Fas antigen (CD95) in pure erythroid cell line AS-E2 is induced by interferon-gamma and tumor necrosis factor-alpha and potentiates apoptotic death

We investigated the expression of Fas antigen (CD95) in the pure erythroid cell line AS-E2 in the presence and absence of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha). TNF-alpha induced apoptosis in AS-E2 cells, whereas IFN-gamma did not. In culture containing no IFN-gamma or TNF-alpha, AS-E2 cells expressed little Fas antigen. However, IFN-gamma and IFN-gamma and TNF-alpha both induced expression of Fas antigen and its mRNA within 24 hours after the stimulation. When anti-Fas monoclonal antibody (IgM) was added to AS-E2 cells after the induction of Fas expression, AS-E2 cells underwent apoptosis as shown by the induction of DNA fragmentation. This apoptotic change was inhibited by an inhibitor of caspase-3-like proteases (Ac-DEVD-CHO) and an inhibitor of CED-3/ICE family proteases (Z-Asp-CH2-DCB) but not by an inhibitor of caspase-1-like proteases (Ac-YVAD-CHO), suggesting a role for caspase-3-like proteases in Fas-receptor signaling. Although AS-E2 cells expressed Fas ligand mRNA, treatment with ZB4, an antibody that inhibits Fas-mediated cell death, failed to suppress IFN-gamma- or TNF-alpha-mediated cytotoxicity. These findings suggest that the late erythroid progenitor cells are negatively regulated by IFN-gamma and TNF-alpha, both of which are capable of inducing functional Fas expression.     

Induction of apoptosis by norcantharidin in human colorectal carcinoma cell lines: involvement of the CD95 receptor/ligand

PURPOSE: Cantharidin, a natural toxin, is the active substance of mylabris and has antitumor effects in man. Norcantharidin, the demethylated analogue of cantharidin, has been used in the treatment of patients with primary hepatoma and those with leukopenia in China. The present study was designed to investigate whether norcantharidin exerts cytotoxic activity against colorectal cancer cells by inducing apoptosis and to examine the possible mechanism in the phenomenon. METHODS: Inhibition of proliferation of norcantharidin on Colo205, HT-29, and SW480 colorectal cancer cells was determined by the trypan blue dye exclusion test. Apoptosis of norcantharidin-treated cells was determined by morphological analysis, agarose gel DNA electrophoresis, and quantitated by flow cytometry after staining with propidium iodide. Cell cycle and the cell surface expression of the CD95/CD95 ligand were evaluated by flow cytometry. Caspase 8-like protease and protein phosphatase 1 and 2A activities were also analyzed. RESULTS: Treatment with norcantharidin of colorectal cancer cells not only inhibited cell proliferation, but also induced apoptosis. Norcantharidin induced apoptosis mainly in two phases: rapid apoptosis in S-phase cells and delayed apoptosis in G2/M arrested cells. Treatment with norcantharidin resulted in an upregulation of the CD95 receptor and CD95 ligand on the cell surface. Furthermore, stimulation with anti-CD95 monoclonal antibody (mAb) resulted in further induction of apoptosis after treatment with norcantharidin. In addition, the apoptosis-inducing effect of norcantharidin was almost completely inhibited by anti-CD95 ligand mAb. Norcantharidin-treated cells showed the activation of caspase 8. Both zVAD-FMK (a broad range caspase inhibitor) and IETD-FMK (a caspase-8 inhibitor) showed apparent inhibition of the apoptosis-inducing effect. Norcantharidin did not show an inhibitory effect on protein phosphatase. CONCLUSIONS: These results suggest that norcantharidin triggers apoptosis in colorectal cancer cell lines via the activation of the CD95 receptor/ligand system, and that this agent may be useful for developing new therapeutic regimens for the treatment of colorectal carcinoma.     

Tumour necrosis factor alpha potentiates ion secretion induced by histamine in a human intestinal epithelial cell line and in mouse colon: involvement of the phospholipase D pathway

BACKGROUND AND AIMS: Patients suffering from inflammatory bowel disease show increased levels of the mast cell products histamine and tumour necrosis factor alpha (TNF-alpha). Treating these patients with antibodies against TNF-alpha diminishes the symptoms of diarrhoea. In this study, the effect of TNF-alpha on ion secretion induced by the mast cell mediator histamine in HT29cl.19A cells and mouse distal colon was investigated and the possible second messengers involved were studied. METHODS: Electrophysiology of filter grown HT29cl.19A cells and isolated mouse distal colon was used to monitor the secretory response to histamine with and without prior exposure to TNF-alpha for 3-24 hours. Phospholipase D (PLD) activity and phosphatidic acid levels were analysed by 32P(i) labelling of HT29cl.19A cells. RESULTS: In both experimental systems TNF-alpha was found to potentiate ion secretion induced by histamine. Phospholipid analysis of HT29cl.19A cells revealed that histamine activates the PLD pathway. Furthermore, TNF-alpha pretreated cells were found to have decreased phosphatidic acid levels, the intermediate product of the PLD pathway, which indicates upregulation of the enzyme phosphatidic acid phosphatase. CONCLUSIONS: The mast cell products TNF-alpha and histamine synergistically stimulate ion secretion in intestinal epithelium via upregulation of the PLD pathway.     

Experimental infection in tumor necrosis factor alpha receptor, interferon gamma and interleukin 4 deficient mice by pathogenic Leptospira interrogans

Only recently, knockout mouse models were applied in studies on the pathogenesis of leptospirosis. Current data suggest an important role of innate immunity receptors and interferon gamma dependant cellular response on protection. It is not clear, however, whether T helper cell polarization influences on outcome of leptospiral infection. We report findings of experimental infection of C57BL/6 (interferon gamma or tumor necrosis factor alpha receptor deficient) and BALB/c (interleukin 4 deficient) mice infected by pathogenic Leptospira interrogans serovar Copenhageni. Specific cytokine gene deficiency had no impact on outcome since all animals survived. TNFR knockout mice, however, exhibited more severe residual renal inflammation during convalescence thus suggesting this cytokine is important in early control of infection, protecting kidneys from relevant pathology.     

Interleukin 10 inhibits macrophage microbicidal activity by blocking the endogenous production of tumor necrosis factor alpha required as a costimulatory factor for interferon gamma-induced activation.

Interleukin 10 (IL-10) inhibits interferon gamma-induced macrophage activation for cytotoxicity against larvae of the human parasite Schistosoma mansoni by suppressing production of the toxic effector molecule nitric oxide (NO). In this study, the mechanism of IL-10 action was identified as inhibition of endogenous tumor necrosis factor alpha (TNF-alpha) production by interferon gamma-activated macrophages. TNF-alpha appears to serve as a cofactor for interferon gamma-mediated activation, since both schistosomulum killing and NO production were inhibited by anti-TNF-alpha antibody, whereas TNF-alpha alone was unable to stimulate these macrophage functions. IL-10 blocked TNF-alpha production by interferon gamma-treated macrophages at the levels of both protein and mRNA synthesis. Addition of exogenous TNF-alpha reversed IL-10-mediated suppression of macrophage cytotoxic activity as well as NO production. Likewise, addition of a macrophage-triggering agent (bacterial lipopolysaccharide or muramyl dipeptide), which induced the production of TNF-alpha, also reversed the suppressive effect of IL-10 on cytotoxic function. In contrast to IL-10, two other cytokines, IL-4 and transforming growth factor beta, which also inhibit macrophage activation for schistosomulum killing and NO production, did not substantially suppress endogenous TNF-alpha production. These results, therefore, describe a separate pathway by which macrophage microbicidal function is inhibited by the down-regulatory cytokine IL-10.     

Glucocorticoids inhibit activation-induced cell death (AICD) via direct DNA-dependent repression of the CD95 ligand gene by a glucocorticoid receptor dimer

Glucocorticoids (GCs) play an important role in the regulation of peripheral T-cell survival. Their molecular mechanism of action and the question of whether they have the ability to inhibit apoptosis in vivo, however, are not fully elucidated. Signal transduction through the glucocorticoid receptor (GR) is complex and involves different pathways. Therefore, we used mice with T-cell-specific inactivation of the GR as well as mice with a function-selective mutation in the GR to determine the signaling mechanism. Evidence is presented for a functional role of direct binding of the GR to 2 negative glucocorticoid regulatory elements (nGREs) in the CD95 (APO-1/Fas) ligand (L) promoter. Binding of GRs to these nGREs reduces activation-induced CD95L expression in T cells. These in vitro results are fully supported by data obtained in vivo. Administration of GCs to mice leads to inhibition of activation-induced cell death (AICD). Thus, GC-mediated inhibition of CD95L expression of activated T cells might contribute to the anti-inflammatory function of steroid drugs.     

Fas antigen expression on CD34+ human marrow cells is induced by interferon gamma and tumor necrosis factor alpha and potentiates cytokine-mediated hematopoietic suppression in vitro

Activation of Fas antigen, a cell surface receptor molecule, by its ligand results in transduction of a signal for cell death. The Fas system has been implicated in target cell recognition, clonal development of immune effector cells, and termination of the cellular immune response. Fas antigen expression on lymphocytes is regulated by interferon gamma (IFN gamma) and tumor necrosis factor alpha (TNF alpha), cytokines that also have inhibitory effects on hematopoiesis. We investigated Fas antigen expression on human marrow cells and the effects of Fas activation on hematopoiesis in vitro. Freshly isolated immature hematopoietic cells, as defined by the CD34 marker, did not express Fas antigen at levels detectable by fluorescent staining. CD34+ cells, which include progenitors and stem cells, showed low levels of Fas expression in culture, even in the presence of growth factors. Stimulation by TNF alpha and IFN gamma markedly increased Fas antigen expression on CD34+ cells. Anti-Fas antibody, which mimics the action of the putative ligand, enhanced IFN gamma- and TNF alpha-mediated suppression of colony formation by bone marrow (BM) in a dose-dependent manner. This effect did not require the presence of accessory cells. Colony formation from mature (CD34+ CD38+) and immature (CD34+ CD38-) progenitor cells and long-term culture initiating cells were susceptible to the inhibitory action of anti-Fas antibody in the presence of IFN gamma and TNF alpha. Apoptosis assays performed on total BM cells and CD34+ cells showed that anti-Fas antibody induced programmed cell death of CD34+ BM cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a tumor necrosis factor alpha (TNF-alpha) inhibitor: evidence of immunological cross-reactivity with the TNF receptor.

Previous studies have shown that urine of febrile patients contains a tumor necrosis factor alpha inhibiting activity (TNF-alpha Inh) when tested in a cytotoxicity assay using the tumor necrosis factor alpha (TNF-alpha)-susceptible cell line L929. In the present study, we investigated the relationship between the TNF-alpha Inh and a potential soluble form of the receptor, as the former has been shown to block TNF-alpha activities by binding to the ligand. We demonstrate that human TNF-alpha is affected to a greater extent than is murine TNF-alpha. This species specificity of the inhibitor correlates with the binding studies of TNF receptor interactions already reported. We raised a polyclonal antibody to TNF-alpha Inh that neutralizes its activity and does not recognize TNF-alpha. Solubilized cross-linked 125I-labeled TNF-alpha receptor complex could be immunoprecipitated by using either anti-TNF-alpha or anti-TNF-alpha Inh antibody, suggesting immunological cross-reactivity between the receptor and the inhibitor. By using fluorescein isothiocyanate-coupled TNF-alpha, it was possible to visualize by fluorescence-activated cell sorter analysis the TNF-alpha receptor on phytohemagglutinin/interleukin 2-activated T cells. A similar increase of immunofluorescence intensity of the activated T cells was observed by using anti-TNF-alpha Inh antibody revealed with a fluorescein isothiocyanate-coupled goat anti-rabbit IgG1 conjugate, suggesting that the TNF-alpha Inh is also expressed as a membrane protein. Taken together, our results suggest that the TNF-alpha Inh originally described might be a soluble form of the TNF receptor itself.     

Glucocorticoid receptor down-regulates c-Jun amino terminal kinases induced by tumor necrosis factor alpha in fetal rat hepatocyte primary cultures

The effect of dexamethasone on Jun N-terminal kinase (JNK) activity was assayed by using fetal hepatocytes in primary culture. The addition of tumor necrosis factor alpha (TNF-alpha) caused an increase in JNK in a dose- and time-dependent manner. We show that activation of JNK by this extracellular signal is inhibited by dexamethasone in a dose-dependent fashion. This inhibitory effect was observed in cells treated for 10 minutes with dexamethasone in the presence of protein phosphatase inhibitors such as orthovanadate or okadaic acid, or in cells previously treated with actinomycin D. Glucocorticoid receptor (GR) can be precipitated with the fusion protein, GST-c-Jun (1-79), bound to agarose beads. However, the inhibitory effect of glucocorticoids on JNK activity was also observed using ATF-2 as substrate. In addition, dexamethasone inhibits JNK phosphorylation induced by TNF-alpha. Finally, we show that GR can also be phosphorylated in tyrosine residues in response to TNF-alpha and epidermal growth factor (EGF) upon ligand-binding. Our results suggest that the anti-inflammatory effect of glucocorticoids on the inflammatory pathways induced by TNF-alpha can be explained, at least in part, by modulating JNK activity through a direct protein-protein interaction; the JNK phosphorylation and tyrosine-phosphorylation state of GR may be regulatory steps also involved in that effect.     

Differential regulation of rheumatoid synovial cell interleukin-12 production by tumor necrosis factor alpha and CD40 signals.

OBJECTIVE: To investigate the roles of tumor necrosis factor alpha(TNFalpha) and the CD40-CD154 interaction in interleukin-12 (IL-12) production by rheumatoid synovial cells (SC). METHODS: Levels of IL-12 (p40 and p70) in synovial tissue and culture supernatants of SC from patients with rheumatoid arthritis (RA), osteoarthritis (OA), and ankylosing spondylitis (AS) were assayed by enzyme-linked immunosorbent assay. Effects of anti-CD154 and anti-TNFalpha antibody on spontaneous and lipopolysaccharide (LPS)-stimulated IL-12 production by SC were examined. Effects of immobilized anti-CD3 treatment and depletion of CD4+ T cells on IL-12 production were also tested. CD154 expression by synovial T cells and intracellular IL-12 production during culture were analyzed by flow cytometry. RESULTS: IL-12 p40 and p70 levels in RA synovial tissue and spontaneous IL-12 p40 production by SC from RA patients were significantly higher than the levels in OA and AS patients. Spontaneous IL-12 production by SC from RA patients significantly decreased after depletion of CD4+ T cells from SC or after application of anti-CD154 antibody, but not by treatment with anti-TNFalpha antibody. Anti-CD3 antibody stimulation increased spontaneous IL-12 p40 production and CD154 expression by synovial T cells. The increment of IL-12 p40 production by anti-CD3 was abrogated by anti-CD154 antibody. IL-12 p40 production was also increased by LPS stimulation. LPS-stimulated IL-12 production was inhibited by anti-TNFalpha antibody, but not by T cell depletion and anti-CD154 antibody treatment. The TNFalpha inhibitor rolipram inhibited LPS-stimulated IL-12 p40 production by RA SC more strongly than spontaneous production. TNFalpha restored LPS-stimulated IL-12 production that had been inhibited by rolipram. CONCLUSION: IL-12 production in RA is regulated by 2 different pathways. One pathway is T cell dependent, predominantly through a CD40-CD154 interaction, while the other is T cell independent, mediated through TNFalpha. Inhibition of IL-12 production by interference with CD40-CD154 interaction and TNFalpha production may be a potential therapeutic strategy for treating RA.     

T cell receptor zeta reconstitution fails to restore responses of T cells rendered hyporesponsive by tumor necrosis factor alpha

Expression and function of the antigen T cell receptor (TCR) play a central role in regulating immune responsiveness. Accordingly, targeting the expression of TCRalphabeta or its associated CD3 subunits profoundly influences T cell development and adaptive immunity. Down-regulation of the invariant TCRzeta chain has been documented in a wide variety of chronic inflammatory and infectious diseases, and is thought to contribute to the paradoxical immune suppression observed in these diseases. Previously, we reported that prolonged exposure of T cell hybridoma clones to tumor necrosis factor alpha (TNF) induces nondeletional and reversible hyporesponsiveness to TCR engagement, associated with down-regulation of TCRzeta chain expression, impaired TCR/CD3 complex assembly, and attenuation of TCR-induced membrane proximal tyrosine phosphorylation. Here, we have tested whether receptor specific T cell responses are rescued in TNF-treated T cell hybridomas by retroviral-mediated expression of zeta-chimeric (C2zeta) receptors or wild-type TCRzeta. Expression of C2zeta receptors at the cell surface is relatively refractory to chronic TNF stimulation. However, C2zeta receptor function depends on association with endogenous TCRzeta chains, whose expression is down-regulated by TNF, and so C2 receptor specific responses are attenuated in TNF-treated T cells. Unexpectedly, overexpression of wild-type TCRzeta maintains cell surface TCR/CD3 complex expression but fails to rescue receptor proximal signaling in TNF-treated T cells, suggesting the existence of hitherto unrecognized mechanisms through which TNF regulates T cell responsiveness. We provide additional evidence that TNF also uncouples distal TCR signaling pathways independently of its effects on TCRzeta expression.     

Astrocytic alterations induced by HTLV type 1-infected T lymphocytes: a role for Tax-1 and tumor necrosis factor alpha

In the neurological disease associated with HTLV-1 infected T lymphocytes infiltrated within the CNS are suspected of playing a prominent role in pathogenesis via inflammatory cytokines and the viral protein Tax-1. We hypothesized that T lymphocytes initiate functional perturbation in astrocytes, resulting in neuronal alteration as glial cells have a crucial role in CNS homeostasis. In particular, astrocytes manage the steady state level of glutamate and continuously provide metabolite precursors to neurons and oligodendrocytes. Using a model system of HTLV-1-infected T cells-astrocytes interaction, we show that after contact with T cells, astrocyte acquire a phenotype typical of gliosis: secretion of proinflammatory cytokines (TNF-alpha, IL-1alpha, IL-6) and matrix metalloproteinases (MMP-9, MMP-3). The concomitant increase in the expression of MMPs and of their endogenous inhibitors (TIMP-1 and TIMP-3) suggests a perturbation in MMP/TIMP balance. This may alter the extracellular matrix and, in turn, the cell environment. At a functional level, glutamate transport and catabolism are impaired in astrocytes. A decrease in glutamate uptake is associated with downregulated expression of glutamate transporters GLAST and GLT1. The expression of astrocytic enzyme of glutamate metabolism is modified with up-regulation of glutamine synthetase and down-regulation of glutamate dehydrogenase. The involvement of Tax-1 in these alterations, directly or indirectly via TNF-alpha, is shown. Altered glutamate uptake and catabolism associated with impairment in cell connectivity via MMP/TIMP imbalance could compromise the functional integrity of the CNS in general and that of neurons and oligodendrocytes in particular.     

Interferon-gamma regulates apoptosis by releasing soluble tumor necrosis factor receptors in a gastric epithelial cell line

BACKGROUND AND AIMS: Interferon (IFN)-gamma and tumor necrosis factor (TNF) are predominant cytokines produced in the gastric mucosa of patients with Helicobacter pylori-infected gastritis. Several studies reported that IFN-gamma and TNF induced the synergistic effect on many cell lines. We attempted to clarify the apoptotic activity and the synergistic effect of IFN-gamma and TNF on the gastric epithelial cell, and whether IFN-gamma relates to soluble TNF receptors (sTNF-R) release from the gastric epithelial cell. METHODS: On the gastric epithelial cell line MKN45, cytotoxic and apoptotic effects of IFN-gamma and TNF were examined. Next, sTNF-R released in response to IFN-gamma and the protective effect of sTNF-R against the cytotoxic activity of TNF and IFN-gamma were examined by blocking the release of sTNF-R with a serine protease inhibitor such as phenylmethylsulfonyl fluoride. RESULTS: Interferon-gamma significantly decreased cell viability, but TNF decreased it only slightly. Interferon-gamma and TNF did not make a synergistic effect on cell viability and apoptosis. Interferon-gamma and TNF induced sTNF-R release from gastric epithelial cells. Phenylmethylsulfonyl fluoride significantly inhibited shedding of sTNF-R and a synergistic effect of TNF and IFN-gamma on apoptosis was observed. CONCLUSION: These results suggest that sTNF-R released by IFN-gamma regulate the injury on the gastric epithelial cell line induced by TNF.     

Characterization of a ceramide-activated protein kinase: stimulation by tumor necrosis factor alpha.

Recent investigations have identified a signal-transduction system involving sphingomyelin and derivatives. In this paradigm, sphingomyelin hydrolysis by a sphingomyelinase generates ceramide, which may be converted to the protein kinase C inhibitor sphingosine or to ceramide 1-phosphate. Ceramide may have second-messenger function because it induces epidermal growth factor receptor phosphorylation, presumably on Thr-669 in A-431 cells. The present studies describe a kinase that may mediate ceramide action. With a 19-amino acid epidermal growth factor receptor peptide containing Thr-669, a membrane-bound activity that phosphorylated the peptide was detected in A-431 cells. Activity was linearly related to ATP (0.3-300 microM) and peptide concentration (0.02-1 mg/ml), possessed a physiologic pH optimum (pH 7.0-7.4), and was Mg(2+)-dependent. Other cations--Ca2+, Mn2+, and Zn(2+)--were ineffective. Natural and synthetic ceramide induced time- and concentration-dependent enhancement of kinase activity. Ceramide (0.5 microM) increased kinase activity 2-fold by 30 s, and activity remained elevated for at least 15 min. As little as 0.001 microM ceramide was effective, and 1 microM ceramide induced maximal phosphorylation. Sphingosine was similarly effective. Because tumor necrosis factor (TNF) alpha rapidly induces sphingomyelin hydrolysis to ceramide during monocytic differentiation of HL-60 cells, its effects on kinase activity were assessed. Kinase activity was increased 1.5-fold at 5 min and 2-fold at 2 hr in membranes derived from TNF-stimulated cells. The effective concentration range was 3 pM-30 nM TNF. Exogenous ceramide induced a similar effect. In sum, these studies demonstrate the existence of an unusual Mg(2+)-dependent ceramide-activated protein kinase that may mediate some aspects of TNF-alpha function.     

Interferon gamma and tumor necrosis factor alpha induce Fas expression and anti-Fas mediated apoptosis in a salivary ductal cell line

BACKGROUND: We previously reported that Fas antigen was strongly expressed on salivary duct epithelial cells and that some salivary infiltrating cells showed the Fas ligand in patients with severe sialoadenitis due to Sj?gren's syndrome (SS). Apoptotic changes were observed in ductal epithelial cells and some infiltrating cells by DNA nick end labeling methods. These findings suggest that the Fas-Fas ligand system may play a role in the pathogenesis of sialoadenitis in SS. OBJECTIVE: To elucidate the mechanism of the de novo expression of ductal Fas antigen in sialoadenitis associated with SS, we investigated the induction of Fas antigen and apoptosis by cytokines in a human salivary duct cell line. METHODS: Human salivary duct cell line (HSG) was cultured with interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta), interleukin 2 (IL-2), interleukin 4 (IL-4), and granulocyte monocyte colony stimulating factor (GM-CSF). The expression of Fas antigen in HSG was examined by immunoperoxidase cell ELISA. The appearance of DNA strand breaks during apoptosis induced by anti-Fas antibody was detected by DNA nick end labeling methods. RESULTS: Unstimulated HSG cells constitutively expressed low levels of Fas antigen. IFN-gamma and TNF-alpha consistently upregulated constitutive levels of Fas. In contrast, IL-1 beta, IL-2, IL-4, and GM-CSF had no effect on Fas levels. HSG cells expressing Fas antigen in response to IFN-gamma or TNF-alpha were susceptible to apoptosis by anti-Fas antibody. CONCLUSION: Our findings suggest that IFN-gamma or TNF-alpha secreted by infiltrating lymphocytes induces ductal Fas expression and ductal apoptosis in sialoadenitis associated with SS.     

Isolation and partial characterization of a nuclear antigen (68/6.3) from the Namalwa cell line (a Burkitt lymphoma)

Summary A nuclear antigen was purified from the 0.01 M Tris-HCl/pH 8 extract of nuclei of the Burkitt tumor Namalwa cell line to electrophoretic homogeneity by DEAE cellulose chromatography, affinity chromatography, and preparative isoelectric focusing. The yield of antigens was 0.02% of the nuclear 0.01 M Tris-HCl/pH 8 extract. On two-dimensional gel electrophoresis, the major antigen separated into two adjacent protein spots with molecular weights of 68,000 and an approximate pI of 6.3 (68/6.3 A and 68/6.3 B). A minor antigen had a molecular weight of 61,000 and pI of 6.0 68/6.0). Fourteen ¹²⁵I-labeled peptides were obtained from the tryptic digest of the major antigen (68/6.3 A and 68/6.3 B). The amino-acid composition analysis of the purified antigens indicated that the amino acids in the highest content were glycine (15%), glutamic acid (11.6%), and serine (9%); the ratio of acidic to basic amino acids was 1.95. In studies on nucleolytic activity, the purified antigen produced a single-stranded and then a double-stranded cleavage of PM 2 and pBR 322 DNA. This antigen is the first purified nuclear antigen that reacts with the HeLa-specific nucleolar antibodies.Abbreviations PBS 8.5mM Na₂HPO₄/1.6mM NaH₂PO₄/0.145 M NaCl/pH 7.2 - BSA bovine serum albumin - PMSF phenylmethylsulfonlyfluoride - IF gel isoelectric focusing gel These studies were supported in part by the Human Tumor Nucleolar Antigen Grant, CA-27534, The Farish Fund, the Pauline Sterne Wolff Memorial Foundation, the Bristol-Myers Fund, and the Cancer Research Program Grant CA-10893, awarded by the National Cancer Institute, DHEW