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. 2006 Jun;8(6):429–436. doi: 10.1593/neo.06169

Induction of the Tumor-Suppressor p16INK4a within Regenerative Epithelial Crypts in Ulcerative Colitis1

Emma E Furth *,†, Karen S Gustafson *, Charlotte Y Dai †,‡,§, Steven L Gibson †,‡,§, Paul Menard-Katcher †,‡,§, Tina Chen †,‡,§, Jim Koh , Greg H Enders †,‡,§
PMCID: PMC1601464  PMID: 16820088

Abstract

p16INK4a is a major tumor-suppressor protein, but its regulation and settings of fuction remain poorly understood. To explore the notion that p16 is induced in vivo in response to replicative stress, we examined p16 expression in tissues from human ulcerative colitis (UC; n = 25) and normal controls (n = 20). p16 was expressed strongly in UC-associated neoplasms (n = 17), as seen previously in sporadic colonic neoplasms. In non-neoplastic UC epithelium, p16 was expressed in 33% of crypts (the proliferative compartment) compared to < 1% of normal controls. p16 expression did not correlate with degree of inflammation but did correlate with the degree of crypt architecture distortion (P = .002)—a reflection of epithelial regeneration. In coimmunofluorescence studies with Ki67, p16 expression was associated with cell cycle arrest (P < .001). Both UC and normal crypts displayed evidence for the activation of the DNA damage checkpoint pathway, and p16 was induced in primary cultures of normal epithelial cells by ionizing irradiation (IR). However, induction by IR displayed delayed kinetics, implying that p16 is not an immediate target of the checkpoint pathway. These findings support a model in which p16 is induced as an “emergency brake” in cells experiencing sustained replicative stress.

Keywords: p16, INK4a, ulcerative colitis, tumor suppressor, DNA damage

Introduction

p16INK4a (p16) is a major tumor-suppressor protein, which is inactivated with particular frequency in solid tumors of the gastrointestinal epithelium [1–3]. The protein binds selectively to cyclin-dependent kinases (Cdks) 4 and 6, and inhibits the activity of these Cdks and Cdk2, the latter by indirect means. Cdk inhibition prevents the phosphorylation of retinoblastoma family proteins and fosters the repression of E2F-regulated genes involved in cell replication. Mice with engineered mutations in the p16 gene develop normally but are tumor-prone [4].

In contrast to our relatively sophisticated understanding of its response pathway, the regulation of p16 remains poorly understood. p16 is expressed at low to undetectable levels in most normal tissues [5–8]. Ultraviolet light can stimulate p16 expression in skin keratinocytes [9,10]. p16 expression is also commonly increased by tissue culture, but the physiologic relevance of this observation has remained unclear [11]. The in vivo settings in which p16 is induced and the steps during neoplastic progression at which p16 intervenes are ill defined. Genetic studies in mouse models suggest that p16 may constrain the long-term proliferation of bone marrow stem cells [12].

p16 inactivation has been shown to occur early in some gastrointestinal neoplasms [3]. Promoter methylation is a common mode of p16 inactivation in tumors and is thought to be a relatively specific event, reflecting selection pressure. p16 methylation is found in colon adenomas and carcinomas [13–16] and in early neoplastic epithelium in Barrett esophagus [17], in some colonic tissues of patients with longstanding ulcerative colitis (UC) [18], and in some cells with preneoplastic features in the mammary epithelium [19–21]. These observations suggest that p16 may be expressed and rate-limiting for proliferation in settings of preneoplasia or early neoplasia.

We have demonstrated previously that p16 is induced in sporadic carcinomas, adenomas, and aberrant crypt foci in the colon [8]. We now extend these studies to UC—an important preneoplastic disease of the colon that is characterized by chronic epithelial damage and ulceration requiring epithelial regeneration. We demonstrate distinct p16 induction within regenerating crypts and neoplasia arising in this setting. These findings suggest that p16 may regulate epithelial regeneration in this context. Recent evidence suggests that DNA damage may be prevalent in settings of early neoplasia and hyperplasia [22,23]. We find evidence for DNA damage checkpoint activation in UC and in normal colon, and demonstrate that p16 is induced by ionizing radiation in diverse normal human epithelial cells in culture, with delayed kinetics. Our findings support a model in which brief replicative stress is insufficient to induce p16, but in which sustained replicative stress may be a common physiologic mediator of p16 induction, in both non-neoplastic and neoplastic cells.

Materials and Methods

Tissue Collection and Histologic Grading

Colonic tissues from 25 UC cases and 20 normal controls were obtained from specimens in the Department of Pathology at the Hospital of the University of Pennsylvania. Ten UC cases had associated polypoid dysplasia, six had associated adenocarcinoma, and three had pseudopolyps. Formalin-fixed paraffin-embedded sections were stained with hematoxylin and eosin and were examined under light microscopy. The degrees of inflammation and crypt distortion, respectively, were graded on a scale of 0 to 3, with 0 representing normal features. For inflammation, (1 = neutrophils infiltrated the mucosa in a spotty fashion; 2 = neutrophils infiltrated between one third and two thirds of the mucosa; 3 = neutrophils infiltrated at least two thirds of the mucosa). Ulceration was considered a marker of acute inflammation. Expansion of the lamina propria by lymphocytes and/or plasma cells was considered a marker of chronic inflammation. Crypt distortion was scored when crypts deviated from their normal straight, regular spacing. A grade of 1 was assigned for branching and/or irregular spacing involving up to one third of crypts, a grade of 2 was assigned for the involvement of one third to two thirds of the crypts, and a grade of 3 was assigned for the involvement of two thirds or more of the crypts. Dysplastic crypts with architecture distortion were scored separately as dysplasia. Similar scoring schemes for UC have been reported and correlated with clinical and endoscopic findings [24].

Antibodies, Immunohistochemistry, and Immunofluorescence

Expression of p16 was assessed in colonic tissues by immunohistochemistry and immunofluorescence using the mouse monoclonal antibody (mAb) JC2, which recognizes the first ankyrin repeat of p16 [7,25]. p16 and Ki67 immunohistochemistry was performed by standard procedures on formalin-fixed paraffin-embedded sections with antigen retrieval (microwave treatment of slides immersed in citric acid buffer), as previously described [8]. Immunohistochemical staining of serine 1981-phosphorylated ataxia telangiectasia mutated (ATM) was a performed on frozen sections using rabbit antibody (Rockland, Gilbertsville, PA). For immunohistochemistry on cultured cells, cells were fixed and permeabilized with acetone/methanol (1:1) for 10 minutes at -20°C, and processed without antigen retrieval. The avidin/biotin detection system (Vector Laboratories, Burlingame, CA) was used with light hematoxylin counterstaining. Ki67 was detected using rabbit antibody (Novocastra Laboratories Ltd., New Castle upon Tyne, UK). Secondary antibodies for immunofluorescence were a CY2-labeled donkey anti-mouse antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) and a CY3-labeled donkey antirabbit antibody (Jackson ImmunoResearch Laboratories). For simultaneous detection of p16 and Ki67 by coimmunofluorescence, CY2 and CY3 signals were collected by separate filters and combined using IPLab Spectrum software (Scanalytics, Fairfax, VA). All cells with staining clearly above background were scored as positive.

Cell Culture and Ionizing Irradiation (IR)

Normal human esophageal epithelial cells were isolated, as previously described [26], from excess human clinical tissues, according to a protocol approved by the institutional review board. Normal primary human bronchial and prostate epithelial cells at passages 3 to 5 were purchased from Clonetics/Biowhittaker (now Cambrex, Walkersville, MD) and cultured under their specifications. Cells were irradiated with 8 Gy using a J. L. Shepard Model 30 Mark I 137Cs irradiator.

Immunoblotting, Northern Blot Analysis, and Flow Cytometry

Immunoblotting [8], Northern blot analysis [27], and flow cytometry [27] were performed as previously described. Northern blot analysis used a probe generated by polymerase chain reaction from p16 exon 1α.

Results

We examined p16 expression by immunohistochemistry using the highly sensitive and specific mAb, JC2 [7,8,25]. Immunohistochemical staining with this antibody has been validated by Western blot analysis and reverse transcription-polymerase chain reaction [8]. Thirty-three specimens from 25 patients with UC were examined together with 20 normal controls. Among the UC cases, 10 had polypoid dysplasia, 6 had carcinoma, and 3 had pseudopolyps.

We observed dramatically higher p16 expression in the non-neoplastic epithelium of UC specimens (Figure 1). Thirty-three percent of UC crypts displayed p16 staining versus less than 1% of crypts from normal controls. (Figure 2, A–C). p16 expression was most prominent in crypts with greater architectural distortion (AD) (Figure 2, D–F). Two percent of crypts in patients without AD displayed p16 staining, compared with about 30% of crypts with mild to moderate AD and with 60% of crypts with severe AD (Figure 2F). The Spearman correlation coefficient for this relationship was highly significant (R = 0.573, P = .002). In contrast, there was no correlation between p16 staining and degree of acute/chronic inflammation (data not shown). p16 staining was further elevated in dysplasia (Figure 2, G and H). Ninety-one percent of dysplasia specimens and all six carcinomas evidenced distinct p16 staining. The staining in these lesions was heterogeneous, with marked differences in intensity between adjacent cells. This pattern is consistent with our previous findings in sporadic colonic neoplasia [8].

Figure 1.

Figure 1

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p16 induction in UC. Normal (Nl) (A and C) and UC (B and D) mucosal samples were stained with hematoxylin and eosin (A and B) or with an antibody directed against p16 (C and D). Note the absence of brown reaction product indicative of p16 staining in the normal colon (C) and the presence of p16 staining in intestinal epithelial crypts in UC (D). Original magnification, x10 to x20.

Figure 2.

Figure 2

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p16 induction in UC in isolated crypts, areas of marked AD, and dysplasia. p16 staining in isolated crypts (A–C), areas of marked crypt AD (D and E), and dysplasia (G and H). Original magnification, x10 to x20. (F) The degree of crypt AD in each patient sample (n = 26) was scored on a scale from 0 (none) to 3 (high) (0, n = 3; 1, n = 5; 2, n = 4; 3, n= 14). The percentage of crypts in each sample that was positive for p16 was scored and graphed. Spearman correlation coefficient (ρ) = 0.57 for trend (P = .002, two-sided).

We performed coimmunofluorescence studies using the robust proliferation marker Ki67 [28–30]. p16 staining was inversely associated with Ki67 staining in UC samples (P < .001; Figure 3). As seen by immunohistochemistry, p16-positive staining often appeared to be confined to the lower portion of the crypt (Figure 3C), although this assessment can be difficult in the presence of crypt AD. These results suggest that, at least at higher levels detectable by immunofluorescence, p16 expression is associated with cell cycle arrest.

Figure 3.

Figure 3

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Paucity of Ki67 staining in p16-expressing UC cells. Samples from three patients identified as p16-positive by immunohistochemistry were assayed by coimmunofluorescence for p16 (green) and Ki67 (red). Nuclei are identified by DAPI staining (blue). (A) Triple-color merged image. Sheets of epithelium are readily identifiable by tightly packed, basally oriented DAPI-stained nuclei. Note the absence of red staining in the nuclei of green-stained cells. (B and C) Two-color merged green and red images. Note that epithelial cells are either red or green, whereas scattered apoptotic mesenchymal cells bind both antibodies nonspecifically and stain yellow. In (C), crypts are outlined by white lines. Note that, in the large crypt with a visible lumen (bottom right), p16-positive cells appear to be located near the base. In addition, the crypt with five p16-positive cells in (A) has both a narrow lumen and a small overall diameter, suggesting that it is near the base. (D) Graph illustrating low Ki67 positivity in p16-positive cells. A total of 1299 cells from 9 x 20 fields were scored. The mean percent Ki67-positive cells per field ± SD were graphed (P < .001; two-tailed t-test). Original magnification, x20.

Recent evidence suggests that early neoplasia and hyperplasia are often accompanied by DNA damage [22,23]. Induction of p53 and p21 has recently been observed in some UC mucosae, in the absence of detectable p53 mutation [31]. These proteins are well-characterized components of DNA damage response, although not specific for it. Furthermore, UV light can induce p16 in skin keratinocytes [10]. We therefore examined UC tissue for evidence of DNA damage checkpoint activation. UC crypts, as well as those from the normal colon, bound an antibody specific for ATM phosphorylated on serine 1981—a marker of ATM activation [32] (Figure 4A). Staining intensity was not dramatically different in UC versus normal samples. We obtained similar results following staining with an antibody directed against another marker of DNA damage checkpoint activation, phosphorylated histone H2AX (data not shown) [22,23]. We reasoned that a brief period of replicative stress in normal colonic transit-amplifying cells might not be sufficient to induce p16, in contrast to a sustained period of replicative stress that occurs while stem cells and/or transit-amplifying cells repopulate denuded mucosae. To assess more directly whether replicative stress can induce p16, we tested whether p16 can be induced in human epithelial cells by IR, the prototype mediator of DNA double-strand breaks. Colonic epithelial cells cannot be cultured under standard conditions [33], and nearly all established colon cell lines have demonstrable lesions in the p16/Rb pathway [34]. We therefore examined p16 expression in several representative primary human epithelial cells amenable to tissue culture. Immunohistochemistry and immunoblotting demonstrated that IR induced p16 in each epithelial cell type tested (bronchi, prostate, and esophagus) (Figure 4, B and C). Induction was not immediate, but required several days to become manifest (Figure 4C) and also appeared to require sustained cell proliferation (data not shown). Northern blot analysis pointed to induction at the mRNA level (Figure 4D). Dual immunofluorescence staining with incorporated BrdU [27] showed that most p16-expressing cells were arrested (data not shown). Flow cytometry demonstrated that most cells were arrested in G1 phase (the typical phase of a p16-mediated arrest; Figure 4E), as opposed to the G2 phase arrest commonly observed following IR treatment of transformed cell types [35].

Figure 4.

Figure 4

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p16 is induced in the setting of DNA damage. (A) Immunohistochemical staining for ATM phosphorylated on serine 1981 in representative frozen sections of normal (NI; n = 6) and UC (n = 6) colon. Arrows mark nuclear staining (brown) that is particularly prominent at the base of crypts. Left to right: ca. x20, x20, x40, and x60 images. No staining was observed in adjacent tissue sections stained without primary antibody (not shown). (B) Early passage bronchial epithelial cells (BEC) and esophageal epithelial cells (EEC) were irradiated with 8 Gy (IR) or were left untreated (con), fixed 6 days later, and subjected to immunohistochemistry for p16 (brown stain). Staining was also performed with a mouse mAb directed against a fungal toxin as negative control (con Ab). Note that some unirradiated cells express detectable p16, as is typically observed in primary cells, and that many irradiated cells develop a flattened senescent morphology (all images, ca. x20). (C) Immunoblotting was performed for p16 before (0) and 3 and 6 days after IR in BEC and prostate epithelial cells (PEC). Extensively passaged mammary epithelia cells typically sustain p16 promoter methylation [52] and serve as negative control (-C). U2-OS cells engineered to induce p16 [27] served as positive control (+C). Load: Coomasie-stained protein. (D) Northern blot analysis of mRNA 3 days after IR in EEC demonstrated increased p16 mRNA. RNA from SaOS2 (+C) and U2-OS (-C) cells served as positive and negative controls, respectively. The same blot was stripped and reprobed for actin, as loading control. (E) EEC cell cycle arrest in G1 and G2/M following IR. EEC were left untreated (con) or were harvested 4 days after IR and analyzed for DNA content by flow cytometry. The percentages of cells in each cell cycle phase are noted. Note that the S-phase fraction was nearly abolished in the IR-treated sample, indicating that few cells were cycling, whereas 63% of the cells remained in G1 phase.

Discussion

We found that p16 is induced in crypts of patients with UC. Induction correlated with degree of crypt AD, rather than with inflammation, implying that epithelial regeneration under stress may be a key factor. p16 expression is generally low in most normal tissues, including the colon [7]. Increased p16 expression has been observed in a number of tumor types, including those from the colon [8], breast [36], endometrium [37], and pancreas [38], and in some aging tissues [5,6], but has rarely been observed in non-neoplastic diseases. Our results underscore that a neoplastic state is not necessary for p16 induction in vivo. The finding that p16 is induced in the proliferative compartment of regenerating epithelium suggests that proliferative stress can induce p16 in vivo and in vitro. Furthermore, we found that ionizing radiation induces p16 expression in diverse normal cultured human epithelial cell types, with delayed kinetics. These results provide further evidence that replicative stress and potential DNA damage perse can incite p16 induction.

p16 is induced in a variety of primary mammalian cells in response to culture stress and limits the long-term proliferation of cultured cells [1,39,40]. Whether or not this induction is artifactual has been controversial [11]. Induction of p16 appears to be governed, in part, by the Ets2 transcription factor, with augmentation by Ras pathway signaling [41] and JunB [42]. Recent surveys of expression in aging mice suggest that these factors likely account for some, but not all, of the normal tissue- and age-related differences in p16 expression [6]. Our findings strengthen evidence that p16 acts as a brake on the proliferation of cells experiencing physiologic levels of replicative stress in vivo.

Evidence for p16 involvement in cell cycle inhibition following DNA damage has previously emerged. p16 is known to be induced by UV light in primary human skin keratinocytes [10,43]. In addition, p16 contributes to G1 arrest following IR in human fibroblasts [44]. p16 was not found to be induced in that study [44], but was noted to increase during the weeks following the treatment of fibroblasts with the DNA-damaging agent, bleomycin [45]. Recent demonstration that preneoplastic and early neoplastic states are commonly characterized by ongoing DNA damage [22,23] and that increased Cdk activity per se can mediate DNA damage [23] points to a potential stimulus for p16 induction in UC. Furthermore, an independent but less extensive study of p16 expression in UC revealed evidence for coexpression in some tissues with p21 and p53, without detected p53 mutation [31]. The absence of p53 mutations suggests that the p53 induction observed is not due to the stabilization of inactive p53, but may reflect functional induction by DNA damage or other stimuli. These authors surmised that the increase in p16 in such settings might be due to feedback regulation by the p53 pathway. However, the potential mechanism here is unclear, and skin cell lines with mutant p53 have been found to induce p16 following UV treatment [9]. We suggest instead that DNA damage may be acting as a common inducerof p53 and p16 in UC. Further investigation will be required to identify the factors regulating p16 expression following DNA damage and to determine whether DNA damage checkpoint pathways are involved. The relatively gradual induction of p16 after IR suggests that this induction may involve additional factors beyond the immediate checkpoint response. Factors such as stem cell phenotype, sustained proliferation, or quantitative differences in DNA damage may account for preferential induction in the UC epithelium versus the normal epithelium. We envision that p16 induction in the setting of replicative stress acts to reduce the number of stressed cells and their proliferation rates, thereby reducing the propensity for genetic change leading to neoplasia and for paracrine signaling by stressed cells. Sustained induction in this setting may mediate cellular senescence [46,47].

Our data suggest that p16 expression in UC likely constrains the proliferation of the principal constituents of crypts, colonic stem cells, and/or their highly proliferative progeny, the transit-amplifying cells [48,49]. There are potential parallels in other tissues. Bmi1 has been implicated as a transcriptional repressor of p16 [50,51]. Bmi1 is preferentially expressed in primitive stem cells of the hematopoietic system, and Bmi1-null mice die of bone marrow failure. Adoptive transfer experiments point to a deficiency of bone marrow stem cells with long-term proliferative potential [50]. Proliferative defect is partially rescued by deletion of the Ink4/Arf locus, implying that p16 may limit the proliferation of bone marrow stem cells. One model to unify these observations and the marked heterogeneity of p16 expression in colonic neoplasms [8,31] would be for p16 to constrain the proliferation of cells experiencing sustained proliferative stress, in each setting.

Acknowledgements

We are grateful to Yasir Suliman and Anil K. Rustgi for help with the culture of primary keratinocytes, and to Rosemarie Mick (Penn's Department of Epidemiology and Biostatistics) for biostatistical support.

Abbreviations

UC

ulcerative colitis

AD

architectural distortion

Footnotes

1

This work was supported by National Institutes of Health (NIH) grant R01 DK6475801 (to G.E.) and used the Morphology Core Facility of the NIH Center for Molecular Studies in Digestive and Liver Diseases at the University of Pennsylvania (grant P30 DK50306).

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