-term exposure to chemopreventive agents can modulate mechanism-relevant biomarkers of protein and mRNA expression of protooncogenes and tumor suppressor genes. Thus, chemopreventive agents (calcium chloride, DFMO, piroxicam and sulindac) that were effective in preventing AOM-induced colon cancer in rats modulated the biomarkers, while agents that were ineffective in preventing colon cancer did not modulate them. In tumors, the protein and mRNA expression of c-myc was decreased, while the expression of p16 and p27 was increased within 7 days. This rapid modulation of gene expression could result from either a direct or an indirect effect. Although, the chemopreventive agents, calcium, DFMO, piroxicam and sulindac that modulated the expression of the genes appear to act through different mechanisms, they have in common the ability to decrease cell proliferation. DFMO and piroxicam strongly decreased cell proliferation, while calcium and sulindac decreased it to a lesser extent. The agents, aspirin, 2-CPR, 9-cis RA, quercetin and rutin that did not affect the expression of the genes, also did not alter the proliferative index in the tumors. The three genes evaluated are involved in the regulation of cell proliferation, in that the expression of c-myc is decreased, while that of p16 and p27 are increased in association with decreased cell proliferation (1820,2830,43). Therefore, the decrease in c-myc and the increase in p16 and p27 expression could be secondary to the decrease in cell proliferation induced by the chemopreventive agents.

There are potentially a number of ways to employ biomarkers in the development of chemopreventive agents. One way is to define a series of biomarkers in animal studies that are modulated by effective dose levels of chemopreventive agents and then to determine whether similar changes occur in humans. Secondly, a biomarker could be used to screen members of a class of chemopreventive agents for relative efficacy using as the end-point their ability to modulate the biomarker. Thirdly, biomarkers that are involved in the carcinogenic process per se, such as those that we examined could be used to evaluate the mechanism of chemopreventive agents. Hence, our results suggest as potential surrogate end-point biomarkers for chemoprevention, the modulation of the mRNA and protein levels of protooncogenes and tumor suppressor genes in tumors by short-term treatment with chemopreventive agents.


    Notes
 
3 To whom correspondence should be addressed Email: ltao{at}mco.edu Back


    Acknowledgments
 
Supported in part by contract nos N01-CN-75102 and NO1-CN-05123 from the National Cancer Institute.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received February 4, 2002; revised April 26, 2002; accepted May 8, 2002.