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.

Navitoclax Combined with Daunorubicin Promotes Apoptosis of Erythroleukemia Cell Lines K562, HEL and TF-1 / 中国实验血液学杂志

OBJECTIVE@#To study the effect of apoptotic drug Navitoclax (NTX) combined with chemotherapy drug Daunorubicin (DNR) on apoptosis of erythroleukemia cells.@*METHODS@#K562, HEL and TF-1 cells in logarithmic growth phase were treated with NTX, DNR and combination of the two drugs. CCK-8 test, Annexin V-DAPI double-staining flow cytometry, real-time RT-PCR were used to detect cell growth, cell apoptosis and expression of BAX, BAK, BCL-2, BCL-xl and BIM respectively. The effects of NTX, DNR and combination of the two drugs on apoptosis of K562, HEL and TF-1 cells were compared and analyzed.@*RESULTS@#NTX combined with DNR could significantly inhibit the growth of K562, HEL and TF-1 cells; Apoptosis detection results showed that the apoptotic rate of K562, HEL and TF-1 cells in combination group was significantly higher than that in NTX and DNR single group; the expression level of apoptosis-related genes BAK and BAX in K562 cells in combination group was significantly higher than that in two single drug groups, and the expression level of anti-apoptotic protein genes BCL-2 and BCL-xl was significantly lower than that in two single drug groups (P＜0.05); the expression level of BAK in HEL cells treated with combined drugs for 24 hours was higher than that in DNR group (P ＜ 0.05); the expression level of BCL-2 in TF-1 cells treated with combined drugs for 24 hours was lower than that in two single drugs groups while the expression level of BAK in 48 hours was the highest in combined drugs group, and the expression level of BCL-2 and BCL-xl in combined drugs group was lower than that in NTX group (P＜0.05).@*CONCLUSION@#NTX combined with DNR can significantly promote the apoptosis of erythroleukemia cell lines K562, HEL and TF-1, and induce the expression of apoptosis-related genes. This study provides a new scheme for the clinical treatment of erythroleukemia.

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.

Effects of bufalin on up-regulating methylation of Wilm's tumor 1 gene in human erythroid leukemic cells / 中国结合医学杂志

<p><b>OBJECTIVE</b>To explore the effects of bufalin on inhibiting proliferation, up-regulating methylation of Wilm' tumor 1 gene (WT1) as well as its possible mechanisms in human erythroid leukemic (HEL) cells.</p><p><b>METHODS</b>The HEL cells were treated with bufalin at various concentrations to observe cellular morphology, proliferation assay and cell cycle. The mRNA and protein expression levels of WT1 were detected by reverse transcription polymerase chain reaction (RT-PCR), Western blot and immunocytochemistry, DNA methylation of WT1 and protein expression levels of DNA methyltransferase 3a (DNMT3a) and DNMT3b were analyzed by methylation-specific PCR, and Western blot respectively.</p><p><b>RESULTS</b>The bufalin was effective to inhibit proliferation of HEL cells in a dose-dependent manner, their suppression rates were from 23.4%±2.1% to 87.2%±5.4% with an half maximal inhibit concentration (IC) of 0.046 μmol/L. Typical apoptosis morphology was observed in bufalin-treated HEL cells. The proliferation index of cell cycle decreased from 76.4%±1.9% to 49.7%±1.3%. The expression levels of WT1 mRNA and its protein reduced gradually with increasing doses of bufalin, meanwhile, the methylation status of WT1 gene changed from unmethylated into partially or totally methylated. While, the expression levels of DNMT3a and DNMT3b protein gradually increased by bufalin treatment in a dose-dependent manner.</p><p><b>CONCLUSIONS</b>Bufalin can not only significantly inhibit the proliferation of HEL cells and arrest cell cycle at G/Gphase, but also induce cellular apoptosis and down-regulate the expression level of WT1. Our results provide the evidence of bufalin for anti-leukemia, its mechanism may involve in increasing WT1 methylation status which is related to the up-regulation of DNMT3a and DNMT3b proteins in erythroid leukemic HEL cells.</p>

Paracrine influence of human perivascular cells on the proliferation of adenocarcinoma alveolar epithelial cells

Understanding the crosstalk mechanisms between perivascular cells (PVCs) and cancer cells might be beneficial in preventing cancer development and metastasis. In this study, we investigated the paracrine influence of PVCs derived from human umbilical cords on the proliferation of lung adenocarcinoma epithelial cells (A549) and erythroleukemia cells (TF-1α and K562) in vitro using Transwell® co-culture systems. PVCs promoted the proliferation of A549 cells without inducing morphological changes, but had no effect on the proliferation of TF-1α and K562 cells. To identify the factors secreted from PVCs, conditioned media harvested from PVC cultures were analyzed by antibody arrays. We identified a set of cytokines, including persephin (PSPN), a neurotrophic factor, and a key regulator of oral squamous cell carcinoma progression. Supplementation with PSPN significantly increased the proliferation of A549 cells. These results suggested that PVCs produced a differential effect on the proliferation of cancer cells in a cell-type dependent manner. Further, secretome analyses of PVCs and the elucidation of the molecular mechanisms could facilitate the discovery of therapeutic target(s) for lung cancer.

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.

Citosina Arabinósido 100 mg y 500 mg en solución / Cytosine Arabinoside 100 mg and 500 mg in solution

FORMA FARMACÉUTICA: bulbo DENOMINACIÓN COMÚN INTERNACIONAL: arabinósido de citosina. COMPOSICIÓN: cada bulbo contiene 100 mg y 500 mg de arabinósido de citosina en solución. CATEGORÍA FARMACOLÓGICA: antineoplásico, agente citotóxico, antimetabolito, analógo de las pirimidinas. FARMACOCINÉTICA: la biodisponibilidad por VO es escasa (menor que 20 %). La distribución es amplia y rápida por los tejidos. Atraviesa las barreras placentarias y hematoencefálica, alcanza el LCR hasta 40‒50 por ciento de la concentración plasmática. Es metabolizado por citidina desaminasa, dando lugar fundamentalmente a arabinósido de uracilo, que es un metabolito inactivo y a trifosfato de aracitidina (activo). La desaminación se produce en el hígado, plasma y tejidos periféricos. Se elimina por la orina (± 80 por ciento) en las primeras 24 h. La vida media de eliminación terminal es 1-3 h. INDICACIONES: leucemia linfocítica y mielocítica aguda y leucemia meníngea. También se emplea en esquemas de segunda o tercera línea de linfomas no Hodgkin y leucemia mieloide crónica. Eritroleucemia. CONTRAINDICACIONES: hipersensibilidad conocida a la citosina. Pacientes con depresión de la médula ósea, enfermedades debilitantes e infecciones virales recientes como varicela o herpes zoster. USO EN POBLACIONES ESPECIALES: LM: datos no disponibles. E: categoría de riesgo D PRECAUCIONES: LM: no se conoce su excreción por la leche humana; no obstante, se recomienda suspender la lactancia materna durante la administración del fármaco. CARCINOGENICIDAD: grupo de riesgo 3. Los efectos depresores de la médula ósea de la citarabina pueden dar lugar a un aumento de la incidencia de infecciones, retardo en la cicatrización y hemorragia gingival. Deben ser cuidadosamente monitoreados los recuentos hemáticos. Si el recuento de leucocitos arroja CAN menor que 1 000 células/mm3 y las plaquetas están por debajo de 50 000 celulas/mm3, el tratamiento debe ser interrumpido. Los valores pueden continuar bajando aún después de que la administración de citarabina sea suspendida. El tratamiento puede reiniciarse cuando existen signos evidentes de recuperación de la médula ósea. Cuando se administran de forma rápida altas dosis por vía IV, los pacientes pueden presentar náusea y vómito durante algunas horas después de la inyección; este problema se presenta en forma menos severa cuando se administra por infusión. En pacientes con enfermedad hepática previa se deberán suministrar dosis menores de citosina, ya que en el hígado ocurre el proceso de detoxificación de este medicamento. Cuando tiene lugar una lisis celular rápida, se deben tomar las debidas precauciones para evitar hiperuricemia y hiperuricosuria y el riesgo de nefropatía por ácido úrico. La neurotoxicidad está asociada con los tratamientos de altas dosis y pueden presentarse como: toxicidad cerebelar aguda o puede ser severa con convulsiones y/o coma, incluso suele ser retardada, hasta 3‒8 días después que el tratamiento haya comenzado. El riesgo de toxicidad cerebelar se incrementa cuando el aclaramiento de creatinina sea inferior a 60 mL/min, edad mayor de 50 años, lesión preexistente del SNC y niveles de fosfatasa alcalina mayor que tres veces el límite superior normal. La conjuntivitis es prevenida y tratada con gotas de solución salina y/o corticosteroides. Como profilaxis, las gotas oculares deben comenzarse de 6 a 12 h antes de iniciar el tratamiento con la citarabina, y continuar hasta 24 h después de haber finalizado esta. El término de altas dosis se define como dosis IV de 2 a 3 g/m2/dosis, cada 12‒24 h, por 4‒12 dosis o de 36 g/m2 en monoterapia, generalmente combinado con otros agentes utilizados en tratamientos con altas dosis de quimioterapia. Puede presentarse el llamado síndrome de la citarabina que se caracteriza por fiebre, mialgia, dolor óseo, dolor torácico, rash maculopapular, astenia y conjuntivitis, puede ocurrir de 6 a 12 h después de la administración de la citarabina. Puede ser tratado de manera eficaz con...(AU)

Evaluation of the effect of miR-26b up-regulation on HbF expression in erythroleukemic K-562 cell line

The major hemoglobin in the fetus is hemoglobin F [alpha2gamma2], whereas in adult humans, hemoglobin A [alpha2beta2] is predominately expressed. Several studies have indicated that expression of the HbF subunit gamma-globin might be regulated post-transcriptionally. This could be done by small non-coding RNAs called microRNAs which target mRNAs in a sequence-specific manner and lead to translational repression or mRNA decay. The aim of this study is to evaluate the effect of miR-26b up-regulation on gamma-globin gene expression in K-562 cell line. These cells were grown in RPMI 1640 and pre miR-26b and were transfected within K-562 cell line using lentiviral vector. After RNA extraction and cDNA synthesis in selected days, miRNA up-regulation was confirmed by miRNA real time PCR and then gamma and beta chain and GATA-1 expression were investigated by RT and QRT-PCR. The viability of cells before transfection was 90%. Three and 7 days after transfection, through the use of relative Q-PCR, the gamma chain expression increased 3.7, 6.8 and 3.8 folds and GATA-1 expression increased 2.1, 6.0 and 8.0 in comparison with untransfected cells. The data suggest that miR-26b can be involved in the increase of gamma-globin gene expression in K-562 cell line. We suggest that miR-26b may be a significant therapeutic target for increasing HbF levels in patients with sickle cell disease and beta-thalassemia

Effects of Danshen Injection () on inhibiting proliferation and inducing apoptosis through down-regulation of mutant JAK2 gene and its protein phosphorylation in human erythroid leukemic cells / 中国结合医学杂志

<p><b>OBJECTIVE</b>To explore the effects of Danshen Injection () on inhibition proliferation, inducing apoptosis and its possible mechanisms on human erythroid leukemic (HEL) cells.</p><p><b>METHODS</b>The commercial Chinese patent medicine of Danshen Injection was extracted and isolated from Chinese herb of Salvia miltiorrhiza bung. The inhibition effects of proliferation were assayed by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) method in HEL cells treated by Danshen Injection at various concentrations for 48 h. The cellular apoptosis was observed in morphology, analyzed by flow cytometry with annexin V and propidium iodide (PI) staining, and examined by DNA degradation ladder on agarose gel electrophoresis. Meanwhile, the expression levels of mutant Janus kinasez (JAK2) gene and phosphorylation-JAK2 (P-JAK2) protein were detected by allele specific-polymerase chain reaction and Western blot.</p><p><b>RESULTS</b>The proliferation of HEL cells was effectively inhibited by Danshen Injection in a dose-dependent manner, with suppression rates from 19.46±2.31% to 50.20±5.21%. Typical apoptosis cells was observed in Danshen Injection treated HEL cells, the rates of annexin V positive cells increased obviously in a dose-dependent manner, as well as the DNA degradation ladder of apoptosis revealed on gel electrophoresis. The expression levels of mutant JAK2 gene and P-JAK2 protein reduced gradually with increasing dosage of Danshen injection.</p><p><b>CONCLUSION</b>Danshen Injection could not only significantly inhibit the proliferation, but also induce apoptosis in HEL cells; down-regulation of the mutant JAK2 gene and P-JAK2 protein expressions are probably one of its molecular mechanisms.</p>

Clinical characteristics and prognoses of 167 acute erythroleukemia patients / 中华血液学杂志

<p><b>OBJECTIVE</b>To observe the biological characteristic and the prognoses in patients with acute erythroleukemia (AEL).</p><p><b>METHODS</b>The results of 167 patients with newly diagnosed AEL, from January 2003 and June 2013 in the First Affiliated Hospital of Soochow University, were reviewed by MICM.</p><p><b>RESULTS</b>Flow cytometry analysis indicated that CD13(96.1%), CD33(95.1%), CD117(87.4%) and CD34 (79.4%) were highly expressed in AEL. 56 of 148 (37.8%) AEL patients had a variety of cytogenetic abnormalities, 27 of 148(18.2%) patients were complex karyotype (abnormalities involving 3 or more chromosomes), the abnormalities of chromosomes 3, 5, 7 and 8 were more frequently involved and the most common one was ＋8, accounting for 35.7% of all abnormal karyotype, followed by 5q- (17.9%). Mutation analysis showed CEBPA mutation ratio of AEL patients was 44.0% (11/25), that of NPM1 as 15.4% (4/26). Initial induced remission rate of AEL was 56.6% (30/53), compared by 33.3% (4/12) of MDSM6. Survival analysis showed that the overall survival in female was better than that in male (P=0.047). The overall survival time of transplantation group is significantly longer than chemotherapy group (P=0.000). The OS of 13-39 years old group was the best, 40-49 years old group took second place, >50 years old group appeared to be the worst.</p><p><b>CONCLUSION</b>AEL had its own unique biological features, and allogeneic hematopoietic stem cell transplantation could significantly improve its poor prognosis.</p>

Suppression of amino acid transporter LAT3 expression on proliferation of K562 cells / 华中科技大学学报（医学）（英德文版）

The activity of the mTOR pathway is frequently increased in acute myeloid leukemia, and is tightly related with cellular proliferation. Leucine is tightly linked to the mTOR pathway and can activate it, thereby stimulating cellular proliferation. LAT3 is a major transporter for leucine, and suppression of its expression can reduce cell proliferation. Here, we show that suppression of LAT3 expression can reduce proliferation of the acute leukemia cell line, K562. We investigated the mRNA and protein expression of LAT3 in several leukemia cell lines and normal peripheral blood mononuclear cells (PBMNCs) using RT-PCR and Western blotting. We also evaluated cell viability using a methyl thiazolyl tetrazolium (MTT) assay after blocking LAT3 expression with either shRNA targeted to LAT3 or a small molecular inhibitor BCH (2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid). LAT3 mRNA and protein expression was detected in leukemia cell lines, but not in normal PBMNCs. Using K562 cells, it was found that cellular proliferation and mTOR pathway activity were significantly reduced when LAT3 was blocked with either shRNA or BCH. Our results suggest that leukemia cell proliferation can be significantly suppressed by blocking LAT3. This finding may lead to a new strategy to develop clinical therapy for the treatment of acute myeloid leukemia.

Interaction of human genes WT1 and CML28 in leukemic cells / 华中科技大学学报（医学）（英德文版）

The molecular pathogenesis of leukemia is poorly understood. Earlier studies have shown both Wilms' tumor 1 suppressor gene (WT1) and CML28 abnormally expressed in malignant diseases of the hematopoietic system and WT1 played an important role in leukemogenesis. However, the relationship between molecular CML28 and WT1 has not been reported. Here we described the use of small interfering RNA (siRNA) against WT1 and CML28 in leukemic cell line K562 to examine the interaction between CML28 and WT1. WT1 and CML28 gene expression in transfected K562 cells was detected by using RQ-PCR and Western blotting. K562 cells transfected with WT1-siRNA could greatly decrease both mRNA and protein expression levels of WT1 and CML28. In contrast, CML28-siRNA did not exert effect on WT1. Further, subcellular co-localization assay showed that the two proteins could co-localize in the cytoplasm of K562 cells, but WT1/CML28 complexes were not detected by using immunoprecipitation. It was suggested that there exists the relationship between CML28 and WT1. CML28 may be a downstream target molecule of WT1 and regulated by WT1, which will provide important clues for further study on the role of CML28 and WT1 in leukemic cells.

Analysis of clinical characteristics and prognosis of 13 cases of acute erythroleukemia / 中国实验血液学杂志

The aim of this study was to investigate the clinical characteristics and prognosis of acute erythroleukemia (AEL, AML-M6). The clinical features and results of morphologic, immunophenotypic, cytogenetic and molecular biologic detections were retrospectively analyzed in 13 cases of AEL from 305 acute leukemia patients hospitalized between October 2007 and October 2012. The results showed that the expression of erythroid and non-erythroid cells increased at the same time. The myeloid antigens mainly expressed CD13/CD33/CD117/CD34, while the erythroid antigens expressed Gly and CD71. The karyotypic detection indicated that there were 1 case with normal karyotype, 3 cases with simple karyotypic abnormality and 2 cases with complex karyotypic abnormality, the other cases were not detected. The molecular biological detection found that the poor prognosis gene existed in 5 cases [38.5% (5/13)], including 3 cases with MLL-MLL fusion gene, 1 case with MLL mutation, and 1 cases with NRAS gene mutation, the abnormal genes were not detected in remainder 8 cases. After chemotherapy with decitabine, the complete remission (CR) rate achieved 53.5% (7/13), partial remission (DR) rate achieved 15.4% (2/13). Finally, 8 patients received allo-HSCT, the median overall survival (OS) was 20.7 months, 3 year survival rate was 79%, 3 year disease-free survival rate was 78%. It is concluded that the acute erythroleukemia is a rare subtype of AML, which is transformed from MDS and has harmful genes and poor prognosis. Allo-HSCT and treatment with decitabine may enhance the survival rate of AEL.

Treatment of leukemia with immunized donor cell infusion after nonmyeloablative haploidentical bone marrow transplantation / 中国实验血液学杂志

This study was purposed to investigate the therapeutic effects of early transfusion of immunized donor lymphocytes after haploidentical transplantation by means of mouse model of nonmyeloablative haploidentical bone marrow transplantation. CB6F1 female mouse was served as recipient and C57BL/6 male mouse was served as donor. Each CB6F1 female mouse was subjected to intravenous transfusion with 1×10(6) erythroleukemia (EL9611) cells at day 4 before transplantation, followed with intraperitoneal injection of Ara-C (0.015 g) respectively at day 2 and day 1, then conditioned for BMT with TBI (450 cGy) at day 1 before transplantation. After conditioning (day 0), each of recipients was transplanted with 6×10(7) mixture of bone marrow and spleen cells from the C57BL/6 mice, and was infused with 6 × 10(7) immunized donor lymphocytes at day 15 after transplantation. All treated animals were evaluated for survival, development of leukemia and aGVHD. The donor CD3(+) cell chimerism and sex determining region Y gene (SRY)in recipients were monitored periodically after transplantation. The results showed tht all mice with only inoculation of 10(6) EL9611 cells survived for 15 ± 1 days (n = 4); all mice of other groups obtained the varying degrees of implantation. SRY could be detected at day 30 and 60 after transplantation. The chimerism of donor CD3(+) cells in mixed bone marrow transplantation (MT) group at day 14, 30 and 60 respectively reached 17.95% ± 12.03%, 37.34% ± 2.78% and 47.06% ± 6.1%. In donor lymphocyte infusion (DLI) group it reached 69.78% ± 12.62%, 75% ± 15.97%, 83.41% ± 16.07% at day 30, 45 and 60 after transplantation. The mice of MT and DLI group survived for 66.66 ± 1.47 days and 78.2 ± 7.82 days. It is concluded that the high tumor burden before transplantation can affect donor cell engraftment and prognosis.Early post-transplanted infusion of immunized lymphocytes from donor can help to improve the therapeutic efficacy and survival.

Role of aquaporin-1 gene in erythroid differentiation of erythroleukemia K562 cells induced by retinoic acid / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the role of aquaporin-1 (AQP1) gene in erythroid differentiation of erythroleukemia K562 cells induced by retinoic acid (RA).</p><p><b>METHODS</b>K562 cells were cultured in the presence of 1 µmol/L RA for varying lengths of time, and γ-globin mRNA expression and hemoglobin content in the cells were detected by real-time PCR (RT-PCR) and ultraviolet spectrophotometry, respectively, to evaluate the erythroid differentiation of K562 cells. RT-PCR and Western blotting were used to examine AQP1 expression in the cells following RA treatment. A retroviral expression vector of AQP1 small interfering RNA (pSUPER-retro-puro-shAQP1) was constructed and transfected into K562 cells to establish a K562 cell line with stable AQP1 down-regulation (K562-shAQP1), in which the changes in γ-globin and hemoglobin expressions after RA treatment were detected.</p><p><b>RESULTS</b>RA treatment significantly increased γ-globin and hemoglobin expressions in K562 cells (P<0.01), causing also significantly enhanced AQP1 mRNA and protein expressions over time (P<0.01). Transfection with the recombinant plasmids pSuper-retro-puro-shAQP1 resulted in stable AQP1 suppression in K562 cells (P<0.01), which showed markedly reduced γ-globin and hemoglobin expressions after RA induction as compared to the control K562 cells (P<0.01).</p><p><b>CONCLUSION</b>K562 cells show a significant increase of AQP1 expression after RA-induced erythroid differentiation, and suppression of AQP1 expression can partially block the effect of RA, suggesting the important role of AQP1 in RA-induced erythroid differentiation of K562 cells.</p>

The influence of the total flavonoids of Hedysarum polybotry on the proliferation, cell cycle, and expressions of p21Ras and proliferating cell nuclear antigen gene in erythroleukemia cell line K562 / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the effect of total flavonoids of Hedysarum polybotry on the proliferation, cell cycle, and expressions of p21(Ras) and proliferating cell nuclear antigen (PCNA) gene in erythroleukemia cell line K562.</p><p><b>METHODS</b>The effect of total flavonoids of Hedysarum polybotry on K562 cell line survival was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) reduction assay. The time- and dose-dependent manner was also observed. The cell cycle and apoptosis were analyzed with flow cytometry (FCM). The immunocytochemistry method was applied to quantitatively analyze the effects of flavonoids of Hedysarum polybotry on changes p21(Ras) and PCNA gene expressions.</p><p><b>RESULTS</b>Flavonoids of Hedysarum polybotry (20-100 μg/mL) significantly inhibited the proliferation of K562 cells in a time- and dose-dependent manner. After K562 cells were cultured for 48 h, total flavonoids of Hedysarum polybotry had no significant effect on the apoptosis of K562 cells but showed significantly inhibition (P<0.01), indicating that total flavonoids of Hedysarum polybotry could induce K562 cells arrested at G(0)/G(1) and G(2)/M phases. Compared with the control group, p21(Ras) and PCNA gene expressions were decreased significantly in K562 cells treated with total flavonoids of Hedysarum polybotry (40 and 80 μg/mL, respectively) for 48 h.</p><p><b>CONCLUSION</b>The inhibitory effect on proliferation of K562 cells was observed in the groups treated with flavonoids of Hedysarum polybotry, which might be related to cells arresting.</p>

Effects of emodin on human erythroleukemia cell line HEL / 中国实验血液学杂志

This study was aimed to investigate the inhibitory effect of emodin on the proliferation of HEL cells, the inducing apoptosis effect of HEL cells and their mechanisms. The proliferation inhibition was detected by MTT method; the change of morphology was observed by AO/EB stains; the cell cycle and apoptosis was analyzed by flow cytometry; the expressions of Akt, P-Akt, P-GSK3β and HSP70 proteins were determined by Western blot. The results indicated that emodin displayed significant anti-proliferative effect on HEL cells in a dose dependent manner(r = 0.99), with IC(50) value of 4.19 µmol/L; AO/EB stains showed that the morphology of HEL cells obviously changed after emodin treatment for 24 hours, and at 24 and 48 hours the apoptosis rates of HEL cells treated by emodin were (27.35 ± 1.68)% and (58.49 ± 1.55)% respectively. Compared with blank control group, the cell ratio in G(0)/G(1) phase increased while that in S phase decreased (p < 0.01); the expression of Akt protein was not changed (p > 0.05), and that of P-Akt, P-GSK3β and HSP70 proteins were down-regulated (p < 0.05). It is concluded that emodin efficiently inhibits the HEL cell proliferation and induces apoptosis of HEL cells, which may be related to the down-regulation of P-Akt, P-GSK3β and HSP70 proteins expression.

TF-1 cell apoptosis-inducing effect of matrine and its effect on SALL4 expression / 中国中药杂志

<p><b>OBJECTIVE</b>To explore the mechanism of matrine (Mat) induced human erythroleukemia TF-1 cell apoptosis and its effect on SALL4 expression.</p><p><b>METHOD</b>Different concentrations of the Mat (0.5, 1.0, 1.5, 2.0 g x L(-1) ) were cultured in vitro in TF-1 cells at different time (24, 48, 72 h). Cell proliferation was assayed by MTT. Cell cycle was determined by flow cytometry (FCM). Cell apoptosis was detected by Annexin V and PI double staining method. SALL4 mRNA expression was detected by reverse transcription RT-PCR (RTT-PCR).</p><p><b>RESULT</b>Administrated with Mat (0.5-2.0 g x L(-1)) after 24, 48, 72 h, the proliferation of TF-1 cells were inhibited (P < 0.01) , and in dose- and time-dependent manner. Half inhibitory concentration (IC50 ) was 1.0 g L(-1) at 48 h. After 48 h that the Mat acted on TF-1 cells, the proportion of G0/G1 phase cells increased while compared with the control group, and S phase cells decreased (P < 0.01). Apoptosis were 8.6% , 11.21%, 15.26% , 17.63%, which showed statistically significant difference (P < 0.01) compared with the control group (5.05%). RT-PCR results showed the ratio between SALL4 mRNA expression and beta-actin (internal reference) expression significantly decreased (P < 0.01) with Mat dose increased.</p><p><b>CONCLUSION</b>In a certain range of concentration and time, Mat can inhibit TFT-1 cells proliferation. The mechanism is to make the cells G0/G1 phase blocked, to inhibit SALL4 gene expression and induce cell apoptosis.</p>