Role for c-myc in activation-induced apoptotic cell death in T cell hybridomas.

Immature T cells and some T cell hybridomas undergo apoptotic cell death when activated through the T cell receptor complex, a phenomenon that is probably related to antigen induced negative selection of developing T cells. This activation-induced apoptosis depends on active protein and RNA synthesis in the dying cells, although none of the genes required for this process have previously been identified. Antisense oligonucleotides corresponding to c-myc block the constitutive expression of c-Myc protein in T cell hybridomas and interfere with all aspects of activation-induced apoptosis without affecting lymphokine production in these cells. These data indicate that c-myc expression is a necessary component of activation-induced apoptosis.

Role of bud6p and tea1p in the interaction between actin and microtubules for the establishment of cell polarity in fission yeast.

BACKGROUND: In many cell types, microtubules are thought to direct the spatial distribution of F-actin in cell polarity. Schizosaccharomyces pombe cells exhibit a regulated program of polarized cell growth: after cell division, they grow first in a monopolar manner at the old end, and in G2 phase, initiate growth at the previous cell division site (the new end). The role of microtubule ends in cell polarity is highlighted by the finding that the cell polarity factor, tea1p, is present on microtubule plus ends and cell tips [1]. RESULTS: Here, we characterize S. pombe bud6p/fat1p, a homolog of S. cerevisiae Bud6/Aip3. bud6Delta mutant cells have a specific defect in the efficient initiation of growth at the new end and like tea1Delta cells, form T-shaped cells in a cdc11 background. Bud6-GFP localizes to both cell tips and the cytokinesis ring. Maintenance of cell tip localization is dependent upon actin but not microtubules. Bud6-GFP localization is tea1p dependent, and tea1p localization is not bud6p dependent. tea1Delta and bud6Delta cells generally grow in a monopolar manner but exhibit different growth patterns. tea1(Delta)bud6Delta mutants resemble tea1Delta mutants. Tea1p and bud6p coimmunoprecipitate and comigrate in large complexes. CONCLUSIONS: Our studies show that tea1p (a microtubule end-associated factor) and bud6p (an actin-associated factor) function in a common pathway, with bud6p downstream of tea1p. To our knowledge, bud6p is the first protein shown to interact physically with tea1p. These studies delineate a pathway for how microtubule plus ends function to polarize the actin cytoskeleton through actin-associated polarity factors.

Gamma interferon induces monocytoid differentiation in the HL-60 cell line.

We investigated the ability of purified, recombinant DNA-derived interferons (IFN) to induce phenotypic changes in cells of the HL-60 promyelocytic leukemia cell line. Changes in cell surface markers detected by monoclonal antibodies as well as morphologic, histochemical, and functional changes were monitored. We found that gamma-IFN, but not alpha- or beta-IFN, induced the expression of antigens characteristic of monocytes and granulocytes (AML-2-23, 63D3, and 61D3), as well as changes in morphology consistent with monocytoid differentiation. These included induction of alpha-naphthyl acetate esterase, increased cell size, and a decrease in azurophilic granules. The gamma-IFN dose dependency and time course of the effect on antigen expression suggest that de novo protein synthesis was induced by gamma-IFN. The activity of gamma-IFN and of mixed-lymphocyte culture supernatant was blocked by a monoclonal antibody to gamma-IFN. Significant augmentation in the ability of the HL-60 cells to mediate antibody-dependent cellular cytotoxicity was induced by gamma-IFN. These findings suggest that gamma-IFN plays a role in the regulation of hematopoiesis.

Microfilament-disrupting agents prevent the formation of apoptotic bodies in tumor cells undergoing apoptosis.

Apoptosis is a form of cell death in which the cell "participates," such that metabolic energy and often protein synthesis are required for the death to occur. Once begun, the process of apoptosis proceeds in an ordered fashion. In the earliest phase DNA fragmentation occurs, accompanied by cell shrinkage and dilation of the endoplasmic reticulum. This is followed by cell fragmentation with the formation of sealed membrane vesicles, termed apoptotic bodies. In the present study we have demonstrated that the fungal metabolite cytochalasin B inhibits cell fragmentation and the formation of apoptotic bodies, probably by its ability to interfere with actin polymerization. This effect was seen when HL-60 cells were pretreated with cytochalasin B and then exposed to one of a number of apoptosis-inducing agents, including UV irradiation, camptothecin, aphidocholin, or PMA plus ionomycin. The observed effect was not peculiar to HL-60 cells, inasmuch as it was also seen for both Molt-4 and U-937 cell lines. Cytochalasin B had no effect on DNA fragmentation occurring in the earliest stage of apoptosis, and it appeared to have no inhibitory effects on nuclear fragmentation. Staurosporin had an effect similar to that seen with cytochalasin B, probably due to its ability to inhibit protein kinase C, which is a known potentiator of microfilament assembly. These data demonstrate that microfilament assembly is necessary for the formation of apoptotic bodies in the later stages of the apoptotic process.

Common and idiosyncratic patterns of cytokine gene expression by Epstein-Barr virus transformed human B cell lines.

Epstein-Barr virus (EBV) transformed human B cells proliferate indefinitely in vitro, and it has been proposed that cytokine-mediated autocrine loops contribute to the maintenance of the lymphoblastoid phenotype. We used a novel multiprobe RNase protection assay to quantify cytokine mRNA species expressed by EBV-transformed lymphoblastoid cell lines (LCL), derived either by the transformation of B cells with B95-8 or wild-type EBV or by the in vitro outgrowth of EBV-associated B cell lymphomas to identify cytokines that are commonly expressed in all LCL and thus more likely to be essential for immortalization of B cells. All 16 LCL expressed high levels of tumor necrosis factor (TNF)alpha, TNFbeta, and transforming growth factor (TGF)beta1 mRNA, while interleukin (IL)-10 transcripts were detected in most LCL but at a lower level. Expression of IL-1alpha, IL-1beta, IL-6, IL-12p35, IL-12p40, IL-13 and IFNgamma mRNA was variable among the LCL tested. Granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-2, IL-4, and IL-5 mRNA were undetectable in all LCL. Furthermore, we found that IL-10, TNFalpha, and TNFbeta mRNA were induced in EBV-negative B cell lines after infection with EBV. These data define common versus idiosyncratic patterns of cytokine expression by LCL and, in the former case, such cytokines as TNFalpha, TNFbeta, and IL-10 emerge as strong candidates that are essential for the autocrine regulation of EBV-immortalized B cells.

The induction of apoptosis by chemotherapeutic agents occurs in all phases of the cell cycle.

A1.1 T-cell hybridoma cells exposed to either actinomycin D (1 microgram/ml), camptothecin (200 ng/ml) or aphidicolin (10 micrograms/ml) for 16 hrs at 37 degrees C die via apoptosis. The cell death was independent of RNA synthesis, in contrast to previous data reported for other forms of apoptosis in murine lymphocyte cells and their derived lines. Each of the three agents described appeared to induce death in all phases of the cell cycle in asynchronously proliferating cells. G1 cells appeared to be more susceptible to the effects of camptothecin and contrasts with other reports which detail its selectivity for S and G2 phase cells. This might indicate that cells are progressing into S phase before dying or, alternatively, cells may indeed be dying in G1. When elutriated synchronised cells were exposed to each of the three cytotoxic agents cell death occurred in all phases of the cell cycle. In view of the fact that G1 and S phase cells did not cycle to any appreciable extent during drug exposure, it was likely that ensuing death, occurred specifically from these phases. G2/M cells, however, moved rapidly into G1 in the presence of each drug, thus making it difficult to determine whether G2/M cells were capable of undergoing drug-induced apoptosis. To overcome this problem, nocodazole (50 ng/ml) was used to block asynchronous cells in M phase. When these cells were exposed to actinomycin D, aphidicolin or camptothecin, cell death ensued via apoptosis.

Dynamics of asexual transmission of a mitochondrial plasmid in Cryphonectria parasitica.

In the chestnut blight fungus Cryphonectria parasitica, as in most fungi, little is known about the efficiency of the asexual transmission of optional mitochondrial plasmids, vertically through conidia, and horizontally through hyphal anastomoses. In this paper, we show that pCRY1, a circular mitochondrial plasmid, is transmitted vertically with 100%-efficiency through conidia. Moreover, the plasmid is transmitted horizontally through hyphal contact from donor strains to vegetatively compatible and most incompatible strains. An allelic difference between the donor and recipient strain, at only one of the five nuclear incompatibility genes that were tested strongly inhibited, but did not absolutely prevent, the transfer of pCRY1 through hyphal fusions. In contrast, allelic differences in any one or several of the other four heterokaryon-compatibility loci suppressed the transmission of the plasmid only partially or not at all. The plasmid was also transmitted among incompatible strains by protoplast fusion without the concomitant transfer of mitochondrial DNA (mtDNA). A comparison of plasmid-bearing with plasmid-free isogenic strains revealed that pCRY1 significantly diminishes the pathogenic potency of some strains of the fungus, but does not affect the virulence of others. Collectively, the observations indicate that the introduction of deleterious mitochondrial genetic elements into natural populations may be a means for managing fungal pathogens.

Apoptosis induced by HIV infection in H9 T cells is blocked by ICE-family protease inhibition but not by a Fas(CD95) antagonist.

Infection of human CD4-positive T lymphocytes by human immunodeficiency virus type 1 (HIV-1) is thought to lead to death of infected cells by apoptosis, although one recent report questions this conclusion. Here we demonstrate that HIV-1-induced apoptosis of the H9 human T cell line is blocked by peptide inhibitors of IL-1 beta converting enzyme (ICE)-family proteases, but not by the antagonistic M3 anti-Fas Ab. Apoptosis occurred in all phases of the cell cycle, not selectively in G2 as a consequence of vpr-mediated cell cycle arrest. We conclude that apoptosis accounts for all cell death related to HIV-1 infection of the human CD4-positive cell line H9, requires an ICE-like protease but is not Fas mediated, and occurs in all phases of the cell cycle.

Activation-induced apoptosis in lymphoid systems.

Lymphocytes become activated when antigen receptors on the cell surface are cross-linked, or when they are exposed to agents that mimic this signal. Although such activation is usually associated with the production of immune mediators (e.g. antibodies, cytokines) and entry into the cell cycle, it can alternatively lead to death via apoptosis. This activation-induced apoptosis was first observed in developing lymphocytes and has been proposed as a mechanism for negative selection, by which immature cells with potential for autoreactivity are eliminated from the maturation pathway. Activation-induced apoptosis has also been observed in normal, mature lymphocytes under some conditions, and this may account for the phenomena of peripheral deletion, in which mature T cells are eliminated upon exposure to high doses of antigen. It may also be an important mechanism whereby CD4+ T cells are depleted in HIV+ individuals. Although the phenomenon of activation-induced apoptosis is not understood, recent studies have begun to implicate specific signal transduction pathways and gene products in the process. Among the latter is the c-myc proto-oncogene, which paradoxically can play an essential role in several forms of apoptosis, including that induced by activation of lymphocytes.

Modulation of class I HLA antigens on HL-60 promyelocytic leukemia cells by serum-free medium: re-induction by gamma-IFN and 1,25-dihydroxyvitamin D3 (calcitriol).

During studies on the effect of different nutrient media on the growth and differentiation of the HL-60 promyelocytic leukemia cell line, we found that the density of the class I HLA antigens is profoundly decreased on cells cultured in a serum-free medium. The ability of recombinant DNA-derived interferons (IFN) and a number of myeloid differentiating agents to induce re-expression of class I HLA antigens and beta-2-m was therefore studied. All three classes (alpha, beta, and gamma) of IFN were capable of re-inducing HLA and beta-2-m, although gamma-IFN was more potent. Of a variety of chemical differentiating agents, only 1,25-dihydroxyvitamin D3 (calcitriol) was found to induce HLA class I antigens. These results suggest that HLA and beta-2-m are not necessarily constitutive cell surface proteins, but instead that their expression is highly inducible.