Oncogenic Actions of the Nuclear Receptor Corepressor (NCOR1) in a Mouse Model of Thyroid Cancer

Studies have suggested that the nuclear receptor corepressor 1 (NCOR1) could play an important role in human cancers. However, the detailed molecular mechanisms by which it functions in vivo to affect cancer progression are not clear. The present study elucidated the in vivo actions of NCOR1 in carcinogenesis using a mouse model (ThrbPV/PV mice) that spontaneously develops thyroid cancer. ThrbPV/PV mice harbor a dominantly negative thyroid hormone receptor b (TRb) mutant (denoted as PV). We adopted the loss-of-the function approach by crossing ThrbPV mice with mice that globally express an NCOR1 mutant protein (NCOR1DID) in which the receptor interaction domains have been modified so that it cannot interact with the TRb, or PV, in mice. Remarkably, expression of NCOR1DID protein reduced thyroid tumor growth, markedly delayed tumor progression, and prolonged survival of ThrbPV/PVNcor1DID/DID mice. Tumor cell proliferation was inhibited by increased expression of cyclin-dependent kinase inhibitor 1 (p21waf1/cip1; Cdkn1A), and apoptosis was activated by elevated expression of pro-apoptotic BCL-Associated X (Bax). Further analyses showed that p53 was recruited to the p53- binding site on the proximal promoter of the Cdkn1A and the Bax gene as a co-repressor complex with PV/NCOR1/histone deacetylas-3 (HDAC-3), leading to repression of the Cdkn1A as well as the Bax gene in thyroids of ThrbPV/PV mice. In thyroids of ThrbPV/PVNcor1DID/DID mice, the p53/PV complex could not recruit NCOR1DID and HDAC-3, leading to de-repression of both genes to inhibit cancer progression. The present studies provided direct evidence in vivo that NCOR1 could function as an oncogene via transcription regulation in a mouse model of thyroid cancer.Fil: Fozzatti, Laura. Consejo Nacional de Invest.cientif.y Tecnicas. Centro Cientifico Tecnol.conicet - Cordoba. Instituto de Inv. Medicas Mercedes y Martin Ferreyra;Fil: Park, Jeong Won.Fil: Li, Zhao.Fil: Willingham, Mark C..Fil: Cheng, Sheue-yann

Antitumor effects of B3-PE and B3-lysPE40 in a nude mouse model of human breast cancer and the evaluation of B3-PE toxicity in monkeys

B3 is a tumor-reactive monoclonal antibody (mAb) that binds to a limited number of normal tissues.Immunotoxins made with B3 coupled to either Pseudomonas exotoxin (PE) or recombinant forms of PE with a deletion of the cell-binding domain (LysPE40) have been shown to cause complete tumor regression in nude mice bearing a rapidly growing A431 (L. H. Pai et al., Proc. Natl. Acad. Sci. USA, 88: 3358-3362, 1991) human epidermoid carcinoma. In this study we show that an immunotoxin composed of mAb B3 when chemically coupled to LysPE40 (B3-LysPE40) led to complete regression of a slowly growing breast cancer, MCF-7, in nude mice when given i.v. every other day for five doses. mAb B3 coupled to native PE also produced significant regression of the MCF-7 tumor. The reactivity of mAb B3 was evaluated using an immunohistochemical method on the two responsive tumors, MCF-7 and A431, and compared with a typical human colon carcinoma specimen that has B3 antigen on its surface. The results showed that both A431 and MCF-7 xenograft tumors have similar reactivity to B3 when compared with the human colon carcinoma specimen. To evaluate the toxicity of B3-PE in primates, Cynomolgus monkeys received escalating doses of B3-PE i.v. on Days 1, 3, and 5. Based on antibody localization studies using frozen sections of normal human and monkey tissue, gastric, trachea, and bladder mucosal injury could have occurred. However, no clinical signs of injury or histological damage to these organs were seen at the doses administered. Chemical hepatitis due to PE was transient and well tolerated at doses up to 50 μg/kg for three doses. The lethal dose was about 100 μg/kg, and the cause of death was liver necrosis, as shown by necropsy. We conclude that m Ab B3, when coupled to PE40 or PE, can produce strong antitumor activity in vivo. The similar level of reactivity of the B3 antibody in our tumor models with a surgical specimen of a human colon carcinoma and the toxicity study in monkeys indicate that therapeutic doses of B3-PE and B3-LysPE40 can be delivered without causing toxicity to normal organs that express B3 antigen. Although both B3-PE and B3-LysPE40 have antitumor activity in nude mice bearing a human xenograft, B3-LysPE40 is better tolerated and should be further evaluated as a therapeutic agent for cancer patients

Novel translocation responses of cytosolic phospholipase A2α fluorescent proteins

AbstractCytosolic phospholipase A2 (cPLA2)α responds to the rise in cytosolic Ca2+ ([Ca2+]i) attending cell stimulation by moving to intracellular membranes, releasing arachidonic acid (AA) from these membranes, and thereby initiating the synthesis of various lipid mediators. Under some conditions, however, cPLA2α translocation occurs without any corresponding changes in [Ca2+]i. The signal for such responses has not been identified. Using confocal microscopy to track fluorescent proteins fused to cPLA2α or cPLA2α's C2 domain, we find that AA mimics Ca2+ ionophores in stimulating cPLA2α translocations to the perinuclear ER and to a novel site, the lipid body. Unlike the ionophores, AA acted independently of [Ca2+]i rises and did not translocate the proteins to the Golgi. AA's action did not involve its metabolism to eicosanoids or acylation into cellular lipids. Receptor agonists also stimulated translocations targeting lipid bodies. We propose that AA is a signal for Ca2+-independent cPLA2α translocation and that lipid bodies are common targets of cPLA2α and contributors to stimulus-induced lipid mediator synthesis

Determination of Point and Nonpoint Source Toxicity in the Clark Fork River Basin Using the Daphnid, Ceriodaphnia Dubia

Ceriodaphnia dubia, a small planktonic daphnid was used to biomonitor point sources of toxicity in wastewater and nonpoint source toxicity in stream samples obtained from the Clark Fork River Basin, MT. Brief descriptions, results and discussions are presented for studies of wastewater from a kraft mill near Frenchtown, MT and potential toxicity of water samples from 19 sites along the Clark Fork River in 1985. In 1987, dilutions of Missoula, MT municipal wastewater fortified with ammonia were tested, as was the wastewater before and after chlorination. Potential toxicity of water samples from eight sites along the upper Clark Fork River were also tested. All studies were cooperative efforts with the Montana Departments of Health and Environmental Sciences and Fish, Wildlife and Parks. Ceriodaphnia appear to be indicators of toxicity in a variety of test conditions such as ammonia in wastewater and metals from past mining activities. The daphnids indicated toxicity from other substances in the wastewater or perhaps the influence of characteristics of the wastewater that increased ammonia toxicity. All example of nonpoint source effects was toxicity in samples from Silver Bow Creek MT, where impaired conditions to aquatic life resulting from the presence of metals have been reported for years. During some of the tests with wastewater, toxicological endpoints were observed using the actual number of daphnids that reproduced in a test, not the average number of young. There was circumstantial evidence in 1985 that copper alone was responsible for the toxicity in Silver Bow Creek. However, the later studies performed under different hydrological conditions found toxicity was probably due to a combination of metals, some of which had not been measurable earlier. For well-defined control of standard conditions during testing, there are indications that waters to be used as reference media for Ceriodaphnia need further research. Nevertheless, the use of daphnids to test the ambient conditions described in this paper should encourage environmental managers to consider approaches with this or similar species in the future

Loss of heterozygosity and SOSTDC1 in adult and pediatric renal tumors

<p>Abstract</p> <p>Background</p> <p>Deletions within the short arm of chromosome 7 are observed in approximately 25% of adult and 10% of Wilms pediatric renal tumors. Within Wilms tumors, the region of interest has been delineated to a 2-Mb minimal region that includes ten known genes. Two of these ten candidate genes, <it>SOSTDC1 </it>and <it>MEOX2</it>, are particularly relevant to tumor development and maintenance. This finding, coupled with evidence that SOSTDC1 is frequently downregulated in adult renal cancer and regulates both Wingless-Int (Wnt)- and bone morphogenetic protein (BMP)-induced signaling, points to a role for SOSTDC1 as a potential tumor suppressor.</p> <p>Methods</p> <p>To investigate this hypothesis, we interrogated the Oncomine database to examine the SOSTDC1 levels in adult renal clear cell tumors and pediatric Wilms tumors. We then performed single nucleotide polymorphism (SNP) and sequencing analyses of <it>SOSTDC1 </it>in 25 pediatric and 36 adult renal tumors. Immunohistochemical staining of patient samples was utilized to examine the impact of <it>SOSTDC1 </it>genetic aberrations on SOSTDC1 protein levels and signaling.</p> <p>Results</p> <p>Within the Oncomine database, we found that SOSTDC1 levels were reduced in adult renal clear cell tumors and pediatric Wilms tumors. Through SNP and sequencing analyses of 25 Wilms tumors, we identified four with loss of heterozygosity (LOH) at 7p and three that affected <it>SOSTDC1</it>. Of 36 adult renal cancers, we found five with LOH at 7p, two of which affected <it>SOSTDC1</it>. Immunohistochemical analysis of SOSTDC1 protein levels within these tumors did not reveal a relationship between these instances of <it>SOSTDC1 </it>LOH and SOSTDC1 protein levels. Moreover, we could not discern any impact of these genetic alterations on Wnt signaling as measured by altered beta-catenin levels or localization.</p> <p>Conclusions</p> <p>This study shows that genetic aberrations near <it>SOSTDC1 </it>are not uncommon in renal cancer, and occur in adult as well as pediatric renal tumors. These observations of <it>SOSTDC1 </it>LOH, however, did not correspond with changes in SOSTDC1 protein levels or signaling regulation. Although our conclusions are limited by sample size, we suggest that an alternative mechanism such as epigenetic silencing of <it>SOSTDC1 </it>may be a key contributor to the reduced SOSTDC1 mRNA and protein levels observed in renal cancer.</p

The spectrum of resistance in SR/CR mice: the critical role of chemoattraction in the cancer/leukocyte interaction

<p>Abstract</p> <p>Background</p> <p>Spontaneous regression/complete resistance (SR/CR) mice are a unique colony of mice that possess an inheritable, natural cancer resistance mediated primarily by innate cellular immunity. This resistance is effective against sarcoma 180 (S180) at exceptionally high doses and these mice remain healthy.</p> <p>Methods</p> <p>In this study, we challenged SR/CR mice with additional lethal transplantable mouse cancer cell lines to determine their resistance spectrum. The ability of these transplantable cancer cell lines to induce leukocyte infiltration was quantified and the percentage of different populations of responding immune cells was determined using flow cytometry.</p> <p>Results</p> <p>In comparison to wild type (WT) mice, SR/CR mice showed significantly higher resistance to all cancer cell lines tested. However, SR/CR mice were more sensitive to MethA sarcoma (MethA), B16 melanoma (B16), LL/2 lung carcinoma (LL/2) and J774 lymphoma (J774) than to sarcoma 180 (S180) and EL-4 lymphoma (EL-4). Further mechanistic studies revealed that this lower resistance to MethA and LL/2 was due to the inability of these cancer cells to attract SR/CR leukocytes, leading to tumor cell escape from resistance mechanism. This escape mechanism was overcome by co-injection with S180, which could attract SR/CR leukocytes allowing the mice to resist higher doses of MethA and LL/2. S180-induced cell-free ascites fluid (CFAF) co-injection recapitulated the results obtained with live S180 cells, suggesting that this chemoattraction by cancer cells is mediated by diffusible molecules. We also tested for the first time whether SR/CR mice were able to resist additional cancer cell lines prior to S180 exposure. We found that SR/CR mice had an innate resistance against EL-4 and J774.</p> <p>Conclusions</p> <p>Our results suggest that the cancer resistance in SR/CR mice is based on at least two separate processes: leukocyte migration/infiltration to the site of cancer cells and recognition of common surface properties on cancer cells. The infiltration of SR/CR leukocytes was based on both the innate ability of leukocytes to respond to chemotactic signals produced by cancer cells and on whether cancer cells produced these chemotactic signals. We found that some cancer cells could escape from SR/CR resistance because they did not induce infiltration of SR/CR leukocytes. However, if infiltration of leukocytes was induced by co-injection with chemotactic factors, these same cancer cells could be effectively recognized and killed by SR/CR leukocytes.</p

Cancer resistance of SR/CR mice in the genetic knockout backgrounds of leukocyte effector mechanisms: determinations for functional requirements

<p>Abstract</p> <p>Background</p> <p>Spontaneous Regression/Complete Resistant (SR/CR) mice are a colony of cancer-resistant mice that can detect and rapidly destroy malignant cells with innate cellular immunity, predominately mediated by granulocytes. Our previous studies suggest that several effector mechanisms, such as perforin, granzymes, or complements, may be involved in the killing of cancer cells. However, none of these effector mechanisms is known as critical for granulocytes. Additionally, it is unclear which effector mechanisms are required for the cancer killing activity of specific leukocyte populations and the survival of SR/CR mice against the challenges of lethal cancer cells. We hypothesized that if any of these effector mechanisms was required for the resistance to cancer cells, its functional knockout in SR/CR mice should render them sensitive to cancer challenges. This was tested by cross breeding SR/CR mice into the individual genetic knockout backgrounds of perforin (Prf^-/-), superoxide (Cybb^-/), or inducible nitric oxide (Nos2^-/).</p> <p>Methods</p> <p>SR/CR mice were bred into individual Prf^-/-, Cybb^-/-, or Nos2^-/-genetic backgrounds and then challenged with sarcoma 180 (S180). Their overall survival was compared to controls. The cancer killing efficiency of purified populations of macrophages and neutrophils from these immunodeficient mice was also examined.</p> <p>Results</p> <p>When these genetically engineered mice were challenged with cancer cells, the knockout backgrounds of Prf^-/-, Cybb^-/-, or Nos2^-/-did not completely abolish the SR/CR cancer resistant phenotype. However, the Nos2^-/-background did appear to weaken the resistance. Incidentally, it was also observed that the male mice in these immunocompromised backgrounds tended to be less cancer-resistant than SR/CR controls.</p> <p>Conclusion</p> <p>Despite the previously known roles of perforin, superoxide or nitric oxide in the effector mechanisms of innate immune responses, these effector mechanisms were not required for cancer-resistance in SR/CR mice. The resistance was functional when any one of these effector mechanisms was completely absent, except some noticeably reduced penetrance, but not abolishment, of the phenotype in the male background in comparison to female background. These results also indicate that some other effector mechanism(s) of granulocytes may be involved in the killing of cancer cells in SR/CR mice.</p

Deweyan tools for inquiry and the epistemological context of critical pedagogy

This article develops the notion of resistance as articulated in the literature of critical pedagogy as being both culturally sponsored and cognitively manifested. To do so, the authors draw upon John Dewey\u27s conception of tools for inquiry. Dewey provides a way to conceptualize student resistance not as a form of willful disputation, but instead as a function of socialization into cultural models of thought that actively truncate inquiry. In other words, resistance can be construed as the cognitive and emotive dimensions of the ongoing failure of institutions to provide ideas that help individuals both recognize social problems and imagine possible solutions. Focusing on Dewey\u27s epistemological framework, specifically tools for inquiry, provides a way to grasp this problem. It also affords some innovative solutions; for instance, it helps conceive of possible links between the regular curriculum and the study of specific social justice issues, a relationship that is often under-examined. The aims of critical pedagogy depend upon students developing dexterity with the conceptual tools they use to make meaning of the evidence they confront; these are background skills that the regular curriculum can be made to serve even outside social justice-focused curricula. Furthermore, the article concludes that because such inquiry involves the exploration and potential revision of students\u27 world-ordering beliefs, developing flexibility in how one thinks may be better achieved within academic subjects and topics that are not so intimately connected to students\u27 current social lives, especially where students may be directly implicated

Impact of sex, MHC, and age of recipients on the therapeutic effect of transferred leukocytes from cancer-resistant SR/CR mice

<p>Abstract</p> <p>Background</p> <p>Spontaneous Regression/Complete Resistant (SR/CR) mice are resistant to cancer through a mechanism that is mediated entirely by leukocytes of innate immunity. Transfer of leukocytes from SR/CR mice can confer cancer resistance in wild-type (WT) recipients in both preventative and therapeutic settings. In the current studies, we investigated factors that may impact the efficacy and functionality of SR/CR donor leukocytes in recipients.</p> <p>Results</p> <p>In sex-mismatched transfers, functionality of female donor leukocytes was not affected in male recipients. In contrast, male donor leukocytes were greatly affected in the female recipients. In MHC-mismatches, recipients of different MHC backgrounds, or mice of different strains, showed a greater negative impact on donor leukocytes than sex-mismatches. The negative effects of sex-mismatch and MHC-mismatch on donor leukocytes were additive. Old donor leukocytes performed worse than young donor leukocytes in all settings including in young recipients. Young recipients were not able to revive the declining function of old donor leukocytes. However, the function of young donor leukocytes declined gradually in old recipients, suggesting that an aged environment may contain factors that are deleterious to cellular functions. The irradiation of donor leukocytes prior to transfers had a profound suppressive effect on donor leukocyte functions, possibly as a result of impaired transcription. The cryopreserving of donor leukocytes in liquid nitrogen had no apparent effect on donor leukocyte functions, except for a small loss of cell number after revival from freezing.</p> <p>Conclusion</p> <p>Despite the functional suppression of donor leukocytes in sex- and MHC-mismatched recipients, as well as old recipients, there was a therapeutic time period during the initial few weeks during which donor leukocytes were functional before their eventual rejection or functional decline. The eventual rejection of donor leukocytes will likely prevent donor leukocyte engraftment which would help minimize the risk of transfusion-associated graft-versus-host disease. Therefore, using leukocytes from healthy donors with high anti-cancer activity may be a feasible therapeutic concept for treating malignant diseases.</p