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Journal of Virology, June 2005, p. 7492-7502, Vol. 79, No. 12
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.12.7492-7502.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Intestinal Dysplasia Induced by Simian Virus 40 T Antigen Is Independent of p53

Jennifer A. Markovics,1 Patrick A. Carroll,1 M. Teresa Sáenz Robles,1 Hannah Pope,1 Craig M. Coopersmith,2 and James M. Pipas1*

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260,1 Department of Surgery, Washington University School of Medicine, St. Louis, Missouri 631102

Received 3 November 2004/ Accepted 22 February 2005


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ABSTRACT
 
Transgenic mice expressing simian virus 40 large T antigen in enterocytes develop intestinal hyperplasia that progresses to dysplasia with age. Hyperplasia is dependent on T antigen binding to the retinoblastoma (pRb) family of tumor suppressor proteins. Mice expressing a truncated T antigen that inactivates the pRb-family, but is defective for binding p53, exhibit hyperplasia but do not progress to dysplasia. We hypothesized that the inhibition of the pRb family leads to entry of enterocytes into the cell cycle, resulting in hyperplasia, while inactivation of p53 is required for progression to dysplasia. Therefore, we examined T antigen/p53 complexes from the intestines of transgenic mice. We found that T antigen did not induce p53 stabilization, and we could not detect T antigen/p53 complexes in villus enterocytes. In contrast, T antigen expression led to a large increase in the levels of the cyclin-dependent kinase inhibitor p21. Furthermore, mice in which pRb was inactivated by a truncated T antigen in a p53 null background exhibited intestinal hyperplasia but no progression to dysplasia. These data indicate that loss of p53 function does not play a role in T antigen-induced dysplasia in the intestine. Rather, some unknown function of T antigen is essential for progression beyond hyperplasia.


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INTRODUCTION
 
Large T antigen (TAg), an oncogene encoded by the small DNA tumor virus simian virus 40 (SV40), is a powerful tool for elucidating the mechanisms of growth regulation and control of cell proliferation. Expression of this viral protein is sufficient to transform multiple primary cell types (reviewed in references 19 and 47), and when expressed ectopically in transgenic mice, it causes neoplasia in numerous tissues (reviewed in reference 47).

Transformation induced by T antigen is accomplished by targeting cellular components. For example, the amino-terminal region of T antigen (Fig. 1) inactivates the retinoblastoma (pRb) family of tumor suppressors via an LXCXE motif (reviewed in reference 37) that mediates binding to pRb proteins and a J domain (56) that interacts with hsc70 and participates in pRb inactivation. T antigen alleviates the growth-suppressive functions of the Rb family by a J-domain-dependent mechanism (52, 54-56, 69) and in the case of murine polyomavirus by both J-domain-dependent and -independent mechanisms (50). The carboxy-terminal region of T antigen binds, stabilizes, and inactivates the tumor suppressor p53 (12, 31, 42) (Fig. 1). Some reports argue that the elimination of pRb and p53 tumor suppressor functions is the only T antigen activity that contributes to transformation (23). However, other reports suggest that T antigen targets additional cellular proteins and that these interactions contribute to transformation as well (1, 16, 31, 46, 62).



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  		FIG. 1. Domain map of SV40 T antigen (amino acids 1 to 708) and the mutant dl1137 (amino acids 1 to 121) indicating the three known transformation-related functions of T antigen: the J domain, the retinoblastoma family binding motif, and the p53-binding domain. The phenotypes indicated are from references 26, 32, and 33.

Studies with transgenic mice have revealed that T antigen is able to induce neoplasia in numerous cell types (reviewed in reference 47). A major complication to the interpretation of the neoplastic phenotype is that the changes induced depend on the cell type or lineage in which T antigen is expressed. For example, T antigen expression in the choroid plexus is sufficient to induce a cell-autonomous uniform hyperplasia that is dependent on intact Rb binding and J domains (9, 48). In this system, choroid plexus cell proliferation is mediated by T antigen inactivation of the pRb proteins while T antigen binding to p53 blocks apoptosis. Furthermore, the Rb and p53 inactivation by T antigen functions in trans to elicit full transformation (6, 57). On the other hand, T antigen is able to induce T-cell lymphomas and pancreatic carcinomas, but in these cases secondary cellular mutations are required as well (11, 58). Unlike choroid plexus neoplasia, the induction of T-cell lymphomas by T antigen does not require a functional J domain (58). Similarly, T antigen induces invasive lens tumors when expressed in the lens fiber of the eye, while expression in the rod photoreceptors leads to cellular degeneration (2). Thus, the consequences of T antigen expression, as well as which specific T antigen functions are required to elicit those consequences, differ widely depending on cell lineages.

In the small intestine, epithelial cells with different states of proliferation and differentiation localize to separated regions of the tissue and can be visualized and isolated for molecular, histological, and biochemical studies (reviewed in references 22, 49, and 61). Pluripotent stem cells are located near the base of the crypts of Lieberkühn and give rise to a population of cells that undergo several rounds of cell division. These progenitors eventually enter a quiescent state and differentiate into one of four lineages: enterocytes or absorptive cells, goblet cells, enteroendocrine cells, and Paneth cells. The first three move from the crypts to an adjacent villus and are subsequently exfoliated when they reach an extrusion zone located near the villus tip. Paneth cells differentiate as they move to the base of the crypt, where they reside for about 3 weeks, finally being eliminated by phagocytosis. The majority of small intestinal epithelial cells are comprised of enterocytes, absorptive cells required for nutrient uptake.

Expression of full-length T antigen in murine enterocytes results in initial hyperplasia which progresses to dysplasia evident by around 4 to 6 months (33). The hyperplasia requires T antigen action on the pRb family of tumor suppressors, since a mutant T antigen that cannot bind pRb proteins exhibits a normal intestine with growth-arrested enterocytes (8). In contrast, expression of a truncated amino-terminal region of T antigen (amino acids 1 to 121) that retains the ability to inhibit pRb proteins but lacks the p53 interaction domain is sufficient to drive enterocytes into the cell cycle, resulting in intestinal hyperplasia (32). However, mice expressing this mutant T antigen exhibit a life-long intestinal hyperplasia with no progression to dysplasia. These results are consistent with a model in which T antigen inhibition of pRb-proteins is sufficient to induce enterocyte proliferation and intestinal hyperplasia, while T antigen inhibition of p53 is essential for progression to dysplasia. To test this hypothesis we have examined the levels and interactions of T antigen and p53 in enriched preparations of enterocytes from transgenic mice.


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MATERIALS AND METHODS
 
Production and maintenance of transgenic mice. FVB/N transgenic mice containing nucleotides –1178 to +28 of the rat intestinal fatty acid binding protein (Fabpi) promoter linked to SV40 TAgwt, SV40 TAgdl1137 (T antigen amino acids 1 to 121), or K-RasVal12 were obtained from Jeff Gordon (Washington University) and have been described previously (32, 33). Fabpi-SV40 TAgdl1137 mice were from pedigree number 18 (32), Fabpi-K-RasVal12 mice were from pedigree number 73, and Fabpi-SV40 TAgwt mice were from pedigree number 103 (33). All pedigrees were maintained by crosses to nontransgenic FVB/N littermates and/or commercially obtained FVB/N mice (Taconic Labs). p53–/– animals were purchased from GenPharm International (Mountain View, CA). Standard genetic crosses were performed to obtain mice with various combinations of the Fabpi-SV40 TAgdl1137 and Fabpi-K-RasVal12 transgenes in p53+/+, p53+/–, or p53–/– background, as described in the text. For simplification purposes, animals are referred in the text as TAgwt (Fabpi-SV40 TAgwt), TAgdl1137 (Fabpi-SV40 TAgdl1137), or K-RasVal12 (Fabpi-K-RasVal12) or their various combinations. The genotype of each mouse was determined using DNA from murine tails and the PCR with protocols detailed elsewhere (8, 32, 33). All routine screenings for several murine pathogens, including hepatitis, minute, lymphocytic, choriomeningitis, ectromelia, polyoma, Sendai, pneumonia, and mouse adenoviruses, enteric bacterial pathogens, and parasites were negative.

Histopathological and morphological analysis. The intestine was removed in its entirety from the abdominal cavity, from the exit of the stomach to the entry into the large intestine. Prior to any further manipulation and without stretching the tissue, the small intestine was measured and its length recorded from the beginning of the duodenum to the end of the ileum. The intestines were then opened along their cephalocaudal axis, washed with phosphate-buffered saline, and rolled from the duodenum to the ileum. Each resulting "Swiss roll" was cut in half, parallel to the proximal/distal axis, and placed in a tissue cassette with the cut edge of one half facing down and the cut edge of the other half facing up. Hematoxylin and eosin (H&E)-stained specimens were graded in a blinded fashion for histopathologic abnormalities, and the samples were categorized as normal, hyperplastic, or dysplastic as described previously, using the following scale: 0 = normal, 1 = minimal to mild dysplasia, 2 = moderate dysplasia, 3 = severe dysplasia, and 4 = adenocarcinoma (33). Hematoxylin and eosin-stained sections of murine intestines were photographed under a Nikon FXA microscope (original magnification x200). Histological data were analyzed using the statistical software program Prism (GraphPad Software, San Diego, CA). Histological scores between groups of transgenic animals were statistically compared by analysis of variance with a Tukey posttest for all possible combinations. A value of P < 0.05 was considered to be statistically significant.

Tissue preparation and fractionation of the intestine. The small intestine was dissected as described above and then subdivided along the axis into three parts of equal length: proximal, middle, and distal. All samples were washed in saline solution. Some intestinal samples were processed as a whole, while other samples were processed to obtain fractions enriched in epithelium from villi, crypts, or cells from the intestinal mesenchyme and muscle as described previously (67), with some modifications. After being cleaned in saline solution, the intestinal samples were placed in 25 ml of phosphate-saline buffer containing 3 mM EDTA and 1 mM dithiothreitol. After incubation at room temperature for 1 h, the material was placed in 10 ml of phosphate-saline buffer containing 1 mM dithiothreitol for fraction collection. Fractions were collected in sequential steps, where the intestinal tissue was shaken gently initially and later vigorously. To assess the quality of the enrichment procedure, each fraction was analyzed microscopically and for the expression of the appropriate molecular markers. The fractions were labeled as enriched in villi (V), crypts (C), or a mixture of villi and crypts (V/C), which included an enrichment of the intervillus zone. The remaining intestinal tissue was examined by microscopy, which indicated absence of epithelium. This fraction was labeled as mesenchyme/muscle (M).

Isolation of primary fibroblasts, cell culture conditions, and establishment of cell lines. Mouse embryo fibroblasts (MEFs) were harvested from 13.5-day-old FVB embryos as described previously (36) and grown in Dulbecco's minimal essential medium supplemented with 10% heat-inactivated fetal calf serum and in the presence of antibiotics (penicillin/streptomycin). Primary cultures of MEFs were transfected with 5 µg of pRSVneoT using Lipofectamine reagent (Life Technologies), according to the manufacturer's instructions. Upon selection in culture with 0.4 mg/ml of G418, transformed colonies (foci) were selected and individually grown and several independent cell lines were established.

Immunohistochemistry. Entire small intestines were removed, flushed with phosphate-buffered saline, prepared into "Swiss rolls" as described above, and fixed for 6 to 12 h in Bouin's or formalin solution. After standard processing, the tissues were embedded in paraffin and 5-µm-thick sections were prepared. If assessment of the proliferative status was required, 42- to 60-day-old mice were given an intraperitoneal injection of 5-bromo-2'deoxyuridine (BrdU; Sigma) (120 mg/kg body weight) and 5-fluoro-2'deoxyuridine (Sigma) (12 mg/kg body weight) 90 min prior to their sacrifice. Sections were stained with appropriate antibodies (p21-rabbit polyclonal M-19 [sc-471] from Santa Cruz Biotechnology [1:50]; goat anti-BrdU sera [1:1,000]) (13). Antigen-antibody complexes were detected with appropriate secondary antibodies and enhancement systems. Anti-p21 incubation was followed by treatment with goat anti-rabbit immunoglobulin G biotin (Vector) (1:250) and streptavidin-peroxidase (Zymed) (1:200) plus development of the peroxidase reaction with DAB substrate (DAKO). Anti-BrdU serum treatment was followed by incubation with donkey anti-goat gold-labeled antibody (Amersham Life Sciences, Arlington, IL) (1:40) and silver stain enhancement (27, 45).

Immunoblot analysis. All samples were lysed on ice using a Tissue Tearor homogenizer (Biospec Products Inc.). The lysis buffer (150 mM NaCl, 50 mM Tris-HCl, 5 mM EDTA, 0.5% NP-40) contained protease and phosphatase inhibitors (5 µg/ml leupeptin, 0.7 µg/ml pepstatin, "Complete" EDTA-free protease inhibitors [Boehringer Manheim], 50 mM NaF, and 1 mM Na-orthovanadate). The concentration of solubilized proteins was quantified with a protein assay dye (Bio-Rad) based on the method published by Bradford (7), and 30 µg of total protein was analyzed by conventional Western blotting techniques. We used three antibodies to detect p53, each targeting different epitopes. Appropriate dilutions of the following primary antibodies were used: p53-mouse monoclonal PAb421 and T antigen-mouse monoclonal PAb419 (25); p53-rabbit polyclonal FL-393 (sc-6243), p53-mouse monoclonal PAb240 (sc-99), MDM2-mouse monoclonal SMP14 (sc-965), p21-rabbit polyclonal M-19 (sc-471), and vimentin-rabbit polyclonal H-84 (sc-5565) (all from Santa Cruz Biotechnology); and alkaline phosphatase-rabbit polyclonal AB904 (Chemicon International), lysozyme-rabbit polyclonal A-0099 (DAKO), and GAPDH-mouse monoclonal G8140-11 (United States Biological). Goat anti-mouse A2554 and goat anti-rabbit A0545 (Sigma) were used as secondary antibodies. The peroxidase reactions were developed with ECL-Plus reagents according to the associated protocol (Amersham Life Sciences).

Immunoprecipitation analysis. Intestinal or mouse embryo fibroblast samples were homogenized in lysis buffer containing protease and phosphatase inhibitors and protein extract concentrations were determined, as described above. A total of 90 µg of total soluble protein whole-cell extracts was immunoprecipitated for T antigen using the mouse monoclonal antibody PAb419 as described previously (54). Input lysate (30 µg), one-sixth of the supernatant, and total immunoprecipitates were denatured in sodium dodecyl sulfate-polyacrylamide gel electrophoresis buffer and electrophoresed through a 10% polyacrylamide gel. Immunoblot analysis was performed using standard procedures.

Reverse transcription-PCR (RT-PCR) analysis. Whole intestine or intestinal fractions were collected, lysed, and homogenized in buffer containing guanidine isothiocyanate, an RNase inhibitor, and the total RNA was extracted using an RNeasy kit (QIAGEN). RNA (0.75 µg) was reverse transcribed into cDNA using a Superscript first-strand synthesis kit (Gibco BRL). The cDNAs were amplified with the PCR using primers specific for SM22{alpha} (5'-ACCAAGCCTTCTCTGCCTCAAC and 5'-CACCATTCTTCAGCCACAC-CTG), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5'-TGCACCACCAACTGCTTAG and 5'-GATGCAGGGATGATGTTC), p53 (5'-CCCCCGCAAAAGAAAAAACC and 5'-GCCAGCAGAGACCTGACAACTATC), intestinal alkaline phosphatase (5'-GAAAGCAGGAAAATCCGTAGGTG and 5'-CCCTCCACAAAG AGATAAAAGCC), L-mannan binding protein (L-MBP) (5'-GCCAAGGGAGAAAAGGGAGAAC and 5'-CCAAAAAAGAGTCAGAGCAGGGG), or EphB3 (5'-TCCAATGTGAATGAGACCTCGC and 5'-AGTTCTTCTGGCTGGTTACAGTGG).

PCR exponential amplifications of the p53 and GAPDH products were obtained as follows: 5 min at 94°C; a series of 22 cycles between 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s; and a final extension step of 7 min at 72°C. The GAPDH reaction rendered a 176-bp product, while the p53 reaction generated two detectable products (431 bp and 527 bp) corresponding to the alternative and regular spliced forms, respectively (24, 68).

Amplification for the alkaline phosphatase, L-MBP, EphB3, and SM22{alpha} reactions used similar conditions, except for annealing temperatures of 57.3°C, 58°C, 59°C, and 57.8°C, respectively. The alkaline phosphatase reaction rendered an expected 525-bp product within exponential range at 30 cycles, while the L-MBP, EphB3, and SM22{alpha} reactions rendered expected product sizes of 563 bp, 487 bp, and 200 bp, respectively, all within exponential range at 25 cycles.


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RESULTS
 
Gordon and colleagues have shown that the expression of SV40 large T antigen in villus enterocytes results in an intestinal hyperplasia that is evident 6 weeks after birth and is dependent on T antigen binding to pRb proteins (8). With age, the phenotype progresses to a dysplasia. The dysplasia becomes apparent between 3 and 9 months after birth and requires sequences within the carboxy-terminal two-thirds of T antigen, a region that includes the p53 interaction surface (32, 33). T antigen binds directly to p53, inhibiting its DNA binding activity and thus blocking its ability to regulate the expression of genes containing p53 binding sequences in their promoter (3, 42, 43). T antigen binding also results in the stabilization of p53 (41). Consequently, SV40-transformed cells typically contain high steady-state levels of p53 bound to T antigen. Thus, if the functional inactivation of p53 plays a role in the intestinal dysplasia induced by T antigen, dysplastic intestines should harbor large amounts of p53 bound to T antigen. Furthermore, this model predicts that the genetic inactivation of p53 should substitute for T antigen action on p53. To test this hypothesis, we undertook a molecular and genetic characterization of the p53 pathway in transgenic mice expressing full-length T antigen or a truncated T antigen defective for p53 interaction in intestinal enterocytes. First we assessed the T antigen-dependent progression to dysplasia.

Transgenic mice expressing wild-type SV40 T antigen exhibit dysplasia, larger crypts, and increased intestinal length and weight. Mice expressing both the wild-type and the truncated version of T antigen developed intestinal hyperplasia by 6 weeks after birth (26, 32; this report). Previously, Gordon and coworkers used immunohistochemistry to show that T antigen expression is limited to the villus enterocytes and that these cells enter the cell cycle (33). As expected based on Gordon's studies we found that mice expressing wild-type T antigen progress to intestinal dysplasia with age (6 to 9 months) while mice expressing the truncated T antigen only display intestinal hyperplasia (Fig. 1).

As reported previously, mice expressing wild-type T antigen had markedly longer intestinal crypts relative to nontransgenic littermates (26). The increase in crypt length was observed all along the intestinal axis, from the duodenum to the ileum of TAgwt mice. As shown in Fig. 2, the mice expressing a truncated T antigen (TAgdl1137) do not show a similar increase and are comparable to nontransgenic mice. Thus, TAgdl1137 shows neither the increased crypt size nor the progression to dysplasia indicative of TAgwt mice.



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  		FIG. 2. Murine intestines expressing T antigen, but not dl1137, exhibit enlarged crypts compared to tissue from nontransgenic littermates. Histological sections from the middle portions of small intestines were prepared and stained with hematoxylin and eosin from control (A, B), TAgdl1137 (C, D), and TAgwt (E, F) animals. The left panels show an original magnification of x200. Enlarged prints of the crypt area are presented on the right panels. Hyperplasia of the TAgdl1137 sample (C) and dysplasia of the TAgwt sample (E) are readily observed. TAgwt and control mice were between 8 and 9 months old, while the TAgdl1137 mouse depicted in this figure was over 14 months old.

We noted that the abdomens of mice expressing wild-type T antigen were enlarged and densely packed with intestines. Measurement of intestinal length revealed a significant increase in the total length of the small intestine in mice expressing wild-type T antigen, at some times almost doubling the normal length of the control littermates (Fig. 3A). These differences were observed in both male and female mice of the TAgwt genotype and were evident as early as 6 weeks of age (Fig. 3A and D). The intestinal length of mice expressing the truncated T antigen was comparable to that of nontransgenic littermates. Both recorded intestinal parameters, length and weight, were found to be increased in TAgwt transgenic mice, resulting in an overall elevated ratio of intestinal weight to length compared to control animals (Fig. 3B and C).



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  		FIG. 3. TAgwt have longer intestines than TAgdl1137 or control mice. (A) The total length of the small intestine was measured and is represented versus the age of the animal. Male (circles) and female (squares) from nontransgenic control (open circles, n = 85; open squares, n = 90), TAgdl1137 (filled circles, n = 21; filled squares, n = 36) and TAgwt (gray-shaded circles, n = 33; gray-shaded squares, n = 52) genotypes were analyzed. (B) Increase in intestinal weight of adult TAgwt (open squares, n = 20) versus control littermates (open squares, n = 16) is evident as the animals age. (C) Ratio between intestinal weight and intestinal length of the small intestine along the life span of adult TAgwt (gray-shaded squares, n = 20) versus control littermates (open squares, n = 16). (D) Measurements of the intestinal length of nontransgenic and TAgwt mice were grouped according to age (0 to 3 months, >3 to 6 months, >6 to 9 months, >9 to 12 months, >12 to 15 months, and >15 months). The average intestine length for each group is depicted, the error bars indicating the standard deviation in the group. Corresponding P values and numbers of animals used in each group are indicated.

Expression of large T antigen does not lead to increased steady-state levels of p53 in the mouse intestine. First we used p53 monoclonal antibody PAb421 in an immunoblot analysis to assess the steady-state levels of p53 in whole intestinal extracts from T antigen-expressing mice or their nontransgenic littermates (Fig. 4). As expected, extracts from T antigen-transformed MEFs contained elevated amounts of p53 while p53 was barely detectable in normal MEF controls (Fig. 4; compare lanes 3 and 4). p53 was undetected in intestinal extracts from control mice, while T antigen-expressing mice displayed a barely observable signal (Fig. 4, lanes 1 and 2). We repeated these experiments using additional anti-p53 antibodies (monoclonal PAb240 and polyclonal p53FL 393) and again failed to detect significant levels of p53 in the intestine. Consistent with this result, we could not detect p53 bound to T antigen in coimmunoprecipitation experiments from whole intestinal extracts (data not shown). Finally, we failed to detect p53 by immunohistochemical examination of intestinal sections (data not shown). Similarly, we could not detect clear signals for the p53-related proteins p63 or p73 in intestine (data not shown). We also assessed the levels of several proteins that are regulated by p53. We did not detect either mdm2 (Fig. 4) or GADD45 (data not shown) in either nontransgenic or T antigen-expressing intestine.



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  		FIG. 4. Steady-state levels of p53-pathway-related proteins in TAgwt mouse intestines or mouse embryo fibroblasts (MEFs). Levels of downstream targets p21 and Mdm2 are also shown. Immunoblot analysis was performed on total soluble protein extracts (30 µg per lane) isolated either from the intestines of 2-month-old nontransgenic or TAgwt transgenic mice or from control nontransformed or TAgwt-transformed MEFs. A total of 1 µg of protein extract from Mdm2-overexpressing SF9 cells was loaded as a control for the Mdm2 immunoblot. These results are representative of experiments performed with at least three pairs of mice.

Large-T-antigen expression leads to elevated p21 levels in intestinal enterocytes. Surprisingly, relative to nontransgenic mice, the intestines from TAgwt mice expressed high levels of the universal cyclin-dependent kinase inhibitor p21WAF1/CIP1 (Fig. 4; compare lanes 1 and 2). As expected, T antigen expression in MEFs resulted in decreased p21 levels (Fig. 4; compare lanes 3 and 4). The large induction of p21, a growth inhibitor, in highly proliferative T antigen-expressing intestine is counterintuitive. Since these experiments were performed on whole intestinal extracts, they represent proteins present in a mixture of cell-types. One possibility is that p21 induction occurs in some cell type other than intestinal enterocytes such as muscle or infiltrating T cells. Therefore, we performed immunohistochemistry experiments to assess the cellular localization of p21. As shown in Fig. 5, nontransgenic mice express little or no p21 in the villi. In contrast, p21 was readily detectable in the villus enterocytes of TAgwt mice. Thus, T antigen expression leads to increased p21 levels in the intestinal epithelium.



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  		FIG. 5. Localization of p21 in murine intestines. p21 is restricted to the proliferative and differentiation zones in nontransgenic mice, while p21 expression extends to the upper portions of the villi in murine intestines expressing T antigen. Sections are from 7.6-month-old male mice. These results corroborate the increase of p21 observed in TAgwt intestines, as shown by immunoblot analysis in Fig. 4. Original magnification was x200 (x400 for TAgwt detail).

Enriched cell populations from fractionated intestine. Extracts obtained from whole intestine contain proteins from a variety of cell types, including the differentiated cells located in the villi, proliferating cells from the crypts, and a variety of hematopoietic cells and fibroblasts in the mesenchyme and muscle. One possible explanation for the inability to detect significant levels of p53 in these extracts is that the p53 signal is diluted by the presence of cell types that do not express T antigen. Therefore, we modified existing protocols for the fractionation of colonic and intestinal epithelium and assessed p53 levels in different enriched cell populations, prepared as described in Materials and Methods.

The enrichment procedure yields four intestinal fractions that we denote as follows: villi (V); villus/crypts (V/C); crypts (C) (diagrammed in Fig. 6A); and muscle/mesenchyme (M). We assessed the success of the enrichment procedure by microscopy and by detection of marker proteins. The greatest enrichment is obtained for the villus fraction. Microscopic examination of this fraction from nontransgenic mice reveals the presence of largely intact villi with very few if any crypts attached (Fig. 6B). Consistent with their phenotype of intestinal hyperplasia, the villi from TAgwt mice are very large and tend to shear during fractionation. Nevertheless, the villus fraction from these mice mostly contains villus enterocytes. Immunoblot analysis indicates that the villus fraction from both T antigen-expressing and nontransgenic mice expresses alkaline phosphatase, a marker of enterocytes, but not lysozyme, a Paneth cell marker, or vimentin, a mesenchymal myofibroblast-specific protein (Fig. 6C).



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  		FIG. 6. p53 protein is not detected in the intestinal epithelium. (A) Micrograph of one villus with crypts attached to its base (original magnification is x40), adjacent to a diagram of the same structures as they are separated during the fractionation process. The epithelial regions denoted between the lines indicate approximate regions of enrichment in each fraction: (V) villus stalks and tips; (V/C) intervillus regions, residual villi and crypts; (C) crypts; (M) remaining mesenchyme and muscle. (B) Micrographs of villi and crypts from 2-month-old nontransgenic or TAgwt mice isolated using the fractionation method described in Materials and Methods, illustrating the enrichment of villus (V) and crypt (C) material. Higher magnification (originally x100) of the crypt fractions illustrates the increase in crypt length in T antigen-expressing mouse intestines compared to nontransgenic controls. (C) Immunoblot analysis from epithelial fractions isolated from nontransgenic or TAgwt mouse intestines or from mouse embryo fibroblasts (MEFs) as control. Alkaline phosphatase, lysozyme, and vimentin are markers of villus enterocytes, crypt Paneth cells, and mesenchymal myofibroblasts, respectively, illustrating the enrichment of these markers in their respective fractions. These results are representative of similar fractions from at least four different pairs of mice.

The V/C fraction is a complex mixture of cell types. Microscopic examination suggests that this fraction contains a region that lies at the crypt-villus interface. This location is of great interest, because it most likely contains cells that are undergoing growth arrest and differentiation. However, to date we have not been able to separate these structures from the significant amounts of contaminating villi and crypts present in this fraction.

Following removal of the villus structures, greatly enriched intact crypts are obtained (Fig. 6B). Consistent with our observations of H&E-stained intestine, the crypts from mice expressing T antigen are much larger than those from their nontransgenic littermates. Finally, after removal of the epithelial cell layer, the underlying mesenchyme and muscle layer were extracted. We did not observe any significant difference between nontransgenic or T antigen-expressing mice in the levels or distribution of marker proteins (Fig. 6C). The sole apparent exception is lysozyme, which is less abundant in TAgwt mice. This is likely due to the fact that the epithelial cell compartment of the crypts is greatly expanded in T antigen-expressing mice. This expansion most likely results in the dilution of the lysozyme-producing Paneth cells.

T antigen was readily detected in intestinal epithelial cells but not in muscle. Consistent with previous studies (33), the greatest levels were present in the V and V/C fractions. This is expected, since the Fabpi promoter that is driving T antigen is specifically expressed in the differentiated enterocytes of the villus epithelium. We also detected low levels of T antigen in the crypt fraction. At present, we do not know whether the presence of T antigen in this fraction represents expression in crypt cells or whether it is due to contaminating differentiated enterocytes.

Fractionated intestinal epithelium lack T antigen/p53 complexes. We examined the levels of p53 and p21 in intestinal fractions from nontransgenic and TAgwt mice (Fig. 6C). As was the case with whole intestine, little or no p53 was detected in any of the fractions from either nontransgenic or TAgwt mice. In contrast, p21 levels were greatly increased in T antigen-expressing fractions (Fig. 6C; compare lanes 5 and 6 to lanes 1 and 2). We could not detect GADD45, mdm2, or p63 in any of the fractions (data not shown).

Next we attempted to enrich T antigen/p53 complexes by immunoprecipitation. T antigen and associated cellular proteins were quantitatively precipitated from extracts of intestinal epithelium and resolved by electrophoresis. p53 associated with T antigen was then detected by immunoblotting with a p53-specific monoclonal antibody. Figure 7 shows that T antigen/p53 complexes are readily detectable in MEFs expressing T antigen (lane 8). However, no p53 was detected in the T antigen immunoprecipitates from intestine (lane 6), although similar levels of T antigen were immunoprecipitated from MEFs or transgenic tissue.



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  		FIG. 7. SV40 large T antigen is not bound to p53 in the intestinal epithelium of transgenic mice. T antigen was immunoprecipitated from 90 µg of total soluble protein extracts isolated from the small intestinal epithelium of either nontransgenic or TAgwt transgenic mice or from T antigen-transformed (TAgwt) MEFs, using a monoclonal antibody PAb419 (25) that specifically recognizes the amino-terminal region of T antigen. Immunoblots were performed on total precipitates, one-sixth of the supernatants, and one-third of the inputs. These results are representative of similar immunoprecipitations from four different pairs of mice or three different clones of TAgwt MEFs.

The absence of p53 in intestinal epithelia could be due to the lack of transcription of the p53 gene or to posttranscriptional control such as increased degradation or inefficient translation. Therefore, we used RT-PCR analysis to assess the levels of p53-specific mRNA in fractionated small intestine. The quality of the fractionation procedure was tested by measuring the mRNA levels of the enterocyte-specific marker, alkaline phosphatase, another villus-tip-specific transcript, L-MBP (63), a crypt-specific marker, EphB3 (4), and the smooth muscle-specific marker, SM22{alpha} (53), by RT-PCR analysis (Fig. 8). The p53 transcript was found in all the fractions of the small intestine. T antigen expression did not alter p53 mRNA levels. The observation that p53 mRNA is readily detectable in both normal and transgenic epithelium while p53 protein is not detected suggests that p53 levels are suppressed in small intestinal epithelia by a posttranscriptional mechanism.



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  		FIG. 8. Analysis of transcriptional levels in fractionated cell populations of the murine small intestine. The middle sections of the small intestine from 3-month-old mice were fractionated and used for RT-PCR as described in Materials and Methods. cDNAs from villus (V), crypt (C), and muscle (M) fractions were amplified using primers for p53, intestinal alkaline phosphatase (Alk. Phos.), L-mannan binding protein (L-MBP), EphB3, SM22{alpha}, and GAPDH as described in Materials and Methods. Nonsaturating PCR conditions were used to ensure linearity of the reaction. The products were resolved on 2.5% NuSieve 3:1 agarose gels and stained with GelStar (BioWhittaker Molecular Applications). These results are representative of similar RT-PCRs of fractions from at least four different pairs of mice.

TAgdl1137 and K-RasVal12 bitransgenic intestines do not progress to dysplasia in a p53 null background. T antigen mutant dl1137 blocks the growth-suppressing functions of the retinoblastoma family and activates E2F-dependent gene expression (54). The intestines of TAgdl1137 mice develop hyperplasia; however, unlike TAgwt mice, they do not progress to dysplasia even in the presence of an activated ras oncogene (14, 32). Since dl1137 lacks the p53 binding domain, a logical hypothesis is that p53 blocks the progression to dysplasia and thus, TAgwt mice progress to dysplasia because T antigen binding to p53 blocks its tumor suppressor function, while TAgdl1137 mice fail to progress beyond hyperplasia because of their inability to inhibit p53. This hypothesis predicts that TAgdl1137 mice should progress to dysplasia in the absence of a functional p53.

To test this hypothesis we compared the phenotypes of TAgdl1137/K-RasVal12 transgenics in p53+/+ and p53–/– backgrounds. We found that the presence or absence of p53 did not influence the proliferative status, amount of apoptosis, or histopathological grade observed in any of these animals (Table 1). Small intestines of the transgenic animals were indistinguishable regardless of their p53 status. The proliferative status was also unaffected. Villus enterocytes reentered the cell cycle in both groups and demonstrated similar levels of S-phase cells. The numbers of apoptotic cells, as determined by a terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay (14), were similar in bitransgenic animals regardless of p53 status. TAgwt/K-RasVal12 mice developed intestinal dysplasia (32) which was unaffected by p53 status. TAgdl1137/K-RasVal12 mice that were homozygous null for p53 did not show signs of intestinal dysplasia at ages of up to 5 months, remaining significantly different from TAgdl1137/K-RasVal12/p53–/– mice, although they did develop nonintestinal tumors typical of mice lacking p53 (data not shown). We conclude that the absence of a functional p53 does not allow TAg dl1137 to progress to dysplasia.


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  		TABLE 1. Histopathology and apoptotic analysis of TAgdl1137/K-RasVal12/p53–/– mouse intestinesa


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DISCUSSION
 
Cell proliferation and growth arrest are mediated by molecular pathways that are activated or repressed by external signals, by cell-cell contact, or by the interaction of cells with substratum or extracellular matrix. The retinoblastoma family and p53 are tumor suppressor proteins that govern two key intersecting pathways that regulate cell cycle entry and exit. Both proteins act, in part, by regulating transcription. For example, members of the pRb-family repress transcription from promoters containing binding sites for the E2F family of transcription factors (reviewed in reference 20). On the other hand, p53 possesses a sequence-specific DNA binding domain and regulates the expression of genes that contain p53 binding sequences in their promoters (43).

Both pRb- and p53-mediated growth control is compromised in most cancers either by genetic mutation or by the presence of viral oncoproteins (reviewed in reference 51). In fact, members of the Polyomaviriadae, Papillomaviridae, and Adenoviridae families of DNA tumor viruses encode proteins that specifically target and inactivate p53 and pRb proteins (29, 66, 70). SV40 large T antigen binds to pRb proteins via an LXCXE motif and, in cooperation with the J domain, relieves pRb-mediated repression of E2F-dependent transcription (47, 56). Similarly, large T antigen binding to p53 blocks p53-dependent transcription and consequently growth arrest and apoptosis (42, 43).

Previously, Gordon and coworkers generated transgenic mice that express SV40 large T antigen specifically in enterocytes, a terminally differentiated cell population located in the intestinal villi. They found that wild-type T antigen induced enterocyte proliferation leading to intestinal hyperplasia evident by 6 weeks after birth that then progressed to dysplasia with age. They also found that progression to dysplasia is accelerated by the presence of an activated ras oncogene and does not require a functional p53 (14, 33). In contrast, mice expressing a mutant large T antigen that is defective for pRb interaction exhibited normal intestinal morphology and growth control (8). Finally, they demonstrated that expression of a truncated T antigen consisting of the J domain and LXCXE motif induced enterocyte proliferation and hyperplasia, but mice expressing this mutant failed to progress to dysplasia even in the presence of the activated ras oncogene (14). Based on these observations, it was hypothesized that inhibition of the pRb family is required for enterocyte entry into the cell cycle and hyperplasia. These data also raise the possibility that progression to dysplasia requires a function in the carboxy-terminal portion of T antigen. Alternatively, differences in T antigen expression levels and/or localization between TAgwt and TAgdl1137 mice may explain the lack of dysplasia in the latter animals.

T antigen action on p53 is not required for progression to intestinal dysplasia. The observation that TAgdl1137 mice, unlike TAgwt mice, do not progress to dysplasia led us to hypothesize that T antigen action on p53 is required for this process. However, two lines of evidence indicate that this is not the case. First, the intestines of TAgdl1137 mice in a p53 null background remain hyperplastic. Since this genetic combination should inactivate both the Rb and p53 pathways, the elimination of p53 in enterocytes is not sufficient to induce the progression towards intestinal dysplasia. Second, we failed to detect the presence of T antigen/p53 complexes in the intestines of transgenic mice. The absence of such complexes along with the absence of p53 stabilization in T antigen-expressing enterocytes leads us to suggest that p53 is not present in this cell type. In support of this supposition, it has been reported that p53 protein remains undetectable in villus enterocytes of normal mice, even when irradiated with gamma rays, although p53 is detected at the base of the crypts in these irradiated mice (15). It is likely that the small amount of p53 we detect in T antigen-expressing intestine is derived from the crypt.

Although T antigen-mediated inactivation of p53 seems to be an essential requirement for full transformation in several cell culture systems and transgenic models (reviewed in reference 47), our results indicate that the p53 protein may not be a key mediator of growth arrest or even play a significant role in the normal, unstressed murine intestinal epithelium. Perhaps p53 function as guardian of the genome is not necessary in a cell type that has only a 3- to 5-day life span. Rather, we speculate that p53 function is critical for genomic integrity in the intestinal stem cells, which only contribute 1 to 3 cells per crypt (49). The fact that these cells are relatively rare and that they do not express T antigen may explain our inability to detect p53 in transgenic intestine.

All three independently developed p53 nullizygous mouse strains do not exhibit spontaneous tumor formation in the small intestine, thereby supporting the lack of a role of p53 in the homeostasis of this regenerative epithelium (18, 30, 44). Additionally, the basal levels of apoptosis observed in the unstressed, homeostatic murine small intestine have been shown to be p53 independent (39) and previous experiments demonstrated the irrelevance of p53 in accelerating the dysplastic phenotype and in changing the apoptotic rates observed in T antigen-expressing intestinal samples (8, 14). In contrast, a number of studies have implicated p53 as a critical factor in stressed tissues (15, 34; reviewed in reference 65). Nevertheless, villus enterocytes do not undergo {gamma}-ray-induced apoptosis (which requires p53) even when expressing T antigen (reference 15 and references therein). These results emphasize the importance of tissue and intratissue cell type specificity and reveal how different mechanisms are required to control cell cycle and growth regulation in distinct cell types.

There are three possible explanations for the failure of TAgdl1137 mice to progress to dysplasia in a p53 null background. First, T antigen may target a protein other than p53 to affect dysplasia. Several reports have indicated the existence of T antigen-transforming activities other than p53 binding, mapping to the carboxy-terminal region (5, 17, 31, 46, 60). Second, T antigen action on p53 may not be equivalent to a total loss of p53 function. Perhaps the T antigen/p53 complex exerts a function necessary for dysplasia. For example, it has recently been shown that mutant p53 proteins that are found in some cancers have a gain-of-function phenotype (35, 40). However, this hypothesis is disfavored by our failure to detect complexes of p53 and wild-type T antigen in intestine. A final possibility is that TAgdl1137 mice fail to progress to dysplasia because the steady-state levels of this mutant protein are lower than wild-type T antigen. While this remains a formal possibility, we think that it is unlikely. Recently we have generated several new founder lines expressing a T antigen mutant similar to dl1137. These lines show a range of T antigen expression levels, but thus far, none have progressed to dysplasia (data not shown).

In at least one other transgenic model system, T antigen-induced tumorigenesis is independent of its action on p53. Expression of a truncated T antigen in pancreatic acinar cells leads to carcinoma (59). However, these results are in clear contrast to T antigen expression in the choroid plexus, where inactivation of p53 plays a critical role in tumor expansion. In this case, T antigen induces quick tumor formation and kills the animals, while dl1137 facilitates dysplasia and tumor formation but at markedly slower rate (10, 48). This delayed rate of tumor growth is abrogated when dl1137 is expressed in a p53 null background, which eliminates p53-dependent apoptosis (57). This indicates the relevance of p53 as a critical regulator of tumorigenesis in the choroid plexus.

T antigen-expressing intestines contain high levels of p21. SV40 T antigen binding to p53 results in its stabilization and inactivation (reviewed in reference 42). As a consequence, activation of genes normally regulated by p53 (e.g., p21) is impaired. Accordingly, upon p53 stabilization by T antigen in primary MEFs, we have observed a reduction in the amount of p21 protein. In clear contrast, an augmentation in the levels of p21 upon expression of T antigen in the intestine was readily observed. This increase in p21 levels occurs largely through increased transcription of the p21 gene (P. G. Cantalupo, M. T. Saenz-Robles, J. M. Markovics, D. Ahuja, R. L. Beermanl, W. H. Patterson, C. Edwards, R. H. Whitehead, and J. M. Pipas, unpublished data).

T antigen efficiently induces enterocyte proliferation and a dramatic expansion of intestinal villi. Thus, it was surprising to find that T antigen-expressing enterocytes contain abundant levels of the cell-cycle inhibitor, p21. It is unclear how enterocyte proliferation proceeds in the presence of these high p21 levels. Furthermore, the mechanism by which T antigen induces increased p21 transcription in intestine is unknown. The lack of p53 stabilization and the concomitant upregulation of p21 suggests that regulatory mechanisms other than those mediated by p53 are responsible for this observed increase in p21 product. In fact, p21 expression is controlled by several factors other than p53 (38), and the p21 protein has been found to be expressed in the intestinal differentiation zone comprised of the upper crypts and lower villus portions, where it may be a marker of differentiating enterocytes (21, 64). However, intestinal cell differentiation does not seem to be perturbed by T antigen expression (26). Thus, at present the significance of increased p21 levels and the mechanism by which this increase is achieved are unclear. Perhaps T antigen expression results in the expansion of the intestinal differentiation zone, thereby increasing the number of enterocytes expressing p21.

Expression of T antigen in enterocytes effects the entire intestinal tissue. T antigen expression in enterocytes correlates with an overall increase of the crypt size and with a notable increase in the normal intestinal length and weight. This suggests that T antigen-expressing enterocytes exert a bystander effect on the surrounding tissue. This is consistent with observations that the expression of T antigen in the villus enterocytes results in an increase in radiosensitivity in the crypt (15).

An increase in crypt size has been previously observed in transgenic animals expressing T antigen in the smooth muscle of the intestine (28). Thus, crypt size can be increased by expression of T antigen in either the villus enterocytes (26; this report) or in the intestinal smooth muscle myocytes (28). Maintenance of the homeostasis, morphology, and architecture of the intestine must be achieved by continuous communication between the tips of the villi, where enterocytes are shed; the base of the crypt, where stem cells proliferate to make new enterocytes; and the underlying muscle layer, which contributes to the final size and length of the intestine (reviewed in references 22 and 61). One intriguing possibility is that this T antigen-dependent bystander effect could be part of a homeostatic mechanism mediated by signals between the different cell populations. How T antigen perturbs normal tissue homeostasis is unclear.


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ACKNOWLEDGMENTS
 
We thank Elizabeth A. Laposata for excellent technical help and mouse husbandry and Javier Lopez for RT-PCR primer design. We also thank Jeffrey I. Gordon (Washington University) for the T-antigen-expressing transgenic mice and for critical comments on the manuscript.

This work was supported by National Institutes of Health grants RO1 GM66202 to C.M.C. and CA098956 and CA40586 to J.M.P.


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FOOTNOTES
 
* Corresponding author. Mailing address: 559 Crawford Hall, Department of Biological Sciences, University of Pittsburgh, Pittsburgh PA 15260. Phone: (412) 624-4691. Fax: (412) 624-4759. E-mail: pipas{at}pitt.edu. Back


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Journal of Virology, June 2005, p. 7492-7502, Vol. 79, No. 12
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.12.7492-7502.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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