Kathleen M. Brundage

Developmental Immunotoxicity of Atrazine in Rodents

There is a substantial literature reporting that the developing immune system is more sensitive to toxic insult and that the measurable phenotype resulting from prenatal/neonatal exposure often differs from that seen in adult exposure models (reviewed in Holladay and Steven, and Smialowicz et al.). Atrazine is a common herbicidal contaminant of groundwater in agricultural areas in the USA. The potential immunotoxicity of atrazine has been extensively studied using adult-exposure models; however, few studies have explored its immunotoxicity in a prenatal and/or lactational exposure model. Prenatal/lactational atrazine exposure affects the function of young adult rodent immune systems in both sex- and age-dependant manners. In our studies, the humoural and cell-mediated immune responses of offspring from atrazine-exposed dams were assessed at two ages, 3 and 6 months of age to test the hypothesis that prenatal/lactational atrazine exposure would cause greater health complications as the mice aged. Male offspring showed a significant immunopotentiation at three moa that was not apparent at 6 months. Three-month-old female offspring showed no significant difference in immune response from controls. However, at 6 months, female litter mates showed a significant depression in their immune function. These results indicate a decreasing trend in immune capacity. Rooney et al. showed a significant depression of the immune function of young male rat exposure prenatally and lactationally to atrazine. These results demonstrate a sex- and age-dependant effect of prenatal exposure to atrazine on the immune system of the adult offspring using two rodent strains.

SLUG is required for SOX9 stabilization and functions to promote cancer stem cells and metastasis in human lung carcinoma

Cancer stem cells (CSCs) are a promising target for cancer therapy, particularly for metastatic lung cancers, but how CSCs are regulated is largely unknown. We identify two proteins, SLUG (encoded by SNAI2 gene) and SOX9, which are associated with advanced stage lung cancers and are implicated in the regulation of CSCs. Inhibition of either SLUG or SOX9 sufficiently inhibits CSCs in human lung cancer cells and attenuates experimental lung metastasis in a xenograft mouse model. Correlation between SLUG and SOX9 levels was observed remarkably, we therefore sought to explore their mechanistic relationship and regulation. SLUG, beyond its known function as an epithelial–mesenchymal transition transcription factor, was found to regulate SOX9 by controlling its stability via a post-translational modification process. SLUG interacts directly with SOX9 and prevents it from ubiquitin-mediated proteasomal degradation. SLUG expression and binding are necessary for SOX9 promotion of lung CSCs and metastasis in a mouse model. Together, our findings provide a novel mechanistic insight into the regulation of CSCs via SLUG-SOX9 regulatory axis, which represents a potential novel target for CSC therapy that may overcome cancer chemoresistance and relapse.

Prenatal cadmium exposure alters postnatal immune cell development and function.

Cadmium (Cd) is generally found in low concentrations in the environment due to its widespread and continual use, however, its concentration in some foods and cigarette smoke is high. Although evidence demonstrates that adult exposure to Cd causes changes in the immune system, there are limited reports of immunomodulatory effects of prenatal exposure to Cd. This study was designed to investigate the effects of prenatal exposure to Cd on the immune system of the offspring. Pregnant C57Bl/6 mice were exposed to an environmentally relevant dose of CdCl(2) (10ppm) and the effects on the immune system of the offspring were assessed at two time points following birth (2 and 7weeks of age). Thymocyte and splenocyte phenotypes were analyzed by flow cytometry. Prenatal Cd exposure did not affect thymocyte populations at 2 and 7weeks of age. In the spleen, the only significant effect on phenotype was a decrease in the number of macrophages in male offspring at both time points. Analysis of cytokine production by stimulated splenocytes demonstrated that prenatal Cd exposure decreased IL-2 and IL-4 production by cells from female offspring at 2weeks of age. At 7weeks of age, splenocyte IL-2 production was decreased in Cd-exposed males while IFN-γ production was decreased from both male and female Cd-exposed offspring. The ability of the Cd-exposed offspring to respond to immunization with a S. pneumoniae vaccine expressing T-dependent and T-independent streptococcal antigens showed marked increases in the levels of both T-dependent and T-independent serum antibody levels compared to control animals. CD4(+)FoxP3(+)CD25(+) (nTreg) cell percentages were increased in the spleen and thymus in all Cd-exposed offspring except in the female spleen where a decrease was seen. CD8(+)CD223(+) T cells were markedly decreased in the spleens in all offspring at 7weeks of age. These findings suggest that even very low levels of Cd exposure during gestation can result in long term detrimental effects on the immune system of the offspring and these effects are to some extent sex-specific.

Prenatal cadmium exposure dysregulates sonic hedgehog and Wnt/β-catenin signaling in the thymus resulting in altered thymocyte development

Cadmium (Cd) is both an environmental pollutant and a component of cigarette smoke. Although evidence demonstrates that adult exposure to Cd causes changes in the immune system, there are limited reports in the literature of immunomodulatory effects of prenatal exposure to Cd. The sonic hedgehog (Shh) and Wnt/beta-catenin pathways are required for thymocyte maturation. Several studies have demonstrated that Cd exposure affects these pathways in different organ systems. This study was designed to investigate the effect of prenatal Cd exposure on thymocyte development, and to determine if these effects were linked to dysregulation of Shh and Wnt/beta-catenin pathways. Pregnant C57Bl/6 mice were exposed to an environmentally relevant dose (10 ppm) of Cd throughout pregnancy and effects on the thymus were assessed on the day of birth. Thymocyte phenotype was determined by flow cytometry. A Gli:luciferase reporter cell line was used to measure Shh signaling. Transcription of target genes and translation of key components of both signaling pathways were assessed using real-time RT-PCR and western blot, respectively. Prenatal Cd exposure increased the number of CD4(+) cells and a subpopulation of double-negative cells (DN; CD4(-)CD8(-)), DN4 (CD44(-)CD25(-)). Shh and Wnt/beta-catenin signaling were both decreased in the thymus. Target genes of Shh (Patched1 and Gli1) and Wnt/beta-catenin (c-fos, and c-myc) were affected differentially among thymocyte subpopulations. These findings suggest that prenatal exposure to Cd dysregulates two signaling pathways in the thymus, resulting in altered thymocyte development.

A Review of the Immunotoxicity of the Pesticide 3,4-Dichloropropionanalide

The pesticide 3,4-dichloropropionanilide (propanil or, alternatively, DCPA) is a member of the acetanilide chemical family and is predominantly used for the control of weeds on commercial rice crops worldwide. This article was written to provide a brief review of the general toxicity of propanil followed by a detailed summary of the immunotoxicity studies that were performed to date in mammalian in vivo and in vitro models. Propanil affects the immune system at organ, cellular, and molecular levels. Studies demonstrated that it produces thymic atrophy and splenomegaly and decreases developing T- and B-cell populations in the thymus and bone marrow. Natural killer (NK) cells and macrophages are critical components of the innate immune system. NK cell cytotoxicity and the ability of macrophages to phagocytose, kill pathogenic bacteria, and produce inflammatory cytokines are suppressed by propanil. Propanil also affects the respiratory burst of macrophages, inhibiting reactive oxygen and nitrogen species production. Molecular mechanisms responsible for propanils effects have begun to be elucidated and include alterations in nuclear factor (NF)-κB transcription factor activity and intracellular Ca2+ signaling. Propanil exposure alters a number of functions of mature T lymphocytes and B lymphocytes that impacts the adaptive immune response. T-cell cytotoxic activity and cytokine production are major T-cell functions inhibited by propanil. The humoral antibody response to model antigens and intact bacteria is differentially affected after propanil exposure. How these changes in innate and adaptive immune responses impact the host response to bacterial challenge or vaccination has begun to be examined.

MicroRNAs 206 and 21 Cooperate To Promote RAS-Extracellular Signal-Regulated Kinase Signaling by Suppressing the Translation of RASA1 and SPRED1

ABSTRACT Despite the low prevalence of activating point mutation of RAS or RAF genes, the RAS–extracellular signal-regulated kinase (ERK) pathway is implicated in breast cancer pathogenesis. Indeed, in triple-negative breast cancer (TNBC), there is recurrent genetic alteration of pathway components. Using short hairpin RNA (shRNA) methods, we observed that the zinc finger transcription factor Krüppel-like factor 4 (KLF4) can promote RAS-ERK signaling in TNBC cells. Endogenous KLF4 bound to the promoter regions and promoted the expression of two microRNAs (miRs), miR-206 and miR-21 (i.e., miR-206/21). Antisense-mediated knockdown (anti-miR) revealed that miR-206/21 coordinately promote RAS-ERK signaling and the corresponding cell phenotypes by inhibiting translation of the pathway suppressors RASA1 and SPRED1. In TNBC cells, including cells with mutation of RAS, the suppression of either RASA1 or SPRED1 increased the levels of GTP-bound, wild-type RAS and activated ERK 1/2. Unlike the control cells, treatment of RASA1- or SPRED1-suppressed cells with anti-miR-206/21 had little or no impact on the level of activated ERK 1/2 or on cell proliferation and failed to suppress tumor initiation. These results identify RASA1 and SPRED1 mRNAs as latent RAS-ERK pathway suppressors that can be upregulated in tumor cells by anti-miR treatment. Consequently, KLF4-regulated miRs are important for the maintenance of RAS-ERK pathway activity in TNBC cells.

Sibling Donor and Recipient Immune Modulation With Atorvastatin for the Prophylaxis of Acute Graft-Versus-Host Disease

PURPOSE Graft-versus-host disease (GVHD) is major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT). Atorvastatin is a potent immunomodulatory agent that holds promise as a novel and safe agent for acute GVHD prophylaxis. PATIENTS AND METHODS We conducted a phase II trial to evaluate the safety and efficacy of atorvastatin administration for GVHD prophylaxis in both adult donors and recipients of matched sibling allogeneic HCT. Atorvastatin (40 mg per day orally) was administered to sibling donors, starting 14 to 28 days before the anticipated first day of stem-cell collection. In HCT recipients (n = 30), GVHD prophylaxis consisted of tacrolimus, short-course methotrexate, and atorvastatin (40 mg per day orally). RESULTS Atorvastatin administration in healthy donors and recipients was not associated with any grade 3 to 4 adverse events. Cumulative incidence rates of grade 2 to 4 acute GVHD at days +100 and +180 were 3.3% (95% CI, 0.2% to 14.8%) and 11.1% (95% CI, 2.7% to 26.4%), respectively. One-year cumulative incidence of chronic GVHD was 52.3% (95% CI, 27.6% to 72.1%). Viral and fungal infections were infrequent. One-year cumulative incidences of nonrelapse mortality and relapse were 9.8% (95% CI, 1.4% to 28%) and 25.4% (95% CI, 10.9% to 42.9%), respectively. One-year overall survival and progression-free survival were 74% (95% CI, 58% to 96%) and 65% (95% CI, 48% to 87%), respectively. Compared with baseline, atorvastatin administration in sibling donors was associated with a trend toward increased mean plasma interleukin-10 concentrations (5.6 v 7.1 pg/mL; P = .06). CONCLUSION A novel two-pronged strategy of atorvastatin administration in both donors and recipients of matched sibling allogeneic HCT seems to be a feasible, safe, and potentially effective strategy to prevent acute GVHD.

Modulating Temporal Control of NF-κB Activation: Implications for Therapeutic and Assay Selection

The activation of transcription factor NF-kappaB (nuclear factor-kappaB) plays a central role in the induction of many inflammatory response genes. This process is characterized by either oscillations or stable induction of NF-kappaB nuclear binding. Changes in dynamics of binding result in the expression of distinct subsets of genes leading to different physiological outcomes. We examined NF-kappaB DNA binding activity in lipopolysaccharide (LPS)-stimulated IC-21 cells by electromobility shift assay and nonradioactive transcription factor assay and interpreted the results using a kinetic model of NF-kappaB activation. Both assays detected damped oscillatory behavior of NF-kappaB with differences in sensitivity and reproducibility. 3,4-Dichloropropionaniline (DCPA) was used to modulate the oscillatory behavior of NF-kappaB after LPS stimulation. DCPA is known to inhibit the production of two NF-kappaB-inducible cytokines, IL-6 and tumor necrosis factor alpha, by reducing but not completely abrogating NF-kappaB-induced transcription. DCPA treatment resulted in a potentiation of early LPS-induced NF-kappaB activation. The nonradioactive transcription factor assay, which has a higher signal/noise ratio than the electromobility shift assay, combined with in silico modeling, produced results that revealed changes in NF-kappaB dynamics which, to the best of our knowledge, have never been previously reported. These results highlight the importance of cell type and stimulus specificity in transcription factor activity assessment. In addition, assay selection has important implications for network inference and drug discovery.

Scalable analysis of flow cytometry data using R/Bioconductor

Flow cytometry is one of the fundamental research tools available to the life scientist. The ability to observe multidimensional changes in protein expression and activity at single‐cell resolution for a large number of cells provides a unique perspective on the behavior of cell populations. However, the analysis of complex multidimensional data is one of the obstacles for wider use of polychromatic flow cytometry. Recent enhancements to an open‐source platform—R/Bioconductor—enable the graphical and data analysis of flow cytometry data. Prior examples have focused on high‐throughput applications. To facilitate wider use of this platform for flow cytometry, the analysis of a dataset, obtained following isolation of CD4+CD62L+ T cells from Balb/c splenocytes using magnetic microbeads, is presented as a form of tutorial. A common workflow for analyzing flow cytometry data was presented using R/Bioconductor. In addition, density function estimation and principal component analysis are provided as examples of more complex analyses. The compendium presented here is intended to help illuminate a path for inquisitive readers to explore their own data using R/Bioconductor (available as Supporting Information).

SOX9 inhibits β-TrCP-mediated protein degradation to promote nuclear GLI1 expression and cancer stem cell properties

ABSTRACT The high mobility group box protein SOX9 and the GLI1 transcription factor play protumorigenic roles in pancreatic ductal adenocarcinoma (PDA). In Kras transgenic mice, each of these factors are crucial for the development of PDA precursor lesions. SOX9 transcription is directly regulated by GLI1, but how SOX9 functions downstream of GLI1 is unclear. We observed positive feedback, such that SOX9-deficient PDA cells have severely repressed levels of endogenous GLI1, attributed to loss of GLI1 protein stability. SOX9 associated with the F-box domain of the SKP1/CUL1/F-box (SCF) E3 ubiquitin ligase component, &bgr;-TrCP (also known as F-box/WD repeat-containing protein 1A), and suppressed its association with SKP1 and GLI1, a substrate of SCF-&bgr;-TrCP. SOX9 also tethered &bgr;-TrCP within the nucleus and promoted its degradation. SOX9 bound to &bgr;-TrCP through the SOX9 C-terminal PQA/S domain that mediates transcriptional activation. Suppression of &bgr;-TrCP in SOX9-deficient PDA cells restored GLI1 levels and promoted SOX9-dependent cancer stem cell properties. These studies identify SOX9–GLI1 positive feedback as a major determinant of GLI1 protein stability and implicate &bgr;-TrCP as a latent SOX9-bound tumor suppressor with the potential to degrade oncogenic proteins in tumor cells.