Decreases in Ikaros activity correlate with blast crisis in patients with chronic myelogenous leukemia.

Gene targeting studies in mice have shown that the lack of Ikaros activity leads to T-cell hyperproliferation and T-cell neoplasia, establishing the Ikaros gene as a tumor suppressor gene in mice. This prompted us to investigate whether mutations in Ikaros play a role in human hematological malignancies. Reverse transcription-PCR was used to determine the relative expression levels of Ikaros isoforms in a panel of human leukemia/lymphoma cell lines and human bone marrow samples from patients with hematological malignancies. Among the cell lines examined, only BV-173, which was derived from a chronic myelogenous leukemia (CML) patient in lymphoid blast crisis, overexpressed the dominant-negative isoform, Ik-6. In 9 of 17 samples of patients in blast crisis of CML, Ikaros activity had been reduced either by drastically reducing mRNA expression (4 of 17) or by overexpressing the dominant-negative isoform Ik-6 (5 of 17). Significantly, expression of Ikaros isoforms seemed normal in chronic phase CML patients and patients with other hematological malignancies. In some cases, overexpression of the dominant-negative Ik-6 protein was confirmed by Western blot analysis, and Southern blot analysis indicated that decreases in Ikaros activity correlated with a mutation in the Ikaros locus. In summary, these findings suggest that a reduction of Ikaros activity may be an important step in the development of blast crisis in CML and provide further evidence that mutations that alter Ikaros expression may contribute to human hematological malignancies.

History and classification of human leukemia-lymphoma cell lines.

Continuous human leukemia-lymphoma cell lines have become indispensable tools in hematological research since the establishment of the first human lymphoma cell line Raji in 1963. We summarize here historical landmarks in the establishment of unique leukemia-lymphoma-derived cell lines from the various cell lineages; their special importance in hematopoietic research is emphasized. The first cell lines were derived from African Burkitt lymphomas and were found to integrate the Epstein-Barr virus in their genome leading to the discovery and isolation of this virus. However, it was later recognized that not every cell line derived from a patient with leukemia-lymphoma represents a malignant cell line as residual normal B-lymphocytes can also be immortalized by EBV infection. During the following 20-30 years many other types of hematopoietic cell lines, commonly derived from hematopoietic neoplasms, were established. These panels of cell lines now span almost the whole spectrum of hematopoietic cell lineages (except for dendritric cells) and the various distinct stages of differentiation along the respective cell axes. From early on, cell lines became important tools for basic and clinical hematological research, initially mainly in the field of immunology, but later expanding to other areas also. It became apparent that leukemia-lymphoma cell lines are of monoclonal origin, are arrested at a discrete maturational stage during differentiation in each lineage, and show sustained and growth factor-independent or -dependent unlimited proliferation. Categorization of cell lines might best be based on the physiological stages of hematopoietic differentiation in the various cell lineages. For an adequate classification, detailed characterizations of both the cell lines and the primary cells from which the cell lines originated are absolutely mandatory. In summary, the availability of large numbers of continuous leukemia-lymphoma cell lines has greatly facilitated clinical and immunobiological studies of normal and malignant hematopoiesis. Human leukemia-lymphoma cell lines will continue to provide exquisite model systems for many biomedical disciplines.

Proposals for the characterization and description of new human leukemia-lymphoma cell lines.

Continuous human leukemia-lymphoma cell lines have become invaluable tools for hematological research as they provide an unlimited amount of cellular material. The first human lymphoma cell line Raji was established in 1963; since then several hundred leukemia-lymphoma cell lines spanning almost the whole spectrum of hematopoietic cell lineages (except for dendritric cells) have been described. The cardinal features of leukemia-lymphoma cell lines are their monoclonal origin, arrest of differentiation, and (growth factor-independent or -dependent) unlimited proliferation. Categorization of cell lines usually follows the physiological stages of hematopoietic differentiation in the various cell lineages. For an adequate classification, a detailed characterization of both primary and cultured cells in absolutely necessary. New cell lines, in particular, must be adequately, characterized; while cell culture data and immunological and cytogenetic features are essential, cell lines should be described in as much detail as possible. In addition to this recommended multiparameter characterization and the obligatory immortality of the culture, authentication of the true origin of the cells, novelty, scientific significance and availability of the cell line for other investigators are of utmost importance. It is still extremely difficult to establish new leukemia-lymphoma cell lines (except for some subtypes), and most attempts fail. Paramount to the lack of our understanding as to why certain cells start to proliferate in culture and others do not (thus implying a random process), is probably the difficulty of mimicking in vitro the physiological in vivo microenvironment. Attempts to improve the efficiency of cell line establishment should focus on examining the appropriateness of the in vitro culture conditions; these conditions should emulate as closely as possible the in vivo situation. In summary, leukemia-lymphoma cell lines have the potential to greatly facilitate diverse studies of normal and malignant hematopoiesis; to that end, these cell lines must be extensively characterized and adequately described.

Comparison of two methods of staining apoptotic cells of leukemia cell lines. Terminal deoxynucleotidyl transferase and DNA polymerase I reactions.

We compared two methods to stain apoptotic cells, one using terminal deoxynucleotidyl transferase (TDT), the other DNA polymerase I, using leukemia cell lines treated with anti-Fas monoclonal antibody (MAb). Both TDT and polymerase I strongly reacted with fragmented nuclei of apoptotic MOLT-16 and Jurkat cells, but only polymerase I strongly reacted with nonfragmented nuclei of early apoptotic cells. Anti-Fas MAb-treated MOLT-4 cells showed morphological changes corresponding to early apoptosis and were strongly positive for polymerase I only. MOLT-16 and Jurkat cells treated with anti-Fas MAb and inhibitors of endonuclease and poly(ADP-ribose) polymerase showed the morphology of early apoptosis but were not strongly stained by TDT. Because DNA polymerase I has nick-translation activity, it is possible that DNA polymerase I reaction is positive in early apoptotic cells by detecting single-strand DNA cleavage, which occurs before extensive oligonucleosomal DNA cleavage and late morphological changes of apoptosis in leukemia cell lines. Although TDT is widely used to stain apoptotic cells, DNA polymerase I may be more applicable in special cases of apoptosis, in which cells undergo single-strand rather than double-strand DNA breaks. However, the procedure has limitations, such as the necessity to use cell smears for comparison with the TDT reaction. (J Histochem Cytochem 46:85-90, 1998)

Induction of hepatocyte growth factor/scatter factor by interferon-gamma in human leukemia cells.

Induction of hepatocyte growth factor/scatter factor (HGF/SF) may be one of the critical steps in organ regeneration, wound healing, and embryogenesis. We previously reported the production of HGF/SF from various human leukemia cell lines and a high level of the growth factor in blood and bone marrow plasma from patients with various types of leukemia. We determined here the effects of hematopoietic cytokines on HGF/SF production in human leukemia cell lines, KG-1, a myeloid cell line, and RPMI-8226, a B cell line. Interferon (IFN)-gamma remarkably stimulated HGF/SF production in both cell lines at concentrations of more than 0.1 or 1 IU/ml. IFN-alpha and IFN-beta were as effective as IFN-gamma in RPMI-8226 cells, but less than IFN-gamma in KG-1 cells. HGF/SF gene expression in KG-1 cells was also up-regulated by IFN-gamma. Granulocyte colony-stimulating factor (G-CSF), granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-5 and IL-6 had no effect on HGF/SF production in the 2 leukemia cell lines. We also determined the effects of HGF/SF inducers known for human fibroblasts on the growth factor production in leukemia cells. Out of phorbol 12-myristate 13-acetate (PMA), cholera toxin, IL-1 beta, and tumor necrosis factor (TNF)-alpha, the former three were as effective as IFN-gamma in KG-1 cells, but only TNF-alpha stimulated HGF/SF production in RPMI-8226 cells, whose effect was less than those of IFN-alpha, IFN-beta, and IFN-gamma. The effect of IFN-gamma in KG-1 cells was synergistic with that of PMA. In contrast with the effect in leukemia cells, HGF/SF induction by IFN-gamma in human skin fibroblasts was much less than that by PMA or cholera toxin. These results indicated that IFN-gamma is a potent inducer of HGF/SF in human leukemia cells. This finding suggests the presence of a homeostatic control mechanism in liver regeneration and repair: hepatic injury, DNA synthesis inhibition, or apoptosis caused by IFN-gamma is subsequently overcome by cytokine-induced HGF/SF, a potent promoter of liver DNA synthesis.

A murine monoclonal antibody (928) recognizing a new epitope formed with a combination of HLA-DPA1*0201 and DPB1*0301 gene products.

A murine monoclonal antibody (mAb), 928, that recognizes a cell surface antigen (928 Ag) on a human Epstein-Barr virus-transformed fetal liver-derived lymphoid progenitor cell line (FL4.4) was generated. The 928 mAb reacted with only FL4.4; it did not react with any other 57 cell lines tested. Two color flowcytometry analysis of peripheral blood mononuclear cells (PBMC) revealed that the 928 mAb reacted with B cell and monocyte fractions from only two individuals out of 63 unrelated donors. Biochemical analyses showed that the 928 Ag composes of two molecules (33 and 34 Kd) and forms a SDS-resistant, noncovalently linked dimer conformation, the feature being similar to that of peptide-bound MHC class II molecules. Treatment of FL4.4 cells with the 928 mAb significantly facilitated homotypic cell aggregation. In addition, treatment of PBMC of the 928 Ag+ donor with recombinant IL-4 augmented the expression of the 928 Ag on CD64+ monocytes. Typing of HLA-DRB1, DPA1 and DPB1 alleles of the 928 Ag expressing and nonexpressing cells revealed that the 928 Ag is expressed only on PBMC of HLA-DPA1*0201 and DPB1*0301 positive donors. Finally, anti-DP antibody precleared 928 Ag from the cell lysate. These results demonstrate that the 928 mAb recognizes a polymorphic determinant of HLA-DPA1*0201-DPB1*0301 gene products. The possibility that amino acids in the groove of the peptide-binding site of HLA-DP molecules are critical for the 928 epitope is discussed.

Selective expression of mRNA coding for the truncated form of erythropoietin receptor in hematopoietic cells and its decrease in patients with polycythemia vera.

The mRNA encoding full-length erythropoietin (EPO) receptor (EPOR-F) comprises exons I through VIII. Another membrane-bound EPOR (EPOR-T) isoform has a truncated cytoplasmic region and is encoded by the mRNA containing unspliced intron VII (EPOR-T mRNA). EPOR-T is believed to have a dominantly negative function against EPOR-F. We show that EPOR-T mRNA is markedly decreased in the blood cells of patients with polycythemia vera (PV). We also show that EPOR-T mRNA is not detected in erythroid/megakaryocytic leukemia cell lines, but is expressed in nonerythroid/nonmegakaryocytic lines, suggesting the presence of a cell type-specific system by which intron VII of the EPOR transcript is spliced. Deregulation of this splicing system in early hematopoietic progenitors possibly explains the profound decrease in EPOR-T mRNA and consequent pathophysiology of PV.

In vitro study using leukemia cell line panel to demonstrate rapid thymic T cell death due to contact with HIV-1 carrier cell clones.

A rapid (within 1 h) and profound cytotoxic cell death of immature TdT+CD4+CD8+ and/or TdT+CD4+ thymic T cell type leukemia cell lines, and of normal thymocyte populations rich in TdT+CD4+CD8+ cells was induced by contact with some human immunodeficiency virus type-1 (HIV-1) carrier T cell clones. This cytotoxic reaction, without requiring a complete viral replication cycle in the thymic T cells, did not occur in any mature CD4+CD8+ and/or CD4+ T cells which are otherwise permissive for virus infection. Although it was not an antigen-specific cytotoxic reaction, the rapid and profound thymic T cell destruction was shown, at the individual clonal level, to be triggered specifically by the binding of CD4 molecules on thymic T cells with gp120/gp160 on HIV-1 carrier clones. The present study suggesting direct elimination of immature T cells by contact with some HIV-1-infected T cells, may provide a novel insight into the mechanism responsible for the mature CD4+ T cell depletion in HIV-1 infection.

Acquired cytotoxic activity of CD8+ HIV-1IIIB carrier immature T cell clones specific to CD4+CD8+ (parental) human T cell clones and normal thymocytes.

By infecting human leukemia cell lines in vitro with HIV-1IIIB' a number of HIV-1 carrier clones were generated. Among them, 5 of 13 CD8+ HIV-1 carrier T cell clones were shown to acquire a rapid cytotoxic activity (within 1 hour) specific to TdT+CD4+CD8+ immature T cells including normal thymocytes. This novel cytotoxic reaction, without requiring virus infection and indicating a rapid T cell precursor elimination during active lymphopoiesis, suggests a mechanism responsible for mature CD4+ T cell depletion in HIV-1 infected individuals.

Interferon-alpha and -gamma differentially reduce rapid immature T-cell death by contact with HIV-1 carrier cell clones in vitro.

The non-antigen specific rapid cytotoxic (CT) death of immature TdT+CD4+CD8+ T cells due to contact with HIV-1 carrier T-cell clones we have found recently is a novel phenomenon. The effects of interferons (IFN) on this CT reaction were studied in vitro. Treatment of the HIV-1 carrier clones, referred to as "effectors," with IFN-alpha but not IFN-gamma, or of the susceptible immature TdT+CD4+CD8+ T cells, referred to as "targets," with IFN-gamma but not IFN-alpha, for 24 hr prior to CT testing was found to reduce the CT reaction. Simultaneously, a down-regulated CD8 expression and an up-regulated antigen expression of both major histocompatibility antigen complex class I (MHC-I) and HIV-1 gp120/gp160 in the IFN-alpha treated effector (gp120+CD8+ HPB-ALL/HIV), and/or simultaneously up-regulated antigen expression of both CD8 and MHC-I in the IFN-gamma treated target (CD4+CD8+ HPB-ALL) were found to be associated with reduced CT reaction. However, altered antigen expression in the IFN-gamma treated effectors or IFN-alpha treated targets did not affect the ultimate degree of CT reaction. This study thus suggests a possible therapeutic efficacy of IFN by reducing the direct elimination of the T-cell precursors in HIV-1 infection.

Genomic analysis of a new mammalian distal-less gene: Dlx7.

We have cloned a new Dlx gene (Dlx7) from human and mouse that may represent the mammalian orthologue of the newt gene NvHBox-5. The homeodomains of these genes are highly similar to all other vertebrate Dlx genes, and regions of similarity also exist between mammalian Dlx7 and a subset of vertebrate Dlx genes downstream of the homeodomain. The sequence divergence between human and mouse Dlx7 in these regions is greater than that predicted from comparisons of other vertebrate Dlx genes, however, and there is little sequence similarity upstream of the homeodomain both between these two genes and with other Dlx genes. We present evidence for alternative splicing of mouse Dlx7 upstream of the homeodomain that may account for some of this divergence. We have mapped human DLX7 distal to the 5' end of the HOXB cluster at an estimated distance of between 1 and 2 Mb by FISH. Both the human and the mouse Dlx7 are shown to be closely linked to Dlx3 in a convergently transcribed orientation. These mapping results support the possibility that vertebrate distal-less genes have been duplicated in concert with the Hox clusters.

Abnormal expression of Evi-1 gene in human leukemias.

The human Evi-1 gene located on chromosome 3q26, encodes a zinc finger protein that functions as a transcription factor. It was frequently overexpressed in leukemias having 3q26 abnormalities such as t(3;3)(q21;q26) and inv(3)(q21 q26), and subjected to structural alteration in t(3;21)(q26;q22). In addition, recent studies indicated that several cases of leukemias without 3q26 abnormalities also expressed Evi-1 gene. In this study we present another case of structural alteration of Evi-1 gene in a case of inv(3)(q21 q26), in which Evi-1 was truncated and a shorter form of Evi-1 protein was expressed upon rearrangement of the gene. We also studied expression of the Evi-1 gene in a variety of leukemias by northern blot analysis. Evi-1 was overexpressed not only in leukemias with 3q26 abnormalities, but, in those without 3q26 abnormalities, especially in blast crisis of CML. Our result also supports an idea that Evi-1 is a relevant oncogene whose overexpression or structural changes might play a crucial role in development of human leukemias.

Autocrine loop through cholecystokinin-B/gastrin receptors involved in growth of human leukemia cells.

The cholecystokinin (CCK)-B/gastrin receptor binds two brain-gut hormones, CCK and gastrin, with high affinities. These peptides have a trophic effect on gastrointestinal cells expressing the receptor in vivo as well as in vitro. Recently, this receptor mRNA was reported to be expressed in immunocytes localized in the lamina propria of normal rat stomach mucosa. Here, we studied the receptor expression in human hematopoietic cells in order to determine whether they play a role in cell growth. The CCK-B/gastrin receptor mRNA was detectable in the polymorphonuclear (PMN) cells but not in the mononuclear cells of normal peripheral white blood cells by reverse transcription-polymerase chain reaction. The receptor transcript was, however, expressed in human leukemia cell lines (14 of 18 cell lines tested) derived from not only myeloid, but also T- and B- lymphoid lineages. The CCK-B/gastrin receptors on several leukemia cell lines were shown to be biologically active by demonstrating ligand-dependent cell proliferation in serum-deprived medium. Interestingly, a human CCK-B/gastrin receptor specific antagonist, YM022, but not its stereotype isoform, selectively inhibited the DNA synthesis of THP-1, MOLT-16, MOLT-14, and CCRF-CEM in the absence of exogenous peptide ligands. Further investigation revealed that these leukemia cell lines and normal PMN cells also expressed gastrin mRNA. These results suggest that growth of human leukemia cells is promoted by an autocrine mechanism through the CCK-B/gastrin receptors.

Metastatic potential of lymphoma/leukemia cell lines in SCID mice is closely related to expression of CD44.

To investigate whether the lymphocyte homing receptors, adhesion molecules regulating normal lymphocyte traffic, influence the dissemination of lymphoma cells, 24 lymphoma/leukemia cell lines were inoculated into SCID mice subcutaneously, and the correlation between the expression of the adhesion molecules and the metastatic potential of the cell lines was examined. Among the six adhesion molecules examined (LFA-1, ICAM-1, CLA, VLA-4, L-selectin and CD44), L-selectin increased the incidence of lymph node metastasis, and CD44 expression was related to both lymph node and organ (hematogenous) metastasis. A monoclonal antibody to the standard form of CD44 (CD44s), Hermes-3, inhibited the local growth and remote metastasis of CD44+ cell lines. Thus, it is concluded that at least CD44s expression is important in both lymphatic and hematogenous metastasis.

Purification and primary amino acid sequence of a novel neutrophil chemotactic factor LECT2.

We purified a neutrophil chemotactic factor from a culture fluid of the PHA-activated human T-cell leukemia SKW-3 cells. The factor showed a 16-kDa basic protein by Tricin-SDS-polyacrylamide gel electorophoresis and analysis of amino acid composition. The primary amino acid sequence revealed that the chemotactic factor was significantly different from other known chemotactic factors, indicating a novel protein designated LECT2. The sequence revealed homology with the myb-induced myeloid protein-1 (Mim-1), which is expressed from gene in immature and normal granulocytes of chicken. Its biological function had not yet been identified. LECT2 and Mim-1 may be involved in the regulation of neutrophil functions in an as yet unidentified way.

Structurally altered Evi-1 protein generated in the 3q21q26 syndrome.

Overexpression of the Evi-1 gene appears to be a consistent feature of the 3q21q26 syndrome, an association of myeloid leukemias/myelodysplastic syndrome with a specific chromosomal aberration involving both 3q21 and 3q26, such as t(3;3)(q21;q26) or inv(3)(q21q26). The rearrangement in 3q26 has been reported to occur near the Evi-1 locus, implicating that it is the critical gene deregulated in the 3q21q26 syndrome. Here we present a structural abnormality of Evi-1 protein in a case with the 3q21q26 syndrome. In this case carrying typical inv(3)(q21q26), the 3q26 breakpoint is located within an intron of the Evi-1 gene, and resulted in overexpression of normally unexpressed, an aberrant form of Evi-1 protein, in which the C-terminal 44 amino acids of wild-type Evi-1 protein were truncated and replaced by five amino acids. The truncated Evi-1 protein is shown to increase AP1 activity when expressed in NIH3T3 cells as its wild-type counterpart. We also show that the origin of this peculiar type of rearrangement of the Evi-1 gene is not an artifact during establishment of the cell line, but is the event that occurred in the primary leukemic cells. Our results strongly support that the primary target for the 3q21q26 syndrome is the Evi-1 gene, and provide the first evidence that the structurally altered Evi-1 gene may be involved in the 3q21q26 syndrome.

Molecular characterization of a chromosome translocation breakpoint t(11;14)(p13;q11) from the cell line KOPT-K1.

Recurrent chromosome translocations involving 11p13 and 14q11 are found in 5-10% of cases of T-ALL. The gene involved in the translocation on chromosome 14 is the T cell antigen receptor alpha or delta. The putative oncogene on chromosome 11 is rhombotin 2 (RBTN2)/translocated in T cell gene 2 (ttg-2), a member of the LIM family of proteins. In this paper we characterize a cell line KOPT-K1 that has a t(11;14)(p13;q11). The breakpoint on chromosome 11 involves an Alu-rich region with the break occurring between two Alu sequences on chromosome 11. In addition, approximately 70 bases from the break on chromosome 11 is a tetranucleotide repeat. Whether either of these structures played a role in the translocation is not known. No heptamer or nonamer sequences, implicated in other rearrangements were found near the breakpoint. The breakpoint on chromosome 11 maps more centromeric than previous translocations of this region. Despite this the RBTN2 gene is highly expressed in KOPT-K1. This cell line will be useful for investigating the role of RBTN2 in leukemogenesis and the mechanism by which the translocation alters the expression of RBTN2.

Hepatocyte growth factor--pleiotropic cytokine produced by human leukemia cells.

Hepatocyte growth factor (HGF) was identified, purified and molecularly cloned as a potent mitogen for mature rat hepatocytes in primary culture. It is one of the largest cytokines and is composed of disulfide-linked subunits of approximately 60 (heavy chain) and 35 kilodaltons (light chain). Recent observations revealed that HGF is mitogenic to various epithelial cells other than hepatocytes and to endothelial cells, and that it also acts as a motogen, morphogen and tumor-suppressor as well as a mitogen. These various biological activities of HGF are presumably transduced through the same receptor, c-Met, which is a member of the tyrosine kinase receptor family. Although it shows multiple biological activities on cells in culture, HGF is most likely the physiological hepatotrophic factor which triggers liver regeneration. It may also function as a renotrophic and pulmotrophic factor after tissue injury. HGF production in the liver, kidney and lung increases after injury to these organs. An elevated HGF level may act as an inducer of compensatory DNA synthesis. The regulation of HGF production is, therefore, important for the control of organ regeneration. HGF is produced mainly by mesenchymal cells such as fibroblasts and vascular smooth muscle cells. Various types of human leukemia cells also secrete HGF both in vitro and in vivo. Some biological activities of HGF on hematopoietic cells, including co-mitogenic activity on myeloid leukemia cell lines, were recently demonstrated. HGF gene expression and the protein production in leukemia and fibroblast cells are modulated by various cytokines and hormones. Those modulators may indirectly affect organ regeneration and other biological processes by controlling HGF production.

Expression of 18.6/CD23 antigen on human lymphoid progenitor cell lines and phorbol 12-myristate 13-acetate (PMA)-induced microglia-shaped cells.

The nature of lymphoid progenitors and factor(s) determining commitment to either the T- or B-lymphocyte pathway are poorly understood in the human system. In this study, we generated a monoclonal antibody (MoAb), 18.6, that recognizes a cell surface antigen on a human lymphoid progenitor cell line (FL4.4). MoAb 18.6 reacted with lymphoid progenitor lines, B lymphoid cell lines, and myelomonocytic cell lines. It did not react with any T cell or erythroid leukemic cell lines. Two color FACS analyses of normal lymphoid tissues showed that MoAb 18.6 reacted with a majority of CD20+ mature B cells and a minority of CD64+ monocytes. Molecules of 3 different sizes with MW of 34, 45, and 68 Kd were precipitated with MoAb 18.6 from the lymphoid progenitor cell line. The 18.6 antigen was not expressed on a fetal liver-derived lymphoid progenitor-like cell line, FL1.4, which has the capacity to differentiate into microglia-shaped cells upon PMA-stimulation. Stimulation of FL1.4 cells with PMA induced expression of the 18.6 antigen within 24 hr and the microglia-shaped cells stained positively with MoAb 18.6. Finally, cloning of a cDNA that encoded the 18.6 antigen revealed that the 18.6 antigen is identical to the CD23 antigen. Taken together, these data suggest that the 18.6/CD23 antigen is expressed on lymphoid precursors at a very early stage of differentiation.