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. Author manuscript; available in PMC: 2007 Jul 17.
Published in final edited form as: Cancer Biol Ther. 2005 Dec 9;4(12):1389–1394. doi: 10.4161/cbt.4.12.2303

p16 Ink4a Inhibits Histologic Progression and Angiogenic Signaling in Min Colon Tumors

Steven L Gibson 1,4, Amelie Boquoi 1,2,3, Tina Chen 1,2,3, Norman E Sharpless 5,6,7, Colleen Brensinger 8, Greg H Enders 1,2,3,4
PMCID: PMC1924686  NIHMSID: NIHMS23712  PMID: 16322687

Abstract

The Ink4a/Arf tumor suppressor locus is widely inactivated in cancer but little is known about the tumor biology of its two products, p16Ink4a (p16) and Arf. Both the p16 and Arf promoters are methylated in a significant fraction of human colon carcinomas, implying a functional role. We have demonstrated previously that Ink4a/Arf-null colon tumors display increased growth and vascularity in C57Bl6 mice carrying the Min (multiple intestinal neoplasia) mutation. We present here an analysis in a mixed genetic background of Min colon tumors (N = 215) in mice with or without selective deficiencies in p16 or Arf, respectively. Absence of Arf did not significantly alter tumor formation. In contrast, tumors in mice lacking p16 were moderately larger and redder. Histological analysis demonstrated that these tumors contained significantly more pockets of necrosis (p = 0.02), a marker of carcinoma in situ; less apoptosis (p = 0.02); and higher red blood cell density (p = 0.02, 0.006 within vessels). Biochemical analyses demonstrated increased levels of vascular endothelial cell growth factor (VEGF, p < 0.01). Exogenous p16 expression in human colon tumor cells in vitro inhibited VEGF production. These results suggest that p16 constrains colon tumor progression, in part through inhibiting angiogenic signaling.

Keywords: p16, Ink4a, colon tumor, tumor progression, VEGF, Min, colorectal carcinoma, angiogenesis

INTRODUCTION

The INK4a/ARF locus is among the most widely inactivated loci in human cancer. 1,2 This locus directs synthesis of two nonhomologous tumor suppressors, p16Ink4a (p16) and Arf. Arf is translated from an alternative reading frame, in a genetic organization virtually without precedent in genomes of higher eukaryotes. The unusual organization of the locus suggests that the two products might be coexpressed and exert some cooperativity in function, despite their transcription from distinct promoters. Much has been learned of the respective response pathways. p16 binds and inhibits cyclin dependent kinases (Cdks) 4 and 6, thereby activating retinoblastoma protein (pRb) family members and inhibiting transcription mediated by E2F. Arf acts in part by stabilizing the p53 tumor suppressor.

In contrast to this progress, the tumor biology of p16 and Arf are poorly understood. p16 and Arf has been shown to be induced in vitro in response to certain stresses such as oncogene activation, but the steps in neoplastic progression at which these proteins intervene remain largely unknown. Colon neoplasia represents an attractive setting in which to examine such issues, due to the availability of human tissue of all tumor stages3 and well characterized animal models.4 Both the p16 and Arf promoters are methylated in a substantial fraction of human colon tumors, often independently, implying functional roles for these proteins.5–14 pRb inactivation is rare in colon carcinoma.15,16 Therefore, where p16 is expressed, it may be functional. We have found that p16 is induced in human colon neoplasia, from its earliest overt stages.17 Expression detectable by immunohistochemistry and immunofluorescence is confined to selected cells and correlates with cell cycle arrest. These observations suggest that p16 may function in a distinct subset of tumor cells to constrain tumorigenesis. Although p16 inactivation is less obligatory in colon neoplasia than in some other tumors, study in this tractable setting has the potential to reveal important principles of p16 regulation and function.

In previous work, we tested genetically the impact of the Ink4a/Arf locus on colon tumorigenesis in a mouse model.18 In a C57Bl6 genetic background, we bred mice bearing the Min (multiple intestinal neoplasia) mutation19 to mice with a targeted deletion of the Ink4a/Arf locus that ablates function of both products.20 Min colon tumors in Ink4a/Arf-null mice grew more rapidly and were more often grossly red, a phenotype that was associated with increased vascularity.18 In addition, these tumors demonstrated features of carcinoma in situ, including pockets of necrosis and areas of cribriform epithelium. To further dissect the contribution of each gene product of the locus, we have now examined the Min colon tumor phenotype in mice selectively deficient in p1621 and Arf,22,23 respectively. Our results implicate p16 as an inhibitor of histologic progression and angiogenic signaling in colon tumors.

METHODS

Mouse husbandry and tumor scoring

Min mice in a C57Bl6 background were purchased from Jackson Laboratories (Bar Harbor, ME, USA). p16-null and Arf-null mice were of mixed 129Sv/FVB/C57Bl6 genetic background (at least 50% C57Bl/6).21–23 Genotyping was determined by polymerase chain reaction (PCR) using tail DNA. Min genotyping was performed as previously described.24 p16 genotyping used the forward primer 5′-GCCAAATAGCGCCACCTAT-3′. The reverse primer 5′-GACTCCATGCTGCTCCAGAT-3′ was specific for the wild type locus, and 5′-GCCGCTGGACCTAATAACTTC-3′ was specific for the ‘knockout’ locus. Arf genotyping used the forward primer 5′-GTCGCAGGTTCTTGGTCACT-3′ and the reverse primer 5′-ATGTTCACGAAAGCCAGAGC-3′. The wild type Arf locus generates a 113 bp product and the ‘knockout’ locus a 202 bp product. To control for potential effects of unlinked loci that differ between genetic backgrounds, parents were mated that bore the Min mutation in one animal and were each heterozygous for the respective knockout allele. In this manner, progeny were scored that had an equal chance of inheriting null or wild type genotypes and unlinked loci. Progeny were sacrificed at three months of age by carbon dioxide inhalation, followed by decapitation. Colons were resected, opened longitudinally, stained with methylene blue, and examined for polyps under a dissecting microscope. An observer blinded to the genotypes measured the maximum tumor diameter and assessed the color on a scale from 0–3, as follows: 0: all white, 1+: any detectable red/pink color, 2+: most of the tumor with discernible red/pink color, and 3+: deep red color.

Histological analysis

Histological analysis was performed as previously described.18 Pockets of necrosis were identified as round-to-oval areas many cell widths in diameter devoid of intact epithelial cells and partially filled with cellular debris and inflammatory cells.25 Apoptosis was assayed using an antibody directed against activated caspase 3 (Cell Signaling Technology, Beverly, MA, USA). Sections were scored for the number of RBCs in areas of highest apparent RBC density outside the tumor stalk, at 20x magnification. This method is modeled after the international standard in the angiogenesis field for scoring maximal vessel density.18,26 RBCs were classified as being either within a visible blood vessel or within the interstitial space (no vessel apparent). Immunohistochemistry for Ki67 used antibodies from Novocastra Laboratories (New Castle upon Tyne, UK).17

Immunoblotting

Whole cell extracts were prepared as described previously,27 using antibodies directed against p16 (JC2 monoclonal),17 pRb (C-15, Santa Cruz Biotechnology, Santa Cruz, CA, USA), VEGF (Ab-1, Labvision, Fremont, CA, USA), and actin (4700 monoclonal, Sigma Chemical Co., St. Louis, MO, USA). Whole cell extracts of tumors were prepared in RIPA buffer,17 and hemoglobin was detected using a rabbit polyclonal antibody (MP Biomedicals, Irvine, CA, USA).

VEGF ELISA assays

VEGF was assayed by ELISA (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Adenoviral-mediated p16 expression and desferoxime treatment

p16 was expressed from a replication-defective adenovirus, as described previously.27 Cells were infected with 50–100 plaque-forming units in serum-free media for 4 hours. Where designated, cells were treated with 130 μM deferoxamine (Sigma) for 24 h.

Real-time PCR

VEGF mRNA levels in total cellular RNA were assayed by real-time PCR on an Applied Biosystems 7000 Sequence Analyzer (Foster City, CA, USA) using Syber green and the following primers set, spanning the exon1-exon2 junction: 5′-GCCTCCGAAACCATGAACTTT-3′ (forward) and 5′GCAGCCTGGGACCACTTG 3′ (reverse). The following primer set was used to assay GAPHDH mRNA levels: 5′-GGTGGTCTCCTCTGACTTCAACA-3′ (forward) and 5′-GTTGCTGTAGCCAAATTCGTTGT-3′ (reverse).

Statistical analyses

Analysis of variance models, backed by Kruskal Wallis tests, were used to compare tumor burden between mouse genotypes. Generalized estimating equation logistic regression analysis was used to analyze differences in tumor color by genotype. The densities of pockets of necrosis, prevalence of apoptosis, and RBC density were analyzed using negative binomial models. Trends in VEGF content per tumor were analyzed by linear regression, based on p16 gene number and tumor color. SAS software 8.2 was employed throughout (SAS Institute, Cary, NC, USA).

RESULTS

We used a mating scheme that ensured that scored progeny of each Ink4a/Arf genotype had an equal chance of inheriting unlinked loci (see methods). Colon tumors were analyzed at three months of age.18 Many tumors are readily visible under a dissecting microscope at this age, which is before the onset of genotype-specific morbidity and mortality. Figure 1A illustrates the scoring of tumor color. Ink4a/Arf wild type (wt) tumors showed modestly more red coloration in the mixed genetic background than in the pure C57Bl6 background18 (see Fig. 1B, below). Against this background, Arf deficiency (heterozygous or null) showed no gross effect on tumor size or color (N = 95, data not shown). In contrast, selective absence of one or both copies of the p16 gene was associated with trends toward larger tumors and increased red coloration (Fig. 1B). We therefore focused our detailed analyses on this tumor set.

Figure 1.

Figure 1

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Trends toward larger and redder Min colon tumors in p16-deficient (heterozygous (HET) and NULL) vs. p16-wild type (WT) mice. (A) Examples of tumor color. Min colon tumors of the designated grades were photographed in situ through a dissecting microscope. W: white, 1+: any detectable red/pink color, 2+: most of the tumor with discernible red/pink color, and 3+: deep red color. Magnification: ca. 10x. (B) Size and color of tumors in p16-heterozygous and –null mice compared to those from p16-wild type littermates. White tumors are marked by open circles. Colored tumors are marked by boxes filled with the designated shade of color (see legend). An orange cross marks the mean tumor area in each group, identified further by connecting lines. The trend toward increased red coloration in p16-null tumors approached but did not reach statistical significance (p = 0.16).

Care was taken in each analysis to match the tumor genotypes well for color and size. Histological analysis demonstrated an increase in p16-null tumors in the density of pockets of necrosis, a feature of carcinoma in situ25 (p = 0.02 Fig. 2). In contrast, p16 null tumor cells displayed significantly less apoptosis (p = 0.02, Fig. 3). This result was somewhat surprising because p16 expression has been primarily associated with cell cycle arrest, rather than death. However, exogenous p16 expression has been found to cause cell death in some settings,28,29 including colon carcinoma cells,30 and the senescent state imposed by p16 can lead to death (see Discussion).31 The overall proliferation index in p16-null tumors, as assessed by Ki67 staining,32,33 was not higher than in p16-wt tumors. In fact, it was moderately lower (29% vs. 40%; Fig. 4, p = 0.04). One potential explanation for this counterintuitive result is that p16 might cause arrest and death of cells undergoing proliferative stress, reducing the proportion of these slowly proliferative cells (see discussion). In any event, this result argues that the p16-null phenotypes observed, including increased vascularity (see below), are not, as might have been surmised, secondary to a higher overall proliferation index.

Figure 2.

Figure 2

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Increased pockets of necrosis in p16-null tumors. Tumors were fixed in formalin, embedded in paraffin, sectioned, stained with hematoxylin and eosin, and scored for pockets of necrosis (black arrows in insert, 30x). All available regions of the sections were scored and the number of necrotic pockets was normalized for area (based on number of 20x fields available for examination). Tumor color is designated by the shade of the bar, as in Figure 1B, and tumors size is designated below.

Figure 3.

Figure 3

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Decreased apoptosis in p16-null tumors. Formalin-fixed sections were stained with an antibody directed against cleaved caspase 3. The number of stained individual cells (outside pockets of necrosis; see black arrows in insert, 40x) was scored and normalized to the area examined. Tumor color and size are designated as before.

Figure 4.

Figure 4

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Lower proliferative index in p16-null tumors. Formalin-fixed sections were stained for Ki67 (see insert, 30x). The percent positive neoplastic epithelial cells was scored. Tumor color and size are designated, as before.

We then investigated whether the trend toward increased red coloration in tumors from p16-deficient animals reflected an increased blood content. Immunoblotting for hemoglobin (Hb) confirmed that red coloration generally correlated with Hb content, as we have shown previously18 (Fig. 5). Histological analysis revealed higher red blood cell (RBC) density in p16-null tumors (p = 0.02, Fig. 6). This difference was particularly striking for RBCs within visible vessels (solid bars, p = 0.006, Fig. 6). Analysis of tumor extracts for vascular endothelial growth factor (VEGF) content showed higher levels in redder tumors (p < 0.01) and distinctly higher levels in p16-deficient tumors compared to wild type tumors matched for color (p < 0.01, Fig. 7). These results identify p16 as a suppressor of colon tumor vascularity and VEGF expression.

Figure 5.

Figure 5

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Hemoglobin content correlates with red tumor color. Protein extracts (10 μg each) from the designated tumors were subjected to immunoblotting for hemoglobin (Hgb). Extracts from lung and normal colon tissue served as positive controls and extracts from mouse embryo fibroblasts (MEFs) served as a negative control. Immunoblotting for actin served as a loading control. The masses (in mg) of the original tumors are designated.

Figure 6.

Figure 6

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Increased red blood cell density in p16-null tumors. Formalin-fixed sections stained with hematoxylin and eosin were scored for the number of red blood cells (RBCs) in the 20x field with highest RBC density (see insert, 30x). Counts were made of RBCs within visible vessels (solid portion of bar) or outside of such (striped portion of bar).

Figure 7.

Figure 7

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Increased VEGF content in p16-deficient tumors. 10 mg of protein extract from each tumor were assayed in triplicate for VEGF content by ELISA. Extracts derived from mouse embryo fibroblasts (MEFs) and normal colon (Nl Col) served as controls. Tumors were matched for color. Tumor area is designated below. The correlation coefficient for p16 genotype was R = −0.63, p < 0.01.

Given that these animals were wild type or null for p16 from conception, absence of p16 might have had long term, indirect effects on tumorigenesis. For example, absence of p16 might favor evolution of a neoplastic clone with enhanced VEGF production. Alternatively, p16 might exert more direct control over VEGF production. To examine the latter possibility, we assayed effects of p16 expression on VEGF levels in two human colorectal carcinoma colon tumor cells in vitro that have silenced p16 expression by methylation and/or mutation.9,11 p16 expression was titrated to achieve hypophosphorylation of pRb in HT29 and HCT 116 cells colorectal carcinoma cells (Fig. 8A, data not shown) and resulted in reduced pRb levels, as has been described previously in many settings (Fig. 8, c.f. Dai et al.31 and references therein). VEGF levels in HT29 cells were stimulated by treatment with the hypoxia mimetic deferoxamine, which stabilizes hypoxia inducible factor 134 and augments VEGF transcription. This induction was largely blocked by p16 expression (Fig. 8A). Likewise, levels of VEGF secreted into the culture media, assayed by ELISA, were stimulated by deferoxamine treatment and inhibited by p16 expression (Fig. 8A). In HCT 116 cells, VEGF production was not further stimulated by DFO treatment (data not shown), and p16 inhibited basal VEGF expression (Fig. 8B). Real-time PCR analysis demonstrated reduced VEGF mRNA levels (Fig. 8B). We have also found that p16 reduces VEGF production in ras-transformed, p53-null mouse colonocytes (the latter a kind gift of Andrei Thomas-Tikhonenko, University of Pennsylvania; data not shown).

Figure 8.

Figure 8

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Acute p16 expression in vitro inhibits VEGF production from human colorectal carcinoma cells. (A) HT29 colorectal carcinoma cells were infected with adenoviruses expressing LacZ (−) or p16 (+) for two days and either cultured under routine conditions (−) or treated with deferoxamine (DFO +) for the last 24 h. Whole cell extracts prepared at the end of this period and culture supernatants harvested from the final 24 h were normalized for cellular protein content. VEGF, p16, pRb, and actin (loading control) were assayed by immunoblotting (top 4 panels). Secreted VEGF in the culture supernatants was assayed by ELISA and expressed as the ratio of the baseline level (i.e., DFO-/p16-; mean ± range from two independent experiments). (B) HCT 116 cells were infected with adenoviruses expressing LacZ (−) or p16 (+) for 48 h, and normalized cell extracts were subjected to immunoblotting for VEGF or actin (above). VEGF mRNA levels were assayed by real-time PCR and normalized to GAPDH mRNA levels (below). The results represent the means ± SD of from three independent experiments, each performed in triplicate.

DISCUSSION

p16 is a major tumor suppressor protein but its impact on tumorigenesis and the steps in neoplastic progression that are countermanded remain largely unknown. Our results indicate that p16 suppresses colon tumor progression in the Min mouse model. p16-null tumors grew modestly faster in the first three months with a trend toward increased red coloration. The density of pockets of necrosis, a marker of carcinoma in situ recognized previously in Ink4a/Arf-null Min colon tumors, was markedly increased in a p16-null background. Moreover, these tumors displayed decreased apoptosis and increased RBC density and VEGF content. Acute p16 expression in vitro inhibited VEGF production from neoplastic human colonocytes, pointing to a relatively direct effect on this major angiogenic factor.

Overall proliferative index was not increased in tumors from p16-null animals, a somewhat surprising finding that underscores the complexity of the in vivo environment. As p16 is a potent cell cycle inhibitor and its expression in human colon tumors is associated with reduced markers of cell proliferation,17,35 p16 might be arresting relatively slowly replicating cells such as stem cells and/or cells experiencing replicative stress and DNA damage. Apoptosis, as judged by caspase 3 activation, was decreased in p16-null tumors, consistent with evidence that p16 can mediate apoptosis in select settings. Indeed, we have observed that acute, high level p16 expression results in substantial cell death in HCT 116 human colorectal carcinoma cells (data not shown) and gradual death, following development of senescent features, in the human osteogenic sarcoma cell line U2-OS.31 Thus, absence of p16 may favor proliferation and survival of slowly replicating cells, thereby reducing the proliferative index. It is also possible that growth of p16-wild type tumors may select for expression of different cooperating lesions that augment cell proliferation. Furthermore, by fostering angiogenic signaling, p16 absence might support, in noncell autonomous fashion, the survival of slowly replicating cells otherwise destined for death in p16-wild type tumors.

Absence of neither p16 nor Arf fully recapitulated the marked tumor progression phenotype observed in Min colon tumors with absence of both proteins.18 This observation would suggest that p16 and Arf act partially redundantly in suppressing colon tumor progression and angiogenesis, as has been seen in other settings.23 A caveat is that the analysis of the selective ‘knockouts’ was performed in a mixed genetic background, rather than the inbred C57Bl/6 background used in our previous study. Indeed, we observed greater heterogeneity in tumor size and color in the mixed genetic background18 (See Fig. 1). Despite this, absence of p16 alone had a marked effect on tumor histology, RBC density, and VEGF content. Thus, our data implicate p16 in colon tumor suppression but do not rule out a significant, possibly partially redundant role for Arf.

p16’s effects in vivo could be complex and indirect. Nonetheless, acute p16 expression inhibited VEGF production, arguing for a direct influence on this key angiogenic factor. Similar data have been obtained following p16 expression in glioma cell lines.36 We have preliminary evidence for effects on additional factors of relevance to angiogenesis (data not shown). Further research will be required to delineate fully the regulation of VEGF and related molecules by p16 and to evaluate the biologic importance of each in colon tumorigenesis. It is, however, noteworthy that administration of antibodies directed against VEGF have proven to extend survival in patients undergoing chemotherapy for advanced colon cancer.37,38 These observations validate VEGF as a biologically relevant factor in colon neoplasia and suggest that p16 might impose significant noncell autonomous constraints on colon tumorigenesis.

Acknowledgments

This work was supported by NIH grant R01 DK64758-01 (to G.H.E.) and utilized the Morphology Core Facility of the NIH Center for Molecular Studies in Digestive and Liver Diseases at the University of Pennsylvania, grant P30 DK50306. N.E.S. is a Sidney Kimmel Scholar for Cancer Research.

ABBREVIATIONS

p16

p16Ink4a/MTS1/Cdkn2a

Arf

p19Arf

pRb

retinoblastoma protein

RBC

red blood cell

VEGF

vascular endothelial growth factor

null

nullizygous

het

heterozygous

wt

wild type

ELISA

enzyme linked immunosorbent assay

References

  • 1.Sharpless NE, DePinho RA. The INK4A/ARF locus and its two gene products. Curr Opin Genet Dev. 1999;9:22–30. doi: 10.1016/s0959-437x(99)80004-5. [DOI] [PubMed] [Google Scholar]
  • 2.Lowe SW, Sherr CJ. Tumor suppression by Ink4a-Arf: Progress and puzzles. Curr Opin Genet Dev. 2003;13:77–83. doi: 10.1016/s0959-437x(02)00013-8. [DOI] [PubMed] [Google Scholar]
  • 3.Kinzler KW, Vogelstein B. Lessons from hereditary colorectal cancer. Cell. 1996;87:159–170. doi: 10.1016/s0092-8674(00)81333-1. [DOI] [PubMed] [Google Scholar]
  • 4.Heyer J, Yang K, Lipkin M, Edelmann W, Kucherlapati R. Mouse models for colorectal cancer. Oncogene. 1999;18:5325–33. doi: 10.1038/sj.onc.1203036. [DOI] [PubMed] [Google Scholar]
  • 5.Baylin SB, Herman JG, Graff JR, Vertino PM, Issa JP. Alterations in DNA methylation: A fundamental aspect of neoplasia. Adv Can Res. 1998;72:141–96. [PubMed] [Google Scholar]
  • 6.Burri N, Shaw P, Bouzourene H, Sordat I, Sordat B, Gillet M, Schorderet D, Bosman FT, Chaubert P. Methylation silencing and mutations of the p14ARF and p16INK4a genes in colon cancer. Lab Invest. 2001;81:217–29. doi: 10.1038/labinvest.3780230. [DOI] [PubMed] [Google Scholar]
  • 7.Esteller M, Tortola S, Toyota M, Capella G, Peinado MA, Baylin SB, Herman JG. Hypermethylation-associated inactivation of p14(ARF) is independent of p16(INK4a) methylation and p53 mutational status. Cancer Res. 2000;60:129–33. [PubMed] [Google Scholar]
  • 8.Gonzalez-Zulueta M, Bender CM, Yang AS, Nguyen T, Beart RW, Van Tornout JM, Jones PA. Methylation of the 5′ CpG island of the p16/CDKN2 tumor suppressor gene in normal and transformed human tissues correlates with gene silencing. Cancer Res. 1995;55:4531–5. [PubMed] [Google Scholar]
  • 9.Herman JG, Merlo A, Lapidus RG, Issa JP, Davidson NE, Sidransky D, Baylin SB. Inactivation of the CDKN2/p16/MTS1 gene is frequently associated with aberrant DNA methylation in all common human cancers. Cancer Res. 1995;55:4525–30. [PubMed] [Google Scholar]
  • 10.Klump B, Hsieh CJ, Holzmann K, Gregor M, Porschen R. Hypermethylation of the CDKN2/p16 promoter during neoplastic progression in Barrett’s esophagus. Gastroenterology. 1998;115:1381–6. doi: 10.1016/s0016-5085(98)70016-2. [DOI] [PubMed] [Google Scholar]
  • 11.Myohanen SK, Baylin SB, Herman JG. Hypermethylation can selectively silence individual p16ink4A alleles in neoplasia. Cancer Res. 1998;58:591–3. [PubMed] [Google Scholar]
  • 12.Toyota M, Ahuja N, Ohe-Toyota M, Herman JG, Baylin SB, Issa JP. CpG island methylator phenotype in colorectal cancer. Proc Natl Acad Sci USA. 1999;96:8681–6. doi: 10.1073/pnas.96.15.8681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Wiencke JK, Zheng S, Lafuente A, Lafuente MJ, Grudzen C, Wrensch MR, Miike R, Ballesta A, Trias M. Aberrant methylation of p16INK4a in anatomic and gender-specific subtypes of sporadic colorectal cancer. Cancer Epidemiol Biomarkers Prev. 1999;8:501–6. [PubMed] [Google Scholar]
  • 14.Guan RJ, Fu Y, Holt PR, Pardee AB. Association of K-ras mutations with p16 methylation in human colon cancer. Gastroenterology. 1999;116:1063–71. doi: 10.1016/s0016-5085(99)70009-0. [DOI] [PubMed] [Google Scholar]
  • 15.Ali AA, Harvey JP, Wildrick DM, Boman BM. Retinoblastoma gene product-associated proteins in human colon cancer cell lines. Biochem Biophys Res Commun. 1993;194:848–54. doi: 10.1006/bbrc.1993.1899. [DOI] [PubMed] [Google Scholar]
  • 16.Ali AA, Marcus JN, Harvey JP, Roll R, Hodgson CP, Wildrick DM, Chakraborty A, Boman BM. RB1 protein in normal and malignant human colorectal tissue and colon cancer cell lines. Faseb J. 1993;7:931–7. doi: 10.1096/fasebj.7.10.8344490. [DOI] [PubMed] [Google Scholar]
  • 17.Dai CY, Furth EE, Mick R, Koh J, Takayama T, Niitsu Y, Enders GH. p16(INK4a) expression begins early in human colon neoplasia and correlates inversely with markers of cell proliferation. Gastroenterology. 2000;119:929–42. doi: 10.1053/gast.2000.17952. [DOI] [PubMed] [Google Scholar]
  • 18.Gibson SL, Dai CY, Lee HW, DePinho RA, Gee MS, Lee WMF, Furth EE, Brensinger C, Enders GH. Inhibition of colon tumor progression by the Ink4a/Arf locus. Cancer Res. 2003;63:742–6. [PubMed] [Google Scholar]
  • 19.Su LK, Kinzler KW, Vogelstein B, Preisinger AC, Moser AR, Luongo C, Gould KA, Dove WF. Multiple intestinal neoplasia caused by a mutation in the murine homolog of the APC gene. Science. 1992;256:668–70. doi: 10.1126/science.1350108. [DOI] [PubMed] [Google Scholar]
  • 20.Serrano M, Lee H, Chin L, Cordon-Cardo C, Beach D, DePinho RA. Role of the INK4a locus in tumor suppression and cell mortality. Cell. 1996;85:27–37. doi: 10.1016/s0092-8674(00)81079-x. [DOI] [PubMed] [Google Scholar]
  • 21.Sharpless NE, Bardeesy N, Lee KH, Carrasco D, Castrillon DH, Aguirre AJ, Wu EA, Horner JW, DePinho RA. Loss of p16Ink4a with retention of p19Arf predisposes mice to tumorigenesis. Nature. 2001;413:86–91. doi: 10.1038/35092592. [DOI] [PubMed] [Google Scholar]
  • 22.Bachoo RM, Maher EA, Ligon KL, Sharpless NE, Chan SS, You MJ, Tang Y, DeFrances J, Stover E, Weissleder R, Rowitch DH, Louis DN, DePinho RA. Epidermal growth factor receptor and Ink4a/Arf: Convergent mechanisms governing terminal differentiation and transformation along the neural stem cell to astrocyte axis. Cancer Cell. 2002;1:269–77. doi: 10.1016/s1535-6108(02)00046-6. [DOI] [PubMed] [Google Scholar]
  • 23.Sharpless NE, Ramsey MR, Balasubramanian P, Castrillon DH, DePinho RA. The differential impact of p16(INK4a) or p19(ARF) deficiency on cell growth and tumorigenesis. Oncogene. 2004;23:379–85. doi: 10.1038/sj.onc.1207074. [DOI] [PubMed] [Google Scholar]
  • 24.Luongo C, Moser AR, Gledhill S, Dove WF. Loss of Apc+ in intestinal adenomas from Min mice. Cancer Res. 1994;54:5947–52. [PubMed] [Google Scholar]
  • 25.DeCostanzo DC, Elias JM, Chumas JC. Necrosis in 84 ovarian carcinomas: A morphologic study of primary versus metastatic colonic carcinoma with a selective immunohisto-chemical analysis of cytokeratin subtypes and carcinoembryonic antigen. Int J Gynecol Pathol. 1997;16:245–9. [PubMed] [Google Scholar]
  • 26.Vermeulen PB, Gasparini G, Fox SB, Toi M, Martin L, McCulloch P, Pezzella F, Viale G, Weidner N, Harris AL, Dirix LY. Quantification of angiogenesis in solid human tumours: An international consensus on the methodology and criteria of evaluation. Eur J Cancer. 1996;32A:2474–84. doi: 10.1016/s0959-8049(96)00379-6. [DOI] [PubMed] [Google Scholar]
  • 27.Mitra J, Dai CY, Somasundaram K, El-Deiry WS, Satyamoorthy K, Herlyn M, Enders GH. Induction of p21(WAF1/CIP1) and inhibition of Cdk2 mediated by the tumor suppressor p16(INK4a) Mol Cell Biol. 1999;19:3916–28. doi: 10.1128/mcb.19.5.3916. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Ausserlechner MJ, Obexer P, Wiegers GJ, Hartmann BL, Geley S, Kofler R. The cell cycle inhibitor p16(INK4A) sensitizes lymphoblastic leukemia cells to apoptosis by physiologic glucocorticoid levels. J Biol Chem. 2001;276:10984–9. doi: 10.1074/jbc.M008188200. [DOI] [PubMed] [Google Scholar]
  • 29.Plath T, Detjen K, Welzel M, von Marschall Z, Murphy D, Schirner M, Wiedenmann B, Rosewicz S. A novel function for the tumor suppressor p16(INK4a): Induction of anoikis via upregulation of the alpha(5)beta(1) fibronectin receptor. J Cell Biol. 2000;150:1467–78. doi: 10.1083/jcb.150.6.1467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Tamm I, Schumacher A, Karawajew L, Ruppert V, Arnold W, Nussler AK, Neuhaus P, Dorken B, Wolff G. Adenovirus-mediated gene transfer of P16INK4/CDKN2 into bax-negative colon cancer cells induces apoptosis and tumor regression in vivo. Cancer Gene Ther. 2002;9:641–50. doi: 10.1038/sj.cgt.7700480. [DOI] [PubMed] [Google Scholar]
  • 31.Dai CY, Enders GH. p16 INK4a can initiate an autonomous senescence program. Oncogene. 2000;19:1613–22. doi: 10.1038/sj.onc.1203438. [DOI] [PubMed] [Google Scholar]
  • 32.Berenzi A, Benetti A, Bertalot G, Rodolfi A, Portolani N, Giulini SM, Pulcini G, Vinco A, Tiberio G. Ki67 immunohistochemical evaluation in colorectal cancer and normal colonic mucosa. Possible clinical applications Pathologica. 1992;84:155–63. [PubMed] [Google Scholar]
  • 33.Diebold J, Dopfer K, Lai M, Lohrs U. Comparison of different monoclonal antibodies for the immunohistochemical assessment of cell proliferation in routine colorectal biopsy specimens. Scand J Gastroenterol. 1994;29:47–53. doi: 10.3109/00365529409090436. [DOI] [PubMed] [Google Scholar]
  • 34.Salceda S, Beck I, Caro J. Absolute requirement of aryl hydrocarbon receptor nuclear translocator protein for gene activation by hypoxia. Arch Biochem Biophys. 1996;334:389–94. doi: 10.1006/abbi.1996.0469. [DOI] [PubMed] [Google Scholar]
  • 35.Wang QS, Papanikolaou A, Nambiar PR, Rosenberg DW. Differential expression of p16(INK4a) in azoxymethane-induced mouse colon tumorigenesis. Mol Carcinog. 2000;28:139–47. doi: 10.1002/1098-2744(200007)28:3<139::aid-mc2>3.0.co;2-v. [DOI] [PubMed] [Google Scholar]
  • 36.Harada H, Nakagawa K, Iwata S, Saito M, Kumon Y, Sakaki S, Sato K, Hamada K. Restoration of wild-type p16 downregulates vascular endothelial growth factor expression and inhibits angiogenesis in human gliomas. Cancer Res. 1999;59:3783–9. [PubMed] [Google Scholar]
  • 37.Ferrara N, Hillan KJ, Gerber HP, Novotny W. Discovery and development of bevacizumab, an anti-VEGF antibody for treating cancer. Nat Rev Drug Discov. 2004;3:391–400. doi: 10.1038/nrd1381. [DOI] [PubMed] [Google Scholar]
  • 38.Hurwitz H, Fehrenbacher L, Novotny W, Cartwright T, Hainsworth J, Heim W, Berlin J, Baron A, Griffing S, Holmgren E, Ferrara N, Fyfe G, Rogers B, Ross R, Kabbinavar F. Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med. 2004;350:2335–42. doi: 10.1056/NEJMoa032691. [DOI] [PubMed] [Google Scholar]

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