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T Torigoe

Publications and source records attributed to T Torigoe.

At least 55 records · Page 3Linked to original sources
PubMed1994

Protein tyrosine kinase p56-Lck regulates lymphocyte function-associated 1 adhesion molecule expression, granule exocytosis, and cytolytic effector function in a cloned T cell.

Elevated levels of p56-Lck kinase activity were achieved in an interleukin 2 (IL-2)-dependent cloned cytolytic T cell CTLL-2 through gene transfer approaches. CTLL-2-Lck cells remained dependent on IL-2 for growth and survival in culture but exhibited markedly elevated, IL-2-independent cytolytic activity against a variety of tumor targets. This immune cell effector function was similar to the non-major histocompatibility complex-restricted cytolytic activity previously described for lymphokine activated killer (LAK) cells, and involved a cytolytic mechanism that was independent of protein synthesis in either the T cells or the tumor targets. Characterization of CTLL-2-Lck cells revealed markedly elevated levels of both the alpha (CD11a) and beta (CD18) chains of the cell adhesion molecule lymphocyte function-associated 1 (LFA-1) and increased binding of these T cells to a recombinant protein representing the extracellular domain of the LFA-ligand, intercellular adhesion molecule 1 (ICAM-1). Antibodies to CD11a partially abrogated cytolytic killing of tumor target cells by CTLL-2-Lck cells, suggesting that the upregulation in LFA protein levels potentially accounts at least in part for the phenotype of these T cells. Gene transfer-mediated elevations in p56-Lck kinase activity in an IL-3-dependent myeloid cell clone 32D.3 also resulted in increased LFA-1 expression, demonstrating that the findings are not unique to CTLL-2 cells. In addition to upregulation of LFA-1 expression, CTLL-Lck cells also exhibited more efficient exocytosis of cytotoxic granules upon activation with Ca(2+)-ionophore and phorbol ester, relative to control transfected and untransfected CTLL-2 cells. The findings functionally link the Lck kinase to a T cell effector pathway involved in cell-mediated cytotoxicity.

Cell Adhesion↗
PubMed1994

Prolactin induces rapid phosphorylation and activation of prolactin receptor-associated RAF-1 kinase in a T-cell line.

The receptor for prolactin (PRL) is a member of the hematopoietic receptor family that also includes the receptors for interleukins 2-7. PRL is synthesized and is secreted by human T lymphocytes and acts as a lymphokine necessary, but not sufficient, for T lymphocyte progression through the G1 phase of the cell cycle. Although data now indicate that PRL serves an immunomodulatory role in vitro and in vivo, the mechanisms of PRL receptor signal transduction in T cells have not been defined. We demonstrate here that PRL induced the phosphorylation of the p72-74 serine/threonine kinase c-Raf-1 in the PRL-dependent rat T-cell line Nb2. Associated with this inducible phosphorylation of Raf-1 was a concentration- and time-dependent activation of in vitro Raf-1 autokinase and substrate kinase activities, which correlated with the PRL-induced proliferation of Nb2 cells. Co-immunoprecipitation studies revealed association of Raf-1 with PRL receptors in Nb2 cells. These results revealed that all isoforms of the PRL receptor (short, intermediate, and long) are expressed in Nb2 cells and associate with Raf-1. In contrast to the PRL-dependent Nb2 cells, phosphorylation and activation of Raf-1 were constitutive in the Nb2-derived, PRL-independent, T-cell line Sp. These studies demonstrate for the first time an association between the PRL receptor and a serine/threonine kinase affiliated with signal transduction.

Animals↗
PubMed1994

Apoptosis regulation by interaction of Bcl-2 protein and Raf-1 kinase.

The Bcl-2 protein is over-produced in many types of human tumors and suppresses apoptosis induced by a wide-variety of stimuli, including chemotherapeutic drugs and gamma-irradiation. The biochemical mechanism of action of the Bcl-2 protein however remains enigmatic. Here we show that Bcl-2 can be co-immunoprecipitated with the serine/threonine-specific Raf-1 kinase both in a mammalian hemopoietic cell 32D.3 and when the two proteins are produced in Sf9 insect cells using recombinant baculoviruses. Though analysis of Raf-1 deletion mutants suggested that the C-terminal half of the protein which contains the catalytic domain is sufficient for co-immunoprecipitation with Bcl-2, Raf-1 does not appear to induce phosphorylation of Bcl-2 protein in 32D.3 and Sf9 cells. Furthermore, a mutant form of Raf-1 that lacks kinase activity could still be co-immunoprecipitated with Bcl-2 in Sf9 cells, suggesting that the interaction of these proteins does not reflect a kinase-substrate relation. Gene transfer experiments using 32D.3 hemopoietic cells demonstrated functional synergy between Bcl-2 and Raf-1 with regards to suppression of apoptosis induced by growth factor withdrawal. Taken together, these observations for the first time functionally link Bcl-2 to a signal transducing protein and suggest that the interaction of the Bcl-2 and Raf-1 proteins may be responsible for their ability to cooperate in the suppression of apoptosis.

Animals↗
PubMed1993

Evidence that protein tyrosine kinase p56-Lck regulates the activity of phosphatidylinositol-3'-kinase in interleukin-2-dependent T-cells.

The relative levels of phosphatidylinositol-3'-kinase (PI3K) activity were measured in interleukin-2 (IL-2)-dependent helper (HT-2) and cytolytic (CTLL-2) T-cell clones that had been stably transfected with expression plasmids encoding either the normal p56-Lck kinase or a mutant version of this kinase, p56-Lck(Y505F), that has constitutively high levels of kinase activity. Stimulation of untransfected T-cells or of transfected T-cells containing increased levels of normal p56-Lck resulted in an approximate doubling of the relative amounts of total cellular PI3K activity. In contrast, T-cells producing the activated version of p56-Lck contained levels of PI3K activity comparable to or slightly higher than those found IL-2-stimulated control cells, even in the absence of IL-2. Assessments of the relative levels of PI3K activity in immunoprecipitates prepared with the use of anti-phosphotyrosine-specific antibodies revealed constitutively high levels of anti-phosphotyrosine-immunoprecipitable PI3K activity in T-cells containing p56-Lck(Y505F), as opposed to T-cells containing normal p56-Lck where increases in anti-PY-immunoprecipitable PI3K activity were IL-2-inducible. IL-2 stimulation of T-cells containing the normal p56-Lck kinase resulted in marked increases in the relative amounts of PI3K activity and p85 that were coimmunoprecipitated when using anti-p56-Lck antibodies. In contrast, PI3K activity and the p85 subunit of PI3K could be coimmunoprecipitated from T-cells producing the activated p56-Lck(Y505F) kinase even in the absence of IL-2 stimulation, implying constitutive association of PI3K with the activated Lck kinase. Taken together with previous studies showing that IL-2 induces rapid increases in the activities of both p56-Lck and PI3K in T-cells, these findings suggest that p56-Lck lies immediately upstream of PI3K in a signal transduction cascade initiated by the binding of IL-2 to its specific receptor on T-lymphocytes.

Amino Acid Sequence↗
PubMed1993

Protooncogene-encoded protein kinases in interleukin-2 signal transduction.

Protooncogenes are the normal forms of cellular genes that when altered in their expression or coding sequences can contribute to neoplastic transformation. As these genes often are important for normal cellular growth control, we explored the possibility that protein kinases encoded by particular protooncogenes could participate in signal transduction pathways regulated by the T cell growth factor, interleukin-2 (IL-2). In this review we summarize our findings to date regarding Raf-1, a serine/threonine-specific kinase that becomes phosphorylated on tyrosine residues and enzymatically activated in response to IL-2 stimulation. In addition, we describe our investigations of Lck and Lyn, two closely related protein tyrosine kinases of the src gene family that physically associate with the IL-2 receptor complex and whose activities are regulated by IL-2 in at least some T cells and B cells, respectively.

Animals↗
PubMed1993

Cellular stress- and transformation-associated cell surface antigens expressed on human and rodent tumor cells.

Stress-induced proteins may have significant roles in anti-tumor resistance. To clarify the immunobiological roles of these proteins, we first developed monoclonal antibody (mAb) H1A that detects the HeLa cell-surface antigens whose expression was enhanced by treatment of the cells with physico-chemical stressors, such as heat, H2O2 and tumor necrosis factor. H1A (IgM) detects several molecules with mol. wt. 30, 43, 75, 90, 100, 120 and 150 kDa in Western blot analysis of HeLa cell lysates. Although the antigen was constitutively expressed on the HeLa cell surface, the cell-surface expression of H1A-defined antigen was rapidly enhanced (within 1 h) after heat treatment of HeLa cells. H1A antigens were also transformation-associated, since 1) the activated oncogene-transformed fibroblasts expressed the antigens, but parental nontransformed cells did not, and 2) certain human neoplastic but not normal cells strongly expressed the antigens. Furthermore, H1A mAb also partly blocked the cytotoxicity of purified protein derivatives-stimulated human T cell receptor gamma delta-type T cells towards HeLa cells. Taken together, these data indicate that H1A-defined stress-inducible proteins may play a vital role in anti-tumor resistance by cytotoxic T cells.

Adenosine Triphosphate↗
PubMed1992

Anergic Th1 cells express altered levels of the protein tyrosine kinases p56lck and p59fyn.

Tolerance in T lymphocytes can result from clonal anergy, or paralysis, of Ag-specific T cells. To investigate the molecular mechanisms responsible for anergy, a system in which tolerance can be induced in vitro was employed. Anergy, as defined by long-lived nonresponsiveness to normal antigenic stimulation for IL-2 production, was produced in cloned murine CD4+ Th1 cells. Here we report that such anergic Th1 cells express constitutively reduced amounts of the protein tyrosine kinase p56lck and constitutively elevated levels of the protein tyrosine kinase p59fyn. Because protein tyrosine phosphorylation is known to be important for the normal induction of IL-2 synthesis, these results suggest that T cell anergy may be maintained, at least in part, by alterations in tyrosine phosphorylation signaling events.

Animals↗
PubMed1992

A strategy for generating monoclonal antibodies against recombinant baculovirus-produced proteins: application to the Bcl-2 oncoprotein.

A strategy is described for production of monoclonal antibodies against recombinant proteins that are produced using the baculovirus expression system and that requires no prior purification of the protein of interest. Crude lysates prepared from cultured Sf9 insect cells infected with recombinant or control baculoviruses are absorbed to nitrocellulose filters and used in a dot-immunobinding assay for screening hybridomas. The monoclonal antibody-producing hybridomas are derived by immunization of mice with a synthetic peptide corresponding to a hydrophilic region in the recombinant protein of interest. By using the baculovirus-produced recombinant protein as the screening antigen and by comparing antibody binding to filters containing control Sf9 lysates, hybridomas are identified that produce monoclonal antibodies with specific reactivity for the recombinant protein of interest and that can then subsequently be used to assist in the large-scale purification of the recombinant protein from baculovirus-infected cells. We applied this method to recombinant 26-kDa human Bcl-2 (B-cell lymphoma/leukemia-2), an integral membrane oncoprotein that regulates programmed cell death ("apoptosis") in hematolymphoid cells through unknown mechanisms. Two mouse monoclonal antibodies were produced that specifically bound the recombinant Bcl-2 baculoprotein in both solution and solid-phase assays.

Amino Acid Sequence↗
PubMed1992

Phenotypic changes induced by interleukin-2 (IL-2) and IL-3 in an immature T-lymphocytic leukemia are associated with regulated expression of IL-2 receptor beta chain and of protein tyrosine kinases LCK and LYN.

We have previously reported the establishment of an interleukin-3 (IL-3)-dependent and phenotypically myeloid cell line (TALL-103/3), obtained by culturing cells from an immature T-lymphoblastic leukemia in the presence of IL-3. These cells differentiated into a T-lymphoid cell line (TALL-103/2) upon removal of IL-3 and incubation in IL-2. Despite the different phenotype, the two cell lines remained karyotypically and genotypically identical. Here, we have analyzed the phenotypic changes and the signaling events induced by these two lymphokines in TALL-103/3 cells by switching them to temporary growth in IL-2 and returning them to IL-3. All four sublines obtained (the myeloid in IL-3 and the lymphoid in IL-2) expressed RNA for CD3, IL-2 receptor (R) alpha, and T-cell receptor (TCR)-gamma and -delta chains. However, cells cultured in IL-3 failed to express detectable levels of the IL-2R beta chain at both the protein and RNA levels, whereas cells exposed to IL-2 always expressed IL-2R beta. In parallel with the changes in IL-2R beta expression, the SRC-like protein tyrosine kinase (PTK) p56 LCK could not be detected in IL-3-dependent cells, but was abundant in the IL-2-dependent cells and underwent markedly increased autophosphorylation in response to IL-2. In contrast, p53/p56 LYN was highly expressed in IL-3-dependent cells, and greatly decreased when these cells were switched to growth in IL-2. LYN kinase autophosphorylation modestly increased in response to IL-3. None of the other kinases in the SRC family that were tested underwent increased autophosphorylation after lymphokine stimulation, indicating the specificity of IL-2 for LCK and of IL-3 for LYN. The TALL-103 cell lines provide a unique system to study the interaction between lymphokines and SRC-family PTKs in signal transduction pathways leading to hematopoietic cell differentiation.

Cell Division↗
PubMed1992

Interleukin-3 regulates the activity of the LYN protein-tyrosine kinase in myeloid-committed leukemic cell lines.

The lymphokine interleukin-3 (IL-3) promotes the growth and survival of immature hematopoietic cells. Previous studies have shown that IL-3 induces rapid increases in protein-tyrosine kinase (PTK) activity in IL-3--dependent cells. Unlike some other hematopoietic growth factor receptors (eg, c-fms and c-kit), however, the known subunits of the IL-3 receptor (IL-3R) lack intrinsic kinase activity. Recently, it was reported that the IL-2R (whose p75 beta-subunit shares sequence homology with a known murine IL-3R subunit and a common beta-subunit of the human IL-3R and granulocyte-macrophage colony-stimulating factor [GM-CSF] receptors) can physically associate with and regulate the activity of the SRC-family PTK, p56-LCK. Because most IL-3--dependent cells contain p53/56-LYN, but not p56-LCK, we explored the effects of IL-3 on the activities of LYN and other SRC-like PTKs in two human leukemic cell lines, AML-193 and TALL-101, which are phenotypically myeloid, and whose in vitro growth is dependent on IL-3. These cells expressed four of the eight known SRC-family proto-oncogenes: lyn, fyn, yes, and hck. When these factor-dependent leukemic cell lines were deprived of lymphokine to achieve cellular quiescence and then restimulated with IL-3, rapid increases (detectable within 1 minute and maximal by 10 minutes) were observed in the activity of the p53/56-LYN kinase, as assessed by in vitro kinase assays. In contrast, no alteration in the activities of other SRC-family PTKs present in these cells was detected after restimulation with IL-3 under the same conditions. This effect of IL-3 reflected an increase in the specific activity of the LYN kinase, because levels of the 53-Kd and 56-Kd LYN proteins were unaltered by IL-3 stimulation, as assessed by immunoblotting. Furthermore, the magnitude of these inducible increases in LYN kinase activity was dependent on the concentration of IL-3, and correlated with IL-3--induced proliferation. The IL-3--induced upregulation of LYN kinase activity may be mediated by the 120-Kd common subunit of the human IL-3 and GM-CSF receptors, because GM-CSF also stimulated marked increases in the activity of the LYN kinase, whereas granulocyte-CSF (G-CSF) did not, despite inducing cellular proliferation. These observations provide the first example of an IL-3--regulable PTK, and strongly suggest that the p53/56-LYN kinase participates in early IL-3--initiated signalling events, at least in some human leukemic cell lines.

Cell Line↗
PubMed1992

Interleukin 2 regulates the activity of the lyn protein-tyrosine kinase in a B-cell line.

Recently, interleukin 2 (IL-2) has been shown to induce increased activity of the p56lck protein-tyrosine kinase (PTK) in T-cell and natural killer cell lines, and evidence for a direct interaction between the p75 subunit of the IL-2 receptor (IL-2R) and this src-family kinase has been reported. Though these findings suggest a central role for lck in IL-2 signal transduction, one problem with this idea is that not all IL-2-responsive cells express the lck gene. For this reason, we examined the effects of IL-2 on the activity of src-like kinases in a pro-B cell line, F7, that lacks p56lck but that displays high-affinity IL-2Rs and vigorously proliferates in response to this lymphokine. Of the eight known src-family PTKs, F7 cells were shown to contain only p53/56lyn, p59fyn, and a small amount of p62yes. Stimulation of resting F7 cells with IL-2 induced a rapid (detectable within 1 min and maximal at 15 min) and concentration-dependent increase in the specific activity of p53/56lyn kinase, as assessed by in vitro kinase assays. This effect of IL-2 on p53/56lyn kinase was specific, since no IL-2-inducible changes were detected in the activities of the p59fyn and p62yes kinases. Furthermore, by using a monoclonal antibody specific for the approximately 75-kDa beta subunit of the IL-2R (referred to as p75/IL-2R beta), evidence for physical association between the lyn kinase and the IL-2R complex was obtained, in that a small proportion of the p53/56lyn kinase in F7 cells, but no detectable p59fyn kinase, was coimmunoprecipitated with p75/IL-2R beta. When combined with the recent evidence that IL-2 regulates p56lck in T cells, these results indicate that some flexibility exists in the ability of various src-like PTKs to participate in IL-2 signal transduction mechanisms and raise the possibility that lineage-specific (T-versus B-cell) responses to IL-2 may be determined at least in part by the repertoire of src-like PTKs expressed in the cell.

Amino Acid Sequence↗
PubMed1992

Gene transfer investigations of p56-LCK function in IL-2-dependent T-cell lines: implications for mechanisms of IL-2-signal transduction.

Recently, the SRC-like non-receptor protein tyrosine kinase p56-LCK has been shown to physically associate with the interleukin-2 receptor (IL-2-R) complex and to undergo rapid elevations in its tyrosine kinase activity upon stimulation of T lymphocytes with IL-2. The functional significance of p56-LCK kinase activation for IL-2-mediated lymphocyte responses, however, has never been directly assessed. Using gene transfer approaches, we have achieved markedly elevated levels of p56-LCK kinase activity in the IL-2-dependent cytolytic T-cell line CTLL-2 and the helper line HT-2. CTLL-2 and HT-2 cells that were stably transfected with expression plasmids encoding either the normal human p56-LCK or a constitutively active version of the mouse p56-LCK kinase (LCK[Y505]) contained striking elevations in the levels of tyrosine phosphorylation on several proteins (34-36, 50-60, 62-68, 77-78, 104-110 kDa), as determined by immunoblot analysis using anti-phosphotyrosine antibodies. CTLL-2 and HT-2 LCK- and LCK(Y505F)-transfected cells remained dependent on IL-2 for their growth and survival in culture despite the findings that (i) IL-2 specifically stimulated elevations in the activity of the endogenous p56-LCK in untransfected CTLL-2 cells without affecting the activities of the other SRC-like kinases in these cells (p59-FYN, p62-YES) and that (ii) IL-2-mediated regulation of p56-LCK correlated with IL-2-driven proliferation of these T cells. Specifically, no elevation in the proliferation (DNA synthesis) or growth of these T cells was found at any of the concentrations of IL-2 examined (0.01-25 U/ml), relative to untransfected and control transfected cells. Furthermore, when cultured in the absence of IL-2, transfected T cells whose relative levels of p56-LCK activity were elevated by approximately 20-50-fold died with the same kinetics as control cells and underwent apoptosis, as defined by uptake of trypan blue dye and DNA fragmentation assays, respectively. Taken together, these data indicate that while IL-2 can up-regulate the enzymatic activity of p56-LCK, elevated levels of p56-LCK tyrosine kinase activity are insufficient to stimulate IL-2-mediated pathways required for T-cell growth and survival. These findings thus imply the existence of other signal-transducing molecules, besides p56-LCK, that physically participate in IL-2R complexes and that are necessary for initiation of the biochemical events ultimately responsible for IL-2's pleiotropic actions on lymphocytes.

Animals↗
PubMed1992

Interleukin 4 inhibits IL-2-induced proliferation of a human T-leukemia cell line without interfering with p56-LCK kinase activation.

Recently we described the establishment in culture and the immunophenotypic and functional characteristics of a human T-leukemia line TALL-103/2 derived from the T-cell receptor (TCR)-gamma/delta subset of T-lymphocytes. TALL-103/2 cells are absolutely dependent on interleukin 2 (IL-2) for their growth and survival in culture and thus provide a model cell line for studies of IL-2 signal transduction in a TCR-gamma/delta T-cell. In this report, we focus on the regulation of SRC-family protein tyrosine kinases (PTKs) by IL-2. TALL-103/2 cells were found to contain p56-LCK, p59-FYN, p62-YES and p53/56-LYN. Stimulation of growth factor-deprived TALL-103/2 cells with IL-2, however, induced increases in the relative activity only of the p56-LCK kinase. This IL-2-mediated increase in LCK kinase activity was manifested both by increased kinase autophosphorylation and by increased phosphorylation of the exogenous substrate enolase during in vitro kinase assays. Furthermore, immunoblot assays determined that the levels of p56-LCK protein were unaltered by IL-2-treatment, indicating that the measured elevations in LCK kinase activity reflected an increase in the specific activity of this PTK. In TALL-103/2 cells, IL-2 stimulated concentration-dependent increases in p56-LCK activity that displayed rapid and transient kinetics: detectable increases occurred within 1 minute after IL-2 stimulation, peaked at 10 minutes, and declined to baseline levels by 30 minutes. Treatment of TALL-103/2 cells with IL-4 abrogated IL-2-initiated proliferation, but did not inhibit IL-2-mediated activation of p56-LCK.(ABSTRACT TRUNCATED AT 250 WORDS)

Dose-Response Relationship, Drug↗
PubMed1992

Effects of p56lck deficiency on the growth and cytolytic effector function of an interleukin-2-dependent cytotoxic T-cell line.

The growth, differentiation, and functional activities of antigen-stimulated T lymphocytes are regulated by the interaction of the T-cell-derived cytokine, interleukin-2 (IL-2), with the high-affinity IL-2 receptor (IL-2R). IL-2R occupancy initiates a rapid increase in intracellular protein tyrosine phosphorylation, suggesting that a receptor-coupled protein tyrosine kinase (PTK) serves as a proximal signaling element for the IL-2R. Previous studies implicated the src-family kinase, p56lck, as a potential IL-2R-linked signal transducer. In this study, we have characterized a spontaneous variant of the IL-2-dependent cytotoxic T-cell line, CTLL-2, which contains no detectable lck-derived mRNA transcripts, protein, or PTK activity. The p56lck-deficient CTLL-2 cells retained strict dependence on IL-2 for both viability and growth, indicating that p56lck activity was not required for the transduction of IL-2-mediated mitogenic signals. However, the p56lck-deficient cells exhibited a moderate decrease in their rate of IL-2-dependent proliferation. In contrast to this relatively modest proliferative defect, the p56lck-deficient cell line displayed a profound reduction in T-cell antigen receptor-dependent cytolytic effector functions. Both the proliferative and the cytolytic defects observed in the p56lck-deficient cells were completely reversed by transfection of these cells with a wild-type lck expression vector. These results indicate that p56lck expression is not obligatory for IL-2-mediated T-cell growth stimulation; however, this PTK plays a central role in the generation T-cell-mediated cytotoxic responses.

Animals↗
PubMed1992

Regulation of SRC-family protein tyrosine kinases by interleukins, IL-2, and IL-3.

Unlike many other growth factor receptors, the known subunits of the receptors for the Interleukins IL-2 and IL-3 lack intrinsic tyrosine kinase activity, and yet increases in the phosphorylation of proteins on tyrosines is a rapid event in hematolymphoid cells following stimulation with these lymphokines. Here we show that IL-2 and IL-3 regulate the activity of specific members of the SRC-family of non-receptor protein tyrosine kinases (PTKs). In IL-2-dependent T-cell lines, IL-2 induced rapid and transient increases in the activity of the p56-LCK kinase without influencing the activities of other SRC-like PTKs (p59-FYN, p62-YES) in these T-lymphocytes. In contrast to IL-2's effects on p56-LCK in T-cells, studies of an IL-2-responsive cell line of the B-cell lineage that lacks p56-LCK revealed that IL-2 specifically regulates the activity of the p53/56-LYN kinase. Thus, some flexibility exists in the ability of various SRC-like PTKs to functionally couple to IL-2 signalling pathways. In several IL-3-dependent myeloid-committed leukemic cell lines, IL-3 was found to specifically regulate the activity of the p53/56-LYN kinase without affecting the activities of other SRC-like PTKs (p59/64-HCK, p59-FYN, p62-YES) in these hematopoietic cells. This finding that p53/56-LYN can be regulated by both IL-2 in B-lineage cells and IL-3 in myeloid-committed cells demonstrates that the same SRC-family PTK can participate in signal transduction events mediated via two independent receptor systems. Taken together, our findings imply that the specific combinations of lymphokine receptors and SRC-like PTKs available for coupling with those receptors are coordinately controlled during the differentiation of hematopoietic cells.

Animals↗
PubMed1991

Tumor rejection antigens on BALB3T3 cells transformed by activated oncogenes.

The clonal expression of tumor rejection Ag (TRA) was analyzed on nine different clones derived from parental BALB3T3 cells that were transfected with activated H-ras, polyoma middle T (PyMT), c-myc, and v-src oncogenes. It was shown that Bras-h clone, which is an activated H-ras oncogene-induced transformant, expressed TRA as assessed in the transplantation study using syngeneic BALB/c mice. This TRA was not detected on parental BALB3T3 nontransformed cells, suggesting that TRA could be expressed in the BALB3T3 cell transformation. Furthermore, the cross-protection experiments indicated that this TRA was also conferred on other BALB3T3 transformants with high anchorage-independent growth potential such as an activated H-ras transformant Bras-d, and PyMT transformants BMT-f, BnMT-11, BnMT-20, except in the case of one H-ras transformant Bnr-12. In contrast, this TRA was not expressed on the transfectants with little or no anchorage independent growth potential such as a PyMT transfectant BnMT-4, a c-myc transfectant Bmyc-7, and a v-src transfectant Bsrc-7. We developed the mAb BRH19 that could react with TRA+ clones but not with TRA- clones. This mAb makes an immunoprecipitate, which is composed of a 50-kDa single polypeptide chain from Bras-h cell lysate. An injection to mice with this antigen could confer the protection against Bras-h challenge. These data indicate that the 50-kDa putative TRA molecule could be expressed in close association with the cell transformation, irrespective of the introduced oncogenes, and there may exist some regulatory mechanisms rather than individually distinct manners for the expression of TRA.

3T3 Cells↗
PubMed1991

Natural killer target molecules associated with the transformation of the oncogene-transfected fibroblast.

Cell surface antigens, the expression of which is highly enhanced along with the transformation of cells, were analyzed. W14 and W31, EJ-ras oncogene-induced transformants of a WKA rat fetus-derived fibroblast WFB, strongly expressed several transformation-associated antigens as defined by monoclonal antibodies 109, 061, and 081. These monoclonal antibodies recognized Mr 86,000, 62,000, and 101,000 molecules, each composed of a single polypeptide chain. The expression of these transformation-associated antigens was negligible on parental WFB cells. Transforming growth factor-beta could enhance the expression of all of these transformation-associated antigens, but platelet-derived growth factor could only enhance the Mr 86,000 kd molecule expression. In the cytotoxicity assays, poly-I:C-induced rat splenic NK cells were cytotoxic to W14 and W31, but not to WFB. The data also showed that the cytotoxicity by these NK cells against NK-sensitive YAC-1 cells was absorbed with the addition of W14, W31, platelet-derived growth factor, or transforming growth factor-beta-stimulated WFB cells. This indicates that NK cells may recognize common target antigens that are expressed among these target cells. It was also indicated that Mr 86,000 and 62,000 molecules were strongly involved in this cytotoxicity, possibly as the target antigens, since F(ab')2 fragments of monoclonal antibodies 109 and 061 strongly inhibited the cytotoxicity. The addition of monoclonal antibody 109, but not 061, inhibited the cytotoxicity even at 60 min after mixing with the effector and target cells, suggesting that the Mr 86,000 molecule may participate in the lethal hit phase of cytotoxicity by NK cells. These data may indicate that some, but not all, transformation-associated antigens are virtually important in the antitumor surveillance mechanisms by the host effector cells, such as NK cells.

Animals↗