M Kim

University of Pittsburgh, Pittsburgh, PA, United States

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Publications (6)21.97 Total impact

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    ABSTRACT: The protein kinase C (PKC) inhibitors bisindolylmaleimide, calphostin C, H-7 and staurosporine were examined for their effect on tumour necrosis factor (TNF) cytotoxic activity. PKC inhibitors potentiated the cytotoxic activity of TNF in TNF-sensitive cell lines (CL8-1 melanoma and WEHI-164 fibrosarcoma), but had no effect on TNF cytolytic activity in TNF-resistant cells (BL6-8 melanoma and MCA-102 fibrosarcoma). The mechanism(s) of PKC inhibitor-mediated potentiation of the cytotoxic activity of TNF was investigated by analysing the effect of PKC inhibitors on TNF-induced arachidonic acid release in TNF-sensitive and -resistant cells. TNF induced the release of arachidonic acid in TNF-sensitive cells but had no effect in TNF-resistant cells. The combination of PKC inhibitor and TNF potentiated the release of arachidonic acid from TNF-sensitive cells, but failed to induce arachidonic acid release in TNF-resistant cells. Kinetic analysis of TNF-induced arachidonic acid release in CL8-1 melanoma cells revealed that it was an early event which preceded TNF tumour lytic activity. TNF was further shown to induce manganese superoxide dismutase (MnSOD) production in TNF-sensitive cells but failed to induce MnSOD activity in TNF-resistant BL6-8 and MCA 102 cells. MnSOD acts as a scaveneger of toxic superoxide radicals and its induction by TNF paralleled arachidonic acid release. Although the PKC-selective inhibitor bisindolylmaleimide potentiated TNF-induced release of arachidonic acid, it blocked TNF-mediated induction of MnSOD in CL8-1 melanoma and WEHI-164 fibrosarcoma cells.(ABSTRACT TRUNCATED AT 250 WORDS)
    Cytokine 09/1995; 7(6):517-25. · 2.52 Impact Factor
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    ABSTRACT: Transfection of BL6-8 (H-2Kb-,H-2Db+) and BL6-2 (H-2Kb-,H-2Db-) melanoma clones with H-2Kb or H-2Kd gene resulted in a stable augmentation of their sensitivity to lysis by NK cells or TNF-alpha that was associated with alterations of various phenotypic properties such as loss of endogenous A- and C-type retrovirus production and expression of melanoma-associated antigen and appearance of cell surface carbohydrates reacting with soybean agglutinin (SBA) and Grifonia simplicifolia 1-B4 (GS1B4) lectins. In contrast, transfection of the same clones with H-2Dd or H-2Ld gene did not reverse their resistance to NK cell- and TNF-mediated cytotoxicity and did not affect the phenotype of melanoma cells. These data suggest that the effect of H-2K gene on NK/TNF sensitivity of BL6 cells is indirect and it is closely associated with H-2K-induced phenotypic changes in these cells. To examine the possible role of the observed alterations of cell surface carbohydrates in augmentation of sensitivity of BL6 melanoma cells to lysis by NK cells or TNF, BL6-8 melanoma cells were transfected with cDNA encoding alpha 1,3-galactosyltransferase (alpha 1,3GT). Although the alpha 1,3GT gene-transfected cells expressed alpha-galactosyl epitopes reacting with GS1B4 lectin and reduced sialylation of cell membrane with unmasking SBA lectin-binding carbohydrates, they did not show an increase in tumor cell sensitivity to lysis by NK cells or TNF, indicating that H-2K gene-induced alterations in cell surface carbohydrate expression are not accountable for the observed increase in NK/TNF sensitivity of BL6 melanoma cells. It is possible that the H-2K gene-induced elimination of the endogenous retroviruses might be responsible for the observed increased sensitivity of BL6 melanoma cells to NK cell- and TNF-mediated cytotoxicity.
    Cellular Immunology 06/1994; 155(2):358-71. · 1.74 Impact Factor
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    ABSTRACT: We have previously demonstrated that transfection of BL6-8 melanoma cells with the H-2Kb gene increased their sensitivity to natural killer (NK)- and natural cytotoxic (NC) cell-mediated cytotoxicity, and resulted in alterations of various cell characteristics such as the loss of melanoma-associated antigen (MAA), and the appearance of Griffonia simplicifolia 1B4 (GS1B4)- and soybean agglutinin (SBA)-lectin-binding sites. In the present study the BL6-8 melanoma clone was transfected with the H-2Kb gene without cotransfection of the neomycin resistance (neor) gene, and transfected cells were isolated on the basis of their low sialylation and ability to bind to SBA-agarose beads. 38 out of 47 analyzed clones were morphologically distinguishable, expressed the H-2Kb gene, lost the MAA and gained SBA- and GS1B4-lectin-binding sites (H-2Kb+, MAA-, SBA+, GS1B4+), whereas only 5 clones were morphologically and phenotypically unchanged (H-2Kb-, MAA+, SBA-, GS1B4-). The remaining 4 clones were morphologically changed, lost MAA and gained SBA- and GS1B4-binding epitopes, but did not express H-2Kb antigen (H-2Kb-, MAA-, SBA+, GS1B4+). Analysis of the NK sensitivity of H-2Kb-gene-transfected clones revealed that H-2Kb+, MAA-, SBA+, GS1B4+ clones were highly sensitive to NK cell lysis, whereas H-2Kb-, MAA+, SBA-, GS1B4- clones were NK resistant. It is of particular note that the 4 clones that did not express H-2Kb antigen but had other phenotypic changes (H-2Kb-, MAA-, SBA+, GS1B4+) were also highly sensitive to NK cells. The NK sensitivity of H-2Kb-transfected cells was closely associated with their increased ability to compete with YAC-1 cells for effector cells in a cold-target inhibition assay. Thus, the data obtained indicate that H-2Kb molecules in BL6-8 melanoma cells are not required for interaction with the effector cells, and H-2Kb-induced alterations in membrane carbohydrates and/or expression of the retrovirus-associated MAA might be important for the increase NK sensitivity of BL6-8 melanoma cells.
    Natural Immunity 01/1994; 13(1):1-14.
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    ABSTRACT: The mechanisms of cytotoxic activity of Griffonia simplicifolia 1-B4 (GS1B4) and wheat germ agglutinin (WGA) lectins against various murine tumour cell lines were studied. Tumour cells that lack lectin-binding carbohydrates were resistant to lysis by these lectins. However, YAC-1 cells that expressed GS1B4 lectin-binding sites showed low sensitivity to lysis. To further analyse the relative importance of cell surface carbohydrates in lectin cytotoxicity, BL6-8 melanoma cells, which do not express the alpha 1,3 galactosyltransferase (alpha 1,3GT) gene and cell surface alpha-galactosyl epitopes reacting with GS1B4 lectin, were transfected with cDNA encoding alpha 1,3GT. After transfection, BL6-8 cells expressed high levels of GS1B4-binding alpha-galactosyl epitopes, but remained resistant to lysis by GS1B4 lectin, suggesting that the presence of lectin-binding epitopes, while essential, is not sufficient for tumour cell lysis and probably some intracellular mechanisms are involved in the regulation of lectin-mediated cytotoxicity. We found that the GS1B4 and WGA lectins induced apoptosis with DNA fragmentation of sensitive, but not resistant, tumour cell lines. DNA fragmentation, as well as tumour cell lysis, was blocked in the presence of the specific inhibitory sugar. To determine whether binding of the lectin to cell surface carbohydrates is sufficient to trigger tumour cell lysis, lectin-sensitive CL8-1 melanoma cells were incubated with GS1B4 lectin immobilized on agarose beads. Although these tumour cells bind to the immobilized lectin, it failed to trigger tumour cell death, suggesting that only soluble lectin is capable of tumour cell lysis and lectin internalization is probably required for their lysis.(ABSTRACT TRUNCATED AT 250 WORDS)
    Glycobiology 11/1993; 3(5):447-53. · 3.54 Impact Factor
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    ABSTRACT: Transfection of the H-2Kb and neor genes into BL6-8 (H-2Kb-, H-2Db+) melanoma clone resulted in various phenotypic changes with appearance of soybean agglutinin (SBA) and Grifonia Simplicifolia 1-B4 (GS1B4) lectin binding carbohydrates and loss of melanoma-associated antigen (MAA). In parallel H-2Kb gene-transfected melanoma cells showed increased sensitivity to TNF lysis. To further delineate the ability of H-2Kb gene to induce the phenotypic changes and TNF sensitivity, BL6-8 melanoma clone was transfected with the H-2Kb gene alone without cotransfection with neor gene and transfected cells were selected for adherence to SBA lectin-conjugated agarose beads. Analysis of isolated clones revealed that 38 of 47 tested clones have been found to be expressing the H-2Kb Ag, SBA, and GS1B4 lectin binding carbohydrates but lost MAA, e.g., H-2Kb+, lectin+, MAA-, and in parallel these cells became sensitive to TNF lysis. Although all clones with high expression of H-2Kb Ag were sensitive to TNF lysis, it seems unlikely that H-2K molecules are directly required for or involved in TNF-induced melanoma cell lysis. This conclusion is based on findings that four H-2Kb-transfected clones selected on SBA-agarose beads did not expressed H-2Kb Ag but manifested increase in SBA and GS1B4 lectin binding and loss of MAA and also became sensitive to TNF lysis. It seems that increase in TNF sensitivity is a part of the broad phenotypic changes induced by the H-2K gene that remained stable even in the clones in which the transfected H-2Kb gene was lost or down-regulated. We believe that the effects of the H-2Kb gene on melanoma cell phenotype and TNF sensitivity are indirect and are probably mediated via its inhibition of the melanoma-associated ecotropic retrovirus production and activation of some repressed cellular genes. Study of the mechanisms responsible for TNF sensitivity of BL6 melanoma cells revealed that the H-2Kb gene transfection resulted in an increase in p55 TNF receptor expression. TNF-induced activation of phospholipase A2 and release of arachidonic acid metabolites was observed only in the H-2Kb transfected, but not in BL6-8 melanoma cells transfected with neor or class II H-21Ak genes. TNF resistance of BL6 melanoma cells appeared to be due to a block in transduction of the lytic signal that was reversed after transfection with H-2Kb gene.
    The Journal of Immunology 11/1993; 151(7):3467-77. · 5.52 Impact Factor
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    ABSTRACT: An H-2Kb negative BL6 melanoma clone (BL6-8) was transfected with plasmids containing either the class I H-2Kb or class II H-2IAk gene in combination with the neor gene. The effects of the transfected genes on the expression of the melanoma-associated antigen (MAA) recognized by the monoclonal antibodies MM2-9B6 and MM2-3C6 and the cell surface carbohydrates recognized by 15 different lectins were studied. The original H-2Kb- clone or clones transfected with neor or class II H-2IAk genes expressed high levels of MAA and very low levels of soybean agglutinin (SBA), Griffonia simplicifolia I-B4 (GSIB4), and peanut agglutinin (PNA) lectin-binding sites. In contrast, clones that expressed high levels of the transfected H-2Kb gene completely lost the expression of MAA. In addition, these clones were characterized by the appearance of high levels of expression of the sugars specifically reacting with SBA, GSIB4, and PNA lectins. When the original BL6-8 clone was transfected with the H-2Kd gene, 25 clones subsequently isolated had relatively low expression of the transfected H-2Kd gene but high expression of the endogenous H-2Kb gene accompanied by an alteration in expression of the MAA and lectin binding identical with patterns common for H-2Kb+ melanoma cells. These changes were not due to the transfection, plasmid construction, or place of insertion, since similar phenotypic characteristics were found in H-2Kb+ but not H-2Kb- clones isolated from the N-methyl-N'-nitro-N-nitrosoguanidine-treated BL6T2 or parental BL6 melanoma lines. In total, 73 BL6 melanoma clones were investigated and all of the 41 H-2Kb+ clones displayed loss of MAA and appearance of SBA, GSIB4, and PNA-binding sugars. None of the 32 H-2Kb- clones showed these changes. This study indicates that the class I H-2Kb gene product might alter several phenotypic properties of BL6 melanoma cells. The mechanisms of these changes remain unknown. We consider that these effects of the class I H-2Kb gene are indirect, involving interactions with the B-tropic ecotropic retrovirus specific for melanomas of C57BL/6 mice origin.
    Cancer Research 11/1991; 51(19):5212-8. · 8.65 Impact Factor