Transcriptomic profile of human erythroleukemia cells in response to Sargassum fusiforme polysaccharide and its structure analysis / 中国天然药物

Sargassum fusiforme (S. fusiforme) has been used as an ingredient in Chinese herbal medicine for thousands of years. However, there are a limited number of studies concerning its therapeutic mechanism. High performance gel permeation chromatography (HPGPC) analysis showed that the average molecular weight of the S. fusiforme polysaccharide, SFPS 191212, is 43 kDa. SFPS 191212 is composed of mannose, rhamnose, galactose, xylose, glucose, and fucose (at a molar ratio: 2.1 : 2.9 : 1.8 : 15.5 : 4.6 : 62.5) with α- and β-configurations. The present research evaluated the anti-tumor potential of the S. fusiforme polysaccharide in human erythroleukemia (HEL) cells in vitro. To explore the SFPS 191212's apoptosis mechanism in HEL cells, transcriptome analysis was performed on HEL cells that were incubated with SFPS 191212. The inhibitory effect of SFPS 191212 on HEL cell growth was also analyzed. It was found that SFPS 191212 inhibited HEL cell proliferation, reduced cell viability in a concentration-dependent manner, and induced an insignificant toxic effect on normal human embryonic lung (MRC-5) cells. Compared with the control group, transcriptome analysis identified a total of 598 differentially expressed genes (DEGs), including 243 up-regulated genes and 355 down-regulated genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed on all DEGs, and 900 GO terms and 52 pathways were found to be significantly enriched. Finally, 23 DEGs were randomly selected and confirmed by quantitative real-time polymerase chain reaction (qRT-PCR). Moreover, SFPS 191212 down-regulated the PI3K/Akt signal transduction pathway. Our results provide a framework for understanding the effect of SFPS 191212 on cancer cells and can serve as a resource for delineating the anti-tumor mechanisms of S. fusiforme.

Effect of Sargassum fusiforme polysaccharide on apoptosis and its possible mechanism in human erythroleukemia cells / 中国天然药物

This study aimed to investigate the effects of Sargassum fusiforme polysaccharide (SFPS I, II, and III) on the apoptosis and regulation of human erythroleukemia (HEL) cells. The effect of different doses of SFPS on HEL cell growth was detected using the Cell Counting Kit-8 method, and apoptosis was detected by Hoechst staining. Cell cycle distribution and apoptosis were detected using flow cytometry. Expression of the cell cycle gene, p53, antiapoptotic genes, Bcl-xL and Bcl-2, and pro-apoptotic genes, Bax, Bad, and Caspase-3, as well as the expression of the corresponding proteins, were detected using real-time quantitative polymerase chain reaction (qPCR) and Western blot. The results showed that SFPS II and III decreased HEL cell viability and induced HEL cell apoptosis. Different concentrations of SFPS (I, II, and III) were detected that induced much less toxic effect in normal human embryonic lung (MRC-5) cells, and SFPS I increased cell proliferation, indicating its favorable selectivity towards cancer cells. The mechanism by which SFPS induced apoptosis was also found to be related to the induction of cell cycle arrest in the G/G phase and the increased expression of apoptosis-related genes and proteins. We concluded that SFPS induces HEL cell apoptosis, possibly via activation of the Caspase pathway, providing the theoretical basis for the development of SFPS-based anti-tumor drug products.

Eritroleucemias: Recategorización según criterios de la organización mundial de la salud / Erythroleukemias. Recategorization according to the World Health Organization's criteria

La leucemia aguda es la enfermedad oncológica más frecuente en la infancia. La leucemia linfoblástica aguda representa el 75% y la mieloblástica aguda el 25% de ellas. La eritroleucemia es una entidad infrecuente, representando menos del 5% de las leucemias mieloblásticas agudas. Su definición ha variado a lo largo del tiempo. La OMS en 2017 define el subtipo de eritroleucemia cuando el porcentaje de eritroblastos representa el 80% de la celularidad total de la médula ósea. El presente trabajo, de tipo analítico, retrospectivo, tuvo como finalidad revisar los hallazgos de morfología, citometría de flujo, citogenética, respuesta al tratamiento y evolución de los casos previamente definidos como eritroleucemia, en nuestro centro, en los últimos 25 años y reclasificar aquellos que no cumplían con los nuevos criterios de la OMS 2017. Entre enero de 1990 y diciembre de 2015, se diagnosticaron 576 casos de leucemia mieloblástica aguda siendo 11 (1.9%) de ellos clasificados como eritroleucemia. Resultaron evaluables 10 casos. La distribución por sexo fue 1:1 y la edad mediana fue 5 (rango: 0.9-14) años. Seis pacientes presentaban antecedentes de síndrome mielodisplásico. Según los nuevos criterios, ninguno de los casos analizados puede ser actualmente definido como eritroleucemia. De acuerdo a la recategorización, fueron definidos como leucemias de subtipos de mal pronóstico, como leucemia aguda indiferenciada, sin diferenciación y megacarioblástica. Solo dos pacientes se encuentran libres de enfermedad, probablemente debido a estos subtipos desfavorables, sumado al antecedente frecuente de mielodisplasia.

Acute leukemia is the most frequent malignant disease in childhood. Acute lymphoblastic leukemia represents 75% and acute myeloblastic leukemia 25% of them. Erythroleukemia is a rare entity, corresponding to less than 5% of acute myeloblastic leukemia. Its definition has changed over the time. WHO in 2017 defines erythroleukemia when the percentage of erythroblasts represent 80% of the total cellularity of the bone marrow aspirate. This analytical and retrospective study was performed with the aim of reviewing morphology, flow cytometry and cytogenetic features, response to treatment and outcome of cases previously defined as erythroleukemia in our center during the last 25 years and, in addition to reclassify those cases which do not meet the new WHO 2017 criteria. From January 1990 to December 2015, 576 patients were diagnosed as acute myeloblastic leukemia and 11 (1.9%) of them were classified as erythroleukemia. Ten cases were evaluable. Sex distribution was 1:1 and the median age at diagnosis was 5 (range: 0.9-14) years. Six of them had presented with previous myelodysplastic syndrome. None of the analyzed cases can be currently defined as erythroleukemia, according to the new criteria. When reclassified, the cases were defined as leukemias of subsets with poor prognosis such as acute undifferentiated leukemia, without differentiation and megakaryoblastic leukemia. Only 2 patients remain leukemia-free and this could be explained both by the unfavorable prognosis of these leukemia subtypes, and the antecedent of myelodysplastic syndrome in most of the cases.

Genetically Engineered In Vitro Erythropoiesis

BACKGROUND: Engineered blood has the greatest potential to combat a predicted future shortfall in the US blood supply for transfusion treatments. Engineered blood produced from hematopoietic stem cell (HSC) derived red blood cells in a laboratory is possible, but critical barriers exist to the production of clinically relevant quantities of red blood cells required to create a unit of blood. Erythroblasts have a finite expansion capacity and there are many negative regulatory mechanisms that inhibit in vitro erythropoiesis. In order to overcome these barriers and enable mass production, the expansion capacity of erythroblasts in culture will need to be exponentially improved over the current state of art. This work focused on the hypothesis that genetic engineering of HSC derived erythroblasts can overcome these obstacles. OBJECTIVES: The objective of this research effort was to improve in vitro erythropoiesis efficiency from human adult stem cell derived erythroblasts utilizing genetic engineering. The ultimate goal is to enable the mass production of engineered blood. METHODS: HSCs were isolated from blood samples and cultured in a liquid media containing growth factors. Cells were transfected using a Piggybac plasmid transposon. RESULTS: Cells transfected with SPI-1 continued to proliferate in a liquid culture media. Fluorescence-activated cell sorting (FACS) analysis on culture day 45 revealed a single population of CD71+CD117+ proerythroblast cells. The results of this study suggest that genetically modified erythroblasts could be immortalized in vitro by way of a system modeling murine erythroleukemia. CONCLUSION: Genetic modification can increase erythroblast expansion capacity and potentially enable mass production of red blood cells.

Effects of p38-2 G4 high-expression on the proliferation and erythriod differentiateon of murine erythroleukemia cells / 解剖学报

Objective To explore the effect of mouse proliferation-associated protein 2G4 (p38-2G4) high-expression on the proliferation and erythriod differentiation of murine erythroleukemia ( MEL ) cells.Methods To establish the recombinant lentivirus vector p 38-2G4-pLJM1, the p38-2G4-pLJM1 was cotransfected into HEK293T cells to obtain lentivirus with pCMV-VSV-G and pCMV-dR8.2.Lentivirus were infected into MEL cells to establish the stably p 38-2G4 high-expressed MEL cells.Western blotting was used to analyse the high-expression efficiency.MTT assay and benzidine staining were applied to detect the cell viability and hemoglobin synthesis of the stable cell line in presence /absence of inducers.Results Western blotting showed that the p38-2G4 high-expression stable cell strain had a higher expression of p38-2G4 as compared to the control group ( MEL) ( P 0.05), but the hemoglobin synthesis had been reduced as compared to the control group (P<0.05).Conclusion p38-2G4 high-expression does not affect the cell viability of MEL cells , but inhibits the erythriod differentiation of MEL cells in three independent experiments .

Expression of NonO protein during murine erythroleukemia cell differentiation induced by sodium butyrate / 解剖学报

Objective To study NonO protein expression changes in murine erythroleukemia ( MEL ) cell differentiation induced by sodium butyrate .Methods Benzidine staining was used to test sodium butyrate-induced erythroid differentiation of MEL cells .We detected NonO protein expression changes in MEL cell differentiation induced by sodium butyrate and NonO protein localization in MEL cells by Western blotting and immunocytochemistry .Furthermore , we applied PCR technique to detect NonO RNA expression in differentiation process .Results We found that NonO protein was upregulated at gene and protein levels in the erythroid differentiation process of MEL cells induced by sodium butyrate and located in cytoplasm and nucleus in MEL cells .Conclusion These results show that NonO protein is closely related with MEL cell differentiation induced by sodium butyrate , which may provide important clues for further study of the mechanism of leukemia .

Comparative proteomic analyses on the differentiation of dimethyl sulfoxide induced murine erythroleukemia cells / 解剖学报

Objective To explore the differentially expressed proteins during erythroid differentiation .Methods The murine erythroleukemia ( MEL) cell were treated by DMSO , and the comparative proteomic was systematically analyzed and identified on different differentiating time points .ratio of cell differentiation and viability were detected by benzidine staining, MTT assay and Ter119 immunofluorescence.Using two-dimensional gel electrophoresis combined with mass spectrometry technology and bioinformatics analysis , we conducted a comparative proteomic analysis on MEL cells during the process of induced differentiation to screen and identify differential proteins .Results The MEL cells induced by 1.2%DMSO for 0 hour, 6hours, 12hours, 24hours, 36hours, 48hours, 72 hours, 96 hours, 120 hours were collected for proteomic analysis, by two-dimensional gel electrophoresis combined with mass spectrometry .A total of 87 kinds of proteins were successfully identified .MEL cells exposed to DMSO at a final concentration of 1.2% for 120 hours reached the highest differentiation rate of 67%.MTT assay showed that 1.0%, 1.2%, 1.4% DMSO had no inhibiting effect on cell vitality.Conclusion DMSO may induce MEL cells to differentiate and have no inhibiting effect on cell vitality .The 87 kinds of differentially expressed proteins from two-dimentional gel electrophoresis may be divided into twelve categories ;the most three parts are 41%enzyme protein, 15%structural protein and 13%regulatory protein.

Aspectos epidemiológicos, clínicos, hematológicos e anatomopatológicos da leucemia eritroide aguda (LMA M6) em gatos / Epidemiological, clinical, hematological, and pathological aspects of acute erythroid leukemia (LMA M6) in cats

Os aspectos epidemiológicos, clínicos, hematológicos e anatomopatológicos da leucemia eritroide aguda (LMA M6) foram estudados em 10 gatos que morreram em consequência dessa condição. Os resultados obtidos diferem daqueles previamente descritos na literatura nos seguintes aspectos: 1) a doença ocorreu na forma de um modelo bimodal relacionado à idade dos gatos afetados, em que 50 por cento tinham 1-3 anos de idade e 50 por cento tinham 10 anos de idade ou mais; 2) quase todos os gatos afetados (87,5 por cento) demonstravam policromasia, possivelmente decorrente de eritropoese extramedular; 3) em todos os casos havia múltiplos focos de células hematopoéticas, principalmente eritropoeticas, em múltiplos órgãos, que incluíam baço (85,7 por cento), linfonodos (71,4 por cento), fígado (57,1 por cento) e rim (28,6 por cento); 4) em alguns casos (28,6 por cento) esses focos podiam ser vistos macroscopicamente, na forma de metástases, mas sempre diferiam histologicamente da medula óssea quanto à proporção dos precursores eritroides envolvidos; 5) em pelo menos um caso ocorreu um continuum patologicum até outra forma de LMA (LMA M4), um fenômeno denominado "infidelidade de linhagem". Esse artigo discute essas diferenças e reforça os critérios fundamentais para se estabelecer o diagnóstico definitivo dessa que é a forma mais importante de leucemia em gatos na nossa região.

The epidemiological, clinical, hematological, and pathological aspects of acute erythroid leukemia (LMA M6) were studied in 10 cats that died from this condition. The results obtained in the present study differ from those previously described in the literature regarding the following aspects: 1) The disease occurred as a bimodal model related to the age of affected cats, in which 50 percent were1-3-years-old and 50 percent were 10 years-old or above; 2) almost all affected cats (87.5 percent) displayed polychromasia, possible resulting from extramedullar erythropoiesis; 3) in all cases there were multiple foci of hematopoietic cells, mainly erythropoietic, in multiple organs including spleen (85.7 percent), lymph nodes (71.4 percent), liver (57.1 percent), and kidney (28.6 percent); 4) in some cases (28.6 percent) these foci could be observed on gross examination as metastasis, but they always histologically differ from bone marrow in regard to the proportion of erythroid precursors involved; 5) at least in one case a continuum patologicum occurred with the condition evolving to another form of LMA (LMA M4), a phenomenon coined as "lineage infidelity". This paper discuss these differences and stresses the fundamental criteria to establish a definitive diagnosis of this condition, which is the most import form of leukemia in cats from our region.

Erythroleukemia Relapsing as Precursor B-cell Lymphoblastic Leukemia / 대한진단검사의학회지

AML relapsing as ALL has rarely been reported. We describe the case of a 62-yr-old man who was diagnosed with erythroleukemia with a complex karyotype and achieved complete hematologic and cytogenetic remission after induction chemotherapy. However, 4 months after the initial diagnosis, he showed relapse with blasts showing a different morphology and immunophenotype and was diagnosed with precursor B-cell ALL. The relapsing precursor B-cell ALL presented with the same leukemic clones as the primary erythroleukemia. Cytogenetic analysis of his bone marrow (BM) at the time of the primary erythroleukemia showed complex karyotypic abnormalities, including monosomy 5 and monosomy 7. At relapse, his BM showed reemergence of these leukemic clones of complex karyotypic abnormalities with clonal switch. To our knowledge, this is the first case of a lineage switch from erythroleukemia to ALL.

Friend erythroleukemia cells induce angiogenesis in chick embryo chorioallantoic membrane and in human umbilical vein endothelial cells

The effects of Friend erythroleukemia cells on angiogenesis were studied in chick embryo chorioallantoic membrane assay and in human umbilical vein endothelial cells. In chorioallantoic membrane assay, the conditioned medium of Friend cells stimulated in vivo angiogenesis to an extent comparable to that observed with Prostaglandin El, used as positive control. Prostaglandin El added to conditioned medium of Friend cells did not further increase angiogenesis. Conditioned medium of Friend erythroleukemia cells also stimulated proliferation of human umbilical vein endothelial cells to an extent comparable to that observed with fetal bovine serum, used as positive control. Conditioned medium and fetal bovine serum together did not affect human umbilical vein endothelial cells proliferation, as compared to that observed when tested separately. These results seem to indicate that Friend erythroleukemia cells produce and secrete factors stimulating angiogenesis. These findings extend and confirm the hypothesis that successful angiogenesis is necessary for development of leukemias.

A Case of Acute Erythroleukemia in a Human Immunodeficiency Virus-Infected Patient / 대한진단검사의학회지

We report a case of acute myelogenous leukemia (AML) developed in a human immunodeficiency virus-infected patient, which is a very rare event. The subtype of AML was initially determined as AML M6a (French-American-British classification), but it was transformed into AML M5b in the course of treatment. Blasts showed very intense staining with periodic acid-Schiff and multiple complex chromosomal abnormalities in the karyotype.

Construction of granulocyte-macrophage colony stimulating factor gene eukaryotic expressing plasmid and identification of its biological activity / 实用儿科临床杂志

Objective To construct mouse granulocyte-macrophage colony stimulating factor (mGM-CSF) gene eukaryotic expressing plasmid pcDNA3-GM-CSF, to transfect the recombinant into erythroleukemia cell line FBL-3, and identify their biological activity.Methods GM-CSF gene eukaryotic expressing plasmid was constructed by subclone and recombinant was transfected into FBL-3 cells by electroporation. After screening by G418 and cloning by limiting dilution,we obtained positive cell clones(FBL-3-GM-CSF). PCR and RT-PCR were used to identify the integration and stable expression of GM-SF gene in FBL-3-GM-CSF cells. The biological activity was confirmed by the hematopoietic progenitor cell proliferative assay and hematopoietic progenitor cell colony formation assay. Results Mouse GM-CSF cDNA was amplified from the prokaryotic expressing plasmid PET-30a(+)-GM-CSF by PCR firstly and BamH Ⅰ and EcoRⅠrestriction sites were introduced. The inserted fragment was cut by BamH Ⅰ and EcoR Ⅰ digestion and ligated into pcDNA3 vector. The pcDNA3-GM-CSF eukaryotic expressing plasmid was constructed. The recombinant was cleared with appropriate endoneucleases and sequenced. The findings showed that the orientation of the insert was correct, while no rearrangement or mutation was found. PCR and RT-PCR assay showed that GM-CSF gene had integrated into FBL-3-GM-CSF cells and stably expressed. The hematopoietic progenitor cell proliferative assay and hematopoietic progenitor cell colony formation assay demonstrated that the cultured supernatant of FBL-3-GM-CSF cells of expressing GM-CSF should obviously stimulate proliferation of murine marrow mononuclear cells, and could stimulate hematopoietic progenitor cell colony formation. The number of colony formation was 54.67?4.83. The rate of colony formation was 0.547 %.Conclusions GM-CSF gene eukaryotic expressing plasmid is constructed successfully. A cell clone, which can express stably GM-CSF gene and possess biological activity,is obtained. Our studies have founded the base for the preparation of GM-CSF gene-modified vaccine of tumor cell and the study of feasibility of immune therapy of leukemia.

Relation of p53 Protein Overexpression to Subtypes of Acute Erythroleukemia / 대한임상병리학회지

BACKGROUND: Acute erythroleukemia (AEL), FAB-M6 is a rare heterogenous disorder diagnosed by myeloblasts more than 30% of nonerythroid cells (NEC). Pure erythroleukemia (Di Guglielmo disease) with an excess of proerythroblasts can be classified as MDS or M0. An aberration of the p53 gene in acute myelogenous leukemia is rare, but related to complex karyotypes with poor prognosis. METHODS: To evaluate heterogenous features, 32 cases of AEL or suspicious AEL were categorized as consisting of more than 50% erythroblasts and M6a with more than 30% myeloblasts of NEC, M6b with more than 30% proerythroblasts of all erythroblasts, and M6c with more than 30% both myeloblasts and proerythroblasts. The relation of the p53 protein overexpression and chromosomal abnormalities to AEL and these subtypes was investigated. RESULTS: There were 18 M6a, 6 M6b, and 8 M6c. The percentage of erythroblasts was M6a 58.7%, 77.7% M6b, and 67.2% M6c. The percentage of myeloblasts in NEC was M6a 53.6%, M6b 4.3%, and M6c 39.2%. The percentage of proerythroblasts in all erythroblasts was M6a 5.6%, M6b 56.2%, and M6c 34.1%. Survivals of M6b and M6c were significantly shorter than M6a (12.0 vs. 2.0 vs. 2.0 months, P=0.003). Five of 11 cases showed complex karyotypes (1 M6a, 2 M6b, 2 M6c), of -5, 5q-, -7, 7q-, -17 and/or 17p-, with shorter survival and poor response. The p53 protein overexpression was M6a 27.3%, M6b 100%, and M6c 83.3%. The p53 protein overexpression was positive in all 5 cases of multiple complex karyotype with frequent treatment failure or shorter survival, but was negative in 5 normal karyotypes. CONCLUSTIONS: The occurrence of complex karyotypes and aberration of the p53 gene frequently observed in M6b and M6c subtypes of acute erythroleukemia would be considered in establishing a new and innovative treatment to target neoplastic proerythroblasts that are resistant to standard therapy for acute myelogenous leukemia.

Erythroleukemia Related to Chronic Ebstein-Barr Virus Infection / 대한소아혈액종양학회지

It is well known that the Ebstein-Barr virus causes not only infectious mononucleosis in the most cases, but also occasionally causes a variety of hematologic disorders such as anemia (hemolytic anemia or aplastic anemia), thrombocytopenia, neutropenia and malignancies such as Burkitt lymphoma and nasopharyngeal carcinoma. Furthermore, recently it is has been reported that it causes leukemia even though very rarely. The authors report a case of erythroleukemia possibly related to chronic Ebstein-Barr virus infection in a 14-year-old boy with brief review of related literatures.

A Case of Secondary Pulmonary Alveolar Proteinosis Accompanied by Acute Erythroleukemia / 대한내과학회지

Pulmonary alveolar proteinosis is a rare disease, which hallmark is a dense accumulation of PAS positive phospholipid material within alveolar sac. Pulmonary alveolar proteinosis is classified as primary form of unknown etiology and secondary form associated with other diseases. We report a case of secondary pulmonary alveolar proteinosis associated with acute erythroleukemia. A C year old male patient complained of nonproductive cough and general weakness, and presented fine inspiratory crackles at both lower lung field. Chest radiographs and high resolution CT scans showd a lobular pattern of ground-grass opacity with interlobular septal thickening in the center field of the both lungs, Bone marrow aspiration and biopsy revealed acute erythroleukemia. Open lung biopsy revealed PAS positive eosinophilic granular material filled in alveoli. He was treated with TAD chemotherapy, but died from multiorgan failure with pneumonia 22days after chemotherapy.