RESUMO
Arsenic toxicity is a global concern to human health causing increased incidences of cancer, bronchopulmonary, and cardiovascular diseases. In human and mouse, inorganic arsenic (iAs) is metabolized in a series of methylation steps catalyzed by arsenic (3) methyltransferase (AS3MT), forming methylated arsenite (MAsIII), dimethylarsenite (DMAIII) and the volatile trimethylarsine (TMA). The methylation of arsenic is coordinated by four conserved cysteines proposed to participate in catalysis, namely C33, C62, C157, and C207 in mouse AS3MT. The current model consists of AS3MT methylating iAs in the presence of the cofactor S-adenosyl-L-methionine (SAM), and the formation of intramolecular disulfide bonds following the reduction of MAsV to MAsIII. In the presence of endogenous reductants, these disulfide bonds are reduced, the enzyme re-generates, and the second round of methylation ensues. Using in vitro methylation assays, we find that AS3MT undergoes an initial automethylation step in the absence of iAs. This automethylation is enhanced by glutathione (GSH) and dithiothreitol (DTT), suggesting that reduced cysteines accept methyl groups from SAM to form S-methylcysteines. Following the addition of iAs, automethylation of AS3MT is decreased. Furthermore, using a Flag-AS3MT immunoprecipitation coupled to MS/MS, we identify both C33 and C62 as acceptors of the methyl group in vivo. Site-directed mutagenesis (C to A) revealed that three of the previously described cysteines were required for AS3MT automethylation. In vitro experiments show that automethylated AS3MT can methylate iAs in the presence of SAM. Thus, we propose that automethylated may represent an active conformation of AS3MT.
Assuntos
Arsênio , Metiltransferases , Animais , Arsênio/metabolismo , Arsênio/toxicidade , Cisteína , Dissulfetos , Glutationa/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos , Espectrometria de Massas em TandemRESUMO
Diffuse large B-cell lymphoma (DLBCL) relapses in approximately 40% of patients following frontline therapy. We reported that STAT6D419 mutations are enriched in relapsed/refractory DLBCL (rrDLBCL) samples, suggesting that JAK/STAT signaling plays a role in therapeutic resistance. We hypothesized that STAT6D419 mutations can improve DLBCL cell survival by reprogramming the microenvironment to sustain STAT6 activation. Thus, we investigated the role of STAT6D419 mutations on DLBCL cell growth and its microenvironment. We found that phospho-STAT6D419N was retained in the nucleus longer than phospho-STAT6WT following IL-4 stimulation, and STAT6D419N recognized a more restricted DNA-consensus sequence than STAT6WT. Upon IL-4 induction, STAT6D419N expression led to a higher magnitude of gene expression changes, but in a more selective list of gene targets compared with STATWT. The most significantly expressed genes induced by STAT6D419N were those implicated in survival, proliferation, migration, and chemotaxis, in particular CCL17. This chemokine, also known as TARC, attracts helper T-cells to the tumor microenvironment, especially in Hodgkin's lymphoma. To this end, in DLBCL, phospho-STAT6+ rrDLBCL cells had a greater proportion of infiltrating CD4+ T-cells than phospho-STAT6- tumors. Our findings suggest that STAT6D419 mutations in DLBCL lead to cell autonomous changes, enhanced signaling, and altered composition of the tumor microenvironment.
Assuntos
Linfoma Difuso de Grandes Células B , Microambiente Tumoral , Humanos , Microambiente Tumoral/genética , Interleucina-4/genética , Interleucina-4/metabolismo , Interleucina-4/farmacologia , Recidiva Local de Neoplasia , Linfoma Difuso de Grandes Células B/patologia , Mutação , Fator de Transcrição STAT6/genética , Fator de Transcrição STAT6/metabolismoRESUMO
Introduction: Evidence suggests that e-cigarette use (vaping) increases cardiovascular disease risk, but decades are needed before people who vape would develop pathology. Thus, murine models of atherosclerosis can be utilized as tools to understand disease susceptibility, risk and pathogenesis. Moreover, there is a poor understanding of how risk factors for atherosclerosis (i.e., hyperlipidemia, high-fat diet) intersect with vaping to promote disease risk. Herein, we evaluated whether there was early evidence of atherosclerosis in an inducible hyperlipidemic mouse exposed to aerosol from commercial pod-style devices and e-liquid. Methods: Mice were injected with adeno-associated virus containing the human protein convertase subtilisin/kexin type 9 (PCSK9) variant to promote hyperlipidemia. These mice were fed a high-fat diet and exposed to room air or aerosol derived from JUUL pods containing polyethylene glycol/vegetable glycerin (PG/VG) or 5% nicotine with mango flavoring for 4 weeks; this timepoint was utilized to assess markers of atherosclerosis that may occur prior to the development of atherosclerotic plaques. Results: These data show that various parameters including weight, circulating lipoprotein/glucose levels, and splenic immune cells were significantly affected by exposure to PG/VG and/or nicotine-containing aerosols. Discussion: Not only can this mouse model be utilized for chronic vaping studies to assess the vascular pathology but these data support that vaping is not risk-free and may increase CVD outcomes later in life.
RESUMO
Diffuse large B cell lymphoma (DLBCL) is successfully treated with combination immuno-chemotherapy, but relapse with resistant disease occurs in ~ 40% of patients. However, little is known regarding relapsed/refractory DLBCL (rrDLBCL) genetics and alternative therapies. Based on findings from other tumors, we hypothesized that RAS-MEK-ERK signaling would be upregulated in resistant tumors, potentially correlating with mutations in RAS, RAF, or associated proteins. We analyzed mutations and phospho-ERK levels in tumor samples from rrDLBCL patients. Unlike other tumor types, rrDLBCL is not mutated in any Ras or Raf family members, despite having increased expression of p-ERK. In paired biopsies comparing diagnostic and relapsed specimens, 33% of tumors gained p-ERK expression, suggesting a role in promoting survival. We did find mutations in several Ras-associating proteins, including GEFs, GAPs, and downstream effectors that could account for increased ERK activation. We further investigated mutations in one such protein, RASGRP4. In silico modeling indicated an increased interaction between H-Ras and mutant RASGRP4. In cell lines, mutant RASGRP4 increased basal p-ERK expression and lead to a growth advantage in colony forming assays when challenged with doxorubicin. Relapsed/refractory DLBCL is often associated with increased survival signals downstream of ERK, potentially corresponding with mutations in protein controlling RAS/MEK/ERK signaling.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Linfoma Difuso de Grandes Células B/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Mutação , Proteínas ras/genética , Proteínas ras/metabolismo , Linhagem Celular Tumoral , Humanos , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Recidiva Local de Neoplasia/genética , Fatores ras de Troca de Nucleotídeo Guanina/genéticaRESUMO
Tungsten is a naturally occurring metal that is increasingly used in industry and medical devices, and is labeled as an emerging environmental contaminant. Like many metals, tungsten accumulates in bone. Our previous data indicate that tungsten decreases differentiation of osteoblasts, bone-forming cells. Herein, we explored the impact of tungsten on osteoclast differentiation, which function in bone resorption. We observed significantly elevated osteoclast numbers in the trabecular bone of femurs following oral exposure to tungsten in male, but not female mice. In order to explore the mechanism(s) by which tungsten increases osteoclast number, we utilized in vitro murine primary and cell line pre-osteoclast models. Although tungsten did not alter the adhesion of osteoclasts to the extracellular matrix protein, vitronectin, we did observe that tungsten enhanced RANKL-induced differentiation into tartrate-resistant acid phosphatase (TRAP)-positive mononucleated osteoclasts. Importantly, tungsten alone had no effect on differentiation or on the number of multinucleated TRAP-positive osteoclasts. Enhanced RANKL-induced differentiation correlated with increased gene expression of differentiated osteoclast markers Nfatc1, Acp5, and Ctsk. Although tungsten did not alter the RANK surface receptor expression, it did modulate its downstream signaling. Co-exposure of tungsten and RANKL resulted in sustained positive p38 signaling. These findings demonstrate that tungsten enhances sex-specific osteoclast differentiation, and together with previous findings of decreased osteoblastogenesis, implicate tungsten as a modulator of bone homeostasis.
Assuntos
Osteoclastos , Tungstênio , Animais , Diferenciação Celular , Feminino , Masculino , Camundongos , Fatores de Transcrição NFATC , Fosfatase Ácida Resistente a Tartarato , Tungstênio/toxicidadeRESUMO
B lymphocytes, or B cells, are important players in immunity that produce antigen-specific immunoglobulins. As a result, they are involved in various immune-linked pathologies. To better understand, prevent, or treat B cell-associated disease and immunotoxicity, we developed an in vitro assay to model early murine B cell differentiation within the bone marrow. This model uses sorted B cell precursors cultured on a supporting stromal cell layer, which over time acquire markers of further differentiated B cells, such as surface antigens and rearranged immunoglobulin light chain. Importantly, we utilized our in vitro model to validate our previous observations that xenobiotics, such as tungsten and organotins, alter B cell development in vivo. Furthermore, gene expression can be modulated in this model using retroviral transduction, making it amenable to investigating signaling pathways involved in disruption of B cell differentiation. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Assessment of early B lymphocyte differentiation in vitro Support Protocol: Isolation of murine bone marrow Alternate Protocol 1: Addition of recombinant interleukin-7 Alternate Protocol 2: Genetic manipulation via retroviral transduction.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Células Precursoras de Linfócitos B/efeitos dos fármacos , Testes de Toxicidade , Animais , Biomarcadores/metabolismo , Linhagem Celular , Separação Celular , Microambiente Celular , Técnicas de Cocultura , Células Alimentadoras , Citometria de Fluxo , Regulação da Expressão Gênica no Desenvolvimento , Imunofenotipagem , Masculino , Camundongos Endogâmicos C57BL , Fenótipo , Células Precursoras de Linfócitos B/imunologia , Células Precursoras de Linfócitos B/metabolismo , Transdução de Sinais , Transdução GenéticaRESUMO
Tungsten is an emerging environmental toxicant associated with several pediatric leukemia clusters, although a causal association has not been established. Our previous work demonstrated that tungsten exposure resulted in an accumulation of pre-B cells in the bone marrow, the same cell type that accumulates in pediatric acute lymphoblastic leukemia (ALL). To better understand the relevant molecular mechanisms, we performed RNA-sequencing on flow sorted pre-B cells from control and tungsten-exposed mice. Tungsten decreased the expression of multiple genes critical for B cell development, including members of the interleukin-7 receptor (IL-7R) and pre-B cell receptor signaling pathways, such as Jak1, Stat5a, Pax5, Syk, and Ikzf3. These results were confirmed in an in vitro model of B cell differentiation, where tungsten arrested differentiation at the pro-B cell stage and inhibited proliferation. These changes were associated with decreased expression of multiple genes in the IL-7R signaling pathway and decreased percentage of IL-7R, phosphorylated STAT5 double-positive cells. Supplementation with IL-7 or overexpression of Pax5, the transcription factor downstream of IL-7R, rescued the tungsten-induced differentiation block. Together, these data support the hypothesis that IL-7R/Pax5 signaling axis is critical to tungsten-mediated effects on pre-B cell development. Importantly, many of these molecules are modulated in ALL.
Assuntos
Linfócitos B/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Fator de Transcrição PAX5/metabolismo , Receptores de Interleucina-7/metabolismo , Compostos de Tungstênio/toxicidade , Animais , Linfócitos B/metabolismo , Linfócitos B/patologia , Regulação para Baixo , Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Fator de Transcrição PAX5/genética , Receptores de Interleucina-7/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
Arsenic exposure has been linked to an increased incidence of atherosclerosis. Previously, we have shown in vitro and in vivo that arsenic inhibits transcriptional activation of the liver X receptors (LXRs), key regulators of lipid homeostasis. Therefore, we evaluated the role of LXRα in arsenic-induced atherosclerosis using the apoE(-/-) mouse model. Indeed, deletion of LXRα protected apoE(-/-) mice against the proatherogenic effects of arsenic. We have previously shown that arsenic changes the plaque composition in apoE(-/-) mice. Arsenic decreased collagen content in the apoE(-/-) model, and we have observed the same diminution in LXRα(-/-)apoE(-/-) mice. However, the collagen-producing smooth muscle cells (SMCs) were decreased in apoE(-/-), but increased in LXRα(-/-)apoE(-/-). Although transcriptional activation of collagen remained the same in SMC from both genotypes, arsenic-exposed LXRα(-/-)apoE(-/-) plaques had increased matrix metalloproteinase activity compared with both control LXRα(-/-)apoE(-/-) and apoE(-/-), which could be responsible for both the decrease in plaque collagen and the SMC invasion. In addition, arsenic increased plaque lipid accumulation in both genotypes. However, macrophages, the cells known to retain lipid within the plaque, were unchanged in arsenic-exposed apoE(-/-) mice, but decreased in LXRα(-/-)apoE(-/-). We confirmed in vitro that these cells retained more lipid following arsenic exposure and are more sensitive to apoptosis than apoE(-/-). Mice lacking LXRα are resistant to arsenic-enhanced atherosclerosis, but arsenic-exposed LXRα(-/-)apoE(-/-) mice still present a different plaque composition pattern than the arsenic-exposed apoE(-/-) mice.
Assuntos
Arsenitos/toxicidade , Aterosclerose/genética , Poluentes Ambientais/toxicidade , Deleção de Genes , Receptores Nucleares Órfãos/genética , Placa Aterosclerótica/genética , Compostos de Sódio/toxicidade , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Aorta/patologia , Apolipoproteínas E/genética , Apoptose/genética , Aterosclerose/induzido quimicamente , Aterosclerose/patologia , Aterosclerose/prevenção & controle , Proliferação de Células/genética , Modelos Animais de Doenças , Metabolismo dos Lipídeos/genética , Lipídeos/sangue , Receptores X do Fígado , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos Knockout , Placa Aterosclerótica/induzido quimicamente , Placa Aterosclerótica/patologia , Placa Aterosclerótica/prevenção & controleRESUMO
Inhibitors of the mammalian target of rapamycin (mTORi) have clinical activity; however, the benefits of mTOR inhibition by rapamycin and rapamycin-derivatives (rapalogs) may be limited by a feedback mechanism that results in AKT activation. Increased AKT activity resulting from mTOR inhibition can be a result of increased signaling via the mTOR complex, TORC2. Previously, we published that arsenic trioxide (ATO) inhibits AKT activity and in some cases, decreases AKT protein expression. Therefore, we propose that combining ATO and rapamycin may circumvent the AKT feedback loop and increase the anti-tumor effects. Using a panel of breast cancer cell lines, we find that ATO, at clinically-achievable doses, can enhance the inhibitory activity of the mTORi temsirolimus. In all cell lines, temsirolimus treatment resulted in AKT activation, which was decreased by concomitant ATO treatment only in those cell lines where ATO enhanced growth inhibition. Treatment with rapalog also results in activated ERK signaling, which is decreased with ATO co-treatment in all cell lines tested. We next tested the toxicity and efficacy of rapamycin plus ATO combination therapy in a MDA-MB-468 breast cancer xenograft model. The drug combination was well-tolerated, and rapamycin did not increase ATO-induced liver enzyme levels. In addition, combination of these drugs was significantly more effective at inhibiting tumor growth compared to individual drug treatments, which corresponded with diminished phospho-Akt and phospho-ERK levels when compared with rapamycin-treated tumors. Therefore, we propose that combining ATO and mTORi may overcome the feedback loop by decreasing activation of the MAPK and AKT signaling pathways.
Assuntos
Arsenicais/farmacologia , Neoplasias da Mama/patologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Retroalimentação Fisiológica/efeitos dos fármacos , Óxidos/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Animais , Antineoplásicos/farmacologia , Trióxido de Arsênio , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Humanos , Camundongos , Fosforilação/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Histone deacetylase inhibitors (HDI) show activity in a broad range of hematologic and solid malignancies, yet the percentage of patients in any given malignancy who experience a meaningful clinical response remains small. In this study, we sought to investigate HDI efficacy in acute myeloid leukemia (AML) cells expressing leukemia-associated fusion proteins (LAFP). HDIs have been shown to induce apoptosis, in part, through accumulation of DNA damage and inhibition of DNA repair. LAFPs have been correlated with a DNA repair-deficient phenotype, which may make them more sensitive to HDI-induced DNA damage. We found that expression of the LAFPs PLZF-RARα, PML-RARα, and RUNX1-ETO (AML1-ETO) increased sensitivity to DNA damage and apoptosis induced by the HDI vorinostat. The increase in apoptosis correlated with an enhanced downregulation of the prosurvival protein BCL2. Vorinostat also induced expression of the cell-cycle regulators p19(INK4D) and p21(WAF1) and triggered a G2-M cell cycle arrest to a greater extent in LAFP-expressing cells. The combination of LAFP and vorinostat further led to a greater downregulation of several base excision repair (BER) enzymes. These BER genes represent biomarker candidates for response to HDI-induced DNA damage. Notably, repair of vorinostat-induced DNA double-strand breaks was found to be impaired in PLZF-RARα-expressing cells, suggesting a mechanism by which LAFP expression and HDI treatment cooperate to cause an accumulation of damaged DNA. These data support the continued study of HDI-based treatment regimens in LAFP-positive AMLs.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Dano ao DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Inibidores de Histona Desacetilases/farmacologia , Proteínas de Fusão Oncogênica/genética , Antineoplásicos/química , Linhagem Celular Tumoral , Reparo do DNA , Regulação Leucêmica da Expressão Gênica , Inibidores de Histona Desacetilases/química , Humanos , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/farmacologia , Leucemia Mieloide Aguda/genética , VorinostatRESUMO
High environmental tungsten levels were identified near the site of a childhood pre-B acute lymphoblastic leukemia cluster; however, a causal link between tungsten and leukemogenesis has not been established. The major site of tungsten deposition is bone, the site of B-cell development. In addition, our in vitro data suggest that developing B lymphocytes are susceptible to tungsten-induced DNA damage and growth inhibition. To extend these results, we assessed whether tungsten exposure altered B-cell development and induced DNA damage in vivo. Wild-type mice were exposed to tungsten in their drinking water for up to 16 weeks. Tungsten concentration in bone was analyzed by inductively coupled plasma mass spectrometry and correlated with B-cell development and DNA damage within the bone marrow. Tungsten exposure resulted in a rapid deposition within the bone following 1 week, and tungsten continued to accumulate thereafter albeit at a decreased rate. Flow cytometric analyses revealed a transient increase in mature IgD(+) B cells in the first 8 weeks of treatment, in animals of the highest and intermediate exposure groups. Following 16 weeks of exposure, all tungsten groups had a significantly greater percentage of cells in the late pro-/large pre-B developmental stages. DNA damage was increased in both whole marrow and isolated B cells, most notably at the lowest tungsten concentration tested. These findings confirm an immunological effect of tungsten exposure and suggest that tungsten could act as a tumor promoter, providing leukemic "hits" in multiple forms to developing B lymphocytes within the bone marrow.
Assuntos
Linfócitos B/efeitos dos fármacos , Medula Óssea/efeitos dos fármacos , Dano ao DNA , Tungstênio/toxicidade , Animais , Linfócitos B/ultraestrutura , Western Blotting , Linhagem da Célula , Ensaio Cometa , Citometria de Fluxo , Masculino , CamundongosAssuntos
Resistencia a Medicamentos Antineoplásicos/genética , Perfilação da Expressão Gênica/métodos , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Proteassoma/farmacologia , Animais , Antineoplásicos/farmacologia , Compostos de Boro/farmacologia , Bortezomib/farmacologia , Glicina/análogos & derivados , Glicina/farmacologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Masculino , Camundongos SCID , Prognóstico , Células U937 , Vorinostat , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Hepatitis C virus (HCV) quasispeciation was studied in two children vertically coinfected with HCV and human immunodeficiency virus type 1 (HIV-1). HCV quasispecies diversification and liver injury were more significant in patient C1, who was immunocompetent with anti-HIV therapy, than in patient C2, who was immunosuppressed, in consistency with modulation of HCV quasispeciation and liver injury by immunocompetence in coinfected children.