OBJECTIVETo investigate the expression of rat protamine (RP) gene in MEL cells and the effect on cell growth.

METHODSEukaryotic expression plasmid pCR-3.1-RP was constructed and transfected into MEL cells. The changes of cell growth rate, mitotic index and colony-forming rate in semi-solid medium were investigated.

RESULTSTransfected MEL cells showed lower growth rate, mitotic index and colony-forming rate. Volumes of cells were reduced and reduction of RNA transcription was observed.

CONCLUSIONThese results suggest that expression of RP in MEL cell may inhibit the cell growth and proliferation.

Animals ; Cell Division ; Leukemia, Erythroblastic, Acute ; genetics ; pathology ; Plasmids ; Protamines ; genetics ; metabolism ; Rats ; Transfection ; Tumor Cells, Cultured

Tanshinone II-A is a derivative of phenanthrene-quinone isolated from Salvia miltiorrhiza BUNGE, a traditional herbal medicine that is known to induce antiinflammatory, anti-oxidative and cytotoxic activity. We have examined cellular effects of Tanshione II-A on HL60 human promyelocytic leukemic cells and K562 human erythroleukemic cells. Tanshione II-A induced a dose- and time-dependent DNA fragmentation into the multiples of 180 bp and specific proteolytic cleavage of poly(ADP-ribose) polymerase in both cell lines. PI-staining and flow cytometry analysis of K562 cells following Tanshione II-A treatment showed an increase of the cells possessing hypodiploid DNA indicative of apoptotic state of cells. Caspase-3 activity was significantly increased during Tanshinone II-A treatment of both HL60 and K562 cells, whereas caspase-1 activity was not changed. These results suggest that Tanshione II-A induced HL60 and K562 cellular apoptosis that may be associated with the selective members of caspase family. Copyright 2000 Academic Press.
Antineoplastic Agents, Phytogenic/pharmacology* ; Antineoplastic Agents, Phytogenic/chemistry ; Apoptosis/physiology* ; Caspases/metabolism* ; Caspases/drug effects* ; Cell Cycle/drug effects ; DNA Fragmentation/drug effects ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal/pharmacology* ; Drugs, Chinese Herbal/chemistry ; Enzyme Activation/drug effects ; HL-60 Cells/pathology ; HL-60 Cells/metabolism ; HL-60 Cells/drug effects ; Human ; Lamiaceae/chemistry ; Leukemia/pathology* ; Leukemia/metabolism ; Leukemia/drug therapy ; Leukemia, Erythroblastic, Acute/pathology ; Leukemia, Erythroblastic, Acute/metabolism ; Leukemia, Erythroblastic, Acute/drug therapy ; Phenanthrenes/pharmacology* ; Phenanthrenes/chemistry ; Tumor Cells, Cultured

OBJECTIVETo explore the role of aquaporin-1 (AQP1) gene in erythroid differentiation of erythroleukemia K562 cells induced by retinoic acid (RA).

METHODSK562 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.

RESULTSRA 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).

CONCLUSIONK562 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.

Aquaporin 1 ; antagonists & inhibitors ; metabolism ; Cell Differentiation ; drug effects ; Humans ; K562 Cells ; Leukemia, Erythroblastic, Acute ; metabolism ; RNA, Messenger ; genetics ; RNA, Small Interfering ; genetics ; Tretinoin ; pharmacology

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.
Antigens, Neoplasm ; metabolism ; Cell Line, Tumor ; Exosome Multienzyme Ribonuclease Complex ; metabolism ; Humans ; K562 Cells ; Leukemia, Erythroblastic, Acute ; metabolism ; Neoplasm Proteins ; metabolism ; Protein Interaction Mapping ; RNA-Binding Proteins ; metabolism ; Subcellular Fractions ; metabolism ; WT1 Proteins ; metabolism

The objective of this study was to investigate the biological characteristics and the therapeutic effect in patients with acute erythroleukemia (AML-M(6)). Morphology, immunophenotype and cytogenetics were retrospectively analyzed in 29 patients with AML-M(6) and were compared with 30 AML-M(2) patients. The results showed that there were immature cells (2% - 10%) and erythroblast, and puncture of bone marrow revealed myelodysplastic features involving multiple hemopoietic lineages in bone marrow of 19 patients. Flow cytometry indicated that the expression frequency of Gly-A in M(6) significantly increased (66.67 +/- 23.86)% and higher than that in M(1), M(2), M(3), M(4) and M(5) (P < 0.01). The expression frequencies of HLA-DR (60.00 +/- 24.79%), CD34 (40.00 +/- 24.79%), CD38 (33.33 +/- 23.86%) in M(6) were high, and the frequencies of myeloid immunophenotypes CD13 (66.67 +/- 23.86%), MPO (33.33 +/- 23.86%), CD33 (46.67 +/- 25.25%), CD15 (33.33 +/- 23.86%), CD117 (46.67 +/- 25.25%) were common as well in M(6). Lymphocytic immunophenotypes CD3, CD4, CD19 were detected in part of patients with M(6), and the expression frequencies of CD4 was 26.67%. The expression frequences of CD38, CD33, CD15, MPO in M(6) were less common than that in M(2) (P < 0.01). In 4 out of 9 M(6) patients the chromosomal abnormatility (44.44%) was seen, in one of which complex chromosome abnormality was found. The complete remmision rate of M(6) patients was 29.41%, and lower than that of M(2) patients (68.18%, P < 0.01). It is concluded that Gly-A is a specific immunophenotype in M(6), which can help to distinguish M(6) from other types of acute myeloid leukemia. Poor clinical therapeutic response may correlated with its biological characteristics.
Adolescent ; Adult ; Antigens, CD ; immunology ; metabolism ; Child ; Chromosome Aberrations ; Female ; Humans ; Immunophenotyping ; Karyotyping ; Leukemia, Erythroblastic, Acute ; immunology ; metabolism ; Male ; Membrane Glycoproteins ; immunology ; metabolism ; Middle Aged ; Retrospective Studies ; Young Adult

OBJECTIVETo explore the mechanism of matrine (Mat) induced human erythroleukemia TF-1 cell apoptosis and its effect on SALL4 expression.

METHODDifferent 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).

RESULTAdministrated 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.

CONCLUSIONIn 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.

Alkaloids ; pharmacology ; Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Gene Expression ; drug effects ; Humans ; Leukemia, Erythroblastic, Acute ; drug therapy ; genetics ; metabolism ; physiopathology ; Quinolizines ; pharmacology ; Transcription Factors ; genetics ; metabolism

OBJECTIVETo explore the effects of Danshen Injection () on inhibition proliferation, inducing apoptosis and its possible mechanisms on human erythroid leukemic (HEL) cells.

METHODSThe 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.

RESULTSThe 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.

CONCLUSIONDanshen 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.

Apoptosis ; drug effects ; Base Sequence ; Cell Proliferation ; drug effects ; DNA Primers ; Down-Regulation ; drug effects ; Humans ; Janus Kinase 2 ; genetics ; metabolism ; Leukemia, Erythroblastic, Acute ; enzymology ; metabolism ; pathology ; Mutation ; Phosphorylation ; Plant Extracts ; pharmacology ; Polymerase Chain Reaction ; Salvia miltiorrhiza ; chemistry

OBJECTIVESearching for more potent and less toxic HMBA-related agents.

METHODSHuman erythroleukemia cell K562, murine erythroleukemia cell (MEL) and its sub-line MEL DS19 were used as target cells to select a cell line which is the most sensitive to HMBA, then analyzed the activity of inducing differentiation of two new designed HMBA derivatives: HMBPA [hexamethylenebi (3-pyridin) amide] and Co-HDTA (ethylenediaminetetra acetic acid cobalt) using cell biology, cytochemical and molecular biology techniques.

RESULTSWe found that the MEL DS19 cells were most sensitive to HMBA (benzidine positive, B+ approximately 76%). Co-HDTA can inhibit the growth of MEL DS19, but induces differentiation just in a small population (B+ 2% approximately 4.5%). Between 0.02 approximately 5 micromol/L, HMBPA induces 3% approximately 8% cells committed to differentiation with little inhibition of cell proliferation. 1 micromol/L HMBPA and 2 mmol/L HMBA together, can obviously increase the percentage of differentiated cell (B+ approximately 72%), inhibit DNA synthesis and accelerate beta-globin transcription.

CONCLUSIONThe new HMBA derivatives may provide potential cancer differentiation inducers.

Acetamides ; chemistry ; pharmacology ; Animals ; Antineoplastic Agents ; pharmacology ; Cell Differentiation ; drug effects ; Cell Division ; drug effects ; Globins ; biosynthesis ; genetics ; Humans ; K562 Cells ; Leukemia, Erythroblastic, Acute ; metabolism ; pathology ; Mice ; Pyridines ; chemistry ; pharmacology ; RNA, Messenger ; genetics ; Tumor Cells, Cultured

OBJECTIVETo observe matrine-induced erythroid differentiation of K562 cells in relation to activation of the apoptotic pathway in vitro.

METHODSK562 cells were cultured in the presence or absence of matrine at different concentrations for 4 days, and the morphological and ultramicrostructural changes of the cells were observed using inverted microscopy and transmission electron microscopy, respectively. The expression of apoptosis-related protein p27kip1 was detected by immunocytochemistry.

RESULTSCompared to untreated K562 cells, the cells treated with matrine at 0.10 g/L exhibited apoptostic characteristics in the cellular morphology and ultramicrostructure, with the expression of p27kip1 protein upregulated in a concentration- and time-dependent manner.

CONCLUSIONMatrine-induced erythroid differentiation of K562 cells is associated with cell apoptosis, and upregulation of p27kip1 protein expression may play a crucial role in the process of apoptosis.

Alkaloids ; pharmacology ; Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; physiology ; Cyclin-Dependent Kinase Inhibitor p27 ; biosynthesis ; Dose-Response Relationship, Drug ; Humans ; Immunohistochemistry ; K562 Cells ; Leukemia, Erythroblastic, Acute ; metabolism ; pathology ; Microscopy, Electron, Transmission ; Quinolizines ; pharmacology ; Signal Transduction ; drug effects ; Time Factors

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.
Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Emodin ; pharmacology ; Glycogen Synthase Kinase 3 ; metabolism ; Glycogen Synthase Kinase 3 beta ; HSP70 Heat-Shock Proteins ; metabolism ; Humans ; Leukemia, Erythroblastic, Acute ; metabolism ; pathology ; Proto-Oncogene Proteins c-akt ; metabolism