The development of Wilms tumor: From WT1 and microRNA to animal models

Wilms tumor recapitulates the development of the kidney and represents a unique opportunity to understand the relationship between normal and tumor development. This has been illustrated by the findings that mutations of Wnt/β-catenin pathway-related WT1, β-catenin, and WTX together account for about one-third of Wilms tumor cases. While intense efforts are being made to explore the genetic basis of the other two-thirds of tumor cases, it is worth noting that, epigenetic changes, particularly the loss of imprinting of the DNA region encoding the major fetal growth factor IGF₂, which results in its biallelic over-expression, are closely associated with the development of many Wilms tumors. Recent investigations also revealed that mutations of Drosha and Dicer, the RNases required for miRNA generation, and Dis3L2, the 3′–5′ exonuclease that normally degrades miRNAs and mRNAs, could cause predisposition to Wilms tumors, demonstrating that miRNA can play a pivotal role in Wilms tumor development. Interestingly, Lin28, a direct target of miRNA let-7 and potent regulator of stem cell self-renewal and differentiation, is significantly elevated in some Wilms tumors, and enforced expression of Lin28 during kidney development could induce Wilms tumor. With the success in establishing mice nephroblastoma models through over-expressing IGF₂ and deleting WT1, and advances in understanding the ENU-induced rat model, we are now able to explore the molecular and cellular mechanisms induced by these genetic, epigenetic, and miRNA alterations in animal models to understand the development of Wilms tumor. These animal models may also serve as valuable systems to assess new treatment targets and strategies for Wilms tumor.     

Role of Wilms tumor 1 (WT1) in the transcriptional regulation of the Mullerian-inhibiting substance promoter

The Wilms tumor 1 (WT1) gene product may regulate the mullerian-inhibiting substance (MIS) gene, because mutations in WT1 can cause persistence of the mullerian duct in men. In the present study, we show by gel shift and chromatin immunoprecipitation assays that WT1 bound to a GC-rich sequence in the murine Mis promoter. Mutation in this site abolished WT1-mediated activation of the Mis promoter. The WT1, SRY box protein 9, and steroidogenic factor 1 could synergistically activate the Mis promoter, and at least two factors were necessary for minimal activation. The WT1 is an essential factor for activation of the Mis promoter; therefore, the persistence of the mullerian duct in patients with Denys-Drash syndrome may result from deregulation of the MIS gene.     

Urotensin-II is a nitric oxide-dependent vasodilator in the pial arteries of the newborn pig

Urotensin-II (UT-II) is a small circular peptide and is described as the most potent endogenous vasoconstrictor in various vascular beds. However, the in vivo effects of UT-II can be either vasoconstriction or vasodilation depending on the species and the tissue investigated. The present study sought to characterize the vasoactive effect of UT-II in the piglet cerebral circulation in vivo. Pial arteries of 99 +/- 6 microm were visualized with intravital microscopy through a closed cranial window in anesthetized newborn piglets. Topical application of UT-II elicited a weak dose-dependent vasodilation of the arteries (0.001 microM: 3 +/- 3 microm, 0.1 microM: 10 +/- 5 microm, 10 microM: 14 +/- 7 microm). Smaller arteries with an initial diameter below 100 microm showed minimal or no vasodilation, while larger arteries between 100 and 120 microm had a pronounced dose-dependent effect. Systemic application of 15 mg/kg Nomega-nitro-L-arginine-methyl ester (L-NAME) completely inhibited the vasodilation. We conclude that UT-II, in contrast to most other vascular beds, is a weak NO-dependent vasodilator in the piglet pial vasculature.     

Nitric oxide inhibits spleen cell proliferative response after burn injury by inducing cytostasis,apoptosis, and necrosis of activated T lymphocytes: role of the guanylate cyclase

We previously showed that an overproduction of nitric oxide (NO) by macrophages was responsible for the collapse of lymphoproliferative responses after burn injury in rats. First, we demonstrate here that 10 days post-burn, the inhibition of splenocyte response to concanavalin-A results from cytostatic, apoptotic, and necrotic effects of NO on activated T cells. This was evidenced by various criteria at the levels of DNA, mitochondria, and plasma membrane. Inhibition of NO synthase by S-methylisothiourea (10 microM) normalized all the parameters. Second, we show that two soluble guanylate cyclase (sGC) inhibitors, LY83583 and ODQ, restored the proliferative response in a concentration-dependent manner. LY83583 (0.5 microM) rescued T cells from apoptosis. Similar results were obtained with KT5823 (5 microM) a specific inhibitor of protein kinase G (PKG). In contrast, neither LY83583 nor KT5823 inhibited NO-induced necrosis. These results suggest that NO blocked T cells in the G1 phase and induced apoptosis through a sGC-PKG-dependent pathway and necrosis through an independent one.     

A mathematical analysis of the interactions between immunogenic tumor cells and cytotoxic T lymphocytes.

Recent developments of biotechnology have enabled us to use immunotherapy against certain kinds of tumors in patients. However, it is reasonable to doubt if the immunotherapy can completely aid the rejection of tumors that have escaped from the immune system. In this paper, we propose a new mathematical model of tumor immunity by tumor-specific cytotoxic T lymphocytes (CTLs), since tumor-specific CTLs play an important role in tumor immunity. Using this model, we have mathematically investigated the interactions between immunogenic tumor cells (TCs) and tumor-specific CTLs and evaluated the availability of immunotherapies for tumors. The findings herein demonstrate that three kinds of dynamics of tumor immunity exist: i.e. (1) TCs continue to proliferate with CTLs; (2) TCs are rejected by CTLs; and (3) TCs equilibrate with CTLs, but with little possibility of the equilibrium. The findings also demonstrate that a sufficient increase in CTLs by immunotherapy can aid the rejection of TCs, but an insufficient increase in CTLs by immunotherapy causes only a transient regression of TCs. Clinically the findings mean that increasing tumor-specific CTLs, e.g., by vaccination or adoptive transfer of tumor-specific CTLs expanded ex vivo, can theoretically aid the rejection of TCs.     

Nitric oxide inhibits proliferation but increases life-span of T lymphocytes in tumour-bearing rats

 Nitric oxide (NO) has been shown to inhibit the proliferation of lymphocytes. However, in tumour-bearing rats treated with the immunomodulator OM 163, the regressing nodules were heavily infiltrated by T lymphocytes, although they contained high levels of NO. We show here that NO, while inhibiting the proliferation of lymphocytes, increased their life-span, pointing to the ambivalence of this molecule in the course of tumour growth and regression. Received: 16 October 1997 / Accepted: 8 January 1998     

Assessment of the number of local cytotoxic T lymphocytes required for degradation of micrometer-size tumor spheroids

Adoptive immunotherapy with human cytotoxic T lymphocytes (CTL) is a promising cancer treatment. Previously we showed that human CTLs against various types of tumors can be efficiently produced by coculturing peripheral blood cells with target cells. The aims of this study were to simulate the interaction of CTLs and micrometer-size tumor tissues in vitro and to assess the required number of CTLs at local tumor sites for degradation of a tumor. Allogeneic CTLs against a human transitional cell carcinoma cell line and autologous CTLs against a renal cell carcinoma cell derived from a surgical specimen were generated. The cytotoxic activities of CTLs against tumor cells in monolayer culture and tumor spheroids formed in U-bottom 96-well culture plates were assessed. Both allogeneic and autologous CTLs showed greater destructive activity than lymphokine activated killer (LAK) cells against target tumor spheroids. CTLs inoculated at E/T ratios of 0.1 to 1 coexisted with the tumor spheroid for 5 to 6 days and then increased in number with apparently lethal activity against the tumor spheroid. In contrast to CTLs, the increase in LAK cell numbers was scarcely observed, and the proliferated LAK cells did not show cytotoxicity against the tumor spheroid. These observations suggest that, when a small number of CTLs reach a local tumor site, they can destroy micrometer-size tumors after considerable local proliferation. This revised version was published online in August 2006 with corrections to the Cover Date.     

Seasonality in expression and distribution of nitric oxide synthase isoforms in the testis of the catfish, Clarias batrachus: Role of nitric oxide in testosterone production

Nitric oxide (NO) is a well-recognized versatile signaling molecule. It is produced by catalytic action of nitric oxide synthase (NOS) on L-arginine in a variety of animal tissues. Existence of different isoforms of NOS has been shown in mammalian testis, but report on their presence in the testis of ectothermic vertebrates is non-existent. This study demonstrates the differential expressions of two isoforms of nitric oxide synthase (neuronal-nNOS and inducible-iNOS) like molecules in different cell types in the testis of seasonally breeding catfish, Clarias batrachus through immunohistochemistry. Positive immunoprecipitation of nNOS and iNOS like molecules were detected in germ cells as well as interstitial cells only in the recrudescing and fully mature fish. The immunoreactions differed in intensity and varied with changing reproductive status. Treatment of adult male fish with NO donor, sodium nitroprusside, and a NOS inhibitor, N-nitro-L-arginine methyl ester (L-NAME) increased and decreased the total nitrate and nitrite concentration in the testis, respectively. Sodium nitroprusside and L-NAME also induced simultaneous decline and rise in the testicular testosterone level, respectively. These findings, thus, suggest that NOS isoforms are expressed variedly in different cell types in the testis of reproductively active fish. This investigation also suggests that NO inhibits testosterone production in the testis.     

Defective nitric oxide-dependent, deaminative cleavage of glypican-1 heparan sulfate in Niemann-Pick C1 fibroblasts

Exit of recycling cholesterol from late endosomes is defective in Niemann-Pick C1 (NPC1) and Niemann-Pick C2 (NPC2) diseases. The traffic route of the recycling proteoglycan glypican-1 (Gpc-1) may also involve late endosomes and could thus be affected in these diseases. During recycling through intracellular compartments, the heparan sulfate (HS) side chains of Gpc-1 are deaminatively degraded by nitric oxide (NO) derived from preformed S-nitroso groups in the core protein. We have now investigated whether this NO-dependent Gpc-1 autoprocessing is active in fibroblasts from NPC1 disease. The results showed that Gpc-1 autoprocessing was defective in these cells and, furthermore, greatly depressed in normal fibroblasts treated with U18666A (3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one), a compound widely used to induce cholesterol accumulation. In both cases, autoprocessing was partially restored by treatment with ascorbate which induced NO release, resulting in deaminative cleavage of HS. However, when NO-dependent Gpc-1 autoprocessing is depressed and heparanase-catalyzed degradation of HS remains active, a truncated Gpc-1 with shorter HS chains would prevail, resulting in fewer NO-sensitive sites/proteoglycan. Therefore, addition of ascorbate to cells with depressed autoprocessing resulted in nitration of tyrosines. Nitration was diminished when heparanase was inhibited with suramin or when Gpc-1 expression was silenced by RNAi. Gpc-1 misprocessing in NPC1 cells could thus contribute to neurodegeneration mediated by reactive nitrogen species.     

豆腐柴根提取物对小鼠T淋巴细胞增殖反应的影响

利用3H-TdR放射性来测定T淋巴细胞增殖强度的方法,研究了豆腐柴根提取物对小鼠T淋巴细胞增殖反应的影响.结果表明,豆腐柴根提取物在一定浓度范围内可促进刀豆蛋白(ConA)诱导T淋巴细胞发生增殖反应,其中2μg.mL-1提取物浓度效果最为明显.该研究为合理开发利用豆腐柴这一野生药物资源提供了科学的实验依据.     

The role of nitric oxide in reducing deformability of Lewis lung tumor cell stimulated by inflammatory cytokines

Highly metastatic cells, especially in the lungs, are known to be resistant to nitric oxide (NO)-mediated cytotoxicity, compared with poorly or non-metastatic cells. However, the precise mechanisms connecting NO and metastasis remain to be determined. To clarify the role of NO in the characteristic changes in NO-resistant cells in response to inflammatory cytokines, we used Lewis lung tumor (LLT) cells, which are known to be highly metastatic NO-resistant cells, and determined the changes in cell deformability and the gene expression profile after the cells were stimulated using cytokine mixture or an NO donor. Both exogenous NO and endogenous NO via inducible NO synthase produced by cytokines decreased cell deformability by enhancing actin polymerization. The expression of several genes associated with actin polymerization was changed so as to increase actin filaments in the cells by enhancing actin polymerization and by suppressing actin depolymerization, actin filament severing, and barbed-end actin filament capping. In conclusion, inflammatory cytokine stimulation reduces deformability of LLT cells and enhances actin polymerization which is mainly controlled by the same genes induced by NO.     

The role of nitrite in nitric oxide homeostasis: A comparative perspective

Nitrite is endogenously produced as an oxidative metabolite of nitric oxide, but it also functions as a NO donor that can be activated by a number of cellular proteins under hypoxic conditions. This article discusses the physiological role of nitrite and nitrite-derived NO in blood flow regulation and cytoprotection from a comparative viewpoint, with focus on mammals and fish. Constitutive nitric oxide synthase activity results in similar plasma nitrite levels in mammals and fish, but nitrite can also be taken up across the gills in freshwater fish, which has implications for nitrite/NO levels and nitrite utilization in hypoxia. The nitrite reductase activity of deoxyhemoglobin is a major mechanism of NO generation from nitrite and may be involved in hypoxic vasodilation. Nitrite is readily transported across the erythrocyte membrane, and the transport is enhanced at low O₂ saturation in some species. Also, nitrite preferentially reacts with deoxyhemoglobin rather than oxyhemoglobin at intermediate O₂ saturations. The hemoglobin nitrite reductase activity depends on heme O₂ affinity and redox potential and shows species differences within mammals and fish. The NO forming capacity is elevated in hypoxia-tolerant species. Nitrite-induced vasodilation is well documented, and many studies support a role of erythrocyte/hemoglobin-derived NO. Vasodilation can, however, also originate from nitrite reduction within the vessel wall, and at present there is no consensus regarding the relative importance of competing mechanisms. Nitrite reduction to NO provides cytoprotection in tissues during ischemia-reperfusion events by inhibiting mitochondrial respiration and limiting reactive oxygen species. It is argued that the study of hypoxia-tolerant lower vertebrates and diving mammals may help evaluate mechanisms and a full understanding of the physiological role of nitrite.     

Copper modulates activities of genistein, nitric oxide, and curcumin in breast tumor cells

Several papers have reported that low level of genistein (<8 microM), the major bioactive component of isoflavones, stimulates the growth of MCF-7 cells. In the present study, we found that genistein-induced growth stimulation of MCF-7 cells is inhibited in the presence of Cu(2+) (5 microM). Genistein induces the release of nitric oxide in MCF-7 cells in a concentration-dependent manner. The release of nitric oxide was inhibited by N(G)-nitro-L-arginine methyl ester, suggesting the possibility of the activation of nitric oxide synthase. The growth of MCF-7 cells also increases in the presence of low levels of sodium nitriprusside (<10 microM), a nitric oxide donor compound, while high levels (>25 microM) are toxic. The sodium nitroprusside-induced growth of MCF-7 cells is drastically suppressed in the presence of Cu(2+) (5 microM). This parallel behavior between Cu(2+)-genistein and Cu(2+)-sodium nitroprusside mixtures suggests that Cu(2+) and/or copper-protein complexes, that may be formed in the media, may be reacting with nitric oxide or nitric oxide-derived reactive species. The products of these reactions may be responsible for the toxic effects of these mixtures. In contrast, the effect of curcumin that inhibits the growth of both estrogen receptor-positive and -negative breast tumor cells appreciably decreased in the presence of Cu(2+). Since copper is known to overwhelmingly bind with proteins, present data suggest that an increase in copper-protein moieties or complexes formed in the serum containing media and their reactions with nitric oxide may be responsible for their toxic effects. Further studies are needed to characterize these reactions.     

Effects of spontaneous or induced brain ischemia on vessel reactivity: the role of inducible nitric oxide synthase

Short episodes of ischemia and reperfusion in various organs may protect the organ itself, and the heart both as an immediate and a delayed effect. The present study investigates whether a systemic protection of vascular function occurs during adaption to ischemia. Brain ischemia was induced by bilateral ligation of the internal carotid arteries in C57BL6 mice, and 24-36 hours later rings of the thoracic aorta were mounted to study in vitro relaxation and contraction, or proteins were extracted for immunoblotting for endothelial nitric oxide synthase (eNOS) or inducible NOS (iNOS). eNOS decreased, while iNOS increased in the aortic wall after carotid artery ligation. In vitro contraction to increasing concentrations of prostaglandin F(2alpha) (PGF(2alpha)) was attenuated, while relaxation to acetylcholine (ACh) was enhanced. The latter was abolished by the iNOS-inhibitor aminoguanidine. When brain ischemia was induced in iNOS deficient mice, an increase of aortic eNOS was found 24 hours later. The ischemia-induced attenuated relaxation to PGF(2alpha) and enhanced relaxation to ACh were abolished. Aortic rings from mice with severe atherosclerosis (apolipoprotein E and low density lipoprotein receptor double knockout (ApoE/LDLr KO) mice) and spontaneous ischemic events in the heart or brain in vivo were also studied. Spontaneous ischemic events in ApoE/LDLr KO animals did not influence iNOS and eNOS in the vessel wall. A reduced contraction to PGF(2alpha) was observed, but relaxation to ACh was unchanged. These findings suggest that induced brain ischemia as a model of delayed, remote preconditioning protects vessel reactivity, and this protection is mediated by iNOS.     

High levels of endogenous nitric oxide produced after burn injury in rats arrest activated T lymphocytes in the first G1 phase of the cell cycle and then induce their apoptosis

Major physical traumas provoke a systemic inflammatory response and immune dysfunction. In a model of thermal injury in rats, we previously showed that an overproduction of nitric oxide (NO) was responsible for the collapse of lymphoproliferative responses. In the present work, we performed a time-course analysis of cell proliferation and cell death parameters in order to establish the sequence of events triggered by the high NO output in Wistar/Han rat splenocytes activated with Con A, 10 days after burn injury. We demonstrate that activated T cells from burned rats never divided whereas normal T cells underwent four division cycles. However, T cells from both burned and normal rat entered the G1 phase as shown by increase of cell size, mitochondria hyperpolarization, and expression of cyclin D1. Burned rat T cells progressed to the late G1 phase as shown by expression of the nuclear Ki-67 antigen, but they never entered the S phase. They underwent apoptosis as shown by morphological parameters, disruption of transmembrane mitochondrial potential, and DNA fragmentation. Persistent accumulation of the p53 protein accompanied these phenomena. NO synthase inhibitors antagonize alterations of cell proliferation and cell death parameters in burned rat T cells and accelerated p53 turnover.     

Acute glucose overload potentiates nitric oxide production in lipopolysaccharide-stimulated macrophages: the role of purinergic receptor activation

The positive effects of high glucose on the cellular productivity of nitric oxide (NO), and the mechanisms of the enhancement, were investigated. Macrophages were shifted from normal-glucose medium (5.5 mM) to high-glucose medium (25 mM) and immediately treated with lipopolysaccharide (LPS). Inducible nitric oxide synthase (iNOS) expression was expressed significantly more quickly, and NO production also increased. High-glucose conditions reduced cell viability at 48 h. Pretreatment with oxidized adenosine triphosphate (o-ATP), the selective purinergic receptor antagonist, strongly reduced LPS-induced iNOS expression, NO production and cell death in cells exposed to high levels of glucose. Apyrase, an ATP-hydrolyzing enzyme, also reduced the effects of high-glucose content. High-glucose content promoted the LPS-induced release of endogenous ATP from RAW 264.7 cells, as measured by luciferin-luciferase assay. In summary, the results revealed that purinergic receptor is important in responding to LPS challenge, increasing LPS-induced NO production and cell death under high-glucose conditions, and promoting the release of ATP from macrophages in high-glucose medium.     

The expression of CCL2 by T lymphocytes of mammary tumor bearers: role of tumor-derived factors

Tumor-associated chemokines, including CC chemokine ligand 2/monocyte chemoattractant protein-1 (CCL2), are thought to play many roles in cancer progression. Here we demonstrate the novel finding that during growth of the D1-7,12-dimethylbenzanthracene-3 mammary tumor in BALB/c mice, there is a dramatic up-regulation of CCL2 in splenic T cells at both the mRNA and protein levels upon stimulation. Of particular relevance is the finding that tumor-infiltrating T cells also produce high levels of CCL2. While a variety of tumor cell lines have been found to produce CCL2, we found no detectable levels of CCL2 protein in supernatants of the cultured mammary tumor cells. Investigation of the mechanisms involved in CCL2 induction showed that treatment of splenic T cells with the tumor-derived factors GM-CSF and phosphatidyl serine (PS) resulted in increased CCL2 production. This increased production may be involved in the downregulation of IFN-gamma by the T cells of tumor-bearing mice previously reported in this model, as treatment of splenic T lymphocytes with CCL2 resulted in a decreased secretion of IFN-gamma by those cells.     

Distribution of intracardiac neurones and nerve terminals that contain a marker for nitric oxide,NADPH-diaphorase,in the guinea-pig heart

There is strong evidence that NADPH-diaphorase can be used as a marker for neurones that employ nitric oxide as a messenger molecule. In the present study, the NADPH-diaphorase activity of intracardiac neurones and nerve terminals in whole-mount stretch preparations and sections of the newborn and adult guinea-pig atria and interatrial septum has been examined histochemically. Together with epicardial, endothelial and endocardial cells, which displayed some NADPH-diaphorase staining, a subpopulation of intracardiac neurones exhibited moderate-heavy labelling for NADPH-diaphorase, while the majority of neurones were only lightly stained or negative. Intracardiac ganglia containing positive neuronal cell bodies were located between the epicardial cells and atrial myocytes in four main regions: in association with the superior and inferior vena cavae, the points of entry of the pulmonary veins, and within the interatrial septum. Nerve terminals exhibiting NADPH-diaphorase activity were seen throughout the atrial tissue, forming basket-like endings around intracardiac neuronal cell bodies; varicose terminals were also observed on atrial myocytes and other non-neuronal structures. A proportion of the nerve fibres was clearly of intrinsic origin, other terminals may well have originated from neuronal cell bodies present outside the heart.