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

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 research the cytotoxicity and in vitro antiproliferative effect of the six flavone compounds extracted from Laggera pterodonta.

METHODThe cytotoxicity on the normal cells and antiproliferative effect on tumor cells were tested by MTT assay, and then the preliminary structure-activity relationship was analysed. The phase distribution of the cell cycle and apoptosis rate were analyzed by flow cytometry.

RESULTThe results of MTT assay showed 5,7,3',4'-tetramethoxy-3-hydroxyflavone and chrysosplenetin B inhibited growth of A549 and Hela cells significantly with a dose dependent mode, while exhibited low cytotoxicity to the two normal cells, Vero and EVC304. Both compounds contain ortho-phenolic methoxyl moietys in their structures. Flow cytometry analysis revealed that Hela cells treated with increasing quantities of chrysosplenetin B increased the percentage of cells in the G2/M phase, and Hela and A549 cells treated with increasing quantites of the 5,7,3',4'-tetramethoxy-3-hydroxyflavone and chrysosplenetin B increased the apoptosis rates.

CONCLUSIONThe 5,7,3',4'-tetramethoxy-3-hydroxyflavone and chrysosplenetin B extracted from L. pterodonta showed high antiproliferative effect on cancer cells with low cytotoxicity on normal cells, and took the effects on A549 and Hela cells through the hold-up of the G2/M phase of the cell cycle and induction of the apoptosis.

Animals ; Asteraceae ; chemistry ; Cell Line ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cercopithecus aethiops ; Flavones ; chemistry ; pharmacology ; Flavonoids ; pharmacology ; Flow Cytometry ; HeLa Cells ; Humans ; Vero Cells

OBJECTIVETo analyze the molecular coining of a fragile site-associated gene.

METHODSGenomic Chinese hamster ovary (CHO) DNA library was constructed using high molecular weight CHO DNA partially digested with MboI restriction enzyme from cultured CHO cells. Screening of genomic DNA library followed the established procedures. Genomic CHO in the positive clones was sequenced. Appropriate primers were designed for the reverse transcriptase-polymerase chain reactions (RT-PCR). The RT-PCR products were cloned into a pCRII TOPO vector and confirmed by DNA sequencing. Antibodies were prepared using synthetic peptides as antigens by immunizing the rabbits. Immunohistochemical analyses were performed to evaluate the expression of the novel gene in different tissues.

RESULTSTo investigate the molecular mechanism underlying the initial events of mdr1a amplification, we cloned 1q31 fragile site DNA. Strikingly, we found that this fragile site contained a novel gene which was designated as a fragile site-associated (FSA) gene. FSA encoded an unusually large mRNA of approximately16 kb. Full-length human FSA cDNA was cloned. FSA mRNA was expressed in many cultured cells and tissue types. Immunohistochemical analyses also revealed an expression pattern of the encoded proteins in postmitotic, well-differentiated epithelial compartments of many organs, including colon, mammary glands, ovary, prostate, and bladder.

CONCLUSIONFSA plays an important role in regulating mammalian epithelial cell growth and differentiation.

Animals ; CHO Cells ; Cell Line ; Chromosome Fragile Sites ; genetics ; Cloning, Molecular ; Cricetinae ; Cricetulus ; HCT116 Cells ; HT29 Cells ; Humans

OBJECTIVETo construct an cell penetrating peptide-based expression vector capable of targeted delivery of proteins into the cell nuclei and study its function of protein transduction.

METHODSThe fusion protein expression vector pET14b-HC(L)NE (pET14-b-His-CPP-Linker-NLS-EGFP) incorporating cell penetrating peptide (CPP), nuclear localization signal(NLS), linker and enhanced green fluorescent protein (EGFP) was constructed based on His-tagged pET14b-HE (pET14b-His-EGFP) by site-directed mutagenesis PCR method. After identification by enzyme digestion and DNA sequencing, the recombinant plasmid was transformed into BL21(DE(3)) strain. The HC(L)NE fusion protein was expressed following IPTG induction and purified with Ni(2+)-NTA affinity chromatography. After dialysis and filtration, the HC(L)NE fusion protein was added into cultured eukaryotic cells. The protein transduction in the living cells was observed under fluorescence microscope and analyzed by Western blotting.

RESULTSEnzyme digestion and DNA sequencing confirmed successful construction of the pET14b-HC(L)NE vector, and the fusion protein efficiently expressed in E. coli. Protein transduction experiments in eukaryotic cells revealed that the fusion protein could rapidly penetrate the cell membrane and reach the cell nucleus, and this internalization was time- and concentration-dependent.

CONCLUSIONThe cell penetrating peptide-based expression vector for targeted protein delivery to the cell nucleus has been successfully constructed, and a transport system that can delivery exogenous proteins or polypeptides into the cytoplasm and cell nucleus is established, which provides an economical and efficient means for functional study of the proteins and polypeptide in cells and targeted drug delivery.

Animals ; Blotting, Western ; COS Cells ; Cell Line ; Cell Membrane ; metabolism ; Cell Nucleus ; metabolism ; Cercopithecus aethiops ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; HeLa Cells ; Humans ; Mice ; Microscopy, Fluorescence ; NIH 3T3 Cells ; Nuclear Localization Signals ; genetics ; Peptides ; genetics ; metabolism ; Protein Transport ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transfection

An antisense approach was attempted to investigate the role of antisense GLUT1 RNA in suppressing tumor cell phenotypes using N-ras-transformed NIH 3T3 cells. The established cell line transformed by ras showed typical biological characteristics of cancer cells, such as increased glucose transport, GLUT1 mRNA contents, and the ability to form colonies on the soft agar. In this system, the plasmids (pMAM-GLUT1(rev)) which can transcribe the antisense GLUT1 RNA were transfected and the accompanying changes in the phenotypes of the ras-transformed cells were observed. The expression of antisense GLUT1 RNA by induction with dexamethasone reduced the glucose transport by 30% (1.97 +/- 0.13 nmoles) after 4 min incubation when compared to the non-induction group of transformed cell (2.85 +/- 0.19 nmoles). Also, the number of colonies sized over 50 microns on the soft agar was reduced significantly in the antisense RNA expressing group compared to non-induction group. These results suggest that the expression of antisense GLUT1 RNA reduced the glucose transport and transforming potential in soft agar possibly by hybridization with GLUT1 mRNA in N-ras-transformed NIH 3T3 cells.
3T3 Cells/metabolism ; Animal ; Base Sequence ; Cell Line, Transformed ; Cell Transformation, Neoplastic/metabolism/*pathology ; *Genes, ras ; Human ; Mice ; Molecular Sequence Data ; Monosaccharide Transport Proteins/*genetics ; Phenotype ; RNA, Antisense/*metabolism ; Support, Non-U.S. Gov't ; Tumor Cells, Cultured/metabolism/pathology

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

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

OBJECTIVETo investigate miR- 125b regulation mechanism by identifying miR-125b target genes and its function in acute myeloid leukemia (AML).

METHODSThe bioinformatics software and database were applied to predict and analyze target genes of miR-125b. The vector contained the target gene 3'-UTR portion cloned into a luciferase reporter construct. A luciferase reporter assay was performed following co-transfection of small molecular miR-125b mimics and target gene wild-type or mutant plasmid into HEK-293T cells. Further in leukemia cell lines NB4 and HL-60, the protein level of target gene was measured by Western blot after overexpression miR-125b. Finally, the viabilities of NB4 and HL-60 cells were measured by CCK-8 assay at 24 h, 48 h, 72 h, 96 h after electroporation.

RESULTSBcl-2-antagonist/killer 1 (Bak1), a pro-apoptotic gene, was a target gene of miR-125b by software predicts. Reporter vector containing the 3'-UTR Bak1 wild and mutation sites were co-transfected with small molecule analogues of miR-125b in HEK-293T cells. Dual luciferase reporter gene assay system showed that miR-125b significantly suppresses the reporter gene activity containing Bak1 3'-UTR by about 53.8% (P<0.05), but it didn't suppresses the reporter gene activity containing 3'-UTR Bak1 mutation. Western blot showed that miR-125b mimics significantly down-regulated the expression of Bak1 in human leukemia cell lines NB4 and HL-60. Meanwhile, the growth rate of cells treated with miR-125b obviously increased compared with that in control by CCK-8 test (P<0.05).

CONCLUSIONOur findings strongly indicated that BAK1 was a downstream target gene of miR-125b, and miR-125b promoted proliferation in human AML cells at least partially by targeting Bak1, so we speculated that miR-125b as an oncogene could be a potential therapeutic target for treating AML.

Cell Line, Tumor ; Cell Proliferation ; Genetic Vectors ; HEK293 Cells ; HL-60 Cells ; Humans ; MicroRNAs ; genetics ; Transfection ; bcl-2 Homologous Antagonist-Killer Protein ; genetics ; metabolism