Lymphotoxin is an autocrine growth factor for Epstein-Barr virus-infected B cell lines.

Because human lymphotoxin (LT) was originally isolated from a lymphoblastoid cell line, we investigated the role of this molecule in three newly established Epstein-Barr virus (EBV)-infected human B cell lines. These lines were derived from acute lymphoblastic leukemia (Z-6), myelodysplastic syndrome (Z-43), and acute myelogenous leukemia (Z-55) patients who had a prior EBV infection. Each lymphoblastoid cell line had a karyotype that was different from that of the original parent leukemic cells, and all expressed B cell, but not T cell or myeloid surface markers. In all three lines, rearranged immunoglobulin heavy chain joining region (JH) bands were found, and the presence of EBV DNA was confirmed by Southern blotting. Z-6, Z-43, and Z-55 cell lines constitutively produced 192, 48, and 78 U/ml LT, respectively, as assessed by a cytotoxicity assay and antibody neutralization. Levels of tumor necrosis factor (TNF) were undetectable. Scatchard analysis revealed that all the cell lines expressed high-affinity TNF/LT receptors with receptor densities of 4197, 1258, and 1209 sites/cell on Z-6, Z-43, and Z-55, respectively. Furthermore, labeled TNF binding could be reversed by both unlabeled TNF, as well as by LT. Studies with p60 and p80 receptor-specific antibodies revealed that the three lines expressed primarily the p80 form of the TNF receptor. When studied in a clonogenic assay, exogenous LT stimulated proliferation of all three cell lines in a dose-dependent fashion at concentrations ranging from 25 to 500 U/ml. Similar results were obtained with [3H]TdR incorporation. Monoclonal anti-LT neutralizing antibodies at concentrations of 25-500 U/ml inhibited cellular multiplication in a dose-dependent manner. It is interesting that in spite of a common receptor, TNF (1,000 U/ml) had no direct effect on Z-55 cell growth, whereas it partially reversed the stimulatory effect of exogenous LT. In addition, TNF inhibited Z-6 and Z-43 cell proliferation, and its suppressive effect was reversed by exogenous LT. Both p80 and p60 forms of soluble TNF receptors suppressed the lymphoblastoid cell line proliferation and their inhibitory effect was partially reversed by LT. Our data suggest that (a) LT is an autocrine growth factor for EBV-transformed lymphoblastoid B cell lines; and (b) anti-LT antibodies, soluble TNF/LT receptors, and TNF itself can suppress the growth of lymphoblastoid cells, probably by modulating or competing with LT.(ABSTRACT TRUNCATED AT 400 WORDS)

Interrelationships of human interferon-gamma with lymphotoxin and monocyte cytotoxin.

Crude preparations of interferon (IFN)-gamma derived from human peripheral blood leukocyte (PBL) cultures induced with 12-O-tetra-decanoylphorbol-13-acetate (TPA) and phytohemagglutinin (PHA) were more cytotoxic to HeLa cells than partially purified nautral or highly purified recombinant human IFN-gamma preparations. Conditioned media from PBL cultures contained, in addition to IFN-gamma, a mixture of cytotoxins, including classic lymphocyte-derived lymphotoxin (LT), and a TPA-induced cytotoxic activity produced by the adherent cell population (presumably monocytes). These two types of cytotoxins, indistinguishable in the mouse L929 cell LT assay, could be differentiated by an antiserum prepared against LT derived from the B lymphoblastoid cell line RPMI 1788. This antiserum neutralized lymphocyte-derived classic LT but failed to neutralize the activity of the monocyte-derived cytotoxin. Processing of conditioned media by sequential chromatography on silicic acid, Con A-Sepharose, and DEAE-Sephacel failed to separate IFN-gamma from the LT activity. However, this procedure did remove the monocyte-derived cytotoxic activity present in the original starting material, leaving predominantly classic LT. This LT showed a slightly basic isoelectric point (pI 7.6) which partially overlapped the more basic pI range of IFN-gamma. The two lymphokine activities also could not be completely separated by fast protein liquid chromatography or molecular sieve chromatography. LT in these partially purified preparations was associated with a protein having an apparent molecular weight of 58,000 on gel filtration. This form dissociated partially into a 20,000 mol wt species after denaturation with 0.1% NaDodSO4. IFN-gamma could be selectively removed from preparations containing both IFN-gamma and LT with the aid of monoclonal antibody to IFN-gamma. The addition of purified LT to purified E. coli-derived recombinant human IFN-gamma resulted in a marked synergistic enhancement of cytotoxicity for HeLa cells.

Identification of human uromodulin as the Tamm-Horsfall urinary glycoprotein.

The primary structure of human uromodulin, a 616-amino acid, 85-kilodalton glycoprotein with in vitro immunosuppressive properties, was determined through isolation and characterization of complementary DNA and genomic clones. The amino acid sequence encoded by one of the exons of the uromodulin gene has homology to the low-density-lipoprotein receptor and the epidermal growth factor precursor. Northern hybridization analyses demonstrate that uromodulin is synthesized by the kidney. Evidence is provided that uromodulin is identical to the previously characterized Tamm-Horsfall glycoprotein, the most abundant protein in normal human urine.

Recombinant human tumor necrosis factor-alpha: effects on proliferation of normal and transformed cells in vitro.

Modulation of the growth of human and murine cell lines in vitro by recombinant human tumor necrosis factor-alpha (rTNF-alpha) and recombinant human interferon-gamma (rIFN-gamma) was investigated. rTNF-alpha had cytostatic or cytolytic effects on only some tumor cell lines. When administered together with rIFN-gamma, rTNF-alpha showed enhanced antiproliferative effects on a subset of the cell lines tested. In contrast to its effects on sensitive tumor cells, rTNF-alpha augmented the growth of normal diploid fibroblasts. Variations in the proliferative response induced by rTNF-alpha were apparently not due to differences in either the number of binding sites per cell or their affinity for rTNF-alpha. These observations indicate that the effects of rTNF-alpha on cell growth are not limited to tumor cells, but rather that this protein may have a broad spectrum of activities in vivo.

Genetic deletion of PKR abrogates TNF-induced activation of IkappaBalpha kinase, JNK, Akt and cell proliferation but potentiates p44/p42 MAPK and p38 MAPK activation.

Double-stranded RNA-dependent protein kinase (PKR), a ubiquitously expressed serine/threonine kinase, has been implicated in the regulation or modulation of cell growth through multiple signaling pathways, but how PKR regulates tumor necrosis factor (TNF)-induced signaling pathways is poorly understood. In the present study, we used fibroblasts derived from PKR gene-deleted mice to investigate the role of PKR in TNF-induced activation of nuclear factor-kappaB (NF-kappaB), mitogen-activated protein kinases (MAPKs) and growth modulation. We found that in wild-type mouse embryonic fibroblast (MEF), TNF induced NF-kappaB activation as measured by DNA binding but deletion of PKR abolished this activation. This inhibition was associated with suppression of inhibitory subunit of NF-kappaB (IkappaB)alpha kinase (IKK) activation, IkappaBalpha phosphorylation and degradation, p65 phosphorylation and nuclear translocation, and NF-kappaB-dependent reporter gene transcription. TNF-induced Akt activation needed for IKK activation was also abolished by deletion of PKR. NF-kappaB activation was diminished in PKR-deleted cells transfected with TNF receptor (TNFR) 1, TNFR-associated death domain and TRAF2 plasmids; NF-kappaB activated by NF-kappaB-inducing kinase, IKK or p65, however, was minimally affected. Among the MAPKs, it was interesting that whereas TNF-induced c-Jun N-terminal kinase (JNK) activation was abolished, activation of p44/p42 MAPK and p38 MAPK was potentiated in PKR-deleted cells. TNF induced the expression of NF-kappaB-regulated gene products cyclin D1, c-Myc, matrix metalloproteinase-9, survivin, X-linked inhibitor-of-apoptosis protein (IAP), IAP1, Bcl-x(L), A1/Bfl-1 and Fas-associated death domain protein-like IL-1beta-converting enzyme-inhibitory protein in wild-type MEF but not in PKR-/- cells. Similarly, TNF induced the proliferation of wild-type cells, but this proliferation was completely suppressed in PKR-deleted cells. Overall, our results indicate that PKR differentially regulates TNF signaling; IKK, Akt and JNK were positively regulated, whereas p44/p42 MAPK and p38 MAPK were negatively regulated.

Evidence that TNF-TNFR1-TRADD-TRAF2-RIP-TAK1-IKK pathway mediates constitutive NF-kappaB activation and proliferation in human head and neck squamous cell carcinoma.

Constitutively activated nuclear factor-kappaB (NF-kappaB) has been associated with a variety of aggressive tumor types, including head and neck squamous cell carcinoma (HNSCC); however, the mechanism of its activation is not fully understood. Therefore, we investigated the molecular pathway that mediates constitutive activation of NF-kappaB in a series of HNSCC cell lines. We confirmed that NF-kappaB was constitutively active in all HNSCC cell lines (FaDu, LICR-LON-HN5 and SCC4) examined as indicated by DNA binding, immunocytochemical localization of p65, by NF-kappaB-dependent reporter gene expression and its inhibition by dominant-negative (DN)-inhibitory subunit of NF-kappaB (IkappaBalpha), the natural inhibitor of NF-kappaB. Constitutive NF-kappaB activation in HNSCC was found to be due to constitutive activation of IkappaBalpha kinase (IKK); and this correlated with constitutive expression of phosphorylated forms of IkappaBalpha and p65 proteins. All HNSCC showed the expression of p50, p52, p100 and receptor-interacting protein; all linked with NF-kappaB activation. The expression of constitutively active NF-kappaB in HNSCC is mediated through the tumor necrosis factor (TNF) signaling pathway, as NF-kappaB reporter activity was inhibited by DN-TNF receptor-associated death domain (TRADD), DN-TNF receptor-associated factor (TRAF)2, DN-receptor-interacting protein (RIP), DN-transforming growth factor-beta-activated kinase 1 (TAK1), DN-kappa-Ras, DN-AKT and DN-IKK but not by DN-TRAF5 or DN-TRAF6. Constitutive NF-kappaB activation was also associated with the autocrine expression of TNF, TNF receptors and receptor-activator of NF-kappaB and its ligand in HNSCC cells but not interleukin (IL)-1beta. All HNSCC cell lines expressed IL-6, a NF-kappaB-regulated gene product. Furthermore, treatment of HNSCC cells with anti-TNF antibody downregulated constitutively active NF-kappaB, and this was associated with inhibition of IL-6 expression and cell proliferation. Our results clearly demonstrate that constitutive activation of NF-kappaB is mediated through the TRADD-TRAF2-RIP-TAK1-IKK pathway, making TNF a novel target in the treatment of head and neck cancer.

Diosgenin inhibits osteoclastogenesis, invasion, and proliferation through the downregulation of Akt, I kappa B kinase activation and NF-kappa B-regulated gene expression.

Diosgenin, a steroidal saponin present in fenugreek (Trigonella foenum graecum) and other plants, has been shown to suppress inflammation, inhibit proliferation, and induce apoptosis in a variety of tumor cells, but through a mechanism that is poorly understood. In the present study, we report that diosgenin inhibits receptor-activated nuclear factor-kappaB ligand-induced osteoclastogenesis, suppresses tumor necrosis factor (TNF)-induced invasion, and blocks the proliferation of tumor cells, all activities known to be regulated by NF-kappaB. Diosgenin suppressed TNF-induced NF-kappaB activation as determined by DNA binding, activation of IkappaBalpha kinase, IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation, and p65 nuclear translocation through inhibition of Akt activation. NF-kappaB-dependent reporter gene expression was also abrogated by diosgenin. TNF-induced expression of NF-kappaB-regulated gene products involved in cell proliferation (cyclin D1, COX-2, c-myc), antiapoptosis (IAP1, Bcl-2, Bcl-X(L), Bfl-1/A1, TRAF1 and cFLIP), and invasion (MMP-9) were also downregulated by the saponin. Diosgenin also potentiated the apoptosis induced by TNF and chemotherapeutic agents. Overall, our results suggest that diosgenin suppresses proliferation, inhibits invasion, and suppresses osteoclastogenesis through inhibition of NF-kappaB-regulated gene expression and enhances apoptosis induced by cytokines and chemotherapeutic agents.

Activation of CPP32-like protease in tumor necrosis factor-induced apoptosis is dependent on mitochondrial function.

Mitochondria have been implicated in apoptosis, however, the precise mechanisms whereby mitochondria exert their effect are not clear. To gain further insights, we generated a panel of cells from ML-1a cells that were rendered respiration deficient by ethidium bromide treatment. Two respiration-deficient clones were subsequently reconstituted by fusion with platelets. Respiration-deficient clones were resistant to TNF-induced apoptosis, whereas ML-1a and reconstituted clones were sensitive. In contrast, inhibition of proliferation and induction of differentiation by TNF were still observed in respiration deficient clones, suggesting a selective requirement of respiration in TNF-induced apoptosis. Furthermore the apoptosis machinery is not completely altered in respiration-deficient cells because they underwent apoptosis after staurosporine treatment. Next, we showed that apoptosis induced by TNF and staurosporine were blocked by z-DEVD-CH2F, an inhibitor of CPP32-like cysteine protease, suggesting the involvement of CPP32-like protease in both apoptosis signaling pathways. Interestingly, TNF activated CPP32-like protease in the parental and reconstituted clones but not in respiration-deficient clones, and staurosporine in all clones. Thus, the apoptosis signaling block in respiration-deficient clones is located at a step before CPP32-like protease activation, which can be bypassed by staurosporine.

Binding and biological effects of tumor necrosis factor alpha on cultured human neonatal foreskin keratinocytes.

Tumor necrosis factor alpha (TNF alpha) localizes to the epidermis when injected in vivo, but its role in the skin has heretofore not been evaluated. As a first approach to assessing the role of TNF alpha in the skin, we evaluated the binding and biological effects of TNF alpha on human neonatal foreskin keratinocytes maintained in culture. We found that TNF alpha at 0.3-1.0 nM inhibited proliferation of keratinocytes in a reversible fashion as demonstrated by a reduction in total DNA content and clonal growth. The antiproliferative effects were most marked when TNF alpha was added in the preconfluent stages of cell growth. Accompanying this antiproliferative effect was a stimulation by TNF alpha of differentiation of keratinocytes as indicated by the stimulation of cornified envelope formation. Keratinocytes specifically bound TNF alpha, reaching maximal binding in 2 h at 34 degrees C or 8 h at 4 degrees C. Much of the apparent binding at 34 degrees C was due to internalization of the TNF alpha. At 4 degrees C the rate of internalization was much less. Confluent keratinocytes showed a single class of high-affinity receptors with 1,250 receptors/cell and a Kd of 0.28 nM. These data suggest a role for TNF alpha in the growth and differentiation of the epidermis.

TNF blockade: an inflammatory issue.

Tumor necrosis factor (TNF), initially discovered as a result of its antitumor activity, has now been shown to mediate tumor initiation, promotion, and metastasis. In addition, dysregulation of TNF has been implicated in a wide variety of inflammatory diseases including rheumatoid arthritis, Crohn's disease, multiple sclerosis, psoriasis, scleroderma, atopic dermatitis, systemic lupus erythematosus, type II diabetes, atherosclerosis, myocardial infarction, osteoporosis, and autoimmune deficiency disease. TNF, however, is a critical component of effective immune surveillance and is required for proper proliferation and function of NK cells, T cells, B cells, macrophages, and dendritic cells. TNF activity can be blocked, either by using antibodies (Remicade and Humira) or soluble TNF receptor (Enbrel), for the symptoms of arthritis and Crohn's disease to be alleviated, but at the same time, such treatment increases the risk of infections, certain type of cancers, and cardiotoxicity. Thus blockers of TNF that are safe and yet efficacious are urgently needed. Some evidence suggests that while the transmembrane form of TNF has beneficial effects, soluble TNF mediates toxicity. In most cells, TNF mediates its effects through activation of caspases, NF-kappaB, AP-1, c-jun N-terminal kinase, p38 MAPK, and p44/p42 MAPK. Agents that can differentially regulate TNF expression or TNF signaling can be pharmacologically safe and effective therapeutics. Our laboratory has identified numerous such agents from natural sources. These are discussed further in detail.

Human immunodeficiency virus type 1 tat gene up-regulates interleukin 4 receptors on a human B-lymphoblastoid cell line.

The human immunodeficiency virus type I (HIV-1) regulatory gene, tat III, is a powerful trans-activator of gene expression from the viral long terminal repeat and is essential for HIV replication. In addition, tat III protein has been shown to be immunosuppressive as indicated by the inhibition of antigen mediated T-cell proliferation. To further test whether tat III might play a direct role in the immunosuppressive effects of HIV-1 in addition to its role in virus replication, we examined the regulation of interleukin 4 (IL-4) receptors on a human B-lymphoblastoid cell line (Raji) transfected with HIV-1 tat gene (Raji-tat III). We used radioligand receptor binding analysis for cell surface expression and Northern blot analysis for the expression of human IL-4 receptor gene in Raji-tat III cells. Control Raji cells expressed 1383 +/- 361 (SE; n = 3) IL-4 binding sites/cell with a dissociation constant (Kd) of 144 +/- 27 pM (n = 3). However, Raji-tat III cells expressed about three times higher IL-4 receptors (4000 +/- 633 IL-4 binding sites/cell; P less than 0.03 compared to Raji cells) with a similar Kd of 273 +/- 90 pM (n = 3; P greater than 0.05 compared to Raji cells). Whereas both Raji and Raji-tat III cells exhibited a single mRNA species (approximately 4 kilobases) of IL-4 receptors by Northern blot analysis, the mRNA level was about 3-fold higher in Raji-tat III cells compared to Raji cells. Cycloheximide inhibited the expression of IL-4 receptors by 50% in about 2 h in both cell types indicating both the half-life of IL-4 receptors and the requirement for protein synthesis for the tat III up-regulation of IL-4 receptors. Since IL-4 under certain circumstances has been shown to be immunosuppressant, our observation that the HIV-1 tat gene up-regulates IL-4 receptors suggests the possibility that the immunosuppressive effects of HIV-1 are mediated at least in part through IL-4 receptors.

Interleukin 4 potentiates the antiproliferative effects of tumor necrosis factor on various tumor cell lines.

In response to a given stimulus, usually a number of cytokines are secreted simultaneously by the immune system. Whether these cytokines are meant to function as a single agent or in combination with others is not understood. Tumor necrosis factor (TNF) has been shown to exhibit antiproliferative effects against a wide variety of tumor cell lines in vitro. In the present report, we investigated the effects of a T-cell-derived cytokine, interleukin 4 (IL-4), on the antiproliferative effects of TNF against different tumor cell lines. The growth characteristics of human breast cancer cells (MDA-MB-330) were minimally affected when the cells were exposed to either TNF or IL-4 alone. However, together these 2 cytokines inhibited cell growth in a dose-dependent manner. The enhancement of the cytotoxic effects of TNF by IL-4 were not just limited to breast tumor cells, but were also observed with human epidermoid carcinoma cells (A-431) and human histiocytic lymphoma cells (U-937). The enhancement of the cytotoxic effect of TNF by IL-4 against various tumor cell lines was found comparable with that by gamma-interferon (IFN-gamma). Interestingly, for certain tumor cell types, IL-4 alone was found to enhance cell proliferation. IL-4 had no effect on the growth-stimulatory activity of TNF on normal human foreskin fibroblasts. Pre-exposure of U-937 cells to IFN-gamma led to a greater than 2-fold induction in TNF receptors, but no modulation of TNF receptors by IL-4 was observed. Moreover, the presence of IFN-gamma was found to further potentiate the antiproliferative effects of TNF and IL-4. These results clearly suggest that IL-4 potentiates the antiproliferative responses of TNF by a mechanism different from that of IFN-gamma. Although it is well known that IL-4 can modulate the production of TNF from macrophages, this is the first report to suggest that IL-4 can also modulate TNF-dependent antiproliferative responses.

Phosphatase inhibitors modulate the growth-regulatory effects of human tumor necrosis factor on tumor and normal cells.

Tumor necrosis factor alpha (TNF-alpha) has been shown to inhibit the growth of tumor cells and stimulate the growth of certain normal cells in vitro. The mechanism by which TNF exerts its cell growth-regulatory effects is not known. In this report, we investigated the effects of phosphatase inhibitors on the cell growth-inhibitory effects of TNF on L-929, a highly sensitive murine connective tissue tumor cell line, and on the growth-stimulatory effects of TNF on normal human fibroblasts. The antiproliferative effects of TNF on L-929 cells were inhibited by orthovanadate, an inhibitor of phosphotyrosine phosphatases, in a dose-dependent manner. Okadaic acid, which is a specific inhibitor of phosphoserine- and phosphothreonine-specific phosphatases, also blocked the growth-inhibitory effects of TNF, suggesting that TNF may function through the activation of certain phosphatases. These inhibitors had no effect on TNF receptors. Addition of phosphatase inhibitor, even 12 h after the treatment of cells with TNF, was sufficient to block the antiproliferative effects of the cytokine, suggesting that the inhibitor is acting at a late event in the pathway of action of TNF. Cells were protected by orthovanadate from the cytotoxic effects of TNF even in the presence of actinomycin D or cycloheximide, thus indicating the lack of a requirement for de novo protein synthesis. Orthovanadate altered the cell morphology from flat spindle shapes to rounded ones. Besides anticellular effects, a phosphatase inhibitor also suppressed the proliferative effects of TNF on human fibroblasts. These results thus suggest that phosphatases may be needed for both proliferative and antiproliferative effects of this cytokine. This is the first report to suggest that phosphatases play a role in the growth-regulatory action of TNF.

Triple helix-forming oligodeoxyribonucleotides targeted to the human tumor necrosis factor (TNF) gene inhibit TNF production and block the TNF-dependent growth of human glioblastoma tumor cells.

Synthetic oligodeoxyribonucleotides (ODNs) designed to selectively inhibit the transcription or translation of specific genes are being used to modulate the activity of the targeted gene. Because multiple copies of mRNA can be transcribed from one actively expressed gene, ODNs that target double-stranded DNA and form triple helices upon binding with the gene itself have an advantage over ODNs that target the gene product (mRNA) in an antisense fashion. For the present studies, we designed four different triple helix-forming phosphodiester ODNs (TFOs) targeted to the tumor necrosis factor (TNF) gene and examined their effect on production of TNF and on cellular growth of tumors in which TNF acts as an autocrine growth factor. The ODNs J-109-50 and J-108-57 were designed to interact with polypurine oligonucleotides corresponding to the binding sites for nuclear factors kB (-237 to -208) and Sp1 (-58 to -33), respectively; J111-51 was designed to interact with a polypurine oligonucleotide in the third intron (+1429 to +1456) of the TNF gene. To enhance the cellular penetration and prevent degradation by cellular nucleases, the TFOs were modified at their 3' ends by either a cholesterol side chain or a propanolamine blocking group. Treatment of the human promonocytic cell line THP-1 with TNF-TFOs at a nontoxic concentration (2 microM) reduced the production of TNF. All of the TNF-TFOs tested were effective, and control-irrelevant TFOs were ineffective in inhibiting TNF production. The activity of the most efficacious TNF-TFOs also correlated with a decrease in TNF mRNA as observed by using reverse transcriptase PCR assays. In several tumors in which TNF acts as an autocrine growth factor, we examined the antiproliferative activity of J111-51. We found that in the human glioblastoma tumor cell line U-251, TNF-induced growth was blocked by J111-51 in a dose-dependent manner. Thus, overall results demonstrate that oligonucleotides directed to the specific regions of TNF can be designed, which may have a potential in cancer therapy.

Effects of growth factors on the antiproliferative activity of tumor necrosis factors.

Tumor necrosis factors (TNFs) are a class of cytokines secreted by activated effector cells involved in host defense against tumor progression. Epidermal growth factor (EGF) and recombinant human transforming growth factor-alpha (rHuTGF-alpha) were shown to interfere with the in vitro antiproliferative effects of recombinant human tumor necrosis factor-alpha (rHuTNF-alpha) and -beta on a human cervical carcinoma cell line, ME-180. The inhibitory effect could be observed at EGF or rHuTGF-alpha concentrations of 100 pg/ml, and was maximal between 1 and 10 ng/ml. This response was not due to down regulation of the TNF receptor or to alteration of the affinity of TNF-alpha for its receptor. Since the antiproliferative effect of recombinant human interferon-gamma was not significantly affected by the presence of EGF or rHuTGF-alpha, the inhibition was specific for recombinant TNFs and was not due solely to enhanced proliferation induced by the growth factors. Neither growth factor had a substantial protective effect on the synergistic cytotoxicity observed when tumor cells were exposed simultaneously to rHuTNF-alpha and recombinant human interferon-gamma. TGF-beta can also interfere with the antiproliferative effects of rHuTNF-alpha in vitro. At concentrations of less than 1 ng/ml, TGF-beta significantly antagonized the cytotoxic effects of rHuTNF-alpha on NIH 3T3 fibroblasts. Since EGF, platelet-derived growth factor, and TGF-beta all enhanced NIH 3T3 cell proliferation, but only TGF-beta interfered with rHuTNF-alpha cytotoxicity, the protective effects of TGF-beta were not related in a simple manner to enhanced cell proliferation. rHuTGF-alpha and TGF-beta did not have a significant protective effect against rHuTNF-alpha-mediated cytotoxicity on two other tumor cell lines, BT-20 and L-929 cells. Based upon these observations we suggest that growth factors might enhance tumor growth in vivo by a combination of distinct mechanisms: (a) by autocrine stimulation tumor cell growth; and/or (b) by interfering with normal effector mechanisms of host defense.

Modulation of the growth of transformed cells by human tumor necrosis factor-alpha and interferon-gamma.

Recombinant human tumor necrosis factor-alpha (rHuTNF-alpha) inhibited growth of the cervical carcinoma cell line, ME-180neo, at doses greater than 50 units/ml, but stimulated the growth of these cells at low doses (0.1-10 units/ml). ME-180neo variants selected for resistance to the cytotoxic effects of rHuTNF-alpha retained the ability to be growth stimulated at all concentrations tested. ME-180neo cells and the rHuTNF-alpha-resistant ME-180neo variants possessed equivalent steady state numbers of TNF-alpha receptors with similar Kd values. Recombinant human interferon-gamma (rHuIFN-gamma) augmented the rHuTNF-alpha-induced cytotoxic response of ME-180neo cells and overcame the resistance of the ME-180neo variants to rHuTNF-alpha cytotoxicity. In separate experiments we were able to show that the number of TNF-alpha binding sites on both rHuTNF-alpha-sensitive and -resistant ME-180neo cells was similar and was increased by treatment with rHuIFN-gamma. These results suggest that the growth stimulation of tumor cells mediated by rHuTNF-alpha can be dissociated from the cytotoxic response and that these responses are not related to the number or affinity of TNF-alpha binding sites.

Oleandrin suppresses activation of nuclear transcription factor-kappaB, activator protein-1, and c-Jun NH2-terminal kinase.

Agents that can suppress the activation of nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) may be able to block tumorigenesis and inflammation. Oleandrin, a polyphenolic cardiac glycoside derived from the leaves of Nerium oleander, is a candidate NF-kappaB and AP-1 modulator. We investigated the effect of oleandrin on NF-kappaB activation induced by inflammatory agents. Oleandrin blocked tumor necrosis factor (TNF)-induced activation of NF-kappaB in a concentration- and time-dependent manner. This effect was mediated through inhibition of phosphorylation and degradation of IkappaBalpha, an inhibitor of NF-kappaB. A proprietary hot water extract of oleander (Anvirzel) also blocked TNF-induced NF-kappaB activation; subsequent fractionation of the extract revealed that this activity was attributable to oleandrin. The effects of oleandrin were not cell type specific, because it blocked TNF-induced NF-kappaB activation in a variety of cells. NF-kappaB-dependent reporter gene transcription activated by TNF was also suppressed by oleandrin. The TNF-induced NF-kappaB activation cascade involving TNF receptor 1/TNF receptor-associated death domain/TNF receptor-associated factor 2/NF-kappaB-inducing kinase/IkappaBalpha kinase was interrupted at the TNF receptor-associated factor 2 and NF-kappaB-inducing kinase sites by oleandrin, thus suppressing NF-kappaB reporter gene expression. Oleandrin blocked NF-kappaB activation induced by phorbol ester and lipopolysaccharide. Oleandrin also blocked AP-1 activation induced by TNF and other agents and inhibited the TNF-induced activation of c-Jun NH2-terminal kinase. Overall, our results indicate that oleandrin inhibits activation of NF-kappaB and AP-1 and their associated kinases. This may provide a molecular basis for the ability of oleandrin to suppress inflammation and perhaps tumorigenesis.

Characterization of the in vitro and in vivo species preference of human and murine tumor necrosis factor-alpha.

The species preference of human and murine tumor necrosis factor-alpha (TNF-alpha) was evaluated in human and murine systems for cytotoxic/cytostatic effects and receptor binding in vitro and murine systems for toxicity and antitumor activity in vivo. The in vitro cytotoxic/cytostatic effects of both species TNF-alpha on human and murine cell lines as well as the receptor binding studies using 125I-labeled recombinant human TNF-alpha demonstrated homologous species preferences. Species preference of TNF-alpha was also apparent in toxicity studies with BALB/c nu/nu and CB6F1 mice, and antitumor responses of CB6F1 mice to s.c. Meth A sarcoma implants. Moreover the growth of Meth A sarcoma implanted i.p. was not inhibited by either human or murine TNF-alpha. These results are discussed in view of the potential for underestimation of the biological potency of TNF-alpha from heterologous sources.

Antitumor activity of the novel human breast cancer growth inhibitor, mammary-derived growth inhibitor-related gene, MRG.

A novel human tumor growth inhibitor was identified by differential cDNA sequencing. The predicted amino acid sequence of this tumor-suppressing factor has a significant sequence homology to mouse mammary-derived growth inhibitor and thus was named mammary-derived growth inhibitor-related gene (MRG). MRG was found to be expressed in normal and benign human breast tissues but not in breast carcinomas. In situ hybridization analysis demonstrated a stage-specific MRG expression as follows. MRG was barely detectable in breast carcinomas, showed partial and weak expression in benign hyperplasia, but was expressed at a high level in normal breast epithelial cells. To determine if MRG can modulate in vivo growth of human breast cancers, we transfected a full-length MRG cDNA into MDA-MB-231 human breast cancer cells and studied the orthotopic growth of MRG transfectants versus control transfectants in the mammary fat pad of athymic nude mice. Overexpression of MRG in human breast cancer cells significantly suppressed cell proliferation in vitro and tumor growth in an orthotopic nude mouse model. These results suggest that MRG has tumor-suppressing activity, and the loss of MRG expression may be involved in the development and progression of breast cancer.

All-trans-retinoic acid upregulates TNF receptors and potentiates TNF-induced activation of nuclear factors-kappaB, activated protein-1 and apoptosis in human lung cancer cells.

Retinoids modulate the growth and differentiation effects of TNF but the mechanism is not understood. In this study, we investigated the effect of all-trans-retinoic acid (ATRA) on the cell surface expression of TNF receptors and receptor-mediated signaling in various human lung cancer cell lines. ATRA treatment of cells that express wild-type p53 (A549 and H460), or null p53 (H1299), or mutant p53 (H596) increased the number of TNF receptors, as determined by the specific binding of 125I-labeled TNF to these cells, in a dose- and time-dependent manner. Treatment with 2 microm ATRA for 24 h at 37 degrees C produced the maximal increase. Scatchard analysis indicated that the increase induced by ATRA was due to an increase in receptor number and not to an increase in affinity. The upmodulation of TNF receptors was also confirmed by covalent receptor-ligand cross-linking studies. The increase in TNF receptors sensitized H596 cells to TNF-induced activation of NF-kappaB, AP-1 and apoptosis. A549 cells, however, were completely resistant to TNF-induced activation of NF-kappaB, AP-1 and apoptosis. Treatment of these cells with as little as 0.5 microM ATRA was effective in converting TNF-resistant cells to TNF-sensitive. Overall our results indicate that ATRA induces the TNF receptors in human lung cancer cells, which sensitizes them to TNF-induced signaling leading to activation of NF-kappaB, AP-1 and apoptosis.