Biomedical subjects

B B Aggarwal

Publications and source records attributed to B B Aggarwal.

At least 127 records · Page 7Linked to original sources

PubMed1995

Phase I study of tumor necrosis factor plus actinomycin D in patients with androgen-independent prostate cancer.

Based on preclinical studies which reveal enhanced antitumor activity of tumor necrosis factor (TNF) when combined with actinomycin D in human prostate cancer cell lines, we performed a phase I clinical study combining TNF and actinomycin D. All patients had metastatic prostatic carcinoma exhibiting androgen-independent growth. Patients were treated with a combination of a short infusion of actinomycin D followed by a TNF infusion daily for five consecutive days. Soluble TNF receptor p60 was not modulated by treatment but p80 receptor increased significantly following treatment with a combination of TNF and actinomycin D (baseline median 3.4 ng/ml) range 2.5-6.6 ng/ml follow up (9.3 ng/ml) range 6-24 ng/ml. We concluded that the maximum tolerated dose of continuous infusion TNF and short infusion actinomycin D is 400 micrograms/m2 of actinomycin D and 400 micrograms/m2 of TNF. The increased soluble receptor isoform (p80) may account for the lack of clinical activity seen in this trial. Should these results be confirmed, a strategy focused on overcoming the upregulation of the TNF soluble receptor will be required before further study of TNF should be considered.

Aged↗

PubMed1995

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.

Base Sequence↗

PubMed1995

Suramin blocks binding of interleukin-4 to its receptors on human tumor cells and interleukin-4-induced mitogenic response.

Suramin, a polysulphonated naphthylurea, has antiproliferative, anticancer, and anti-HIV activities and has been shown to prevent binding of a variety of growth factors to their respective receptors. In the current study we have investigated the effects of suramin on binding of interleukin-4 (IL-4) to its receptors and IL-4-induced biological response. We found that suramin prevented the binding of 125I-labeled IL-4 to its receptor in a dose-dependent manner. The concentration of suramin that caused 50% inhibition of IL-4 binding (IC50) ranged between 55 and 70 microM. This effect was observed on two human renal cell carcinoma cell lines (PM-RCC and WS-RCC), a human T lymphoma cell line (H9), and a human premyeloid cell line (TF-1). Cross-linking experiments provided direct evidence that suramin prevented binding of 125I-labeled IL-4 to its receptors. Radiolabeled IL-4 specifically cross-linked with major proteins of approximately 145 and 65-70 kDa in both PM-RCC and H9 cell lines. Suramin prevented cross-linking to both affinity cross-linked IL-4 binding proteins. Gel filtration results indicated that suramin caused aggregation of 125I-labeled IL-4. Suramin had a cytostatic rather than cytotoxic effect on H9 cells, and it inhibited IL-4-induced proliferation of TF-1 cells. These data indicate that suramin may be a useful drug in the abrogation of IL-4-induced effects, and this property should be further explored in IL-4-mediated pathologic states.

Antigens, CD↗

PubMed1994

Role of tumor necrosis factor receptors in the activation of nuclear factor kappa B in human histiocytic lymphoma U-937 cells.

Tumor necrosis factor (TNF) has been shown to mediate numerous cellular responses through its interaction with two distinct types of receptor. However, the relationship between TNF receptor and the biological response is not well understood. Modulation of the number of cell surface receptors by various agents has shown a lack of direct correlation with biological responses to the cytokine. In this report, we used several approaches to investigate the relationship between TNF receptor number and an early response in human histiocytic lymphoma U-937 cells. When we examined the activation of the nuclear transcription factor kappa B (NF-kappa B), an event mediated by TNF within 10-15 min, we discovered a correlation between TNF receptor occupancy up to a certain threshold and the extent of activation of the transcription factor. In addition, by kinetically down-regulating TNF receptor expression with phorbol esters, cycloheximide, or trypsin, we determined that receptors were necessary for transduction of the TNF signal. However, 10-25% of total receptors were sufficient for optimum induction of the NF-kappa B signal. When examined in different cell lines, the activation of an early biological response was found to be related not only to the TNF receptor number but also to the type of TNF receptor. These results, overall, suggest that although TNF receptors are essential for induction of NF-kappa B, a small percentage is sufficient to fully transduce this signal.

Cycloheximide↗

PubMed1994

Transfection of cells with transforming growth factor-alpha leads to cellular resistance to the antiproliferative effects of tumor necrosis factor.

Tumor necrosis factor (TNF) is a growth-modulatory cytokine that inhibits the growth of certain cell lines, stimulates the growth of some, and has no effect on the growth of still others. The molecular basis for this differential regulation of growth by TNF is not understood. We postulate that the growth of normal or tumor cells is determined by the balance between growth-stimulatory and -inhibitory signals. In the present study, we demonstrate that the transfection of cells with the transforming growth factor (TGF)-alpha gene induces resistance to TNF. Colon carcinoma cell lines that express elevated levels of TGF-alpha were also found to be resistant to this cytokine. Exogenous addition of the growth factor was also effective in decreasing the antiproliferative effects of TNF. Transfection of cells with the TGF-alpha gene led to downmodulation of TNF receptors but an increase in intracellular glutathione levels. Thus, these results support our hypothesis that expression of growth factors by certain tumor cells can lead to resistance to antiproliferative agents such as TNF.

3T3 Cells↗

PubMed1994

Role of sulfhydryl groups in induction of cell surface down-modulation and shedding of extracellular domain of human TNF receptors in human histiocytic lymphoma U937 cells.

The interaction of different polypeptide growth factors with their cell surface receptors, which are typically rich in cysteine, is modulated by sulfhydryl (SH) reagents. Because the extracellular domain of both human TNFRs are likewise rich in cysteine residues, we examined the effect of SH reagents on these receptors in human histiocytic lymphoma U937 cells, which express both p60 and p80 forms. Iodoacetamide induced down-regulation of cell surface TNFRs in a dose- and time-dependent manner. Down-regulation was complete at 10 mM IAA for 1 h at 37 degrees C. The decrease was minimal at 4 degrees C. The down-modulation was also observed with other cell-permeable and -impermeable thiol reagents. The expression of other cell surface molecules such as CD3, CD11b, CD23, and CD25 were not affected. IAA induced the down-regulation of TNFR on cells of both epithelial and myeloid origin. With the use of receptor-specific Abs, we found that the kinetics of down-modulation of the p60 and p80 receptors by IAA were very similar. We also found that down-modulation was not a result of internalization but rather of the shedding of the receptors, as ascertained by receptor-ligand cross-linking analysis, immunoassay, Western blot analysis, and ligand-blot analysis. When TNFRs with a molecular mass of 80 kDa disappeared from the cell surface, a 42-kDa polypeptide appeared in the medium. Thus, we demonstrate that SH reagents down-regulate TNFRs by inducing shedding of both receptors from the cell surface, most likely by activating an endopeptidase that cleaves the receptor.

Cell Membrane↗

PubMed1994

Reconstitution of nuclear factor kappa B activation induced by tumor necrosis factor requires membrane-associated components. Comparison with pathway activated by ceramide.

Tumor necrosis factor (TNF) is known to induce the activation of a nuclear transcription factor, nuclear factor kappa B (NF-kappa B), in a wide variety of cell types. The post-receptor binding events that culminate in TNF-dependent NF-kappa B activation are not understood. To dissect this pathway, we developed a reconstitution system consisting of membrane, cytosolic, and post-nuclear fractions. Our results indicate that when incubated with the post-nuclear fraction derived from TNF-untreated cells, the membrane fraction from TNF-treated cells causes the activation of NF-kappa B with kinetics similar to that observed in intact cells. Under these conditions, the cytosolic fraction has no effect. This activation is tyrosine kinase-dependent since erbstatin completely abolished the effect. Furthermore, as revealed by immunoblotting, no degradation of the inhibitory subunit of NF-kappa B was observed. In this reconstitution system, we can also demonstrate the activation of NF-kappa B by ceramide, but this activation is not tyrosine kinase-dependent. Overall, our results indicate that intermediates required for NF-kappa B activation by TNF or ceramide are membrane-bound, but the mechanism of activation by TNF is most likely different from that of ceramide.

Cell Line↗

PubMed1994

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.

Antibodies↗

PubMed1994

Identification of a protein kinase associated with the cytoplasmic domain of the p60 tumor necrosis factor receptor.

Tumor necrosis factor (TNF) has been shown to bind two distinct receptors, designated p60 and p80, with high affinity, resulting, within minutes, in phosphorylation of several proteins. The receptors themselves do not exhibit protein kinase activity nor have any associated proteins been identified. We employed the glutathione-S-transferase (GST) fusion protein system consisting of the cytoplasmic domain of p60 (GST-p60CD delta 1) as a probe to help us identify receptor-associated proteins from human histiocytic lymphoma U-937 cells. We found that a protein of approximately 52 kDa (pp52) bound to GST-p60CD delta 1 from [35S]methionine- and 32P-labeled cells. The associated protein was phosphorylated on serine and threonine residues. Furthermore, we identified serine/threonine kinase activity associated with p60CD delta 1 that required either Mn2+ or Mg2+ for optimal activity. The preferred substrates for this kinase, in addition to p60CD delta 1, included casein and histone H1, but not histone H2B, myelin basic protein, enolase, or the cytoplasmic domain of p80. As was the case in U-937 cells, p60CD delta 1-associated kinase activity was also identified in human breast adenocarcinoma MCF-7 cells and human foreskin fibroblasts. TNF stimulation of MCF-7 and foreskin fibroblasts for 5-15 min induced approximately 50 and 240% increases in phosphorylation of p60CD delta 1, respectively. Thus, our results provide the first evidence for protein kinase activity that is specifically associated with the cytoplasmic domain of the p60 form of the TNF receptor and causes its phosphorylation. This p60 TNF receptor-associated protein and the associated kinase described here are referred to as p60-TRAP and p60-TRAK, respectively.

Base Sequence↗

PubMed1994

Physical and functional association of a serine-threonine protein kinase to the cytoplasmic domain of the p80 form of the human tumor necrosis factor receptor in human histiocytic lymphoma U-937 cells.

Tumor necrosis factor (TNF) binds two distinct cell surface receptors designated p60 and p80. Our previous studies indicate that a protein kinase from U-937 cells binds to and phosphorylates the p60 receptor. While the p80 receptor is phosphorylated in vivo, no association of a protein kinase has been described. We employed a fusion protein comprising of glutathione S-transferase and the cytoplasmic domain of the p80 receptor (GST-p80CD) to identify cellular proteins that might associate with this receptor. From 35S- and 32P-labeled cells, a protein of 59 kDa bound specifically to GST-p80CD. In vitro kinase reactions indicated that serine/threonine protein kinase activity associated with GST-p80CD and causes its phosphorylation. Additionally, a 59-kDa phosphoprotein was also identified after kinase reactions of proteins bound to GST-p80CD. This kinase activity required either Mg2+ or Mn2+ for optimal activity, and it phosphorylated myelin basic protein, histone H2B, and also the cytoplasmic domain of the p60 receptor. Treatment of cells with TNF increased the p80 receptor-associated kinase activity by 200%. In summary, our results provide evidence of a novel ligand-activated serine/threonine protein kinase that associates with the cytoplasmic domain of the p80 receptor and causes the phosphorylation of both forms of the TNF receptor. This p80 TNF receptor-associated protein and the associated kinase described here are referred to as p80-TRAP and p80-TRAK, respectively.

Base Sequence↗

PubMed1994

pp60v-src kinase overexpression leads to cellular resistance to the antiproliferative effects of tumor necrosis factor.

While some tumor cells are sensitive to the antiproliferative effects of tumor necrosis factor (TNF), others are resistant. The molecular basis for cellular resistance to TNF is not completely understood. Previously we have shown that transfection of cells with an oncogene HER2/neu/erb B2, a receptor tyrosine kinase, leads to resistance to the anticellular effects of TNF [(1988) Proc. Natl. Acad. Sci. USA 85, 5102-5106]. In the present study, we demonstrate that the overexpression of another oncogenic tyrosine kinase, pp60v-src also induces resistance to TNF. In contrast to HER2, however, pp60v-src transfection of cells did not lead to down-modulation of TNF receptors but rather to decreased intracellular glutathione levels. The pp60v-src-induced cellular resistance to TNF could be abrogated by interferon-gamma. Thus, these results indicate that the resistance of certain tumors to TNF may also be due in part to the overexpression of pp60v-src oncogene.

3T3 Cells↗

PubMed1994

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.

3T3 Cells↗

PubMed1994

Differential roles of two types of the TNF receptor in TNF-induced cytotoxicity, DNA fragmentation, and differentiation.

Two different types of TNF receptors, the p60 receptor with a molecular mass of 60 kDa and the p80 receptor with a mass of 80 kDa, have been identified. TNF exhibits a wide variety of biologic actions, but which receptor is responsible for these biologic actions is not well characterized. In the present study, we examined the roles of the p60 and p80 receptors in three different TNF-induced biologic actions: 1) cytotoxicity; 2) DNA fragmentation; and 3) differentiation to macrophages. Analysis of TNF actions on various tumor cell lines revealed that TNF-induced cytotoxicity occurred in cells that expressed the p60 receptor, irrespective of expression of the p80 receptor. In contrast, DNA fragmentation and differentiation were observed only in cells that expressed both receptor types. Additionally, the specific Ab to each receptor were used to examine the roles of both receptors in the myelogenous leukemia cell line, ML-1a. Anti-p60 Ab alone showed cytotoxicity but little DNA fragmentation and differentiation, and anti-p80 Ab alone showed no effects. When these Abs were added in the presence of TNF, each independently, and almost completely, inhibited TNF-induced DNA fragmentation and differentiation. We also found that both Abs together synergistically induce differentiation and DNA fragmentation. These results indicate that signals through the p60 receptor are essential to induce cytotoxicity, but signals through both the p60 and p80 receptors are necessary, and act synergistically, for DNA fragmentation and differentiation.

Cell Differentiation↗

PubMed1994

Staurosporine induces the cell surface expression of both forms of human tumor necrosis factor receptors on myeloid and epithelial cells and modulates ligand-induced cellular response.

Staurosporine, an inhibitor of protein kinase C, is commonly used to inhibit the growth factor-induced signal transduction pathway at the post-receptor level. In this report, we examined the effect of staurosporine on the constitutive expression of tumor necrosis factor (TNF) receptors in K562, a human erythroblastoid leukemic cell line. Exposure of these cells to staurosporine enhanced cell surface expression of TNF receptors by almost 7-fold in a dose- and time-dependent fashion. Maximum induction occurred at a concentration of 20 nM of the agent for 16 h at 37 degrees C. Induction of the TNF receptor was found to be temperature-dependent. No induction was observed at 22 or at 4 degrees C, suggesting the role of cell metabolism. Scatchard analysis indicated an increase in receptor number without any change in receptor affinity. TNF receptors were induced by staurosporine on a wide variety of human cells of both epithelial (primarily p60 receptors) and myeloid (mainly p80 receptor) origin. Receptor-specific antibodies showed that both TNF receptors were induced. The induction was abolished by inhibitors of protein synthesis, thus suggesting the de novo synthesis of the receptor. Furthermore, we found that staurosporine had no effect on the internalization or shedding of the receptor, but it induced the mRNA for both forms of the TNF receptor. Inhibitors of tyrosine kinases had no effect on the induction of TNF receptors. Modulation of the receptor number by staurosporine correlated with the enhancement of antiproliferative effects of TNF against different tumor cells. Thus, overall these results indicate that protein kinase C may be involved in the signal transduction of TNF not only at the postreceptor level but also at the receptor level.

Alkaloids↗

PubMed1994

TNF induces internalization of the p60 receptor and shedding of the p80 receptor.

As is true for other peptide hormones, TNF causes the down-modulation of its own receptor. The process by which down-regulation occurs and the particular role of each of two recently identified receptors, however, are not understood. In this report we used Abs specific to p60 and p80 TNFR types to examine the ligand-induced down-regulation in histiocytic cell line U-937. These cells express both types of TNFRs, but the amount of p80 is two to three times greater than that of the p60 receptor. Treatment of U-937 cells with the ligand led to maximum down-modulation of TNFR within 30 min, and this decrease was found to be a result of receptor number and not affinity. When examined for the receptor type, approximately 90% of the p60 receptor and 35% of the p80 receptor was down-regulated by the cytokine. Rapid internalization of TNF in U-937 cells is mediated through p60, because this process was inhibited only by anti-p60 Ab and was also inhibited in cells (PMA-pretreated) that primarily express the p80 receptor. In contrast to p60, we observed that the ligand-induced shedding of the p80 receptor into the medium results in the down-regulation. Interestingly, however, the signal for shedding of the p80 receptor appears to be mediated through the p60 receptor, because anti-p60 Ab inhibited the shedding. Overall, our results provide evidence that ligand-induced down-modulation of TNFR is a result of the internalization of p60 and of the shedding of the p80 receptor and that the signaling for both is mediated through the p60 receptor.

Cell Line↗

PubMed1994

Curcumin is a non-competitive and selective inhibitor of phosphorylase kinase.

Recently, we reported that curcumin (diferuloylmethane) inhibits the growth of several different kinds of tumor cells. In order to investigate the mechanism of this inhibition, we examined the effects of curcumin on different protein kinases: highly purified protein kinase A (PkA), protein kinase C (PkC), protamine kinase (cPK), phosphorylase kinase (PhK), autophosphorylation-activated protein kinase (AK) and pp60c-src tyrosine kinase. While all kinases tested were inhibited by curcumin, only PhK was completely inhibited at relatively lower concentrations. At around 0.1 mM curcumin, PhK, pp60c-src, PkC, PkA, AK, and cPK were inhibited by 98%, 40%, 15%, 10%, 1%, and 0.5%, respectively. Lineweaver-Burk plot analysis indicated that curcumin is a non-competitive inhibitor of PhK with a Ki of 0.075 mM. Overall, our results indicate that curcumin is a potent and selective inhibitor of phosphorylase kinase, a key regulatory enzyme involved in the metabolism of glycogen. This has important implications for the anti-proliferative effects of curcumin.

Binding, Competitive↗

PubMed1994

Both type I and type II interferons down-regulate human tumor necrosis factor receptors in human hepatocellular carcinoma cell line Hep G2. Role of protein kinase C.

It is well known that interferon-gamma (IFN-gamma; type II) potentiates various responses of human tumor necrosis factor (TNF) in a wide variety of cells and that this potentiation is accompanied by the up-regulation of TNF receptor synthesis. In the present studies we examined the regulation of TNF receptors by type I and type II IFNs in a hepatocellular carcinoma cell line, HEP G2. Exposure of these cells to IFN-gamma led to a decrease in TNF receptor number (4029 vs. 2719 sites/cell) without any change in the receptor affinity (0.96 nM vs. 1.1 nM). The effect was time and dose-dependent. Like IFN-gamma, IFN-alpha and IFN-beta (type I) down-modulated the TNF receptors on these cells. The effect of IFNs on the TNF receptors was inhibited by staurosporin, a protein kinase C (PK-C) inhibitor. Furthermore, by the use of receptor-specific antibodies, we found that the IFN-dependent decrease was primarily due to the p60 form of the TNF receptor. Our results presented are the first to demonstrate that IFNs can also down-modulate TNF receptors in certain cells and that this effect is mediated through PK-C.

Alkaloids↗

PubMed1994

Cell density-dependent regulation of cell surface expression of two types of human tumor necrosis factor receptors and its effect on cellular response.

Tumor necrosis factor (TNF) is a multipotential cytokine known to regulate the growth of a wide variety of normal and tumor cells. It has been shown that the density of cells in culture can modulate the growth regulatory activities of TNF, the mechanism of which, however, is not understood. In this report, we investigated the effect of cell density on the expression of TNF receptors. The receptors were examined on epithelial cells (e.g., HeLa), which primarily express the p60 form, and on myeloid cells (e.g., HL-60) known to express mainly the p80 form. We observed that binding of TNF to both cell lines decreased with increase in cell density. Scatchard analysis of binding on HeLa and HL-60 cells revealed a 4- to 5-fold reduction in the number of TNF receptors without any significant change in receptor affinity in both cell types at high density. The decrease in TNF receptor numbers at high cell density was also observed in several other epithelial and myeloid cell lines. The downmodulation at high cell density was unique to TNF receptors, since minimum change in other cell surface proteins was observed as revealed by fluorescent activated cell sorter analysis. Neutralization of binding with antibodies specific to each type of the receptors revealed that both the p60 and p80 forms of the TNF receptor were equally downmodulated. A decrease in leucine incorporation into proteins was observed with increase in cell density, suggesting a reduction in protein synthesis. Since inhibition of protein synthesis by cycloheximide also leads to a decrease in TNF receptors, it is possible that the density-dependent reduction in TNF receptor number is due to an overall decrease in protein synthesis. The density-dependent decrease in TNF receptors was accompanied by a decrease in intracellular reduced glutathione levels. A reduction in the number of receptors on TNF sensitive tumor cells induced by cell-density correlated with increase in resistance to the cytokine.

Cell Count↗