Inhibition of Intestinal Polyposis with Reduced Angiogenesis in Apc Mice Due to Decreases in c-Myc Expression

The c-myc oncogene plays an important role in tumorigenesis and is frequently deregulated in many human cancers, including gastrointestinal cancers. In humans, mutations of the adenomatous polyposis coli (Apc) tumor suppressor gene occur in most colorectal cancers. Mutation of Apc leads to stabilization of B-catenin and increases in B-catenin target gene expression (c-myc and cyclin D1), whose precise functional significance has not been examined using genetic approaches. Apc mice are a model of familial adenomatous polyposis and are heterozygous for an Apc truncation mutation. We have developed a model for examining the role of c-Myc in Apc-mediated tumorigenesis. We crossed c-myc mice to Apc to generate Apc c-myc animals. The compound Apc c-myc mice were used to evaluate the effect of c-myc haploinsufficiency on the Apc phenotype. We observed a significant reduction in tumor numbers in the small intestine of Apc c-myc mice compared with control Apc c-myc mice. In addition, we observed one to three polyps per colon in Apc c-myc mice, whereas only two lesions were observed in the colons of Apc mice that were haploinsufficient for c-myc . Moreover, reduction in c-myc levels resulted in a significant increase in the survival of these animals. Finally, we observed marked decreases in vascular endothelial growth factor, EphA2, and ephrin-B2 expression as well as marked decreases in angiogenesis in intestinal polyps in Apc c-myc mice. This study shows that c-Myc is critical for Apc-dependent intestinal tumorigenesis in mice and provides a potential therapeutic target in the treatment of colorectal cancer. (Mol Cancer Res 2007;5(12):1296–303) Introduction Mutation and inactivation of the adenomatous polyposis coli (Apc) tumor suppressor gene is an early event in the development of colorectal cancer (1). Inactivation of Apc occurs in f80% of adenomatous polyps and it is also responsible for the genetic disorder familial adenomatous polyposis, which leads to the formation of adenomatous polyps in the small and large intestine that results in colon cancer (2-4). It has been shown that the major function of Apc is to degrade cytosolic levels of h-catenin, thus preventing formation of the h-catenin/T-cell factor-4 complex and effectively blocking h-catenin/T-cell factor-4–meditated transcription (5). Interestingly, mice lacking T-cell factor-4 display no proliferating cells in their intestinal crypts, suggesting that this signaling pathway is essential for intestinal proliferation (6). Multiple downstream targets for h-catenin have been identified, including cyclin D1 and c-myc , two factors that are critical for cell cycle traverse and other biological processes (7, 8). Indeed, previous studies have shown that c-myc expression is repressed by Apc and is increased by overexpression of h-catenin in human colon cancer cells (7, 9). Although these two h-catenin target genes are likely to be important for colorectal tumorigenesis, the exact functional significance of these targets has yet to be clearly defined. The c-myc gene encodes a transcription factor with a basic helixloop-helix leucine zipper domain that mediates heterodimerization with its obligate DNA-binding partner Max. This basic helix-loop-helix leucine zipper domain is also essential for direct DNA binding. Myc activates or represses the transcription of a wide array of target genes that elicit a variety of biological responses, including cell growth, proliferation, differentiation, metabolism, and apoptosis (10-12). In addition, the role of Myc during development has been examined in several tissues, including the gastrointestinal tract (13). Previous data from the Trumpp laboratory showed that c-Myc is essential for intestinal crypt formation but is dispensable for maintenance and homeostasis of the adult intestinal epithelium (13). These data were surprising because c-Myc is generally essential for cell cycle traverse. Thus, the role of c-Myc during development and homeostasis of the intestine remains to be discovered. Additionally, c-Myc has also been shown to modulate the expression of various angiogenic factors, including vascular endothelial growth factor (VEGF; refs. 14-16). Moreover, c-Myc has been shown to down-regulate antiangiogenic factors, such as thrombospondin-1 (14, 17, 18). The importance of Myc in cancer is evident by its increased expression in many human cancers, including colon, breast, prostate, and lung (19). Furthermore, Myc overexpression in various cell types in mice Received 5/24/07; revised 7/31/07; accepted 8/7/07. Grant support: NIH grant RR017698. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Troy A. Baudino, Department of Cell and Developmental Biology and Anatomy, University of South Carolina School of Medicine, 6439 Garners Ferry Road, Building #1, C-57, Columbia, SC 29209. Phone: 803-7331562; Fax: 803-733-1533. E-mail: [email protected] Copyright D 2007 American Association for Cancer Research. doi:10.1158/1541-7786.MCR-07-0232 Mol Cancer Res 2007;5(12). December 2007 1296 Research. on March 30, 2017. © 2007 American Association for Cancer mcr.aacrjournals.org Downloaded from induces tumors that display increased proliferation, delayed differentiation, and increased angiogenesis (16, 20). Indeed, c-Myc levels are increased in the tumors of Apc mice and deletion of c-myc results in reduced tumorigenesis due to increases in apoptosis (21). However, these previous studies from the Gerner and Trumpp laboratories did not examine other biological processes in which Myc is involved, specifically angiogenesis. Therefore, because c-Myc expression is increased in the Apc mouse model of familial adenomatous polyposis, it is possible that this leads to increases in c-Myc target gene expression and promotes progression through the adenomacarcinoma sequence and subsequent metastasis. Thus, the aim of the present study was to evaluate the role of c-Myc on tumorigenesis in the Apc murine model. Results c-myc Haploinsufficiency Increases the Survival of Apc Mice and Greatly Decreases Tumor Incidence and Size It has been previously shown that c-myc is a h-catenin target gene, and in Apc mice, the levels of c-myc are significantly increased when compared with wild-type (WT) animals (7, 9). Moreover, it has been previously shown that complete deletion of c-myc in the intestinal tract does not perturb normal intestinal homeostasis but does disrupt the formation of intestinal crypts (13). In addition, recent studies from Sansom et al. (22) show that c-myc deletion blocks tumorigenesis in Apc-deficient mice in the small intestine, suggesting that Myc is a critical mediator of neoplasia following Apc loss. Furthermore, recent studies from the Gerner laboratory showed that c-myc loss (2-fold reduction in protein) resulted in reduced tumorigenesis in Apc mice due to increased levels of apoptosis (21). Thus, to further investigate the role of c-Myc in Apc-mediated tumorigenesis, we crossed Apc mice with c-myc animals to generate Apc c-myc mice. We then followed the life span of these animals, with mice being humanely sacrificed when moribund. Mice that were WT for c-myc and Apc had a median life span of 169 days, whereas animals that were heterozygous for c-myc and Apc lived an average of 291 days (Fig. 1). Amazingly, many of the animals that were Apc c-myc were still alive at 1 year of age (Fig. 1). Having observed the dramatic increase in survival of Apc c-myc animals, we next examined the intestines of agematched (12 weeks) animals to determine if the observed increase in survival was due to a decrease in polyp number and/ or size or if there was another underlying cause. Indeed, in 12-week-old age-matched animals, we observed a significant reduction in the total number of polyps, as well as marked decreases in polyp numbers of various sizes, with the largest decreases observed in polyps that were >2.0 mm in size (Fig. 2A). In addition, similar reductions in polyp numbers were observed in moribund animals (Fig. 2B). Moreover, whereas most of the Apc c-myc mice displayed between one and three colonic polyps with an 80% incidence rate, only two of the c-myc animals displayed a colonic polyp (10% incidence rate; Fig. 2C). Furthermore, gross analyses of whole intestine or H&E; staining of intestinal sections further showed the dramatic reduction in polyp number and size with the loss of only a single c-myc allele (Fig. 2D and E) Taken together, these data suggest that c-Myc is playing a critical role in the initial development and growth of these intestinal and colonic polyps and that by simply reducing c-Myc levels we observe a marked reduction in polyp number and a dramatic increase in survival. Cell Proliferation Is Reduced in the Intestinal Polyps of Apc c-myc Mice Myc has been shown to play a critical role in cell cycle traverse and apoptosis during development as well as in cancer. Indeed, previous studies from Ignatenko et al. (21) showed that c-myc loss in Apc mice resulted in marked increases in apoptosis. However, recent studies from Alan Clarke’s laboratory have shown that Myc deficiency shows remarkably little effect on cell proliferation but does cause a block in apoptosis in the small intestine in the absence of Apc (22). Nonetheless, having observed a dramatic reduction in polyp size and number in animals that were heterozygous for c-myc , we chose to examine the effect of c-myc reduction on cell proliferation in the intestinal polyps of Apc c-myc mice. Therefore, we isolated intestines from 12-week-old agematched animals and examined intestinal polyps for the presence of phospho-histone H3–positive cells (Fig. 3A). Computational analyses of immunofluorescent images showing staining for nuclei (4¶,6-diamidino-2-phenylindole, blue) or phospho-histone H3 (red) showed a significant difference in levels of cell proliferation in intestinal polyps present in Apc c-myc mice when compared with animals that were Apc c-myc (Fig. 3A and B). Apoptosis Is Increased in the Intestinal Polyps of Apc c-myc but not in Normal Intestinal Crypts To examine apoptotic levels, we did terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) analyses on intestinal sections from Apc c-myc and Apc c-myc mice. Not surprisingly, we did not see any differences in the levels of apoptosis in the normal intestinal crypts of Apc mice that are WT (12.8%) or haploinsufficient for c-myc (12.7%; Fig. 4A-C). These data are similar to those observed in recent studies examining c-myc loss in the small intestine (13) as well as our previous studies examining FIGURE 1. Survival curve of Apc mice that are WT or haploinsufficient for c-myc . Loss of a single allele of c-myc results in a dramatic increase in the life span of Apc animals. Apc mice that are WT for c-myc (gray) or heterozygous for c-myc (black ) are shown. Mice that were WT for c-myc and Apc had a median life span of 169 d (n = 65), whereas animals that were heterozygous for c-myc had a median life span of 291 d (n = 37). Reduced c-Myc Delays Tumorigenesis in Apc Mice Mol Cancer Res 2007;5(12). December 2007 1297 Research. on March 30, 2017. © 2007 American Association for Cancer mcr.aacrjournals.org Downloaded from c-myc loss in the whole E9.5 murine embryo (14). However, the number of TUNEL-positive nuclei was significantly increased (f30%) in the intestinal polyps of Apc mice following the loss of one allele of c-myc when compared with Apc c-myc mice (f5%; Fig. 4D-F). Having observed significant changes in levels of cell proliferation and apoptosis between Apc c-myc and Apc c-myc polyps, we chose to delve deeper into the cause of these changes. It has been shown that c-Myc can affect cell cycle progression by modulating the expression of cyclins or by altering cyclin activity, specifically cyclin D1 and cyclin E. Cyclins, along with cyclin-dependent kinases, are critical for G1-S transition during cell cycle progression. It has been shown that c-myc is capable of directly inducing cyclin E expression as well as affecting cyclin E activity (23). Therefore, we examined expression of cyclin D1 and cyclin E in normal intestinal tissue as well as in polyps of different sizes from animals that were either Apc c-myc or Apc c-myc by real-time PCR (Fig. 5B and C). We also examined the different-sized polyps for changes in c-myc expression, as amplification or overexpression of oncogenes is common in many cancers due to selective pressure (Fig. 5A). Examination of cyclin D1 levels in normal intestinal tissue as well as in intestinal polyps of equal size displayed no difference in expression levels regardless of c-Myc status (Fig. 5B). Although surprising, these results were not entirely unexpected, as cyclin D1 is also a direct h-catenin target gene (8). In addition, we examined c-myc and cyclin E expression (Fig. 5A and C). Although c-myc expression was significantly reduced in normal intestinal tissue in Apc c-myc mice, as well as in polyps of equal sizes, there was a marked increase in c-myc expression in the larger polyps of Apc c-myc mice (Fig. 5A). Moreover, the pattern of cyclin E expression seemed to correlate with the observed changes in c-myc expression in the different-sized polyps (Fig. 5A and C). Taken together, these data suggest that Myc is controlling cell cycle traverse through regulation of cyclin E levels and/or cyclin E activity. Reduction in c-Myc Levels Correlates with a Decrease in the Expression of Angiogenic Factors c-Myc has been shown to act as a master regulator of angiogenesis as well as apoptosis and cell cycle traverse (10, 14). Dysregulation of any of these processes could be the reason for the overall decrease in polyp numbers that we observed in Apc c-myc animals (Fig. 2A, B, and D). Indeed, we observed a decrease in the levels of proliferation in intestinal polyps from Apc mice that were heterozygous for c-myc when compared with mice that were WT for c-myc (Fig. 3A and B). In addition, we also observed an increase in apoptosis in the polyps of Apc animals that were heterozygous for c-myc (Fig. 4D-F). It is doubtful that these modest changes in proliferation or apoptosis could account for the dramatic increases in survival or decreases in polyp size and number that we observed. This reasoning is based on our previous studies examining c-myc loss on teratoma formation in nude mice (14). Whereas c-myc loss had no significant effect on FIGURE 2. Intestinal and colonic polyp numbers are reduced in Apc c-myc animals. A. Intestinal polyps were counted according to size in 12-wkold age-matched animals that were either WT (black columns ) or heterozygous (gray columns ) for c-myc (n = 10 for each group). We observed a 67% reduction in polyps <0.5 mm in size, a 75% reduction in polyps 0.5 to 2.0 mm in size, and a 90% reduction in polyps >2.0 mm in size in c-myc animals. B. Intestinal polyps were counted according to size in moribund animals that were either WT (black columns ; n = 29) or heterozygous (gray columns ; n = 20) for cmyc . Again, notice the marked reduction in the number of polyps present in c-myc animals. C. Total numbers of colonic polyps were counted in moribund animals. There was a >90% reduction in the number of colonic polyps per mouse in animals that were heterozygous for c-myc . In fact, only two c-myc animals (n = 20) displayed any colonic polyps. D. Representative images of intestines isolated from age-matched (12 wk) Apc mice that were either WT (left ) or heterozygous (right ) for c-myc . Notice the marked decrease in the number and size of polyps present in c-myc animals. E. Representative H&Estained; sections for Apc mice that were WT (left ) or heterozygous (right ) for c-myc . Notice the decrease in polyp size (white arrows ) in c-myc animals. Yekkala and Baudino Mol Cancer Res 2007;5(12). December 2007 1298 Research. on March 30, 2017. © 2007 American Association for Cancer mcr.aacrjournals.org Downloaded from proliferation or apoptosis in embryonic stem cells, tumor formation was markedly reduced due to dysregulation of angiogenic factors (14). Thus, we next examined the expression of VEGF as well as several other factors involved in angiogenesis, including Eph receptor tyrosine kinases, which are deregulated in many human cancers, and their respective ligands, the ephrins (24, 25). We first examined the expression of VEGF in the whole animal using serum isolated from retroorbital eye bleeds. Using serum isolated from WT, Apc c-myc, Apc c-myc , or Apc c-myc age-matched animals (8 weeks), we examined VEGF expression by ELISA (Fig. 6A). Loss of a single allele of c-myc resulted in a decrease in circulating VEGF in Apc mice when compared with Apc mice that were WT for c-myc (Fig. 6A). In the absence of the Apc mutation, c-myc loss displayed no effect on circulating VEGF protein levels (Fig. 6A). We next did real-time PCR analyses on normal intestinal tissue or intestinal polyps isolated from Apc mice that were WT or heterozygous for c-myc (Fig. 6B). These real-time PCR analyses showed marked decreases in the expression of VEGF in polyps isolated from c-myc animals when compared with polyps isolated from animals that were WT for c-myc (Fig. 6B). Having observed increases in c-myc expression correlating with polyp size, we decided to examine VEGF expression in polyps of equal size (Fig. 6C). In the polyps that were WT for c-myc , we observed a large increase in the levels of VEGF, going from 2-fold over normal to >7-fold over normal levels in the largest polyps (Fig. 6C). Increases were also observed in the polyps that were heterozygous for c-myc , although the overall levels were not as high as those observed in theWT polyps (Fig. 6C). Furthermore, in addition to the marked decrease in VEGF expression, we also observed significant differences in EphA2 and ephrin-B2 expression when comparing WT polyps with c-myc polyps (Fig. 6D). However, no differences in expression were observed for EphB4 or ephrin-A1 (Fig. 6D). Taken together, these data further support our hypothesis that c-Myc functions as a master regulator of cytokines, such as VEGF, to regulate cell growth, proliferation, and angiogenesis, and when c-Myc expression is deregulated or amplified, this leads to cancer. Reduction in c-Myc Levels Restores Spleen Size and Hematocrit in Apc Animals It has been well documented that Apc mice display splenomegaly and a decreased hematocrit when compared with FIGURE 4. Effect of c-myc haploinsufficiency on apoptosis in the normal intestinal crypts and polyps of Apc mice. A, B, D, and E. TUNEL-positive nuclei were quantified in the normal intestinal crypts or polyps of WT (black columns ) or c-myc (gray columns ) mice and expressed as percentage of total number of nuclei counted using Image-Pro Plus software in the representative 40 confocal images. There is no significant difference in the percentage of TUNEL-positive nuclei in the normal intestinal crypts between Apc c-myc and Apc c-myc (C), but the percentage of TUNEL-positive nuclei is dramatically increased with loss of one allele of c-myc in polyps isolated from Apc intestines (F). n = 5 mice for each group; n = 5 crypts or polyps from each mouse. Bar, 50 Am. FIGURE 3. Cell proliferation is reduced in intestinal polyps of Apc c-myc animals. A. Immunofluorescent images showing phosphohistone H3 (P-Histone H3)-positive cells (red ) in intestinal polyps from WT (left ) or c-myc animals (right ) that are Apc. Sections were counterstained with 4¶,6-diamidino-2-phenylindole (blue ) to visualize nuclei. B. Cell proliferation is reduced in intestinal polyps present in Apc c-myc mice. Graph showing the percentage of cells undergoing proliferation in Apc c-myc (black columns ) or Apc c-myc (gray columns ) mice. Data were obtained from z-series confocal sections (n = 10) of multiple intestinal tumors (n = 5) from multiple mice (n = 5 per group) and quantified using Image-Pro Plus software. Reduced c-Myc Delays Tumorigenesis in Apc Mice Mol Cancer Res 2007;5(12). December 2007 1299 Research. on March 30, 2017. © 2007 American Association for Cancer mcr.aacrjournals.org Downloaded from WT animals. Therefore, we examined the effect that c-myc loss had on spleen size and hematocrit, as well as examining WBC counts in the animals used in this study (12 weeks). Although we observed no detectable differences in the hematocrit levels of animals that were WT or heterozygous for c-myc in the absence of the Apc mutation, animals that were Apc c-myc displayed a significantly reduced hematocrit (Fig. 7A). However, loss of one allele of c-myc in animals that were Apc returned their hematocrit levels to normal (Fig. 7A). Moreover, WBC counts were also normal in Apc c-myc mice, whereas Apc mice that were WT for c-myc displayed WBC counts that were f2-fold greater than those observed in WT animals (Fig. 7B). Finally, mice that were Apc c-myc displayed enlarged spleens, with an average weight off260 mg, whereas Apc mice that were c-myc displayed normal-sized spleens (f75 mg; Fig. 7C). These data clearly show that loss of only a single c-myc allele can restore hematocrit levels in Apc mice and protect these animals from tumorigenesis and the observed corresponding splenomegaly. Discussion The aim of this study was to evaluate the importance of the oncogene c-Myc in Apc-mediated intestinal tumorigenesis using the Apc mouse as a model. The Apc gene is defective in most colorectal cancers, including both inherited and sporadic forms, but the mechanisms through which Apc regulates tumorigenesis remain poorly understood. It has been previously shown that Apc normally controls cytosolic levels of h-catenin by proteasome degradation, preventing formation of the h-catenin/T-cell factor-4 complex and effectively blocking h-catenin/T-cell factor-4–meditated transcription (5). One of the downstream targets for h-catenin that has been identified is c-myc (7, 8). Indeed, previous studies have shown that the c-myc promoter is repressed by Apc in human colorectal cancer cells and c-myc expression is increased by overexpression of h-catenin (7). Thus, it seems that mutation of Apc leads to an increase in h-catenin activity and this leads to an increase in c-Myc expression and subsequently an increase in the expression of c-Myc target genes. Indeed, recent data from Sansom et al. (22) further support this through demonstration that c-Myc is required for activation of Wnt pathway target genes following Apc loss. Myc activates or represses the transcription of a wide array of genes, including proangiogenic and antiangiogenic factors (14-16). Indeed, Myc overexpression in various cell types in mice induces tumors that display increased proliferation, delayed differentiation, and increased angiogenesis (16, 20). To better understand the role that c-Myc plays in Apc-mediated tumorigenesis, we examined tumor formation and survival in Apc animals that were either WT or heterozygous for c-myc . Our results clearly showed that c-Myc is critical for Apc-mediated tumorigenesis, as loss of a single allele of c-myc resulted in a dramatic increase in survival (Fig. 1). Additionally, FIGURE 5. c-myc and cyclin E , but not cyclin D1 , expression is increased in the intestinal polyps of Apc mice. A. Normal intestinal tissue or polyps of equal sizes (0.5 mm, 0.5-2.0 mm, or >2.0 mm) were examined for c-myc expression by real-time PCR. All samples are normalized to ARPP P0 and then expression is plotted as fold of normal WT or c-myc tissue. c-myc expression is reduced in Apc c-myc (gray columns ) when compared with Apc mice that are WT for c-myc (black columns ). However, in the larger polyps, c-myc expression is amplified. B. Cyclin D1 expression is unchanged regardless of c-myc expression. C. Cyclin E expression is reduced in intestinal polyps that are heterozygous for c-myc . Notice that the reduction in cyclin E expression correlates with levels of c-myc expression. Statistical significance between samples as determined by Student’s t test (n = 4). Yekkala and Baudino Mol Cancer Res 2007;5(12). December 2007 1300 Research. on March 30, 2017. © 2007 American Association for Cancer mcr.aacrjournals.org Downloaded from c-myc animals displayed markedly fewer overall polyps than their WT littermates, as well as showing dramatic decreases in the number of larger polyps (>2.0 mm; 90% decrease; Fig. 2A and B). Surprisingly, we only observed a 2-fold change in the level of proliferation within the intestinal polyps following the loss of a single c-myc allele (Fig. 3A and B). We also examined the normal intestinal crypts as well as polyps for apoptosis. We observed no differences in the apoptotic indices in the normal intestinal crypts between WT and c-myc heterozygotes in Apc mice (Fig. 4A-C). However, the number of TUNEL-positive cells was significantly increased in adenomatous polyps in animals that were heterozygous for c-myc (Fig. 4D-F). These results are interesting, as previous studies from Alan Clarke’s laboratory showed that there is a decrease in levels of apoptosis in the small intestine following loss of both Apc and c-myc in the adult animal (22). Furthermore, previous studies from our laboratory and others showed that c-myc loss has no effect on levels of apoptosis either in E9.5 embryos (14) or in the normal intestinal epithelium (13). These effects on proliferation and apoptosis are due to changes in c-myc expression in the polyps of Apc mice. Indeed, when we examined c-myc expression in intestinal polyps from Apc c-myc mice, we observed a significant decrease in c-myc expression in the smaller polyps when compared with those isolated from Apc mice that are WT for c-myc (Fig. 5A). However, in the larger polyps, the difference in c-myc expression is not as great (Fig. 5A). This increase in c-myc expression that is observed in the larger polyps of the Apc c-myc mice could be due to loss of the WT Apc allele or could also be due to increases in c-Myc expression due to the hypoxic environment of the larger polyps. Indeed, several recent articles have shown that hypoxia can increase c-Myc expression and/or transcriptional activity (26, 27). In addition to the observed proliferative and apoptotic changes in the Apc c-myc mice, the polyps that did form in these animals (Apc c-myc ) showed marked decreases in vascularization when compared with Apc c-myc mice and the blood vessels present in the polyps from Apc c-myc animals seem to be nonfunctional (data not shown). Finally, these decreases in vascularization seem to be due to significant reductions in levels of VEGF, EphA2 , and ephrin-B2 expression (Fig. 6B-D). Taken together, these studies show that c-Myc is an important downstream target of h-catenin and that c-Myc functions as an FIGURE 6. Reduced levels of c-myc result in decreased expression of angiogenic factors in tumors in the small intestine. A. Levels of VEGF present in the serum of age-matched (8 wk) WT (solid black columns ), c-myc (solid gray columns ), or Apc mice that were either WT for c-myc (black hatched columns ) or heterozygous for c-myc (gray hatched columns) are shown. Notice the increase in VEGF levels in Apc mice that are WT for c-myc (n = 5 per group). B. Normal intestinal tissue or intestinal polyps were examined for VEGF expression by real-time PCR. Total RNA was isolated from normal intestinal tissue from Apc c-myc (solid black columns ) or pooled intestinal polyps (black hatched columns) or normal tissue from Apc mice that were heterozygous for c-myc (solid gray columns ) or pooled intestinal polyps (gray hatched columns ). All samples were normalized to ARPP P0 and then expression was plotted as fold of normal WT tissue. Significance between samples as determined by Student’s t test (n = 5). C. Normal intestinal tissue or polyps of equal sizes (0.5 mm, 0.5-2.0 mm, or >2.0 mm) were examined for VEGF expression by real-time PCR. Notice that VEGF expression is reduced in Apc c-myc (gray columns ) when compared with Apc mice that are WT for c-myc (black columns ). However, in the larger polyps, VEGF expression is increased, similar to that pattern observed for c-myc expression. Statistical significance between samples as determined by Student’s t test (n = 4). D. Expression of several Ephs and ephrins in normal intestinal tissue and polyps. Total RNA was isolated and examined for EphA2, EphB4, ephrinA1 , and ephrin-B2 expression. As in B, all samples were normalized to ARPP P0 . c-myc expression only alters the expression of EphA2 and ephrin-B2 . Asterisks, significance between the sample and all other samples as determined by Student’s t test (n = 5). Reduced c-Myc Delays Tumorigenesis in Apc Mice Mol Cancer Res 2007;5(12). December 2007 1301 Research. on March 30, 2017. © 2007 American Association for Cancer mcr.aacrjournals.org Downloaded from important mediator of polyp growth and colorectal cancer initiation by regulating factors that are essential for angiogenesis to allow for adequate vascularization, supply of nutrients, and tumorigenesis. These studies further strengthen the importance of c-Myc as a target for novel therapeutic modalities in the treatment of colorectal cancers as well as other cancers with deregulated Myc expression. Materials and Methods Animal Studies Apc mice on a pure C57BL/6 background were mated to c-myc mice on a mixed C57BL/6 SV129 background, and the resulting pups were screened for the Min mutation (28) and for the c-myc –null gene (14) by PCR as previously described. Animals that were homozygous for the WT c-myc gene and heterozygous for the Min mutation served as controls. Animals were housed and bred within the Animal Resources Facility at the University of South Carolina School of Medicine. Primer sequences for genotyping are available on request. Polyp Number Determination Intestinal adenomas and colonic adenomas were scored for numbers and size (diameter) at postnatal day 84 (age matched) or when the animal was moribund. The mice were weighed and sacrificed by cervical dislocation and blood and serum were obtained. In addition, the small and large intestines and colon were isolated. The intestinal tract was rinsed with cold PBS using a blunt-end syringe and opened along the longitudinal axis. The opened intestine was spread flat between sheets of filter paper and fixed in fresh 4% paraformaldehyde. Paraformaldehyde-fixed intestinal sections were rinsed in deionized water and stained with 0.2% methylene blue and polyps were counted at 20 magnification under a dissecting microscope using tweezers to pick through the intestinal villi and identify polyps. Polyps were categorized as >0.5 mm, 0.5 to 2.0 mm, or >2.0 mm in size. After polyps were counted, intestinal sections were placed in 70% ethanol for further analysis. For each experimental group, the incidence of tumors, defined as the number of mice with tumors/number of mice in the group, the mean number of tumors/mouse F SD, and the mean tumor diameter (mm) in the group F SD were calculated for the intestine and colon separately. Student’s t test was used to compare means of each group; differences were considered to be significant if P values were <0.05. For histology, segments of intestine and colon were paraffin embedded; 5-Am sections were cut and stained with H&E.; In addition, whole mount images were taken at 20 to show differences in polyp size and number. Measurement of Hematocrit and Spleen Weight For measurement of hematocrit and WBC counts, retroorbital eye bleeds (200 AL) were done on WT, Apc c-myc, Apc c-myc , and Apc c-myc agematched animals (12 weeks) and samples were analyzed using the VetScan HM2 Hematology System (Abaxis). Animals were then sacrificed and their spleens were isolated and weighed (n = 5). In addition, their small and large intestines and colon were isolated as described above for further analyses. ELISA Measurement of VEGF Retro-orbital bleeds were done as described above on agematched animals (8 weeks) and blood samples were allowed to coagulate and samples were centrifuged at 14,000 g for 10 min to isolate blood serum. Serum was then assayed for VEGF protein levels using an ELISA assay specific for murine VEGF as described by the manufacturer (R&D; Systems). FIGURE 7. Reduced c-myc expression in Apc mice restores spleen size and a normal hematocrit. A. Blood was isolated from agematched animals (12 wk) that were either Apc c-myc, Apc c-myc, Apc c-myc , or Apc c-myc and hematocrit was determined. B.WBC counts were restored in Apc mice on c-myc loss. C. Loss of c-myc eliminated splenomegaly in Apc mice. Following eye bleeds, hematocrit and WBC counts were done using a VetScan HM2 Hematology System. Following blood isolation, animals were humanely sacrificed and spleens were isolated and weighed. Asterisks, significance between the sample and all other samples as determined by Student’s t test (n = 5). Yekkala and Baudino Mol Cancer Res 2007;5(12). December 2007 1302 Research. on March 30, 2017. © 2007 American Association for Cancer mcr.aacrjournals.org Downloaded from Real-time PCR Analyses Total RNA was isolated from normal intestinal tissue or intestinal tumor tissue using Trizol reagent (Invitrogen) as described by the manufacturer. Real-time PCR analyses were done using a Bio-Rad reverse transcription-PCR kit for probes and the Bio-Rad iQ5 Real-time Thermocycler (Bio-Rad) as described by the manufacturer. We used primer/probe sets that were specific for murine c-myc, cyclin D1, cyclin E, VEGF, EphA2, EphB4, ephrin-A1, ephrin-B2 , and ARPP P0. ARPP P0 served as our internal control for normalization. Primer and probe sequences are available on request. Tissue Isolation and Cell Proliferation Analyses Immunofluorescent analyses were done on intestines isolated from 12-week-old Apc c-myc and Apc c-myc animals. Briefly, intestines were fixed in fresh 4% paraformaldehyde overnight at 4jC and 120-Am-thick sections were cut using a vibratome (Oxford Instruments). Sections were permeabilized using PBS containing 0.01 mol/L glycine and 0.1% Triton X-100 for 1 h at room temperature. The sections were then blocked using PBS containing 5% bovine serum albumin for 1 h at room temperature. Antibody against phosphohistone H3 (Santa Cruz Biotechnology) was used at a 1:200 concentration. Sections were incubated with primary antibody in 1% blocking buffer (1% bovine serum albumin/PBS) overnight at 4jC. Sections were washed and incubated with donkey antirabbit FITC secondary antibody (Zymed) at a 1:100 concentration in 1% blocking buffer for 2 h at room temperature. In addition, sections were stained with Texas red phalloidin for actin visualization and nuclei were visualized through staining with 4¶,6-diamidino-2-phenylindole (Molecular Probes). Intestinal sections were imaged using a Leica LSM 510 laser scanning microscope. Image-Pro Plus software was used to quantify total number of nuclei and nuclei that were phosphohistone H3 positive per representative 40 high magnification field. Five polyps were analyzed from five individual mice for each genotype (Apc c-myc and Apc c-myc ). Cell Death Detection In situ Using TUNEL Apoptotic cells in the intestinal polyps were visualized using the TUNEL method (Roche Molecular Biochemicals) as described by the manufacturer. Briefly, deparaffinized tissue sections were permeabilized using Triton X-100 (Sigma). The sections were then incubated with TUNEL reaction mixture containing terminal deoxynucleotidyl transferase and fluorescein-dUTP, at 37jC for 30 min, rinsed in PBS for 10 min, mounted, and analyzed using a Leica LSM 510 laser scanning microscope. Image-Pro Plus software was used to quantify the number of TUNEL-positive nuclei. 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BaudinoMice Due to Decreases in c-Myc ExpressionMin/+ApcinInhibition of Intestinal Polyposis with Reduced Angiogenesis Updated versionhttp://mcr.aacrjournals.org/content/5/12/1296Access the most recent version of this article at: Cited articleshttp://mcr.aacrjournals.org/content/5/12/1296.full.html#ref-list-1This article cites 28 articles, 11 of which you can access for free at: Citing articles/content/5/12/1296.full.html#related-urlsThis article has been cited by 17 HighWire-hosted articles. Access the articles at: E-mail alertsrelated to this article or journal.Sign up to receive free email-alerts SubscriptionsReprints [email protected] atTo order reprints of this article or to subscribe to the journal, contact the AACR Publications [email protected] atTo request permission to re-use all or part of this article, contact the AACR Publications Research.on March 30, 2017. © 2007 American Association for Cancermcr.aacrjournals.orgDownloaded from