Objective: To investigate the effect of CD33-targeted bi-specific and tri-specific T-cell engagers on T-cell proliferation and explore their cytotoxicity on leukemia cells. Methods: The CD33-targeted bi-specific T-cell engager (CD33-BiTE) and tri-specific T-cell engager (CD33-TriTE) expression vectors were successfully constructed and expressed through a eukaryotic cell expression system. CD33-BiTE and CD33-TriTE were purified by affinity chromatography. The effects of CD33-BiTE and CD33-TriTE on T cells were analyzed through in vitro experiments. Results: ① CD33-BiTE and CD33-TriTE were successfully constructed and purified and could compete with flow cytometry antibodies for binding to the target cells. ② After 12 days of co-culture with CD33-BiTE and CD33-TriTE, the number of human T cells were expanded to 33.89±19.46 and 81.56±23.62 folds, respectively. CD33-TriTE induced a stronger proliferation of T cells than CD33-BiTE (P<0.05) . ③ Both CD33-BiTE and CD33-TriTE induced specific dose-dependent cytotoxicity on CD33(+) leukemia cells. ④ Compared to CD33-TriTE, leukemia cells were prone to express PD-L1 when co-cultured with T cells and CD33-BiTE. CD33-TriTE induced powerful cytotoxicity on leukemia cells with high PD-L1 expression. Conclusion: CD33-BiTE and CD33-TriTE expression vectors were constructed, and fusion proteins were expressed in eukaryotic cells. Our results support the proliferative and activating effects of BiTE and TriTE on T cells. Compared to that of CD33-BiTE, CD33-TriTE induced a stronger proliferative effect on T cells and a more powerful cytotoxicity on leukemia cells with high PD-L1 expression.
B7-H1 Antigen/pharmacology* ; Humans ; Leukemia, Myeloid, Acute/metabolism* ; Sialic Acid Binding Ig-like Lectin 3/pharmacology* ; T-Lymphocytes

The object of this study was to explore the effects of Panax notoginosides (PNS) on proliferation and differentiation of human CD34(+) stem/progenitor cells. CD34(+) cells were isolated from human bone marrow by using immune beads of Dynal M- 450 system. The cells were exposed to PNS at different concentrations in both liquid and semi-solid culture for 14 days. The cells were marked with monoclonal antibodies and analyzed by flow cytometry after culture. The CFU-Mix colony formation from CD34(+) cells was assayed. The results showed that: (1) The yield of CD34(+) cells after being selected by immune beads were (1.03 +/- 0.74)% out of bone marrow nuclear cells with purity of 86% - 93%. (2) PNS (10 - 25 mg/L) stimulated the proliferation of CD34(+) cells, and raised the colony numbers of CFU-Mix obviously in vitro. PNS 25 mg/L was the optimal concentration to promote proliferation of CD34(+) cells, the increasing rate of CFU-Mix colony was (34.7 +/- 16.0)%. (3) The differentiation of CD34(+) cells was induced by exposure to PNS (25, 50 and 100 mg/L) in liquid culture for 14 days. The percentages of CD33(+) and CD15(+) cells were increased after PNS exposure, which were significantly higher than those of control (P < 0.01), however CD71(+) and G-A(+) cells were no obviously difference after PNS treatment. In conclusion, Panax notoginosides not only promote the proliferation of CD34(+) cells, but also induce the differentiation committed to granulocytes.
Antigens, CD ; analysis ; Antigens, CD34 ; analysis ; Antigens, Differentiation, Myelomonocytic ; analysis ; Cell Differentiation ; drug effects ; Cell Division ; drug effects ; Ginsenosides ; pharmacology ; Hematopoietic Stem Cells ; cytology ; drug effects ; Humans ; Lewis X Antigen ; analysis ; Sialic Acid Binding Ig-like Lectin 3

In order to investigate the potential of human ERMAP gene in erythroid cell differentiation, K562 cells were induced to erythroid lineage by Ara-C and to macrophage lineage by TPA, human ERMAP mRNA was detected by fluorescent quantitative PCR. The results showed that human ERMAP mRNA increased while K562 cells were induced to erythroid lineage after treatment with Ara-C at 2.5 x 10(-6) mmol/L/L and 1.0 x 10(-6) mmol/L/L. Human ERMAP mRNA not changed while K562 cells were induced to macrophage lineage after treatment with TPA at 2.0 x 10(-6) mmol/L/L and 1.0 x 10(-6) mmol/L/L. It is concluded that human ERMAP gene plays an important role in differentiation and proliferation of erythroid cells.
Antigens, CD ; analysis ; Antigens, Differentiation, Myelomonocytic ; analysis ; Blood Group Antigens ; genetics ; Butyrophilins ; Cell Differentiation ; drug effects ; genetics ; Cytarabine ; pharmacology ; Erythrocytes ; cytology ; metabolism ; ultrastructure ; Flow Cytometry ; Gene Expression ; drug effects ; Humans ; K562 Cells ; Macrophages ; cytology ; metabolism ; ultrastructure ; Microscopy, Electron ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, Transferrin ; analysis ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sialic Acid Binding Ig-like Lectin 3 ; Tetradecanoylphorbol Acetate ; pharmacology ; Time Factors

To study the effect of interleukin-15 (IL-15) on the proliferation, differentiation and apoptosis of MDS CD34(+) cells, CD34(+) cells of high enrichment were separated by MACS system, and cultured in liquid media with different concentration of IL-15 in treated group and without IL-15 in the control group. Apoptosis of hematopoietic precursors was assayed by propidium iodine staining and cell by FCM, and the other MDS CD34(+) cells were stained by cytochemical staining after culture. The results showed that after culture with IL-15 the proliferation and differentiation of MDS CD34(+) cells were obviously promoted. It was found the every lineage of mature cells developed, the expressions of cell surface antigens CD71, CD33 and CD19 all increased in the MDS CD34(+) cell treated with IL-15. It is suggested that IL-15 stimulates the proliferation and differentiation of MDS CD34(+) cells, and partly shows anti-apoptosis effects which may be applicable to the therapy MDS.
Antigens, CD ; immunology ; Antigens, CD19 ; immunology ; Antigens, CD34 ; immunology ; Antigens, Differentiation, Myelomonocytic ; immunology ; Apoptosis ; drug effects ; Bone Marrow Cells ; drug effects ; immunology ; pathology ; Cell Cycle ; drug effects ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Flow Cytometry ; Humans ; Interleukin-15 ; pharmacology ; Microscopy, Fluorescence ; Myelodysplastic Syndromes ; blood ; immunology ; pathology ; Receptors, Transferrin ; immunology ; Sialic Acid Binding Ig-like Lectin 3

OBJECTIVETo explore the synergic effect of heparan sulfate (HS) and cytokines on the growth of human umbilical cord blood (UCB) hematopoietic cells.

METHODSHematopoietic cells from human UCB were cultured with (1) cytokines (rhIL-3, rhIL-1beta, rhIL-6 and SCF) or (2) SN (supernatant from cultured human marrow stromal cells) and cytokines, or (3) SN, or (4) HS and cytokines. Cellular proliferation, CFU-GM yields and changes of cell immunophenotype were observed.

RESULTSHematopoietic cell proliferation reached peak at the 14th day. The number of total nucleated cells increased 134.5-, 171.3-, 81.5- and 167.2-fold in (1), (2), (3) and (4) groups, respectively, at the 21th day, the (3) group significantly decreased. CD(34)(+) cells increased at the 7th day and reached peak, at the 14th day with a percentage of 68.4%, 82.5%, 69.8% and 79.3%, and at the 21th day 56.2%, 71.7.%, 12.3% and 73.3%, respectively. CD(33)(+) cells reached peak at the 14th day and increased by 80.2%, 68.6%, 81.6% and 70.3%, respectively, and remained these levels at the 21th day. CD(38)(+) cells increased by 66.6%, 73.8%, 70.4% and 71.9% at the 7th day and remained this level at the 14th and the 21th day. From the first week of culture, the percentage of CFU-GM increased in all of the four groups, at the second week of culture, it increased by 250%, 279%, 217% and 273%, and at the third week still increased by 151%, 240%, 145% and 231%, respectively.

CONCLUSIONThe combination of SN and cytokines have a synergic effect in promoting proliferation of hematopoietic cell from human UCB. The synergic effect remained the same when SN was replaced by heparan sulfate.

ADP-ribosyl Cyclase ; analysis ; ADP-ribosyl Cyclase 1 ; Antigens, CD ; analysis ; Antigens, CD34 ; analysis ; Antigens, Differentiation, Myelomonocytic ; analysis ; Cell Division ; drug effects ; Cells, Cultured ; Cytokines ; pharmacology ; Drug Synergism ; Fetal Blood ; cytology ; drug effects ; immunology ; Heparitin Sulfate ; pharmacology ; Humans ; Infant, Newborn ; Interleukin-1 ; pharmacology ; Interleukin-3 ; pharmacology ; Leukocytes, Mononuclear ; cytology ; drug effects ; immunology ; Membrane Glycoproteins ; Recombinant Proteins ; pharmacology ; Sialic Acid Binding Ig-like Lectin 3 ; Time Factors

OBJECTIVE12-0-tetradecanoylphorbol-13 acetate (TPA) plays an important role in precipitating cell differentiation for various tumor cells, especially leukemic cells. Changes of many genes may be involved in this process. The purpose of this study was to observe the relationship between the EGR1mRNA expression and cell differentiation during TPA-induced K562 cell differentiation.

METHODSIncubation of human K562 cells in vitro was applied to cultivate K562 cells. The cells were treated in two different ways. K562 cells of experiment group were treated with TPA and those of control group were treated without TPA. Using morphology (Wright's staining and NSE staining) and flow cytometry (FCM), the investigators observed the differentiation characteristics of K562 cells, cell-cycle and the differentiation antigen expressions of CD33 and CD14 on cell membranes. RT-PCR was carried out to assay EGR1 mRNA expression.

RESULTSAfter treated with TPA for 7 d, the morphology of K562 cells obviously tended to mature differentiation, like monocytes. The differentiation rate of induced K562 cells was up to 95% in experiment group and 4.5% in control group, respectively. Using SPSS software, the above result showed statistical significance (P < 0.01). Using NSE staining, K562 cells showed positive reaction. Some of them were densely stained. The positive rate was up to 86%. More than half of the positive cells could be inhibited by NaF. The inhibiting rate of NaF was up to 58.72%, showing statistical difference when compared with that of control group. FCM analysis showed that most of K562 cells stimulated by TPA underwent G1/S phase cell-cycle arrest. The composing rate of cell-cycle in TPA-treated group showed that (53.7 +/- 1.25)% of cells were at G0 + G1 phase and (44.3 +/- 1.32)% were at S phase (P < 0.05). The level of CD33 expression on cell membranes was mildly decreased from 0.997% to 0.893% (P > 0.05). However, the level of CD14 expression was significantly increased from 0.049% to 0.387% (P < 0.05).

CONCLUSIONK562 cells could express EGR1mRNA during TPA-induced differentiation, which suggested that EGR1mRNA might participate in the process of K562 cells differentiating into monocyte/macrophages, and might play an important role in precipitating and maintaining cell differentiation for leukemic cells.

Antigens, CD ; metabolism ; Antigens, Differentiation, Myelomonocytic ; metabolism ; Carcinogens ; pharmacology ; Cell Cycle ; drug effects ; genetics ; Cell Differentiation ; drug effects ; genetics ; Cell Division ; drug effects ; genetics ; Cell Membrane ; chemistry ; drug effects ; DNA-Binding Proteins ; genetics ; Early Growth Response Protein 1 ; Flow Cytometry ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Immediate-Early Proteins ; genetics ; K562 Cells ; Lipopolysaccharide Receptors ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Sialic Acid Binding Ig-like Lectin 3 ; Tetradecanoylphorbol Acetate ; pharmacology ; Transcription Factors ; genetics

To establish a mouse model bearing transplantable human chronic myeloid leukemia for hematopoietic stem cell transplantation to treat leukemia, 4 - 5-week-old female BALB/c nude mice were given cyclophosphamide 2 mg/mouse at day -2, -1, and then the human chronic myeloid leukemia K562 cells were engrafted into the mice at day 0 by injection via tail vein or peritoneal cavity. PB and BM cells were collected, the CD45, CD13, and CD33 antigens were delected by using FCM, the bcr/abl fusion gene mRNA was examined by RT-PCR. The results showed that transplantable leukemic mice could be yielded from 4 - 5-week-old nude mice either by injection through tail vein or peritoneal cavity when the total number of inoculated tumor cells was more than 2 x 10(5) per mouse, whether being pretreated with 2 mg CTX/mouse or not. The transplanted mice could survive 30 - 60 day with leukemia. In conclusion, the mouse model bearing leukemia can be established by inoculation 2 x 10(5) K562 cells into immunodeficient BALB/c nude mice.
Animals ; Antigens, CD ; blood ; Antigens, Differentiation, Myelomonocytic ; blood ; Antineoplastic Agents, Alkylating ; pharmacology ; CD13 Antigens ; blood ; Cyclophosphamide ; pharmacology ; Female ; Flow Cytometry ; Fusion Proteins, bcr-abl ; genetics ; Gene Expression Regulation, Leukemic ; drug effects ; Humans ; K562 Cells ; Leukemia, Experimental ; blood ; genetics ; pathology ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; blood ; genetics ; pathology ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Sialic Acid Binding Ig-like Lectin 3 ; Transplantation, Heterologous

To explore the regulation mechanism of survivin gene, the NB4 and HL-60 cells were used in experiments, the cell culture in vitro and cell morphological observation were performed and survivin mRNA expression was detected by semi-quantitative RT-PCR. The results showed that the survivin expression in NB4 cell was positive. By treatment of 1 micromol/L ATRA, cell differentiation antigen CD11b was gradually increased ([chi = 47.002, P = 0.000) and CD33 was gradually decreased (chi = 1.614, P = 0.806) with time. Simultaneously, survivin mRNA expression was down-regulated and the cell cycle was arrested at G(0)-G(1) phase (chi = 58.566, P = 0.000). ATRA could down-regulate the survivin mRNA expression of HL-60 cell, but G-CSF, GM-CSF and PHA could up-regulate the survivin expression of HL-60 cell. The cytokine could regulate survivin expression in gene transcription level. The up-regulation of survivin expression was observed while HL-60 cell was stimulated by PHA. The survivin gene expression could be blocked by the survivin antisense oligonucleotide. The survivin mRNA expression of NB4 cell was inhibited by 100 nmol/L-1000 nmol/L survivin antisense oligonucleotide in a dose-dependent manner. The survivin mRNA expression in the NB4 cell was obviously inhibited in 600 nmol/L survivin AS-ODN groups (38%) while the AS-ODN dose increases, the inhibition rate does not descend, but was not inhibited in the control groups, liposomes groups and ODN groups. After NB4 cell was treated by survivin AS-ODN, the typical morphological changes for the apoptosis emerged in NB4 cell. These changes were not found in control groups. It is concluded that PHA, GM-CSF and G-CSF can up-regulate the survivin gene expression, but survivin AS-ODN and ATRA can down-regulate survivin gene expression. The cell cycle arrest at G(0)-G(1) phase while the survivin gene expression was down-regulated by ATRA. It suggested that the survivin gene expression is very related to cell cycle. The morphological changes of cell apoptosis can be observed when the survivin gene expression of NB4 cell was suppressed.
Antigens, CD ; analysis ; Antigens, Differentiation, Myelomonocytic ; analysis ; Apoptosis ; drug effects ; genetics ; CD11b Antigen ; analysis ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Down-Regulation ; drug effects ; genetics ; Flow Cytometry ; Fluorescent Antibody Technique ; Gene Expression Regulation, Neoplastic ; Granulocyte Colony-Stimulating Factor ; pharmacology ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; HL-60 Cells ; Humans ; Inhibitor of Apoptosis Proteins ; Microtubule-Associated Proteins ; genetics ; Neoplasm Proteins ; genetics ; Oligonucleotides, Antisense ; genetics ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Sialic Acid Binding Ig-like Lectin 3 ; Tretinoin ; pharmacology ; Up-Regulation ; drug effects ; genetics