RESUMEN
BACKGROUND: Approximately 15% to 24% of essential thrombocythemia (ET) and 25-35% of primary myelofibrosis cases carry a mutation in the calreticulin (CALR) gene. Sanger sequencing, qPCR, high resolution melt or targeted next generation sequencing usually used to detect these mutations are expensive and require costly equipment. Nevertheless, type 1 CALR mutations are detectable by using polymerase chain reaction (PCR) and agarose gel electrophoresis. AIM: To offer the use of the allele-specific reverse transcription (RT) PCR for rapid low-cost detection of the type 2 mutation in the CALR gene. MATERIALS AND METHODS: Allele-specific primers designed for detecting type 2 mutation (5-bp insertion; c.1154_1155 ins TTGTC) of the CALR gene were used for allele-specific RT-PCR analysis of cDNA of the patient with JAK2-, MPL-negative ET, whose mutation in CALR gene has been identified by Sanger sequencing. RT-PCR samples were analyzed by agarose gel electrophoresis. RESULTS: The type 2 mutation (K385fs*47 ins5) in CALR gene was detected by Sanger sequencing in JAK2- and MPL-negative ET patient. The cDNA obtained was then re-analyzed by using allele-specific RT-PCR with newly designed primers. Normal and type 2 mutation alleles of the CALR gene were detected by gel electrophoresis. The results of allele-specific RT-PCR were consistent with the data of Sanger sequencing. CONCLUSION: Allele-specific RT-PCR analysis may be used for the fast low-cost detection of the major type 2 mutation (ins 5) of the CALR gene in patients with MPNs.
Asunto(s)
Trastornos Mieloproliferativos , Neoplasias , Trombocitemia Esencial , Alelos , Calreticulina/genética , ADN Complementario , Humanos , Janus Quinasa 2/genética , Mutación , Trastornos Mieloproliferativos/diagnóstico , Trastornos Mieloproliferativos/genética , Neoplasias/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trombocitemia Esencial/genéticaRESUMEN
For more than 35 years after Chornobyl catastrophe, about 5 million people in Ukraine, Republic of Belarus and Russian Federation inhabit the territories that are residually contaminated with long-lived radionuclides such as 137Cs, 90Sr. The previous studies of the Reference Laboratory operating at RE Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology allowed specifying the effects of the protracted low dose irradiation on the state of the hematopoietic and lymphoid tissues resulting in the increased proportion of the B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma and acute myeloid leukemia among the patients referred from the contaminated areas of Ukraine. Since the beginning of 2020, these effects of radiation were superimposed by the factors associated with COVID-19 pandemic. SARS-CoV-2 infection is associated with the significant impact on hematopoiesis and immune system. Particular attention should be given to the role of such combined burden in the development of the immunodeficiency-associated lymphoid neoplasms. The extensive studies of the combined effects of low dose irradiation and COVID-19 within the large affected populations could be made a priority in future endeavors of epidemiologists and oncohematologists.
Asunto(s)
COVID-19/epidemiología , Neoplasias Hematológicas/epidemiología , Radiación Ionizante , SARS-CoV-2/patogenicidad , COVID-19/complicaciones , COVID-19/virología , Accidente Nuclear de Chernóbil , Neoplasias Hematológicas/complicaciones , Neoplasias Hematológicas/virología , Humanos , Dosis de RadiaciónRESUMEN
The 2017 revision of WHO Classification of tumors of hematopoietic and lymphoid tissues contains separate chapters on the immunodeficiency-associated lymphoproliferative disorders. In this mini-review, the brief description of pathological, immunophenotypical and clinical features of lymphoid neoplasms associated with primary immune disorders, HIV infection, those arising in post-transplant setting and other lymphoproliferative disorders (excluding those induced by radiation) is given. The heterogeneous spectrum of these lymphoid malignancies is specified by the nature of those factors that are capable to induce immune suppression or chronic antigenic stimulation of immune system. Taking into account the full swing of SARS-CoV-2 pandemic and our ignorance of the ability of this virus to induce the sustained stimulation of immune system, we could not exclude the high risk of autoimmune diseases and lymphoid neoplasms in the long-term post-pandemic period. In this context, the role of angiotensin-converting enzyme 2 as well as some recently reported cell receptors for SARS-CoV-2 cell entry should be considered as far as some of them (CD147, CD26) could be tumor-associated antigens.
Asunto(s)
COVID-19/epidemiología , Linfoma/epidemiología , Trastornos Linfoproliferativos/epidemiología , Enzima Convertidora de Angiotensina 2/fisiología , Antígenos/fisiología , COVID-19/complicaciones , Infecciones por VIH , Humanos , Síndromes de Inmunodeficiencia/complicaciones , Síndromes de Inmunodeficiencia/epidemiología , Inmunofenotipificación , Linfoma/complicaciones , Trastornos Linfoproliferativos/complicaciones , Trasplante de Órganos/efectos adversos , Pandemias , Receptores de TrasplantesRESUMEN
Placental-like alkaline phosphatase (PLAP) is expressed by many tumors and can be detected in sera of patients with various cancers. Its aberrant expression has been considered to be potentially useful as tumor marker. However, the biological background of the role of this aberrant alkaline phosphatase (AP) in cancer is still unclear. The expression of various forms of AP in cells of chronic myeloid leukemia (CML) has not yet been studied. AIM: To analyze the expression patterns of various AP forms in cells originated from CML patients in blast crisis and to modify their expression by vitamin E. MATERIALS AND METHODS: RNA extracted from leukemic cells was converted to cDNA and real-time reverse transcription polymerase chain reaction was performed using SYBR Green protocol with primers to tissue non-specific alkaline phosphatase (TNAP), intestinal alkaline phosphatase and CCAAT-enhancer-binding proteins alpha (C/EBPα). To analyze the modulation of expression of APs and C/EBPα, CML cells were incubated with 100 µM vitamin E. RESULTS: We have observed the aberrant expression of mRNA intestinal alkaline phosphatase in CML cells that upon sequencing demonstrated the significant alignment with PLAP sequence while no gene homology with tissue placental alkaline phosphatase (PAP) was revealed. Vitamin E decreases mRNA PLAP expression and increases mRNA TNAP expression. Moreover, along with down-regulation of aberrant PLAP and up-regulation of TNAP, vitamin E increases C/EBPα mRNA expression. CONCLUSION: The loss of TNAP in CML may contribute to pathogenesis of this disease. PLAP may be considered as a putative target in differentiation therapies in myeloid neoplasms. Our findings suggest the potential role of vitamin E as the inducer of differentiation potential of leukemic cells in CML.
Asunto(s)
Fosfatasa Alcalina/genética , Biomarcadores de Tumor/genética , Crisis Blástica/enzimología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Isoenzimas/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Vitamina E/farmacología , Crisis Blástica/genética , Crisis Blástica/patología , Línea Celular Tumoral , Regulación hacia Abajo , Humanos , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Regulación hacia ArribaRESUMEN
According to the modern concept, leukemic stem cells (LSC) in acute myeloid leukemia (AML) are distinct from the bulk of leukemic cells in bone marrow and peripheral blood of AML patients. Nevertheless, LSC are responsible for managing all the hierarchy of the bulk of leukemic blast populations. This mini-review provides brief information on the distinctive features of LSC and blast cells in cytologically recognized types of AML. The study of different phenotypes of LSC and blast cells in AML with the aid of up-to-date flow cytometric techniques is important both for the deep insight into the mechanisms of leukemogenesis and development of novel strategies of target therapy. The urgent need for extending the diagnostic panel of monoclonal antibodies used for diagnosing AML is beyond doubt.
Asunto(s)
Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Biomarcadores , Médula Ósea/metabolismo , Médula Ósea/patología , Humanos , Inmunofenotipificación , Clasificación del Tumor , FenotipoRESUMEN
BACKGROUND: Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder associated with the activity of BCR-ABL fusion oncogene. Tyrosine kinase inhibitors are the current treatment of CML, but secondary mutations finally contribute to therapy resistance and blast crisis of the disease. The search for the novel compounds for the effective control of CML is now in the spotlight. The progression of CML to blast crisis is correlated with down-modulation of C/EBP alpha. Therefore, C/EBP alpha may be considered as a putative target in differentiation therapies in myeloid leukemias. The aim of the study was to assess the potential of vitamin E as the possible inducer of C/EBP alpha expression in BCR-ABL-positive CML K562 cells. MATERIALS AND METHODS: RNA extracted from K562 cells cultured with valproic acid or vitamin E was converted to cDNA, RT-PCR reactions were carried out using HotStarTaq DNA polymerase with primers for C/EBP alpha and granulocyte colony-stimulating factor receptor (G-CSFR). RESULTS: We have not found detectable expression of C/EBP alpha in K562 cells. Upon 48-h culture with vitamin E at a dose of 100 µM, K562 cells expressed both C/EBP alpha and G-CSFR. CONCLUSION: Vitamin E restored the expression of C/EBP alpha mRNA in chronic myelogenous leukemia K562 cells. In this setting, G-CSFR expression in vitamin E treated K562 cells seems to suggest the activation to granulocytic differentiation. It should be further elucidated whether such effects of vitamin E on C/EBP alpha transcription factor are direct or mediated indirectly due to antioxidant properties of vitamin E.
Asunto(s)
Antineoplásicos/farmacología , Proteína alfa Potenciadora de Unión a CCAAT/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Receptores de Factor Estimulante de Colonias de Granulocito/genética , Vitamina E/farmacología , Proteína alfa Potenciadora de Unión a CCAAT/biosíntesis , Línea Celular Tumoral , Proteínas de Fusión bcr-abl/genética , Regulación Leucémica de la Expresión Génica , Humanos , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocito/biosíntesis , Activación Transcripcional/efectos de los fármacos , Ácido Valproico/farmacologíaRESUMEN
Chornobyl impact on the health of adult population in Ukraine, Belarus and Russian Federation was a subject of several studies. However, the studies of the effects of Chornobyl on leukemia in adult populations in post-Soviet countries are scarce and the results are contradictory up to present. The results of the epidemiological studies of the oncohematological consequences of Chornobyl accident are briefly reviewed with particular focus on pre-Chornobyl and post-Chornobyl trends in leukemia incidence in Ukraine, Belarus and Russian Federation as well as in small territories of these countries with various levels of radionuclide contamination. This article is a part of a Special Issue entitled "The Chornobyl Nuclear Accident: Thirty Years After".
Asunto(s)
Accidente Nuclear de Chernóbil , Neoplasias Hematológicas/epidemiología , Neoplasias Hematológicas/etiología , Neoplasias Inducidas por Radiación/epidemiología , Adulto , Humanos , Incidencia , República de Belarús/epidemiología , Federación de Rusia/epidemiología , Ucrania/epidemiologíaRESUMEN
Exposure to ionizing radiation is associated with increasing risk of various types of hematological malignancies. The results of major studies on association of leukemias and radiation exposure of large populations in Japan and in Ukraine are analyzed. The patterns of different types of leukemia in 295 Chernobyl clean-up workers diagnosed according to the criteria of up-to-date World Health Organization classification within 10-25 years following Chernobyl catastrophe are summarized. In fact, a broad spectrum of radiation-related hematological malignancies has been revealed both in Life Span Study in Japan and in study of Chernobyl clean-up workers in Ukraine. The importance of the precise diagnosis of tumors of hematopoietic and lymphoid tissues according to up-to-date classifications for elucidating the role of radiation as a causative factor of leukemias is emphasized. Such studies are of high importance since according to the recent findings, radiation-associated excess risks of several types of leukemias seem to persist throughout the follow-up period up to 55 years after the radiation exposure.
Asunto(s)
Leucemia/etiología , Neoplasias Inducidas por Radiación/etiología , Accidente Nuclear de Chernóbil , Accidente Nuclear de Fukushima , Humanos , Leucemia/epidemiología , Linfoma/epidemiología , Linfoma/etiología , Neoplasias Inducidas por Radiación/epidemiologíaRESUMEN
Classical and up-to-date models of hematopoietic lineage determination are briefly reviewed with the focus on myeloid-based models challenging the existence of the common progenitor for T cells, B cells and NK cells. The analysis of immunophenotype of leukemic blast cells seems to be a promising approach for interpreting some controversies in the schemes of normal hematopoiesis. The literature data as well as our own findings in the patients with various types of acute leukemias are in favor of the concept postulating that common myeloid-lymphoid progenitors giving rise to T and B cell branches retain the myeloid potential. The similarity of some immunophenotypic features of blast cells in pro-B acute lymphoblastic leukemia and acute monoblastic leukemia is consistent with monocyte origin postulated in the studies of normal hematopoiesis. Study of acute leukemias may be the challenging area of research allowing for new insight into the origin of hematopoietic cell lineages.
Asunto(s)
Hematopoyesis , Leucemia/patología , Células Madre Neoplásicas/patología , Animales , Linfocitos B/citología , Linfocitos B/patología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/patología , Humanos , Células Asesinas Naturales/citología , Células Asesinas Naturales/patología , Células Mieloides/citología , Células Mieloides/patología , Linfocitos T/citología , Linfocitos T/patologíaRESUMEN
UNLABELLED: Photodynamic therapy (PDT) is considered as a possible alternative approach to overcoming multidrug resistance (MDR). Analysis of cross-resistance to PDT in cells with different MDR pathways and resistance levels seems to be advantageous for elucidating the general mechanisms of cancer cell resistance to various treatment modalities. AIM: The aim of the study was to clarify whether the Jurkat/A4 leukemia cells with MDR phenotype are cross-resistant to PDT. METHODS: Human T-cell acute lymphoblastic leukemia line Jurkat and Jurkat/A4 subline with MDR phenotype were used. 5-Aminolevulinic acid (ALA) and Photolon (a complex of chlorine-e6 and polyvinylpyrrolidone; PL) or gold nanocomposite of PL were applied as photosensitizers. The cells were pretreated with photosensitizers and exposed to laser radiation at corresponding wavelengths. The phototoxicity was assessed in trypan blue exclusion test. The hypodiploid cell fraction was analyzed by flow cytometry of propidium iodide-stained cells. Expression of genes related to PDT resistance was analyzed by microarray technique with Affymetrix U133A chips. RESULTS: ALA-mediated PDT resulted in dose-dependent cell death in both lines, the relative photodynamic efficacy in Jurkat/A4 cells being inferior to that in the parental Jurkat cells. There was no correlation between phototoxicity and apoptosis induction both in Jurkat and Jurkat/A4 cells. PL-mediated general phototoxicity in Jurkat cells amounted up to 75% at the maximal photosensitizer dose with about 40% of apoptotic death fraction. PL-phototoxicity in Jurkat/A4 cells was considerably lower. In contrast to Jurkat cells, PL-gold composite did not increase the efficacy of photosensitization as compared to free PL in Jurkat/A4 cells. CONCLUSIONS: Multidrug-resistant Jurkat/A4 cells exhibit reduced sensitivity to phototoxic effect in comparison with parental Jurkat cells independently of nature of the photosensitizer being assayed.
Asunto(s)
Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Fenotipo , Fotoquimioterapia , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Resistencia a Múltiples Medicamentos/genética , Resistencia a Antineoplásicos/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Células Jurkat , Fármacos Fotosensibilizantes/farmacologíaRESUMEN
BACKGROUND: Genetic mechanisms that result in the development and progression of B-cell chronic lymphocytic leukemia (B-CLL) are mainly unknown. We have analyzed gene expression patterns in Ukrainian B-CLL patients with the aim of identifying B-CLL involved / associated genes in order to shed light on the biology of this pathological entity. MATERIAL AND METHODS: The samples of the peripheral blood and bone marrow of 44 Ukrainian B-CLL patients with no characteristics indicative of unfavorable course of the disease such as CD38 were analyzed morphologically and immunocytochemically according to the new WHO classification. Total RNA was isolated, and gene expression levels were determined by microarray method comparing with the sample from 17 healthy donors. RESULTS: We investigated interactions using the Ingenuity Pathway Analysis (IPA) software and found 1191 network eligible up-regulated genes and 3398 Functions/Pathways eligible up-regulated genes, 1225 network eligible down-regulated genes and 2657 Functions/Pathways eligible down-regulated genes. CONCLUSION: In B-CLL patients, gene networks around MYC, HNF1A and HNF4A, YWHAG, NF-κB1 and SP1 are identified as up-regulated; CEBPA, YWHAG, SATB1 and RB1 -- as down-regulated. G protein coupled receptor signaling, arachidonic acid and linoleic acid metabolisms, calcium signaling, metabolism of xenobiotics by cytochrome P450 are found out as significant up-regulated pathways. EIF2 and Cdc42 signaling, regulation of eIF4 and p70S6k signaling, protein ubiquitination pathway and oxidative phosphorylation are the most significant down-regulated pathways obtained in our study. The involvement of NF-κB gene network and upregulated levels of G protein coupled receptor signaling pathway, which has an important role in transcription of NF-κB, are important and need further examination.
Asunto(s)
Accidente Nuclear de Chernóbil , Perfilación de la Expresión Génica , Leucemia Linfocítica Crónica de Células B/genética , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Humanos , Ucrania , Población Blanca/genéticaRESUMEN
BACKGROUND: The complete medical consequences of the long-term exposure of population to ionizing radiation in post-Chernobyl period are still a controversial issue. The molecular biological analysis of malignant diseases of hematopoietic and lymphoid tissues in contaminated territories requires the precise diagnosis based on criteria of novel classifications. AIM: To analyze the relative gene expression of six apoptosis-related genes in different types of tumors of hematopoietic and lymphoid tissues in patients living in areas of Ukraine contaminated with radionuclides in post-Chernobyl period. MATERIAL AND METHODS: The samples of the peripheral blood and bone marrow of 189 Ukrainian leukemia patients and 16 patients with reactive lymphocytosis were analyzed morphologically and immunocytochemically for precise delineation of the main forms and cytological variants of hematological malignancies according to new WHO classification. Expression of six apoptosis-related genes was analyzed in the individual samples of 9 different groups of malignant diseases of hematopoietic and lymphoid tissues and one group of patients with reactive lymphocytosis by quantitative RT-PCR. Expression of genes was assessed relative to that in control group of healthy donors. RESULTS: Up-regulation of six analyzed apoptosisrelated genes is observed in all groups of leukemia. In most groups of leukemia being analyzed, BCL-2 up-regulation level is superior to that of BAX. Prominent MYC up-regulation is observed in B-lymphoblastic leukemia/lymphoma, non-Hodgkin's lymphoma, and T-lymphoblastic leukemia/lymphoma groups. In myelodysplastic/myeloproliferative neoplasms, the striking up-regulation of Fas-1 and P38MAPK is evident. Practically all the groups of leukemia are characterized by stable high ratios of P53 up-regulation. CONCLUSION: In Ukrainian patients, up-regulation of six analyzed apoptosis-related genes is observed practically in all types of malignant diseases of hematopoietic and lymphoid tissues under study. Microarray-based analysis of these samples would be of great importance in terms of elucidating genomic interactions in leukemias and their possible association with ionizing radiation.
Asunto(s)
Apoptosis/genética , Accidente Nuclear de Chernóbil , Leucemia/genética , Médula Ósea/efectos de la radiación , Exposición a Riesgos Ambientales , Regulación Leucémica de la Expresión Génica , Humanos , Leucemia/epidemiología , Leucemia/patología , Tejido Linfoide/citología , Tejido Linfoide/patología , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-myc/biosíntesis , Proteínas Proto-Oncogénicas c-myc/genética , Radiación Ionizante , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/genética , Ucrania/epidemiología , Proteína X Asociada a bcl-2/biosíntesis , Proteína X Asociada a bcl-2/genética , Receptor fas/biosíntesis , Receptor fas/genética , Proteínas Quinasas p38 Activadas por Mitógenos/biosíntesis , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
The data on the verified cases of mature B-cell neoplasms (chronic lymphocytic leukemia - CLL, B-prolymphocytic leukemia, non-Hodgkin's lymphoma in leukemization phase and multiple myeloma - MM; 146 cases in total) in the consecutive group of Ukrainian clean-up workers within 10-25 years after Chernobyl accident are summarized. B-cell neoplasms represent the most prevalent group among all diagnosed neoplasms of hematopoietic and lymphoid tissues in clean-up worker patients under study (49.4%). MM percentage in the patients of Chernobyl clean-up worker group turned out to be significantly higher than in the patients of the general populations studied at the same period. While the percentage of B-CLL is similar in clean-up worker patients and patients of general population, the trend towards younger age of patients with mature B-cell neoplasms in clean-up worker group is evident. The current concepts on the possible association between mature B-cell neoplasms (mainly B-CLL) and radiation exposure are briefly outlined. Only the precise diagnosis of hematopoietic malignancies combining with large-scale analytical epidemiological studies with careful dose assessment and long-term follow-up may represent the basis for resolving the question whether mature B-cell neoplasms may be radiogenic.
Asunto(s)
Linfocitos B/patología , Accidente Nuclear de Chernóbil , Neoplasias Hematológicas/patología , Neoplasias Inducidas por Radiación/patología , Antígenos de Superficie/metabolismo , Neoplasias Hematológicas/epidemiología , Humanos , Masculino , Persona de Mediana Edad , Células Mieloides/patología , Neoplasias Inducidas por Radiación/epidemiología , Linfocitos T/patología , Ucrania/epidemiologíaRESUMEN
BACKGROUND: While multidrug resistance of cancer cells is a well-known phenomenon, little is known on the cross resistance between cytotoxic chemotherapeutical agents and unrelated substances such as natural flavonoids. AIM: To compare the effects of cytotoxic drug, vepeside and natural flavonoid, quercetin in Jurkat cells and their multidrug-resistant subline Jurkat/A4, in particular to analyze the effector mechanisms of apoptosis and the profiles of several pro- and antiapoptotic proteins in these cells upon exposure to vepeside or quercetin. METHODS: Apoptosis and poly (ADP-ribose) polymerase cleavage were assessed by flow cytometry. Expression of apoptosis-related proteins was analyzed by Western blotting. RESULTS: Jurkat/A4 cells are less sensitive to antiproliferative effects of quercetin as compared with the parental Jurkat cell line. While vepeside as well as quercetin initially induces apoptosis in both cell lines, the following survival of the exposed cells is essentially different. In resistant Jurkat/A4 cells, vepeside or quercetin treatment activates significantly less caspase-9 and -3 as compared with that in the parental cells. The expression of Bad and BNip1 proteins in Jurkat/A4 cells is lower than in the parental cell line. At the same time, XIAP and CAS levels in Jurkat/A4 cells increase. Upon apoptosis induction, XIAP and CAS levels in Jurkat cells decrease, this effect being negligible in resistant cells. CONCLUSION: Multidrug-resistant Jurkat/A4 cells exhibit reduced sensitivity to cytotoxic effects of quercetin. The expression profile of Jurkat/A4 cells is characterized by the increased levels of XIAP and CAS representing the endogenous inhibitors of apoptosis.
Asunto(s)
Antineoplásicos/farmacología , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Resistencia a Antineoplásicos/fisiología , Etopósido/farmacología , Expresión Génica/efectos de los fármacos , Quercetina/farmacología , Western Blotting , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Separación Celular , Resistencia a Múltiples Medicamentos , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Células JurkatRESUMEN
AIM: To obtain polyclonal antibodies against recombinant proteins recognizing Bcr domain and fusion region of Bcr-Abl and analyze the patterns of intracellular distribution of Bcr and Bcr-Abl proteins in K562 cells of chronic myelogenous leukemia. METHODS: The coding sequences of DH and PH domains of Bcr-Abl were cloned, and the recombinant proteins were expressed in E. coli. The rabbit polyclonal antibodies were produced and used for immunocytochemical study of Bcr and Bcr-Abl localization in K562 cells. RESULTS: The gene constructs containing sequences coding for DH and PH domains of Bcr-Abl have been obtained. The antibodies with relative specificity to corresponding recombinant proteins differ by the patterns of their intracellular reactivity with Bcr- and Bcr-Abl related structures. While Bcr protein is located predominantly perinuclearly, antibody against hybrid Bcr-Abl protein is reacted with the structures in cell periphery, namely on cell membranes. CONCLUSION: Antibodies against DH and PH domains of Bcr-Abl react with proteins located differently in chronic myelogenous leukemia cells. The difference in intracellular localization of Bcr and Bcr-Abl may be attributable to the different domains interacting with different multiprotein complexes.
Asunto(s)
Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Proteínas Proto-Oncogénicas c-bcr/metabolismo , Animales , Anticuerpos , Especificidad de Anticuerpos , Western Blotting , Línea Celular , Membrana Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Fusión bcr-abl/química , Proteínas de Fusión bcr-abl/inmunología , Proteínas de Fusión bcr-abl/metabolismo , Humanos , Inmunohistoquímica , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Neoplasias , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-bcr/química , Proteínas Proto-Oncogénicas c-bcr/inmunología , Conejos , Proteínas Recombinantes/metabolismoRESUMEN
The apoptotic index, cell cycle progression and caspase-3 activation in K-562 cells induced to differentiate by DMSO or quercetin have been studied. Quercetin treatment of K-562 cells was accompanied by cell cycle arrest in G2/M and apoptosis with caspase-3 activation. In contrast, DMSO-induced differentiation was accompanied by the complete cell cycle arrest in G1/G0 with negligible caspase-3 activation. In spite of the appearance of benzidine-positive cells and the decreased CD71 level in K-562 cells after exposure to quercetin, the analysis of 1H NMR spectra revealed the overall balance in favor of apoptosis, namely the increase in the content of NMR-visible mobile lipid domains and the decreased intensity of choline-containing metabolites.
Asunto(s)
Apoptosis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Dimetilsulfóxido/farmacología , Células Eritroides/efectos de los fármacos , Metabolismo de los Lípidos/efectos de los fármacos , Quercetina/farmacología , Caspasa 3/metabolismo , Técnicas de Cultivo de Célula , Ciclo Celular/efectos de los fármacos , Colina/metabolismo , Células Eritroides/metabolismo , Células Eritroides/patología , Ácidos Grasos/metabolismo , Citometría de Flujo , Humanos , Células K562 , Espectroscopía de Resonancia MagnéticaRESUMEN
The current problems of malignant growth biology, in particular the molecular background of the specific microenvironment of tumor cells and their interaction with stromal cells, which mediates the behavior of tumors and the tumor-host interrelationship were the subject of the International Conference entitled "Tumor Hypoxia and Malignant Progression", a meeting held at the House of Scientists of the NAS of Ukraine in Kyiv, Ukraine, October 1 st to 4 th , 2008. The meeting was hosted by the R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology of the NAS of Ukraine (IEPOR), and was dedicated to the 90 th Anniversary of the National Academy of Sciences of Ukraine. Over the last years, scientists have focused extensively on the problem of tumor hypoxia as a factor promoting tumor progression. It is known that hypoxia, as a constituent of the tumor cell microenvironment as well as aerobic glycolysis, are important features of malignant tumors. The direct correlation between high levels of hypoxia and tumor aggressiveness has been shown in numerous studies. Therefore, hypoxia is regarded as a factor of unfavorable prognosis. There is a number of different methods available for the evaluation of the level of hypoxia, some of which are being applied in the clinical setting. The stimulating impact of hypoxia and hypoxia-associated proteins on neoangiogenesis and vasculogenesis in tumor tissue has been demonstrated. Several studies have focused on the development of agents capable of blocking hypoxia-associated signaling pathways and vasculogenesis in tumor. Recently, the direct association between hypoxia-dependent signaling pathways and expression of factors that mediate inflammation in tumor tissue, in particular tumor-associated macrophages has been shown. To summarize, a better understanding of the relationships between hypoxia-associated signaling pathways, metabolic peculiarities and inflammatory factors that positively influence tumor progression may elucidate not only how the aggressive tumor phenotype is formed but also may assist in the development of new approaches for the treatment of cancer patients.
Asunto(s)
Hipoxia de la Célula/fisiología , Invasividad Neoplásica/patología , Neoplasias/patología , Animales , Progresión de la Enfermedad , HumanosRESUMEN
UNLABELLED: The search for the substances sensitizing cancer cells to apoptosis induction by chemotherapeutic agents is a task of high importance in the modern strategy of anticancer therapy. THE AIM of the study was to investigate the apoptogenic and apoptosis-modulating activities of fucoidan (sulfated polysaccharide) isolated from far-eastern brown seaweeds Fucus evanescens in two human malignant lymphoid cell lines, MT-4 and Namalwa. METHODS: Apoptosis was assessed morphologically and quantified by flow cytometry analysis of cells stained with propidium iodide. Caspase-3 activation was assayed by flow cytometry with the aid of labeled monoclonal antibodies. RESULTS: The fucoidan at 500 microg/ml was not cytotoxic in MT-4 or Namalwa cells even in the setting of long-term presence in culture medium up to 14 days. Nevertheless, pretreatment of MT-4 but not Namalwa cells with fucoidan followed by the exposure to DNA topoisomerase II inhibitor etoposide led to about two-fold increase in the relative apoptotic index as compared with etoposide alone. Apoptosis enhancement of MT-4 cells by fucoidan was not accompanied by further increase in the number of the cells with active form of caspase-3. CONCLUSION: The present findings demonstrate for the first time that fucoidan enhances etoposide induced caspase-dependent cell death pathway in MT-4 but not Namalwa cell line. The mechanisms of such enhancement do not seem to be related directly to caspase-3 activation.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Etopósido/administración & dosificación , Linfocitos/efectos de los fármacos , Polisacáridos/administración & dosificación , Algas Marinas/química , Apoptosis/efectos de los fármacos , Caspasa 3/efectos de los fármacos , Caspasa 3/metabolismo , Línea Celular Tumoral , Activación Enzimática/efectos de los fármacos , Citometría de Flujo , Fucus/química , HumanosRESUMEN
AIM: The precise mechanisms of apoptosis induced by various selenium compounds are not well understood. Therefore, the apoptogenic activity of two inorganic selenium compounds, sodium selenite and sodium selenate, in human T-cell acute lymphoblastic leukemia MT-4 cells was compared focusing on their effects on cell cycle progression and activation of proapoptotic Bax protein. METHODS: Apoptosis and cell cycle distribution of MT-4 cells exposed to inorganic selenium compounds was assessed by flow cytometry upon propidium iodide staining. Bax expression was analyzed by flow cytometry of cells labeled with anti-Bax monoclonals. Extent of DNA damage was assessed by Comet analysis. RESULTS: Sodium selenite induced apoptosis in dose-dependent mode starting from concentrations of 10 microM, while sodium selenate was much less toxic inducing apoptotic cell death only at 300 microM. Sodium selenite but not sodium selenate caused a slight arrest in G2/M phase of cell cycle. The cytotoxicity of sodium selenite was accompanied by DNA damaging effects visualized in DNA comet assay. Nevertheless, the drastic increase in Bax flow cytometry intensity was evident only in selenite-induced apoptosis. CONCLUSION: Sodium selenite induced apoptosis is accompanied by increased Bax expression.
Asunto(s)
Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Compuestos de Selenio/administración & dosificación , Proteína X Asociada a bcl-2/efectos de los fármacos , Línea Celular Tumoral , Ensayo Cometa , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Humanos , Proteína X Asociada a bcl-2/metabolismoRESUMEN
The technique of proton magnetic resonance spectroscopy (1H MRS) was used as the sensitive express method for early specific detection of the apoptotic cells. The technique allows recognition of the changes in signal intensities corresponding to methylene (CH2) and methyl (CH3) protons of the mobile lipid domains (MLD) and choline, which are characteristic of apoptotic rather than of necrotic cells. A strong linear correlation between MLD content (calculated as CH2/CH3 signal intensity ratio) and the number of apoptotic cells in Namalwa or MT4 cell lines has been shown for any inducer of apoptosis used in the study. MLD content estimated by 1H MRS technique correlated significantly with apoptotic cells numbers (r = 0.992) recorded by conventional techniques. The increase in MLD content was registered as early as 60 min after the addition of etoposide coinciding with the time course of caspase-3 activation.