HGF/SF and its receptor c-MET play a minor role in the dissemination of human B-lymphoma cells in SCID mice.

The MET protooncogene, c-MET, encodes a cell surface tyrosine kinase receptor. The ligand for c-MET is hepatocyte growth factor (HGF), also known as scatter factor (SF), which is known to affect proliferation and motility of primarily epithelial cells. Recently, HGF/SF was also shown to affect haemopoiesis. Studies with epithelial and transfected NIH3T3 cells indicated that the HGF/SF-c-MET interaction promotes invasion in vitro and in vivo. We previously demonstrated that HGF/SF induces adhesion of c-MET-positive B-lymphoma cells to extracellular matrix molecules, and promoted migration and invasion in in vitro assays. Here, the effect of HGF/SF on tumorigenicity of c-MET-positive and c-MET-negative human B-lymphoma cell lines was studied in C.B-17 scid/scid (severe combined immune deficient) mice. Intravenously (i.v.) injected c-MET-positive (BJAB) as well as c-MET-negative (Daudi and Ramos cells) B-lymphoma cells formed tumours in SCID mice. The B-lymphoma cells invaded different organs, such as liver, kidney, lymph nodes, lung, gonads and the central nervous system. We assessed the effect of human HGF/SF on the dissemination of the B-lymphoma cells and found that administration of 5 microg HGF/SF to mice, injected (i.v.) with c-MET-positive lymphoma cells, significantly (P = 0.018) increased the number of metastases in lung, liver and lymph nodes. In addition, HGF/SF did not significantly influence dissemination of c-MET-negative lymphoma cells (P = 0.350 with Daudi cells and P= 0.353 with Ramos cells). Thus the effect of administration of HGF/SF on invasion of lymphoma cells is not an indirect one, e.g. via an effect on endothelial cells. Finally, we investigated the effect of HGF/SF on dissemination of c-MET-transduced Ramos cells. In response to HGF/SF, c-MET-transduced Ramos cells showed an increased migration through Matrigel in Boyden chambers compared to wild-type and control-transduced Ramos cells. The dissemination pattern of c-MET-transduced cells did not differ from control cells in in vivo experiments using SCID mice. Also no effect of HGF/SF administration could be documented, in contrast to the in vitro experiments. From our experiments can be concluded that the HGF/SF-c-MET interaction only plays a minor role in the dissemination of human B-lymphoma cells.

Hepatocyte growth factor/scatter factor (HGF/SF) affects proliferation and migration of myeloid leukemic cells.

Hepatocyte growth factor (HGF), also known as scatter factor (SF), is produced by mesenchymal cells, including bone marrow (BM) stromal cells, and has mitogenic and motogenic effects on a variety of cell types. Recently, a role has been assigned to HGF/SF and its receptor, c-MET, in both normal and malignant hemopoiesis. We investigated the function of HGF/SF on hemopoietic mononuclear cells (MNC) from patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) with circulating blasts. In contrast to results with normal MNC, HGF/SF alone stimulated the proliferation and colony formation of MNC from these patients. MNC from some (4/13) of the AML patients also produced HGF/SF (0.1-0.2 ng/ml/day), while we could not detect HGF/SF in cultures from normal MNC. Furthermore, it appeared that HGF/SF induced migration of leukemic cells in Boyden using KG1a cells as a model for leukemic blasts. The membranes dividing the two compartments of the Boyden chambers were coated with fibronectin. HGF/SF significantly promoted migration in 3/5 samples of MDS patients and in 5/7 samples of AML patients. Supernatant of human BM stromal cells, which is chemoattractive for normal human hemopoietic progenitor cells, also promoted migration of MNC from 4/5 MDS patients and 6/7 AML patients. Since HGF/SF is one of the growth factors produced by BM stromal cells, a neutralizing antibody directed against HGF/SF was added to the BM stroma supernatant, which reduced migration significantly in 2/3 MDS and in 3/6 AML responders to BM stroma supernatant. In conclusion, HGF/SF promotes proliferation and migration of hemopoietic cells from AML and MDS patients in vitro and may therefore contribute to the malignant potential of these cells.

Hepatocyte growth factor/scatter factor (HGF/SF) is produced by human bone marrow stromal cells and promotes proliferation, adhesion and survival of human hematopoietic progenitor cells (CD34+).

The fate of hematopoietic progenitor cells (HPCs) in the bone marrow (BM) microenvironment is determined by two different interactions: 1) they adhere (via integrins) to both extracellular matrix molecules and BM stromal cells; and 2) stromal cells produce cytokines that influence their survival, proliferation, differentiation, and mobilization. The ligands for the protein tyrosine kinase receptors c-KIT and FLT3/FLK2, stem cell factor (SCF), and FL are produced by BM stromal cells and are known to affect several facets of hematopoiesis. We studied another protein tyrosine kinase receptor, c-MET, and its ligand hepatocyte growth factor (HGF), also known as scatter factor (SF), which play a similar role in hematopoiesis. c-MET mRNA is expressed in immature human BM HPCs (CD34+CD33- or CD34+CD38-), but not in more mature HPCs (CD34+CD33+ or CD34+CD38+). The ligand HGF/SF is predominantly produced by BM stromal cells at both the mRNA and protein levels. We confirmed functionally that HGF/SF alone has no effect on proliferation of HPCs, but that when combined with granulocyte/macrophage colony-stimulating factor (GM-CSF) or interleukin-3 it acts as a synergistic proliferative factor, although not as potently as kit-ligand or FLT-3/FLK-2 ligand. Furthermore, HGF/SF promotes adhesion of HPCs to immobilized fibronectin. HGF/SF-induced adhesion to fibronectin is probably caused by activation of the integrins alpha4beta1 and alpha5beta1, insofar as we were able to block this interaction by using monoclonal blocking antibodies directed against these integrin subunits. Addition of the tyrosine-phosphorylation inhibitor genistein inhibited HGF/SF-induced adhesion, supporting the idea that HGF/SF-induced effects are the result of signaling via the receptor c-MET after ligand binding. The enhanced adhesion of HGF/SF to fibronectin proved to be beneficial for the maintenance of the colony-forming potential of HPCs. HGF/SF alone and especially in combination with fibronectin prolongs survival of GM colony-forming cells in liquid culture. Our data indicate that HGF/SF is a polyfunctional cytokine in the BM microenvironment. It is produced by human BM stromal cells and directly or indirectly promotes proliferation, adhesion, and survival of human HPCs.

Hepatocyte growth factor/scatter factor promotes adhesion of lymphoma cells to extracellular matrix molecules via alpha 4 beta 1 and alpha 5 beta 1 integrins.

Hepatocyte growth factor (HGF)/scatter factor (SF) is the ligand for a tyrosine kinase cell surface receptor encoded by the MET protooncogene (c-MET). HGF/SF can induce proliferation and motility in epithelial cells and promotes invasion of carcinoma cells and NIH3T3 fibroblasts transfected with both HGF/SF and c-MET genes. Our results show that HGF/ SF and c-MET also play a role in adhesion and invasion of human lymphoma cells. c-MET mRNA is expressed in hemopoietic cells, such as hemopoietic progenitor cells (CD34+ cells) in bone marrow (BM) and mobilized peripheral blood, immature B cells in cord blood and BM, and germinal center B-centroblasts. In normal peripheral blood B cells, which are c-MET-, c-MET expression was induced by PMA, ConA, HGF/ SF, and Epstein-Barr virus (EBV) infection. Using immunohistochemistry, we detected c-MET on the cell surface of large activated centroblasts in lymph nodes from patients with B-non-Hodgkin's lymphoma and Hodgkin's disease. In the latter group, c-MET expression correlated well with the presence of EBV. Because HGF/SF and c-MET promote metastasis of carcinoma cells, we studied the effects of c-MET stimulation by HGF/SF of B-lymphoma cells on properties relevant for metastasis, ie, adhesion, migration, and invasion. HGF/SF stimulated adhesion of the c-MET+ B-cell lines to the extracellular matrix molecules fibronectin (FN) and collagen (CN) in a dose dependent manner. However, adhesion to laminin was not affected by HGF/SF. Adhesion to FN was mediated by beta 1-integrins alpha 4 beta 1 (VLA4) and alpha 5 beta 1 (VLA5) since blocking antibodies against beta 1- (CD29), alpha 4-(CD49d), or alpha 5- (CD49e) integrin subunits, completely reversed the effect of HGF/SF. Furthermore, HGF/SF induced adhesion was abrogated by addition of genistein, which blocks protein tyrosine kinases, including c-MET. Addition of HGF/SF resulted in a sixfold increase in migration of c-MET B-lymphoma cells through Matrigel, compared to medium alone. In rat fibroblast cultures, HGF/SF doubled the number of c-MET+ B-lymphoma cells that invaded the fibroblast monolayer. In these adhesion, migration and invasion assays HGF/SF had no effect on c-MET- cell lines. In conclusion, c-MET is expressed or can be induced on immature, activated, and certain malignant B cells. HGF/SF increased adhesion of c-MET+ B-lymphoma cells to FN and CN, mediated via beta 1-integrins alpha 4 beta 1 and alpha 5 beta 1, and furthermore promoted migration and invasion.

The value of flow cytometric analysis of platelet glycoprotein expression of CD34+ cells measured under conditions that prevent P-selectin-mediated binding of platelets.

In the present study, we show by adhesion assays and ultrastructural studies that platelets can bind to CD34+ cells from human blood and bone marrow and that this interaction interferes with the accurate detection of endogenously expressed platelet glycoproteins (GPs). The interaction between these cells was found to be reversible, dependent on divalent cations, and mediated by P-selectin. Enzymatic characterization showed the involvement of sialic acid residues, protein(s). The demonstration of mRNA for the P-selectin glycoprotein ligand 1 (PSGL-1) in the CD34+ cells by polymerase chain reaction (PCR) analysis suggests that this molecule is present in these cells. Under conditions that prevent platelet adhesion, a small but distinct subpopulation of CD34+ cells diffusely expressed the platelet GPIIb/IIIa complex. These cells were visualized by immunochemical studies. Furthermore, synthesis of mRNA for GPIIb and GPIIIa by CD34+ cells was shown using PCR analysis. The semiquantitative PCR results show relatively higher amounts of GPIIb mRNA than of PF4 mRNA in CD34+CD41+ cells in comparison with this ratio in platelets. This finding is a strong indication that the PCR results are not caused by contaminating adhering platelets. MoAbs against GPIa GPIb alpha, GPV, P-selectin, and the alpha-chain of the vitronectin receptor did not react with CD34+ cells. The number of CD34+ cells expressing GPIIb/IIIa present in peripheral blood stem cell (PBSC) transplants was determined and was correlated with platelet recovery after intensive chemotherapy in 27 patients. The number of CD34+CD41+ cells correlated significantly better with the time of platelet recovery after PBSC transplantation (r = .83, P = .04) than did the total number of CD34+ cells (r = .55). Statistical analysis produced a threshold value for rapid platelet recovery of 0.34 x 10(6) CD34+CD41+ cells/kg. This study suggests that if performed in the presence of EDTA the flow cytometric measurement of GPIIb/IIIa on CD34+ cells provides the most accurate indication of the platelet reconstitutive capacity of the PBSC transplant.

Clonality in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) comprises a group of acquired hematological disorders which is characterized by a progressive peripheral blood pancytopenia of one or more cell lineages. A high percentage of blast cells in either bone marrow or peripheral blood predisposes for the transformation to acute myeloid leukemia. The clinical presentation with pancytopenia suggest that all cell lineages are affected by MDS. The first experiments with X-linked restriction fragment length polymorphism (RFLP) indicated that MDS is a stem cell disorder since the clonal deletions could be detected in all cell lineages. During the 1st decade several new molecular biological techniques such as polymerase chain reaction, and fluorescent in situ hybridization (FISH) were applied to study molecular aberrations in subpopulations of cells. Molecular aberrations in all subpopulations would indicate that MDS is a stem cell disorder. The clonal studies in MDS are equivocal. Studies involving the expression of chromosomal abnormalities (standard karyotyping and FISH) in different cell lineages suggest that the pluripotent stem cell is not affected in MDS since the lymphoid cells usually do not express the abnormal karyotype. Results obtained by RFLP vary widely. Some studies indicate that the lymphoid lineage is not involved, while other studies find a polyclonal expression of the polymorphic genes in lymphoid cells. One study using PCR demonstrated mutations in the ras-oncogenes in T-cells as well as myeloid cells, suggesting that a common ancestor of myeloid and lymphoid cells is affected by MDS. This review discusses the different experimental approaches carried out to solve the discussion whether MDS is a stem cell disorder.

Transfer of tumor immunity by both CD4+ and CD8+ tumor infiltrating T lymphocytes activated in vivo by IL-2 therapy of tumor bearing mice.

DBA/2 mice were inoculated i.p. with syngeneic SL2 lymphoma or P815 mastocytoma on day 0 and treated i.p. with 20,000 units IL-2/day on day 10-14. This treatment is curative for 70-90% of the tumor bearing mice. Peritoneal cells and/or spleen cells were isolated from responding mice at the last day of IL-2 therapy. The in vivo antitumor activity of these cells was tested in Winn Assays (i.p.) and by adoptive transfer (i.v.) into mice injected s.c. with tumor previously. Peritoneal exudate cells isolated on day 14 (PEC14) from mice cured of SL2 tumor were highly effective in Winn Assays. Up to 5 x 10(7) SL2 cells could be eliminated in naive mice when injected i.p. together with 2 x 10(7) PEC14. Adoptive transfer (i.v.) of PEC14, without the addition of IL-2, into mice s.c. injected with SL2 tumor cells 1 or 3 days earlier, also prevented outgrowth of the tumor cells. T cells isolated from the spleens were less effective. Only at E:T ratios of 100:1 and 10:1 was tumor outgrowth inhibited. The adoptive transfer of PEC14 resulted in a long lasting immunity of the recipient mice. Furthermore, it was shown that depletion of both the CD8+ and CD4+ T cells from the suspensions used in the transfer studies, resulted in a significant decrease of tumor inhibition. However, the effect was not abrogated completely. PEC14 isolated from IL-2-treated DBA/2 mice cured of P815 tumor, protected naive mice against P815 tumor at E:T ratio 20:1. Adoptive transfer (i.v.) of these PEC14 into mice bearing s.c. P815 did not have an antitumor effect. In conclusion, low dose i.p. IL-2 therapy predominantly induces locally both CD4+ and CD8+ T cells with a strong antitumor activity in vivo. The potency of the IL-2-induced immunity seems related to the type of tumor used.

Effector cells of low-dose IL-2 immunotherapy in tumor bearing mice: tumor cell killing by CD8+ cytotoxic T lymphocytes and macrophages.

The presence and cytotoxicity of tumor infiltrating cells is described in mice during effective immunotherapy with interleukin 2 (IL-2). DBA/2 mice were inoculated i.p. with 2 x 10(4) tumor cells on day 0 and treated with daily i.p. injections with 20,000 units IL-2 on days 10-14. Mice bearing a large syngeneic i.p. tumor burden (SL2 lymphoma, P815 mastocytoma, L5178Y lymphoma, or L1210 lymphoma) could be cured by i.p. immunotherapy with these low doses of IL-2. In the peritoneal cavity of these mice an infiltrate of mononuclear cells was present. Similar numbers of lymphocytes (10(6)-10(7)) and macrophages (+/- 10(7)) were present in control tumor bearing mice and IL-2 treated tumor bearing mice. The ratio of CD4+/CD8+ T lymphocytes in the peritoneal cavity of mice rejecting the SL2 tumor was smaller than 0.5, whereas this ratio is about 2 in naive mice. In the spleens of IL-2 treated tumor bearing mice only a minor decrease of CD4+/CD8+ ratio was observed from 2.1-2.4 to 0.9-1.9. T cells isolated from the peritoneal cavity of mice inoculated with SL2 tumor cells and treated with IL-2, were highly cytotoxic to SL2 cells: at E:T ratio 2:1 the cytotoxicity index was 37 +/- 3. This cytotoxicity was specific and mediated by CD8+ T lymphocytes. Macrophages that were present in the peritoneal cavity of mice treated with IL-2 were also highly cytotoxic. The C.I. of these cells was 63-76% at E:T ratio 1:1. Cytotoxic macrophages were also present in untreated tumor bearing mice. The i.p. injections of IL-2 (20,000 units/day) caused a four-fold increase in the local NK-activity in the peritoneal cavity in naive mice. These IL-2 injections did not generate LAK-activity in vivo. Specificity of the in vivo tumor rejection was tested by injection SL 2 i.p. on day 0 and P815 i.p. on day 10, or vice versa, followed by IL-2 treatment. Only the tumor cells that were injected on day 0 were rejected. These in vivo experiments point to specific tumor rejection. In conclusion, both cytotoxic macrophages and CTL's are present in a sufficient number and with sufficient cytotoxicity to explain the killing of tumor cells in the peritoneal cavity. The CTL-activity seems of decisive importance for tumor rejection as this is induced by IL-2.