Irreversible pan-ErbB tyrosine kinase inhibitor CI-1033 induces caspase-independent apoptosis in colorectal cancer DiFi cell line

The epidermal growth factor receptor (EGFR) is overexpressed in the majority of colorectal carcinomas and represents a target for therapeutic interventions with signal transduction inhibitors. We investigated the ability of CI-1033 to induce apoptosis and inhibition of proliferation in the colorectal cancer cell lines DiFi and Caco-2, which both express high levels of EGFR. While in Caco-2 cells CI-1033 treatment at a concentration 0.1 μ M for 72 hours demonstrated only antiproliferative (53.7 ± 4.3%) but no pro-apoptotic effects, treatment of DiFi cells resulted in a reduced proliferation rate (31.4 ± 3.1%) and in apoptosis (44.2 ± 8.9%). In order to define proteins involved in the regulation of apoptosis, we aimed to determine differences in the proteome profile of both cell lines before and after treatment with CI-1033. Cellular proteins were analyzed by 2-D gel electrophoresis followed by computational image analysis and mass spectrometry. Our data show that DiFi cells differ from Caco-2 cells in nine significantly upregulated proteins, and their potential role in apoptosis is described. We demonstrate that induction of apoptosis was triggered via caspase-independent pathways. Overexpression of leukocyte elastase inhibitor (LEI) and translocation of cathepsin D to the cytosol accompanied by upregulation of other defined proteins resulted in Bax-independent AIF translocation from mitochondria into the nucleus and apoptosis. Definition of these proteins can pave the way for functional studies and contribute to a better understanding of the effects of CI-1033 and the pathways of caspase-independent cell death.     

Effects of combinations of transforming growth factor-beta 1 and tumor necrosis factor on induction of differentiation of human myelogenous leukemic cell lines

Effects of transforming growth factor-beta 1 (TGF-beta 1), either alone or in combination with TNF, on the induction of differentiation of human myelogenous leukemic cell lines were examined. TGF-beta 1 alone induced differentiation of a human monocytic leukemia U-937 line into the cells with macrophage characteristics. When combined with TNF, TGF-beta 1 synergistically or additively induced differentiation associated properties. A human myeloblastic leukemia cell line, ML-1, differently responded to TGF-beta 1 in induction of differentiation. FcR activity and phagocytic activity induced by TNF were suppressed by TGF-beta 1. However, nitroblue tetrazolium reducing activity was synergistically induced by combinations of TGF-beta 1 and TNF. Scatchard analysis of TNF receptors indicated that the number of binding sites and dissociation constant of TNF for its receptors on U-937 or ML-1 cells were not changed by treatment with TGF-beta 1. Although IFN-gamma, IL-6, granulocyte CSF, and granulocyte-macrophage CSF-induced nitroblue tetrazolium reducing activity of U-937 cells, only IFN-gamma, and TNF induced it synergistically in combination with TGF-beta 1. Synergism between TGF-beta 1 and TNF was also observed in inhibition of growth of U-937 and ML-1 cells. Although TGF-beta 1 induction of differentiation of other monocytoid leukemic THP-1 cells was similar to that of U-937 cells, TGF-beta 1 only slightly induced differentiation of promyelocytic leukemic HL-60 cells, either alone or in combination with TNF. Our observations indicate that TGF-beta 1 strongly modulates differentiation and proliferation of human myelogenous leukemia cells, macrophage precursors.     

Purification of guinea pig tumor necrosis factor (TNF): comparison of its antiproliferative and differentiative activities for myeloid leukemic cell lines with those of recombinant human TNF

Recently, we suggested that the effect of differentiation inducing factor (D-factor) which is found in the supernatant of macrophages, and induced the differentiation of a mouse myeloid leukemic cell line, M1, into macrophage-like cells, may be a result of the cooperative effects of tumor necrosis factor (TNF) and interleukin 1 (IL-1). In this study, we purified guinea pig (G.P.) TNF secreted from peritoneal macrophages and compared the antiproliferative and differentiative effects of the G.P. TNF with those of recombinant human TNF (rHuTNF). The purification scheme consisted of ultrafiltration, gel filtration-high performance liquid chromatography (HPLC), DEAE-HPLC, and reverse-phase HPLC. The cytotoxic activity of the purified substance was approximately 1.5 x 10(8) U/mg. The isoelectric point was 5.2. The molecular weight was 40 to 45 kDa as estimated by gel filtration and 18 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The NH2-terminal amino acid sequence was determined to be Ser-Ala-Ser-Gln-Asn-Asp . . . . Approximately 76 or 71% homology between G.P. TNF and mouse or human TNF exists in the NH2-terminal 21 residues. The purified G.P. TNF and rHuTNF demonstrated D-factor activity only in the presence of recombinant human IL-1 alpha in M1 cells. We also determined the effect of TNF on two human myeloid leukemic cell lines (THP-1 and U937). The purified G.P. TNF and rHuTNF inhibited the growth of U937 cells, but did not induce their differentiation. In THP-1 cells, TNF slightly inhibited the growth and induced differentiation. In mouse cell lines G.P. TNF was more effective than rHuTNF for differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tumor necrosis factor-alpha mediates both apoptotic cell death and cell proliferation in a human hematopoietic cell line dependent on mitotic activity and receptor subtype expression

The TF-1 human erythroleukemic cell line exhibits opposing physiological responses toward tumor necrosis factor-alpha (TNF) treatment, dependent upon the mitotic state of the cells. Mitotically active cells in log growth respond to TNF by rapidly undergoing apoptosis whereas TNF exposure stimulates cellular proliferation in mitotically quiescent cells. The concentration-dependent TNF-induced apoptosis was monitored by cellular metabolic activity and confirmed by both DNA epifluorescence and DNA fragmentation. Moreover, these responses could be detected by measuring extracellular acidification activity, enabling rapid prediction (within approximately 1.5 h of TNF treatment) of the fate of the cell in response to TNF. Growth factor resupplementation of quiescent cells, resulting in reactivation of cell cycling, altered TNF action from a proliferative stimulus to an apoptotic signal. Expression levels of the type II TNF receptor subtype (p75TNFR) were found to correlate with sensitivity to TNF-induced apoptosis. Pretreatment of log growth TF-1 cells with a neutralizing anti-p75TNFR monoclonal antibody inhibited TNF-induced apoptosis by greater than 80%. Studies utilizing TNF receptor subtype-specific TNF mutants and neutralizing antisera implicated p75TNFR in TNF-dependent apoptotic signaling. These data show a bifunctional physiological role for TNF in TF-1 cells that is dependent on mitotic activity and controlled by the p75TNFR.     

Effect of fibroblast-derived differentiation inducing factor on the differentiation of human monocytoid and myeloid leukemia cell lines

A fibroblast-derived differentiation inducing factor (F-DIF) purified from medium conditioned by a human fibroblast cell line (WI-26VA4) induced differentiation of human monocytic leukemia cell lines (U-937, THP-1) into cells with macrophage characteristics. F-DIF alone induced the differentiation of ML-1 cells only marginally, but it synergistically increased the differentiation when combined with TNF. Interferon-gamma, tumor necrosis factor, GM-CSF, interleukin-1 and interlukin-4 synergistically enhanced the differentiation of U-937 cells when combined with F-DIF.     

Induction of tumor necrosis factor-alpha and its receptors during differentiation in myeloid leukemic cells along the monocytic pathway. A possible regulatory mechanism for TNF-alpha production

We examined the characteristics of tumor necrosis factor (TNF) receptors expressed on immature mouse myeloid leukemic cells (M1), M1 cells induced to differentiate into macrophages, and macrophage cells (Mm1 cells) by binding studies with radioiodinated TNF. Scatchard analysis of TNF binding revealed that a single class of high affinity receptor was present and that 750-1,100 receptors were expressed on each immature M1 cell. The number of TNF receptors was increased 1.5-2-fold on differentiated M1 cells and 4-5-fold on Mm1 cells with no change in affinity. The addition of interferon-gamma (IFN-gamma) up-regulated the expression of TNF receptors in differentiated M1 cells and Mm1 cells, while immature M1 cells were insensitive to IFN-gamma. The number of TNF receptors on the differentiated cells was increased 4-5-fold by the treatment with IFN-gamma with no change in the binding constant. The affinity of TNF receptors to human TNF-alpha (Kd = 1.7-2.8 nM) was lower than that to murine TNF-alpha (Kd = 0.2-0.7 nM). The assays for cell growth and [3H]thymidine incorporation suggested that no relation exists between the sensitivity of the cells to TNF-alpha and the number of TNF receptors. Enhancement of TNF-mediated cytotoxicity by the treatment with IFN-gamma did not correlate with increases in the number of TNF receptors. Cytolytic assays using L929 cells demonstrated that the amount of constitutive and lipopolysaccharide (LPS)-induced secretion of TNF-alpha was markedly increased during differentiation. Both the constitutive expression and IFN-gamma-mediated superinduction of TNF receptors, and the constitutive and LPS-induced secretion of TNF-alpha were closely related to the extent of cellular differentiation along the monocytic pathway. The time course of LPS-induced TNF-alpha activity showed a rise-and-decline profile with a peak at 2 h. On the other hand, the time course of the number of cell surface TNF receptors showed a decline-and-rise profile, a mirror image of the TNF-alpha activity time course profile in the supernatant. Anti-TNF-alpha antibody treatment blocked the LPS-induced down-regulation of TNF receptors and increased TNF-alpha mRNA accumulation. We discussed "an autoinhibitory system" in which an internalization of secreted TNF-alpha mediated by its own receptors is involved not only in decreasing TNF-alpha activity in the supernatant but also in reducing TNF-alpha mRNA expression.     

Tumor necrosis factor and vascular endothelial growth factor induce endothelial integrin repertories, regulating endovascular differentiation and apoptosis in a human extravillous trophoblast cell line

Angiogenesis is crucial in human development. Extravillous trophoblast (EVT) cells mimic endothelial cells in angiogenesis during endovascular differentiation, inducing a remodeling of spiral arteries that increases blood flow toward the intravillous space. We have previously shown that tumor necrosis factor (TNF) alpha regulates expression of ITGA6 and ITGA1, which are involved in cell survival, in the human EVT cell line TCL1. To further investigate endovascular differentiation, we examined the effects of vascular endothelial growth factor (VEGF), TNF, and extracellular matrix (ECM) on TCL1 cells. Seeded on Matrigel, TCL1 cells show tube-like formation that specifically recalls morphological changes in endothelial cells. Anti-ITGAV/ITGB3 antibodies significantly reduced the size of the capillary network (P < 0.05) on Matrigel and also suppressed TNF-induced apoptosis (P < 0.05) in TCL1 cells. VEGF induced expression of ITGAV/ITGB3 subunits and protein aggregation, as in the case of TNF, which in turn, induces synthesis of VEGF in TCL1 cells. Soluble FLT1 suppressed these activities in TCL1 cells, indicating that signals involving VEGF axis are essential for endovascular differentiation. These results suggest that TNF, VEGF, and ECM collaboratively regulate EVT behavior, including cell survival and endovascular differentiation, through integrin signaling during establishment and maintenance of successful human pregnancies.     

SYNERGISTIC REGULATORY EFFECTS OF TNFα, IL-1αAND IFNα ON THE GROWTH AND DIFFERENTIATION OF DAUDI LYMPHOMA CELLS

The regulatory effects of the combined treatment of tumour necrosis factorα (TNFα), interleukin-1α (IL-1α) and interferonα(IFNα) on the growth and differentiation of Daudi lymphoma cells were investigated. By means of anti-BrdU monoclonal antibodies and [³H-thymidine] incorporation a reduced proliferation rate was shown both through a combi-nation of TNFα with either IL-1α or IFNα and, above all, through simultaneous treatment with the three cytokines. In parallel, the degree of differentiation was evaluated via morphological criteria and detection of Fc receptors (FcR) and appeared higher after treatment with the three cytokines. Our results provide evidence of the increased sensitivity of this cell line to this combined treatment supporting the existence of a synergistic interaction in inducing the antiproliferative and differentiative effects.     

Characteristics of alpha-adrenoceptors in two human colorectal cancer cell lines.

The DiFi and HT-29 human colorectal cancer cell lines were characterized and compared with respect to binding properties of alpha adrenoceptors present on the cell surface. Both cell lines possessed alpha-1 and alpha-2 adrenoceptors of high affinity; however, DiFi cells were rich in alpha-1 adrenoceptors, whereas HT-29 cells were rich in alpha-2 adrenoceptors. In each cell line, specificity of radioligand binding to alpha-1 or alpha-2 adrenoceptors was proved via competition studies using non-tritiated drugs. We believe this to be the first characterization of alpha-1 adrenoceptors in cell line HT-29 and of alpha-1 and alpha-2 adrenoceptors in DiFi cells. Differences between these cell lines in alpha adrenoceptor expression are discussed in relation to colon carcinogenesis. The high level of alpha-1 adrenoceptors seen in DiFi cells should make this cell line useful in studies of the function and regulation of this adrenoceptor subtype.     

RNAi‐mediated silencing of insulin receptor substrate‐4 enhances actinomycin D‐ and tumor necrosis factor‐α‐induced cell death in hepatocarcinoma cancer cell lines

Insulin receptor substrate‐4 (IRS‐4) transmits signals from the insulin‐like growth factor receptor (IGF‐IR) and the insulin receptor (IR) to the PI3K/AKT and the ERK1/2 pathways. IRS‐4 expression increases dramatically after partial hepatectomy and plays an important role in HepG2 hepatoblastoma cell line proliferation/differentiation. In human hepatocarcinoma, IRS‐4 overexpression has been associated with tumor development. Herein, we describe the mechanism whereby IRS‐4 depletion induced by RNA interference (siRNA) sensitizes HepG2 cells to treatment with actinomycin D (Act D) and combined treatment with Act D plus tumor necrosis factor‐α (TNF‐α). Similar results have been obtained in HuH 7 and Chang cell lines. Act D therapy drove the cells to a mitochondrial‐dependent apoptotic program involving cytochrome c release, caspase 3 activation, PARP fragmentation and DNA laddering. TNF‐α amplifies the effect of Act D on HepG2 cell apoptosis increasing c‐jun N‐terminal kinase (JNK) activity, IκB‐α proteolysis and glutathione depletion. IRS‐4 depleted cells that were treated with Act D showed an increase in cytochrome c release and procaspase 3 and PARP proteolysis with respect to control cells. The mechanism involved in IRS‐4 action is independent of Akt, IκB kinase and JNK. IRS‐4 down regulation, however, decreased γ‐glutamylcysteine synthetase content and cell glutathione level in the presence of Act D plus TNF‐α. These results suggest that IRS‐4 protects HepG2 cells from oxidative stress induced by drug treatment. J. Cell. Biochem. 108: 1292–1301, 2009. © 2009 Wiley‐Liss, Inc.     

Effect of interleukin-8 on production of tumor-associated substances and autocrine growth of human liver and pancreatic cancer cells

We have previously reported that human liver cancer cell lines produce interleukin-8 (IL-8) at high levels. Those tumor cells appeared to express two kinds of IL-8 receptor on their surface. In order to analyze the role of IL-8 on the biological characteristics of those tumor cells, we suppressed IL-8 production from human liver (HuH-7 and HuCC-T1) and pancreatic cancer cell lines (HuP-T4) by treatment with IL-8 antisense oligonucleotides. Suppression of IL-8 production resulted not only in inhibition of cell growth, but also in an increase in the concentrations of some tumor-associated substances such as carbohydrate antigen 19-9 (CA19-9) in the medium. These data indicate that IL-8 produced by human liver and pancreatic tumors may act as an autocrine growth factor and may control the production of some tumor-associated substances. Furthermore, surface expression of sialyl-Lewis^a, which is a ligand for ELAM-1 on human umbilical vein endothelial cells (HUVEC), HuCC-T1 and HuP-T4 cells was decreased and the attachment of these tumor cells to HUVEC was inhibited by treatment with IL-8 antisense oligonucleotide. Since the soluble form of CA19-9 (sialyl-Lewis^a) was shown to inhibit the tumor cell binding to HUVEC, the decrease in release of CA19-9 into the medium and increase in the expression of sialyl-Lewis^a on the cell surface may suggest that IL-8 production from the tumor cells enhances metastatic potential by augmenting the binding activity of the tumor cells to HUVEC. These data demonstrate that a cytokine produced by tumor cells may function as an autocrine growth factor and affect tumor cell dissemination. Received: 4 February 1998 / Accepted: 6 May 1998     

Signalling pathway of tumor necrosis factor in normal and tumor cells

Summary Several aspects of the activity and effects of tumor necrosis factor (TNF) were investigated to gain further insight into its cytotoxic mechanism. The relation between number of TNF receptors and TNF susceptibility of both tumor cells and normal cells was studied, utilizing a specific binding assay. Among the tumor cells, a fairly close correlation (r=0.855) was observed between receptor number and sensitivity to TNF. No cytotoxic effect by TNF was observed on any of the normal cells tested, even though TNF receptors were shown to be present, and cell proliferation was apparently stimulated by TNF in some cases. TNF internalization and intracellular distribution were studied by pulse-labelling and Percoll density gradient centrifugation. In L-M (murine tumorigenic fibroblasts, highly sensitive to TNF cytotoxicity) cells and HEL (human embryonic lung cells, non-sensitive to TNF cytotoxicity) cells, receptor-bound ¹²⁵I-labelled recombinant human TNF was rapidly internalized and delivered to lysosomes within 15–30 min, and this was followed by degradation and release into the culture medium. The presence of either a cytoskeletal disrupting agent or a lysosomotropic agent was observed to inhibit the cytotoxic effect of TNF, thus also indicating that TNF internalization, followed by delivery to lysosomes, is essential in the cytolytic mechanism of TNF.As observed by [³H]uridine incorporation, TNF did not affect RNA synthesis in L-R cells (TNF-resistant cell lines derived from L-M cells) and HEL cells, but markedly stimulated (by 3.5 times) RNA synthesis in L-M cells.     

Tumor necrosis factor increases the number of epidermal growth factor receptors on human fibroblasts

Tumor necrosis factor (TNF) was shown previously to stimulate the growth of human FS-4 fibroblasts. Here we show that human recombinant TNF can increase the binding of epidermal growth factor (EGF) to these cells. Incubation with TNF resulted in a 40-80% increase in the number of EGF-binding sites with no apparent change in receptor binding affinity. The increase in EGF binding was apparent 8-12 h after the addition of TNF. TNF also increased the amount of EGF receptor protein immunoprecipitated from cells labeled with [35S]methionine. Stimulation of EGF receptor protein synthesis was demonstrable 2-4 h following TNF treatment. TNF increased EGF binding with a dose-response relationship similar to that reported earlier for the mitogenic action. Increased expression of EGF receptors, due to enhanced synthesis of the EGF receptor protein, may be functionally related to the mitogenic action of TNF in human fibroblasts.     

BG-1 ovarian cell line: An alternative model for examining estrogen-dependent growth in vitro

Summary Examination of estrogen-responsive processes in cell culture is used to investigate hormonal influence on cancer cell growth and gene expression. Most experimental studies have used breast cancer cell lines, in particular MCF7 cells, to investigate estrogen responsiveness. In this study we examined an ovarian cancer cell line, BG-1, which is highly estrogen-responsive in vitro. This observation, plus the fact that the cells are of ovarian rather than mammary gland origin, makes it an attractive alternative model. 17β-Estradiol, epidermal growth factor, and insulin-like growth factor induced proliferation of BG-1 and MCF7 cells. Viability was dependent on these growth factors in BG-1 cells, but not in MCF7 cells. Therefore, we examined the differences between these two cell lines with respect to estrogen and growth factor receptors. BG-1 cells have twice as many estrogen receptors as MCF7 cells, and BG-1 cells have higher insulin-like growth factor-1 and epidermal growth factor receptor levels than MCF7 cells. This may also explain why BG-1 cells proliferate 56% more robustly in serum and show more serum dependence in culture. In both BG-1 and MCF7 cells, epidermal growth factor receptor number is low (<20 000/cell), while insulin-like growth factor-1 receptor level was highest in estrogen receptor positive cell lines. For example, insulin-like growth factor-1 receptor was higher in BG-1 and MCF7 cells than in estrogen receptor negative cells (HeLa>MDA-MB-435>HBL100). In conclusion, BG-1 cells are an excellent model for understanding hormone responsiveness in ovarian tissue and an alternative for examining estrogen receptor-mediated and insulin-like growth factor-1/epidermal growth factor/estrogen cross-talk processes because of their sensitivity to these factors.     

Effect of recombinant tumor necrosis factor on HL-60 cells: cell-cycle specificity and synergism with actinomycin D

The tumor necrosis factor (TNF) exhibits a multitude of activities depending on the type of target cells. We characterized the cytostatic and cytotoxic effects of recombinant TNF, alone and in combination with actinomycin D (AMD), on the human leukemic cell line HL-60. Because HL-60 cells, when triggered to monocytic differentiation by phorbol esters, are known to produce and secrete TNF, their sensitivity to the factor could indicate an autocrine function of TNF in this cell system. Indeed, HL-60 cells were affected by TNF; their doubling time was increased by about 50% and progression through the cell cycle was perturbed. Initially, (up to 8 h) TNF induced a temporary arrest in G2 while later (24-48 h) it delayed progression through the G1 phase. Also, a transient increase in RNA content peaking at 6-8 h was apparent. The cytotoxicity of TNF alone was low. Thus, TNF may be involved in the regulation of the cell cycle of HL-60 cells during early stages of their differentiation. The cytotoxicity of TNF was markedly potentiated in the presence of AMD; the effect was AMD but not TNF concentration-dependent. Whereas at 20 and 50 ng/ml of AMD alone nonviable cells did not exceed 20% during the first 24 h of treatment, their proportion increased to 80 and 90%, respectively, in the presence of TNF. The most sensitive were cells in the S phase of the cell cycle. The observed synergistic effect of TNF and AMD does not appear to be caused by the action of TNF increasing the permeability of the cell membrane to AMD. The results indicate that HL-60 cells, ordinarily resistant to the cytotoxic action of TNF, can be rendered sensitive by treatment with AMD. This implies that a combination of TNF and AMD may be considered in oncology for treatment of tumors otherwise nonresponding to TNF alone.     

Lovastatin induces differentiation of Mono Mac 6 cells

The proliferation of human monocytic Mono Mac 6 cells was significantly retarded by treatment with lovastatin (LOV, 10 μM) for 72 h. Treatment of Mono Mac 6 cells with LOV increased surface protein expression of monocyte-associated CD14 and the integrin-chain CD11b towards levels found in isolated human blood monocytes. These effects were dose-dependent and completely reversed by the isoprenoid precursor mevalonate (MVA). LOV failed to induce growth retardation and upregulation of CD11b or CD14 in the less mature premonocytic U937 cell line. While CD11b expression was comparable in Mono Mac 6 cells treated with LOV (10 μM), TNF (100 U ml^?1) or LPS (10 ng ml^?1), upregulation of CD14 by LOV was less pronounced. Basal CD23 expression was unaffected by LOV but markedly reduced by treatment with TNF or LPS. Moreover, LOV enhanced Mono Mac 6 adhesiveness to human umbilical vein endothelial cells to levels found in isolated human blood monocytes, probably due to the increased CD11b and CD14 expression. In conclusion, LOV can induce differentiation of monocytic cells which is reflected by the retardation of growth, expression of CD14 and CD11b, and enhanced adhesiveness.     

Tumor necrosis factor modulates epidermal growth factor receptor phosphorylation and kinase activity in human tumor cells. Correlation with cytotoxicity

Tumor necrosis factor (TNF) is a cytokine which induces cytotoxicity in some but not all tumor cells. Initial studies of five tumor cell lines demonstrated that TNF was able to rapidly (within 30 min) modulate tyrosine protein kinase activity of epidermal growth factor (EGF) receptors on tumor cell lines which were sensitive to the cytotoxic effects of TNF but not alter EGF receptor kinase activity in TNF-resistant tumor cells. Two tumor cell lines (ME-180 cervical carcinoma and T24 bladder carcinoma) which have been shown to express similar TNF-binding characteristics but differ in their sensitivity to the cytotoxic actions of TNF were chosen for further characterization. Treatment of TNF-sensitive ME-180 cells with 1 nM TNF resulted in a 3-fold stimulation of EGF receptor tyrosine protein kinase activity within 10 min which correlated with increased phosphorylation of EGF receptor protein itself. In addition, dose-response studies indicate that similar concentrations of TNF modulate both ME-180 cell growth and EGF receptor kinase activity. Treatment of TNF-resistant T24 cells showed that TNF had no significant effect on their growth, EGF receptor tyrosine protein kinase activity, or phosphorylation of EGF receptor protein although EGF receptor kinase activity was stimulated by EGF. Quantitation of receptors expressed on the surface of ME-180 and T24 cells demonstrated a 3-fold difference between the number of EGF-binding sites on T24 (100,000) versus ME-180 cells (300,000), suggesting the relative abundance of EGF receptor does not solely account for differential effects of TNF on EGF receptor activation in these two cell lines. Phosphoamino acid analysis of EGF receptor from 32P-equilibrated ME-180 cells demonstrated that TNF-induced phosphorylation of amino acids which was quantitatively similar to that of EGF but distinct from the effects of phorbol ester. However, unlike EGF, TNF was unable to stimulate EGF receptor kinase activity in ME-180 cell lysates. The kinetics of EGF receptor activation and the metabolic consequence of activation of EGF receptor activity by TNF appear to be distinct from those induced by EGF. These results suggest that TNF-induced modulation of EGF receptor occurs through a unique mechanism and may play a role in the cytotoxic actions of TNF.