Gene transfer of pro-IL-18 and IL-1beta converting enzyme cDNA induces potent antitumor effects in L1210 cells.

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.

Production of matrix metalloproteinase-9 by cord blood CD34+ cells and its role in migration.

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.

Autocrine and possible intracrine regulation of HL-60 cell proliferation by macrophage colony-stimulating factor.

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.

Expression of membrane-associated macrophage colony-stimulating factor (M-CSF) in Hodgkin's disease and other hematologic malignancies.

We have identified a membrane-bound form of M-CSF (m-M-CSF) from an established human leukemic J6-1 cell line. To further understand its biological significance, we studied the expression of this membrane-associated growth factor in the lymph nodes of lymphoma patients and bone marrow smears from patients with hematologic diseases by immunohistochemical staining using anti-M-CSF MAb. We detected a high incidence of m-M-CSF expression in 75% (9/12) of the lymph node sections from patients with Hodgkin's Disease (HD). The antigens were detected primarily in large clusters of mononuclear Hodgkin's cells and the extracellular matrix (EM) surrounding them. In one HD patient with abundant multinucleated Reed-Sternberg (R-S) cells, all of them were intensely stained with anti-M-CSF MAb. In non-Hodgkin's lymphomas (NHL), the incidence (17.6 %) of m-M-CSF expression was lower (3/17). Yet, no m-M-CSF antigens were detected in the lymph nodes from six cases of non-hematologic malignancies and other diseases. A high response also was detected in bone marrow smears obtained from patients with hematologic malignancies, which include myeloid leukemias (32.5%), lymphomas with bone marrow metastasis (50%) and myelodysplastic syndromes (MDS) (37.5 %). By comparison, only 6.8 % of bone marrow smears from non-malignant hematologic diseases and 2.7% of lymphoid leukemias showed positive staining with anti-M-CSF MAb. Our results showed that high expression of m-M-CSF antigens is linked to some types of lymphomas, especially HD. and myeloid leukemias, and may play a role in the development of these hematologic malignancies.

Enhancement of J6-1 human leukemic cell proliferation by membrane-bound M-CSF through a cell-cell contact mechanism II. Role of an M-CSF receptor-like membrane protein.

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".