Growth of the endometrial adenocarcinoma cell line AN3 CA is modulated by tumor necrosis factor and its receptor is up-regulated by estrogen in vitro.

We demonstrate that tumor necrosis factor (TNF) has a biphasic effect on the growth of the human endometrial adenocarcinoma cell line AN3 CA in vitro. Low levels (0.2-5 pg/ml) of TNF were moderately growth stimulatory (up to 20% enhancement), while levels over 100 pg were growth inhibitory (up to 45% inhibition). Northern blot analysis showed expression of the 75-kilodalton (kDa) TNF receptor mRNA, but no expression of the 55-kDa TNF receptor mRNA or TNF mRNA. The growth of these cells was not directly affected by physiological concentrations (10(-7)-10(-9) M) of 17 beta-estradiol (E2). However, [125I]TNF binding studies and Scatchard analysis showed that 18-h coculture with 10(-8) M E2 increased the number of TNF receptors expressed on these cells 3-fold. Quantitative mRNA analysis confirmed that 75-kDa TNF receptor mRNA expression increased within 4 h of incubation with E2. These observations suggest an interaction between the endocrine and the immune systems, with an important implication for the homeostasis of endometrial tissues.

Purification of rabbit tumor necrosis factor.

Rabbit tumor necrosis factor (TNF) was purified and shown by SDS-PAGE to be a single protein of 18 kDa. TNF in 355 ml rabbit serum was precipitated with ammonium sulfate, and purified by repeated DEAE-Sephadex and Sephacryl S-200 chromatographies, and the final fractionation on Blue-Sepharose 6B. By this procedure its yield was 22% and its specific activity was 2.4 X 10(7) U/mg protein. The sequence of the N-terminal 20 amino acids was determined.

Spontaneous release of interleukin-6 by primary cultures of lymphoid and tumor cell populations purified from human ovarian carcinoma.

Interleukin-6 (IL-6) is a cytokine that has been implicated as a growth factor in human ovarian carcinoma, yet the in vivo source of IL-6 in patients remains undefined. We measured IL-6 by ELISA in cell-free ascites (CFA) of 19 patients with ovarian carcinoma. IL-6 was detectable in all samples (mean level 3.3 ng/ml). To identify the cellular source of IL-6, we measured this cytokine by ELISA in 24-48 h supernatants of cultured lymphocyte-, macrophage-, and tumor cell-enriched populations purified from three solid ovarian carcinomas by centrifugal elutriation. All cell populations spontaneously released IL-6; however, tumor cells and tumor-associated macrophage released levels of IL-6 that greatly exceeded those released by tumor-associated lymphocytes. Kinetic studies revealed that IL-6 was detectable at 6 h and that levels increased in all cultures examined over a 48 h time course. These data suggest that both tumor and infiltrating host cells may be the source of the high levels of IL-6 found in carcinomatous ascites. Furthermore, although all three cell types examined may contribute to IL-6 production in patients with ovarian carcinoma, tumor cells are perhaps the most clinically significant source.

A 20 amino acid synthetic peptide of a region from the 55 kDa human TNF receptor inhibits cytolytic and binding activities of recombinant human tumour necrosis factor in vitro.

Tumour Necrosis Factor (TNF) and Lymphotoxin (LT) can exert a wide range of effects on cells and tissues and they are important effector molecules in cell mediated immunity. All these effects are induced subsequent to the binding of these cytokines to specific membrane receptors. Recently, two of these membrane receptors of 55 and 75 kDa, have been identified which share some amino acid (AA) homology in their N-terminal extracellular domains but differ in their intracellular domains. We synthesized two synthetic 20 AA peptides from hydrophilic regions of the N-terminal extracellular domains of the 55 kDa receptor; peptide A shares homology with both 55 and 75 kDa receptors, peptide B is unique. We found peptide B inhibits both the binding and cytolytic activity of recombinant human TNF when tested on murine L929 cells in vitro. Polyclonal antiserum generated against peptide B will block binding of 125I-labelled TNF to these cells in vitro. However, peptide A and antiserum prepared against peptide A are without effect in these same assay systems. These data suggest that the 20 AA sequences from AA 175 to 194 in the N-terminal extracellular domain of the 55 kDa TNF receptor are expressed on the cell surface and are involved in the binding of TNF.

Effect of recombinant human tumor-necrosis-factor-alpha and dequalinium chloride on human ovarian-cancer cell-lines in-vitro.

Due to preferential uptake and retention, the small molecular weight lipophilic, cationic antimicrobial agent dequalinium chloride (DECA) displays potent in vitro and in vivo antitumor activity against carcinoma cells. The primary mechanism of DECA activity is directed against the mitochondria where it disrupts cellular energy production. One of the direct antitumor effects of tumor necrosis factor (TNF) is also targeted against the mitochondria. The ability of DECA to synergize this effect was examined in vitro against a panel of human ovarian cancer cell lines. The data from single agent and combined drug exposure were analyzed by the isobologram methods of Tsai et al (Cancer Res 49: 2390-2397, 1989). We demonstrate that TNF and DECA strongly synergize in vitro at clinically achievable doses for TNF and potentially clinically achievable doses for DECA. The degree of synergy varied with the cell line tested with UCI-101 being the least responsive and PA-1 cells displaying the greatest synergistic effect. DECA treatment also prolonged animal survival in mice bearing the PA-1 intraperitoneal ovarian carcinoma xenograft. Single agent DECA (5 mg/kg; qod) increased animal survival by 37% (p=0.002) whereas recombinant human TNF (0.5 mug/mouse; qod) increased survival by 12% (p=0.27) in those animals treated 3 days post tumor injection. Sequential DECA/TNF enhanced animal survival by 45% (p=0.0002) in similarly treated animals. DECA, as a mitochondrial poison is an agent capable of potentiating the effects of tumor necrosis factor against ovarian cancer cell lines.

Tumour necrosis factor (TNF) binding proteins (soluble TNF receptor forms) with possible roles in inflammation and malignancy.

Inhibitors of Tumour Necrosis Factor (TNF) may be necessary for protection of the host against harmful systemic manifestations of this cytokine such as in the septic syndrome and inflammatory conditions. TNF-binding proteins (TNF-BP) have been identified and shown to be the soluble extracellular domains of two transmembrane TNF receptors produced by proteolytic cleavage. TNF-BP inactivates TNF by formation of high affinity complexes thereby reducing the binding of TNF to target cell membrane receptors. In addition, TNF is stabilized in complex with TNF-BP, and under certain conditions the complex may act as a slow releaser of biologically active TNF. TNF can induce the release of TNF-BP in vivo which might neutralize the bioactivity of TNF. Cytokine control by natural and recombinant cytokine inhibitors such as TNF-BP could be a promising therapeutic approach in chronic inflammatory disorders to shift the balance between a cytokine-induced response and counteracting "anticytokines". A local production of TNF-BP in some tumour tissues may inactivate TNF for the benefit of the tumour. In some leukemias e.g. B-cell chronic lymphocytic leukemia, where TNF can act as a growth factor for the malignant cells, TNF-BP may be growth inhibitory.

Involvement of two types of TNF receptor in TNF-alpha induced neutrophil apoptosis.

We previously demonstrated that tumor necrosis factor-alpha (TNF-alpha) induces rapid human neutrophil apoptosis. In this paper, we examined which of the TNF receptors, p55 kDa TNF receptor (55-R) or p75 kDa TNF receptor (75-R), or both are involved in this process using specific rabbit antisera. Antibodies to 55-R (anti55-R) or 75-R (anti75-R) alone did not induce neutrophil apoptosis. Further addition of cycloheximide and anti-rabbit immunoglobulin to anti55-R but not to anti75-R accelerated apoptosis, although anti75-R augmented the capacity of anti55-R to do so. These results suggest that 55-R is a prerequisite for TNF-alpha induced neutrophil apoptosis.

Mechanism of release of soluble forms of tumor necrosis factor/lymphotoxin receptors by phorbol myristate acetate-stimulated human THP-1 cells in vitro.

The mechanism involved in the release of the soluble forms of 55 and 75 kDa TNF and lymphotoxin (LT) membrane receptors was studied in a continuous human monocytic cell line, THP-1, in vitro. THP-1 cells were found to spontaneously release soluble forms of both 55 and 75 kDa TNF/LT receptors. Release was up-regulated by PMA, and optimal release was achieved at 10(-8) M PMA. Serine protease inhibitors such as PMSF,3,4 dichloroisocoumarin, N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) were found to inhibit the production of both soluble TNF/LT receptors. PMSF (2 mM) also blocked receptors shedding from paraformaldehyde-fixed THP-1 cells coincubated with conditioned media from PMA-stimulated THP-1 cells. Colchicine at 1 and 10 microM stimulated the production of both soluble TNF/LT receptors, but the PMA-induced release of both soluble TNF/LT receptors was inhibited. It appears that the PMA-induced release of soluble TNF/LT receptors involves serine proteases in the extracellular space where the soluble parts of the TNF/LT receptors are cleaved directly off the cell membrane.

Primary structure of two mRNAs encoding putative salmon alpha-subunits of pituitary glycoprotein hormone.

1. By random sequence analyses, we isolated from the cDNA library of salmon pituitary glands two clones, the deduced amino acid sequences corresponding to the C-terminal region of which are almost the same as those of the alpha subunits of mammalian glycoprotein hormones. 2. Comparison of the nucleotide sequences and deduced amino acid sequences from these two clones with those of mammalian species revealed that the two newly-isolated cDNAs corresponded to mRNAs encoding the putative salmon pre-alpha subunit of glycoprotein hormones. 3. Homology in the nucleotide sequences of these two clones suggested that corresponding mRNAs may be encoded by separate genes which probably evolved from a common ancestral gene.

Combination antitumor therapy with rabbit tumor necrosis factor and chemo- and immuno-therapeutic agents against murine tumors.

The antitumor activity of partially purified rabbit tumor necrosis factor (TNF) in combination with lentinan or chemotherapeutic agents was tested. Partially purified TNF was obtained from TNF-containing rabbit sera by salt precipitation and ion-exchange chromatography. Its specific activity, determined in vitro as its cytotoxic activity against L929 cells in the presence of actinomycin D, was 10(5) U/mg protein or about 30-fold that of the crude rabbit sera. The growths of MH134 hepatoma in C3H/He mice and Lewis lung carcinoma in C57BL/6 mice were partially inhibited by intratumoral administration of a suboptimal dose of the TNF preparation. Additional treatment with lentinan, actinomycin D, mitomycin C or adriamycin significantly increased the antitumor activity. In particular, combination therapy with TNF and lentinan was very effective against MH134 hepatoma without having detectable side-effects, showing that lentinan expanded the therapeutical potential of TNF. These results are discussed in relation to our previous results on combination antitumor therapy.

Release of soluble TNF/LT receptors from a human ovarian tumor cell line (PA-1) by stimulation with cytokines in vitro.

We have demonstrated the presence of the 55- and 75-kDa receptor for tumor necrosis factor (TNF) and lymphotoxin (LT) (TNF-R) in serum, and ascites from women with ovarian cancer. The present studies were initiated to begin to examine the possible cellular source of these receptors in women with ovarian cancer. Human ovarian tumor cells (PA-1) were cocultured for 24-48 hr with various levels of recombinant human cytokines (IL-1 beta, IL-4, IFN-gamma) and the supernatants were assayed by ELISA for the soluble forms of each receptor. PA-1 cells spontaneously release the 55-kDa TNF-R and low levels of the 75-kDa TNF-R. The release of both 55- and 75-kDa TNF-R was stimulated when PA-1 cells were cultured with IL-1 beta and IFN-gamma but unaffected by IL-4. The level of 55-kDa TNF-R was elevated slightly over spontaneous release but the level of 75-kDa TNF-R increased dramatically. IFN-gamma was the most potent stimulator of receptor release particularly of the 75-kDa TNF-R. IFN-gamma also induced increased expression of cell membrane TNF-R measured by binding of 125I-labeled TNF. Membrane TNF-Rs which were induced by IFN-gamma were the 75-kDa type, because TNF binding was blocked by anti-75-kDa TNF-R antibody. These data suggest that IFN-gamma selectively induced release and expression of 75-kDa TNF-Rs.

Antitumor effect of systemic administration of novel recombinant tumor necrosis factor (rTNF-S) with less toxicity than conventional rTNF-alpha in vivo.

This study reported that acute toxicity (expressed as the LD50 value) of rTNF-SAM1 and rTNF-SAM2 which we constructed was considerably lower than that of rTNF-alpha (1). Following this finding, the antitumor effects of systemic administration of these novel recombinant tumor necrosis factors (TNF-S) (named rTNF-SAM1 and rTNF-SAM2) were examined in vivo on murine tumors (Meth A fibrosarcoma, MH134 hepatoma, and B16 melanoma). Both rTNF-SAM1 and rTNF-SAM2 could be administered systemically to tumor-bearing mice at doses up to 3 X 10(4) to 10(5) U. Growth of all tumors was significantly inhibited by systemic administration of rTNF-SAM1 or rTNF-SAM2 at a dose of greater than 3 X 10(4) U; at this dosage conventional rTNF-alpha could not be administered systemically to any of the mice strains due to its toxicity. These results confirm our previous finding that the rTNF-SAM group can be administered with safety at higher doses than rTNF-alpha, and revealed that these novel rTNF-S could be more promising antitumor drugs than rTNF-alpha in view of their lower toxicity compared with antitumor effect.

Production in Escherichia coli of human thymosin beta 4 as chimeric protein with human tumor necrosis factor.

We have produced thymosin beta 4 protein in Escherichia coli as a chimeric protein with tumor necrosis factor (TNF). The protein was abundantly expressed, was immunoreactive against both anti-thymosin beta 4 and anti-TNF antibodies, and retained cytotoxicity in a TNF assay using mouse L929 fibroblasts. This latter characteristic enabled the easy and simple purification of thymosin beta 4 merely by following the TNF activity. The chimeric protein was designed to have an Asp-Pro bridge between thymosin beta 4 and TNF which could be specifically cleaved under suitable acidic conditions to release the thymosin beta 4 from the chimeric protein. These results indicate that the expression system in E.coli of a chimeric protein composed of thymosin beta 4 and TNF is appropriate for obtaining an abundant amount of the beta 4 peptide, especially since its purification from tissues is usually difficult because of limited yield and obscurity of its biological activity.

Transforming growth factor-beta 1 down-regulates expression of membrane-associated lymphotoxin and secretion of soluble lymphotoxin by human lymphokine-activated killer T cells in vitro.

Lymphokine-activated T killer lymphocytes (T-LAK) are important effector cells in various diseases of tissue destructive reactions. They require stimulation with various cytokines to proliferate and mature into function effector cells. We have examined the role of various endogenously and exogenously added cytokines, and tumor necrosis factor (TNF)/lymphotoxin (LT) receptors in this process in vitro. The present report is a continuation of these studies. We found that human T-LAK cells express membrane-associated LT (mLT) but not TNF, and secrete high amount of soluble LT (sLT) but low levels of TNF. When added to the initial cultures or immature T-LAK cells, transforming growth factor-beta 1 (TGF-beta 1) suppressed both mLT expression and sLT secretion by 30-40%. Coculture of mature T-LAK cells with TGF-beta 1 caused 35% down-regulation of both mLT expression and sLT secretion after 18 h of incubation. Kinetic experiments indicated reduction of LT mRNA synthesis could occur in as little as 1 h when cocultured with 5 ng/ml of TGF-beta 1. TGF-beta 1 also reduced mLT induced T-LAK cell cytolytic activity on L929 cells in vitro. It appears TGF-beta 1 can down-regulate LT mRNA syntheses, mLT expression, and sLT secretion of human T-LAK cells in vitro.

Studies of membrane-associated and soluble (secreted) lymphotoxin in human lymphokine-activated T-killer cells in vitro.

The expression, release, and cytolytic activity of membrane-associated lymphotoxin was examined in cultures of phytohemagglutinin-P (PHA-P) and interleukin 2 (IL-2)-stimulated human T-lymphokine-activated killer (T-LAK) cells in vitro. Lymphotoxin (LT/TNF-beta) was identified on the membrane of T-LAK cells using flow cytometry. The membrane form of LT (mLT) is detected not only on T-LAK cells but also on LT-secreting human B-cell lymphoid cell lines, RPMI 1788 and Raji, but not on U937 or K562 cells. Maximum expression of mLT on T-LAK cells and the secretion of LT into the supernatant depended on the concentration of IL-2. Expression of mLT on T-LAK cells was reduced by stimulation with PHA-P; however, supernatant LT levels greatly increased. Both expression of mLT and release of soluble LT was reduced after incubation of T-LAK cells with actinomycin D (ActD) or cycloheximide (CHx). Paraformaldehyde-fixed T-LAK cells caused cytolysis of WEHI 164 cells in vitro, which was blocked by anti-LT but not anti-TNF antibody. These data support the concept that mLT may be an intermediate form to secreted LT, and that the mLT form is cytolytically active.

Lymphocyte and monocyte-induced motility of MCF-7 cells by tumor necrosis factor-alpha.

A potentially important tumor-host interaction is increased tumor-cell invasiveness in response to motility factors derived from stromal and lymphoid cells. Conditioned medium of IL-2-stimulated lymphocytes and fractions enriched in either T cells, natural killer (NK) cells, or monocytes induced motility in MCF-7 breast carcinoma cells. ELISA and antibody neutralization studies demonstrated that this effect was due to tumor necrosis factor-alpha (TNF-alpha) secretion by the lymphoid cells or the enriched fractions. Unstimulated leukocytes in direct contact with MCF-7 cells also induced motility that was inhibited by anti-TNF-alpha antiserum. Time-lapse video microscopy of cells exposed to 10 ng/ml TNF-alpha showed that motility was independent of its toxic effects. Immunoperoxidase showed that MCF-7 cells expressed both the 55-kDa and the 75-kDa TNF-alpha receptors (TNFR). Antiserum against the 55-kDa TNFR, like TNF-alpha, induced motility in MCF-7 cells. This was most likely due to cross-linking of the 55-kDa TNFR monomers, since the monomeric F(ab) did not produce this effect. Our results raise the possibility that TNF-alpha-induced motility is one mechanism by which tumor cells overcome the potential anti-tumor immune function of lymphocytes and macrophages in peri-tumoral infiltrates.

Prostaglandin-E2 regulation of tumor necrosis factor receptor release in human monocytic THP-1 cells.

Recent in vitro studies indicate that tumor necrosis factor (TNF) production in human monocytic THP-1 cells is suppressed by action of arachidonic acid metabolite prostaglandin-E2 (PGE2). PGE2 stimulation of human monocytic cell line THP-1 demonstrates that PGE2 not only regulates TNF activity at production levels, but does so through the release of two soluble TNF receptors (BP-55, BP-75) as well. PGE2 can thus exert a regulatory effect on TNF biologic activity by interfering with its ability to reach cell membrane receptors. THP-1 cells were activated with PGE2 for either 2- or 6-hr time periods, and the supernatants subsequently tested by ELISA to quantitate the levels of soluble receptor released. In addition, we examined mechanisms of receptor shedding by investigating the rate of membrane internalization and the role of serine proteases. PGE2-stimulated THP-1 cells showed soluble 55- and 75-kDa TNF receptor release levels which exceeded that of spontaneous release at both 2- and 6-hr activation periods. The numbers of both membrane TNF receptors were significantly upregulated as well in PGE2-activated cells, whereas the levels of 55- and 75-kDa TNF receptor mRNA levels remained unchanged. Thus, PGE2 induces TNF receptor release primarily at posttranscriptional levels. Inhibition of serine proteases with Pefabloc, a phenylmethylsulfonyl fluoride analog, resulted in the inhibition of both spontaneous and PGE2-stimulated release. Treatment of THP-1 cells with N-ethylmaleimide and low-temperature incubation, both known to block membrane internalization, also blocked spontaneous and PGE2-induced release. Internalization and cleavage by protease are therefore critical factors in PGE2-induced release of soluble TNF receptor shedding.

Detection of a countertranscript in promyelocytic leukemia cells HL60 during early differentiation by TPA.

We have isolated several cDNA clones corresponding to the mRNAs expressed during early phase of differentiation of promyelocytic leukemia cells HL60 by TPA. Two interrelated clones were examined, one pHH81 and the other pHH58 possessing inserts of 180 and 730 base pairs, respectively. Northern blot analyses of poly(A) RNAs from induced cells revealed that the clone pHH81 hybridized with 4.3kb RNA, while the clone pHH58 hybridized with 4.3kb and, in addition 0.7kb RNA. Sequence determination of those cDNA clones and extensive Northern blot analyses revealed that the inserts of these clones were derived from 4.3kb mRNAs. 0.7kb RNA was hybridized with only 5' upstream region of the clone pHH58, especially with the strand designed to detect anti-sense RNA. Thus we concluded that 0.7kb RNA is a countertranscript of 4.3kb RNA expressed during early differentiation of HL60 cells.

The regulation of TNF receptor mRNA synthesis, membrane expression, and release by PMA- and LPS-stimulated human monocytic THP-1 cells in vitro.

The regulation of the 55-kDa TNF receptor (TNF-R) mRNA synthesis, membrane expression, and TNF binding factor (BF) release was examined in resting and activated human monocytic THP-1 and human promyelocytic leukemia HL-60 cells in vitro. Cells were activated with phorbol myristate acetate (PMA) and bacterial lipopolysaccharide (LPS). TNF alpha cytolytic activity in the supernatant of THP-1 cells stimulated by PMA began to appear at 4 hr, reached a peak at 8 hr, and declined by 12 hr. For THP-1 cells stimulated with LPS, the peak of TNF alpha activity appeared at 4 hr and then declined. TNF alpha-binding sites on the cell membrane were down-regulated within 1 hr after PMA and LPS treatment and then reappeared 12 hr later. Fifty-five-kilodalton TNF-R mRNA expression during this time period did not correlate with the level of membrane TNF-binding site expression. Additional studies indicated the presence of a 30-kDa TNF-BF in the supernatants which appeared after 24 hr. These data suggest that activated THP-1 and HL-60 cells are capable of releasing TNF-BF into the supernatant and this material may be involved in the control of secreted TNF alpha activities.