Inhibition of erythropoietin production by cytokines and chemotherapy may contribute to the anemia in malignant diseases.
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Anemia↗
Biomedical subjects
J Fandrey
Publications and source records attributed to J Fandrey.
Prevalence of paraneoplastic erythropoietin production by renal cell carcinomas.
The prevalence of increased serum immunoreactive erythropoietin (Epo) was determined in a prospective study of 49 patients with renal cell carcinoma. Measured by a monoclonal antibody based commercial enzyme-linked immunoassay, the Epo concentration was above the normal range, determined in nonanemic humans, in four of the renal carcinoma patients. Since three of these were anemic, their increased Epo level was considered to be appropriate. The high estimate of serum Epo (218 U/l) in the fourth patient, who was not anemic, was not confirmed when tested by radioimmunoassay. Thus, in contrast with earlier studies, our results indicate that increased Epo is not a clear serological renal cell carcinoma marker. In addition, when monolayer cell cultures of 14 different established human renal carcinoma lines were screened, none of these released immunoreactive Epo in measurable amounts.
Adult↗
Serum immunoreactive erythropoietin in hyper- and hypothyroidism: clinical observations related to cell culture studies.
Laboratory experiments have demonstrated that tetra- and triiodothyronine (T4, T3) enhance hypoxia-induced erythropoietin (Epo) production. In the present study serum immunoreactive Epo was measured in 29 patients with hyperthyroidism and in 10 patients with hypothyroidism. Epo levels were inversely correlated to the blood haemoglobin concentration [Hb] in both groups of patients. However, Epo levels at given [Hb] were significantly higher in the hyperthyroid state. In vitro studies confirmed that T4 and T3 stimulate Epo synthesis in the human liver cell line HepG2. This stimulating effect persisted for at least 1 day after the removal of T4 and T3 from the cultures. Thus, while thyroidal disorders affect steady-state levels of circulating Epo, it seems unlikely that thyroid hormones play a major role in abrupt adjustments of Epo production, such as the diurnal changes.
Carcinoma, Hepatocellular↗
Thyroid hormones enhance hypoxia-induced erythropoietin production in vitro.
Effects of thyroid hormones on the production of erythropoietin (Epo) were investigated in isolated perfused rat kidneys and in the human hepatoma cell line, HepG2. Epo protein was measured by radioimmunoassay. L-triiodothyronine and L-thyroxine stimulated hypoxia-induced Epo formation both in the kidney and in HepG2 cells in a dose-dependent fashion. Quantitation of Epo mRNA by competitive polymerase chain reaction (PCR) showed that hypoxic HepG2 cells had three-fold higher Epo messenger RNA levels when treated with thyroid hormones for 3 hours. Measurements of oxygen consumption revealed that this effect was not due to an increase in the degree of hypoxia. Thus, apart from the known direct effect on erythroid precursors, thyroid hormones appear to stimulate erythropoiesis by a noncalorigenic increase in Epo production.
Animals↗
In vivo and in vitro regulation of erythropoietin mRNA: measurement by competitive polymerase chain reaction.
The regulation of erythropoietin (Epo) production was investigated by competitive polymerase chain reaction, a highly sensitive and accurate means of measuring Epo mRNA levels. Co-amplification of the test sample with added mutant Epo cDNA template corrects for variability in the efficiency of amplification. Epo mRNA levels were determined in tissues of normal rats and in animals with varying degrees of anemia. Reduction of the hematocrit level from 0.40 to 0.15-0.20 resulted in a 300-fold increase in kidney Epo mRNA, which comprised 80% of the total Epo mRNA versus 20% from the liver. In contrast, very low levels detected in lung and spleen were not significantly increased by anemia. The human hepatoma cell line, Hep3B, secretes high levels of Epo in response to hypoxia. This regulation is, to a large extent, transcriptional. When Hep3B cells were incubated in the presence of decreasing O2 tension from 160 to 7 mm Hg, there was a monotonic increase in Epo mRNA to 50 to 100 times the normoxic level. Hyperoxia did not suppress basal expression. When cells were incubated at a PO2 of 7 mm Hg, induction of Epo mRNA was first noted at 30 minutes and was maximal at 5 to 6 hours. After Epo mRNA was boosted by a 4-hour hypoxic incubation, cells were then exposed to normoxia, which shut off further transcription of the Epo gene. The decay of Epo mRNA levels closely followed first order kinetics with a half-life of 2 hours, an effective measurement of message stability.
Animals↗
Regulation of the erythropoietin gene.
Erythropoietin (Epo), the hormone that stimulates red blood cell production, is induced by hypoxia. We have utilized the human hepatoma cell line, Hep3B, to investigate the regulation of the Epo gene. We present evidence that the oxygen sensor in Hep3B cells is a heme protein. Hypoxic and cobalt induction of Epo protein is paralleled by a 50- to 100-fold increase in Epo mRNA which we have accurately quantified by means of an assay based on competitive polymerase chain reaction. This increase in Epo mRNA is due primarily to increased transcription. Transfection experiments utilizing the sensitive luciferase reporter gene show that the minimal portions of the Epo gene required for hypoxic induction include a 53 bp promoter element and a 43 bp enhancer located downstream from the polyadenylation site. Gel shift experiments show that these two regions cross-compete for specific DNA binding proteins. The enhancer contains a hexanucleotide direct repeat with a two bp insert which footprints with nuclear extracts from Hep3B cells and, when mutated, results in loss of hypoxic induction. This sequence is likely to bind to a member of the steroid/thyroid hormone receptor family of DNA binding proteins. These enhancer and promoter elements appear to cooperate in enabling the Epo gene to respond to hypoxia in a physiologically appropriate manner.
Erythropoietin↗
Immunocytochemical demonstration of erythropoietin in hypoxic human hepatoma cultures.
The possibility of demonstrating erythropoietin at the light microscopic level was examined in homogenous cultures of the erythropoietin-producing human hepatoma cell lines HepG2 and Hep3B. Immunoperoxidase staining was applied in combination with several mono- and polyclonal antibodies. Sufficiently strong colour responses were obtained with all three polyclonal antibodies and with one of three monoclonal antibodies raised against recombinant human erythropoietin. The staining intensity was increased in hypoxic versus nonhypoxic hepatoma cultures. Intracellular erythropoietin immunoreactivity was confirmed by Western blot analysis of HepG2 extracts. The effect of oxygen supply on erythropoietin gene expression was confirmed by competitive polymerase chain reaction of erythropoietin mRNA and by radioimmunoassay of secreted erythropoietin.
Animals↗
Microelectrode measurements of pericellular PO2 in erythropoietin-producing human hepatoma cell cultures.
On the basis of Fick's law of gas diffusion, it has been proposed that cells in conventional monolayer cultures may be severely hypoxic. Because knowledge of the cellular O2 availability is important for the interpretation of biochemical and toxicological cell culture work, microelectrode measurements of the pericellular PO2 were carried out using the erythropoietin (Epo)-producing human hepatoma cell lines Hep G2 and Hep 3B as an in vitro model. In confluent hepatoma cultures grown in polystyrene dishes and incubated in air with 5% CO2, the pericellular steady-state PO2 was < 1 mmHg. The rates of the production of immunoreactive Epo and lactate were high due to a misproportion between O2 supply and O2 requirements. Epo production decreased when shaken instead of static cultures were studied, or when the O2 concentration in the gas atmosphere was increased gradually up to 95%. In cultures grown on gas-permeable supports, pericellular and gas PO2 values were very similar, with increased Epo production at lowered PO2. In agreement with mathematical models, our experimental data make PO2 measurements desirable for studies of O2-dependent biological functions in cell cultures.
Carcinoma, Hepatocellular↗
Erythropoietin production and pH.
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Adult↗
Interleukin 1 beta and tumour necrosis factor alpha induce a macrophage-type of nitric oxide synthase in rat renal mesangial cells.
Treatment of mesangial cells with interleukin 1 beta (IL-1 beta) or tumour necrosis factor alpha (TNF alpha) has been shown to increase cGMP formation, most probably due to induction of nitric oxide synthase. Here we report that maximum stimulation of cGMP formation over a 24-h period required the presence of IL-1 beta or TNF alpha during the first 18 h of induction. N4-monomethyl-L-arginine (L-NMMA) was a potent inhibitor of cytokine-induced cGMP formation while N4-nitro-L-arginine (L-NNA) was less active. Formation of nitric oxide was detected in the cytosol of cytokine-treated mesangial cells by activation of purified soluble guanylate cyclase and was stimulated by tetrahydrobiopterin, but not by calcium calmodulin. Treatment of cells with IL-1 beta or TNF alpha markedly attenuated the contractile response to a subsequent challenge with angiotensin II. Furthermore, conditioned medium from IL-1 beta-treated cells increased cGMP in untreated control cells.
Amino Acid Oxidoreductases↗
Monokines inhibiting erythropoietin production in human hepatoma cultures and in isolated perfused rat kidneys.
The blood level of erythropoietin (Epo) is often anomalously low in anemic patients with inflammatory or malignant diseases. Therefore, we studied effects of pure recombinant immunomodulatory peptides on Epo formation in cultures of the human hepatoma cell line, HepG2. Interleukin (IL)-1 beta, IL-1 alpha, and tumor necrosis factor alpha lowered Epo production with half-maximal inhibition at 2, 5, and 20 U/ml, respectively. IL-6, transforming growth factor beta 2 and interferon gamma did not inhibit. Furthermore, IL-1 beta (10 U/ml) proved to block Epo formation in isolated serum-free perfused rat kidneys. Proposedly, monokines play a role in the pathogenesis of Epo deficiency in various diseases.
Animals↗
Inhibition of erythropoietin production by phorbol ester is associated with down-regulation of protein kinase C-alpha isoenzyme in hepatoma cells.
The role of protein kinase C (PKC) in the control of erythropoietin (Epo) production was studied using the human hepatoma cell line HepG2. Inhibition of PKC by staurosporine and the selective PKC inhibitor CGP 41251 significantly reduced Epo formation. No inhibition occurred with the inactive staurosporine derivative CGP 42700. Treatment with phorbol 12-myristate 13-acetate (PMA) for 24 h dose-dependently inhibited Epo formation, thus suggesting that down-regulation of PKC might be responsible for this inhibition. Immunoblotting experiments showed that incubation of HepG2 cells with PMA for 24 h resulted in a selective and almost complete down-regulation of PKC-alpha. Thus, PKC-alpha may play a permissive role in Epo synthesis in HepG2 cells.
Alkaloids↗
Interleukin-1 and tumor necrosis factor-alpha inhibit erythropoietin production in vitro.
The effects of several immunomodulatory peptides (recombinant, human) on the in vitro production of erythropoietin (Epo) were studied in cultures of the human hepatoma cell line Hep G2. A dose-dependent decrease of up to 60% in Epo production was induced by interleukin-1 beta, interleukin-1 alpha, and tumor necrosis factor-alpha (in that order of potency). In contrast, moderately increased Epo levels resulted with interleukin-6 or interferon-gamma treatment at high concentrations. Concomitant measurements of the production of alpha-fetoprotein indicated that the observed effects were specific for Epo. Hence, we suspect a modulating role of the immune system in the in vivo control of Epo production and postulate that interleukin-1 and tumor necrosis factor-alpha are involved in some of the cases of lowered blood Epo levels in association with renal diseases, chronic inflammation, and malignancies.
Carcinoma, Hepatocellular↗
Theophylline and magnesium inhibit the contraction elicited with ciclosporin and angiotensin II in mesangial cell cultures.
One serious side effect of ciclosporin (CS) therapy is its acute nephrotoxicity characterized by a marked decrease in glomerular filtration rate (GFR). A main determinant of GFR is the ultrafiltration coefficient which is thought to be regulated by the contractile state of the cells of the mesangium. CS enhances contractions of mesangial cells elicited with angiotensin II. By way of a lowered ultrafiltration coefficient this effect of CS may be partly responsible for its acute nephrotoxicity. We have therefore examined the effect of compounds that may attenuate the enhanced contractile response of the smooth-muscle-like mesangial cells. Prostaglandin E2 (10(-8)-10(-5) mol/l) and dibutyryl cyclic AMP (10(-4) mol/l) reduced the number of contracting cells from more than 50% to about 10%. The clinically used agent theophylline (10(-5) g/ml) decreased the number of contractions of CS-pretreated mesangial cells from 80.0 to 28.4% (p less than 0.001). Additionally, a marked decrease in the percentage of contractions (83.3 to 33.3%; p less than 0.001) was observed when the concentration of magnesium chloride was elevated from 0 to 2 mmol/l. The finding that the enhanced contractile response of CS-pretreated mesangial cells can be overcome by theophylline and/or high magnesium levels may be of clinical interest with a view to acute CS nephrotoxicity.
Angiotensin II↗
[Studies on cultured rat mesangial cells using cyclosporin A and magnesium--is magnesium nephroprotective in cyclosporin A therapy?].
One serious side effect of Cyclosporine A therapy is its acute nephrotoxicity characterized by a marked decrease in the glomerular filtration rate. A main determinant of the glomerular filtration rate is the ultrafiltration coefficient which is thought to be regulated by the contractile state of the cells of the mesangium. Cyclosporine A enhances contractions of mesangial cells elicited with angiotensin II. By way of lowered ultrafiltration coefficient this effect of Cyclosporine A may be partly responsible for its acute nephrotoxicity. Hypomagnesaemia is often associated with Cyclosporine A therapy. Profound tubular magnesium wasting by Cyclosporine A has been claimed its cause. We have investigated the effect of low and high magnesium concentration on the contractility of mesangial cells pretreated with Cyclosporine A. Without magnesium 80% of the cells contracted upon addition of angiotensin II. A marked decrease in the contractility was seen when the magnesium concentration was elevated to 2 mmol/l (34%). From these observations we conclude that magnesium serum levels even in the high normal range might be protective against the decrease of the glomerular filtration rate seen with CsA therapy.
Angiotensin II↗
Potentiation of angiotensin II-stimulated phosphoinositide hydrolysis, calcium mobilization and contraction of renal mesangial cells upon down-regulation of protein kinase C.
Long-term pretreatment of rat mesangial cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) down-regulated protein kinase C activity and potentiated the angiotensin II-induced inositol trisphosphate (InsP3) formation. This increased response to angiotensin II occurred without a significant change in the receptor number or Kd value of angiotensin II binding to the cells. The biologically inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate was without effect on angiotensin II-stimulated InsP3 generation. Long-term pretreatment with TPA also increased the angiotensin II-induced mobilization of Ca2+ and the subsequent contraction of mesangial cells.
Angiotensin II↗
Role of cytochrome P450 in the control of the production of erythropoietin.
Effects of agents affecting cytochrome P450 were studied on the production of erythropoietin (Epo) in cultures of the human hepatoma cell line HepG2. Epo was measured by radioimmunoassay of the culture media after 24 h of incubation. The addition of phenobarbital or 3-methylcholanthrene, which induce cytochrome P450, significantly enhanced the formation of Epo. Likewise, the thyroid hormones T3 and T4 stimulated the rate of the production of Epo. On the other hand, the formation of Epo was lowered following the addition of diethyldithiocarbamate or cysteamine chloride, which inhibit cytochrome P450. These findings support the idea that O2 sensitive hemoproteins of the microsomal mixed-functional oxidases play a role in the control of the synthesis of Epo.
Carcinoma, Hepatocellular↗
Modulation of the production of erythropoietin by cytokines: in vitro studies and their clinical implications.
The etiology of the anemia of chronic disorders is complex. Factors which clearly contribute to the suppression of erythropoiesis are (a) reduced iron availability and (b) stimulation of the synthesis of immunomodulatory peptides such as IL-1, TNF-alpha and IFN-gamma, which inhibit the proliferation of erythrocytic progenitors in the bone marrow. The question as to whether lack of EPO is of general importance in the pathogenesis of the anemia of chronic inflammatory and malignant diseases is still a subject of controversy. The present in vitro studies show that IL-1 and TNF-alpha, but not IFN-gamma significantly lower the pO2-dependent formation of EPO in HepG2 cultures. In addition, clinical examples are given of anomalously low or high EPO levels in association with diseases involving the immune system. It is proposed that monokines and related immunomodulatory peptides could play a role in the control of the production of EPO.
Erythropoietin↗