Yuziro Namba

Cloning, mapping and expression of PEBP2αC, a third gene encoding the mammalian Runt domain

Abstract PEBP2/CBF is a heterodimeric transcription factor composed of α and β subunits. Previously, we reported two distinct mouse genes, PEBP2αA and PEBP2αB, which encode the α subunit. PEBP2αB is the homologue of human AML1, encoding the acute myeloid leukemia 1 protein. AMLI and human PEBP2/CBFβ were detected independently at the breakpoints of two characteristic chromosome translocations observed frequently in two subtypes of acute myeloid leukemia. The PEBP2α proteins contain a 128-amino-acid (aa) region highly homologous to the Drosophila melanogaster segmentation gene runt. The evolutionarily conserved region, named the Runt domain, harbors DNA-binding and heterodimerizing activities. In this study, we identified the third Runt-domain-encoding gene, PEBP2αC, which maps to lp36.11-p36.13 in the human chromosome and encodes a 415-aa protein. PEBP2αC forms a heterodimer with PEBP2β, binds to the PEBP2 site and transactivates transcription, similar to PEBP2αA and PEBP2αB.

Induction of human glioma-specific cytotoxic T-lymphocyte lines by autologous tumor stimulation and interleukin 2

SummaryTwo human glioma-specific cytotoxic T-lymphocyte (G-S-CTL) lines were established by autologous tumor stimulation (ATS) with the aid of lectin free interleukin 2 (IL 2). Coculture of patients peripheral blood lymphocytes and autologous irradiated glioma cells and subsequent addition of partially purified IL 2 enhanced the tumoricidal activity of the lymphocytes. These CTL lines possessed cross-cytotoxic activity against autologous and allogeneic glioma cells and exhibited low cytotoxic activity against non-glial tumor cells. They did not lyse autologous lymphoblasts. This phenomenon suggested the existence of a common gliomaspecific antigen recognized by the CTL lines.T-cell subset depletion test revealed that the major surface phenotype of G-S-CTL line, responsible for cytotoxic activity was OKT 3 positive, OKT 4 negative and OKT 8 positive.G-S-CTL lines were composed of a low proportion of OKT 8 positive subpopulation after primary ATS and successive propagation with IL 2. The proportion of OKT 8 positive subpopulation was increased by secondary ATS, which enhanced the cytotoxic activity to glioma cells more effectively.

Immunocytology of cultured IgM-forming cells of mouse. II. Purification of phagocytic cell factor and its role in antibody formation.

Abstract A factor required for the proliferation of IgM-forming tumor cells of mouse was purified approximately 1500-fold from culture supernatant of phagocytic cells through conventional protein fractionation procedures. The isoelectric point of the factor was pH 6.1 ± 0.1 as measured by isoelectric focusing. Its molecular weight was estimated to be approximately 5 × 10 4 daltons. These characteristics of the factor were not identical with those of the stimulating factor for granulocyte and macrophage colony formation. Antiserum against the purified factor inhibited the growth-promoting activity of the factor. The effect of the antiserum on the normal antibody response of mouse to sheep red blood cells was examined in the tissue culture system. The antiserum inhibited only the late stage in the formation of direct plaque-forming cells.

Regulatory factors of lymphocyte-lymphocyte interaction. I. Con A-induced mitogenic factor acts on the late G1 stage of T-cell proliferation.

DNA synthesis in murine lymphocytes was augmented by a soluble factor in the supernatant of serum‐free cultures of syngeneic spleen cells activated with concanavalin A (Con A). This so‐called mitogenic factor (MF), which is probably identical with interleukin II, partially purified by DEAE‐cellulose and Sephadex G‐75 chromatography, is a fairly homogeneous molecule of 17–25 × 103 daltons. By using partially purified MF, the role of MF in lymphocyte proliferation was investigated.

Scintigraphic detection of neural-cell-derived small-cell lung cancer using glioma-specific antibody

Radiolabeled GA-17, a murine monoclonal antibody that reacts specifically with glioma cells, bound to a small-cell lung cancer (SCLC) cell line NCI-H69 derived from neural cells, both in vitro and in vivo. The affinity constant of GA-17 F (ab′)2 fragment binding to NCI-H69 was 1.02×108/M while that to the glioma cell line U87MG was 1.22×108/M. Iodine-125-labeled GA-17 F (ab′)2 fragments injected i.v. localized well in NCI-H69 cells xenografted in nude mice. The percentage of the injected dose per gram accumulated in the xenografted tumor was 6.87±1.34%g−1 (mean±SD,n=5) 24 h after injection. On the other hand, control monoclonal F (ab′)2 fragments accumulated in the xenografted tumor at 0.75±0.30%g−1. The tumor-to-blood ratio was 1.8 for NCI-H69, while that of control F (ab′)2 was 0.60. In conclusion, the radiolabeled GA-17 F (ab′)2 fragment is expected to be useful clinically to visualize the small-cell lung cancer and in radioimmunotherapy.

Human glioma-specific antigens detected by monoclonal antibodies.

Three murine monoclonal antibodies, designated GA-17, GB-4, and GC-3, were prepared by the hybridization of murine myeloma cells (NS-1) and spleen cells of BALB/c mice immunized with the crude membrane fraction of cultured human gliosarcoma cells (GI-1). Two of them (GA-17 and GB-4) reacted exclusively with the membrane of glioma cells, and the other (GC-3) reacted with the membrane of glioma cells and a T cell line (MOLT-4). Although these antibodies reacted with almost all of the gliomas, the reactions differed. GA-17 reacted equally well with all glioblastoma (17 cases) and low-grade astrocytoma (10 cases), whereas GB-4 reacted poorly with 7 cases of glioblastoma and GC-3 did not react with 7 cases of low-grade astrocytoma. The antigens, exclusively expressed on the cell surface, were analyzed by surface labeling with 125I followed by a cell lysis and immunoprecipitation with these antibodies. The findings obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that GA-17, GB-4, and GC-3 reacted with Mr 140,000-145,000, Mr 160,000, and Mr 145,000-150,000 proteins, respectively. Some evidence has been obtained indicating that these antigens are composed of the same polypeptide chain (Mr 120,000) with the carbohydrate chains being different.

Interleukin 2 induces rapid phosphorylation of cellular proteins in murine T-lymphocytes

When quiescent murine T‐lymphocyte cells were stimulated by the addition of interleukin 2 (IL‐2), they reinitiated DNA synthesis after a lag period of 5 h. Under these conditions, rapid but transient phosphorylation of two cellular proteins with M r, values of 27000 and 26000 was detected; maximal phosphorylation occurred within 10–15 min after the addition of IL‐2. The protein of M r, 27 000 contained phosphoserine, while the protein of M r 26000 contained phosphothreonine.

Synthesis of Sterols and 5‐Lipoxygenase Products are Required for the G1‐S Phase Transition of Interleukin‐2‐Dependent Lymphocyte Proliferation

A murine killer T cell line, G‐CTLL 1, whose proliferation depends on the presence of interleukin 2 (IL‐2), was used to analyze the mechanism of IL‐2 action with respect to sterol synthesis and arachidonate metabolism. De novo sterol synthesis was substantially enhanced much earlier than DNA synthesis, and the rate reached a maximum at 13 hr after the addition of IL‐2. Compactin, which is a potent competitive inhibitor of 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase (HMG CoA reductase, the enzyme in the rate‐limiting step of the sterol synthesis), inhibited the IL‐2‐induced DNA synthesis. The addition of mevalonate, the product of HMG CoA reductase, prevented the inhibition of DNA synthesis by compactin, suggesting that the supply of a sufficient amount of sterol is an essential prerequisite for IL‐2 action. The IL‐2‐induced DNA synthesis was also inhibited by AA861, a specific inhibitor of arachidonate 5‐lipoxygenase, and by other lipoxygenase inhibitors such as nordihydroguaiaretic acid and esculetin. In contrast, indomethacin, an inhibitor of arachidonate cyclooxygenase, had no effect. These findings suggest that synthesis of 5‐lipoxygenase products is also a prerequisite. The inhibition of DNA synthesis was effectively inhibited only when compactin or lipoxygenase inhibitors were added early enough to block the synthesis of sterols or 5‐lipoxygenase products; addition of the reagents after 3 hr decreased the inhibition with time. Therefore, about 3 hr after the addition of IL‐2, several drastic intracellular changes are assumed to begin and to lead to DNA synthesis.

Mechanism of Interferon Gamma-Induced Protection of Human Gliosarcoma Cells from Lymphokine-Activated Killer Lysis: Division of Lymphokine-Activated Killer Cells into Natural Killer- and T-Like Cells

The mechanism by which interferon gamma (IFN-gamma) decreases the susceptibility of the established cultured gliosarcoma line Gl-1 to lymphokine-activated killer (LAK) lysis was analyzed. The results of monolayer depletion and lectin-dependent cellular cytotoxicity assays by LAK cells revealed that the resistance to LAK lysis of IFN-gamma-treated Gl-1 cells is manifested at the stage of LAK cell target recognition alone. We have also divided LAK cells into populations of phenotypically natural killer (NK)- and T-like cells with monoclonal antibodies and complement, respectively. We have used these cells to examine the mechanism of IFN-gamma-induced protection of Gl-1 cells from LAK lysis in cold target inhibition, monolayer depletion, and direct binding assays. The results revealed that NK-like cells do not recognize IFN-gamma-treated Gl-1 cells as efficiently as they do untreated targets, whereas T-like cells show the opposite tendency. In conclusion, we have demonstrated that the IFN-gamma induced protection of tumor cells from LAK lysis is predominantly regulated by the target recognition of NK-like cells. On the other hand, IFN-gamma-treated tumor cells may bind to T-like cells but fail to trigger them to initiate further stages for lysis as effectively as NK-like cells.

Detection of Altered Adhesion Molecule Expression in Experimental Tumors by a Radiolabeled Monoclonal Antibody

Adhesion molecules play a major role in the processes of invasion and metastasis of malignant tumors. Their expression within tumors has been reported to be quantitatively and qualitatively altered according to the invasiveness and metastatic potential of the tumor. The present study tested whether the intratumoral expression of integrin α3 can be detected by a radiolabeled monoclonal antibody. The in vitro hinding study with four different human cancer cells showed that radioiodinated GA17 antibody recognizing integrin “3 bound specifically to these cells to varying degrees, according to the antigen density on each cell. The biodistribution study with 125I– and 111In–labeled antibodies showed specific localization of radiolabeled GA17 to the xenografts. However, the in vivo tumor localization was not proportional to the antigen density calculated in vitro, and antibody metabolism varied among the tumors, as was also confirmed by in vitro radionuclide retention assay. The intratumoral distribution of radioactivities varied reflecting the antigen expression within the tumor. These results indicate that 1) integrin α3 was expressed in various kinds of tumors and could he localized by the radiolabeled antibody, and 2) the expression of integrin “3 and the metabolism of the radiolabeled antibody after binding to the antigen within the tumor were variable among the tumors, which affected the radionuclide distribution characteristics. The expression of adhesion molecules within these tumors was noninvasively detected by a radiolabeled antibody. It may be possible to use integrin a3, when it is overexpressed, as a target of therapy with antibodies radiolabeled with a or β emitters.