Role of EGR-1 in thapsigargin-inducible apoptosis in the melanoma cell line A375-C6.

Induction of apoptosis by diverse exogenous signals is dependent on elevation of intracellular Ca2+. This process of cell death can be blocked by actinomycin D, indicating that it requires gene transcription events. To identify genes that are required for apoptosis, we used thapsigargin (TG), which inhibits endoplasmic reticulum-dependent Ca(2+)-ATPase and thereby increases cytosolic Ca2+. Exposure to TG led to induction of the zinc finger transcription factor, EGR-1, and apoptosis in human melanoma cells, A375-C6. To determine the functional relevance of EGR-1 expression in TG-inducible apoptosis, we employed a dominant negative mutant which functionally competes with EGR-1 in these cells. Interestingly, the dominant negative mutant inhibited TG-inducible apoptosis. Consistent with this observation, an antisense oligomer directed against Egr-1 also led to a diminution of the number of cells that undergo TG-inducible apoptosis. These results suggest a novel regulatory role for EGR-1 in mediating apoptosis that is induced by intracellular Ca2+ elevation. We have previously shown that in these melanoma cells, EGR-1 acts to inhibit the growth arresting action of interleukin-1. Together, these results imply that EGR-1 plays inducer-specific roles in growth control.

A novel repressor, par-4, modulates transcription and growth suppression functions of the Wilms' tumor suppressor WT1.

The tumor suppressor WT1 represses and activates transcription. The loss and/or imbalance of the dual transcriptional activity of WT1 may contribute to Wilms' tumor. In this study, we identified par-4 (for prostate apoptosis response) as a WT1-interacting protein that itself functions as a transcriptional repressor. par-4 contains a putative leucine zipper domain and is specifically upregulated during apoptosis of prostate cells (S. F. Sells, D. P. Wood, Jr., S. S. Joshi-Barve, S. Muthukkumar, R. J. Jacob, S. A. Crist, S. Humphreys, and V. M. Rangnekar, Cell Growth Differ. 5:457-466, 1994). The leucine repeat domain of par-4 was shown to interact with the zinc finger DNA binding domain of WT1. Immunoprecipitation-Western blot (immunoblot) analyses demonstrated in vivo WT1-par-4 interactions. par-4 was ubiquitously expressed, and the protein was found in both the nucleus and the cytoplasm. Functionally, par-4 inhibited transcription activated by WT1, but not by the related protein EGR1. Inhibition of WT1-mediated transcription was dependent on the domain of par-4 that mediates its physical association with WT1. In addition, par-4 augmented WT1-mediated repression, possibly by contributing an additional repression domain. Consistent with these results, par-4 functioned as a transcriptional repressor when brought to a promoter via a heterologous DNA binding domain. Significantly, par-4, but not a mutant unable to interact with WT1, rescued growth suppression caused by WT1. Thus, we identified a novel repressor that modulates transcription as well as growth suppression functions of WT1.

Expression and function of the leucine zipper protein Par-4 in apoptosis.

The prostate apoptosis response-4 (par-4) gene was identified by differential screening for genes that are upregulated when prostate cancer cells are induced to undergo apoptosis. The par-4 gene is induced by apoptotic signals but not by growth-arresting, necrotic, or growth-stimulatory signals. The deduced amino acid sequence of par-4 predicts a protein with a leucine zipper domain at its carboxy terminus. We have recently shown that the Par-4 protein binds, via its leucine zipper domain, to the zinc finger domain of Wilms' tumor protein WT1 (R. W. Johnstone et al., Mol. Cell. Biol. 16:6945-6956, 1996). In experiments aimed at determining the functional role of par-4 in apoptosis, an antisense par-4 oligomer abrogated par-4 expression and activator-driven apoptosis in rat prostate cancer cell line AT-3, suggesting that par-4 is required for apoptosis in these cells. Consistent with a functional role for par-4 in apoptosis, ectopic overexpression of par-4 in prostate cancer cell line PC-3 and melanoma cell line A375-C6 conferred supersensitivity to apoptotic stimuli. Transfection studies with deletion mutants of Par-4 revealed that full-length Par-4, but not mutants that lacked the leucine zipper domain of Par-4, conferred enhanced sensitivity to apoptotic stimuli. Most importantly, ectopic coexpression of the leucine zipper domain of Par-4 inhibited the ability of Par-4 to enhance apoptosis. Finally, ectopic expression of WT1 attenuated apoptosis, and coexpression of Par-4 but not a leucine zipperless mutant of Par-4 rescued the cells from the antiapoptotic effect of WT1. These findings suggest that the leucine zipper domain is required for the Par-4 protein to function in apoptosis.

Activation of syk in an immature B cell line does not require lyn activity.

BKS-2 is an immature B cell lymphoma that undergoes apoptotic cell death when signaled via its surface IgM receptor. To study the signaling components of surface IgM mediated apoptosis in B lymphoma cells, we generated mutants of BKS-2 that were resistant to anti-IgM induced apoptosis. One mutant cell line, 1.B5, did not undergo apoptotic cell death upon treatment with anti-IgM antibodies and also did not exhibit elevation of intracellular Ca2+ in response to cross-linking of surface IgM. This appeared to be due to a defect in protein tyrosine kinase (PTK) activity since fewer proteins were tyrosine phosphorylated in the mutant cells stimulated with anti-IgM when compared to wild type BKS-2. Subsequently, we showed that protein tyrosine kinases lyn and blk were inducibly tyrosine phosphorylated in the wild type BKS-2 but not in 1.B5 mutant cells in response to anti-IgM. Also the kinase activity of lyn was elevated in the wild type but not in mutant cells upon triggering through surface IgM. Furthermore, tyrosine phosphorylation of CD19, a known substrate of lyn, was inducible in anti-IgM stimulated BKS-2 cells but severely reduced in 1.B5 cells. In contrast, kinase activity of another src kinase, blk, was increased on anti-IgM stimulation in both wild type and mutant cells. Surprisingly, syk, a non-src protein tyrosine kinase important for surface IgM mediated signaling, was tyrosine phosphorylated in the lyn deficient mutant cells as well as in the wild type BKS-2 cells. Furthermore, anti-IgM induced increase in kinase activity of syk was similar in the mutant and wild type cells. Thus, in contrast to other studies that propose syk to be a downstream target of src family kinases, syk may act upstream of lyn in immature B cells. Consistent with a functional syk, its target, phospholipase gamma2 (PLC-gamma2) was normally tyrosine phosphorylated in mutant cells.

Rapamycin, a potent immunosuppressive drug, causes programmed cell death in B lymphoma cells.

Rapamycin, a potent immunosuppressive drug that prevents rejection of organ transplants in many animals, caused profound growth inhibition in an immature B cell lymphoma, BKS-2, at very low concentrations (2 ng/ml). Similar growth inhibition was also observed in a series of B cell lymphomas (i.e., L1.2, NFS.1.1, and WEHI-279) as well as in thymoma cells. The cell death induced by rapamycin in BKS-2 lymphoma was found to be via programmed cell death, or apoptosis. In contrast to rapamycin, neither FK506 nor CsA affected the normal growth of these cells. FK506, but not CsA antagonized the effect of rapamycin and rescued the BKS-2 cells from undergoing apoptosis. Further, suboptimal concentrations of anti-IgM antibodies and rapamycin acted synergistically in causing the growth inhibition of BKS-2 cells and this inhibitory effect was also completely reversed by FK506. Thus, rapamycin appeared to inhibit lymphoma growth by binding to FK506 binding protein. These results indicate that rapamycin should be evaluated as an effective immunosuppressive therapeutic agent to prevent the incidence of lymphoma after transplantations.

Effect of cyclosporin A on murine experimental salmonellosis.

Investigations were undertaken to evaluate the effect of cyclosporin A (CyA) on primary infection of mice with Salmonella typhimurium. Further, its effect on delayed-type hypersensitivity (DTH) and antibody response to porin, an outer membrane protein of S. typhimurium, has been characterized under different CyA regimen. When mice were infected i.p. with 5 x 10(4) live medium virulent organisms, the bacterial growth increased by about 2 log10 in CyA treated mice. CyA administered daily for 2 weeks following immunization with porin caused profound suppression of DTH and both IgM and IgG antibody responses. Given a single dose of CyA 24 h before DTH elicitation, the drug had a marked suppressive effect on DTH, but not on the antibody response to porin. Treatment on days 0-5 following immunization had no effect on the manifestation of the DTH response nor on the anti-porin IgM response whereas an enhanced level of IgG was observed in this group.

Involvement of CD5 in Th1 and Th2 contact-mediated rescue of anti-mu and ionomycin induced growth inhibition in a B cell lymphoma.

Cross-linking of membrane Ig receptors by anti-mu antibodies (Ab) or treatment with ionomycin induced complete growth arrest and subsequent apoptotic cell death in an immature B cell lymphoma, BKS-2. The growth-inhibitory signals delivered by anti-mu and ionomycin were overcome by anti-CD3-activated Th2 clones D10.G4 and F1 and by Th1 cell clone S53. In this report the Th-mediated growth reversal in BKS-2 cells was shown to require contact-dependent interactions when the inhibition was caused by immobilized anti-mu or ionomycin. Th2 cells in transwells (lymphokines) failed to protect BKS-2 cells from the growth-inhibitory effect of immobilized anti-mu or ionomycin. Monoclonal antibodies to CD5 or CD40 ligands on activated Th cells partially inhibited the Th2 contact-dependent growth reversal of BKS-2 cells whereas simultaneous addition of both antibodies effectively prevented the delivery of contact-mediated growth signal. In contrast, anti-class I or class II Ab did not affect Th cell mediated growth reversal of BKS-2 cells. These data demonstrated that noncognate physical interaction with Th cells was essential for the recovery of BKS-2 cells when the latter were growth-arrested by strong inhibitory stimuli such as immobilized anti-mu and ionomycin. Further CD5 as well as CD40 ligands on Th cells are important for signal transduction in this type of T-B interaction.

Elevation of cytosolic calcium is sufficient to induce growth inhibition in a B cell lymphoma.

Recently, we have described that anti-IgM antibodies profoundly inhibited the growth of BKS-2, an immature B cell lymphoma. In this report, we demonstrated that ionomycin alone at very low concentrations (20 nM) inhibited the growth of BKS-2 cells completely. The levels of intracellular Ca2+ induced by the inhibitory concentrations of ionomycin were comparable to those in anti-IgM-treated cells. The growth inhibition caused by ionomycin was reversed by phorbol 12-myristate 13-acetate and lipopolysaccharide. In addition, the immunosuppressants, cyclosporin A and FK506 conferred significant protection from the negative signal induced by ionomycin. However, either cyclosporin A, FK506 or lipopolysaccharide was not found to have direct effect on ionomycin-induced Ca2+ mobilization in BKS-2 cells. Also, ionomycin augmented the anti-IgM-induced growth arrest in these cells. Furthermore, BKS-2 cells that were exposed to anti-IgM or ionomycin underwent apoptosis as characterized by DNA fragmentation. Thus, the characteristics of growth inhibition induced by ionomycin and anti-IgM appeared to be similar in that phorbol 12-myristate 13-acetate, lipopolysaccharide, cyclosporin A and FK506 caused significant reversal from such negative signals and both ionomycin and anti-IgM induced apoptosis in these cells. Altogether, these results showed that the elevation of intracellular Ca2+ alone was sufficient to inhibit the growth of some B lymphoma cells.

Cyclosporin A blocks surface IgM-mediated growth inhibition in an immature B lymphoma, BKS-2.

Cyclosporin A (CSA) is an immunosuppressive drug, which blocks selective activation pathways in T and B cells. Antigen receptor-mediated signaling events have been shown to be very sensitive to CSA. Signaling through the surface IgM receptor had been shown to induce growth inhibition in immature B cell lymphoma cells. In this report, we demonstrate that CSA caused significant reversal of growth inhibition induced by an anti-mu antibody in an immature B lymphoma, BKS-2. Time-course experiments indicated that CSA was completely effective when added as late as 4 h after the addition of ligand. CSA was also found to have no direct effect on anti-mu-induced Ca2+ elevation. These results suggest that the likely target for CSA lies downstream from the initial generation of second messengers, such as Ca2+.

Detection of porin antigen in serum for early diagnosis of mouse infections with Salmonella typhimurium.

The monoclonal antibodies to porin, an outer membrane protein isolated from Salmonella typhimurium and sandwich enzyme linked immunosorbent assay (ELISA) has made possible the detection of porin from sera of S. typhimurium-infected mice. The specificity of the monoclonal antibodies was ascertained based on their cross-reactivity with porins isolated from S. typhi, Shigella flexneri and Escherichia coli and lipopolysaccharide (LPS) of S. typhimurium and E. coli. Serum samples were found to be positive for porin as early as 3 days after intravenous and 5 days after oral infection. In addition, a positive correlation was observed between the bacterial load and the concentration of porin detected in the sera. On the other hand, analysis of sera for anti-porin antibody showed diametrically opposite time kinetics with antigenaemia. These results indicate that porin accumulates in the serum of infected mice much earlier than the appearance of antibodies to porin. Thus detection of porin holds promise for early diagnosis of typhoid.

Mechanism of protective immunity induced by porin-lipopolysaccharide against murine salmonellosis.

Investigations were undertaken to characterize the protective immunity induced by porin-lipopolysaccharide (LPS) against Salmonella typhimurium infection in mice. Mice immunized with porin-LPS showed higher levels of antiporin immunoglobulin G than mice which received porin alone. Further, T cells from porin-LPS-immunized mice showed an augmented proliferative response to porin in vitro compared with the response of T cells from porin-injected animals. The passive transfer of anti-LPS antibodies conferred significant protection (17%), while antiporin serum failed to protect mice against lethal challenge, indicating the protective ability of anti-LPS antibodies. However, the transfer of serum obtained from porin-LPS-immunized mice resulted in better protection (30%) than did anti-LPS or antiporin antibodies alone. In contrast to LPS, monophosphoryl lipid A completely failed to induce protection against lethal infection. However, comparable to the effect of LPS, injection of porin with monophosphoryl lipid A enhanced antibody response and the protective ability of porin (81.25%). The transfer of T cells from porin-LPS-immunized mice provided higher levels of protection (47%) against lethal challenge than did T cells from porin-immunized mice (23%). The combination of T cells and serum from porin-immunized mice transferred 36% protection. However, a combination of T cells and serum from porin-LPS-immunized mice conferred the highest level of protection (92%), which was reflected by the number of survivors (100%) in the porin-LPS-immunized group. These results demonstrate that besides the protective effect of anti-LPS antibodies, the ability of LPS to augment humoral and cell-mediated immune responses to porin confers effective protection against Salmonella infection.

The ability of B cells and dendritic cells to present antigen increases during ontogeny.

The immune response to polysaccharide (PS) Ags in mice is delayed during ontogeny even when administered in a thymus-dependent (TD) form. In this study, Neisseria meningitidis group C PS-tetanus toxoid conjugate (MCPS-TT) vaccine was used to examine whether the delay in the development of Ab responses to TD PS conjugate vaccines in neonatal mice is due to defective Ag presentation. The results show that B cells and dendritic cells (DC) from 3- and 7-day-old mice were severely defective in presenting TT and MCPS-TT to Ag-specific T cell clones. The ability of these cells to present Ag reaches adult levels by 4 wk. The development of anti-MCPS and anti-TT Abs in neonatal mice parallels the functional ability of their APC to present Ag. DC from neonatal mice expressed very low levels of MHC class II, costimulatory molecules B7.1, B7.2, and CD11c but high levels of monocyte-specific markers F4/80 and CD11b and granulocyte marker, Ly6G. Significant changes in the expression of these markers were observed as the age of the mice increased. MHC class II, B7.1 and B7.2, and CD11c all increased with age, reaching adult levels between 3 and 4 wk, concurrent with the function of APC. These results demonstrate that one reason neonates fail to produce high titers of anti-PS Abs even when immunized in a TD form is that their B cells and DC are not fully functional.

Activation of lyn, blk, and btk but not syk in CD72-stimulated B lymphocytes.

CD72 is a B cell-specific glycoprotein that has been shown to be important for activation of mature B cells. Previously we showed that some of the early signaling events, such as calcium mobilization and phospholipase-gamma activation, were similar in B cell Ag receptor (BCR)- and CD72-stimulated B cells and that BCR- but not CD72-mediated early signaling events were blocked by protein kinase A activation. The present report shows that CD72 ligation induces a variety of tyrosine-phosphorylated proteins, most of which were of the same molecular mass as those seen in anti-IgM-treated B cells, except for a 72-kDa protein. Further analysis showed that the tyrosine kinases lyn and blk were activated in CD72-ligated B cells. Interestingly, the non-src kinase syk was not activated in CD72-stimulated cells whereas the tec family kinase btk was activated in both CD72- and BCR-stimulated B cells. Furthermore, B cells from xid mice were unresponsive to CD72-induced proliferation, indicating an essential role for btk in CD72-induced signaling events. Surprisingly, tyrosine phosphorylation of phospholipase C-gamma2 was normal in CD72-stimulated cells in spite of a lack of activation of syk. Furthermore, B cell proliferation through CD72 was blocked by the immunosuppressive agents cyclosporin A and FK506, indicating the important role for Ca2+-regulated activation events similar to BCR-stimulated cells. We propose that btk can substitute for syk in inducing phospholipase C-gamma2 tyrosine phosphorylation and initiating calcium mobilization in CD72-stimulated B lymphocytes.

Interleukin-1 induces growth arrest by hypophosphorylation of the retinoblastoma susceptibility gene product RB.

Interleukin-1 (IL-1) causes G0/G1 phase growth arrest in human melanoma cells, A375-C6. Because hypophosphorylation of the retinoblastoma susceptibility gene product, RB, is one of the key events responsible for G0/G1 phase growth arrest, we investigated whether IL-1 altered the phosphorylation status of RB protein in these cells. Exposure to IL-1 caused a time-dependent increase in hypophosphorylated RB that correlated with an accumulation of cells arrested in the G0/G1 phase. The ability of IL-1 to cause hypophosphorylation of RB and growth arrest was abrogated by the SV40 large T antigen, which binds preferentially to hypophosphorylated RB, but not by the K1 mutant of the T antigen, which is defective in binding to RB. Furthermore, the cells were protected from IL-1-inducible growth inhibition by ectopic expression of dominant-negative mutants of the Rb gene, or the transcription factor E2F-1, which is a downstream target of RB. These results suggest that hypophosphorylated RB mediates the growth arrest induced by IL-1.

Suppression by Trypanosoma brucei rhodesiense of the capacities of human T lymphocytes to express interleukin-2 receptors and proliferate after mitogenic stimulation.

We studied the suppressive effects induced in phytohemagglutinin (PHA)-stimulated human peripheral blood mononuclear cells (PBMC) by purified blood forms of Trypanosoma brucei rhodesiense. The parasite was found to markedly impair lymphocyte proliferation (measured in terms of [3H]thymidine incorporation). The extent of this effect increased with parasite concentration and was not due to mitogen absorption, depletion of medium nutrients, or PBMC killing by the parasite. Significant reductions in interleukin-2 receptor (IL-2R) expression, determined by flow cytometric analysis, were also observed in PHA-stimulated PBMC cultured in the presence of T. b. rhodesiense as evidenced by marked decreases in the surface density of the receptor. Concomitant decreases in the percentage of IL-2R+ cells were recorded in approximately half of the experiments. A discrete, dimly stained subpopulation of IL-2R+ cells were consistently demonstrable whether or not a reduction in the percentage of IL-2R+ cells occurred. Living, but not glutaraldehyde-fixed, parasites suppressed IL-2R expression. In kinetic studies, a low but reproducible level of suppression of IL-2R was demonstrable as early as 6 h after PHA stimulation; the extent of this effect became considerably more pronounced as additional culture time elapsed. Levels of IL-2 biological activity in cocultures of T. b. rhodesiense with PHA-stimulated PBMC were comparable with or higher than those present in control cultures lacking the parasite. Therefore, insufficient levels of this cytokine would be an unlikely explanation for the noted suppression of IL-2R expression and lymphoproliferation. These effects of T. b. rhodesiense could represent an important component of the mechanism by which immunosuppression develops in African sleeping sickness.

Role of Egr-1 gene expression in B cell receptor-induced apoptosis in an immature B cell lymphoma.

Ligation of B cell receptor (BCR) on BKS-2, an immature B cell lymphoma by anti-IgM antibodies (Ab) caused apoptosis. Here we report that signaling through B cell receptor in wild type BKS-2 cells down-regulated the expression of Egr-1, a zinc finger-containing transcription factor. A reduction in the level of Egr-1 mRNA could be demonstrated as early as 30 min after the ligation of BCR on BKS-2 cells. Immunocytochemical and Western blot analysis revealed that the expression of EGR-1 protein was also inhibited by anti-IgM treatment. Antisense oligonucleotides to Egr-1 caused growth inhibition and apoptosis in BKS-2 cells, suggesting that expression of Egr-1 is important for the survival of these B lymphoma cells. In contrast to wild type BKS-2 cells, the mutant 1. B5 cell line, which is refractory to B cell receptor-mediated growth-inhibitory signals, showed an increased expression of Egr-1 upon treatment with anti-IgM. These results implicate a role for Egr-1 in blocking B cell receptor-mediated apoptosis in immature B cells.

Modulation of anti-IgM-induced B cell apoptosis by Bcl-xL and CD40 in WEHI-231 cells. Dissociation from cell cycle arrest and dependence on the avidity of the antibody-IgM receptor interaction.

The demise of B cell progenitors expressing functional IgM receptors for self appears to be the main mechanism by which B cell tolerance is accomplished. The genetic mechanisms that regulate the death process during this critical step of B cell development are still poorly understood. We have studied the regulation of apoptosis in WEHI-231 lymphoma cells after treatment with a panel of anti-IgM mAbs as an in vitro model of clonal B cell deletion. We showed that a product of bcl-x, Bcl-xL, can inhibit anti-IgM-induced apoptosis but not cell cycle arrest in a dose-dependent manner. Bcl-xL was efficient in protecting B cells from low but not high avidity anti-IgM mAbs. In contrast to that observed with Bcl-xL, CD40 stimulation was efficient in inhibiting both cell cycle arrest and apoptosis after IgM cross-linking regardless of the binding avidity of the anti-IgM Ab. Moreover, activation through IgM receptors but not CD40 induced up-regulation followed by rapid down-modulation of Bcl-xL. Thus, the capacity of Bcl-xL to modulate anti-IgM-induced apoptosis in WEHI-231 cells is highly dependent on the avidity of the Ab-IgM receptor interaction.

Early growth response-1-dependent apoptosis is mediated by p53.

The early growth response-1 (EGR-1) protein is an anti-proliferative signal for certain tumor cells and is required for apoptosis induced by stimuli that elevate intracellular Ca2+. We present evidence that EGR-1 transactivates the promoter of the p53 gene and up-regulates p53 RNA and protein levels. Inhibition of p53 function with dominant-negative p53 mutants abrogates EGR-1-dependent apoptosis. These findings establish a direct functional link between EGR-1 and the p53-mediated cell death pathway and suggest that mutant forms of p53 in tumor cells may provide resistance to the anti-proliferative effects of EGR-1.

EGR-1 induction is required for maximal radiosensitivity in A375-C6 melanoma cells.

Exposure to ionizing radiation leads to induction of the immediate-early gene, early growth response-1 (Egr-1). Previous studies have suggested distinct cell type- and inducer-specific roles for EGR-1 protein in cellular growth inhibition. The present study was undertaken to determine the functional role of EGR-1 in growth inhibition caused by exposure of tumor cells to ionizing radiation. Exposure to ionizing radiation caused induction of EGR-1 protein in human melanoma cells A375-C6. Inhibition of either the function of EGR-1 protein by stable transfection with a dominant-negative mutant or the expression of EGR-1 by transient transfection with an antisense oligomer resulted in a diminished growth-inhibitory response to ionizing radiation. Because previous studies have suggested that mutations in the tumor-suppressor gene p53 confer radio-resistance, we examined the p53 status of A375-C6 cells. Interestingly, both the parental and the transfected A375-C6 cells showed trisomy for wild-type p53 alleles. Exposure to ionizing radiation resulted in induction of p53 protein that localized to the nucleus in A375-C6 cells. These data suggest that inhibition of EGR-1 function confers radio resistance despite the induction of wild-type nuclear p53. Thus, EGR-1 is required for the growth-inhibitory response to ionizing radiation in A375-C6 cells.

Ionizing radiation-inducible apoptosis in the absence of p53 linked to transcription factor EGR-1.

The tumor suppressor protein p53 is a pivotal regulator of apoptosis, and prostate cancer cells that lack p53 protein are moderately resistant to apoptotic death by ionizing radiation. Genes encoding the transcription factor early growth response-1 (EGR-1) and cytokine tumor necrosis factor-alpha (TNF-alpha) were induced upon irradiation of prostate cancer cells, and inhibition of EGR-1 function resulted in abrogation of both TNF-alpha induction and apoptosis. Induction of the TNF-alpha gene by ionizing radiation and EGR-1 was mediated via a GC-rich EGR-1-binding motif in the TNF-alpha promoter. Because TNF-alpha induces apoptosis in prostate cancer cells, these findings suggest that, in the absence of p53, ionizing radiation-inducible apoptosis is mediated by EGR-1 via TNF-alpha transactivation.