Effect of nuclear factor kappaB inhibition on tumor cell sensitivity to natural killer-mediated cytolytic function.

Inhibition of the transcription factor NF-kappaB has been reported to increase cell sensitivity to TNF and some cytotoxic drugs. We investigated the effect of NK-kappaB inhibition on the susceptibility of tumor cells to freshly isolated, nonactivated, human NK cells and to a TCRgamma/delta T cell clone displaying an MHC-unrestricted "NK-like" lysis. Using electrophoretic mobility shift assay, we first demonstrated that NF-kappaB/DNA binding activity was induced in target cells following coculture with NK cells or TCRgamma/delta T cell clone. To investigate the effect of target cell NF-kappaB inhibition on NK-mediated lysis, we blocked NF-kappaB translocation by introducing a human cDNA coding for a mutated IkappaB-alpha. Interestingly, our results indicated that inhibition of NF-kappaB did not induce any increase in either granzyme-dependent non-MHC-restricted cytotoxicity mediated by fresh non-stimulated NK cells and by TCR gamma/delta T cell clone or in CD95-mediated lysis. These results emphasize that NF-kappaB expressed in target cells does not play a role in the molecular process related to the control of target cell susceptibility to NK-mediated lysis and suggest that the NF-kappaB pathway is not a general mechanism for controlling the cytotoxic response.

Adenovirus-mediated wild-type-p53-gene expression sensitizes TNF-resistant tumor cells to TNF-induced cytotoxicity by altering the cellular redox state.

We have shown that the loss of p53 function contributed to resistance of tumor cells to TNF-induced cytotoxicity. In the present study, we evaluated the effect of wild-type p53 (wt-p53) expression on TNF sensitivity, by introducing wt-p53 into MCF7/Adr cells in which p53 was deleted, via a recombinant adenovirus encoding p53 (Ad-p53). Our results indicate that infection with Ad-p53 (50-100 viral particles per cell) resulted in pronounced cytotoxicity, whereas infection with 10 viral particles per cell, which was weakly toxic for the MCF7/Adr cells, sensitized these cells to TNF-induced cell death. Moreover, expression of wt-p53 in MCF7/Adr cells induced the production of reactive oxygen intermediates (ROIs) and caused glutathione (GSH) depletion, indicating disturbances in the cellular redox state. Additional treatment of cells with the anti-oxidant and glutathione (GSH) precursor N-acetylcysteine (NAC) resulted in inhibition of p53-induced ROIs production and in partial restoration of intracellular GSH levels, which was associated with the ability of NAC to inhibit p53-modulated TNF-induced cytotoxicity. Interestingly, Ad-p53 was able to inhibit TNF-induced MnSOD mRNA expression in MCF7/Adr cells, which might contribute to the sensitization of cells to the cytotoxic action of TNF. Taken together, our data strongly suggest that wt-p53 expression sensitizes TNF-resistant MCF7 cells with p53 deletion to TNF-induced cell death by a pathway that is dependent on ROIs production.

Adenovirus-mediated transfer of wild-type p53 gene sensitizes TNF resistant MCF7 derivatives to the cytotoxic effect of this cytokine: relationship with c-myc and Rb.

Tumor suppressor p53 is a nuclear transcription factor that blocks cell cycle progression and induces apoptosis. We have previously shown that the MCF7 resistance to the cytotoxic action of TNF correlates with p53 mutations. In the present study, we used a recombinant adenovirus carrying a wild-type p53 gene (Adwtp53) in order to investigate the effect of wt p53 transfer on modulation of cell resistance to the cytotoxic action of TNF. Our data indicate that infection of TNF resistant MCF7 cells (1001 and MCF7/Adr) with Adwtp53 resulted in the restoration of wt p53 expression and function as respectively revealed by the yeast assay and the induction of p53 inducible genes MDM2 and p21. Furthermore, the restoration of p53 function significantly sensitized TNF resistant cells to TNF cytotoxic action. This correlated with a significant down-regulation of c-myc in both TNF-resistant cell lines and a decrease of Retinoblastoma protein (Rb) in 1001 clone. In contrast, the effect of p53 seems to be independent from Bcl-2 and Bax protein level regulation. The present study suggests that the combination of TNF and Adwtp53 may be a potential strategy to sensitize mutant p53 TNF-resistant tumors to the cytotoxic action of this cytokine.

Analysis of human breast adenocarcinoma MCF7 resistance to tumor necrosis factor-induced cell death. Lack of correlation between JNK activation and ceramide pathway.

Considerable progress has been made in the understanding of tumor necrosis factor (TNF) signaling; however, the molecular and biochemical basis of tumor resistance to the cytotoxic action of TNF are still not definitively identified yet. Although a role of c-Jun N-terminal kinase (JNK) pathway has been suggested as an effector in TNF signaling, its exact relative contribution and its interaction with ceramide pathway and tumor resistance to TNF remain unknown. The relationship between JNK activation and human breast adenocarcinoma MCF7 resistance acquisition to the cytotoxic action of TNF was therefore investigated. We demonstrate that TNF triggers JNK activation in both TNF-sensitive MCF7 cells and its resistant derivative, RA1/1001. In addition, when MCF7 cells were stably transfected with mitogen-activated protein kinase kinase 4 (MKK4) dominant-negative cDNA or transiently transfected with a dominant-negative c-Jun mutant (TAM 67), their susceptibility to the cytotoxic action of TNF remains comparable with control cells. We also demonstrated that JNK activation does not require ceramide generation since in MCF7 cells transfected with a dominant-negative derivative of FADD (FADD-DN), which are resistant to the cytotoxic action of TNF, TNF induced JNK activation in the absence of ceramide generation. Furthermore, our data indicate that exogenous permeable synthetic ceramide C-6 induced the killing of MCF7 cells transfected with MKK4 dominant-negative cDNA. These results provide strong evidence indicating that tumor acquisition of resistance to the cytotoxic action of TNF may occur either independently or at a level downstream of JNK activation and suggest that JNK activation is not linked to ceramide pathway in TNF-mediated apoptosis.

Alteration of the sphingomyelin/ceramide pathway is associated with resistance of human breast carcinoma MCF7 cells to tumor necrosis factor-alpha-mediated cytotoxicity.

The interference of tumor necrosis factor-alpha (TNF) signaling processes with the acquisition of tumor resistance to TNF was investigated using the TNF-sensitive human breast carcinoma MCF7 cell line and its established TNF-resistant variant (R-A1). The resistance of R-A1 cells to TNF correlated with a low level of p55 TNF receptor expression and an absence of TNF signaling through TNF receptors. Stable transfection of wild-type p55 receptor in R-A1 resulted in enhancement of p55 expression and in partial restoration of TNF signaling, including nuclear factor-kappaB (NF-kappaB) activation. However, the transfected cells remained resistant to TNF-induced apoptosis. Northern blot analysis revealed a comparable induction of manganous superoxide dismutase and A20 mRNA expression in p55-transfected cells and in sensitive MCF7 cells, making it unlikely that these genes are involved in the resistance to TNF-mediated cytotoxicity. While TNF significantly stimulated both neutral and acidic sphingomyelinase (SMase) activities with concomitant sphingomyelin (SM) hydrolysis and ceramide generation in MCF7, it failed to trigger these events in TNF-resistant p55-transfected cells. In addition, the basal SM content was significantly higher in sensitive MCF7 as compared to the resistant counterparts. Furthermore, the TNF-resistant cells tested could be induced to undergo cell death after exposure to exogenous SMase or cell-permeable C6-ceramide. This study also shows that TNF failed to induce arachidonic acid release in p55-transfected resistant cells, suggesting that an alteration of phospholipase A2 activation may be associated with MCF7 cell resistance to TNF. Our findings strongly suggest a role of ceramide in the mechanism of cell resistance to TNF-mediated cell death and may be relevant in elucidating the biochemical nature of intracellular messengers leading to such resistance.

Impairment of Fas-antigen expression in adriamycin-resistant but not TNF-resistant MCF7 tumor cells.

Anti-cancer drugs and cytotoxic cytokines such as members of the TNF/Fas-ligand family play a predominant role in apoptosis induction in tumor cells, and are critical in cancer therapy. In this study we used the human breast-carcinoma cell line MCF7, its derivatives MCF7Adr (resistant to adriamycin) and R-A1 (resistant to TNF), to determine the impact of acquired drug and cytokine resistance on susceptibility to Fas-induced cytotoxicity and Fas-antigen expression. While MCF7 and R-A1 cells were killed by anti-Fas in the presence of IFN-gamma, MCF7Adr was found to be resistant to Fas-mediated apoptosis. This resistance was correlated with a loss of surface Fas-protein expression. Fas-gene transfer in MCF7Adr resulted in high sensitivity to Fas-mediated cytotoxicity, indicating that the Fas signalling pathway is virtually intact in this cell line. Over-expression of the MDR1 gene in MCF7 following gene transfer did not affect Fas expression and anti-Fas sensitivity, suggesting that the P-gp-mediated multidrug-resistance phenotype is not directly involved in the loss of Fas expression, contrary to what has been observed by others in T-cell lines. Furthermore, the down-regulation of Fas expression and subsequent resistance to anti-Fas were observed in drug-resistant human ovarian-carcinoma IGR-OV1/VCR cells and leukemic lymphoblast CEM/VLB cells, suggesting that the alteration of Fas expression following drug-resistance selection is not restricted to one cell type.

Protein kinase A phosphorylation of RhoA mediates the morphological and functional effects of cyclic AMP in cytotoxic lymphocytes.

We have observed that stimulation of human natural killer cells with dibutyryl cAMP (Bt2cAMP) reproduced the effects of ADP ribosylation of the GTP binding protein RhoA by Clostridium botulinum C3 transferase: both agents induced similar morphological changes, inhibited cell motility and blocked the cytolytic function. We demonstrate here that cAMP-dependent protein kinase A (PKA) phosphorylates RhoA in its C-terminal region, on serine residue 188. This phosphorylation does not affect the ability of recombinant RhoA to bind guanine nucleotides, nor does it modify its intrinsic GTPase activity. However, treatment of cells with Bt2cAMP results in the translocation of membrane-associated RhoA towards the cytosol. Experiments using purified membrane preparations indicated that Rho-GDP dissociation inhibitor, which can complex phosphorylated RhoA in its GTP-bound state, was the effector of this translocation. Taken together, these data suggest that PKA phosphorylation of RhoA is a central event in mediating the cellular effects of cAMP, and support the existence of an alternative pathway for terminating RhoA signalling whereby GTP-bound RhoA, when phosphorylated, could be separated from its putative effector(s) independently of its GTP/GDP cycling.

Regulation of interleukin-2 and interleukin-4 receptor expression in human and ape lymphoid cell lines.

In this study, we have analyzed the expression and regulation of receptors for IL-2 (alpha and beta chains) and IL-4 in four lymphoid cell lines established from leukemic cells. The gibbon ape cell line MLA 144 was the only one to express constitutively the IL-2R beta chain and IL-4R, whereas the NK-like YT cells express only IL-2R beta. The two other cell lines in this study, PEER and HSB2, are derived from T lymphocytes, and express neither IL-4R, IL-2R beta, nor IL-2R alpha unless stimulated. We report here that those receptors that are constitutively expressed, i.e., IL-2R beta on YT cells and IL-2R beta or IL-4R on MLA cells, are down-regulated by stimulation with PHA + PMA. In contrast, RNase protection experiments showed that PHA + PMA stimulation of T cell lines induces mRNA for all three receptors in PEER cells, and only IL-2R alpha and IL-4R in HSB-2. Thus each of these three receptors is subjected to a different regulation, which in addition varies depending on the lineage (or differentiation stage) of the cells. This was further supported by the finding that IL-1 alpha or TNF-alpha regulates these receptors differently. These two cytokines have no effect on IL-2R beta and IL-4R in MLA and YT, but induce IL-2R alpha in YT. In contrast, they do not induce either chains of the IL-2R in the T cell lines PEER or HSB-2, but TNF induces IL-4R mRNA in HSB2 cells, and IL-1 does so in both cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)

ADP-ribosylation of the ras-related, GTP-binding protein RhoA inhibits lymphocyte-mediated cytotoxicity.

The Rho proteins are identified as a subgroup of the Ras superfamily of low molecular weight GTP-binding proteins. We have studied the expression of these proteins in human cytotoxic natural killer cells and found that RhoA is the most abundantly expressed member of the Rho family. The Rho proteins are specific substrates for ADP-ribosylation catalyzed by the C3 exoenzyme from Clostridium botulinum. We report here that introduction of recombinant C3 in electropermeabilized natural killer cells or in cytotoxic T lymphocytes resulted in a dose-dependent inhibition of their cytolytic function. Furthermore, a single substrate is efficiently ADP-ribosylated by C3 in extracts from cytotoxic cells. Biochemical analyses indicate that this substrate is RhoA, and subcellular fractionation experiments demonstrate that it is essentially present in the cytosol of the cells. Western blot analysis, however, revealed that a small proportion of the Rho protein can be found associated with the cell membrane as well as with the cytotoxic granules. These results indicate that the low molecular weight GTP-binding protein RhoA is present in cytotoxic lymphocytes and plays a critical role in cell-mediated cytotoxicity.

A hemopoietic specific gene encoding a small GTP binding protein is overexpressed during T cell activation.

We have isolated, from a human B cell line cDNA library, a cDNA (Gx) encoding a small G protein identical to rac 2, a member of the ras superfamily. Gx/rac 2 gene is expressed as a unique mRNA of 1,7 Kb in peripheral blood lymphocytes, in purified B and T cells, in thymus as well as in several B and T cell lines. It is not expressed in many other tissues analysed including liver, brain, lung, heart and kidney. Upon in vitro stimulation with phytohemagglutinin A, peripheral blood lymphocytes show a clear increase of the Gx/rac 2 mRNA after 6 hours; a 30-50 fold accumulation is reached at 24 hours and persists thereafter. Purified T lymphocytes exhibit a similar increase in Gx/rac 2 mRNA expression upon mitogenic stimulation. Therefore, the expression of the Gx/rac 2 gene appears to be restricted to cells of the hemopoietic lineage and to be strongly stimulated during T cell activation. Gx/rac 2 protein must fulfill a specific role in activated T cells that could provide a new model for studying the function of small G proteins.

Functional consequences of cAMP accumulation in human natural killer cells. Implications for IL-2 and IL-4 signal transduction.

To approach the mechanisms whereby IL-2 activates human NK cells, we have compared the effects of IL-4 and of Bt2cAMP on this activation. Both agents block completely the proliferation induced by IL-2 on highly purified CD3-negative human NK cells. We also report that the net LAK response of PBL is inhibited by IL-4 and cAMP. However, kinetics analysis showed that IL-4 appears to inhibit an early stage of IL-2-induced activation of NK cells. IL-4 does not affect the cytotoxicity of freshly isolated NK cells against the K562 target and is ineffective on IL-2-preactivated cells. In contrast, cAMP primarily blocks the lytic effector phase, whether cells have been cultured in IL-2 or not, and its effect appears independent of time of addition. These differences between the activity of IL-4 and cAMP suggested that cAMP was not directly involved in IL-4 signal transduction in human NK cells. Consistent with this interpretation, we did not observe any variation in the level of intracellular cAMP concentrations when NK cells were stimulated with IL-4, or when they are stimulated with IL-2 or IL-2 plus IL-4. In addition, we also demonstrate that NK cell cytotoxic activation induced by IL-2 is still demonstrable in the presence of Bt2cAMP under conditions in which NK cell proliferation is blocked. These results clearly indicate that the differentiative effect of IL-2 on NK cells is independent of cell proliferation. Furthermore, the p70-75 IL-2R-initiated signal transduction pathway that leads to increased cytotoxicity appears not to be susceptible to inhibition by cAMP in human NK cells.

Expression and regulation of interleukin-1 mRNA and interleukin-1 receptors in human B-cell lines.

Long-term human lymphoid B-cell lines have been described to produce a number of growth factors, including interleukin-1 (IL-1) which may be of importance in the autocrine growth regulation of these cells and may participate in their antigen presenting function. In this report, we have analyzed the production of IL-1 by 12 established B-cell lines at the level of mRNA expression. Among the 5 cell lines containing an IL-1 message, three expressed exclusively IL-1 alpha, one contained traces of IL-1 beta, and only one line contained both. The steady-state level of IL-1 alpha mRNA in these cells could be drastically increased by a short culture of the cells with the protein synthesis inhibitor cycloheximide or with PMA. PMA, however, induced a transient increase in mRNA which could be stabilized by Ca2+ ionophore. Furthermore, only constitutively produced IL-1 mRNA are increased by these compounds which do not induce de novo transcription of silent IL-1 genes in these lines. These data provide the basis for further investigations on the regulation of IL-1 mRNA expression in human B cells. In addition, we studied expression of IL-1 receptors in these lines and observed that only cells which produce IL-1, displayed IL-1 receptors at their surface, as detected by radiolabeled IL-1 binding experiments. These data strongly suggest an autocrine role for IL-1 in these lines. IL-1 mRNA and IL-1 receptors were reciprocally regulated by PMA, which increased IL-1 mRNA and lowered the number of receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

[C3 stimulates the proliferation of human pre-B Raji cells]. / Le C3 stimule la prolifération des cellules humaines pré-B de la lignée Raji.

In a defined medium in which transferrin (3 micrograms/ml) was the only source of exogenous proteins, Raji cells of the human pre-B lymphoblastoid cell line died within 48 h after forming polykaryons. The simple addition of purified C3 at a concentration equal to or higher than 3 micrograms/ml allowed Raji cells to divide. This preliminary report provides a defined system for studying the mitogenic effect of human C3 or C3 fragments upon proliferation of human B-cells lines.

Possible role of sialic acid and endogenous neuraminidase in T-cell proliferation.

The proliferation of T cells in MLR with strains C57Bl/6 and DBA2, mouse spleen and lymph node cells was increased when the inhibitors of endogenous neuraminidase was added in the culture. This is in favor of participation of this enzyme and implicitly of terminal sialic acid in the proliferation of T-cells. Ths possibility that the suppressive activity of T-cells might have been abolished is discussed.

3H-glucosamine and 3H-fucose incorporation by murine peritoneal macrophages "non-divided" during differentiation in vitro.

Normal murine peritoneal macrophages were maintained in culture for 24 hours and 6 days. These cells continuously incorporated 3H-glucosamine and 3H-fucose in their membrane components. For both precursors culture ages, these components may be considered to undergo turnover according to a given rhythm with a 24 hours doubling time. Treatment with neuraminidase did not modify the doubling time, although a slight increase of incorporation of radioactivity was observed. Variations of 3H-fucose and 3H-glucosamine incorporations were observed. Variations of 3H-fucose and 3H-glucosamine incorporations were observed between 24 hours old and 6 days old cultures: 6 days old cultures incorporated more glucosamine than 24 hours old cultures whereas no difference in fucose incorporation was observed between the two cultures.