OBJECTIVETo detect mutations of human telomeric repeat binding factor 1 (TERF1) gene in 11 malignant hematopoietic cell lines, which have positive telomerase activity, and evaluate the significance of the mutations.

METHODSGenome structure of TERF1 was predicted by using biology information program, and verified by PCR and sequencing. Telomerase activity was detected by telomeric repeat amplification (TRAP)-ELISA. PCR and sequencing were used to detect mutation of each exon of TERF1 in 11 cell lines, including myelogenous leukemia cell lines K562, HL-60, U-937, NB4, THP-1, HEL and Dami; lymphoblastic leukemia cell lines 6T-CEM, Jurkat and Raji and MDS-RAEB cell line MUTZ-1. Five DNA samples from healthy volunteers were detected as normal controls.

RESULTSTERF1 gene has 10 exons and spans 38.6 kb. All the 11 cell lines showed positive telomerase activity. No mutation was found in all exons of TERF1 in the 11 cell lines. However, 4 variants were found in intron1, 2 and 8 near exon1, exon2 and exon9, respectively. The variants in MUTZ-1 was different from those in leukemia cell lines; but no difference was found between the variants in myelogenous and lymphoblastic leukemia cell lines.

CONCLUSIONTERF1 mutation is probably not among the main factors of the gene dysfunction in malignant hematopoietic diseases.

Base Sequence ; Cell Line, Tumor ; DNA Mutational Analysis ; Enzyme-Linked Immunosorbent Assay ; Exons ; genetics ; HL-60 Cells ; Hematologic Neoplasms ; genetics ; metabolism ; pathology ; Humans ; Jurkat Cells ; K562 Cells ; Mutation ; Polymerase Chain Reaction ; Telomerase ; metabolism ; Telomere-Binding Proteins ; genetics ; metabolism ; U937 Cells

This study was aimed to examine the correlation of the cytotoxic effects induced by two types of TNF-α to cell cycle. Hoechst 33342 and PI were used to detect the morphological changes in the cell death induced by the two types of TNF-α. TdT and PI co-staining was performed to determine the phase of cell cycle of apoptotic cells. L929 cells in different phases of cell cycle were further synchronized and their sensitivity to the two types of TNF-α was observed. Our results showed that the apoptosis of HepG2 cells triggered by tm-TNF-α mainly occurred in G(1) phase while in HL-60, Raji and K562 cell lines it mainly took place in S phase. The apoptosis of L929 cells induced by tm-TNF-α mainly occurred in S phase while the apoptosis induced by s-TNF-α mainly appeared in G(1) phase. L929 cells were sensitive to s-TNF-α when synchronized in G(1) phase (cytotoxicity 49.8%) while their sensitivity to tm-TNF-α was highest in S phase (45.7%) and G(1)/S phase (cytotoxicity 40.6%). It was concluded that tm-TNF-α-induced apoptosis of different target cells took place in different phases of cell cycle. The apoptosis of the specific cell line induced by the two types of TNF-α occurred in different phases of cell cycle. The sensitivity of the specific cell line to the two types of TNF-α was correlated with the phase of cell cycle.
Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Line, Tumor ; HL-60 Cells ; Hep G2 Cells ; Humans ; K562 Cells ; Membrane Proteins ; metabolism ; pharmacology ; Tumor Necrosis Factor-alpha ; metabolism ; pharmacology

OBJECTIVETo investigate the role of MHC class II Transactivator (C II TA) in expression of HLA molecules in five human malignant hematological cell lines.

METHODSThe expressions of HLA molecules and C II TA protein were detected by immunohistochemistry and flow cytometry. The expression of C II TA gene was detected by RT-PCR. The response of peripheral T cells after stimulation by Jurkat cells was detected by mixed lymphocyte reaction.

RESULTThe HLA II-positive tumor cells expressed the C II TA and IFN-gamma induced the expression of HLA I, II in tumor cells, which were able to express C II TA constitutively.

CONCLUSIONThere is a correlation between the inability of the tumor cells in response to IFN-gamma for HLA expression and the deficiency in the inducible expression of C II TA.

Cell Line, Tumor ; Genes, MHC Class II ; genetics ; HL-60 Cells ; HLA Antigens ; biosynthesis ; genetics ; Humans ; K562 Cells ; Leukemia ; metabolism ; pathology ; Nuclear Proteins ; biosynthesis ; genetics ; Trans-Activators ; biosynthesis ; genetics ; U937 Cells

To investigate the pattern of human ERMAP gene expression in different cell lines, 15 cell lines derived from hematopoietic tumor, somatic tumor and normal tissue were chosen and were cultured; cells were harvested after culture for 12, 24, 36, 48, 60 and 72 hours; the expression of the human ERMAP was detected by using fluorescent quantitative PCR. The results showed that human ERMAP gene expression was positive in K562 cell line at interval of 12 and 24 hours, and the expression levels were (5.092 +/- 2.331) x 10(6) cps/microl RNA, (5.328 +/- 3.916) x 10(6) cps/microl RNA, respectively; ERMAP gene expression was also positive in ECV304 cell line at interval of 24 hours, and the expression level was (0.84 +/- 0.12) x 10(6) cps/microl RNA; and its expression was negative in other 13 cell lines. It is concluded that human ERMAP gene expression in ECV304 cell line was found first, and its expression in K562 cell line was further confirmed.
Blood Group Antigens ; genetics ; Butyrophilins ; Cell Line ; Cell Line, Tumor ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; HL-60 Cells ; Hematologic Neoplasms ; genetics ; pathology ; Humans ; Jurkat Cells ; K562 Cells ; RNA, Messenger ; genetics ; metabolism

OBJECTIVETo explore the effect of DAPK overexpression on the biological behaviors and caspase-3 expression in HL-60 cells.

METHODSThe expression of DAPK mRNA was detected by RT-PCR leukemia cell lines K562, Molt4, U937, and HL-60 cells. HL-60 cells were transfected by a eukaryotic expression vector pReceiver-M29-DAPK via LipofectamineTM 2000, and the impact of DAPK overexpression on cell apoptosis, cell cycle, cell differentiation and caspase-3 expression were analyzed.

RESULTSDAPK mRNA expression was positive in K562, Molt4 and U937 cells but negative in HL-60 cells. Significantly increased cell apoptosis was observed in pReceiver-M29-DAPK-transfected HL-60 cells by flow cytometry and Hoechst33342 staining. The cell cycle distribution and differentiation showed no significant changes after the transfection. The expression of caspase-3 was significantly increased in the cells after transfection.

CONCLUSIONDAPK gene overexpression promotes apoptosis of HL-60 cells without affecting the cell cycle and differentiation. Caspase-3 may be involved in the regulation of cell apoptosis.

Apoptosis ; Caspase 3 ; metabolism ; Cell Cycle ; Cell Differentiation ; Cell Line, Tumor ; Death-Associated Protein Kinases ; metabolism ; HL-60 Cells ; Humans ; RNA, Messenger ; metabolism ; Transfection ; U937 Cells

This study was aimed to investigate the inhibitory effect of flavonoids of puerarin (PR) in different concentrations on proliferation of 4 kinds of acute myeloid leukemia (AML) cell lines (Kasumi-1, HL-60, NB4 and U937), and to explore its possible mechanism. The MTT method was used to detected the inhibitory effect of PR on proliferation of AML cell lines. The flow cytometry was adopted to determine the change of cell cycle in vitro. The results showed that a certain concentration of PR could inhibit the proliferation of these 4 cell lines effectively in time-and dose-dependent manners, and the intensity of inhibition on 4 kinds of AML cell lines was from high to low as follows: NB4>Kasumi-1>U937>HL-60. Meanwhile, PR could also change cycle process, cell proportion in G1/G0 phase decreased, cells in S phase increased and Sub-diploid peak also appeared. It is concluded that PR can selectively inhibit the proliferation of 4 AML cell lines and block cell cycle process, especially for NB4 cells.
Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; HL-60 Cells ; Humans ; Isoflavones ; pharmacology ; Leukemia, Myeloid, Acute ; classification ; pathology ; U937 Cells

OBJECTIVEThe SH2 domain containing inositol 5'-phosphatase (SHIP) is predominately expressed in hematopoietic cells, and is a crucial negative regulator in the development of hematopoietic cells. This paper is to evaluate the role of the SHIP gene in human leukemogenesis.

METHODSExpression of SHIP gene in bone marrow and/or peripheral blood from 32 patients with acute myeloid leukemia (AML), 9 with acute lymphoblastic leukemia (ALL), as well as human hematopoietic cell lines was analyzed by reverse transcription-polymerase chain reaction (RT-PCR), single strand conformational polymorphism (SSCP) and DNA sequencing.

RESULTSRT-PCR showed that all samples expressed SHIP gene. Mutations of SHIP gene were detected in 7 (22%) of 32 AML patients and one (12%) of 9 ALL patients. Interestingly, two missense mutations that had been observed in a AML patient at diagnosis disappeared after complete remission (CR). In addition, in vitro Akt phosphorylation was prolonged and increased following IL-3 stimulation of this patient's cells.

CONCLUSIONOur data demonstrate for the first time the mutation of SHIP gene in acute leukemia and suggest a possible role of the mutation of this gene in the development of acute leukemia. SHIP may serve as a tumor suppressor by negatively regulating the PI3K/Akt signaling pathway in hematopoietic cells.

Blotting, Western ; Cell Line, Tumor ; DNA Mutational Analysis ; HL-60 Cells ; Humans ; Inositol Polyphosphate 5-Phosphatases ; Interleukin-3 ; pharmacology ; K562 Cells ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; Mutation ; Oncogene Protein v-akt ; metabolism ; Phosphoric Monoester Hydrolases ; genetics ; metabolism ; Phosphorylation ; drug effects ; Polymorphism, Single-Stranded Conformational ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; U937 Cells

The aim of the research was to ascertain different expression levels of miRNA-17-19b in different cell lines and to observe the changes of miR-17-19b expression in the cell line K562 with high expression level of miR-17-19b after 5-azacytidine (5-aza) treatment. Total RNA was extracted from K562, HL-60, NB-4 and HeLa cell lines, white blood cells of peripheral blood from patient with chronic myeloid leukemia (CML) and mobilized white blood cells of peripheral blood from normal persons, respectively. Total RNA was polyadenylated by poly (A) polymerases and the expressions of miR-17-19b in the cell lines and the above mentioned cells were detected by SYBR-green real-time PCR. The K562 cell line was treated with 2.5 µmol/L 5-aza for 24, 48 and 72 hours, then were collected at 96 hours. The changes of miR-17-19b expression were determined by real-time PCR after 5-aza treatment. K562 cell line proliferation was observed after inhibition of miR-19a function. The results showed that the expression levels of miRNA-17-19b in K562 cells and white blood cells of peripheral blood from CML patients were higher than those in mobilized white blood cells of peripheral blood from normal person. The expression level of miR-17-19b in K562 cells with high expression of miR-17-19b was down-regulated after 5-aza treatment. The proliferation of K562 cells was inhibited through suppression of miR-19a function. It is concluded that expression level of miR-17-19b is higher in K562 cell line and white blood cells of peripheral blood from CML patients than that in white blood cells of peripheral blood from normal person. Expression of miR-17-19b is inhibited in K562 cell line after 5-aza treatment. Inhibition of miR-19a in vitro can suppress the proliferation of K562 cell line.
Antimetabolites, Antineoplastic ; pharmacology ; Apoptosis ; Azacitidine ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Down-Regulation ; HL-60 Cells ; Humans ; K562 Cells ; MicroRNAs ; genetics

Human solid tumors are less well oxygenated than normal tissues. This leads to resistance to radiotherapy and anticancer chemotherapy. Many of genes encoding proteins induced by hypoxia are potential products for modifying the radiation response of normal or malignant tissues. Radiosensitivity or radioresisitance of the solid tumor cell lines can be exploited in overcoming the resistance to radiotherapy for cancer treatment. In this study, we investigated the radiosensitivity or radioresistance of the solid tumor cell line induced by hypoxia after radiation and the effect of hypoxia or/and irradiation on the expression of cell cycle regulatory proteins in the NIH3T3, MCF7 and HepG2 cell line. In normal cell line, NIH3T3, the cell death induced by irradiation was decreased by undergoing hypoxic treatment before radiation in the FACS and LDH assays. The percentage of cell death resulted in a significant increase by 70~80% in the NIH3T3 and HepG2 cell line after irradiation or hypoxia, but the cell death was significantly decreased after irradation under hypoxia in the HepG2 cells. However there is no difference in the cell death of MCF7 cells after same insults. DNA fragmentation was observed in only HepG2 cells under hypoxia by Heochst-PI nuclear staining, DNA gel electrophoresis and FACS analysis. In human breast cancer cell line MCF7, the level of E2F was more increased in hypoxia than normoxia, but was decreased after gamma-irradiation. However, it was shown that the effects of radiation can be changed by reoxygenation condition after hypoxia in human hepatoma cell line HepG2. The expression of E2F-1 was observed to decrease in HepG2 when cells were exposed to gamma-radiation after hypoxia. While investigating in MCF7 cells, the level of E2F-1 was increased under hypoxia. However, the expression of p53 decreased in hypoxia, though was increased after irradiation in the NIH3T3, MCF7 and HepG2 cell line. The increase of E2F-1 expression in MCF7 cells may be associated with hypoxic resistance in hypoxia-mediated apoptosis of tumors. The level of E2F may contribute to some critical factors of cellular repair function associated with DNA damage, and to deciding whether the cells will pass through cell cycle arrest or apoptosis.
Anoxia* ; Apoptosis ; Breast Neoplasms ; Carcinoma, Hepatocellular ; Cell Cycle Checkpoints ; Cell Cycle Proteins* ; Cell Cycle* ; Cell Death ; Cell Line ; Cell Line, Tumor* ; DNA ; DNA Damage ; DNA Fragmentation ; Drug Therapy ; Electrophoresis ; Hep G2 Cells ; Humans ; MCF-7 Cells ; Oxygen ; Radiation Tolerance ; Radiotherapy

Structural modifications were performed with natural product of oleanolic acid to search for novel anticancer drugs. Ten oleanolic acid derivatives were designed and obtained by the reaction of oxidation, acylation or hydrolyzation, etc. The cytotoxic activity of derivatives was evaluated against HeLa, HepG2 and BGC-823 cells in vitro by MTT assay, gefitinib and etoposide used as a positive control. The results showed that compound 5a was particularly active to inhibit HepG2 cells growth, and anti-tumor activity of compound 7 on HeLa cells was significantly stronger than oleanolic acid. They are worthy to be studied further.
Antineoplastic Agents ; chemical synthesis ; chemistry ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; HeLa Cells ; Hep G2 Cells ; Humans ; Oleanolic Acid ; analogs & derivatives ; chemical synthesis ; chemistry ; pharmacology