Objective To explore the function of fluorescent probe JC-1 in detecting the changes of mitochondrial membrane potential(△?m)in early apoptotic cells.Methods After 2-ME was used to induce MUTZ-1 cell apoptosis,cells were dyed with fluorescent probe JC-1,and then the changes of △?m in the early stage of apoptotic cells were analyzed by flow cytometry or detected under fluorescent microscope. Results The control cells with high △?m are those forming JC-1 aggregates in the inner membrane of mitochondria,thus showing orange-red fluorescence.2-ME caused decrease of △?m in MUTZ-1 cells,in which JC-1 maintains monomeric form,thus showing only green fluorescence.The decreases of △?m were in a time-dependent manner,which were significantly higher than those in control group(P

The total RNA was extracted from ginseng leaves of Panax ginseng. The Cu/Zn-SOD gene was amplified via RT-PCR and the pET-28(a)-Cu/Zn-SOD expression vector was constructed. The pET-28 (a)-Cu/Zn-SOD recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent cells and was induced by IPTG in order to select optimal induction of expression conditions. The target protein was purified by the nickel ions (Ni ) affinity chromatography and the target protein enzyme activity was determinated by the xanthine oxidase method. The similarity of the Cu/Zn-SOD gene sequences and the Cu/Zn-SOD gene sequences of Korean ginseng in NCBI was 99. 00%. The target protein expression level was about 44.42%, and the molecular weight was 16.30 kDa after the pET-28(a)-Cu/Zn-SOD recombinants were induced by IPTG. The purified Cu/Zn-SOD protease activity reached 10,596.69 U x mg(-1). The P. ginseng pET-28(a)-Cu/Zn-SOD prokaryotic expression vector was built by the method of molecular biology, which provided the foundation for studying the Cu/Zn-SOD biology function.
Cloning, Molecular ; Escherichia coli ; genetics ; Gene Expression ; Genetic Engineering ; methods ; Genetic Vectors ; genetics ; Panax ; enzymology ; genetics ; Sequence Analysis ; Superoxide Dismutase ; genetics ; isolation & purification ; metabolism

To study the effects and possible mechanism of Vitamin K(2) (VK(2)) in the treatment of MDS-JSN04 cells, the changes of morphologic features of MDS-JSN04 cells were investigated by cytomorphology, the apoptosis of MDS-JSN04 cells was observed by transmission electron microscope; cellular proliferation was determined by the MTT assay; cell apoptosis, cell cycle shift and expression of myeloid-specific differentiation antigen (CD11b, CD13) were analyzed by flow cytometry (FCM). The expression of apoptosis-related genes bcl-2, survivin and bax were detected by retrotranscriptase polymerase chain reaction (RT-PCR); the activity of caspase-3 was determined by chemiluminescence assay. The results showed that the typical apoptotic morphological features appeared in cells treated with VK(2) for 72 hours; VK(2) induced apoptosis of MDS-JSN04 cells and in a dose-and-time-dependent manner, G(0)/G(1) cell arrest and significantly down-regulated the expression of bcl-2 and survivin, but had no effect on the expression of bax; the activity of caspase-3 significantly increased. It is concluded that VK(2) induces apoptosis of MDS-JSN04 cells through activating caspase-3 pathways and the apoptosis-related genes bcl-2, survivin may play an important role in this process.
Apoptosis ; drug effects ; CD11b Antigen ; analysis ; CD13 Antigens ; analysis ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Flow Cytometry ; Gene Expression Regulation, Neoplastic ; Humans ; Inhibitor of Apoptosis Proteins ; Luminescent Measurements ; methods ; Microscopy, Electron, Transmission ; Microtubule-Associated Proteins ; genetics ; Myelodysplastic Syndromes ; genetics ; metabolism ; pathology ; Neoplasm Proteins ; genetics ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Vitamin K 2 ; pharmacology ; bcl-2-Associated X Protein ; genetics

OBJECTIVETo in vitro study the inhibition effect and possible mechanism of As2S3 nanoparticles (As2S3 nano) on human MDS cell line MUTZ-1 and to compare with that of traditional As2S3.

METHODMUTZ-1 cells were treated with As2S3 nano and traditional regular-sized particles (TRSP) at different concentrations. The cell growth inhibition rate was determined by MTT assay, cell apoptosis by morphology and flow cytometry (FCM), cell cycle by FCM and the activity of caspase-3 by chemiluminescence assay.

RESULTSTreatment of As2S3 nano and TRSP at concentrations of 2, 4, 8 and 16 micromol/L for 48 h could lead to a significant dose-dependent decrease of MUTZ-1 cells and induce apoptosis. The percentages of inhibition were 48.9%, 75.9%, 89.4% and 96.5% in As2S3 nano vs 14.5%, 25.4%, 34.7% and 51.5% in TRSP and apoptosis rates were (12.9 +/- 1.9)%, (19.2 +/- 2.2)%, (30.1 +/- 2.5)% and (45.9 +/- 2.3)% in As2S3 nano vs (5.3 +/- 1.8%)%, (11.1 +/- 2.6)%, (19.3 +/- 2.3)% and (25.5 +/- 2.5)% in TRSP respectively. There was statistically significant difference in these two groups (P < 0.01). The proportion of cell in G2/M phase and the activity of caspase-3 of MUTZ-1 cells treated with A2S32 nano were significantly higher than those treated with control group and As2S3 TRSP groups (P < 0.01).

CONCLUSIONSAs2S3 nanoparticles and TRSP can inhibit the proliferation of MUTZ-1 cells and induce apoptosis, which maybe through activating caspase-3 pathways and increasing the proportion of G2/M phase. As2S3 nanoparticles can produce a much better antitumor effect than As2S3 TRSP do.

Apoptosis ; drug effects ; Arsenicals ; administration & dosage ; pharmacology ; Caspase 3 ; metabolism ; Cell Cycle ; drug effects ; Cell Line ; Cell Proliferation ; drug effects ; Humans ; Myelodysplastic Syndromes ; metabolism ; pathology ; Nanoparticles

OBJECTIVETo study the effect of 2-methoxyestradiol(2-ME) on telomerase activity expression and apoptosis in human myelodysplastic syndrome cells line MUTZ-1.

METHODSMUTZ-1 cells were incubated with different concentrations of 2-ME, apoptosis rate and cell cycle were measured by flow cytometry (FCM). Telomerase activity in MUTZ-1 cells was examined by telomeric repeat amplification protocol-Enzyme linked immunosorbent assay (TRAP-ELISA).

RESULTSThe FCM analysis showed that cells in G0/G1 phase and S phase were decreased, while in G2/M phase increased after exposed to 1,2 and 4 micromol/L of 2-ME for 12 hours (P < 0.05). 1 and 2 micromol/L of 2-ME had no notable effect on MUTZ-1 cells as compared with the control group (P > 0. 05). Cells incubated with 1, 2 and 4 micromol/L of 2-ME for 36 hours were induced apoptosis, the percentage of apoptosis was between (12.87 +/- 0.86)% and (21.82 +/- 1.71)% with a dose- and time- dependent manner. Telomerase activity was significantly inhibited in these concentration and negatively correlated with cell number in G2/M phase (r = -0.979, P = 0.021) and increased apoptosis (r = -0.970, P = 0.030 ), respectively. Moreover, the inhibition effect of telomerase activity was enhanced in a dose- and time- dependent manner.

CONCLUSIONS2-ME-induced apoptosis and inhibition of telomerase activity provide a possible mechanism for explaining the 2-ME's anticancer activity.

Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Line ; Estradiol ; analogs & derivatives ; pharmacology ; Humans ; Myelodysplastic Syndromes ; enzymology ; pathology ; Telomerase ; metabolism

The study was aimed to investigate the mechanism of proliferation inhibition and apoptosis of MDS-RAEB MUTZ-1 cells induced by 2-methoxyestradiol (2-ME), the cell proliferation was determined by MTT assay, apoptosis rate was determined with annexinV-FITC/PI double staining and cell cycle was analyzed by flow cytometry (FCM) after MUTZ-1 cells were treated with different concentrations of 2-ME; the changes of morphologic features of MUTZ-1 cells were observed with Wright-Giemsa's staining; lactate dehydrogenase was determined by Beckman Counter; and agarose gel electrophoresis was used to verify whether 2-ME can induce apoptosis of MUTZ-1 cells. The results showed that 2-ME inhibited the proliferation of MUTZ-1 cells in a dose-and time-dependent manner and caused a sustained arrest at G(2)/M phase in MUTZ-1 cells; the typical apoptotic morphological features appeared in MUTZ-1 cells; the production of lactate dehydrogenase was up-regulated and the marked DNA ladder pattern of internucleosomal fragmentation was observed. It is concluded that the mechanism of proliferation inhibition and apoptosis of MUTZ-1 cells induced by 2-ME is probably related with the G(2)/M cell cycle arrest; 2-ME may be a potentially adjunctive anticancer drug useful to treat myelodysplastic syndrome.
Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Estradiol ; analogs & derivatives ; pharmacology ; Humans ; L-Lactate Dehydrogenase ; metabolism ; Myelodysplastic Syndromes ; pathology

This study was aimed to establish a cytokine-independent human myelodysplastic cells line from bone marrow of a patient with MDS-CMML. This cell line was incubated in mixed culture of RPMI 1640 and DMEM with 15% bovine serum, but without cytokines; its biological characteristics were identified by morphology, surface marker profiles, cell proliferation, differentiation and apoptosis. The results showed that the established cell line could not depend on cytokines for long-term survival and growth, and could differentiate into colony-forming unit-macrophage, colony-forming unit-megakaryocyte. In conclusion, a cytokine-independent human myelodysplastic syndrome cell line, named MDS-JSN04 (MDS Nanjing Jiansu 04), was established. Its partial biological characteristics were identified and clarified.
Antigens, CD19 ; analysis ; Antigens, CD20 ; analysis ; Apoptosis ; drug effects ; Bone Marrow Cells ; metabolism ; pathology ; ultrastructure ; CD79 Antigens ; analysis ; Cell Differentiation ; drug effects ; Cell Line ; Cell Proliferation ; drug effects ; Culture Media ; pharmacology ; Cytokines ; pharmacology ; Flow Cytometry ; HLA-DR Antigens ; analysis ; Humans ; Lewis X Antigen ; analysis ; Male ; Microscopy, Electron, Scanning ; Microscopy, Electron, Transmission ; Middle Aged ; Myelodysplastic Syndromes ; metabolism ; pathology ; Sialic Acid Binding Ig-like Lectin 2 ; analysis ; Time Factors

The purpose of this study was to explore the mechanism underlying the regulation of 2-methoxyestradiol (2-ME)-induced cell apoptosis by mcl-1 and bax gene in myelodysplastic syndrome (MDS). The MUTZ-1 cells were pretreated with 2-ME; then the activity of caspases-3 was determined by fluorescent colorimetry; the mRNA expressions of apoptosis-related genes (mcl-1) and bcl-2-related X protein (bax) were determined by RT-PCR. The results showed that as compared with control, the 2-ME enhanced the activity of caspase-3 in MUTZ-1 cells in a dose-and time-dependent manners (p<0.05); along with increasing of 2-ME concentration, the expression of intracellular mcl-1 mRNA reduced (p<0.05), meanwhile the expression level of mcl-1 mRNA negatively correlated to the activity of caspase-3 at the corresponding time points (r=-0.992, p<0.01), but the expression of bax mRNA did not show significant change (p>0.05). It is concluded that 2-ME can regulate the apoptosis of MDS cells through the pathway of down-regulating the expression of mcl-1 mRNA and activating the caspase-3.
Apoptosis ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Estradiol ; adverse effects ; analogs & derivatives ; Humans ; Myelodysplastic Syndromes ; metabolism ; pathology ; Myeloid Cell Leukemia Sequence 1 Protein ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; bcl-2-Associated X Protein ; metabolism

To investigate the purging effect of CD3AK/iNOS on primary leukemic cells from chronic myeloid leukemia patients in vitro, amphotropic packaging cell line PA317 transfected with the whole length of iNOS gene was cultivated, amplified and screened by G418. The viral titer was determined by the NIH3T3 cells. Human peripheral blood mononuclear cells were isolated and activated by anti-CD3 monoclonal antibody in vitro. CD3AK cells were incubated with viral supernatant and selected by G418. Resistant clones were assayed for iNOS gene expression by RT-RCR. The content of nitric oxide and the activity of iNOS in the culture supernatant of CD3AK/iNOS were evaluated by the method of Griess. After BMMNC or PBMNC from CML patients were co-cultured with CD3AK/iNOS, CD3AK/Neo and CD3AK/iNOS respectively, the expression of bcr/abl fusion gene was detected by serial dilution semi-quantitative net RT-PCR assay. The results showed that anti-G418 positive packaging cell line PA317 transfected with the whole length of iNOS gene clones could stably synthesize and excrete recombinant retroviral vectors. The titer of recombinant retroviral vectors was 1.0 x 10(5) CFU/ml. After being transfected by recombinant retroviral supernatant, the iNOS cDNA was expressed in CD3AK/iNOS. The content of NO and activity of iNOS that synthesized and excreted by CD3AK/iNOS were notably increased, compared with those of CD3AK. There were statistically significant differences in NO content and iNOS activity between two groups. After BMMNC or PBMNC from CML patients were co-cultured with CD3AK/iNOS, CD3AK/Neo and CD3AK/iNOS respectively, the expression of bcr/abl fusion gene in all of them was down-regulated by serial dilution semi-quantitative RT-PCR assay. It is concluded that construction of CD3AK/iNOS can markedly increase the content of NO and the activity of iNOS, which can be more efficient in in vitro purging leukemia cells for autologous hematopoietic stem cell transplantation.
Adult ; Aged ; Animals ; CD3 Complex ; immunology ; Cell Line ; Cytotoxicity, Immunologic ; Female ; Fusion Proteins, bcr-abl ; genetics ; Humans ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; immunology ; pathology ; Male ; Mice ; Middle Aged ; NIH 3T3 Cells ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type II ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Cells, Cultured

This study was aimed to investigate the cytogenetic changes of MDS cell line (MUTZ-1) with chromosome 5q deletion. R-banding analysis was used to identify chromosome aberrations in MDS cell line and Vysis Spectra Vysion M-FISH was used to further characterize chromosomal complex karyotype. The results indicated that M-FISH exhibited obvious chromosomal aberrations with high frequency including translocation, insertion, breakage and rearrangement, deletion and increasement of chromosome number, the complex karyotype of MUTZ-1 was shown as 50, xx, der (1) t (1;2), ins (1;14), +der (2) t(2;19), der (3) t (3;5), der (3) (3::5::22), 5q-, der (6) t (3;6), der (7) (18::7::17), +8, +der (9) t (1;9), der (10) t (1;10), +11, +12, der (?13) (10::13::5::8), der (14) t (8;14), der (14) t (14, 15), der (15) t (15;21) x 2, +17, +18, -21, -22. It is concluded that M-FISH analysis revealed obvious changes in complex karyotype of MDS cell line MUTZ-1, and the M-FISH technique can increase accuracy of detection for chromosomal complex karyotype, and help diagnosis and prognostic evaluation of MDS.
Child, Preschool ; Chromosome Aberrations ; Chromosome Deletion ; Chromosomes, Human, Pair 5 ; Female ; Humans ; In Situ Hybridization, Fluorescence ; methods ; Karyotyping ; Myelodysplastic Syndromes ; genetics ; Translocation, Genetic ; Tumor Cells, Cultured