RESUMO
Arsenic exposure is correlated with atherosclerosis in epidemiological studies and in animal models. We have previously shown that arsenic exposure enhanced the atherosclerotic plaque size, increased the plaque lipid content, and decreased the plaque smooth muscle cell and collagen contents in the apolipoprotein E knockout (apoE-/-) mice. However, the percentage of plaque-resident macrophages, the primary drivers of atherosclerosis remained unchanged. Therefore, we hypothesized that although arsenic does not change the quantity of macrophages, it alters the macrophage transcriptome towards a proatherogenic state. To test this hypothesis, we used bone marrow-derived macrophages, polarized them to either interferon-γ (IFN-É£) stimulated, proinflammatory or interleukin-4 (IL-4) stimulated, alternatively activated macrophages in the presence or absence of 0.67 µM (50 ppb) arsenic and performed RNA sequencing. Arsenic exposure altered the gene expression of the macrophages in a subtype-specific manner. Most differentially expressed genes (88%) were altered specifically in either IFN-É£- or IL-4-stimulated macrophages, whereas in the remaining 12% of genes that changed in both cell types, did so in opposite directions. In IL-4-stimulated macrophages, arsenic significantly downregulated the genes involved in cholesterol biosynthesis and the chemokines CCL17/CCL22, whereas in IFN-É£-stimulated macrophages, the genes associated with the liver X receptor (LXR) pathway were downregulated by arsenic. Using a bone marrow transplant experiment, we validated that the deletion of LXRα from the hematopoietic compartment rescued arsenic-enhanced atherosclerosis in the apoE-/- mouse model. Together, these data suggest that arsenic modulates subtype-specific transcriptomic changes in macrophages and further emphasize the need to define macrophage heterogeneity in atherosclerotic plaques in order to evaluate the proatherogenic role of arsenic.
Assuntos
Arsênio , Aterosclerose , Placa Aterosclerótica , Animais , Camundongos , Arsênio/metabolismo , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-4/genética , Interleucina-4/metabolismo , Camundongos Knockout , Aterosclerose/induzido quimicamente , Aterosclerose/genética , Aterosclerose/metabolismo , Placa Aterosclerótica/metabolismo , Macrófagos/metabolismo , Expressão Gênica , Apolipoproteínas E/genética , Camundongos Endogâmicos C57BLRESUMO
Relapse occurs in 10-40% of Burkitt lymphoma (BL) patients that have completed intensive chemotherapy regimens and is typically fatal. While treatment-naive BL has been characterized, the genomic landscape of BL at the time of relapse (rBL) has never been reported. Here, we present a genomic characterization of two rBL patients. The diagnostic samples had mutations common in BL, including MYC and CCND3. Additional mutations were detected at relapse, affecting important pathways such as NFκB (IKBKB) and MEK/ERK (NRAS) signaling, glutamine metabolism (SIRT4), and RNA processing (ZFP36L2). Genes implicated in drug resistance were also mutated at relapse (TP53, BAX, ALDH3A1, APAF1, FANCI). This concurrent genomic profiling of samples obtained at diagnosis and relapse has revealed mutations not previously reported in this disease. The patient-derived cell lines will be made available and, along with their detailed genetics, will be a valuable resource to examine the role of specific mutations in therapeutic resistance.
Assuntos
Linfoma de Burkitt/genética , Genômica/métodos , Mutação , Recidiva Local de Neoplasia/genética , Adulto , Linfoma de Burkitt/tratamento farmacológico , Linfoma de Burkitt/patologia , Linhagem Celular Tumoral , Ciclina D3/genética , Humanos , Masculino , Análise de Sequência de DNA , Adulto Jovem , Proteína X Associada a bcl-2/genéticaRESUMO
Tungsten is a naturally occurring metal that increasingly is being incorporated into industrial goods and medical devices, and is recognized as an emerging contaminant. Tungsten preferentially and rapidly accumulates in murine bone in a concentration-dependent manner; however the effect of tungsten deposition on bone biology is unknown. Other metals alter bone homeostasis by targeting bone marrow-derived mesenchymal stromal cell (MSC) differentiation, thus, we investigated the effects of tungsten on MSCsin vitroandin vivoIn vitro, tungsten shifted the balance of MSC differentiation by enhancing rosiglitazone-induced adipogenesis, which correlated with an increase in adipocyte content in the bone of tungsten-exposed, young, male mice. Conversely, tungsten inhibited osteogenesis of MSCsin vitro; however, we found no evidence that tungsten inhibited osteogenesisin vivo Interestingly, two factors known to influence adipogenesis are sex and age of mice. Both female and older mice have enhanced adipogenesis. We extended our study and exposed young female and adult (9-month) male and female mice to tungsten for 4 weeks. Although tungsten accumulated to a similar extent in young female mice, it did not promote adipogenesis. Interestingly, tungsten did not accumulate in the bone of older mice; it was undetectable in adult male mice, and just above the limit of detect in adult female mice. Surprisingly, tungsten enhanced adipogenesis in adult female mice. In summary, we found that tungsten alters bone homeostasis by altering differentiation of MSCs, which could have significant implications for bone quality, but is highly dependent upon sex and age.
Assuntos
Adipogenia/efeitos dos fármacos , Medula Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Fêmur/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Caracteres Sexuais , Tungstênio/toxicidade , Envelhecimento/metabolismo , Animais , Medula Óssea/metabolismo , Relação Dose-Resposta a Droga , Poluentes Ambientais/farmacocinética , Feminino , Fêmur/metabolismo , Fêmur/patologia , Técnicas In Vitro , Masculino , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Osteogênese/efeitos dos fármacos , Tungstênio/farmacocinéticaRESUMO
The number of individuals exposed to high levels of tungsten is increasing, yet there is limited knowledge of the potential human health risks. Recently, a cohort of breast cancer patients was left with tungsten in their breasts following testing of a tungsten-based shield during intraoperative radiotherapy. While monitoring tungsten levels in the blood and urine of these patients, we utilized the 66Cl4 cell model, in vitro and in mice to study the effects of tungsten exposure on mammary tumor growth and metastasis. We still detect tungsten in the urine of patients' years after surgery (mean urinary tungsten concentration at least 20 months post-surgery = 1.76 ng/ml), even in those who have opted for mastectomy, indicating that tungsten does not remain in the breast. In addition, standard chelation therapy was ineffective at mobilizing tungsten. In the mouse model, tungsten slightly delayed primary tumor growth, but significantly enhanced lung metastasis. In vitro, tungsten did not enhance 66Cl4 proliferation or invasion, suggesting that tungsten was not directly acting on 66Cl4 primary tumor cells to enhance invasion. In contrast, tungsten changed the tumor microenvironment, enhancing parameters known to be important for cell invasion and metastasis including activated fibroblasts, matrix metalloproteinases, and myeloid-derived suppressor cells. We show, for the first time, that tungsten enhances metastasis in an animal model of breast cancer by targeting the microenvironment. Importantly, all these tumor microenvironmental changes are associated with a poor prognosis in humans.
Assuntos
Neoplasias da Mama/patologia , Neoplasias Pulmonares/secundário , Microambiente Tumoral , Compostos de Tungstênio/toxicidade , Animais , Biópsia , Carga Corporal (Radioterapia) , Neoplasias da Mama/sangue , Neoplasias da Mama/metabolismo , Neoplasias da Mama/urina , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quelantes/uso terapêutico , Feminino , Humanos , Mediadores da Inflamação/metabolismo , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/urina , Mamografia , Camundongos Endogâmicos BALB C , Invasividade Neoplásica , Medição de Risco , Fatores de Risco , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Compostos de Tungstênio/sangue , Compostos de Tungstênio/urinaRESUMO
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
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 , CamundongosRESUMO
Cytidine (CR) deaminase is a key enzyme in the catabolism of cytosine nucleoside analogues, since their deamination results in a loss of their pharmacological activity. In this report we have investigated the importance of CR deaminase with respect to the antineoplastic action of inhibitors of DNA methylation, 5-aza-2'-deoxycytidine (5-AZA-CdR) and zebularine. Zebularine has a dual mechanism of action, since it can also inhibit CR deaminase. The objective of our study was to investigate the importance of zebularine as an inhibitor of CR deaminase with respect to the antineoplastic action of 5-AZA-CdR. Using an in vitro clonogenic assay, we investigated the antineoplastic action of 5-AZA-CdR and zebularine, alone and in combination on wild type 3T3 murine fibroblasts and corresponding V5 cells transduced with CR deaminase gene to express a very high level of CR deaminase activity. The V5 cells were much less sensitive to 5-AZA-CdR than the wild type 3T3 cells. The addition of zebularine significantly enhanced the antineoplastic action of 5-AZA-CdR on V5 cells, but not 3T3 cells. Enzymatic analysis on CR deaminase purified from the V5 cells showed that zebularine is a competitive inhibitor of the deamination of 5-AZA-CdR. These in vitro observations are in accord with our in vivo study in mice with L1210 leukemia, which showed that zebularine increased the antileukemic activity of 5-AZA-CdR. Pharmacokinetic analysis also showed that zebularine increased the plasma level of 5-AZA-CdR during an i.v. infusion in mice. Our results indicate that the major mechanism by which zebularine enhances the antineoplastic action of 5-AZA-CdR is by inhibition of CR deaminase. These findings provide a rationale to investigate 5-AZA-CdR in combination with zebularine in patients with advanced leukemia.
Assuntos
Antineoplásicos/farmacologia , Azacitidina/análogos & derivados , Citidina Desaminase/antagonistas & inibidores , Citidina/análogos & derivados , Inibidores Enzimáticos/farmacologia , Leucemia Experimental/tratamento farmacológico , Células 3T3 , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Azacitidina/farmacocinética , Azacitidina/farmacologia , Azacitidina/uso terapêutico , Citidina/farmacologia , Citidina/uso terapêutico , Decitabina , Sinergismo Farmacológico , Inibidores Enzimáticos/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBARESUMO
BACKGROUND: The inactivation of tumor suppressor genes (TSGs) by aberrant DNA methylation plays an important role in the development of malignancy. Since this epigenetic change is reversible, it is a potential target for chemotherapeutic intervention using an inhibitor of DNA methylation, such as 5-aza-2'-deoxycytidine (DAC). Although clinical studies show that DAC has activity against hematological malignancies, the optimal dose-schedule of this epigenetic agent still needs to be established. METHODS: Clonogenic assays were performed on leukemic and tumor cell lines to evaluate the in vitro antineoplastic activity of DAC. The reactivation of TSGs and inhibition of DNA methylation by DAC were investigated by reverse transcriptase-PCR and Line-1 assays. The in vivo antineoplastic activity of DAC administered as an i.v. infusion was evaluated in mice with murine L1210 leukemia by measurement of survival time, and in mice bearing murine EMT6 mammary tumor by excision of tumor after chemotherapy for an in vitro clonogenic assay. RESULTS: Increasing the DAC concentration and duration of exposure produced a greater loss of clonogenicity for both human leukemic and tumor cell lines. The reactivation of the TSGs (p57KIP2 in HL-60 leukemic cells and p16CDKN2A in Calu-6 lung carcinoma cells) and the inhibition of global DNA methylation in HL-60 leukemic cells increased with DAC concentration. In mice with L1210 leukemia and in mice bearing EMT6 tumors, the antineoplastic action of DAC also increased with the dose. The plasma level of DAC that produced a very potent antineoplastic effect in mice with leukemia or solid tumors was > 200 ng/ml (> 1 microM). CONCLUSION: We have shown that intensification of the DAC dose markedly increased its antineoplastic activity in mouse models of cancer. Our data also show that there is a good correlation between the concentrations of DAC that reduce in vitro clonogenicity, reactivate TSGs and inhibit DNA methylation. These results suggest that the antineoplastic action of DAC is related to its epigenetic action. Our observations provide a strong rationale to perform clinical trials using dose intensification of DAC to maximize the chemotherapeutic potential of this epigenetic agent in patients with cancer.
Assuntos
Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Azacitidina/análogos & derivados , Epigênese Genética/efeitos dos fármacos , Animais , Azacitidina/administração & dosagem , Azacitidina/farmacocinética , Linhagem Celular Tumoral , Proteínas Inibidoras de Quinase Dependente de Ciclina/metabolismo , Metilação de DNA/efeitos dos fármacos , Decitabina , Relação Dose-Resposta a Droga , Esquema de Medicação , Impressão Genômica/efeitos dos fármacos , Células HL-60 , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Ensaio Tumoral de Célula-TroncoRESUMO
Tumor suppressor genes that have been silenced by aberrant DNA methylation are potential targets for reactivation by novel chemotherapeutic agents. The potent inhibitor of DNA methylation and antileukemic agent, 5-aza-2'-deoxycytidine (5-AZA-CdR, Decitabine), can reactivate silent tumor suppressor genes. One hindrance to the curative potential of 5-AZA-CdR is its rapid in vivo inactivation by cytidine deaminase (CD). An approach to overcome this obstacle is to use 5-AZA-CdR in combination with zebularine (Zeb), a potent inhibitor of CD. Zeb also possesses independent antineoplastic activity due to its inhibition of DNA methylation. We tested the capacity of 5-AZA-CdR and Zeb alone and in combination to inhibit growth and colony formation of different leukemic cell lines. 5-AZA-CdR and Zeb in combination produced a greater inhibition of growth against murine L1210 lymphoid leukemic cells, and a greater reduction in colony formation by L1210 and human HL-60 myeloid leukemic cells, than either agent alone. The ability of these agents to reactivate the tumor suppressor gene, p57KIP2, was also tested using RT-PCR. The combination produced a synergistic reactivation of p57KIP2 in HL-60 leukemic cells. A methylation-specific PCR assay showed that this combination also induced a significantly greater demethylation level of the p57KIP2 promoter than either drug alone. The in vivo antineoplastic activity of the agents was evaluated in mice with L1210 leukemia. A greater increase in survival time of mice with L1210 leukemia was observed with the combination than with either agent alone using three different dose schedules. The enhanced activity observed with 5-AZA-CdR plus Zeb in both murine and human leukemic cells lines provides a rationale for the clinical investigation of these drugs in patients with advanced leukemia. The probable mechanism of this drug interaction involves inhibition of CD by Zeb and the complementary inhibition of DNA methylation by both agents.
Assuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Azacitidina/análogos & derivados , Azacitidina/uso terapêutico , Citidina Desaminase/antagonistas & inibidores , Leucemia L1210/tratamento farmacológico , Nucleosídeos de Pirimidina/farmacologia , Animais , Linhagem Celular Tumoral , Citidina/análogos & derivados , Metilação de DNA/efeitos dos fármacos , Decitabina , Relação Dose-Resposta a Droga , Interações Medicamentosas , Humanos , Leucemia L1210/enzimologia , Leucemia L1210/metabolismo , Masculino , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ensaio Tumoral de Célula-TroncoRESUMO
Epigenetic changes, such as aberrant DNA methylation that silences tumor suppressor genes (TSGs), can play an important role in the development of leukemia. The DNA methylation inhibitor, 5-aza-2'-deoxycytidine (5-AZA-CdR), can reactivate these silent TSGs and is an interesting agent to investigate for therapy of leukemia. It has been reported that the effectiveness of 5-AZA-CdR to reactivate TSG can be enhanced by inhibitors of histone deacetylase (HDIs). HDIs can convert a compact chromatin structure to an open configuration that facilitates gene expression. An interesting HDI is phenylbutyrate (PB), which has shown some clinical activity for the therapy of leukemia. In this report we have investigated the antineoplastic activity of 5-AZA-CdR and PB alone and in combination on murine L1210 lymphoid leukemic cells. The in vitro treatment of 5-AZA-CdR and PB in combination produced a greater inhibition of growth, DNA synthesis, and also a greater reduction on colony formation on both L1210 and human HL-60 leukemic cells as compared to either drug alone. The combination also produced a synergistic activation of the TSG, p15CDN2B, in the L1210 cells. In mice with L1210 leukemia the combination showed enhanced antineoplastic activity. We also observed an enhancement of the antineoplastic activity of this combination in mice with L1210 leukemia. These data provide a rationale to investigate 5-AZA-CdR and PB in patients with advanced leukemia.
Assuntos
Antineoplásicos/farmacologia , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Leucemia/patologia , Fenilbutiratos/farmacologia , Animais , Antineoplásicos/uso terapêutico , Azacitidina/uso terapêutico , Proteínas de Ciclo Celular/metabolismo , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p15 , DNA/biossíntese , Replicação do DNA/efeitos dos fármacos , Decitabina , Sinergismo Farmacológico , Genes Supressores de Tumor , Células HL-60 , Humanos , Leucemia/tratamento farmacológico , Masculino , Camundongos , Fenilbutiratos/uso terapêutico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida , Proteínas Supressoras de Tumor/metabolismoRESUMO
BACKGROUND: Inactivation of genes that suppress neoplasia by aberrant DNA methylation is a key event that occurs during the development of leukemia. The inhibitor of DNA methylation, 5-aza-2'-deoxycytidine (5AZA), which can re-activate these genes, is under clinical investigation for therapy of leukemia. The objective of this study was to determine the concentrations of 5AZA that will re-activate target silent genes in human leukemic cell lines. MATERIALS AND METHODS: RT-PCR was used to evaluate the effect of concentrations of 1 to 100 ng/ml of 5AZA on the re-activation of p15 and p73 in KG1a myeloid leukemic cells and E-cadherin in HL-60 myeloid leukemic cells. The effect of 5AZA on inhibition of growth, DNA synthesis and colony formation in these cell lines was also investigated. RESULTS: The extent of activation of the target genes was dependent on the concentration of 5AZA. For p15, pronounced activation was observed at 10 ng/ml or greater. For p73 and E-cadherin significant activation was observed at 100 ng/ml of 5AZA. Maximal inhibition of growth, DNA synthesis and colony formation occurred at 100 ng/ml. CONCLUSION: The in vitro antineoplastic and gene re-activation activity of 5AZA is dependent on the concentration of this analog. These data may be helpful in the design of the optimal dose-schedule of 5AZA for the clinical therapy of leukemia.