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.     

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.     

Tensin4 (TNS4) is upregulated by Wnt signalling in adenomas in multiple intestinal neoplasia (Min) mice

Apc^Min/+ mice are regarded as a standard animal model of colorectal cancer (CRC). Tensin4 (TNS4 or Cten) is a putative oncogene conferring features of stemness and promoting motility. Our objective was to assess TNS4 expression in intestinal adenomas and determine whether TNS4 is upregulated by Wnt signalling. Apc^Min/+ mice (n = 11) were sacrificed at approximately 120 days old at the onset of anaemia signs. Small intestines were harvested, and Swiss roll preparations were tested for TNS4 expression by immunohistochemistry (IHC). Individual polyps were also separately collected (n = 14) and tested for TNS4 mRNA expression and Kras mutation. The relationship between Wnt signalling and TNS4 expression was tested by Western blotting in the human CRC cell line HCT116 after inhibition of β‐catenin activity with MSAB or its increase by transfection with a Flag β‐catenin expression vector. Overall, 135/148 (91.2%) of the total intestinal polyps were positive for TNS4 expression by IHC, whilst adjacent normal areas were negative. RT‐qPCR analysis showed approximately 5‐fold upregulation of TNS4 mRNA in the polyps compared to adjacent normal tissue and no Kras mutations were detected. In HCT116, β‐catenin inhibition resulted in reduced TNS4 expression, and conversely, β‐catenin overexpression resulted in increased TNS4 expression. In conclusion, this is the first report linking aberrant Wnt signalling to upregulation of TNS4 both during initiation of intestinal adenomas in mice and in in vitro models. The exact contribution of TNS4 to adenoma development remains to be investigated, but the Apc^Min/+ mouse represents a good model to study this.     

Immunomodulatory effects of turmeric: Proliferation of spleen cells in mice

Experiments were conducted to examine the effects of turmeric on spleen cell proliferation. Cell suspensions of spleen cells from young and aged mice were treated with or without conconavalin A (Con-A) as a proliferation stimulant, and with and without turmeric (20 mg/mL) in different concentrations. Spleen cells from young mice that received turmeric showed significant increase in spleen cell proliferation (P < 0.05), while spleen cells from aged mice that received turmeric showed no significant increase in T lymphocytes. The data indicates that turmeric increases the ability of spleen cells in young mice to proliferate, in vitro.     

Motor abnormalities in weaver mutant mice

Summary Weaver mutant mice lose mainly cerebellar granule cells. Motor abnormalities included less activity in a hole-board test and a loss of balance in two equilibrium tests. It was noted that the weaver mutants also displayed paw clasping responses when held by the tail of a sort never seen in normal mice. The role of the cerebellum in these abnormalities is discussed.     

Motor abnormalities in staggerer mutant mice

Summary Staggerer mutant mice spontaneously lose both Purkinje and granule cells of the cerebellum. Motor abnormalities included less activity for an exploratory task and loss of balance in two equilibrium tests. It was observed that staggerer mutants exhibited paw clasping and limb crossing responses when held in the air by the tail, of a sort never seen in normal adult mice. The role of the cerebellum in these abnormalities is discussed.     

The development of duodenal microadenomas in FAP patients: the human correlate of the Min mouse

The morphological changes associated with the adenoma-carcinoma sequence are well documented in the colorectum. Small intestinal carcinogenesis is thought to progress through a similar adenoma-to-carcinoma pathway, but there is a relative dearth of studies examining the associated morphological changes. The best-known mouse model of intestinal neoplasia, the multiple intestinal neoplasia (Min) mouse, has been criticized as a genetic model of intestinal neoplasia, as the majority of its tumours occur in the small intestine. We examined pancreatico-duodenal resection specimens from seven familial adenomatous polyposis (FAP) patients. Serial sections of these were stained with haematoxylin and eosin for beta-catenin and its downstream target CD44, for BMPR1a, lysozyme, carbonic anhydrase II, and with MIB-1. Individual dysplastic crypts were isolated and mutations in the FAP (APC) gene compared between the top and bottom of the crypt. We found that: (a) duodenal microadenomas are extremely common in FAP patients; (b) these grow in the core of duodenal villi, forming lesions similar to those described in the Min mouse; (c) many lesions arise as monocryptal adenomas and grow by a process of crypt fission and branching; (d) migrating adenomatous cells lose their dysplastic phenotype as they migrate up the crypt villous axis; and (e) Paneth cells lose positional information. In conclusion: (a) the morphological similarity of adenomas in the Min mouse and human suggest the Min mouse is a good model of FAP; (b) duodenal adenomas in FAP originate in monocryptal adenomas and follow the 'bottom-up' rather than the 'top-down' model of morphogenesis; (c) early microadenomas show evidence of cellular differentiation; (d) defects in the positioning of Paneth cells suggests disruption of the EphB2:EphB3 receptor system.     

The amount of white pulp in the spleen; a morphometrical study done in methacrylate-embedded splenectomy specimens

This report deals with a morphometrical study on 92 surgically removed human spleens, to investigate the composition of spleens which are considered to be normal, i.e. spleens which had ruptured in traffic accidents or which had been incidentally removed during abdominal operations. A comparison was made with 16 spleens with hypersequestration of platelets and to 11 with hyper-sequestration of erythrocytes. Methyl-methacrylate embedding was used because of the superiority of this technique over conventional paraffin embedding. Significant differences were found between both 'normal' groups as to the absolute and relative amount of white pulp as well as the perifollicular red pulp zone. Based also on the few morphometrical reported studies in the literature, spleens removed during abdominal surgery form the best control group. Traumatic rupture of the spleen in traffic accidents might specifically occur in spleens which already contained a stimulated lymphatic compartment. A probably non-specific increase of white pulp was found in splenomegaly of varied aetiology. An expected influence of age on weight and composition of the spleen was not found in our study. The spleen changes in weight and composition only up to 5 years of age. Significant involution at older age was not found in ours nor in other reported larger series.     

Antimetastatic action and toxicity on healthy tissues of Na[trans-RuCl4(DMSO)Im] in the mouse

The ruthenium-dimethylsulfoxide complex Na(trans-RuCl₄(DMSO)Im] was given i.v. to mice bearing MCa mammary carcinoma and its effects on tumor growth and on healthy host tissues were studied by macroscopic examination of primary tumor growth, by survival time, and by histological analysis using light microscopy and SEM. Either by means ofvivo-vivo bioassays or by microscopic examination it appeared that the growth of lung tumors was markedly reduced, whereas the growth of the i.m. primary tumor was much less affected. These effects account for the prolongation of survival time and for the cure rate observed. The favourable effect on survival time was also influenced by the lack of significant cytotoxicity for normal tissues such as lung and kidney epithelia, muscle and liver cells, splenocytes and bone marrow. It thus appears that the selective interaction with tumor cells in the lungs cannot simply be attributed to a selectively higher localization of the compound at this site, nor to a modification of the histological structure of primary tumor. These results highlight the pharmacologic properties of this compound for the control of solid tumor metastases, an effect that was shown to be similarly exerted on advanced tumor metastases.     

多器官功能障碍综合征小鼠脾脏树突状细胞与炎症因子变化的关系

目的探讨多器官功能障碍综合征（MODS）病程中脾脏树突状细胞（DC）变化对炎症因子的影响与作用。方法采用腹腔注射酵母多糖的方法复制小鼠MODS模型，分为正常对照组和MODS组。采用流式细胞技术检测脾脏DC免疫表型，运用免疫组化技术检测脾脏中促炎因子IL-1β与抗炎因子IL-10的表达，计量观察DC与炎症因子在病程中的变化并做相关分析。结果伤后6h组，脾脏中CD11c^＋／MHC-Ⅱ’DC数目与IL-18’细胞数目均明显增多，至24h组达峰值。自48h组，CD11c^＋／MHC-Ⅱ’DC与IL-1β’细胞含量开始下降，在6天组和10～12天组降至正常组或低于正常组含量；而同时CD11c^＋／MHC-Ⅱ^-DC和IL-10阳性细胞含量则相对增多，至10～12天组阳性细胞含量显著升高达到峰值。结论DC的活性在MODS病程早期与IL-1β表达呈正相关性，在病程晚期与IL-10表达呈负相关，提示脾脏DC活性变化与促炎因子和抗炎因子均有密切的相关性。     

Nivalenol-induced apoptosis in thymus, spleen and Peyer''s patches of mice

ICR:CD-1 male mice were orally administered with Nivalenol(NIV) at the dose levels of 5, 10 and 15 mg/kg body weight, and examined at 12, 24 and 48 hours after inoculation (HAI), respectively, to elucidate the process of development of apoptosis in the thymus, spleen and Peyer's patch. There were no signs of clinical disorders and no changes in body and organ weights until 48 HAI except for that the thymus weight significantly decreased at 48 HAI. Immunohistochemically, the number of apoptotic lymphocytes evaluated by in situ detection for fragmented DNA showed a dose-dependent increase at 12 HAI in both the thymus and the Peyer's patch, while it became to increase at 24 HAI in the spleen. Dead lymphocytes in the thymus, spleen and Peyer's patch showed ultrastructural characteristics of apoptosis. Moreover, the DNA ladder was first detected by agarose gel electrophoresis at 12 HAI in the thymus of 15 mg/kg-group. The results clearly indicate that NIV is able to induce apoptosis in the lymphoid tissues of mice.     

交感神经对小鼠小肠T淋巴细胞亚型及其增殖的影响

采用化学阻断剂6-羟多巴胺(6-OHDA)损毁交感神经,研究了交感神经对小鼠小肠局部细胞免疫作用的内在机理。将30只雄性昆明小鼠随机分为2组,一组连续5d腹腔注射6-OHDA,另一组用作对照。采用免疫组织化学染色和细胞培养方法检测两组动物小肠CD4+、CD8+T淋巴细胞数量及肠系膜淋巴结T淋巴细胞体外增殖能力。结果表明,试验组的CD4+T细胞数量比对照组少6.5%～51.5%,十二指肠、空肠段差异极显著(P<0.01);CD8+T细胞数量比对照组增加5.6%～22.7%,十二指肠段差异显著(P<0.01);CD4+/CD8+T淋巴细胞的比值总体呈下降趋势。肠系膜淋巴结T淋巴细胞转化率降低,在ConA浓度为3μg/ml时比对照组降低了9.2%,差异显著(P<0.05)。以上结果提示:交感神经通过调节小鼠小肠T淋巴细胞增殖以及亚型CD4+、CD8+T淋巴细胞的数量,影响肠黏膜细胞免疫的调控。     

Hormonal and morphological study of the pituitaries in reeler mice

Reelin is a neuronal glycoprotein that plays a crucial role in brain layer formation during prenatal development. The reeler mutant mouse lacks Reelin, leading to abnormalities in the neuronal layering of cerebral cortex and cerebellum, producing ataxia, tremor and abnormal locomotion. Reeler mice are reported to have growth retardation and most of them are sterile or unable to bring up their newborns. Since the brain is one of the main regulator of pituitary hormone secretion and no information was reported regarding pituitary function and structure in these mutant mice, we studied pituitary endocrine activity and morphology in reeler mice. Mice were classified in three groups as reeler homozygote (RHM), reeler heterozygote (RHT) or control (CO). Pituitary hormone blood levels were assessed by enzyme immunoassay (EIA) and immunoradiometric assay (IRMA). Animals and their pituitaries were weighted and pituitaries were studied by histology, immunohistochemistry and electron microscopy. Results showed statistically significant differences in body weight and in adrenocorticotropic hormone (ACTH) and luteinizing hormone (LH) blood levels between the three groups. In contrast, growth hormone (GH) blood levels showed a high individual variation and no decrease in reeler groups compared with CO. Morphological studies revealed no differences in pituitary cell types except that somatotrophs appeared to be slightly smaller in RHM and RHT. Although it seems that pituitary hypofunction is not responsible for growth retardation, more studies are needed to obtain a deeper insight into the endocrine status of these mutant mice to elucidate the cause of their low body weight and reproductive behaviour.     

Telomerase activity in cells of 9-day-old spleen colonies formed by bone marrow of normal and thymectomized mice

Telomerase activity was measured in spleen colonies, in progeny of individual 9-day-old splenic CFU formed by the bone marrow from normal (physiological aging) and thymectomized mice. Cells of spleen colonies expressed telomerase activity. No correlation was found between telomerase activity in spleen colony cells and age of animals. Thymectomy of bone marrow donors had no effect on telomerase activity. Our results suggest that the thymus plays a role in cell aging.Translated from Byulleten Eksperimentalnoi Biologii i Meditsiny, Vol. 138, No. 11, pp. 567–569, November, 2004     

Cytological characteristics of lymphoid organs of pure-line mice

The relative weights, karyocyte counts, and cytological composition of the thymus, spleen, and mesenteric lymph node were determined in 101/H, CC 57W, A2G, CBA/Lac, B6W^Y, and C57BL/6 mice. Interlinear differences in the numbers of the various types of cells are evaluated.Central Scientific-Research Laboratory and Department of Pathophysiology, Tomsk Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR D. D. Yablokov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 88, No. 8, pp. 207–208, August, 1979     

Mutated K-rasAsp12 promotes tumourigenesis in ApcMin mice more in the large than the small intestines, with synergistic effects between K-ras and Wnt pathways

K‐ras mutations are found in 40–50% of human colorectal adenomas and carcinomas, but their functional contribution remains incompletely understood. Here, we show that a conditional mutant K‐ras mouse model (K‐ras^Asp12/Cre), with transient intestinal Cre activation by β‐Naphthoflavone (β‐NF) treatment, displayed transgene recombination and K‐ras^Asp12 expression in the murine intestines, but developed few intestinal adenomas over 2 years. However, when crossed with Apc^Min/+ mice, the K‐ras^Asp12/Cre/Apc^Min/+ offspring showed acceleration of intestinal tumourigenesis with significantly changed average lifespan (P < 0.05) decreased to 18.4 ± 5.4 weeks from 20.9 ± 4.7 weeks (control Apc^Min/+ mice). The numbers of adenomas in the small intestine and large intestine were significantly (P < 0.01) increased by 1.5‐fold and 5.7‐fold, respectively, in K‐ras^Asp12/Cre/Apc^Min/+ mice compared with Apc^Min/+ mice, with the more marked increase in adenoma prevalence in the large intestine. To explore possible mechanisms for K‐ras^Asp12 and Apc^Min co‐operation, the Mitogen‐activated protein kinase (Mapk), Akt and Wnt signalling pathways, including selected target gene expression levels, were evaluated in normal large intestine and large intestinal tumours. K‐ras^Asp12 increased activation of Mapk and Akt signalling pathway targets phospho‐extracellular signal‐regulated kinase (pErk) and pAkt, and increased relative expression levels of Wnt pathway targets vascular endothelial growth factor (VEGF), gastrin, cyclo‐oxygenase 2 (Cox2) and T‐cell lymphoma invasion and metastasis 1 (Tiam1) in K‐ras^Asp12/Cre/Apc^Min/+ adenomas compared with that of Apc^Min/+ adenomas, although other Wnt signalling pathway target genes such as Peroxisome proliferator‐activated receptor delta (PPARd), matrix metalloproteinase 7 (MMP7), protein phosphatase 1 alpha (PP1A) and c‐myc remained unchanged. In conclusion, intestinal expression of K‐ras^Asp12 promotes mutant Apc‐initiated intestinal adenoma formation in vivo more in the large intestine than the small intestine, with evidence of synergistic co‐operation between mutant K‐ras and Apc involving increased expression of some Wnt‐pathway target genes.