Inactivation of p21WAF1/cip1 enhances intestinal tumor formation in Muc2-/- mice

In the Apc1638(+/-) mouse model of intestinal tumorigenesis, targeted inactivation of the cyclin-dependent kinase inhibitor p21(WAF1/cip1) is highly effective in enhancing Apc-initiated tumor formation in the intestine. Because p21(WAF1/cip1) plays a critical role in regulating intestinal cell proliferation, maturation, and tumorigenesis, we examined whether its inactivation would enhance tumor formation in a different mouse model of colon cancer. Therefore, we mated p21(-/-) mice with mice carrying a genetic deficiency of the Muc2 gene, which encodes the major gastrointestinal mucin. Muc2(-/-) mice develop tumors in the small and large intestine and the rectum, but in contrast to tumors in Apc1638(+/-) mice, this does not involve increased expression or nuclear localization of beta-catenin. We found that inactivation of p21(WAF1/cip1) significantly increased the frequency and size of intestinal tumors in Muc2 knockout mice and also led to development of more invasive adenocarcinomas. This enhanced tumorigenesis significantly decreased mouse life span. Further, inactivation of p21(WAF1/cip1) increased cell proliferation, decreased apoptosis, and decreased intestinal trefoil factor expression in the mucosa of both the small and large intestine. Surprisingly, reduced expression of p27(kip1) was also observed in the Muc2(-/-), p21(+/-), and p21(-/-) mice. In contrast, the expression of c-myc was significantly elevated. Thus, p21 modulates the formation of tumors whose initiation does (Apc) or does not (Muc2) involve altered beta-catenin-Tcf4 signaling, but which may converge on common elements downstream of this signaling pathway.     

Increased proliferation of CD8+ T cells in SAP-deficient mice is associated with impaired activation-induced cell death

Defective signaling lymphocyte activation molecule (SLAM)-associated protein (SAP) is responsible for the human X-linked lymphoproliferative syndrome. Defects in T helper 2, natural killer, natural killer T and B cells have been demonstrated in SAP-deficient humans and mice, and increased proliferation of CD8+ T cells has been observed. In the current study, we investigated the properties of CD8+ T cell proliferation and activation-induced cell death (AICD), using OT-I T cell receptor (TCR)-transgenic mice on either wild-type (WT) or SAP-/- background. Interestingly, we found that ovalbumin peptide-activated SAP-/- CD8+ T cells have lower AICD compared to their WT counterparts. Furthermore, the induction of p73, a key mediator of TCR-induced apoptosis through the mitochondrial apoptotic pathway, was significantly reduced at both the mRNA and protein levels in the activated mutant cells. Meanwhile, a reduced level of activated caspase 9 was observed in the mutant cells. We conclude that reduced AICD in activated SAP-/- CD8+ T cells is associated with impaired p73 induction, indicating that the initiation of the mitochondrial apoptotic pathway might be impaired. Our data demonstrate an intrinsic defect in SAP-/- CD8+ T cells and shed light on the increased responsiveness of CD8+ T cells in SAP-/- mice.     

Regional localization of the gene coding for sphingolipid activator protein SAP-1 on human chromosome 10

Sphingolipid activator protein SAP-1 is required for the enzymatic hydrolysis of GMI ganglioside and sulfatide. The gene coding for SAP-1 was previously mapped to human chromosome 10 using monospecific antibodies prepared against SAP-1 in synteny analysis of somatic cell hybrids. In this study, we used a cDNA probe for SAP-1 and in situ hybridization to regionally localize theSAP1 gene to the long arm of chromosone 10, region q21–22. Additional mapping data using cell hybrids containing partial chromosome 10 and skin fibroblasts with trisomy 10p are consistent with the in situ hybridization mapping results.     

Eye defects in receptor protein-tyrosine phosphatase alpha knock-down zebrafish.

Receptor protein-tyrosine phosphatase alpha (RPTP alpha) is highly expressed in the developing retina of different species, but little is known about its function there. Here, we report that injection of antisense morpholinos in zebrafish embryos reduced RPTP alpha expression to almost nondetectable levels up to 3 days postfertilization (dpf). RPTP alpha was detectable again from 4 dpf onward. RPTP alpha knock-down resulted in smaller eyes. Examination of sections of the retina at different developmental stages demonstrated that already at 28 hours postfertilization (hpf) fewer cells were present in the retina of RPTP alpha-morpholino-injected embryos. At 3 dpf, the layered organization of the retina was absent. In addition, the morphology and labeling with an axon specific antibody, acetylated tubulin, demonstrated that most cells appeared to be undifferentiated. Strikingly, at 5 dpf the lamination of the retina was partially restored, concomitant with re-expression of RPTP alpha protein. Although cells in the retina were now differentiated, the layering of the retina remained disrupted and significant gaps were observed in the amacrine cell layer. Therefore, knock-down of RPTP alpha protein provides evidence that RPTP alpha is essential for normal retinal development.     

急性胰腺炎患者高迁移率族蛋白B1表达增强

 目的 探讨高迁移率族蛋白B1（HMGB1）在急性胰腺炎（AP）中的表达及其临床意义。方法 选择轻症急性胰腺炎（MAP）20例,重症急性胰腺炎无器官功能障碍（SAP-1）10例,伴器官功能障碍（SAP-2）10例,健康志愿者20例,用RT-PCR检测血液中HMGB1 mRNA水平、用ELISA检测血清中HMGB1、TNF-α和IL-6的浓度,分析血清HMGB1水平与急性胰腺炎病情严重程度的关系。结果 MAP组、SAP-1组、SAP-2组血清中HMGB1的浓度分别为3.5 ± 0.8、5.7 ± 1.6 和8.3 ± 1.7 ng/mL,明显高于对照组的1.6 ± 0.4 ng/mL; HMGB1 mRNA的表达分别为0.56±0.11,0.71±0.07和0.89±0.09,明显高于对照组的0.25±0.04。HMGB1的表达均随病情加重而增加。结论 HMGB1在重症急性胰腺炎的病理过程中起十分重要的作用,并且可能是其发生器官功能障碍的一种关键性的细胞因子。     

Intestinal epithelial cell-specific CD98 expression regulates tumorigenesis in Apc(Min/+) mice

The transmembrane glycoprotein CD98 regulates integrin signaling that in turn controls cell proliferation and survival. CD98 expression is upregulated in various carcinomas, including colorectal cancer. Recently, by generating gain- and loss-of-function mouse models featuring genetic manipulation of CD98 expression specifically in intestinal epithelial cells (IECs), we have explored the crucial role of CD98 in the regulation of intestinal homeostasis and inflammation-associated tumorigenesis. In the present study, we investigated the contribution of CD98 to intestinal tumorigenesis in Apc(Min/+) mice and the underlying mechanism of action. Mice featuring IEC-specific CD98 overexpression (Tg animals) were crossed with Apc(Min/+) mice, and the characteristics of intestinal adenoma formation were assessed. Compared with Apc(Min/+) mice, Tg/Apc(Min/+) animals exhibited increases in both intestinal tumor incidence and tumor size; these parameters correlated with enhanced proliferation and decreased apoptosis of IECs. IEC-specific CD98 overexpression resulted in increased synthesis of the oncogenic proteins c-myc and cyclin-D1 in Apc(Min/+) mice, independently of the Wnt-APC-β-catenin pathway, suggesting the implication of CD98 overexpression-mediated Erk activation. IEC-specific CD98 overexpression enhanced the production of proinflammatory cytokines and chemokines that are crucial for tumorigenesis. We validated our results in mice exhibiting IEC-specific CD98 downregulation (CD98(flox/+)VillinCre animals). IEC-specific CD98 downregulation efficiently attenuated tumor incidence and growth in Apc(Min/+) mice. The reduction of intestinal tumorigenesis upon IEC-specific CD98 downregulation was caused by the attenuation of IEC proliferation and cytokine/chemokine production. In conclusion, we show that CD98 exerts an oncogenic activity in terms of intestinal tumorigenesis, via an ability to regulate tumor growth and survival.     

c-Jun NH2-terminal kinase 1 plays a critical role in intestinal homeostasis and tumor suppression

The c-Jun NH(2)-terminal kinase (JNK) signal transduction pathway plays important roles in cellular processes and stress. However, the role of JNK1 in intestinal homeostasis and tumorigenesis is unknown. Therefore, we used a JNK1 knockout mouse model to characterize intestinal cell maturation and tumorigenesis. In addition, colon cancer cell lines were used to validate the role of JNK1 and to elucidate the underlying molecular mechanisms in vitro. To our surprise, we found that mice with targeted inactivation of JNK1 spontaneously developed intestinal tumors. The normal mucosa in JNK1-deficient mice showed decreased cell differentiation and increased cell proliferation. This tumorigenesis was closely linked to the down-regulation of p21(WAF1/cip1), a cyclin-dependent kinase inhibitor, in intestinal epithelial cells. Immunohistochemical staining showed that JNK1 was highly expressed in the differentiation compartment of the intestinal mucosa and that the expression of JNK1 was significantly decreased in both human colonic and mouse intestinal tumors. In the colon cancer cell lines, JNK1 expression was up-regulated during spontaneous differentiation, corresponding to the up-regulation of p21(WAF1/cip1). Moreover, butyrate-induced p21 expression was linked to phosphorylation of JNK1. Reduced JNK1 expression by small interfering RNA suppressed butyrate-induced apoptosis. We concluded that JNK1 plays a critical role in the regulation of homeostasis and in the suppression of tumor formation in the intestine, which was linked to the altered expression of p21(WAF1/cip1).     

Thermo-stability and antitumor activity on colon cancer cell lines of monoclonal anti-CEA antibody-saporin immunotoxin.

Eight saporin peaks were obtained from the purification of seed extracts of Saponaria officinalis L. Saporin peak No. 6 (SAP-6) showed the highest activity in the inhibition of protein synthesis (98%) in an in vitro translation study. An immunotoxin (IT) was prepared from SAP-6 conjugated to a monoclonal anti-CEA antibody 26/5/1 (mab B) using N-succinimidyl pyridyl dithiopropionate (SPDP) and 2-iminothiolane as a cross linker. Under thermal stability study by a DSC (differential scanning calorimetry), the IT showed a denature temperature of 75 degrees C. In in vitro translation studies, the purified IT showed the same activity as SAP-6 at 10(-7) M and 10(-9) M protein concentration at 0, 30 and 60-min incubation effects with mab B and SAP-6 not conjugated at 24-hr incubation periods on human promyelocytic cell line HL 60 and on human colon adenocarcinoma cell lines which were SW 403, LoVo and LS 174 T. SAP-6, mab B and IT had no cytotoxic effect on HL-60. The IT showed a higher cytotoxic effect than SAP-6 in CEA-positive cell lines. The IT demonstrated the highest cytotoxic effect of 51% inhibition of control at 10(-7) M on the LS 174 T.     

Helicobacter hepaticus infection promotes colon tumorigenesis in the BALB/c-Rag2(-/-) Apc(Min/+) mouse

Adenomatous polyposis coli (APC) mutations are linked to human and mouse colorectal cancers. The Apc multiple intestinal neoplasia (Min) mouse mutation causes adenomas to develop throughout the small and large intestines. The BALB-Min (C.B6-Apc(Min/+)) congenic strain was generated by backcrossing into BALB/c the Apc(Min) allele from C57BL/6J-Apc(Min/+) mice. BALB-Min mice have a low tumor multiplicity (27.4 small intestine tumors/mouse) and a relatively long life span (>1 year) that makes them amenable to long-term studies. To investigate the interplay of the adaptive immune system and intestinal tumorigenesis, the immunodeficient compound mutant strain BALB-RagMin (C.Cg-Rag2(-/-) Apc(Min/+)) was generated. BALB-RagMin mice had a significant increase in tumors in the small, but not large, intestine relative to their BALB-Min counterparts (43.0 versus 24.0 tumors/mouse, respectively). The results suggest that the adaptive immune system plays a role in either the elimination or the equilibrium phase of cancer immunoediting in the small intestine in this model. We investigated the effect of the enterohepatic bacterial pathogen Helicobacter hepaticus on liver and intestine tumorigenesis in BALB-RagMin mice. H. hepaticus-infected BALB-RagMin mice developed moderate hepatitis, moderate typhlitis, and mild colitis. There were no differences in small intestine and cecal tumor multiplicity, regionality, or size relative to that in uninfected mice. However, H. hepaticus-infected BALB-RagMin mice had a significant increase in colon tumor incidence relative to uninfected BALB-RagMin mice (23.5% versus 1.7%, respectively). The data suggest that H. hepaticus, which is present in many research colonies, promotes colon tumorigenesis in the BALB-RagMin mouse and that it has the potential to confound colon tumorigenesis studies.     

Interleukin-23 is sufficient to induce rapid de novo gut tumorigenesis,independent of carcinogens,through activation of innate lymphoid cells

Chronic inflammation has been associated with increased risk for developing gastrointestinal cancer. Interleukin-23 (IL-23) receptor signaling has been correlated with inflammatory bowel disease pathogenesis, as well as promotion of tumor growth. However, little is known about the relative potential for IL-23-directed causality in gut tumorigenesis. We report that IL-23 transgene expression was sufficient to induce rapid (3–4 weeks) de novo development of intestinal adenomas with 100% incidence. Initiation of tumorigenesis was independent of exogenous carcinogens, Helicobacter colonization, or pre-existing tumor-suppressor gene mutations. Tumorigenesis was mediated by Thy1⁺IL-23R⁺ innate lymphoid cells (ILC3), in part, through IL-17 responses as tumor development was inhibited in RAG^−/− × IL-17^−/− double knockout mice. Remarkably, IL-23 initiation of tumorigenesis by resident ILCs consistently occurred before recruitment of conspicuous inflammatory infiltrates. Our results reveal an explicit role for IL-23-mediated initiation of gut tumorigenesis and implicate a key role for IL-23R⁺ ILC3 in the absence of overt cellular infiltrate recruitment.     

Identification of a novel putative gastrointestinal stem cell and adenoma stem cell marker, doublecortin and CaM kinase-like-1, following radiation injury and in adenomatous polyposis coli/multiple intestinal neoplasia mice

In the gut, tumorigenesis arises from intestinal or colonic crypt stem cells. Currently, no definitive markers exist that reliably identify gut stem cells. Here, we used the putative stem cell marker doublecortin and CaM kinase-like-1 (DCAMKL-1) to examine radiation-induced stem cell apoptosis and adenomatous polyposis coli (APC)/multiple intestinal neoplasia (min) mice to determine the effects of APC mutation on DCAMKL-1 expression. Immunoreactive DCAMKL-1 staining was demonstrated in the intestinal stem cell zone. Furthermore, we observed apoptosis of the cells negative for DCAMKL-1 at 6 hours. We found DNA damage in all the cells in the crypt region, including the DCAMKL-1-positive cells. We also observed stem cell apoptosis and mitotic DCAMKL-1-expressing cells 24 hours after irradiation. Moreover, in APC/min mice, DCAMKL-1-expressing cells were negative for proliferating cell nuclear antigen and nuclear beta-catenin in normal-appearing intestine. However, beta-catenin was nuclear in DCAMKL-1-positive cells in adenomas. Thus, nuclear translocation of beta-catenin distinguishes normal and adenoma stem cells. Targeting DCAMKL-1 may represent a strategy for developing novel chemotherapeutic agents.     

Microbiological and histological study of the gastrointestinal tract of germ-free mice infected with Helicobacter trogontum

Helicobacter spp. have been the focus of considerable research because of the role of this genus in gastrointestinal diseases. We infected NIH germ-free mice with Helicobacter trogontum, a recently described intestinal bacterium of rats, in order to study the distribution of this bacterium in the gastrointestinal tract and the histopathological changes it can induce in this host. Sixteen mice were challenged with a single dose of H. trogontum (test group) and killed one and six weeks after inoculation (eight animals at each point). Eight animals were challenged with 0.85% saline alone (control group) and killed at the same time points (four at each point). Fragments from the gastric and intestinal mucosa were obtained for microbiological and histological examination. H. trogontum was isolated from the cecum and colon of all test mice and also from the gastric mucosa of several of them. All infected animals presented histological changes in at least one region of the bowel. Alterations in the gastric mucosa were also observed mainly in the six-week-infected group. The predominant histological change observed was a moderate diffuse inflammatory infiltrate of mononuclear cells in the lamina propria, often accompanied by a mild infiltration of polymorphonuclear cells. Two animals presented focal infiltration of inflammatory cells in the liver, although no bacteria were found in the liver of any animal. H. trogontum is an intestinal species that is able to elicit inflammatory responses in other regions of the gastrointestinal tract such as the gastric mucosa and the liver of gnotobiotic mice.     

Intestinal overexpression of IL-18 promotes eosinophils-mediated allergic disorders

Baseline eosinophils reside in the gastrointestinal tract; however, in several allergic disorders, excessive eosinophils accumulate in the blood as well in the tissues. Recently, we showed in vitro that interleukin (IL)-18 matures and transforms IL-5-generated eosinophils into the pathogenic eosinophils that are detected in human allergic diseases. To examine the role of local induction of IL-18 in promoting eosinophil-associated intestinal disorders, we generated enterocyte IL-18-overexpressing mice using the rat intestinal fatty acid-binding promoter (Fabpi) and analysed tissue IL-18 overexpression and eosinophilia by performing real-time polymerase chain reaction, Enzyme-Linked Immunosorbent Assay and anti-major basic protein immunostaining. Herein we show that Fabpi-IL-18 mice display highly induced IL-18 mRNA and protein in the jejunum. IL-18 overexpression in enterocytes promotes marked increases of eosinophils in the blood and jejunum. Our analysis shows IL-18 overexpression in the jejunum induces a specific population of CD101⁺ CD274⁺ tissue eosinophils. Additionally, we observed comparable tissue eosinophilia in IL-13-deficient-Fabpi-IL-18 mice, and reduced numbers of tissue eosinophils in eotaxin-deficient-Fabpi-IL-18 and IL-5-deficient-Fabpi-IL-18 mice compared with Fabpi-IL-18 transgenic mice. Notably, jejunum eosinophilia in IL-5-deficient-Fabpi-IL-18 mice is significantly induced compared with wild-type mice, which indicates the direct role of induced IL-18 in the tissue accumulation of eosinophils and mast cells. Furthermore, we also found that overexpression of IL-18 in the intestine promotes eosinophil-associated peanut-induced allergic responses in mice. Taken together, we provide direct in vivo evidence that induced expression of IL-18 in the enterocytes promotes eotaxin-1, IL-5 and IL-13 independent intestinal eosinophilia, which signifies the clinical relevance of induced IL-18 in eosinophil-associated gastrointestinal disorders (EGIDs) to food allergens.     

The phosphatase PRL-3 affects intestinal homeostasis by altering the crypt cell composition

Expression of the phosphatase of regenerating liver-3 (PRL-3) is known to promote tumor growth in gastrointestinal adenocarcinomas, and the incidence of tumor formation upon inflammatory events correlates with PRL-3 levels in mouse models. These carcinomas and their onset are associated with the impairment of intestinal cell homeostasis, which is regulated by a balanced number of Paneth cells and Lgr5 expressing intestinal stem cells (Lgr5+ ISCs). Nevertheless, the consequences of PRL-3 overexpression on cellular homeostasis and ISC fitness in vivo are unexplored. Here, we employ a doxycycline-inducible PRL-3 mouse strain to show that aberrant PRL-3 expression within a non-cancerous background leads to the death of Lgr5+ ISCs and to Paneth cell expansion. A higher dose of PRL-3, resulting from homozygous expression, led to mice dying early. A primary 3D intestinal culture model obtained from these mice confirmed the loss of Lgr5+ ISCs upon PRL-3 expression. The impaired intestinal organoid formation was rescued by a PRL inhibitor, providing a functional link to the observed phenotypes. These results demonstrate that elevated PRL-3 phosphatase activity in healthy intestinal epithelium impairs intestinal cell homeostasis, which correlates this cellular mechanism of tumor onset with PRL-3-mediated higher susceptibility to tumor formation upon inflammatory or mutational events.

Key messages

? Transgenic mice homozygous for PRL-3 overexpression die early.

? PRL-3 heterozygous mice display disrupted intestinal self-renewal capacity.

? PRL-3 overexpression alone does not induce tumorigenesis in the mouse intestine.

? PRL-3 activity leads to the death of Lgr5+ ISCs and Paneth cell expansion.

? Impairment of cell homeostasis correlates PRL-3 action with tumor onset mechanisms.

     

P-选择素对ApcMin/+小鼠肠道肿瘤的作用

目的: 研究P-选择素(P-selectin)在Apc^Min/+小鼠肠道肿瘤中的作用。方法: 采用P-selectin 基因缺失的基因工程小鼠和肠道肿瘤模型Apc^Min/+小鼠杂交,计数Apc^Min/+小鼠与Apc^Min/+ P-selectin ^-/-杂交小鼠小肠及大肠肿瘤的数目,并测量其肿瘤体积,研究P-selectin对Apc^Min/+小鼠肠道肿瘤的作用。结果: 与Apc^Min/+小鼠相比,Apc^Min/+P-selectin^-/-杂交小鼠在9周龄时肠道肿瘤数目与总负荷明显减少。结论: P-selectin 缺失能够显著抑制Apc^Min/+小鼠肠道肿瘤的生长。