Impaired in vitro interferon, blastogenic, and natural killer cell responses to viral stimulation in acquired immune deficiency syndrome.

The in vitro immune response to herpes simplex virus (HSV), type 1, strain 539, HSV type 2, strain 316D, and cytomegalovirus was studied in 20 patients (14 with acquired immune deficiency syndrome, four with the acquired immune deficiency syndrome-related symptom complex, and two sexually active asymptomatic homosexuals) and 18 heterosexual healthy controls. Peripheral blood mononuclear cells were cultured with 2 X 10(5) plaque-forming units of heat-inactivated viruses, their lymphocyte blastogenic responses were measured after 5 days in culture by [3H]-thymidine incorporation, their interferon production was measured after 24 hr and 5 days, and natural killer (NK) cell activation was measured after 24 hr and 5 days of culture. Blastogenic responses to viruses were significantly low for only HSV, type 1:1.75 X 10(3) cpm in patients' cells compared to 6.36 for controls. Interferon responses to all three viruses were significantly low at both 24 hr and 5 days; e.g., HSV, type 1:139 IU/ml in patients' cells compared to 777 for controls at 24 hr. NK cell responses of patients were lower than those of controls when tested fresh and after 24 hr of incubation: 6.1 versus 11.7% and 9.2 versus 16.8% target cell lysis, respectively. Exposure to viruses boosted NK cell responses of both patients' and controls' cells, but boosting was generally greater among the normal rather than the patients' cells. The abnormalities of response were present in all three patient groups. Addition of interleukin-2 in vitro increased the patient and control blastogenic and NK responses but did not augment the interferon responses. The in vitro responses to both HSV, type 1, and HSV, type 2, correlated significantly with our conventional assays of the percentage and absolute level of T4+-helper lymphocytes in the blood and the blastogenic responses to mitogens, such as phytohemagglutinin, pokeweed mitogen, and concanavalin A. This system should be useful for the study of host defense in acquired immune deficiency syndrome patients and those in high-risk groups, and also for the in vitro evaluation of immunomodulators.

Adenoviral transgene expression of MMAC/PTEN in human glioma cells inhibits Akt activation and induces anoikis.

The MMAC/PTEN tumor suppressor gene encodes for a phosphatase that recently has been shown to have phosphotidylinositol phosphatase activity, implicating its possible involvement in phosphatidylinositol 3'-kinase-mediated signaling. To investigate possible alterations in growth factor-mediated signal transduction, an adenovirus containing MMAC/PTEN, Ad-MMAC, previously shown to inhibit growth and tumorigenicity in glioma cells, was used to acutely express the transgene. Human glioma cells infected with Ad-MMAC but not with control adenoviruses exhibited an inhibition of phosphorylation of both activating residues of Akt, Ser-473, and Thr-308, along with Akt's serine/threonine kinase activity, without significantly altering Akt expression. The effects of functional MMAC/PTEN expression were relatively specific, because members of several other growth factor-mediated signaling pathways showed no altered responses. The presence of MMAC/PTEN also inhibited phosphorylation of BAD, although no evidence of apoptosis in the in situ treated cells was observed. However, U251 glioma cells infected with Ad-MMAC were induced to undergo anoikis at a significantly higher rate than U251 cels treated with control viruses or mock infected with media. These results demonstrate that the acute administration of MMAC/PTEN results in the inhibition of Akt-mediated signaling, growth inhibition, and anoikis, implying that loss of MMAC/PTEN increases cellular proliferation and significantly augments a cell's survival potential during cellular processes that are associated with malignancy.

The PTEN/MMAC1/TEP tumor suppressor gene decreases cell growth and induces apoptosis and anoikis in breast cancer cells.

The PTEN/MMAC1/TEP (PTEN) tumor suppressor gene at 10q23.3 is mutated in multiple types of sporadic tumors including breast cancers and also in the germline of patients with the Cowden's breast cancer predisposition syndrome. The PTEN gene encodes a multifunctional phosphatase capable of dephosphorylating the same sites in membrane phosphatidylinositols phosphorylated by phosphatidylinositol 3'-kinase (PI3K). We demonstrate herein that loss of PTEN function in breast cancer cells results in an increase in basal levels of phosphorylation of multiple components of the P13K signaling cascade as well as an increase in duration of ligand-induced signaling through the P13K cascade. These alterations are reversed by wild-type but not phosphatase inactive PTEN. In the presence of high concentrations of serum, enforced expression of PTEN induces a predominant G1 arrest consistent with the capacity of PTEN to evoke increases in the expression of the p27Kip1 cyclin dependent kinase inhibitor. In the presence of low concentrations of serum, enforced PTEN expression results in a marked increase in cellular apoptosis, a finding which is consistent with the capacity of PTEN to alter the phosphorylation, and presumably function, of the AKT, BAD, p70S6 kinase and GSK3 alpha apoptosis regulators. Under anchorage-independent conditions, PTEN also induces anoikis, a form of apoptosis that occurs when cells are dissociated from the extracellular matrix, which is enhanced in conjunction with low serum culture conditions. Together, these data suggest that PTEN effects on the PI3K signaling cascade are influenced by the cell stimulatory context, and that depending on the exposure to growth factors and other exogenous stimuli such as integrin ligation, PTEN can induce cell cycle arrest, apoptosis or anoikis in breast cancer cells.

Molecular and biologic characterization of a newly established Philadelphia-positive acute lymphoblastic leukemia cell line (Z-33) with an autocrine response to GM-CSF.

We have recently established a new Philadelphia chromosome (Ph1)-positive acute lymphoblastic leukemia (ALL) cell line, designated Z-33. This line has L2 morphology, ultrastructural characteristics of lymphoblasts and typical B lineage surface markers identical to those observed in the Ph1-positive ALL patient from whom the line was derived. In addition, a rearranged immunoglobulin heavy-chain gene (JH) band was found in Z-33 cells by Southern blot analysis, confirming B cell clonality. Cytogenetic analysis of the cell line revealed t(9;22)(q34;q11.2). Polymerase chain reaction (PCR)-amplified cDNA from Z-33 cells demonstrated an e1-az BCR-ABL junction, and the p190BCR-ABL protein was detected in them by the immune complex kinase assay. Z-33 cells produce interleukin (IL)-1 beta, IL-6, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), tumor necrosis factor (TNF)-alpha, and transforming growth factor (TGF)-beta, Neither IL-1 beta, G-CSF, TNF-alpha, nor their corresponding antibodies affected the cell line's growth. In contrast, anti-GM-CSF neutralizing antibodies suppressed Z-33 colony formation, and GM-CSF stimulated it in a dose-dependent fashion. In addition, receptor studies with biotinylated GM-CSF demonstrated specific binding to Z-33 cells, indicating that the cells express GM-CSF receptors. Taken together, our data suggest that the Ph1-positive Z-33 ALL cells produce GM-CSF, express GM-CSF receptors, and show an autocrine proliferative response to this cytokine.

Overexpression of edg-2/vzg-1 induces apoptosis and anoikis in ovarian cancer cells in a lysophosphatidic acid-independent manner.

Lysophosphatidic acid (LPA) is one of the major growth factors in ascites from ovarian cancer patients and appears to play an important role in proliferation, survival, and invasion of ovarian cancer cells. Recently, several groups have shown that Edg-2, which belongs to the G-protein coupled receptor family, is a functional LPA receptor. Northern blot analysis showed that most ovarian cancer cell lines express Edg-2. Edg-2 expression was especially high in the cisplatin-resistant and slowly proliferating 2780cp cell line and was almost absent from the cisplatin-sensitive and rapidly proliferating A2780 cell line. We thus assessed whether Edg-2 could contribute to changes in cell viability, cell proliferation, or cisplatin resistance. Stable overexpression of Edg-2 in A2780 cells induced an exogenous LPA-independent decrease in proliferation but did not alter cisplatin sensitivity. The LPA-independent decrease in growth rate induced by overexpression of Edg-2 could be explained, at least in part, by Edg-2-induced apoptosis rather than by effects on cell cycle progression. In agreement with the results in stably transfected A2780 cells, transient expression of Edg-2 in Jurkat T cells also induced apoptosis. When cells were separated from the extracellular matrix, they underwent a specialized form of apoptosis called anoikis, which is particularly important in survival of cells in the circulation during metastasis. A2780 cells engineered to overexpress Edg-2 were particularly sensitive to anoikis. These observations suggest that Edg-2 may be a negative regulator for ovarian epithelial cell growth and metastasis.

In vitro and in vivo antitumor effect of the anti-CD26 monoclonal antibody 1F7 on human CD30+ anaplastic large cell T-cell lymphoma Karpas 299.

CD26 is a M(r) 110,000 surface glycoprotein with diverse functional properties, including having a potentially significant role in tumor development, and antibodies to CD26 mediate pleomorphic cellular functions. In this report, we show that binding of soluble anti-CD26 monoclonal Ab 1F7 inhibits the growth of the human CD30+ anaplastic large cell T-cell lymphoma cell line Karpas 299 in both in vitro and in vivo experiments. In vitro experiments show that 1F7 induces cell cycle arrest at the G1-S checkpoint, associated with enhanced p21 expression that is dependent on de novo protein synthesis. Furthermore, experiments with a severe combined immunodeficient mouse tumor model demonstrate that 1F7 treatment significantly enhances survival of tumor-bearing mice by inhibiting tumor formation. Our data therefore suggest that anti-CD26 treatment may have potential clinical use for CD26+ hematological malignancies.

Fas antigen signals proliferation of normal human diploid fibroblast and its mechanism is different from tumor necrosis factor receptor.

Recent cloning of the cDNA for Fas/Apo-1 and its ligand has revealed that they belong to the tumor necrosis factor (TNF) receptor and TNF family, respectively, and play an important role in apoptosis (programmed cell death). Like TNF, antibodies against the Fas antigen (anti-Fas) have been shown to be cytotoxic to Fas-expressing cells. Whether Fas, like TNF receptor, also mediates proliferation of normal human diploid fibroblasts (HDF), is not known. In this study, we show that HDF expresses Fas antigen and the engagement of this antigen signals proliferation of these cells in a dose-dependent manner. Unlike TNF receptor, however, Fas-mediated proliferation of HDF could not be blocked by orthovanadate, a tyrosine phosphatase inhibitor. The difference in the signaling was further evident from our observation that TNF induced the expression of interleukin-6 but anti-Fas did not. Overall, our results demonstrate for the first time that besides cell killing, Fas also mediates proliferation of HDF and that its mechanism is different from that of TNF receptor.

Qualitative and quantitative differences in the cellular responses mediated through Fas antigen and tumor necrosis factor receptor.

Like tumor necrosis factor (TNF), antibodies against the Fas antigen (anti-Fas) are cytotoxic to some and induce proliferation of other Fas-expressing cells. In this study, we compared cellular responses mediated through TNF with anti-Fas using a T cell line (Jurkat) and a macrophage cell line (U-937). These two cell types differed in that the Jurkat cells expressed higher levels of Fas antigen than U-937 cells, whereas the latter expressed higher levels of the p80 form of the TNF receptor than Jurkat cells. Treatment for 72 h with anti-Fas inhibited the growth of both Jurkat and U-937 cells, the 50% inhibitory concentrations (IC50) being 10 and 100 ng/ml, respectively. Under similar conditions, the IC50 for TNF was > 100 and 0.8 ng/ml for Jurkat and U-937 cells, respectively. Like TNF, the cytotoxic effects of anti-Fas were potentiated by cycloheximide, showing they did not require protein synthesis. Interestingly, in the presence of cycloheximide, the kinetics of cell killing was more rapid for TNF than anti-Fas (50% inhibition occurred at 3 versus 6h). Treatment of both cell types with anti-Fas led to time-dependent DNA fragmentation, but TNF-induced DNA fragmentation occurred only in the presence of cycloheximide. Pretreatment of cells with TNF led to resistance to TNF but not to anti-Fas, suggesting that the receptors for the two are not cross-modulated. Furthermore, TNF activated the nuclear transcriptional factor NF-kappa B in both cell types, whereas anti-Fas had no effect. Overall, our results demonstrate that anti-Fas and TNF transduce over-lapping and nonoverlapping signals in macrophage-like and T cell lines through distinct pathways.

Leukemia inhibitory factor binds to human breast cancer cells and stimulates their proliferation.

Leukemia inhibitory factor (LIF) is a cytokine that was originally described as a differentiation factor of a murine myeloid leukemia cell line and subsequently found to be an important mediator of embryonic development. Although extensively studied in the hematopoietic system, its effects on solid tumors are generally unknown. In the present study we investigated the role of LIF in human breast cancer cells. Using the reverse transcriptase-polymerase chain reaction, we found that the human breast carcinoma MCF-7 cell line expressed the message for both LIF receptor and its signal-transducing protein gp130, suggesting that these receptors might be biologically active. Binding studies with radiolabeled LIF demonstrated that MCF-7 cells interacted with this cytokine, and the ligand binding was specific and time, dose, and temperature dependent. In addition, a Scatchard analysis of the data revealed a single class of high-affinity (Kd 0.27 nM) receptors with a density of approximately 430 sites per cell. MCF-7 cells exposed to LIF internalized and degraded the ligand. LIF stimulated the growth of MCF-7 as well as other estrogen-dependent and independent breast cancer cell lines, but the effect on normal breast epithelial lines was less significant. Likewise, it stimulated colony formation by breast cancer cells obtained from five different breast cancer patients in a dose-dependent fashion. These results overall suggest that human breast tumor cells express functional LIF receptors that play a role in breast cancer cell proliferation.

Lysophospholipid growth factors in the initiation, progression, metastases, and management of ovarian cancer.

Levels of lysophosphatidic acid (LPA) and lysophosphatidylcholine (LPC) are elevated in the plasma and ascites of ovarian cancer patients, but not in most other tumor types. LPA increases cell proliferation, cell survival, resistance to cisplatin, cell shrinkage, and production of vascular endothelial growth factor, urokinase plasminogen activator, and LPA itself in ovarian cancer cells, but not in normal ovarian surface epithelial cells. PSP24 and members of the endothelial differentiation gene (EDG) family (EDG1, EDG2, EDG4, and EDG7) of G protein-coupled receptors mediate LPA signaling. Ovarian cancer cell lines do not express EDG1 mRNA, have variable EDG2 mRNA and protein levels, and frequently exhibit levels of EDG4 mRNA and protein, suggesting that EDG4 may contribute to the deleterious effects of LPA in ovarian cancer. In contrast, activation of the EDG2 LPA receptor on ovarian cancer cells may lead to apoptosis and counter the effects of other LPA receptors. Thus, the development of agonists and antagonists for the appropriate spectrum of LPA receptors may alter proliferation, apoptosis, or response to therapy of ovarian cancer cells. Indeed, over 60% of all current drugs target the G protein-coupled family of receptors, making the LPA receptor family a "drugable" target. LPC, although not as thoroughly studied, increases cellular proliferation and mediates multiple other functions through unique signaling pathways.

Synergism between alpha-interferon and interleukin-2-activated killer cells: in vitro studies.

The effects of a combination of recombinant alpha-interferon (IFN-alpha) and interleukin-2 (IL-2)-activated human killer cells (lymphokine-activated killer or LAK cells) on Hs294T (IFN-sensitive) and A375P (IFN-resistant) human melanoma cell lines were evaluated. Pretreatment of target cells with IFN-alpha for at least 1 day increased their susceptibility to the lytic activity of LAK cells. The combination of the two agents in sequence (IFN-alpha followed by LAK cells) resulted in a true synergystic killing of both IFN-alpha-sensitive and resistant tumor cells. No synergy was observed when the sequence was reversed (LAK cells followed by IFN-alpha). When peripheral blood mononuclear cells were incubated simultaneously with IFN-alpha and IL-2, LAK cell generation and antitumor activity was markedly inhibited when tested against both IFN-treated and -non-treated tumor cells. These studies may be used to plan clinical trials of combination cytokine therapy for human cancer.

Suramin inhibits tumor cell cytotoxicity mediated through natural killer cells, lymphokine-activated killer cells, monocytes, and tumor necrosis factor.

Suramin, a polysulfonated naphthylurea, is an antitrypanosomal and antifilarial drug. Because of its anti-reverse transcriptase activity and antiproliferative activity, suramin is also used for the treatment of acquired immunodeficiency syndrome and cancer. In spite of these uses, very little is known about its effects on the immune system. In this report, we investigated the effects of suramin on peripheral blood mononuclear cells. We found that natural killer (NK) cell-mediated cytotoxicity against human erythroblastoid cell line K562 was completely inhibited by suramin in a dose-dependent manner. It also completely blocked lymphokine-activated killer (LAK) cell-mediated cytotoxicity against the human B lymphoblastoid cell lines Raji and Daudi. The cytotoxicity against the human melanoma tumor cell line A-375 mediated by unstimulated and stimulated monocytes was also suppressed by suramin. Maximum inhibition of monocyte-mediated cytotoxicity was observed when suramin was present during both the activation and the effector phases of cytotoxicity. Besides its effects on cell-mediated cytotoxicity, suramin also inhibited the cytotoxic effects of tumor necrosis factor (TNF) against different tumor cell lines. Furthermore, we found that suramin interferes with the binding of TNF with its receptor. Thus our results indicate that suramin overall downregulates the immune system by inhibiting cell-mediated and TNF-mediated cytotoxicity against different tumor cells.

Tumor necrosis factor and lymphotoxin. Qualitative and quantitative differences in the mediation of early and late cellular response.

Tumor necrosis factor (TNF) is a 17-kDa protein produced by monocytes and a wide variety of other cell types in response to endotoxin and other cytokines. In contrast, lymphotoxin (LT) is a 25-kDa glycoprotein produced only by lymphocytes activated by mitogens. These two cytokines are 28% identical in their amino acid sequences. As they have common cell surface receptors, it is generally assumed that all cellular responses mediated through TNF are also mediated by LT and vice versa. In this report we tested this assumption, comparing the effect of TNF and LT on mediation of early (activation of the transcription factor NF-kappa B) and late (reduction of nitro blue tetrazolium, NBT) cellular responses in the human myelomonoblastic leukemic cell line ML-1a. Both qualitative and quantitative differences were found. LT was found to display 5-10 times more potent antiproliferative effects against murine fibroblasts than TNF. However, in ML-1a cells at concentrations wherein TNF activated NF-kappa B, LT did not. Higher concentrations (1,000-10,000 fold) of LT could activate NF-kappa B, but the activated complex was short lived (less than 1 h versus greater than 6 h when activated by TNF) and required longer treatment (15 min versus less than 5 min). TNF induced NBT-reducing activity in a dose-dependent manner, whereas LT was essentially inactive. Since both TNF and LT have been shown to bind to a common receptor, we tested whether the TNF-induced effects could be blocked by LT. LT inhibited both the early and late TNF-mediated cellular responses. By using receptor-blocking antibodies we found that both p60 and p80 forms of TNF receptors were functional for NBT-reducing activity, but TNF-dependent NF-kappa B activation required only the p60 receptor. Furthermore, we found that both TNF and LT bound with higher affinity to the p80 than to the p60 receptor. Thus, our overall results indicate that there are qualitative and quantitative differences in the action of TNF and LT, and these could be noted quite early in their signaling.

Retinoids downregulate both p60 and p80 forms of tumor necrosis factor receptors in human histiocytic lymphoma U-937 cells.

Because retinoids are known to modulate the growth and differentiation effects of tumor necrosis factor (TNF), we investigated the effect of all-trans-retinoic acid (RA) on the cell surface expression of TNF receptors in human histiocytic lymphoma U-937 cells. RA decreased the specific binding of 125I-labeled TNF to these cells in a dose- and time-dependent manner. The maximal decrease occurred when cells were treated with 1 mumol/L RA for 24 hours at 37 degrees C. Scatchard analysis of the binding indicated that the decrease by RA was caused by a decrease in receptor number and not by a decrease in affinity. The downmodulation of TNF receptors was also confirmed by covalent receptor-ligand cross-linking studies. Receptor-mediated internalization of the ligand was also found to be decreased on treatment of cells with RA. Northern blot analysis also indicated a decrease in the transcript of the receptor. By using antibodies specific to either the p60 or p80 form of the TNF receptor, we found that both receptors were downregulated by RA. RA treatment also decreased TNF receptors on acute monocytic leukemia cell line THP-1. Other analogues of RA, specifically 9-cis-RA, (E)-4-[2-(5,6,7,8- tetrahydro-2-naphthalenyl)-1-propenyl]-benzoic acid (TTNPB), and 3-methyl-TTNPB, which differ in their specificity towards different RA receptors, were also active in downregulating TNF receptors. 3-Methyl-TTNPB, which is more specific for the RXR form of the RA receptor, was found to be most potent. The downregulation of TNF receptors by RA correlated with the downmodulation of the antiproliferative effects of TNF against U-937 cells. Overall, our results indicate that RA downmodulates both the p60 and p80 form of the TNF receptor on cells of myeloid origin, which correlates with the cellular response.

TNF and its receptor antibody agonist differ in mediation of cellular responses.

TNF binds to two distinct receptors designated p60 and p80. Because Abs to the p60 receptor (anti-p60) can mimic TNF, we therefore compared the cellular signaling of TNF with that of anti-p60. We demonstrate both qualitative and quantitative differences between TNF and anti-p60. HepG2 cells, which express the p60 receptor, were found to be completely resistant to TNF but highly sensitive to the antiproliferative effects of anti-p60. In contrast, normal fibroblasts were found to be several fold more sensitive to TNF than to anti-p60. Several other epithelial cell lines that also express primarily the p60 receptor showed quantitative differences in mediation of cellular responses by TNF and anti-p60. The blocking of the p60 receptor by TNF had no effect on the response of HepG2 cells to anti-p60, suggesting a difference in their binding sites. Anti-p60, however, inhibited the effect of TNF on fibroblasts. Ab against the p80 receptor had no effect by itself or on the effect of TNF and anti-p60. The difference in the response to TNF and anti-p60 could not be correlated to the differences in the level of expression of p60 receptor on these cells. Furthermore, cycloheximide potentiated the TNF-mediated effect but not that mediated through anti-p60, thus also indicating a difference in the mechanism of action of these two agents. Overall, these results demonstrate that TNF and anti-p60, although both working through the p60 receptor, differ in their cellular signaling.

In vitro selection of NIH-3T3 cells for resistance to lymphotoxin induces resistance to activated macrophages and enhances tumorigenicity in vivo.

It is well known that expression of certain growth factors leads to tumorigenesis. However, the role of growth inhibitory molecules in this process is less certain. During the last few years several cytokines with growth inhibitory properties have been identified. In spite of the production of these cytokines by the body's immune system, the growth and progression of the tumor continue. In order to understand the mechanisms by which tumor escapes the host defense system, we have used lymphotoxin (LT), a lymphocyte-derived cytokine that is known to selectively inhibit the growth of certain tumor cells. The effect of LT was investigated on NIH-3T3 mouse fibroblast cells that are highly sensitive to its cytotoxic effects and are also tumorigenic in nude mice. On exposure to 10 units/ml of LT, 50% of these cells are killed within 24 h. A stable variant of NIH-3T3 cells that is completely resistant (LT-R) to even 10,000-fold higher concentration of the cytokine than that of sensitive cells (LT-S) was isolated in vitro by repeated exposure to LT. Both LT-S and LT-R displayed similar characteristics when grown both as a monolayer and in soft agar. No significant difference in LT receptor number or affinity between the two cell types was observed. It was not possible to overcome the resistance to LT by the addition of interferon-gamma but the resistance could be overcome by the presence of various chemotherapeutic agents suggesting a difference in the mechanism of action of these two agents.(ABSTRACT TRUNCATED AT 250 WORDS)

Fractionated extract of Astragalus membranaceus, a Chinese medicinal herb, potentiates LAK cell cytotoxicity generated by a low dose of recombinant interleukin-2.

Success with rIL-2 immunotherapy of human cancer appears to depend on the administration of high doses which are frequently associated with excessive toxicity. Future use of rIL-2 will require certain modifications based on the use of lower doses of rIL-2 without significant loss of antitumor efficacy. We tested in vitro the possibility of potentiating the activity of rIL-2 in terms of LAK cell generation. We hypothesized that co-incubation of LAK cell precursors with a Chinese herbal extract (F3) of Astragalus membranaceus, (an immune modulator currently under study in our laboratory), along with a low concentration of rIL-2, would generate levels of LAK cell activity equivalent to those generated by high concentrations of rIL-2 alone. We found (1) a 10-fold potentiation of rIL-2 activity manifested by tumor cell-killing activity of 80% resulting from LAK cell generation with F3 plus 100 u/ml of rIL-2 versus 76% generated by 1,000 u/ml of rIL-2 alone; (2) a significant reduction in the number of effector LAK cells required for equicytotoxic reaction following LAK cell generation with F3 plus rIL-2 compared to rIL-2 alone. We conclude that potentiation of antitumor activity mediated by rIL-2 in low concentrations is possible by the concomitant use of another immune modulator such as Astragalus membranaceus.

Tumor necrosis factor alpha and gamma-interferon enhancement of anti-epidermal growth factor receptor monoclonal antibody binding to human melanoma cells.

Previous studies have demonstrated that the expression of tumor-associated antigens can be regulated by cytokines. The purpose of this study was to determine whether tumor necrosis factor alpha (TNF alpha) and gamma-interferon (IFN gamma) were capable of modulating epidermal growth factor receptor (EGFr) immunorecognition on a human melanoma cell line in vitro. DX-3 melanoma cells treated for 24-72 h with various concentrations of each cytokine were incubated with an anti-EGFr monoclonal antibody (Mab) (A108) that recognizes an extracellular domain of the receptor, and differences in binding were analyzed by flow cytometry and radioimmunoassay. A dose- and time-dependent enhancement in EGFr immunorecognition was measurable in TNF alpha- and IFN gamma-treated cells. Combinations of these cytokines enhanced the recognition of EGFr on DX-3 cells to a level greater than that achieved with either TNF alpha or IFN gamma alone. Scatchard analysis of receptor binding curves revealed that there was no significant change in Mab affinity between control and cytokine-treated DX-3 melanoma cells, whereas a 1.5- to 1.8-fold enhancement in the number of Mab binding sites was measurable in TNF alpha- and IFN gamma-treated cells, respectively, when compared with controls. Immune complex kinase assay of EGFr showed threefold higher tyrosine kinase activity in TNF alpha-treated cells, but no change in kinase activity was observed following IFN gamma treatment.(ABSTRACT TRUNCATED AT 250 WORDS)