The aim of study was to investigate the killing effect of double suicide gene system mediated by retroviral vector on K562 cells in vivo and ex vivo. CDglyTK gene was transfected into PA317 cells by using lipofectamine. K562 cells were infected with viral supernatant. K562/CDglyTK cells were treated with 5-fluorocytosine (5-FC) and/or ganciclovir (GCV). Mice were randomly divided into three groups: tumor formation, tumor inhibition and tumor therapy. Each mouse was implanted with K562/CDglyTK cells or K562 cells. The results indicated that the killing effect of 5-FC in combination with GCV on K562/CDglyTK was more significant than using 5-FC or GCV alone. In vivo study showed that after being injected subcutaneously with K562 cells and K562/CDglyTK cells, there was not obvious difference in tumor formation rate of mice, 5-FC + GCV could suppress tumor formation of the K562/CDglyTK cells. After being treated with 5-FC and GCV, the median tumor volume of mice implanted with K562/CDglyTK cells decreased obviously, compared with the control group. Their median survival was significantly prolonged. It is concluded that double suicide genes are more effective for killing effect on K562 cells in vivo and in ex vivo. It may be applicable to clinical gene therapy.
Cytosine Deaminase ; genetics ; Flucytosine ; pharmacology ; Ganciclovir ; pharmacology ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; Genetic Vectors ; genetics ; Humans ; K562 Cells ; Protein-Tyrosine Kinases ; genetics ; Receptor Protein-Tyrosine Kinases ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; genetics ; Recombination, Genetic ; Retroviridae ; genetics

The objective of study was to investigate the combined effect of tyrosine-kinase inhibitor (imatinib) and p15 gene on the proliferation, cell cycle and apoptosis of chronic myeloid leukemia cell line K562. p15 gene was amplified from peripheral blood mononuclear cells by RT-PCR, and confirmed by DNA sequencing, then the recombinant p15-pcDNA3.1 vector was constructed and transfected into K562 cell line by Lipofectine. After screening with G418, p15-pcDNA3.1-K562 cell clone stably expressing P15 was isolated. P15 protein was identified by Western blot. The cell survival rate was determined by MTT, cell cycle and apoptosis were detected by flow cytometry. The results showed that partial deletion of p15 gene in K562 cells was verified by DNA sequencing, leading to the function of P15 protein to be lost. The expression of P15 protein can be detected by Western blot in p15-pcDNa3.1-K562 cells. A strong inhibition of cell proliferation was observed in p15-pcDNA3.1-K562 cells as compared with that of the control K562 cell. The cells of G(0)/G(1) phase in p15-pcDNA3.1-K562 cells increased apparently, and S phase cells declined signifcantly. Cell cycle was arrested in G(0)/G(1) phase. The percentage of apoptotic cells greatly increased after transfection with p15-pcDNA3.1-K562 cells combined with imatinib, and cell survival rate notably declined. It is concluded that the imatinib in combination with the expression of p15 gene has a synergistic effect on the inhibition of K562 cell proliferation and promotion of its apoptosis.
Apoptosis ; drug effects ; Base Sequence ; Cell Proliferation ; drug effects ; Cyclin-Dependent Kinase Inhibitor p15 ; genetics ; Humans ; K562 Cells ; Molecular Sequence Data ; Protein-Tyrosine Kinases ; antagonists & inhibitors ; Transfection

To investigate the relationship between LRP15 gene promoter region methylation and its gene expression in acute leukemia patients, the status of LRP15 gene promoter region methylation was detected by MS-PCR and the gene expression was detected by RT-PCR in bone marrow samples from leukemia patients. The results indicated that the LRP15 gene expression was 47.6% in complete remission (CR) patients and 16.7% in non-CR patients respectively, while LRP15 gene promoter region methylation was 38.1% in CR group and 72.2% in non-CR group respectively. No relationship was found between LRP15 gene promoter region methylation and its expression (P = 0.0087). It is concluded that the methylation in LRP15 gene promoter region may not be the only reason for LRP15 gene silence.
DNA Methylation ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Leukemia, Myeloid, Acute ; genetics ; pathology ; Male ; Neoplasm Proteins ; biosynthesis ; genetics ; metabolism ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; pathology ; Promoter Regions, Genetic ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

The study was purposed to investigate the polymorphism of killer cell immunoglobulin-like receptor (KIR) gene of the patients with leukemia and to explore the correlation between the KIR gene and susceptibility of leukemia. The KIR genotype of 50 patients with leukemia and 60 healthy controls in northern. Hans were analyzed by PCR-SSP. The results indicated that the present known 18 KIR genes were detected and identified. The frequencies of KIR 3DL3, 3DL2 and 2DL4 were 100% in all subjects, with the most frequent genotype KIR 3DP1 (0.86) followed by 2DP1, 2DL3, 3DL1, 2DL1, 3DS1, 2DL5, 2DS4, 2DS2, 1D, 2DS5, 2DL2, 2DS1, 2DS3 and 3DP1v in leukemia successively. Compared with the control, the KIR 3DL1 (0.60) and 2DL1 (0.57) were significantly lower in the leukemia patient group than that in the control group (1.00) (P < 0.01). It is concluded that the polymorphism of KIR gene is associated with susceptibility of leukemia in Hans. There may be a negative correlation between pathogenesis of leukemia and KIR 3DL1, KIR 3DS1, KIR 2DL1, KIR 2DL5 genes.
Adolescent ; Adult ; Child, Preschool ; Female ; Genetic Predisposition to Disease ; genetics ; Genotype ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; Male ; Middle Aged ; Polymorphism, Genetic ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; Receptors, Immunologic ; genetics ; Receptors, KIR ; Receptors, KIR2DL1 ; Receptors, KIR2DL3 ; Receptors, KIR2DL4 ; Receptors, KIR3DL1 ; Receptors, KIR3DL2 ; Receptors, KIR3DS1

The aim of study was to investigate the expression of CD36 in leukemia cells and to explore its significance in diagnosis and differential diagnosis for leukemia in patients. Blood samples from 133 cases of leukemias were analyzed by CD45/SSC double parameters and multi-color flow cytometry in order to determine the CD36 and other leukocyte differentiation antigens. The results show that the CD36 positive rate was 21.8% (29/133) in 133 cases of leukemia, 41.9% (26/62) in 62 cases of AML (acute myeloid leukemia), and none of the 54 cases of lymphocytic leukemia was positive for this antigen. The positive rate of CD36 in M(4) (8/10), M(5) (12/12) and M(6) (3/3) was significantly higher than that in M(1) (0/9), M(2) (3/12), M(3) (0/16) (all P < 0.001). The percent of positive cells of CD36 in M(5a) was significantly higher than in M(5b) (P = 0.001). A significantly negative regression was found between CD36 and CD117 in AML (r = -0.751, P = 0.005). And a significantly positive regression was found between CD36 and CD14 in M(4) and M(5b) (r = 0.870, P = 0.011). In monocyte lineage involved leukemia (MLIL), the positive rate of CD36 (92.6%, 25/27) was significantly higher than that of CD14 (48.1%, 13/27)(P = 0.001). None of the 7 cases with M(5a) was positive for CD14, but 4 of 5 cases of M(5b) were positive. The positive rate of CD117 in M(5a) was significantly higher than that of in M(5b)(P = 0.01). The positive rate of CD34 in M(5) was low (33.3%, 4/12). It is concluded that the combination of CD36 with lymphoid and myeloid antigens is helpful to the diagnosis and differential diagnosis of lymphoid, myeloid and MLIL. The positive rate of CD36 is higher than that of CD14 in MLIL.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; CD36 Antigens ; analysis ; Child ; Child, Preschool ; Female ; Flow Cytometry ; methods ; Humans ; Immunophenotyping ; Infant ; Leukemia, Monocytic, Acute ; immunology ; Leukemia, Myeloid, Acute ; immunology ; Lipopolysaccharide Receptors ; analysis ; Male ; Middle Aged ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; immunology ; Proto-Oncogene Proteins c-kit ; analysis

The study was purposed to investigate the effect of deguelin on expression of nucleoporin 98 (nup98) in leukemia K562 cells. MTT assay was used to assess the effects of deguelin on cell proliferation. FCM and RT-PCR were used to analyze the changes of nup98 mRNA and protein in K562 cells after treating with deguelin. The results showed that deguelin inhibited the proliferation of K562 cells in a time- and dose-dependent manner; mean fluorescence intensity of nup98 in blank group (34.22 +/- 1.63) was significantly higher than that in control group (2.83 +/- 0.02, P < 0.01), 10 nmol/L deguelin could significantly inhibit the expression of nup98 protein; 10 nmol/L could not inhibit the expression of nup98 mRNA, 20, 40, 80, 160 nmol/L deguelin could significantly inhibit the expression of nup98 mRNA in a dose-dependant manner. It is concluded that deguelin inhibits the proliferation of K562 cell through inhibiting the expression of nup98, and may be considered as a new target for therapy of acute leukemia.
Antineoplastic Agents, Phytogenic ; pharmacology ; Cell Proliferation ; Dose-Response Relationship, Drug ; Humans ; K562 Cells ; Nuclear Pore Complex Proteins ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Rotenone ; analogs & derivatives ; pharmacology

To study the incidence, the types of fusion genes and the clinical significance of rearrangements of mixed lineage leukemia (MLL) gene in acute leukemia (AL), the rearrangements of MLL gene of 60 patients with AL were detected by fluorescence in situ hybridization (FISH) and 6 types of common fusion genes resulting from the rearrangements of MLL gene were detected by nested RT-PCR. The results showed that 7 out of 60 AL patients were found the rearrangements of MLL gene, the incidence of which was 11.67%. 2 out of 7 patients were diagnosed as AML-M(5), 5 patients were diagnosed as B-ALL. The fusion genes of the 2 AML-M(5) patients who had the rearrangements of MLL gene were MLL/AF(9). Among 5 B-ALL patients, 2 patients were confirmed to express MLL/ENL, 1 patient was confirmed to express MLL/AF(4), the other 2 patients did not express the fusion genes. It is concluded that FISH is a fast, specific and sensitive method to detect the rearrangements of MLL gene in AL patients and nested RT-PCR is a convenient and feasible method to detect the types of fusion genes resulting from the rearrangements of MLL gene. The detection of MLL gene rearrangement is of great importance in predicting prognosis and guiding therapy in AL.
Adolescent ; Adult ; Child ; Child, Preschool ; Female ; Gene Rearrangement ; Humans ; In Situ Hybridization, Fluorescence ; Infant ; Leukemia, Myeloid, Acute ; genetics ; Male ; Middle Aged ; Myeloid-Lymphoid Leukemia Protein ; genetics ; Oncogene Proteins, Fusion ; genetics ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; methods

The study was aimed to establish a K562/NOD-SCID leukemia mouse model and to explore its growth characteristics. Nude mice exposed to total body irradiation were inoculated subcutaneously with K562 cells, then the local K562 tumor was taken out and the tumor tissues without necoosis were selected for preparing single cell suspension which was inoculated into irradiated NOD/SCID mice by intraperitoneal injection. The results indicated that the systemic disseminated leukemia model was established successfully by intraperitoneal injection. On the fourth week after inoculation the leukemia cells were found on peripheral blood smear, and the leukemia cell infiltration was observed in liver, spleen and bone marrow. On the brink of death, the count of peripheral blood WBC was 8 - 10 times as much as that before inoculation. The leukemia cells on peripheral blood smear accounted to 20% - 30% of the total WBCs on average. The local tumors appeared in the abdominal cavity or on the greater omentum, less were involved in other organs. It is concluded that the established K562/NOD-SCID mouse model with leukemia well imitates the process of leukemia in human body, so it is a good model for the research on the effects of new drugs and target or gene therapy.
Animals ; Disease Models, Animal ; Female ; Humans ; K562 Cells ; Leukemia ; Male ; Mice ; Mice, Inbred NOD ; Mice, Nude ; Mice, SCID ; Neoplasm Transplantation ; Whole-Body Irradiation

MRE11 plays an important role in the signal transduction of DNA damage response, therefore this study was purposed to explore the relationship between hMRE11 focus formation and DNA double-strand breaks (DSBs) caused by etoposide (VP-16) in human promonocytic cells U937. After U937 cells were treated with VP-16, the drug-induced DSBs were assessed by pulsed-field gel electrophoresis (PFGE), the gene transcription levels of hMRE11 were evaluated by RT-PCR, the nuclear focus formation of hMRE11 protein was examined using immunofluorescence technique, the cell cycle in parallel was analyzed by flow cytometry. The results showed that the percentage of U937 cells with DSBs induced by VP-16 raised from 13.0 +/- 2.3% in VP-16 2 microg/ml to 32.0 +/- 4.3% in VP-16 20 microg/ml (P < 0.01) along with increase of VP-16 dose. No difference of the hMRE11 mRNA level in U937 cells following the treatment with 100 microg/ml VP-16 at different times was discovered (P > 0.05). The hMRE11 protein was abundantly and uniformly distributed in the nuclei of untreated U937 cells outside of nucleoli, however, it formed discrete nuclear foci following VP-16 treatment. The mean value of nuclear foci increased by 5 to 20 times following the drug dosing (P < 0.01). An average of 5 nuclear foci per positive nucleus were observed at a dose of 2 microg/ml, and it was increased to an average of over 14 nuclear foci per positive nucleus after treating with VP-16 20 microg/ml. The percentage of nuclei containing hMRE11 nuclear foci also increased from less than 10% after treatment wiht VP-16 2 microg/ml to over 50% after VP-16 20 microg/ml (P < 0.01) following treatment with VP-16. After U937 cells were treated with 100 microg/ml VP-16 for 2 hours and fixed at 4, 8, 12 and 24 hours, the percentage of nuclei with hMRE11 nuclear foci increased to 61.54 +/- 3.6% (the control U937 cells: 0.47 +/- 1.17%, P < 0.01) at 8 hours, with a subsequent decrease in the percentage of nuclear foci-positive cells by 24 hours. The ratio of S-phase U937 cells at 8 hours after being treated with 100 microg/ml VP-16 for 2 hours was 47.55 +/- 2.35%, and that without 100 microg/ml VP-16 was 21.95 +/- 2.91% (P < 0.05). It is concluded that the nuclear focus formation of hMRE11 protein may be a response to DNA damage induced by topoisomerase II inhibitor VP-16 in human promonocytic cell line U937.
Antineoplastic Agents, Phytogenic ; pharmacology ; DNA Damage ; DNA Repair ; genetics ; physiology ; DNA-Binding Proteins ; biosynthesis ; genetics ; metabolism ; physiology ; Dose-Response Relationship, Drug ; Etoposide ; pharmacology ; Humans ; MRE11 Homologue Protein ; Protein Binding ; RNA, Messenger ; biosynthesis ; genetics ; Signal Transduction ; Topoisomerase II Inhibitors ; U937 Cells

This study was aimed to explore the expression of survivin in leukemia cells and to investigate the effect of GM-CSF on survivin expression. The survivin expressions in 37 previously untreated leukemia patients and 10 normal persons as well as in three kinds of leukemia cell lines (K562, HL-60, U937) were analyzed by RT-PCR. The HL-60 cells were treated by the proper concentration of GM-CSF, and the changes of survivin mRNA and protein expression levels were detected by using RT-PCR and Western blot respectively. The results indicated that the positive rate of survivin gene in 37 leukemia patients was 67.6% (25/37) and significantly higher than that in normal control (20.0%). The expression level was higher in ALL cells than that in AML cells (73.3% vs 63.6%). Moreover, three kinds of leukemia cell lines all expressed survivin. After treating with the proper concentration of GM-CSF for 2 days, the expression of survivin obviously increased and the expression level of survivin mRNA was up-regulated by 26%, the expression level of survivin protein was up-regulated by 49%. It is concluded that the survivin gene extensively express in leukemia and its cell lines, and its expression can be obviously increased by GM-CSF.
Adolescent ; Adult ; Aged ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; HL-60 Cells ; Humans ; Inhibitor of Apoptosis Proteins ; K562 Cells ; Leukemia, Myeloid, Acute ; metabolism ; Male ; Microtubule-Associated Proteins ; biosynthesis ; genetics ; Middle Aged ; Neoplasm Proteins ; biosynthesis ; genetics ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; metabolism ; U937 Cells