RESUMO
Mantle cell lymphoma (MCL) is an aggressive B-cell non-Hodgkin lymphoma having a poor overall survival that is in need for the development of new therapeutics. In this study, we report the identification and expression of a new isoform splice variant of the tyrosine kinase receptor AXL in MCL cells. This new AXL isoform, called AXL3, lacks the ligand-binding domain of the commonly described AXL splice variants and is constitutively activated in MCL cells. Interestingly, functional characterization of AXL3, using CRISPR inhibition, revealed that only the knock down of this isoform leads to apoptosis of MCL cells. Importantly, pharmacological inhibition of AXL activity resulted in a significant decrease in the activation of well-known proproliferative and survival pathways activated in MCL cells (ie, ß-catenin, Ak strain transforming, and NF-κB). Therapeutically, preclinical studies using a xenograft mouse model of MCL indicated that bemcentinib is more effective than ibrutinib in reducing the tumor burden and to increase the overall survival. Our study highlights the importance of a previously unidentified AXL splice variant in cancer and the potential of bemcentinib as a targeted therapy for MCL.
Assuntos
Linfoma de Célula do Manto , Proteínas Tirosina Quinases , Humanos , Adulto , Animais , Camundongos , Tirosina Quinase da Agamaglobulinemia , Linfoma de Célula do Manto/tratamento farmacológico , Linfoma de Célula do Manto/genética , Linfoma de Célula do Manto/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , ApoptoseRESUMO
The Ca2+ ion is a universal second messenger involved in many vital physiological functions including cell migration and development. To fulfil these tasks the cytosolic Ca2+ concentration is tightly controlled, and this involves an intricate functional balance between a variety of channels and pumps of the Ca2+ signalling machinery. Among these proteins, plasma membrane Ca2+ ATPases (PMCAs) represent the major high-affinity Ca2+ extrusion systems in the cell membrane that are effective in maintaining free Ca2+ concentration at exceedingly low cytosolic levels, which is essential for normal cell function. An imbalance in Ca2+ signalling can have pathogenic consequences including cancer and metastasis. Recent studies have highlighted the role of PMCAs in cancer progression and have shown that a particular variant, PMCA4b, is downregulated in certain cancer types, causing delayed attenuation of the Ca2+ signal. It has also been shown that loss of PMCA4b leads to increased migration and metastasis of melanoma and gastric cancer cells. In contrast, an increased PMCA4 expression has been reported in pancreatic ductal adenocarcinoma that coincided with increased cell migration and shorter patient survival, suggesting distinct roles of PMCA4b in various tumour types and/or different stages of tumour development. The recently discovered interaction of PMCAs with basigin, an extracellular matrix metalloproteinase inducer, may provide further insights into our understanding of the specific roles of PMCA4b in tumour progression and cancer metastasis.
RESUMO
Endoplasmic reticulum (ER) calcium homeostasis plays an essential role in cellular calcium signaling, intra-ER protein chaperoning and maturation, as well as in the interaction of the ER with other organelles. Calcium is accumulated in the ER by sarco/endoplasmic reticulum calcium ATPases (SERCA enzymes) that generate by active, ATP-dependent transport, a several thousand-fold calcium ion concentration gradient between the cytosol (low nanomolar) and the ER lumen (high micromolar). SERCA enzymes are coded by three genes that by alternative splicing give rise to several isoforms, which can display isoform-specific calcium transport characteristics. SERCA expression levels and isoenzyme composition vary according to cell type, and this constitutes a mechanism whereby ER calcium homeostasis is adapted to the signaling and metabolic needs of the cell, depending on its phenotype, its state of activation and differentiation. As reviewed here, in several normal epithelial cell types including bronchial, mammary, gastric, colonic and choroid plexus epithelium, as well as in mature cells of hematopoietic origin such as pumps are simultaneously expressed, whereas in corresponding tumors and leukemias SERCA3 expression is selectively down-regulated. SERCA3 expression is restored during the pharmacologically induced differentiation of various cancer and leukemia cell types. SERCA3 is a useful marker for the study of cell differentiation, and the loss of SERCA3 expression constitutes a previously unrecognized example of the remodeling of calcium homeostasis in tumors.
Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Biomarcadores Tumorais/análise , Neoplasias da Mama/enzimologia , Sinalização do Cálcio , Carcinoma/enzimologia , Diferenciação Celular , Linhagem Celular Tumoral , Neoplasias do Plexo Corióideo/enzimologia , Neoplasias Gastrointestinais/enzimologia , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Homeostase , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Leucemia Promielocítica Aguda/metabolismo , Neoplasias Pulmonares/enzimologia , Megacariócitos/citologia , Megacariócitos/metabolismo , Especificidade de Órgãos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/análise , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genéticaRESUMO
BACKGROUND: Remodeling of Ca2+ signaling is an important step in cancer progression, and altered expression of members of the Ca2+ signaling toolkit including the plasma membrane Ca2+ ATPases (PMCA proteins encoded by ATP2B genes) is common in tumors. METHODS: In this study PMCAs were examined in breast cancer datasets and in a variety of breast cancer cell lines representing different subtypes. We investigated how estrogen receptor alpha (ER-α) and histone deacetylase (HDAC) inhibitors regulate the expression of these pumps. RESULTS: Three distinct datasets displayed significantly lower ATP2B4 mRNA expression in invasive breast cancer tissue samples compared to normal breast tissue, whereas the expression of ATP2B1 and ATP2B2 was not altered. Studying the protein expression profiles of Ca2+ pumps in a variety of breast cancer cell lines revealed low PMCA4b expression in the ER-α positive cells, and its marked upregulation upon HDAC inhibitor treatments. PMCA4b expression was also positively regulated by the ER-α pathway in MCF-7 cells that led to enhanced Ca2+ extrusion capacity in response to 17ß-estradiol (E2) treatment. E2-induced PMCA4b expression was further augmented by HDAC inhibitors. Surprisingly, E2 did not affect the expression of PMCA4b in other ER-α positive cells ZR-75-1, T-47D and BT-474. These findings were in good accordance with ChIP-seq data analysis that revealed an ER-α binding site in the ATP2B4 gene in MCF-7 cells but not in other ER-α positive tumor cells. In the triple negative cells PMCA4b expression was relatively high, and the effect of HDAC inhibitor treatment was less pronounced as compared to that of the ER-α positive cells. Although, the expression of PMCA4b was relatively high in the triple negative cells, a fraction of the protein was found in intracellular compartments that could interfere with the cellular function of the protein. CONCLUSIONS: Our results suggest that the expression of Ca2+ pumps is highly regulated in breast cancer cells in a subtype specific manner. Our results suggest that hormonal imbalances, epigenetic modifications and impaired protein trafficking could interfere with the expression and cellular function of PMCA4b in the course of breast cancer progression.
Assuntos
Neoplasias da Mama/enzimologia , Sinalização do Cálcio/efeitos dos fármacos , Receptor alfa de Estrogênio/metabolismo , Inibidores de Histona Desacetilases/farmacologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Neoplasias da Mama/patologia , Sinalização do Cálcio/genética , Linhagem Celular Tumoral , Conjuntos de Dados como Assunto , Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor alfa de Estrogênio/genética , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , ATPases Transportadoras de Cálcio da Membrana Plasmática/genéticaRESUMO
Plasma membrane Ca(2+) ATPases (PMCAs) are intimately involved in the control of intracellular Ca(2+) concentration. They reduce Ca(2+) in the cytosol not only by direct ejection, but also by controlling the formation of inositol-1,4,5-trisphosphate and decreasing Ca(2+) release from the endoplasmic reticulum Ca(2+) pool. In mammals four genes (PMCA1-4) are expressed, and alternative RNA splicing generates more than twenty variants. The variants differ in their regulatory characteristics. They localize into highly specialized membrane compartments and respond to the incoming Ca(2+) with distinct temporal resolution. The expression pattern of variants depends on cell type; a change in this pattern can result in perturbed Ca(2+) homeostasis and thus altered cell function. Indeed, PMCAs undergo remarkable changes in their expression pattern during tumorigenesis that might significantly contribute to the unbalanced Ca(2+) homeostasis of cancer cells. This article is part of a Special Issue entitled: Calcium and Cell Fate. Guest Editors: Jacques Haiech, Claus Heizmann, Joachim Krebs, Thierry Capiod and Olivier Mignen.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Membrana Celular/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Animais , Homeostase , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/genéticaRESUMO
We have previously presented co-expression of the plasma membrane calcium ATPase isoforms 4b (PMCA4b) and 1b (PMCA1b) in colon carcinoma cells, and selective upregulation of PMCA4b during differentiation initiated by short chain fatty acids or post-confluent growth. Here we show that the induction of PMCA4b expression is a characteristic feature of the post-confluency-induced differentiation of both enterocyte-type and goblet cell-type colon cancer cells. Vitamin D3 (1,25(OH)2D3) is a well-known regulator of intestinal Ca(2+) absorption and of basic cell functions such as growth and differentiation in various cell types. As PMCA proteins are involved both in intestinal Ca(2+) absorption and adenocarcinoma cell differentiation, we investigated the effect of 1,25(OH)2D3 on PMCA expression in enterocyte-like colon carcinoma cells, and monitored its effect on the expression of various differentiation markers. 1,25(OH)2D3 stimulated PMCA1b, but not PMCA4b expression without modulating the expression of the majority of the differentiation markers examined. Caco-2 cells differentiated in post-confluent cultures present normal enterocyte-like intestinal epithelial phenotype. To better understand the role of PMCA proteins in vectorial Ca(2+) transport by enterocytes, we also studied their subcellular localization in mature polarized Caco-2 cells. Both PMCA isoforms were located to the basolateral membrane, and the PMCA-specific immunofluorescent signal was significantly higher in vitamin D3-treated cells, underlining the 1,25(OH)2D3-induced upregulation of PMCA (presumably 1b isoform) expression in differentiated Caco-2 cells. We suggest that while PMCA1b has a housekeeping function in colon cancer cells, PMCA4b participates in the reorganization of the Ca(2+) signalling machinery during cell differentiation. The subcellular localization of PMCA1b and its selective 1,25(OH)2D3-dependent upregulation indicate that this isoform may have a specific role in 1,25(OH)2D3-stimulated intestinal Ca(2+) absorption.
Assuntos
Calcitriol/farmacologia , Cálcio/metabolismo , Regulação Neoplásica da Expressão Gênica , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Células CACO-2 , Sinalização do Cálcio , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Polaridade Celular , Ativação Enzimática/efeitos dos fármacos , Células HT29 , Humanos , Fenótipo , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Ativação Transcricional/efeitos dos fármacosRESUMO
AIMS: Sarco/Endoplasmic Reticulum Calcium ATPase-type calcium pumps (SERCA enzymes) control cell activation by sequestering calcium ions from the cytosol into the endoplasmic reticulum. Although endoplasmic reticulum calcium signalling plays an important role in the regulation of choroid plexus epithelial function, SERCA expression in the choroid plexus has not been investigated so far. METHODS: In this work we investigated the expression of the SERCA3-type calcium pump in choroid plexus epithelial cells grown in vitro, and in normal and hyperplastic choroid plexus tissue, in choroid plexus papillomas displaying various degrees of atypia, and in choroid plexus carcinoma by immunohistochemistry in situ. RESULTS: Whereas normal choroid plexus epithelial cells express SERCA3 abundantly, SERCA3 expression is strongly decreased in papillomas, and is absent in choroid plexus carcinoma, while expression in hyperplastic epithelium is high, similarly to normal epithelium. SERCA3 expression was detected also in normal primary choroid plexus epithelial cells grown in vitro, and expression was markedly enhanced by short-chain fatty acid-type cell differentiation inducing agents, including valproate. CONCLUSION: These observations show that SERCA3 is a new phenotypic marker of normal choroid plexus epithelial differentiation, and that SERCA3 constitutes an early tumour marker 'by loss of expression' in the choroid plexus that may be useful to distinguish hyperplastic processes from papillomas. Endoplasmic reticulum calcium homeostasis becomes anomalous, due to loss of SERCA3 expression, already in benign neoplastic lesions of the choroid plexus epithelium.
Assuntos
Neoplasias do Plexo Corióideo/metabolismo , Plexo Corióideo/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Células Epiteliais/metabolismo , Humanos , Papiloma do Plexo Corióideo/metabolismo , Cultura Primária de CélulasRESUMO
Accurate characterisation of gastrointestinal stromal tumours (GIST) is important for prognosis and the choice of targeted therapies. Histologically the diagnosis relies on positive immunostaining of tumours for KIT (CD117) and DOG1. Here we report that GISTs also abundantly express the type 3 Sarco/Endoplasmic Reticulum Calcium ATPase (SERCA3). SERCA enzymes transport calcium ions from the cytosol into the endoplasmic reticulum and play an important role in regulating the intensity and the periodicity of calcium-induced cell activation. GISTs from various localisations, histological and molecular subtypes or risk categories were intensely immunopositive for SERCA3 with the exception of PDGFRA-mutated cases where expression was high or moderate. Strong SERCA3 expression was observed also in normal and hyperplastic interstitial cells of Cajal. Decreased SERCA3 expression in GIST was exceptionally observed in a zonal pattern, where CD117 staining was similarly decreased, reflecting clonal heterogeneity. In contrast to GIST, SERCA3 immunostaining of spindle cell tumours and other gastrointestinal tumours resembling GIST was negative or weak. In conclusion, SERCA3 immunohistochemistry may be useful for the diagnosis of GIST with high confidence, when used as a third marker in parallel with KIT and DOG1. Moreover, SERCA3 immunopositivity may be particularly helpful in cases with negative or weak KIT or DOG1 staining, a situation that may be encountered de novo, or during the spontaneous or therapy-induced clonal evolution of GIST.
Assuntos
Tumores do Estroma Gastrointestinal , Humanos , Tumores do Estroma Gastrointestinal/diagnóstico , Tumores do Estroma Gastrointestinal/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Cálcio , Retículo Endoplasmático/metabolismo , Imuno-Histoquímica , Proteínas Proto-Oncogênicas c-kit/metabolismoRESUMO
Ca(2+) signaling plays an important role in B cell survival and activation and is dependent on Ca(2+) trapped in the endoplasmic reticulum (ER) and on extracellular Ca(2+). Epstein-Barr virus (EBV) can immortalize B cells and contributes to lymphomagenesis. Previously, we showed that the ER Ca(2+) content of Burkitt lymphoma cell lines was increased following infection with immortalization-competent virus expressing the full set of EBV latency genes (B95-8). In contrast, infection with an immortalization-deficient virus (P3HR-1) not expressing LMP-1 is without effect. LMP-1 protein expression was sufficient to increase the ER Ca(2+) content and to increase the cytosolic Ca(2+) concentration ([Ca(2+)](cyt)). In this follow-up study, we showed that the resting [Ca(2+)](cyt) of P3HR-1-infected cells was decreased, implying that EBV not only modified the ER homeostasis but also affected the cytosolic Ca(2+) homeostasis. Furthermore, even if the store-operated calcium entry (SOCE) of these cells was normal, the [Ca(2+)](cyt) increase after thapsigargin + CaCl(2) stimulation was blunted. In contrast, the resting [Ca(2+)](cyt) of B95-8 infected cells was not changed, even if their SOCE was increased significantly. When expressed alone, LMP-1 induced an increase of the SOCE amplitude and the expression of the protein allowing this influx, Orai1, showing the effect of EBV on SOCE of B cells are mediated by LMP-1. However, other hitherto unidentified EBV processes, unmasked in P3HR-1 infected cells, counteract this LMP-1-dependent increase of SOCE amplitude to impair a general and potentially toxic increase of [Ca(2+)](i). Thus, EBV infection modifies the cellular Ca(2+) homeostasis by acting on the ER and plasma membrane transporters.
Assuntos
Linfócitos B/metabolismo , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Transformação Celular Viral/fisiologia , Herpesvirus Humano 4/metabolismo , Proteínas da Matriz Viral/metabolismo , Linfócitos B/virologia , Cálcio/farmacologia , Canais de Cálcio/genética , Linhagem Celular Tumoral , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Inibidores Enzimáticos/farmacologia , Herpesvirus Humano 4/genética , Humanos , Proteína ORAI1 , Tapsigargina/farmacologia , Proteínas da Matriz Viral/genéticaRESUMO
Mantle cell lymphoma (MCL) is a non-Hodgkin lymphoma that remains incurable with the treatment options available today. In the present study, we have identified the dihydroorotate dehydrogenase (DHODH), an essential enzyme for the de novo biosynthesis of pyrimidine-based nucleotides, to be overexpressed in MCL in comparison to healthy peripheral blood mononuclear cells (PBMC). In vitro inhibition of the DHODH activity using a newly developed DHODH inhibitor, namely (R)-HZ05, can induce MCL cell death in the nanomolar range independently than the P53 status of the investigated cell lines. Moreover, the combination of (R)-HZ05 with tyrosine kinase inhibitor shows the synergistic activity on cell death. Pre-clinical investigation on the efficacy of (R)-HZ05 shows that it can be prolonged animal lifespan similar to ibrutinib. (R)-HZ05 use in combination with tyrosine kinase inhibitor demonstrated a superior efficacy on tumor burden reduction and survival than either drug alone. We have demonstrated that the depletion of the pyrimidine nucleotide pool, using DHODH inhibitor, represents a new therapeutic strategy that may benefit MCL patients.
RESUMO
The non-classical HLA class I molecule HLA-G is expressed in trophoblasts where it contributes to maternal-fetal tolerance. HLA-G has been implicated in the control of trophoblast invasion, uterine vascular remodeling, and maintenance of a local immunosuppressive state. Understanding HLA-G biology at the maternal-fetal interface is therefore a critical issue in reproduction. In this regard, we review here: (i) the effects of HLA-G on decidual leucocytes and stromal cells, (ii) the contribution of trogocytosis in HLA-G expression on decidual cells, (iii) its interaction with the ILT2, ILT4 and KIR2DL4 receptors, (iv) the link between HLA-G polymorphism and pregnancy disorders, and (v) the expression of newly-described HLA-G isoforms at the maternal-fetal interface.
Assuntos
Decídua/imunologia , Antígenos HLA-G/imunologia , Complicações na Gravidez/genética , Gravidez/imunologia , Trofoblastos/imunologia , Animais , Feminino , Predisposição Genética para Doença , Antígenos HLA-G/genética , Humanos , Polimorfismo Genético , Complicações na Gravidez/imunologia , Transdução de Sinais , TrogocitoseRESUMO
BACKGROUND: Calcium signaling plays an important role in B lymphocyte survival and activation, and is critically dependent on the inositol-1,4,5-tris-phosphate-induced release of calcium stored in the endoplasmic reticulum (ER). Calcium is accumulated in the ER by Sarco/Endoplasmic Reticulum Calcium ATPases (SERCA enzymes), and therefore these enzymes play an important role in ER calcium homeostasis and in the control of B of cell activation. Because Epstein-Barr virus (EBV) can immortalize B cells and contributes to lymphomagenesis, in this work the effects of the virus on SERCA-type calcium pump expression and calcium accumulation in the endoplasmic reticulum of B cells was investigated. RESULTS: Two Sarco-Endoplasmic Reticulum Calcium transport ATPase isoforms, the low Ca2+-affinity SERCA3, and the high Ca2+-affinity SERCA2 enzymes are simultaneously expressed in B cells. Latency type III infection of Burkitt's lymphoma cell lines with immortalization-competent virus expressing the full set of latency genes selectively decreased the expression of SERCA3 protein, whereas infection with immortalization-deficient virus that does not express the EBNA2 or LMP-1 viral genes was without effect. Down-modulation of SERCA3 expression could be observed upon LMP-1, but not EBNA2 expression in cells carrying inducible transgenes, and LMP-1 expression was associated with enhanced resting cytosolic calcium levels and increased calcium storage in the endoplasmic reticulum. Similarly to virus-induced B cell immortalisation, SERCA3 expression was also decreased in normal B cells undergoing activation and blastic transformation in germinal centers of lymph node follicles. CONCLUSION: The data presented in this work indicate that EBV-induced immortalization leads to the remodelling of ER calcium homeostasis of B cells by LMP-1 that copies a previously unknown normal phenomenon taking place during antigen driven B cell activation. The functional remodelling of ER calcium homeostasis by down-regulation of SERCA3 expression constitutes a previously unknown mechanism involved in EBV-induced B cell immortalisation.
Assuntos
Linfócitos B/metabolismo , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas da Matriz Viral/metabolismo , Linfócitos B/citologia , Linfócitos B/virologia , Western Blotting , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patologia , Linfoma de Burkitt/virologia , Linhagem Celular Tumoral , Transformação Celular Viral , Antígenos Nucleares do Vírus Epstein-Barr/genética , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Homeostase , Humanos , Imuno-Histoquímica , Ativação Linfocitária , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Proteínas da Matriz Viral/genética , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Imatinib and retinoids induce apoptosis in FIP1L1/PDGFRalpha-positive EoL-1 leukemia cells. Although imatinib induces complete remission in most FIP1L1/PDGFRalpha-positive patients, response to imatinib is sometimes suboptimal. In order to enhance the potency of the molecularly targeted therapy of eosinophilic leukemia, we investigated the effect of retinoids combined with tyrosine kinase inhibitors on EoL-1 cells. We demonstrate that retinoids combined with tyrosine kinase inhibitors lead to enhanced apoptosis induction in EoL-1 cells. Our results suggest that tyrosine kinase inhibitors combined with retinoids may constitute a valuable therapeutic approach for sensitive neoplasias that may display enhanced anti-leukemic potency when compared to single drug treatments.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Síndrome Hipereosinofílica , Western Blotting , Linhagem Celular Tumoral , Humanos , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Tirosina Quinases/antagonistas & inibidores , Tretinoína/administração & dosagemRESUMO
Hypereosinophilic syndrome (HES) has recently been recognized as a clonal leukemic lesion, which is due to a specific oncogenic event that generates hyperactive platelet-derived growth factor receptor-alpha-derived tyrosine kinase fusion proteins. In the present work, the effect of retinoids on the leukemic hypereosinophilia-derived EoL-1 cell line and on primary HES-derived cells has been investigated. We show that all-trans-retinoic acid (ATRA) inhibits eosinophil colony formation of HES-derived bone marrow cells and is a powerful inducer of apoptosis of the EoL-1 cell line. Apoptosis was shown in the nanomolar concentration range by phosphatidylserine externalization, proapoptotic shift of the Bcl-2/Bak ratio, drop in mitochondrial membrane potential, activation of caspases, and cellular morphology. Unlike in other ATRA-sensitive myeloid leukemia models, apoptosis was rapid and was not preceded by terminal cell differentiation. Use of isoform-selective synthetic retinoids indicated that retinoic acid receptor-alpha-dependent signaling is sufficient to induce apoptosis of EoL-1 cells. Our work shows that the scope of ATRA-induced apoptosis of malignancies may be wider within the myeloid lineage than thought previously, that the EoL-1 cell line constitutes a new and unique model for the study of ATRA-induced cell death, and that ATRA may have potential for the management of clonal HES.
Assuntos
Apoptose/efeitos dos fármacos , Hipersensibilidade a Drogas/patologia , Eosinófilos/efeitos dos fármacos , Síndrome Hipereosinofílica/patologia , Receptores do Ácido Retinoico/metabolismo , Tretinoína/farmacologia , Linhagem Celular Tumoral , Hipersensibilidade a Drogas/etiologia , Hipersensibilidade a Drogas/metabolismo , Eosinófilos/metabolismo , Eosinófilos/patologia , Células HL-60 , Humanos , Síndrome Hipereosinofílica/tratamento farmacológico , Síndrome Hipereosinofílica/metabolismo , Receptor alfa de Ácido Retinoico , Transdução de Sinais , Células-Tronco/efeitos dos fármacos , Células-Tronco/patologia , Fatores de Poliadenilação e Clivagem de mRNA/biossínteseRESUMO
In this work we demonstrate a differentiation-induced up-regulation of the expression of plasma membrane Ca2+ATPase (PMCA) isoforms being present in various gastric/colon cancer cell types. We found PMCA1b as the major isoform in non-differentiated cancer cell lines, whereas the expression level of PMCA4b was significantly lower. Cell differentiation initiated with short chain fatty acids (SCFAs) and trichostatin A, or spontaneous differentiation of post-confluent cell cultures resulted in a marked induction of PMCA4b expression, while only moderately increased PMCA1b levels. Up-regulation of PMCA4b expression was demonstrated both at the protein and mRNA levels, and closely correlated with the induction of established differentiation markers. In contrast, the expression level of the Na+/K+-ATPase or that of the sarco/endoplasmic reticulum Ca2+ATPase 2 protein did not change significantly under these conditions. In membrane vesicles obtained from SCFA-treated gastric/colon cancer cells a marked increase in the PMCA-dependent Ca2+ transport activity was observed, indicating a general increase of PMCA function during the differentiation of these cancer cells. Because various PMCA isoforms display distinct functional characteristics, we suggest that up-regulated PMCA expression, together with a major switch in PMCA isoform pattern may significantly contribute to the differentiation of gastric/colon cancer cells. The analysis of PMCA expression may provide a new diagnostic tool for monitoring the tumor phenotype.
Assuntos
Diferenciação Celular/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Animais , Transporte Biológico , Células COS , Células CACO-2 , Cálcio/metabolismo , Cálcio/farmacocinética , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Chlorocebus aethiops , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Ácidos Graxos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HT29 , Células HeLa , Humanos , Imuno-Histoquímica , Isoenzimas/genética , Isoenzimas/metabolismo , Microssomos/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND: Endoplasmic reticulum (ER) calcium storage and release play important roles in B lymphocyte maturation, survival, antigen-dependent cell activation and immunoglobulin synthesis. Calcium is accumulated in the endoplasmic reticulum (ER) by Sarco/Endoplasmic Reticulum Calcium ATPases (SERCA enzymes). Because lymphocyte function is critically dependent on SERCA activity, it is important to understand qualitative and quantitative changes of SERCA protein expression that occur during B lymphoid differentiation and leukemogenesis. METHODS: In this work we investigated the modulation of SERCA expression during the pharmacologically induced differentiation of leukemic precursor B lymphoblast cell lines that carry the E2A-PBX1 fusion oncoprotein. Changes of SERCA levels during differentiation were determined and compared to those of established early B lymphoid differentiation markers. SERCA expression of the cells was compared to that of mature B cell lines as well, and the effect of the direct inhibition of SERCA-dependent calcium transport on the differentiation process was investigated. RESULTS: We show that E2A-PBX1+ leukemia cells simultaneously express SERCA2 and SERCA3-type calcium pumps; however, their SERCA3 expression is markedly inferior to that of mature B cells. Activation of protein kinase C enzymes by phorbol ester leads to phenotypic differentiation of the cells, and this is accompanied by the induction of SERCA3 expression. Direct pharmacological inhibition of SERCA-dependent calcium transport during phorbol ester treatment interferes with the differentiation process. CONCLUSION: These data show that the calcium pump composition of the ER is concurrent with increased SERCA3 expression during the differentiation of precursor B acute lymphoblastic leukemia cells, that a cross-talk exists between SERCA function and the control of differentiation, and that SERCA3 may constitute an interesting new marker for the study of early B cell phenotype.
Assuntos
Expressão Gênica , Leucemia de Células B/genética , Leucemia de Células B/patologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Cálcio/metabolismo , Diferenciação Celular/genética , Linhagem Celular Tumoral , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Humanos , Leucemia de Células B/metabolismo , Gradação de Tumores , Estadiamento de Neoplasias , Proteína Quinase C/genéticaRESUMO
Calcium homeostasis of the endoplasmic reticulum (ER) is involved in intracellular signaling pathways and is implicated in major cell functions such as cell growth, differentiation, protein synthesis and apoptosis. The accumulation of calcium in the ER is performed by specific sarco/endoplasmic reticulum calcium transport ATPases (SERCA iso-enzymes). The expression of biochemically distinct SERCA isoforms is cell type dependent and developmentally regulated. This review summarizes pertinent data about the modulation of the expression of SERCA enzymes during the differentiation of normal and tumor cells. These data support the implication of SERCA pumps and especially SERCA3 in the differentiation program of cancer and leukemia cells. During the multi-step process of colon carcinogenesis, the decrease of SERCA3 expression seems to be linked to enhanced APC/ss-catenin/TCF4 signaling and deficient Sp1-like factor-dependent transcription.
Assuntos
Neoplasias do Colo/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Diferenciação Celular , Neoplasias do Colo/patologia , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/patologia , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Isoenzimas/genética , Ativação LinfocitáriaRESUMO
Permanent synovial sarcoma cell lines are invaluable tools for understanding of the biology of this tumor. The present study reports the establishment of a new human cell line, PDSS-26, derived from a surgical specimen of a poorly differentiated synovial sarcoma. PDSS-26 has a doubling time of a 72 hours and grows as a monolayer of spindle cells that retain immunoreactivity for bcl-2 and vimentin. Karyotypic analysis revealed a rearrangement involving chromosomes 17 and 18, at the breakpoints q11.2 and q11.2, respectively, as the only structural aberrations. Analysis by reverse transcriptase polymerase chain reaction showed the presence of the SYT-SSX1 fusion transcript in both the primary tumor and the cell line. Cytoplasmic PTEN staining was detected by immunohistochemistry in both the PDSS-26 cell line and in original tumor, whereas no mutation was identified by automatic sequencing. Thus, PDSS-26 cells could be useful for future functional studies.
Assuntos
Aberrações Cromossômicas , Proteínas de Fusão Oncogênica/análise , Sarcoma Sinovial/genética , Sarcoma Sinovial/patologia , Células Tumorais Cultivadas , Adulto , Cromossomos Humanos Par 17 , Cromossomos Humanos Par 18 , Feminino , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , PTEN Fosfo-Hidrolase , Monoéster Fosfórico Hidrolases/análise , Opinião Pública , Translocação Genética , Proteínas Supressoras de Tumor/análiseRESUMO
The expression of the plasma membrane Ca2+ ATPase (PMCA) isoforms is altered in several types of cancer cells suggesting that they are involved in cancer progression. In this study we induced differentiation of MCF-7 breast cancer cells by histone deacetylase inhibitors (HDACis) such as short chain fatty acids (SCFAs) or suberoylanilide hydroxamic acid (SAHA), and by phorbol 12-myristate 13-acetate (PMA) and found strong upregulation of PMCA4b protein expression in response to these treatments. Furthermore, combination of HDACis with PMA augmented cell differentiation and further enhanced PMCA4b expression both at mRNA and protein levels. Immunocytochemical analysis revealed that the upregulated protein was located mostly in the plasma membrane. To examine the functional consequences of elevated PMCA4b expression, the characteristics of intracellular Ca2+ signals were investigated before and after differentiation inducing treatments, and also in cells overexpressing PMCA4b. The increased PMCA4b expression - either by treatment or overexpression - led to enhanced Ca2+ clearance from the stimulated cells. We found pronounced PMCA4 protein expression in normal breast tissue samples highlighting the importance of this pump for the maintenance of mammary epithelial Ca2+ homeostasis. These results suggest that modulation of Ca2+ signaling by enhanced PMCA4b expression may contribute to normal development of breast epithelium and may be lost in cancer.
Assuntos
Inibidores de Histona Desacetilases/farmacologia , Ésteres de Forbol/farmacologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Regulação para Cima/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Cálcio/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Ácidos Hidroxâmicos/farmacologia , Indóis/farmacologia , Células MCF-7 , Maleimidas/farmacologia , Ácidos Pentanoicos/farmacologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , VorinostatRESUMO
Cellular calcium signaling plays important roles in several signal transduction pathways that control proliferation, differentiation and apoptosis. In epithelial cells calcium signaling is initiated mainly by calcium release from endoplasmic-reticulum-associated intracellular calcium pools. Because calcium is accumulated in the endoplasmic reticulum by sarco/endoplasmic reticulum calcium ATPases (SERCA), these enzymes play a critical role in the control of calcium-dependent cell activation, growth and survival. We investigated the modulation of SERCA expression and function in human lung adenocarcinoma cells. In addition to the ubiquitous SERCA2 enzyme, the SERCA3 isoform was also expressed at variable levels. SERCA3 expression was selectively enhanced during cell differentiation in lung cancer cells, and marked SERCA3 expression was found in fully differentiated normal bronchial epithelium. As studied by using a recombinant fluorescent calcium probe, induction of the expression of SERCA3, a lower calcium affinity pump, was associated with decreased intracellular calcium storage, whereas the amplitude of capacitative calcium influx remained unchanged. Our observations indicate that the calcium homeostasis of the endoplasmic reticulum in lung adenocarcinoma cells presents a functional defect due to decreased SERCA3 expression that is corrected during pharmacologically induced differentiation. The data presented in this work show, for the first time, that endoplasmic reticulum calcium storage is anomalous in lung cancer cells, and suggest that SERCA3 may serve as a useful new phenotypic marker for the study of lung epithelial differentiation.