Fecal microbiota diversity in survivors of adolescent/young adult Hodgkin lymphoma: a study of twins.

BACKGROUND: Adolescent/young adult Hodgkin lymphoma (AYAHL) survivors report fewer exposures to infections during childhood compared with controls, and they have functional lymphocyte aberrations. The gut microbiota plays a central role in immunity. METHODS: We investigated whether fecal microbial diversity differed between 13 AYAHL survivors and their unaffected co-twin controls. Pyrosequencing of fecal bacterial 16S rRNA amplicons yielded 252 943 edited reads that were assigned to species-level operational taxonomic units (OTUs) and standardised for sequencing depth by random sampling. Microbial diversity was compared within vs between twin pairs and by case-control status. RESULTS: The number of unique OTUs was more similar within twin pairs compared with randomly paired participants (P=0.0004). The AYAHL cases had fewer unique OTUs compared with their co-twin controls (338 vs 369, P=0.015); this difference was not significant (169 vs 183, P=0.10) when restricted to abundant OTUs. CONCLUSION: In this small study, AYAHL survivors appear to have a deficit of rare gut microbes. Further work is needed to determine if reduced microbial diversity is a consequence of the disease, its treatment, or a particularly hygienic environment.

Comparison of genetic divergence and fitness between two subclones of Helicobacter pylori.

Helicobacter pylori has a very plastic genome, reflecting its high rate of recombination and point mutation. This plasticity promotes divergence of the population by the development of subclones and presumably enhances adaptation to host niches. We have investigated the genotypic and phenotypic characteristics of two such subclones isolated from one patient as well as the genetic evolution of these isolates during experimental infection. Whole-genome genotyping of the isolates using DNA microarrays revealed that they were more similar to each other than to a panel of other genotyped strains recovered from different hosts. Nonetheless, they still showed significant differences. For example, one isolate (67:21) contained the entire Cag pathogenicity island (PAI), whereas the other (67:20) had excised the PAI. Phenotypic studies disclosed that both isolates expressed adhesins that recognized human histo-blood group Lewis(b) glycan receptors produced by gastric pit and surface mucus cells. In addition, both isolates were able to colonize, to equivalent density and with similar efficiency, germ-free transgenic mice genetically engineered to synthesize Lewis(b) glycans in their pit cells (12 to 14 mice/isolate). Remarkably, the Cag PAI-negative isolate was unable to colonize conventionally raised Lewis(b) transgenic mice harboring a normal gastric microflora, whereas the Cag PAI-positive isolate colonized 74% of the animals (39 to 40 mice/isolate). The genomic evolution of both isolates during the infection of conventionally raised and germ-free mice was monitored over the course of 3 months. The Cag PAI-positive isolate was also surveyed after a 10 month colonization of conventionally raised transgenic animals (n = 9 mice). Microarray analysis of the Cag PAI and sequence analysis of the cagA, recA, and 16S rRNA genes disclosed no changes in recovered isolates. Together, these results reveal that the H. pylori population infecting one individual can undergo significant divergence, creating stable subclones with substantial genotypic and phenotypic differences.

Pre-steady-state kinetic studies of Saccharomyces cerevisiae myristoylCoA:protein N-myristoyltransferase mutants identify residues involved in catalysis.

MyristoylCoA:protein N-myristoyltransferase (Nmt, EC 2.3.1.97), a member of the GCN5 acetyltransferase (GNAT) superfamily, is an essential eukaryotic enzyme that catalyzes covalent attachment of myristate (C14:0) to the N-terminal Gly of proteins involved in myriad cellular functions. The 2.5 A resolution structure of a ternary complex of Saccharomyces cerevisiae Nmt1p with a bound substrate peptide (GLYASKLA) and nonhydrolyzable myristoylCoA analogue [Farazi, T. A., et al. (2001) Biochemistry 40, 6335] was used as the basis for a series of mutagenesis experiments designed to define the enzyme's catalytic mechanism. The kinetic properties of an F170A/L171A Nmt mutant are consistent with the proposal that their main chain amides, located in a beta-bulge structure conserved among GNATs, function as an oxyanion hole to polarize the thioester carbonyl of bound myristoylCoA prior to subsequent nucleophilic attack. Removal of the two C-terminal residues (M454 and L455) produces a 300--400-fold reduction in the chemical transformation rate and converts the rate-limiting step from a step after the transformation to the transformation event itself. This finding is consistent with the main chain C-terminal carboxylate of L455 functioning as a catalytic base that abstracts a proton from the N-terminal Gly ammonium of the bound peptide to generate the nucleophilic amine. Mutating N169 and T205 in concert reduces the rate of the chemical transformation, supporting their role as components of an H-bonding network that facilitates attack of the Gly1 amine and stabilizes the tetrahedral intermediate.

Impaired prostate tumorigenesis in Egr1-deficient mice.

The transcription factor early growth response protein 1 (EGR1) is overexpressed in a majority of human prostate cancers and is implicated in the regulation of several genes important for prostate tumor progression. Here we have assessed the effect of Egr1 deficiency on tumor development in two transgenic mouse models of prostate cancer (CR2-T-Ag and TRAMP). Using a combination of high-resolution magnetic resonance imaging and histopathological and survival analyses, we show that tumor progression was significantly impaired in Egr1-/- mice. Tumor initiation and tumor growth rate were not affected by the lack of Egr1; however, Egr1 deficiency significantly delayed the progression from prostatic intra-epithelial neoplasia to invasive carcinoma. These results indicate a unique role for Egr1 in regulating the transition from localized, carcinoma in situ to invasive carcinoma.

Bacterial exposure induces and activates matrilysin in mucosal epithelial cells.

Matrilysin, a matrix metalloproteinase, is expressed and secreted lumenally by intact mucosal and glandular epithelia throughout the body, suggesting that its regulation and function are shared among tissues. Because matrilysin is produced in Paneth cells of the murine small intestine, where it participates in innate host defense by activation of prodefensins, we speculated that its expression would be influenced by bacterial exposure. Indeed, acute infection (10-90 min) of human colon, bladder, and lung carcinoma cells, primary human tracheal epithelial cells, and human tracheal explants with type 1-piliated Escherichia coli mediated a marked (25-50-fold) and sustained (>24 h) induction of matrilysin production. In addition, bacterial infection resulted in activation of the zymogen form of the enzyme, which was selectively released at the apical surface. Induction of matrilysin was mediated by a soluble, non-LPS bacterial factor and correlated with the release of defensin-like bacteriocidal activity. Bacteria did not induce matrilysin in other cell types, and expression of other metalloproteinases by epithelial cells was not affected by bacteria. Matrilysin was not detected in germ-free mice, but the enzyme was induced after colonization with Bacteroides thetaiotaomicron. These findings indicate that bacterial exposure is a potent and physiologically relevant signal regulating matrilysin expression in epithelial cells.

Inducible gene knockouts in the small intestinal and colonic epithelium.

We have developed two systems for performing Cre-mediated recombination of target genes in the rapidly self-renewing mouse small intestinal and colonic epithelium. When expression of Cre recombinase is placed directly under the control of transcriptional regulatory elements from a fatty acid-binding protein gene (Fabp), deletion of loxP flanked (floxed) DNA sequences is initiated as early as embryonic day 13.5, well before completion of intestinal morphogenesis. By embryonic day 16.5, Fabp-Cre also directs recombination in all cell layers of the transitional epithelium that lines the renal calyces and pelvis, ureters, and bladder. Fabp-Cre expression and recombination are maintained in both epithelia throughout adulthood. The second system allows recombination to be induced only in the gut and at any period during adulthood. This system uses Fabp regulatory elements to direct expression of a reverse tetracycline-regulated transactivator (rtTA). Another transgene encodes Cre under the control of tet operator sequences and a minimal promoter from human cytomegalovirus (tetO-P(hCMV)-Cre). In the absence of a doxycycline inducer, no basal recombination is detectable in the gut of adult tri-transgenic mice containing Fabp-rtTA, tetO-P(hCMV)-Cre, plus a floxed reporter gene. After 4 days of oral administration of doxycycline, recombination of the reporter is apparent in the small intestinal, cecal, and colonic epithelium. After doxycycline is withdrawn, the recombined locus persists for at least 60 days, indicating that recombination has occurred in epithelial cell progenitors that have long residency times in the proliferative units of the intestine (crypts of Lieberkühn). This inducible system should have a number of applications for examining gene function at selected times in postnatal life, under selected physiologic or pathophysiologic conditions.

The structure of myristoyl-CoA:protein N-myristoyltransferase.

Protein N-myristoylation is a covalent modification that occurs co-translationally in eukaryotes. Myristate, a rare 14 carbon saturated fatty acid (C14:0), is attached, via an amide linkage, to the N-terminal glycine of a subset of eukaryotic and viral proteins by myristoyl-CoA:protein N-myristoyltransferase (Nmt). Genetic and biochemical studies have established that Nmt is a target for development of a new class of fungicidal drugs. The enzyme is also a potential target for development of antiviral and antineoplastic agents. The structure of Saccharomyces cerevisiae Nmt1p has been determined recently with bound substrate analogs. The Nmt fold resembles the fold of members of the GCN5-related N-acetyltransferase superfamily. The structure reveals how Nmt's myristoyl-CoA and peptide substrates are recognized and bound, and what elements control the enzyme's ordered kinetic mechanism. Acyl transfer occurs through the nucleophilic addition-elimination reaction: an oxyanion hole formed by main chain atoms polarizes the thioester carbonyl and stabilizes the transition state while deprotonation of the ammonium of the Gly acceptor appears to be mediated by Nmt's C-terminal carboxylate. The use of main chain carboxylate atoms as general base catalyst is a novel feature.

Helicobacter pylori attaches to NeuAc alpha 2,3Gal beta 1,4 glycoconjugates produced in the stomach of transgenic mice lacking parietal cells.

Helicobacter pylori infection of the human stomach is associated with altered acid secretion, loss of acid-producing parietal cells, and, in some hosts, adenocarcinoma. We have used a transgenic mouse model to study the effects of parietal cell ablation on H. pylori pathogenesis. Ablation results in amplification of the presumptive gastric epithelial stem cell and its immediate committed daughters. The amplified cells produce sialylated oncofetal carbohydrate antigens that function as receptors for H. pylori adhesins. Attachment results in enhanced cellular and humoral immune responses. NeuAc alpha 2,3Gal beta 1,4 glycoconjugates may not only facilitate persistent H. pylori infection in a changing gastric ecosystem, but by promoting interactions with lineage progenitors and/or initiated cells contribute to tumorigenesis in patients with chronic atrophic gastritis.

Bcl-2 inhibits ischemia-reperfusion-induced apoptosis in the intestinal epithelium of transgenic mice.

Little is known about the effects of ischemia-reperfusion on the inductive, commitment, or execution phases of apoptosis. We have created a genetically defined model to study the response of small intestinal epithelial cells to ischemia-reperfusion injury as a function of their proliferative status and differentiation. Occlusion of the superior mesenteric artery for 20 min in adult FVB/N or C57BL/6 mice results in the appearance of TUNEL-positive apoptotic cells in the jejunal epithelium within 4 h, with a maximum response occurring at 24 h. Stimulation of apoptosis is greater in postmitotic, differentiated epithelial cells located in the upper portions of villi compared with undifferentiated, proliferating cells in the crypts of Lieberkühn (7-fold vs. 2-fold relative to sham-operated controls). Comparisons of p53(+/+) and p53(-/-) mice established that the apoptosis is p53 independent. To further characterize this response, we generated FVB/N transgenic mice that express human Bcl-2 in epithelial cells distributed from the base of crypts to the tips of their associated villi. The fivefold elevation in steady-state Bcl-2 concentration is not accompanied by detectable changes in the levels or cellular distributions of the related anti-apoptotic regulator Bcl-xL or of the proapoptotic regulators Bax and Bak and does not produce detectable effects on basal proliferation, differentiation, or death programs. The apoptotic response to ischemia-reperfusion is reduced twofold in the crypts and villi of transgenic mice compared with their normal littermates. These results suggest that both undifferentiated and differentiated cells undergo a commitment phase that is sensitive to Bcl-2. Forced expression of Bcl-2 also suppressed the p53-dependent death that occurs in proliferating crypt epithelial cells following gamma-irradiation. Thus suppressibility by Bcl-2 operationally defines a common feature of the apoptosis induced in the crypt epithelium by these two stimuli.

A transgenic mouse model of metastatic prostate cancer originating from neuroendocrine cells.

A transgenic mouse model of metastatic prostate cancer has been developed that is 100% penetrant in multiple pedigrees. Nucleotides -6500 to +34 of the mouse cryptdin-2 gene were used to direct expression of simian virus 40 T antigen to a subset of neuroendocrine cells in all lobes of the FVB/N mouse prostate. Transgene expression is initiated between 7 and 8 weeks of age and leads to development of prostatic intraepithelial neoplasia within a week. Prostatic intraepithelial neoplasia progresses rapidly to local invasion. Metastases to lymph nodes, liver, lung, and bone are common by 6 months. Tumorigenesis is not dependent on androgens. This model indicates that the neuroendocrine cell lineage of the prostate is exquisitely sensitive to transformation and provides insights about the significance of neuroendocrine differentiation in human prostate cancer.

Forced expression of Id-1 in the adult mouse small intestinal epithelium is associated with development of adenomas.

Ids are dominant-negative helix-loop-helix (HLH) proteins that play overlapping yet distinct roles in antagonizing basic HLH transcription factors. Although Ids affect myogenesis, neurogenesis, and B-cell development, little is known about their in vivo functions in epithelia. We have examined the effects of forced expression of Id-1 in the small intestinal epithelium of adult chimeric mice. 129/Sv embryonic stem cells, transfected with DNA containing Id-1 under the control of transcriptional regulatory elements that function in all intestinal epithelial cell lineages, were introduced into C57Bl/6 (B6) blastocysts heterozygous for the ROSA26 marker. The B6 ROSA26/+ intestinal epithelium of the resulting adult chimeras produces Escherichia coli beta-galactosidase, allowing identification of this internal control cell population. Chimeras produced from nontransfected embryonic stem cells served as additional controls. Immunohistochemical studies of the control chimeras indicated that the small intestinal epithelium supports a complex pattern of endogenous Id expression. Id-1 is restricted to the cytoplasm; levels do not decrease as descendants of multipotent intestinal stem cells differentiate. Id-2 and Id-3 are only detectable in nuclei; levels increase markedly as epithelial cells differentiate. Forced expression of Id-1 in the 129/Sv epithelium results in a decline in Id-2 and Id-3 to below the limits of immunodetection. A subset of chimeric-transgenic mice lacked growth factor- and defensin-producing Paneth cells in their 129/Sv epithelium and also developed intestinal adenomas. These changes were not present in normal control chimeras. Adenomas were composed of proliferating beta-Gal-positive and -negative epithelial cells, suggesting that they arose through cooperative interactions between 129/Sv(Id-1) and B6 ROSA26/+ cells. These chimeras provide a model for studying how perturbations in Id expression affect tumorigenesis.

Effects of forced expression of an NH2-terminal truncated beta-Catenin on mouse intestinal epithelial homeostasis.

beta-Catenin functions as a downstream component of the Wnt/Wingless signal transduction pathway and as an effector of cell-cell adhesion through its association with cadherins. To explore the in vivo effects of beta-catenin on proliferation, cell fate specification, adhesion, and migration in a mammalian epithelium, a human NH2-terminal truncation mutant (DeltaN89 beta-catenin) was expressed in the 129/Sv embryonic stem cell-derived component of the small intestine of adult C57Bl/6-ROSA26 left and right arrow 129/Sv chimeric mice. DeltaN89 beta-Catenin was chosen because mutants of this type are more stable than the wild-type protein, and phenocopy activation of the Wnt/Wingless signaling pathway in Xenopus and Drosophila. DeltaN89 beta-Catenin had several effects. Cell division was stimulated fourfold in undifferentiated cells located in the proliferative compartment of the intestine (crypts of Lieberkühn). The proliferative response was not associated with any discernible changes in cell fate specification but was accompanied by a three- to fourfold increase in crypt apoptosis. There was a marked augmentation of E-cadherin at the adherens junctions and basolateral surfaces of 129/Sv (DeltaN89 beta-catenin) intestinal epithelial cells and an accompanying slowing of cellular migration along crypt-villus units. 1-2% of 129/Sv (DeltaN89 beta-catenin) villi exhibited an abnormal branched architecture. Forced expression of DeltaN89 beta-catenin expression did not perturb the level or intracellular distribution of the tumor suppressor adenomatous polyposis coli (APC). The ability of DeltaN89 beta-catenin to interact with normal cellular pools of APC and/or augmented pools of E-cadherin may have helped prevent the 129/Sv gut epithelium from undergoing neoplastic transformation during the 10-mo period that animals were studied. Together, these in vivo studies emphasize the importance of beta-catenin in regulating normal adhesive and signaling functions within this epithelium.

Epithelial attachment alters the outcome of Helicobacter pylori infection.

Genetically defined in vivo models are needed to assess the importance of target cell attachment in bacterial pathogenesis. Gastric colonization by Helicobacter pylori in human populations is common and persistent, and has various outcomes including peptic ulcers and cancer. The impact of attachment on the course of infection was examined in transgenic mice expressing a human receptor for H. pylori in their gastric epithelium. Persistent infection by a clinical isolate occurred at comparable microbial densities in transgenic and nontransgenic littermates. However, microbial attachment in transgenic mice resulted in production of autoantibodies to Lewisx carbohydrate epitopes shared by bacteria and acid-secreting parietal cells, chronic gastritis, and parietal cell loss. This model should help identify bacterial and host genes that produce attachment-related pathology.

Stimulation of activin receptor II signaling pathways inhibits differentiation of multiple gastric epithelial lineages.

Activins are TGFbeta family members known to mediate a variety of developmental events. We examined the effects of activins on the self-renewing epithelial lineages present in gastric units of the adult mouse stomach. These lineages are descended from multipotent stem cells located in the midportion of each unit. The stem cell and its immediate descendants can be identified by their morphological features. Studies of knockout mice lacking activins A or B, and/or activin type II receptors (ActRII) revealed that ActRII-mediated signaling is not required for normal gastric epithelial morphogenesis or homeostasis. Mice homozygous for a null allele of the alpha-inhibin gene (inha[m1/m1]) develop gonadal sex cord stromal tumors that secrete large amounts of activins A and B. Analysis of inha(m1/m1) mice, with or without gonads, established that supraphysiological levels of activins block differentiation of preparietal to acid-producing parietal cells, differentiation of neck cells to pepsinogen-producing zymogenic cells, and terminal differentiation of mucus-producing pit cells. ActRII mRNA is normally present in pit, parietal, and zymogenic cells. inha(m1/m1)actRII(m1/m1) compound homozygotes develop activin-secreting gonadal tumors but have no abnormalities in their gastric epithelium, indicating that persistent stimulation of ActRII-dependent signaling pathways produces pleiotrophic effects on gastric epithelial differentiation. When a lineage-specific promoter is used to ablate mature parietal cells with an attenuated diphtheria toxin A fragment in transgenic mice, there is increased proliferation of the multipotent gastric stem cell and its committed daughters and subsequent development of gastric neoplasia. Parietal cell loss in inha(m1/m1) mice is not associated with this proliferative response. These different responses to parietal cell loss suggest that stimulation of ActRII-dependent signaling pathways in inha(m1/m1) animals affects the proliferative activity of the stem cell and its immediate descendents. This finding may have therapeutic significance.

Notes from some crypt watchers: regulation of renewal in the mouse intestinal epithelium.

The mouse intestinal epithelium undergoes rapid renewal throughout life, thereby requiring continuous coordination of its cellular proliferation, differentiation, and death programs. Recent advances in our understanding of this process have highlighted some of the molecules that regulate renewal and their potential roles in gut neoplasia.

Examining the role of Paneth cells in the small intestine by lineage ablation in transgenic mice.

The Paneth cell lineage is one of four epithelial lineages derived from the adult mouse small intestine's multipotent stem cell. Mature Paneth cells secrete antimicrobial peptides (cryptdins), growth factors, as well as two gene products, a secreted phospholipase A2 and matrilysin, that has been implicated as modifiers of adenoma formation in mice containing a mutation in the tumor suppressor Apc. Immature Paneth cells are located just above and below the cell layer, in intestinal crypts, that has been proposed to contain the multipotent stem cell. Paneth cells differentiate during a downward migration to the crypt base. The location and direction of Paneth cell migration, their high density and long residency time at the crypt base, and the nature of their secreted gene products, suggest that they may influence the structure and/or function of the stem cell niche. Paneth cell ablation can therefore be viewed as an experimental manipulation of the cellular microenvironment that purportedly contains the stem cell and its immediate descendants. Two types of ablation experiments were performed in transgenic mice. Nucleotides -6500 to +34 of the mouse cryptdin-2 gene (CR2) were used to express an attenuated diphtheria toxin A fragment. Light and electron microscopic immunohistochemical analyses of several pedigrees of postnatal day 28 to 180 animals established that ablation of Paneth cells is accompanied by an increase in the proportion of undifferentiated crypt base columnar cells. These cells normally co-exist with Paneth cells. The ablation does not produce a detectable effect on the proliferation or terminal differentiation programs of the other three lineages or on host-microbial interactions. The last conclusion is based on the ability of crypts to remain free of microbes detectable by Gram and Warthin-Starry stains and by retention of the normal crypt-villus distribution of components of the diffuse gut-associated lymphoid tissue. CR2-directed expression of simian virus 40 large T antigen also results in a loss of mature Paneth cells but produces a marked amplification of crypt cells having a morphology intermediate between Paneth and granule goblet cells. EM immunohistochemical analyses suggest that intermediate cells can differentiate to mature goblet cells but not to Paneth cells, as they migrate up the crypt-villus axis. Our findings suggest that (i) stemness in the crypt is not defined by instructive interactions involving the Paneth cell; (ii) expressing a Paneth cell fate may require that precursors migrate to the crypt base; (iii) antimicrobial factors produced by Paneth cells are not required to prevent colonization of small intestinal crypts; and (iv) this lineage does not function to maintain the asymmetric crypt-villus distribution of components of the diffuse gut-associated lymphoid tissue.

Molecular mechanics of calcium-myristoyl switches.

Many eukaryotic cellular and viral proteins have a covalently attached myristoyl group at the amino terminus. One such protein is recoverin, a calcium sensor in retinal rod cells, which controls the lifetime of photoexcited rhodopsin by inhibiting rhodopsin kinase. Recoverin has a relative molecular mass of 23,000 (M[r] 23K), and contains an amino-terminal myristoyl group (or related acyl group) and four EF hands. The binding of two Ca2+ ions to recoverin leads to its translocation from the cytosol to the disc membrane. In the Ca2+-free state, the myristoyl group is sequestered in a deep hydrophobic box, where it is clamped by multiple residues contributed by three of the EF hands. We have used nuclear magnetic resonance to show that Ca2+ induces the unclamping and extrusion of the myristoyl group, enabling it to interact with a lipid bilayer membrane. The transition is also accompanied by a 45-degree rotation of the amino-terminal domain relative to the carboxy-terminal domain, and many hydrophobic residues are exposed. The conservation of the myristoyl binding site and two swivels in recoverin homologues from yeast to humans indicates that calcium-myristoyl switches are ancient devices for controlling calcium-sensitive processes.

Bi-transgenic mice reveal that K-rasVal12 augments a p53-independent apoptosis when small intestinal villus enterocytes reenter the cell cycle.

Studies in cell culture systems have indicated that oncogenic forms of Ras can affect apoptosis. Activating mutations of Ras occur in approximately 30% of all human tumors and 50% of colorectal carcinomas. Since these mutations appear at early or intermediate stages in multistep journeys to neoplasia, an effect on apoptosis may help determine whether initiated cells progress towards a more neoplastic state. We have tested the effects of K-rasVal12 on apoptosis in transgenic mice. A lineage-specific promoter was used to direct expression of human K-rasVal12, with or without wild-type (wt) or mutant SV-40 T antigens (TAg), in postmitotic villus enterocytes, the principal cell type of the small intestinal epithelium. Enterocytes can be induced to reenter the cell cycle by TAgWt. Reentry is dependent upon the ability of TAg to bind pRB and is associated with a p53-independent apoptosis. Analyses of K-rasVal12 x TAgWt bi-transgenic animals indicated that K-rasVal12 can enhance this apoptosis threefold but only in cycling cells; increased apoptosis does not occur when K-rasVal12 is expressed alone or with a TAg containing Glu107,108two head right arrow Lys107, 108 substitutions that block its ability to bind pRB. Analysis of bi-transgenic K-rasVal12 x TAgWt mice homozygous for wild-type or null p53 alleles established that the enhancement of apoptosis occurs through a p53-independent mechanism, is not attributable to augmented proliferation or to an increase in abortive cell cycle reentry (compared to TAgWt mice), and is not associated with detectable changes in the crypt-villus patterns of expression of apoptotic regulators (Bcl-2, Bcl-xL, Bak, and Bax) or mediators of epithelial cell-matrix interactions and survival (e.g., alpha5beta1 integrin and its ligand, fibronectin). Coexpression of K-rasVal12 and TAgWt produces dysplasia. The K-rasVal12-augmented apoptosis is unrelated to this dysplasia; enhanced apoptosis is also observed in cycling nondysplastic enterocytes that produce K-rasVal12 and a TAg with a COOH-terminal truncation. The dysplastic epithelium of K-rasVal12 x TAgWt mice does not develop neoplasms. Our results are consistent with this finding: (a) When expressed in initiated enterocytes with a proliferative abnormality, K-rasVal12 facilitates progression to a dysplastic phenotype; (b) by diminishing cell survival on the villus, the oncoprotein may impede further progression; and (c) additional mutations may be needed to suppress this proapoptotic response to K-rasVal12.

gamma-Ray-induced apoptosis in transgenic mice with proliferative abnormalities in their intestinal epithelium: re-entry of villus enterocytes into the cell cycle does not affect their radioresistance but enhances the radiosensitivity of the crypt by inducing p53.

The radiosensitivity of proliferating crypt epithelial cells makes the gut a major limiting factor in the use of radiotherapy for treatment of abdominal cancers. As post-mitotic epithelial cells migrate from mouse small intestinal crypts to the base of adjacent villi, they rapidly lose their ability to undergo apoptosis in response to ionizing irradiation (IR). To determine whether this radioresistance reflects withdrawal from the cell cycle, we used a lineage-specific promoter to direct expression of wild type Simian virus 40 T antigen (SV40 TAg(Wt)) to villus, but not crypt, enterocytes in FVB/N transgenic mice. SV40 TAg(Wt) induced, pRB-dependent, re-entry into the cell cycle is not associated with the acquisition of IR-stimulated apoptosis 4 h or 24 h after 6 Gy or 12 Gy of gamma-irradiation. Co-expression of SV40 TAg(Wt) and K-ras(val12) produces dysplasia in cycling villus enterocytes but no shift towards apoptotic responsiveness to IR. These findings suggest that the radioresistance of villus enterocytes is not simply due to their cell cycle arrest and may be a reflection of their microenvironment. Remarkably, reentry of villus enterocytes to the cell cycle increases the radiosensitivity of the crypt epithelium without changing Bcl-2, Bcl-xL, Bak, or Bax expression. This effect is only manifest after IR and, based upon results obtained with mutant SV40 TAgs, depends upon reaching a critical level of proliferation in villus enterocytes. Like the normal crypt response to IR, the villus-derived enhancement of IR-stimulated crypt apoptosis is associated with an induction of p53 and Raf-1, and is dependent upon p53. Unlike the normal crypt response to IR, the p53 induction involves cells distributed throughout the crypt and the apoptotic response is not confined to the lower half of the crypt. These results indicate that signals initiated by cycling enterocytes can be transmitted to the crypt epithelium to induce p53 and influence their IR-induced apoptosis. Understanding the underlying signaling pathways may provide clues about how to modify a normal crypt's radiosensitivity for therapeutic benefit.