The Related Retinoblastoma (pRb) and p130 Proteins Cooperate to Regulate Homeostasis in the Intestinal Epithelium
2005; Elsevier BV; Volume: 281; Issue: 1 Linguagem: Inglês
10.1074/jbc.m509053200
ISSN1083-351X
AutoresKevin M. Haigis, Julien Sage, Jon Glickman, Sarah A Shafer, Tyler Jacks,
Tópico(s)Cancer-related Molecular Pathways
ResumopRb, p107, and p130 are related proteins that play a central role in the regulation of cell cycle progression and terminal differentiation in mammalian cells. Nevertheless, it is still largely unclear how these proteins achieve this regulation in vivo. The intestinal epithelium is an ideal in vivo system in which to study the molecular pathways that regulate proliferation and differentiation because it exists in a constant state of development throughout an animal's lifetime. We studied the phenotypic effects on the intestinal epithelium of mutating Rb and p107 or p130. Although mutating these genes singly had little or no effect, loss of pRb and p107 or p130 together produced chronic hyperplasia and dysplasia of the small intestinal and colonic epithelium. In Rb/p130 double mutants this hyperplasia was associated with defects in terminal differentiation of specific cell types and was dependent on the increased proliferation seen in the epithelium of mutant animals. At the molecular level, dysregulation of the Rb pathway led to an increase in the expression of Math1, Cdx1, Cdx2, transcription factors that regulate proliferation and differentiation in the intestinal epithelium. The absence of Cdx1 function in Rb/p130 double mutant mice partially reverted the histologic phenotype by suppressing ectopic mitosis in the epithelium. These studies implicate the Rb pathway as a regulator of epithelial homeostasis in the intestine. pRb, p107, and p130 are related proteins that play a central role in the regulation of cell cycle progression and terminal differentiation in mammalian cells. Nevertheless, it is still largely unclear how these proteins achieve this regulation in vivo. The intestinal epithelium is an ideal in vivo system in which to study the molecular pathways that regulate proliferation and differentiation because it exists in a constant state of development throughout an animal's lifetime. We studied the phenotypic effects on the intestinal epithelium of mutating Rb and p107 or p130. Although mutating these genes singly had little or no effect, loss of pRb and p107 or p130 together produced chronic hyperplasia and dysplasia of the small intestinal and colonic epithelium. In Rb/p130 double mutants this hyperplasia was associated with defects in terminal differentiation of specific cell types and was dependent on the increased proliferation seen in the epithelium of mutant animals. At the molecular level, dysregulation of the Rb pathway led to an increase in the expression of Math1, Cdx1, Cdx2, transcription factors that regulate proliferation and differentiation in the intestinal epithelium. The absence of Cdx1 function in Rb/p130 double mutant mice partially reverted the histologic phenotype by suppressing ectopic mitosis in the epithelium. These studies implicate the Rb pathway as a regulator of epithelial homeostasis in the intestine. Normal adult tissues maintain a constant cell number by regulating the relative amounts of proliferation and apoptosis. The intestinal epithelium is a highly proliferative tissue; the human small intestinal epithelium undergoes ∼1011 mitoses per day (1Potten C.S. Morris R.J. J. Cell Sci. 1988; : 45-62Crossref Google Scholar). In this tissue homeostasis is preserved through strict regulation of proliferation, differentiation, migration, and exfoliation. The position of a cell within the crypt/villus axis directly reflects its cell cycle and differentiation states. Cell proliferation normally occurs in the undifferentiated cells near the base of the crypts of Lieberkühn. The progeny of these undifferentiated cells migrate out of the crypt and into the villus and in the process exit the cell cycle and become terminally differentiated. Differentiation of the intestinal epithelium is controlled by several transcription factors including hairy and enhancer of split 1 (Hes1), atonal 1 (Atoh1/Math1), and E74-like factor 3 (Elf3/Ese1) (2Sancho E. Batlle E. Clevers H. Curr. Opin. Cell Biol. 2003; 15: 763-770Crossref PubMed Scopus (173) Google Scholar). The spatial orientation of cells within the crypt/villus axis and the high rate of cell turnover within the tissue provide a unique opportunity to study the development of the intestinal epithelium throughout an organism's lifetime. pRb, p107, and p130 comprise a family of cell cycle regulators known as the “pocket proteins.” The predominant function of the pocket proteins is to control the G1/S transition through negative regulation of the E2F family of transcription factors (3Classon M. Harlow E. Nat. Rev. Cancer. 2002; 2: 910-917Crossref PubMed Scopus (609) Google Scholar). In addition, this protein family plays an important role in regulating other cellular processes, such as terminal differentiation and senescence (4Liu H. Dibling B. Spike B. Dirlam A. Macleod K. Curr. Opin. Genet. Dev. 2004; 14: 55-64Crossref PubMed Scopus (84) Google Scholar). The regulation of differentiation by pRb appears to occur independently of E2Fs (5Sellers W.R. Novitch B.G. Miyake S. Heith A. Otterson G.A. Kaye F.J. Lassar A.B. Kaelin Jr., W.G. Genes Dev. 1998; 12: 95-106Crossref PubMed Scopus (288) Google Scholar); instead, pRb promotes differentiation through interaction with tissue-specific transcription factors. For example, pRb interacts with CCAAT enhancer-binding protein α to promote adipocyte differentiation and core binding factor α1 to promote osteoblast differentiation (6Classon M. Kennedy B.K. Mulloy R. Harlow E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10826-10831Crossref PubMed Scopus (123) Google Scholar, 7Thomas D.M. Carty S.A. Piscopo D.M. Lee J.S. Wang W.F. Forrester W.C. Hinds P.W. Mol. Cell. 2001; 8: 303-316Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). The pRb-related proteins p107 and p130 also appear to play a role in differentiation. Mice mutant for p107 and p130 have cartilage and bone differentiation defects and fail to develop terminally differentiated keratinocytes (8Cobrinik D. Lee M.H. Hannon G. Mulligan G. Bronson R.T. Dyson N. Harlow E. Beach D. Weinberg R.A. Jacks T. Genes Dev. 1996; 10: 1633-1644Crossref PubMed Scopus (378) Google Scholar, 9Ruiz S. Segrelles C. Bravo A. Santos M. Perez P. Leis H. Jorcano J.L. Paramio J.M. Development. 2003; 130: 2341-2353Crossref PubMed Scopus (47) Google Scholar). In addition, the pocket proteins directly interact with histone deacetylases and regulate their ability to alter chromatin structure around specific promoters (10Luo R.X. Postigo A.A. Dean D.C. Cell. 1998; 92: 463-473Abstract Full Text Full Text PDF PubMed Scopus (839) Google Scholar, 11Brehm A. Miska E.A. McCance D.J. Reid J.L. Bannister A.J. Kouzarides T. Nature. 1998; 391: 597-601Crossref PubMed Scopus (1080) Google Scholar). In adipocytes a complex consisting of pRb and histone deacetylase 3 prevents differentiation by inhibiting the expression of peroxisome proliferator-activated receptor γ (12Fajas L. Egler V. Reiter R. Hansen J. Kristiansen K. Debril M.B. Miard S. Auwerx J. Dev. Cell. 2002; 3: 903-910Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). Given the importance of the pocket proteins in regulating cell cycle progression and differentiation, it is not surprising that mutations in the RB gene are frequent in many human tumor types (3Classon M. Harlow E. Nat. Rev. Cancer. 2002; 2: 910-917Crossref PubMed Scopus (609) Google Scholar). RB mutations are common is tumors such as retinoblastoma, osteosarcoma, and small cell lung cancer. In contrast to RB, it is unclear whether P107 or P130 act as tumor suppressor genes. Although P130 expression is lost in a subset of ovarian carcinomas and non-small cell lung cancers, a causal relationship between P130 mutations and these types of cancer has not been established (13D'Andrilli G. Masciullo V. Bagella L. Tonini T. Minimo C. Zannoni G.F. Giuntoli 2nd, R.L. Carlson Jr., J.A. Soprano D.R. Soprano K.J. Scambia G. Giordano A. Clin. Cancer Res. 2004; 10: 3098-3103Crossref PubMed Scopus (50) Google Scholar, 14Caputi M. Groeger A.M. Esposito V. De Luca A. Masciullo V. Mancini A. Baldi F. Wolner E. Giordano A. Clin. Cancer Res. 2002; 8: 3850-3856PubMed Google Scholar). Studies of mice with targeted alleles of the genes encoding Rb, p107, and p130 have uncovered functional relationships between the pocket proteins. Although p107-/- and p130-/- mice are viable and fertile, mutation of p107 and p130 in combination is embryonic lethal (8Cobrinik D. Lee M.H. Hannon G. Mulligan G. Bronson R.T. Dyson N. Harlow E. Beach D. Weinberg R.A. Jacks T. Genes Dev. 1996; 10: 1633-1644Crossref PubMed Scopus (378) Google Scholar). In addition to displaying functional redundancy, the pocket proteins exhibit striking functional compensation. For example, in the absence of p130, p107 expression is up-regulated in peripheral T lymphocytes, and this up-regulation results in increased association of p107 with E2F4 (15Mulligan G.J. Wong J. Jacks T. Mol. Cell. Biol. 1998; 18: 206-220Crossref PubMed Scopus (70) Google Scholar). Clearly, the pocket proteins function in a complex, multi-tiered molecular pathway to protect cells from inappropriate cell cycle progression. The functions of pRb, p107, and p130 in the intestinal epithelium remain largely uncharacterized. However, studies of the laboratory mouse have provided some insight into the function of the pocket proteins in this tissue. Enterocytes expressing SV40 large T antigen, which binds to and inactivates all of the pocket proteins, reenter the cell cycle and display an increase in p53-independent apoptosis (16Hauft S.M. Kim S.H. Schmidt G.H. Pease S. Rees S. Harris S. Roth K.A. Hansbrough J.R. Cohn S.M. Ahnen D.J. Wright N.A. Goodlad R.A. Gordon J.I. J. Cell Biol. 1992; 117: 825-839Crossref PubMed Scopus (57) Google Scholar, 17Chandrasekaran C. Coopersmith C.M. Gordon J.I. J. Biol. Chem. 1996; 271: 28414-28421Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Interestingly, the expression of large T antigen did not appear to induce the de-differentiation of enterocytes (16Hauft S.M. Kim S.H. Schmidt G.H. Pease S. Rees S. Harris S. Roth K.A. Hansbrough J.R. Cohn S.M. Ahnen D.J. Wright N.A. Goodlad R.A. Gordon J.I. J. Cell Biol. 1992; 117: 825-839Crossref PubMed Scopus (57) Google Scholar). Nevertheless, these studies were limited to assessing the function of the pocket proteins in differentiated enterocytes and not any of the other cell types in the epithelium, thereby preventing a broad study of cell cycle exit and differentiation. In addition, large T antigen has multiple cellular targets and may not fully inactivate the three pocket proteins. Additional studies are required to appreciate the function of the pocket proteins in the intestinal epithelium. Homeostasis in the intestinal epithelium is achieved in part through regulation of proliferation and differentiation, making it an ideal system for studying the molecular pathways that control these processes in vivo. By using mice with conditional and germ-line null mutations in the pocket protein genes, we have evaluated their role in regulating homeostasis in this tissue. We found that mutation of the pocket protein genes alters the development of the small intestinal and colonic epithelium. This study implicates the pocket proteins as regulators of both proliferation and differentiation in the intestinal epithelium. Mouse Strains—Cdx1 knock-out mice (18Subramanian V. Meyer B.I. Gruss P. Cell. 1995; 83: 641-653Abstract Full Text PDF PubMed Scopus (302) Google Scholar) were the generous gift of Dr. Jacqueline Deschamps (Hubrecht Laboratory, The Netherlands). The Fabpl[email protected]-Cre transgenic mouse strain (19Saam J.R. Gordon J.I. J. Biol. Chem. 1999; 274: 38071-38082Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar) was obtained from the Mouse Models of Human Cancer Consortium Repository. Rb2lox and p130 mutant mice have been described previously (8Cobrinik D. Lee M.H. Hannon G. Mulligan G. Bronson R.T. Dyson N. Harlow E. Beach D. Weinberg R.A. Jacks T. Genes Dev. 1996; 10: 1633-1644Crossref PubMed Scopus (378) Google Scholar, 20Sage J. Miller A.L. Perez-Mancera P.A. Wysocki J.M. Jacks T. Nature. 2003; 424: 223-228Crossref PubMed Scopus (447) Google Scholar). For each animal, the entire intestinal tract was removed, flushed with 1× PBS, 4The abbreviations used are: PBSphosphate-buffered salineBrdUrdbromodeoxyuridine. and fixed overnight in 10% neutral-buffered formalin. Tissue for histologic analysis was removed from the identical location in all animals. For colonic sections, tissue was taken from the medial colon, ∼4 cm proximal to the rectum. For small intestinal sections, tissue was taken within 1 cm of the ileo-cecal junction. In our experience with Fabpl-Cre mice, nearly 100% of the crypts in this region of the small intestine are Cre-positive. For cell cycle analysis, animals were injected with 30 mg/kg 5-bromo-2-deoxyuridine (BrdUrd) 1 h before sacrifice. phosphate-buffered saline bromodeoxyuridine. Immunohistochemistry—Fluorescence immunohistochemistry was performed using a modified citric acid unmasking protocol. Briefly, slides were deparaffinized in xylene and rehydrated through ethanol. Slides were microwaved for 25 min in citrate buffer, pH 6.0, and then cooled in running tap water. After incubation in 1× PBS plus 0.1% Tween 20 for 10 min, slides were blocked in 10% native donkey serum for 1 h at room temperature. Primary antibody (diluted 1:100 in 1× PBS plus 0.1% Tween 20) was added, and slides were incubated overnight at 4 °C. The following day slides were washed three times in 1× PBS plus 0.1% Tween 20. Secondary antibody (diluted 1:200 in 1× PBS plus 0.1% Tween 20) was added, and slides were incubated for 1 h at 37 °C. Slides were again washed, counterstained with propidium iodide or 4′,6-diamidino-2-phenylindole, and mounted in 1× PBS. For BrdUrd immunohistochemistry, samples were deparaffinized and microwaved in citrate followed by standard detection with 3,3-diaminobenzidine using a kit from Vector Laboratories (Burlingame, CA). Samples stained for BrdUrd were counterstained with hematoxylin. Primary antibodies used were as follows: rabbit α-Rb C15 (Santa Cruz Biotechnology), rabbit α-p107 C18 (Santa Cruz Biotechnology), rabbit α-p130 C20 (Santa Cruz Biotechnology), goat α-l-FABP N20 (Santa Cruz Biotechnology), rabbit α-mucin2 H300 (Santa Cruz Biotechnology), rabbit α-lysozyme (ICN), rabbit α-chromogranin A (Abcam), rabbit α-Ki67 (Novocastra), goat α-Mcm6 C20 (Santa Cruz Biotechnology), rabbit α-phospho-histone H3 (Cell Signaling Technology), rabbit α-cleaved caspase 3 (Cell Signaling Technology), and mouse α-BrdUrd (BD Biosciences). Specificity of Rb, p107, p130, and Cdx1 antibodies was confirmed by hybridizing to tissue sections from intestine lacking the relevant protein (data not shown). In all cases each antibody detected specific nuclear staining. In some instances, nonspecific background staining was detected in the cytoplasm. Secondary antibodies for immunofluorescence were raised in donkey and labeled either with Alexa Fluor 488 or Alexa Fluor 594. Western Blotting—Western blots were performed on purified intestinal epithelium. The purification was performed with a protocol modified from Whitehead et al. (21Whitehead R.H. Demmler K. Rockman S.P. Watson N.K. Gastroenterology. 1999; 117: 858-865Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Briefly, intestines were removed, flushed with ice-cold 1× PBS, and opened lengthwise. Tissues were then incubated in 3 mm EDTA, 50 μm dithiothreitol for 1 h on ice at 4 °C. Tissues were gently washed in ice-cold 1× PBS then transferred to new 1× PBS. Intestinal epithelium was dislodged by vigorous shaking. Epithelial cells were isolated by centrifugation at 1500 rpm for 5 min. After decanting of supernatant, cells were resuspended in Nonidet P-40 buffer (100 mm NaCl, 100 mm Tris-HCl, pH 8.0, 1% Nonidet P-40) and incubated on ice for 10 min. Samples were then centrifuged to remove insoluble matter and quickly frozen in liquid nitrogen. Polyacrylamide gels (8 or 12%) were loaded with 25 or 40 μg of purified epithelial protein. Western blots were detected with ECL Plus (Amersham Biosciences). Primary antibodies were as follows: mouse α-Rb (BD Pharmingen) at 1:400 dilution, rabbit α-p107 C18 (Santa Cruz Biotechnology) at 1:1000 dilution, rabbit α-p130 C20 (Santa Cruz Biotechnology) at 1:1000 dilution, mouse α-actin C4 (Abcam) at 1:3000 dilution, rabbit α-Cdx1 CPSP (22Silberg D.G. Furth E.E. Taylor J.K. Schuck T. Chiou T. Traber P.G. Gastroenterology. 1997; 113: 478-486Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar) at 1:4000 dilution, and mouse α-Cdx2 (BioGenex) at 1:1000 dilution. RNA Isolation and Taqman Analysis—Before RNA isolation, intestinal epithelium was purified using the modified EDTA protocol (see “Western blotting” above). RNA was isolated using Trizol® reagent according to the manufacturer's instructions (Invitrogen). cDNA was generated from 1 μg of total epithelial RNA using Superscript™ III reverse transcriptase (Invitrogen). Quantitation of p107 and Cdx1 expression was performed using a Taqman® gene expression assay (Applied Biosystems). Quantitative PCR reactions were run and scored on an ABI Prism 7000 Sequence detection system. For each genotypic class, RNA from at least 2 animals was analyzed. All reactions were done in quadruplicate. To normalize p107 and Cdx1 expression levels between samples, its expression relative to TATA box-binding protein (Tbp) was assessed. For the control group, Rb2lox/2lox, the average expression level relative to Tbp was determined. Then, for each of the experimental groups, each single reaction was compared with the average of the control to generate a mean relative expression level for that group. Statistical analysis was performed using the Mstat computer program (mcardle.oncology.wisc.edu/mstat). Quantitative Analysis of Tissue Kinetics—The heights of the Ki67- and BrdUrd-positive zones were determined by counting the number of cell positions from the bottom of the crypt that the highest-staining cell occupied. For phospho-histone H3 staining, entopic cell positions were defined (based on staining in wild-type colon) as the bottom 10 cell positions of the crypt. Ectopic cell positions were defined as any positive staining above the tenth position. For each assay, at least 50 crypts, originating from tissue sections of 2–3 different mice, were scored for each genotype. Only crypts for which a good longitudinal section was obtained were scored. Statistical analysis was performed using the Mstat computer program. Little is known about the role of the pocket proteins in regulating homeostasis in the intestinal epithelium. To understand the function of the pocket proteins in this context, we first analyzed the pattern of expression within the crypts and villi of pRb, p107, and p130. In the colon, nuclear pRb was found in all epithelial cells, with higher expression in cells toward the bottom of the crypt (Fig. 1A). In contrast to the ubiquitous expression of pRb, p107 was expressed predominantly in the lower half of the crypt, whereas p130 is expressed in the upper portion of the crypt and the epithelium lining the lumen (Fig. 1, B and C). The expression patterns of pRb, p107, and p130 were similar in the small intestinal epithelium. The undifferentiated cells in the small intestinal crypt expressed pRb and p107 (Fig. 1, D and E), whereas the differentiated cells in the villi expressed Rb and p130 (Fig. 1, D and F). Although immunohistochemistry allowed us to assess the localization of pocket proteins within the epithelium, it did not give us an indication of the relative expression levels of these proteins. We analyzed by Western blot the expression of pRb, p107, and p130 in purified intestinal epithelium to determine whether/how functional compensation occurs in this tissue. Because loss of Rb function is lethal during embryogenesis (23Clarke A.R. Maandag E.R. van Roon M. van der Lugt N.M. van der Valk M. Hooper M.L. Berns A. te Riele H. Nature. 1992; 359: 328-330Crossref PubMed Scopus (897) Google Scholar, 24Jacks T. Fazeli A. Schmitt E.M. Bronson R.T. Goodell M.A. Weinberg R.A. Nature. 1992; 359: 295-300Crossref PubMed Scopus (1521) Google Scholar, 25Lee E.Y. Chang C.Y. Hu N. Wang Y.C. Lai C.C. Herrup K. Lee W.H. Bradley A. Nature. 1992; 359: 288-294Crossref PubMed Scopus (1127) Google Scholar), we utilized a conditional allele of Rb (Rb2lox) (20Sage J. Miller A.L. Perez-Mancera P.A. Wysocki J.M. Jacks T. Nature. 2003; 424: 223-228Crossref PubMed Scopus (447) Google Scholar) and a transgenic mouse strain that expresses Cre recombinase in the intestinal epithelium (Fabpl-Cre) (19Saam J.R. Gordon J.I. J. Biol. Chem. 1999; 274: 38071-38082Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). In this strain of mice, Cre is expressed mosaically within the intestinal epithelium, with little or no expression in the proximal small intestine and relatively high expression in the distal small intestine and colon. It is this mosaic Cre expression that may account for the ability to detect pRb, albeit at reduced levels, in “Rb-mutant” intestinal epithelium (Fig. 1G). We found that pRb and p130 levels remained constant, even when one or another pocket protein was mutated (Fig. 1G). By contrast, the levels of p107 increased dramatically upon mutation of Rb or p130 (Fig. 1G). In Rb/p130 double mutant intestine, the levels of p107 were even higher (Fig. 1G). This increase p107 expression was evident not only at the protein level but also in RNA isolated from mutant intestinal epithelium (Fig. 1H). These data indicate that in the intestinal epithelium, loss of Rb and/or p130 function is associated with compensatory up-regulation of p107 and that this compensation most likely occurs at the level of p107 transcription. We went on to examine the intestinal phenotype of mice with mutations in Rb and/or p107 and p130. Fabpl-Cre;Rb2lox/2lox mice exhibited a histologically normal intestinal epithelium (Fig. 2, C and D). Given the mosaic nature of the Cre expression in Fabpl-Cre mice, we confirmed by immunohistochemistry that the histologically normal crypts were indeed deficient for pRb (data not shown). We also confirmed that loss of Rb function produces histologically normal crypts by injecting Rb-/-;Rosa26/+ embryonic stem cells into wild-type blastocysts to generate chimeric mice (Fig. 2C, inset). In these chimeric animals the lacZ+, Rb-mutant crypts are histologically normal. The failure of Rb-mutant intestinal epithelium to exhibit even a subtle histological phenotype may be due to functional overlap with p107 and/or p130. The intestinal epithelium in mice singly mutant for p107 or p130 was not markedly different from wild type at the histologic level. In the small intestinal epithelium there was no apparent phenotype due to mutation of p107 or p130 (Fig. 2, F and J). The colonic epithelium from p107- and p130-mutant mice displayed focal hyperplasias that manifested as a minor increase in the depth of otherwise normal crypts (Fig. 2, E and I). Nevertheless, these hyperplasias were extremely mild and did not progress to neoplasia, even in animals greater than 18 months of age. Mice double mutant for Rb and p107 or p130 developed significant hyperplasia in the small intestine and colon. This hyperplasia was present throughout the regions of the intestinal epithelium in which Cre was expressed and did not develop as focal lesions. In the small intestine the dysplasia was associated with severe dysmorphogenesis of the crypt/villus axis (Fig. 2, H and L). In the colon the crypts were elongated, and the surface epithelium often displayed a serrated border. In both the small intestine and colon, the orientation of the crypts relative to the muscularis was skewed such that it was often difficult to obtain perfectly longitudinal sections (see Fig. 2L). Age of onset was extremely variable in both classes of double mutant animals. This is probably due to the mixed genetic background of the animals. In general, the hyperplasia was detectable by 6 months of age. Interestingly, the hyperplasia did not appear to significantly affect the health of the animals, which routinely lived 18–24 months. In no cases did the hyperplasia progress to neoplasia. The presence of chronic hyperplasia in Rb/p130 double mutant mice indicates that these pocket proteins play an important role in regulating development and homeostasis of the intestinal epithelium. This observation led us to analyze the cellular and molecular bases for the phenotype of the mutant animals. We chose to focus our efforts on characterizing the phenotype of Rb/p130 double mutant animals because their phenotype was significantly more robust than that of Rb/p107 mutant animals. Mutation of the pocket proteins has been found to affect the proliferative and apoptotic indices of many different tissues, for example in the retina (26MacPherson D. Sage J. Kim T. Ho D. McLaughlin M.E. Jacks T. Genes Dev. 2004; 18: 1681-1694Crossref PubMed Scopus (199) Google Scholar). We used immunohistochemistry to analyze cell cycle kinetics in the intestinal epithelium of our mutant mice. Ki67 is expressed by cells that remain active in the cell cycle. In wild-type mice, Ki67-positive cells were restricted to the bottom 7 cell positions in the colonic crypt (Fig. 3A). The Ki67-positive zone was expanded to 11 cell positions in Rb and p130 mutant colon and 18 cell positions in double mutant colon (Fig. 3A). Thus, as reported for a variety of other cell types, mutation of the Rb pathway caused a delay in the ability of colonic epithelial cells to exit the cell cycle and enter G0. Loss of Rb function enhances apoptosis in a cell autonomous manner in the peripheral nervous system, lens, and retina (26MacPherson D. Sage J. Kim T. Ho D. McLaughlin M.E. Jacks T. Genes Dev. 2004; 18: 1681-1694Crossref PubMed Scopus (199) Google Scholar, 27MacPherson D. Sage J. Crowley D. Trumpp A. Bronson R.T. Jacks T. Mol. Cell Biol. 2003; 23: 1044-1053Crossref PubMed Scopus (125) Google Scholar). Furthermore, inactivation of the entire Rb family by expression of large T antigen in the intestinal epithelium leads to an increase in apoptosis (17Chandrasekaran C. Coopersmith C.M. Gordon J.I. J. Biol. Chem. 1996; 271: 28414-28421Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Surprisingly, we found no difference in the apoptotic indices between wild-type, Rb mutant, p130 mutant, or double-mutant colons by staining for activated caspase 3 (CC3) (data not shown). It appears, therefore, that the induction of apoptosis in response to pRb loss is a tissue-specific phenomenon. A common consequence of Rb inactivation is ectopic entry into S-phase (24Jacks T. Fazeli A. Schmitt E.M. Bronson R.T. Goodell M.A. Weinberg R.A. Nature. 1992; 359: 295-300Crossref PubMed Scopus (1521) Google Scholar). Inactivation of all pocket proteins through expression of large T antigen in differentiated enterocytes leads to ectopic S-phase entry in small intestinal villi (16Hauft S.M. Kim S.H. Schmidt G.H. Pease S. Rees S. Harris S. Roth K.A. Hansbrough J.R. Cohn S.M. Ahnen D.J. Wright N.A. Goodlad R.A. Gordon J.I. J. Cell Biol. 1992; 117: 825-839Crossref PubMed Scopus (57) Google Scholar). To test for ectopic S-phase entry in mutant colons, we assayed for incorporation of BrdUrd. In wild-type colons, BrdUrd-positive cells were found only within the bottom nine cell positions of the crypt (Fig. 3B). In all of the mutant colons (both single and double mutants), however, BrdUrd-positive cells extended to ∼15 cell positions from the bottom of the crypt (Fig. 3B). Cells that enter S-phase at ectopic positions within the crypt might also progress to mitosis. To determine whether the ectopic BrdUrd staining in mutant colons leads to ectopic cell cycling, we measured the mitotic index by immunostaining for phosphorylated histone H3. As with BrdUrd, phosphorylated histone H3-positive cells could be found at higher cell positions in mutant colons compared with wild-type colons (Fig. 3, C and D). At entopic cell positions (1Potten C.S. Morris R.J. J. Cell Sci. 1988; : 45-62Crossref Google Scholar, 2Sancho E. Batlle E. Clevers H. Curr. Opin. Cell Biol. 2003; 15: 763-770Crossref PubMed Scopus (173) Google Scholar, 3Classon M. Harlow E. Nat. Rev. Cancer. 2002; 2: 910-917Crossref PubMed Scopus (609) Google Scholar, 4Liu H. Dibling B. Spike B. Dirlam A. Macleod K. Curr. Opin. Genet. Dev. 2004; 14: 55-64Crossref PubMed Scopus (84) Google Scholar, 5Sellers W.R. Novitch B.G. Miyake S. Heith A. Otterson G.A. Kaye F.J. Lassar A.B. Kaelin Jr., W.G. Genes Dev. 1998; 12: 95-106Crossref PubMed Scopus (288) Google Scholar, 6Classon M. Kennedy B.K. Mulloy R. Harlow E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10826-10831Crossref PubMed Scopus (123) Google Scholar, 7Thomas D.M. Carty S.A. Piscopo D.M. Lee J.S. Wang W.F. Forrester W.C. Hinds P.W. Mol. Cell. 2001; 8: 303-316Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 8Cobrinik D. Lee M.H. Hannon G. Mulligan G. Bronson R.T. Dyson N. Harlow E. Beach D. Weinberg R.A. Jacks T. Genes Dev. 1996; 10: 1633-1644Crossref PubMed Scopus (378) Google Scholar, 9Ruiz S. Segrelles C. Bravo A. Santos M. Perez P. Leis H. Jorcano J.L. Paramio J.M. Development. 2003; 130: 2341-2353Crossref PubMed Scopus (47) Google Scholar, 10Luo R.X. Postigo A.A. Dean D.C. Cell. 1998; 92: 463-473Abstract Full Text Full Text PDF PubMed Scopus (839) Google Scholar), there was no significant difference in the mitotic index between wild-type, single or double mutant colon (Fig. 3C). There was a striking difference, however, in the number of ectopic mitoses between wild-type and mutant colons (Fig. 3D). Our analysis of the tissue kinetics in mutant epithelium revealed that a
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