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Background Temporal lobe epilepsy (TLE) is the most common drug-resistant epilepsy in adults, with pathological mechanisms remaining to be fully elucidated. Fibroblast Growth Factor 13 (FGF13) encodes an intracellular protein involved in microtubule stabilization and regulation of voltage-gated sodium channels (VGSCs) function. FGF13 mutation has been identified in patients with inherent seizure, suggesting a potential association between FGF13 and the etiology of TLE. Here, we set to explore the pathological role of FGF13 in the etiology of TLE. Results We found that the expression of FGF13 was increased in the cortical lesions and CA1 region of sclerotic hippocampus and correlated with the seizure frequency in TLE patients. Also, Fgf13 expression was increased in the hippocampus of chronic TLE mice generated by kainic acid (KA) injection. Furthermore, Fgf13 knockdown or overexpression was respectively found to attenuate or potentiate the effects of KA on axonal length, somatic area and the VGSCs-mediated current in the hippocampal neurons. Conclusions Taken together, these findings suggest that FGF13 is involved in the pathogenesis of TLE by modulating microtubule activity and neuronal excitability.

Nucleotides are new players in the intercellular communication network. P2X7 is a member of the P2X family of receptors, which are ATP-gated plasma membrane ion channels with diverse biological functions. Abnormal expression and dysfunction of P2X7 have been reported in leukemias. Here, we report a new P2X7 mutant (an A(559)-to-G substitution causing N187D P2X7) cloned from J6-1 leukemia cells. The characteristics of N187D P2X7 were studied by establishing stably transfected K562 cell lines. Our results show that N187D P2X7 required a higher concentration of agonist for its activation, leading to Ca(2+) influx (EC(50) = 293.3 ± 6.6 μm for the mutant and 93.6 ± 2.2 μm for wild-type P2X7) and ERK phosphorylation, which were not caused by differential cell-surface expression or related to high ATPase activity on the cell surface and in the extracellular space. K562 cells expressing this N187D mutant showed a proliferative advantage and reduced pro-apoptosis effects in vitro and in vivo. Furthermore, elevated angiogenesis and CD206-positive macrophage infiltration were found in tumor tissues formed by K562-M cells. In addition, higher expression of VEGF and MCP1 could be detected in tumor tissues formed by K562-M cells. Our results suggest that N187D P2X7, representing mutants hyposensitive to agonist, might be a positive regulator in the progression of hematopoietic malignancies.

The theory of evolution of tumor cell population has been established for nearly 40 years. It was widely accepted for research and clinical anti-tumor treatment. Recently, it was suggested that cancer stem cells are the unit of evolution. Considering recent advances on genesis of tumor and leukemia with ecological and evolutionary views, this article reviews origin and evolution of leukemia stem cells. Over the last few years, clinical and experimental data suggest there are two paths for the origin of leukemia stem cells: from a transformed hematopoietic stem cell or progenitor. The mechanisms of leukemia stem cell formation and clonal evolution were elucidated. Sub-clonal mutations and clonal architectures in leukemia were studied and a mosaic evolution pattern is described. Random evolution or non-inherited mutations of leukemia cells would accelerate the progression of malignant disease. Finally, the mosaic or network mechanism for leukemogenesis is also discussed.

The cDNA encoding N-terminal three immunoglobin-like domains of human M-CSFR was linked to His-tag and endoplasmic reticulum retention sequence (KDEL) before being inserted into the genome of tobacco plant, Nicotiana tabacum cv. NC-89, by Agrobacterium tumefaciens-mediated transformation. The insertion and expression of target gene were confirmed by PCR, ELISA, and Western blot. The recombinant M-CSFsR reached a maximum expression level of 1.92% of total soluble protein in transgenic tobacco plant leaf tissues. The recombinant M-CSFsR could be purified through a one-step IMAC process and its bioactivity was confirmed by the inhibition of colony formation of J6-1 cells. The results suggested that we successfully expressed a high level of bioactive human M-CSFsR in tobacco plants.

The P2X7 nucleotide receptor is an adenosine 5'-triphosphate (ATP) -gated ion channel, which is widely expressed in cells of hematopoietic origin and functions as a non-selective cation channel permeable to Na+, Ca2+, etc upon stimulation. Here, we investigated P2X7 expression in 11 human hematopoietic cell lines, representing different lineages, as well as bone marrow mononuclear cells (BMMC) samples from 87 leukemia and 10 myelodysplastic syndrome (MDS) patients. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry results showed that both P2X7 mRNA and protein were detected in eight cell lines with a non-lineage-specific manner. Samples from 69 leukemia and 9 MDS patients were P2X7 positive at mRNA level. Moreover, both positive rates and relative expression levels were significantly higher in acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), and MDS groups than that in normal donor group. The expression levels varied among AML subtypes with higher levels being observed in M4, M5, and M6 groups but not in M1 or M2 group. Furthermore, after one course of standard induction therapies, the remission rate in high P2X7 expression group was lower than that in either P2X7 negative group or low P2X7 expression group. Cytoplasmic free calcium increase was detected in five of eight P2X7+ cell lines as well as P2X7+ normal donor and patient samples tested, but not in three Epstein-Barr virus (EBV) positive cell lines (J6-1, Namalwa, and LCL-H) in Locke's solution upon stimulation by extracellular ATP or the more potent and specific agonist, 2',3'-O-(4-benzoyl)benzoyl-ATP (BzATP). The possible mechanisms causing the loss of P2X7 function were discussed.

The human cationic antimicrobial peptide hCAP-18/LL-37 is the unique cathelicidin identified in human to date. It has broad spectrum of antimicrobial activities and LPS-neutralizing activity and is involved in angiogenesis. Both purified and synthetic LL-37 or its derivatives were used in the study on LL-37. However, production of LL-37 in Escherichia coli has not been established. In this study, its precursor instead of the mature peptide was adopted for expression to avoid the lethal effect of recombinant LL-37 on host cells. A thrombin recognition site was introduced between the cathelin-like domain and LL-37 domain by overlap PCR to construct fragment encoding modified precursor (mhCAP-18) to facilitate the final release of the recombinant peptide. Then mhCAP-18 was fused in-frame to thioredoxin gene under the control of inducible T7 promoter to construct expression vector pET-mhCAP-18. The soluble form fusion protein was expressed in E. coli and purified by Chelating Sepharose column chromatography. Thrombin digestion of the fusion protein yielded recombinant GSLL-39, which was then purified by cation-exchange chromatography. Recombinant GSLL-39, which has two extra residues on its N-terminus when compared with its native counterpart, showed similar antimicrobial activities against both Gram-negative and Gram-positive bacteria.

The transmigration of hematopoietic progenitor cells is a crucial step in the homing of transplanted stem cells into bone marrow (BM) microenvironment; however, the molecular basis for this is not fully understood. Matrix metalloproteinases (MMPs), which are implicated in the migration of leukocytes, are important in degrading components of extracellular matrix molecules. In this study, using zymographic analysis and enzyme-linked immunosorbent assay (ELISA), we investigated the production of MMP-9 in CD34(+) cells from cord blood (CB) and BM, compared their spontaneous migration across a reconstituted basement membrane-coated filter in transwell, and studied the role of MMP-9 in the transmigration. Zymography and ELISA showed that MMP-9 is produced by freshly isolated CD34(+) stem/progenitor cells obtained from CB. CB CD34(+) cells showed significantly higher migrational capacity than BM CD34(+) cells ( p=0.008). Furthermore, the migrational ability of CB CD34(+) cells over the extracellular matrix (ECM) was significantly inhibited by the inhibitor of MMP, o-phenanthroline and anti-MMP-9 monoclonal antibody (73.3+/-11.8% and 37.5+/-10.4% inhibition, respectively). Our results strengthen the potential role of MMP-9 in the higher migrational capacity of CB CD34(+) cells, which may be beneficial to homing of these cells to the BM environment.

We report in a murine model of acute lymphoid leukemia L1210 the potent antitumor efficiency of a combinatorial delivery of pro-IL-18 gene modified L1210 (Lp18) and IL-1beta converting enzyme (ICE) gene modified L1210 (LpICE). Live leukemia cells Lp18 or Lp18 plus LpICE showed apparently reduced leukemogenicity with a survival rate of 40 or 50% at 50 days after intraperitoneal (i.p.) inoculation of a lethal dose of cells, respectively. Combination of Lp18 and LpICE was capable of inhibiting accumulation of bloody ascites, synergistically superior to Lp18 or LpICE alone. All surviving mice were rechallenged with parental L1210 cells at day 50, and all survived up to day 80, suggesting that gene-modified cells induced immune protection. Moreover, NK cytotoxicity and CTL activity were both enhanced in mice injected with Lp18, especially Lp18 plus LpICE. Levels of IFN-gamma were not altered significantly by inoculation of Lp18 or Lp18 plus LpICE. Our results demonstrate that IL-18 is a useful candidate gene in gene therapy of lymphoma or lymphoid leukemia, and ex vivo combinatorial delivery of Lp18 plus LpICE either as a single approach or as an adjunct to concomitant radiotherapy or chemotherapy, may be more efficient in a situation of minimal residual disease.

Objective: To investigate the functions of nM-CSF in malignant cells. Methods: recombinant M-CSF was targeted into cell nucleus by employing a eukaryotic expression plasmid vector pCMV/myc/nuc. The constructed plasmid was transfected into cells of EBV transformed lymphoblastoid cell line (LCL). RT-PCR, Western blot and immunofluorescent staining showed that recombinant M-CSF was localized into LCL cell nucleus. The transgenic cells showed elevated proliferation potential, enhanced resistance to apoptosis and increased ability ofin vitro migration. Conclusion: Nucleus presenting M-CSF might act as a promoting factor in the processes of cell malignancy.

LL-37/hCAP-18 is an important part of host defense. Several diseases in human are characterized by impairment in the function of LL-37/hCAP-18 peptide. We examined the expression of LL-37/hCAP-18 in a panel of hematopoietic cell lines representing multiple cell lineages. LL-37/hCAP-18 expression at mRNA level was detected and varied among six of nine cell lines. The level of Raji cells was about eight folds higher than that of Ramos cells. However, only two cell lines, J6-1 and U937, expressed protein products. We also investigated LL-37/hCAP-18 protein in nine leukemia and three idiopathic thrombocytopenic purpura (ITP) patients via immunocytochemical staining. The rate of LL-37/hCAP-18 positive cells ranged from 60 (ITP) to 0.5% (M5). These data suggested that the low translation efficiency of LL-37/hCAP-18 expresses in some leukemia cells might be one of the reasons that leukemia patients were susceptible to infection.

The importance of IL-18, although clearly established in solid tumors, has not been fully elucidated in human hematopoietic neoplasms. Here we examined the mRNA and protein for IL-18 in eight human hematopoietic cell lines representing different lineages and neoplasms including leukemia, lymphoma and others. Our results revealed that IL-18 mRNA was expressed in these cells and that the corresponding protein was found in the cytoplasm. Seven of eight cell lines were also found to express two subunits of the IL-18 receptor (IL-18R) at varied levels. Furthermore, 29 out of 51 leukemia patients tested were observed to express IL-18R with 18/29 (62%) co-expression of both receptor and ligand. By blocking the IL-18 loop using specific antisense oligodeoxynucleotide (ASON) for IL-18 mRNA or anti-human IL-18R monoclonal antibody (McAbR), we were not able to demonstrate a marked inhibition on the most leukemic cell lines growth. Moreover, the potential proliferation in vitro of primary AML cells co-expressing IL-18 and its receptor was not significantly enhanced by recombinant human IL-18, suggesting that IL-18 is not apparently implicated in the proliferation of the leukemia cells via an autocrine loop. Additionally, we also found the effective modulating effect of M-CSF, IFN-alpha and TNF-alpha on IL-18R expression, implying an important in vivo effect of cytokines on IL-18-induced reaction. Moreover, the modulation of IL-18R expression was possibly irrelevant to IFN-gamma secretion induced by these cytokines.

Objective: To explore the effects of nuclear M-CSF on the process of tumorigenesis. Methods: Functional part of M-CSF cDNA was inserted into an eukaryotic expression plasmid pCMV/myc/nuc, which can add three NLS to the C-terminal of the expressed protein and direct the protein into the cell nuclei. The constructed plasmid was transferred into NIH3T3 cells and the cell clones were selected by G-418 selection. Cell clones stable expressing target protein were identified by RT-PCR, ABC immunohistochemistry assay and Western blot. Cell growth kinetics analyses through growth curves, cell doubling time, MTT test and anti-sense oligodeoxynucleotide (ASODN) inhibiting cell growth test were performed to identify cells proliferation potential. Results: The transfected cells showed elevated proliferation potential over the control cells. Conclusion: Abnormal appearance of M-CSF in nucleus could enhance cell proliferation, which suggests that cytokine isoforms within cell nucleus might play transcription factor-like role.

The cDNA of human interleukin-18 (hIL-18) was successfully inserted into the genome of tobacco plant, Nicotiana tabacum cv. NC-89, using Agrobacterium tumefaciens-mediated transformation. Insertion and translation of hIL-18 in transformants were confirmed by PCR, ELISA, and Western blot, respectively. The transformed extracts contained the recombinant hIL-18 protein up to 0.05% of total soluble protein. Activity of the recombinant hIL-18 in plant cells was confirmed by the induction of IFN-γ on IL-18-responsive J6-1 cells by the extracts obtained from the transformants. The expression level of hIL-18 (351 ng g−1 tobacco tissue) obtained in the present study may be sufficient to induce responses/effects in vivo.

Little is known about the clinical significance of interleukin (IL)-18, a novel immunoregulatory cytokine, in acute myeloid leukemia (AML). Using reverse transcriptase polymerase chain reaction (RT-PCR) analysis, levels of IL-18 mRNA were assessed in bone marrow mononuclear cells (BMMC) from 47 adult patients with de novo or CR AML in order to explore the clinical significance of IL-18. The relationship between expression levels and the established prognostic factors such as age, cytogenetic aberrations, CD34 expression and FAB subtypes was investigated. Either disease status, age or CD34 expression were found to significantly correlate with the expression of IL-18. With respect to FAB cytotypes, expression of IL-18 gene in M4/M5 (n=15) was statistically higher than that in other subtypes (n=32, P

The abnormal expression of macrophage colony stimulating factor (M-CSF) isoforms, i.e. membrane bound M-CSF (m-M-CSF) and intracellular M-CSF (c-M-CSF), and their receptor were reported in some leukemia and tumor cells. Furthermore, the nuclear localization of them may be related to poor prognosis and metastasis, while the mechanism is uncertain. We previously reported that m-M-CSF and its receptor played auto-juxtacrine and adhesion molecule role in human leukemia cell line J6-1. In this paper, we show that HL-60 cells highly express M-CSF and its receptor. The localization of positive reactions was mainly in cytoplasma and nuclear in HL-60 cells. In cytoplasma and nuclear, three isoforms of M-CSF were found with molecular weight (MW) of 20, 16 and 14 kDa, while one type of m-CSF receptor (M-CSFR) was discovered with MW of 120 kDa. Immunoprecipitation assay showed that these ligands could exist separately or binding with their receptor. Monoclonal antibody (McAb) against M-CSF and anti-sense oligodeoxynucleotides (ASON) blocking M-CSF expression inhibited the proliferation of HL-60 cells. McAb and ASON regulated the expression of cyclin D1/E, CDK2/4 and p16. Simultaneous administration of both McAb and ASON inhibited the proliferation of HL-60 cells and modulate the expression of cyclins at greater degrees. Our results suggested an autocrine and possible an intracrine loop of M-CSF/M-CSFR in HL-60 cells.

Objective: To investigate the serum levels of soluble macrophage colony-stimulating factor receptor (M-CSFsR) in normal subjects and patients with hematological diseases and its clinical implications in hematological diseases. Methods: The concentration of M-CSFsR was determined by ELISA. The serum M-CSFsR was identified and characterized by immunoprecipitation and Western blotting. Results: The mean serum level of M-CSFsR of 123 normal individuals was 0.48 ng/ml ± 0.41 ng/ml. Immunoprecipitation and Western blotting assay revealed a ∼ 90kD band of serum M-CSFsR. The mean serum M-CSFsR level of 60 patients with acute lymphoblastic leukemia (ALL), 36 patients with acute myeloblastic leukemia (AML), 13 patients with myelodysplastic syndrome (MDS) and 42 patients with aplastic anemia (AA) .were 0.22 ng/ml±0.23 ng/ml, 0.17 ng/ml±0.16 ng/ml, 0.19 ng/ml±0.16 ng/ml and 0.23 ng/ml±0.21 ng/ml, respectively, which were significantly lower than that of normal subjects (P=0.002 ,P

Objective: To study the potential role of cellular macrophage colony-stimulating factor (cM-CSF) and cellular macrophage colony-stimulating factor receptor (cM-CSF-R) with breast cancer and hepatoma and search the way for clinical application. Methods: Frozen surgical specimens from 48 breast cancer patients, including 29 cases of histological grade II and 19 eases of grade III, and 16 hepatoma patients were investigated by Avidin Biotin Complex (ABC) immunohistochemical assay with anti-M-CSF monoclonal antibody (Mab) and anti-M-CSF-R Mab. Pathohistological examination was performed as well. Results: cM-CSF and cM-CSF-R were detected in tested specimens. The expression levels of cM-CSF and cM-CSF-R in grade III group were higher than in grade II group and more higher than control group hyperplasia of breast. Hepatoma tissues also showed higher expression level of cM-CSF and cM-CSF-R than normal adult and fetal liver. Conclusion: Breast cancer and hepatoma tissues presented higher expression levels of cM-CSF and cM-CSF-R than control and expression level might be related with tumor’s process.

Feedback and feedforward widely exist in life system, both of them are the basic processes of control system. While the concept of feedback has been widely used in life science, feedforward regulation was systematically studied in neurophysiology, awaiting further evidence and mechanism in molecular biology and cell biology. The authors put forward a hypothesis about the feedforward regulation of membrane bound macrophage colony stimulation factor (mM-CSF) on the basis of their previous work. This hypothesis might provide a new direction for the study on the biological effects of mM-CSF on leukemia and solid tumors, and contribute to the study on other membrane bound cytokines.

The binding of recombinant human macrophage colony-stimulating factor (M-CSF) soluble receptor (rh-M-CSF-sR) to membrane-bound macrophage colony stimulating factor (m-M-CSF) and the internalization and recycling of rh-M-CSF-sR/m-M-CSF complexes mediated by m-M-CSF were studied with a model of J6-1 cell line. The results indicated that m-M-CSF bound rh-M-CSF-sR with high affinity (kd = 1.78×1012 mol/L) and mediated a temperature- and energydependent internalization of rh-M-CSF-sR, and that internalized rh-M-CSF-sR could return to the cell surface in an m-M-CSF-bound state, suggesting that m-M-CSF may have a capability to mediate the internalization and recycling of rh-M-CSF-sR/m-M-CSF complexes. In addition, the half-lives of cell-associated M-CSF and its receptor of stimulated normal human cord blood mononuclear cells (CBMCs) and 4 leukemic cell lines were measured by indirect immunofluorescence and flow cytometry. The results showed that the half-lives of the various kinds of M-CSF isoforms and M-CSF-R in 4 leukemic cells, except those of membrane-bound M-CSF receptor (M-CSF-mR) of K562 and HL-60 cells, were longer than those of corresponding M-CSF isoform and its receptor in stimulated normal human CBMCs, suggesting that there is a slow degradation rate of cell-associated M-CSF and its receptor in leukemic cells.

Membrane-bound macrophage colony-stimulating factor (m-M-CSF) is the membrane form M-CSF by alternative splicing. J6-1 leukemic cell line spontaneously forms cell clusters, whose growth depends on the auto-juxtacrine mediated by m-M-CSF and its receptor (M-CSFR). In this study, M-CSFR isolated from J6-1 cells and recombinant human M-CSF soluble receptor (rh-M-CSFsR) were used to study their effects on J6-1 cells. Both receptors inhibited cell proliferation. Use of M-CSFR monoclonal antibodies, M-CSFR or rh-M-CSFsR to block either M-CSFR or m-M-CSF on cell surface inhibited the cluster forming process, while both receptors stimulated cells adhering to culture plate. Furthermore, M-CSFR and/or rh-M-CSFsR caused multiple cellular changes including cytoplasmic pH, multinuclear cell ratio, antigen expression and cell diameter. A [Ca 2+ ] rise was induced within 90 s by both receptors. Western blot experiments showed that rh-M-CSFsR caused tyrosine phosphorylation on multiple cytoplasmic proteins of 45 kDa and 55–90 kDa, which could be blocked by H7. These observations suggested that m-M-CSF and M-CSFR mediate J6-1 cell intercellular adhesion with bi-directional signal transduction, and Ca 2+ , protein tyrosine kinases, PKC and/or other H7 sensitive kinase(s) involve in the counter-directional signal transduction.

Objective: To verify the antigen association of MAF-J6-1 receptor with M-CSFR and to further study the role of M-CSF and its receptor mediated juxtaerine in promoting leukemic cell proliferation. Methods: Monoclonal antibody (McAb) of MAF-J6-1R RE2 and polyclonal antibody (PolyAb) of rhM-CSFR were prepared. The specificity of MeAh RE2 to M-CSFR was confirmed by indirect ELISA, cross-neutralizing assay with J6-1 cell colony formation and neutralization test by ELISA. Results: The reactive activity of purified RE2 to M-CSFR was over 1: 16000. The inhibitory activity of M-CSFR and MAF-J6-1R could be blocked by RE2 and antI-M-CSFR antibody. The reactivity of RE2 to M-CSFR could be reduced by M-CSFR. Conclusion: The specificity of RE2 to M-CSFR was confirmed and the antigen association of MAF-J6-1R with M-CSFR was proved, It suggests that M-CSF and its receptor mediated auto-juxtaerine stimulation could be an operative mechanism in either leukemia or nonhematological malignancies.

Objective: To investigate the potential role of macrophage colony-stimulating factor (M-CSF) and macrophage colony-stimulating factor receptor (M-CSF-R) on the growth of human hepatoma cells. Methods: Specimens of different origin, including tissues of human hepatocellular carcinoma (HCC), human fetal liver (FL) and normal liver (NL), the hepatoma cell lines, as well as the peripheral blood mononuclear cells (PBMC) from patients with HCC or liver metastatic tumor (LMT), were used to detect the expression levels of M-CSF and M-CSF-R by ABC immunohistochemistry staining and reverse transcription polymerase chain reaction methods the expression levels of M-CSF and M-CSF-R. Influence of monoclonal antibody against M-CSF (B5) or M-CSF-R (RE2) on proliferation ability of hepatoma cell linesin vitro was also studied. Results: The results showed that hepatoma tissues produced elevated levels of both M-CSF and M-CSF-R compared with those of fetal liver (P

We have isolated an M-CSF-like membrane-associated growth factor from human leukemic J6-1 cells that can enhance the growth and colony formation of J6-1 cells in vitro . Indirect evidence suggests that this membrane-associated M-CSF-like growth factor may do so by stimulating a corresponding receptor co-expressed on the adjacent J6-1 cells. The objective of this study is to isolate the putative receptor in J6-1 cells by virtue of its ability to bind and thus “block” the growth of J6-1 cells. Based on this approach, we have isolated from the J6-1 cell membrane an inhibitory activity that can inhibit the clonal growth of J6-1 cells. The activity of this inhibitor can be readily neutralized by either anti-M-CSFR MAb or anti-M-CSFR antiserum, suggesting that it is related to M-CSFR, a product of c-fms proto-oncogene. Judging from Sephadex G-200 gel filtration, the molecular weight (MW) of this putative M-CSFR-like inhibitor was estimated to be approx. 150–180 kDa, comparable with that of M-CSFR. The specificity of M-CSFR-like protein to recognize and block membrane-bound M-CSF also was implicated by its ability to upregulate the steady-state levels of c-fms mRNA in J6-1 cells. Besides its antiproliferative activity in vitro , treatment of J6-1 cells with the putative receptor protein before inoculation effectively blocked the growth and tumor formation in vivo by J6-1 cells in a nude mouse model. These findings suggest that the growth and tumor development by J6-1 leukemic cells may involve a contact-mediated “juxtacrine mechanism”.

Evolutionary medicine can give rational explanation for metabolism diseases via ecology and evolutionary theory. Recently, the view of somatic cell macroevolution was used in the study on the genesis and development of tumors, which provided new insight in the research work on tumors. In this article, the well-adopted tumor therapy strategy, "Dancing with Cancer", was analyzed preliminarily from the point of co-evolution game theory, based on the non-classical immunology theory and genome theory. The importance of increasing host fitness by changing host life-style to enhance tolerance was emphasized, which is the basis of the Dancing with Cancer strategy. On the other hand, the spreading tumor cells are not equally malignant and spreading tumors should be treated as other chronic diseases. Finally, basic and clinical research should be strengthened to improve the efficiency of the "Dancing with Cancer" strategy.

Density-dependent cell proliferation and cluster formation are growth phenotypes frequently associated with leukemia cells. The secretion of autocrine growth factor, such as granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 1 (IL-1), has been implicated as one possible mechanism in leukemogenesis. In many cases, however, leukemia cells do not appear to produce autocrine growth stimulators. J6-1 is an established human myeloid leukemia cell line that exhibits both density-dependent and cluster-forming growth characteristics. The effect of direct cell-cell contact on J6-1 cell proliferation was investigated. We have isolated from J6-1 cells a membrane-bound factor (designated as MAF-J6-1) that promoted the colony formation by both J6-1 cells and mouse bone marrow CFU-GM. The growth-promoting activity of MAF-J6-1 can be neutralized by either anti-macrophage-CSF (M-CSF or CSF-1) or anti-MAF-J6-1 monoclonal antibodies (MAb), suggesting that MAF-J6-1 is related to M-CSF. Using an immunoblot analysis with anti-MAF-J6-1 MAb, the MW of this membrane-associated factor was estimated to be 80 kDa. Both antibodies also induced a modest growth inhibition on J6-1 cells in vitro. Similarly, addition of exogenous recombinant human M-CSF augmented the colony formation by J6-1 cells, an effect also neutralized by both antibodies. Using an in situ hybridization technique, J6-1 cells were found to express a high level of c-fms proto-oncogene, which encodes the receptor for the M-CSF. Taken together, our results suggest that the membrane-bound MAF-J6-1 promote J6-1 cell proliferation and cluster formation through a 'juxtacrine' mechanism.

Relapse, which puzzled several generations of hematologists, is the bottle-neck of radical treatment for leukemias. The progress of Human Microbiome Project at the beginning of 21st century suggested that human body was a super-organism constituted by the core of human cells and symbiotic microorganisms. The elucidation and characterization of endogenous retrovirus and prion protein suggested the possible effects of co-evolutional microorganisms on human health. Recently, the elucidation of the roles of tunneling nanotubes in intercellular communication and transportation suggested a novel way for cellular communication and transport of oncogenic materials. The role and significance of in vivo cell fusion have been studied in more detail. On the other hand, donor cell leukemia was reported. All of these approaches provide novel insights for studying the mechanism of leukemia relapse. Based on previous work, the authors suggest the hypothesis: there are two possible mechanisms for the relapse of leukemias: the minimal residual disease (MRD) and intercellular transportation of oncogenic materials.

Neuronal synapse is the critical structure of neuronal network. Immune system is mainly consisted of invisible network. Recently, evidence showed that leukocyte synapses between immune cells named as immunological synapses (IS), were formed under some functional conditions to form temporal local network. In fact, they are dynamic structures, which can be classified into synapse and kinase. Different leukocytes have different synapses. Inflammatory and leukemic cells showed special patterns of IS. Similar structure is also observed in some viral infected lymphocytes, which is called virological synapse (VS). This is one of the mechanisms for viral transmission, not only enhancing the transmission efficiency but also mediating the escape from antibody neutralization, leading persistent infection. Recently the flower-like poly synapses was reported by French scientists. This is a multi-tunneling nanotube flower-like structure on cell surface. We had observed this kind of structure in EB virus infected human leukemic cell line J6-2. In this paper, the structure and function of leukocyte synapses are reviewed combined with authors' own work. Their significance is discussed.

Immune and hemopoiesis are one of basic project of experimental hematology. Immune function is a essential activity of white blood cells. It was puzzled for the diversity and complexity of immune response. Polarized immune response of immune cells was discovered 30 years ago, which facilitates the study on differentiation of lymphocyte. Recently recognition on multifunctional polarized immune response of lymphocyte and monocyte/macrophage would promote to elucidate the regulatory network of immune cells, diversity and complexity of immune response as well as the study on hemopoiesis. In this paper the approach of multifunctional polarized immune response of lymphocyte, monocyte/macrophage and dendritic cells were reviewed, and their role, especially in cytokine storm and tumor pro-inflammation condition were discussed.

With the development of science, the methods and the views of scientific research changed from analyses to syntheses. Recently, more attention has been paid to bio-diversity and complexity. According to the study on M-CSF and its receptor for years, the author suggests that, the multi-level of bio-diversity also appears at the bio-macromolecular level. Probability of bio-diversity is one of the bases for bio-complexity. Cellular sociology and topobiology are important aspects in bio-complexity, and should be developed. If taking Chinese traditional medicine together with the advantage from Reductionism, joining the study on complexity, Chinese scientist would make a chair in the international scientific society.

The success of yielding induced pluripotent stem (iPS) cells from human somatic cells demonstrates the important role of reprogramming in the formation of stem/progenitor cells and initiates the exploration of the origin of leukemia stem cells. In our previous work, we have found two types of leukemia, bona fide leukemia and non-bona fide leukemia. Different leukemias originate from different leukemia stem/progenitor cells which are critical to the genesis and evolution of leukemia. Bona fide leukemia and non-bona fide leukemia originate from leukemia stem cells and progenitor cells, respectively. Recent research suggests that different types of leukemia are influenced by the reprogramming state of their origin cells.

Autoinhibitory activity has been discovered in murine T lymphocyte leukemia models derived from 615 mice in our lab. It was designated 615 mice leukemia associated inhibitor (LAI-615) . To further confirm whether LAI activity could ~ be found in human leukemia, 6 ALL cases and 2 AML cases were examined. The results showed that 5/6 of ALL and 1/2 of AML cases had detectable LAI activity. The different sensitivities of LAI activity were also found between autologous bone marrow cells and human leukemic cell lines, which indicate that autoinhibitory activity might have individual specificity.

Up to date, eight types of human herpes viruses have been identified, all of which are ubiquitous, and usually establish latent infection in the host after primary infection. Since most of the herpes viruses are maintained in an asymptomatic form, they are often neglected. However, under some circumstances, these herpes viruses can cause fatal or severe diseases. Furthermore, the association of herpes viruses with hematopoietic malignancies is attracting researchers' attention. With the extensive development of hematopoietic stem cell and organ transplantation, reports regarding transplantation failure and complication caused by infection of human herpes virus has been increasing. Cytokine storm was firstly suggested as the mechanism of graft-versus-host diseases. In recent years, which has also been applied in the pathogenesis research of inflammation, and is supposed to play an important role in severe virus infection. In this paper, through discussing the possible role of latent infection of human herpes virus in the failure or complication of bone marrow or hematopoietic stem cell transplantation, and in refractory leukemia, the function and significance of latent infection of human herpes virus and the cytokine storm it caused were investigated.

The membrane form of macrophage colony–stimulating factor (mM-CSF) is an alternative splicing variant of this cytokine. Although its high expression was detected in hematopoietic malignancies, its physiologic and pathologic roles in hematopoietic system have not been established. In this report, stable transfectant clones expressing mM-CSF (Namalwa-M and Ramos-M) were obtained, which showed reduced proliferation potential in vitro. Moreover, the in vivo study showed that Namalwa-M and Ramos-M exhibited enhanced oncogenicity in tumor size in nude mice model, which could be inhibited by M-CSF monoclonal antibody. A remarkable increase in infiltrating macrophage and the vessel densities was found in tumor tissues formed by lymphoma cell lines that stably expressed mM-CSF, which suggested the involvement of macrophages in this process. The in vitro results using coculture system showed that macrophages could promote Namalwa-M and Ramos-M proliferation and activate extracellular signal-regulated kinase/mitogen-activated protein kinase signal pathway. In addition, the expression of murine origin vascular endothelial growth factor, basic fibroblast growth factor, and hepatocyte growth factor was elevated in Namalwa-M formed tumor tissues. These results suggested that mM-CSF should be a positive regulator in the development of hematopoietic malignancies by abnormally activating infiltrating macrophages, which in turn promote the malignant development. Thus, mM-CSF may be a critical linker between macrophages and malignant cells in the development of hematopoietic malignancies. [Cancer Res 2008;68(14):5639–47]

As pioneer of tumor stem cell research, leukemia stem cell research has not only important theoretical significance, but also clinical application potential. The survival and development of stem cells are directly impacted by their microenvironment. The research on leukemia stem cells and their microenvironment are now becoming a hot topic. The author presumes that stem cells are a population with heterogenecity and hierarchy; any single cell from the population is difficult to form a clone; the interaction between the leukemia stem cell and its microenvironment can be described by the concept of leukemia stem cell niche. In this article, the leukemia cell population with heterogenecity and hierarchy as well as leukemia stem cell niche were summarized and discussed.

J6-1 cell line is the first leukemic cell line established in China. It is a multi-clone cell line infected with both EBV and HHV-6. Many cytokines, receptors and other genes were cloned from J6-1 cell line since its establishment 30 years ago. Valuable information on leukemic characteristics and functions were obtained from the studies on this cell line, which could be categorized into several research subjects. These achievements implied the unique research value of multi-clone cell lines. This comment focuses attention on research advance of the J6-1 leukemic cell line in 30 years, including heterogeneity and multi-cloning of J6-1 cells, survival mechanism of J6-1 cell populations, abnormal intercellalar communication of J6-1 cells with its significance and inspiration from J6-1 cell line.

Murine beta-defensin 2 (MBD2) is not only chemotactic for immature dendritic cells but also activates them by Toll-like receptor 4. We have previously demonstrated that vaccine with MBD2 elicited potent antileukemia responses in the L1210 murine model. Interleukin-18 (IL-18) is an essential cytokine for the generation of Th1 response and natural killer cells and cytotoxic T lymphocytes (CTL) activation. As MBD2 and IL-18 appear to function on different components required by an effective antitumor immune response including both innate and adaptive immunity, we investigated whether combinatorial delivery of MBD2 and IL-18 transduced L1210 cells could elicit synergistic antileukemia effects. First, we constructed a single plasmid vector carrying both pro-IL-18 and IL-1beta converting enzyme (ICE) genes, and found that transfection of this vector into L1210 cells resulted in efficient secretion of bioactive IL-18. Combinatorial delivery of MBD2 and pro-IL-18-ICE modified L1210 cells conferred a superior inhibition of leukemogenicity over either L1210-MBD2 or L1210-pro-IL-18-ICE alone; moreover, the survived mice developed long-lasting protective immunity as determined by rechallenge experiments. This combined vaccine also elicited the most marked therapeutic effect, CTL activity and interferon-gamma production. These results suggest that the combination of MBD2 and IL-18 induces more effective antileukemia activity and provides a promising strategy for cancer therapy.

Murine β-defensin 2 (MBD2) is a small antimicrobial peptide of the innate immune system. Recent study showed that MBD2 could not only recruit immature dendritic cells but also activate them by Toll-like receptor 4 and thus may provide a critical link between the innate immune system and the adaptive immune response. In this report, we examined the antileukemia activity of MBD2 in a murine model of acute lymphoid leukemia (ALL) L1210. L1210 cells were engineered to secrete biologically functional MBD2. MBD2-modified L1210 (L1210-MBD2) showed significantly reduced leukemogenecity, resulting in a 80% rate of complete leukemia rejection. Inoculation of mice with L1210-MBD2 induced enhanced CTL and natural killer (NK) activity and augmented interleukin-12 and IFN-γ production. All the recovered mice from the inoculation showed a protective immunity to the following challenge with parental L1210 cells and generate leukemia-specific memory CTL. Vaccines with irradiated L1210-MBD2 cells could cure 50% leukemia-bearing mice. Depletion of CD8+ T cells but not CD4+ T cells completely abrogated the antileukemia activity of MBD2. Interestingly, NK cells were also required for the MBD2-mediated antileukemia response, although ALL generally display a high degree of resistance to NK-mediated lysis. Our results suggest that MBD2 can activate both innate and adaptive immunity to generate potent antileukemia response, and MBD2 immunotherapy warrants further evaluation as a potential treatment for ALL. (Cancer Res 2006; 66(2): 1169-76)

Evidence has indicated that low doses of anti-tumor regimens can induce cell apoptosis in vitro, although different regimens induce apoptosis by different mechanism and pathway. In recent years, new tumor treatment strategy has been mainly focused on inducing tumor cell apoptosis. The present review discusses the advantages and disadvantages of inducing tumor cell apoptosis. The benefit of inducing apoptosis is not to cause inflammatory reaction, but as its disadvantage, it inhibits immune responses, and the phagocytosis of apopotic bodies may result in horizontal transfer of genes (including oncogenes and other oncogenic materials), which can be one of the causes of tumor relapse. This paper proposes that the tumor treatment strategy should be turn into promoting tumor cell necrosis and inducing anti-tumor immune responses.

Regulation of P2X7 receptor expression is of interest because activation of this receptor by extracellular ATP triggers a wide variety of cell functions in leukocytes. However, its expression and modulation in human peripheral blood mononuclear cells (PBMC) and monocytes remain unclear. RT-PCR was used to detect the constitutive level of P2X7 receptor and the levels upon stimulation with bacteria, bacterial product, mitogen and various cytokines in human PBMC and monocytes. P2X7 receptor mRNA was detected in PBMC and monocytes. P2X7 receptor expression in PBMC was up-regulated by interleukin-2, -4, -6 (IL-2, IL-4, IL-6) tumour necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS) and heat-inactivated Staphylococcus aureus Cowan strain I (SAC). However, interferon-gamma (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF) and phytohemagglutinin-M (PHA-M) had little effect on the expression of P2X7 receptor. Furthermore, LPS and M-CSF could up-regulate P2X7 receptor expression in monocytes, while IFN-gamma, TNF-alpha and GM-CSF had weak effects, but pretreatment with these inducers could not further enhance LPS-stimulated P2X7 receptor expression in monocytes. The results obtained demonstrate that inflammatory stimuli drive P2X7 expression, thus supporting the hypothesis that P2X7 receptor may play a role in the inflammatory responses against bacteria infection, which need further verification.

Earlier studies indicate that J6-1 human leukemic cells proliferate and propagate via the membrane-bound macrophage colony-stimulating factor (M-CSF)-mediated auto-juxtacrine mechanism. Matrix metalloproteinases (MMPs) can modulate the activity of cell membrane molecules and influence many cellular behaviors. Therefore, we hypothesized that MMP may also be involved in the membrane-bound M-CSF-mediated juxtacrine mechanism. First, we investigated whether blocking of membrane-bound M-CSF by neutralizing antibody to M-CSF or M-CSF receptor and adding of exogenous M-CSF are able to influence MMP-9 release. Next, we determined whether MMP-9 participated in J6-1 cells proliferation and influence the shedding of membrane-bound M-CSF and its receptor. Current studies show that blockade of the interaction between membrane-bound M-CSF and M-CSF receptor by antibody to M-CSF or M-CSF receptor promotes MMP-9 release. Moreover, we demonstrated that because of M-CSF mediated juxtacrine, lack of MMP-9 promotes J6-1 cell proliferation, in which a decrease in the shedding of cell-surface M-CSFR is involved. Hence, we suggest that membrane-bound M-CSF inhibit MMP-9 release and down-regulated MMP-9 contribute to juxtacrine stimulating in leukemic cell growth.

DNA vaccine against M-CSFR(J6-1) (macrophage colony-stimulating factor receptor cloned from the J6-1 leukemic cell line) has shown both protective and therapeutic effects. In this study, to explore the adjuvant effects of LL-37 to M-CSFR(J6-1) DNA vaccines, we constructed genetically fused vaccines encoding M-CSFR(J6-1) and LL-37(pF). After immunizing BALB/c mice, specific humoral and cellular immune responses were detected. Compared with pR (encoding the extracellular region of M-CSFR(J6-1)), pF was more effective in inducing humoral and cytotoxic immune response, prolonging survival of mice challenged with SP2/0-CSFR(J6-1) tumor cells, and inducing IFN-gamma and IL-4 release by splenocytes. In this study, we also constructed pLL37 (encoding the mature LL-37) and coadministrated pLL37 and pR to see whether the genetic fusion was necessary. We found that compared with pR alone, pLL37+pR could not prolong survival of mice challenged with SP2/0-CSFR(J6-1) tumor cells. Our results suggest that when genetically fused with M-CSFR(J6-1), LL-37 could enhance adaptive immune response against M-CSFR(J6-1) in a murine model challenged with tumor cells bearing M-CSFR(J6-1).

IL-23 is a novel cytokine composed of an IL-12 p40 and a p19 subunit. We analyzed human peripheral blood mononuclear cells (PBMC) and hematopoietic cell lines for constitutive expression of IL-23 and IL-12 subunits and expression following exposure to various stimuli, and investigated the mechanisms of their induction by LPS and SAC. IL-23 p19 and IL-12 p35 mRNAs were expressed in fresh PBMC, and expression of all IL-23 and IL-12 subunits was up-regulated by LPS and SAC. LPS-induced increase of IL-23 and IL-12 subunits expression was CD14-dependent, while CD14 was not involved in SAC-induced p19 transcription. Both LPS- and SAC-induced subunits expression required p38 MAPK pathway. PHA effected an increase of p19 mRNA in both CD4+ and CD8+ T cells, whereas p35 was minimally regulated by PHA. IFN-gamma primed monocytes for LPS stimulation of p19, p35 and p40 expression. p19 mRNA was detectable in most hematopoietic cell lines tested but p35 distribution was more restricted. A phorbol diester enhanced p19, p35 and p40 expression in EBV-positive cell lines. Our results suggest that both the subunits of IL-23 are tightly regulated; the expression pattern and regulation mode of IL-23 p19 is similar to as well as distinct from that of IL-12 p35.

Similar to matrix metalloproteinases (MMP-9/-2), IL-18 was overexpressed in some hematologic malignancies such as acute myeloid leukemia (AML), which is associated with a poor clinical outcome. To establish a possible functional relationship between IL-18 and MMPs in myeloid leukemia, we used semi-quantitative PCR and zymographic analysis to examine whether IL-18 stimulates human myeloid leukemia cell line HL-60 to produce MMPs and/or specific tissue inhibitors (TIMPs), and to degrade extracellular matrix (ECM) gel in vitro. In the ECM invasion assay IL-18 significantly up-regulated transmigration of HL-60 cells, which in turn was inhibited by a synthetic MMP inhibitor: O-phenanthroline (o-PE), anti-MMP-9, anti-MMP-2 as well as anti-IL-18 monoclonal antibody (McAb), respectively, suggesting that induction of gelatinases by IL-18 leads to ECM degradation by these cells. Moreover, IL-18 could significantly increase MMP-9 but not MMP-2 production at both mRNA and/or protein level, slightly up-regulate TIMP-1 mRNA, and clearly induce TIMP-2 mRNA secretion. We postulate that IL-18 may in part play a role in the clinical aggressiveness of human myeloid leukemia by stimulating MMP-9 production.

The cDNA of human interleukin-18 (hIL-18) was successfully inserted into the genome of tobacco plant, Nicotiana tabacum cv. NC-89, using Agrobacterium tumefaciens-mediated transformation. Insertion and translation of hIL-18 in transformants were confirmed by PCR, ELISA, and Western blot, respectively. The transformed extracts contained the recombinant hIL-18 protein up to 0.05% of total soluble protein. Activity of the recombinant hIL-18 in plant cells was confirmed by the induction of IFN-gamma on IL-18-responsive J6-1 cells by the extracts obtained from the transformants. The expression level of hIL-18 (351 ng g(-1) tobacco tissue) obtained in the present study may be sufficient to induce responses/effects in vivo.

The term "functional redundancy" has been used as key words in foreign literature recently. However, it is rarely appeared in Chinese literature. Functional redundancy is an important characteristic of cytokines and transcription factors. The author gives personal opinion on this term for discussion.

To study the expression of gelatinases, including matrix metalloproteinase-9 (MMP-9, gelatinase B) and matrix metalloproteinase-2 (MMP-2, gelatinase A), in CD(34)(+) cells and leukemic cell lines, and explore the significance of gelatinase in migrating and homing capacity of CD(34)(+) cells, as well as the role of gelatinase in leukemia pathogenesis.CD(34)(+) cells were isolated from umbilical cord blood and normal bone marrow by Mini MACS system. By zymogram analysis, MMP-2 and MMP-9 were detected in the serum free condition medium of CD(34)(+) cells and cell lines.One brilliant band with molecular weight of 92 x 10(3) was detected in condition medium of cord blood CD(34)(+) cells. No band was detected in condition medium of bone marrow CD(34)(+) cells. Brilliant bands with molecular weight of 92 x 10(3) and 72 x 10(3) were detected in the condition medium of U937, KG-1a and HL-60 cell lines, but not in that of HEL, Namalva, CEM, K562 and LCL-H cell lines. In the condition media of J6-1 and J6-2 cells only the 92 x 10(3) band was detected.Cord blood CD(34)(+) cells produced MMP-9, but bone marrow CD(34)(+) cells did not, partly explains the fact that cord blood CD(34)(+) cells possessed higher migrating capacity in comparison with bone marrow CD(34)(+) cells. The expression of MMP-9 and MMP-2 in leukemic cell lines varied.

DNA vaccine against macrophage colony-stimulating factor receptor (M-CSFR) has shown both protective and therapeutic effects. In this study, we explore the possibility of using DNA vaccines against both M-CSFR and membrane-bound macrophage colony-stimulating factor (mM-CSF) to achieve better effects.Three plasmids were constructed by inserting either extracellular and transmembrane region of mM-CSF (pM), or extracellular region of M-CSFR (pR), or extracellular region of M-CSFR linked with extracellular and transmembrane regions of mM-CSF by a (Gly Gly Ser)2 flexible linker (pF), into pcDNA3.1. A SP2/0 cell line stably expressing pF (SP2/0-F) was established to evaluate humoral and cytotoxic immune responses as well as therapeutic and preventive effects induced by pM, pR, pF or pM+pR vaccination in BALB/c mice. The mechanisms of these vaccinations were also studied by monitoring the release of interleukin (IL)-4 and interferon (IFN)-g by splenocytes upon activation.Vaccination against two epitopes had better effects than against a single epitope while vaccination by pM+pR had the greatest effects on inducing humoral and cytotoxic immune responses, prolonging survival of mice challenged with SP2/0-F, and inducing IL-4 and IFN-g release by splenocytes.Our results suggest that co-immunization of M-CSFR and mM-CSF DNA vaccines is better than M-CSFR-mM-CSF fusion DNA vaccine.

Macrophage colony-stimulating factor (M-CSF) plays important roles in hematopoietic and immunologic systems. Some isoforms or mutations have been demonstrated including membrane-bound and cellular M-CSF, which associated with some leukemia, lymphoma and other solid tumors. We previously reported that the M-CSF-like membrane-associated factor (MAF-J6-1) and its receptor was found from human leukemic cell line J6-1. In this report, the cDNA of MAF-J6-1 and its receptor were cloned. The cDNA sequence of MAF-J6-1 shows a 768 bp open reading frame (ORF) with 99.2% homology to m-M-CSF, but six site mutations, including two synonymous mutations and four missense mutations. The cDNA of MAF-J6-1-R has a 2916 bp ORF shared 99.6% homology with M-CSF-R, but 13 site mutations, including six synonymous mutations and seven missense mutations. At the same time, a 1662 bp mutant s-M-CSF cDNA, which has 10 site mutations including three synonymous mutations and seven missense mutations, was cloned from J6-1 cells. The cDNAs of MAF-J6-1 and MAF-J6-1-R were inserted into a mammalian expression plasmid pTARGET and were expressed in COS-7 cells that demonstrated by their specific MAb. COS-7 cells transfected with MAF-J6-1-R show obvious protein tyrosine kinase (PTK) activity. Our present work shows that MAF-J6-1 and its receptor are mutations of M-CSF and its receptor.