Erythropoietin production in long-term cultures of human renal carcinoma cells : The role of cell population density

The present studies report the maintenance of erythropoietin (Ep) production in long-term cultures of a human renal carcinoma from a patient with erythrocytosis. The renal carcinoma cells were grown and maintained in monolayer cultures for 7 months. They were serially passaged every 2-3 weeks when the cultured cells reached confluency. Ep levels measured with a sensitive radioimmunoassay in the spent culture media of the cells in the stage of semiconfluent or confluent density were less than 20 and 30 mU/ml, respectively, throughout the period of 15 successive passages. However, when the renal carcinoma cells were maintained in culture without passage after reaching confluency, Ep levels in the spent media of these cells reproducibly showed an exponential increase to more than 300 mU/ml at the time of saturation density. The importance of cell population density in Ep production by the renal carcinoma cell cultures was further confirmed by the observation that the cultures with higher seeding density reached confluency earlier and began an exponential increase in Ep production sooner than those cultures with lower seeding density.     

Inhibitory effects of tetradecanoylphorbol acetate and diacylglycerol on erythropoietin production in human renal carcinoma cell cultures

A human renal carcinoma from a patient with an erythrocytosis, serially transplanted into athymic nude mice, was grown in primary monolayer cell cultures. After reaching confluency the cultured cells formed multicellular hemicysts (domes) which became more abundant as the cultures approached saturation density. Erythropoietin (Ep) production by this renal carcinoma in culture was only slightly increased at the time of semiconfluency but showed a marked increase in Ep levels in the culture medium after the cultures reached confluency, in parallel with an increase in dome formation. The phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) showed a significant dose-related inhibitory effect on Ep production and dome formation in the renal carcinoma cell cultures, suggesting an important role of protein kinase C, the only known receptor for TPA, in inhibiting the expression of differentiated phenotypes in the renal carcinoma cells. TPA also suppressed Ep secretion over a period of 96 h, indicating a time course of suppression of this differentiated function of the renal carcinoma cells in culture. This hypothesis was further supported by the observation that diacylglycerol, the endogenous activator of protein kinase C, likewise inhibited Ep production and dome formation in the renal carcinoma cell cultures. These studies suggest a role of the inositol-lipid second messenger path and protein kinase C in the regulation of Ep production.     

Effect of membrane dialysis and filtration-sterilization on erythropoietin activity.

Most erythropoietin (Ep) preparations contain non-erythropoietin contaminants. The use of such hormone concentrates raises important questions regarding interpretations of results derived from in vivo and especially from in vitro studies. By sterilizing various Ep preparations with Nalgene, Millipore, or Selas silver filtration, or even after conventional membrane dialysis, variable responses were noted when the Ep was assayed with mouse bone marrow cells in vitro (i.e. by stimulating the production of erythroid colonies from CFU-e and BFU-e) and in vivo (i.e., by using the exhypoxic, polycythemic mouse bioassay for Ep). The utility and limitations of such preparative procedures are discussed.     

Effect of Erythropoietin on the interaction of Concanavalin A with rat erythrocytes

Effect of Erythropoietin (Ep) on the interaction of Concanavalin A (Con A) with rat erythrocytes was studied using ¹²⁵I-labelled Con A. Binding of Con A to erythrocytes was dependent on time and cell concentration. Starvation caused an elevation of the lectin binding capacity of red cells which again came down towards the normal level on Ep administration to starved rats. Binding of Con A to erythrocytes decreased linearly with increasing concentration of Ep. Specificity of binding was confirmed by inhibition studies with -methyl-D-mannopyranoside (Me Man) Cells from the starved rats compared to those from normal and Ep treated animals were less prone to inhibition by this sugar analog. Positive cooperative binding of Con A to rat erythrocyte was observed at low concentration of Con A but was absent at higher lectin concentrations. Starvation caused an increase in the number of binding sites per cell which returned to normal level after Ep treatment. Under identical conditions, binding affinities were not much changed in these cells. Cells from the starved animals were more susceptible to agglutination compared to those from normal and Ep-treated rats. Microviscosity and cholesterol/phospholipid ratio of red cell membrane decreased in the starved animals which retraced its way back towards the normal level after Ep treatment.     

SAR studies of 4-pyridyl heterocyclic anilines that selectively induce autophagic cell death in von Hippel-Lindau-deficient renal cell carcinoma cells

We recently identified a class of pyridyl aniline thiazoles (PAT) that displayed selective cytotoxicity for von Hippel-Lindau (VHL) deficient renal cell carcinoma (RCC) cells in vitro and in vivo. Structure-activity relationship (SAR) studies were used to develop a comparative molecular field analysis (CoMFA) model that related VHL-selective potency to the three-dimensional arrangement of chemical features of the chemotype. We now report the further molecular alignment-guided exploration of the chemotype to discover potent and selective PAT analogues. The contribution of the central thiazole ring was explored using a series of five- and six-membered ring heterocyclic replacements to vary the electronic and steric interactions in the central unit. We also explored a positive steric CoMFA contour adjacent to the pyridyl ring using Pd-catalysed cross-coupling Suzuki-Miyaura, Sonogashira and nucleophilic displacement reactions to prepare of a series of aryl-, alkynyl-, alkoxy- and alkylamino-substituted pyridines, respectively. In vitro potency and selectivity were determined using paired RCC cell lines: the VHL-null cell line RCC4 and the VHL-positive cell line RCC4-VHL. Active analogues selectively induced autophagy in RCC4 cells. We have used the new SAR data to further develop the CoMFA model, and compared this to a 2D-QSAR method. Our progress towards realising the therapeutic potential of this chemotype as a targeted cytotoxic therapy for the treatment of RCC by exploiting the absence of the VHL tumour suppressor gene is reported.     

Heteroconjugate antibody-directed killing of autologous human renal carcinoma cells by in vitro-activated lymphocytes

Tumor cell lysis can be enhanced significantly in vitro when heteroconjugate (HC) antibodies (anti-CD3 x anti-tumor mAb) are used to specifically direct lymphocyte effector cells to the tumor cell target. In order to effectively utilize HC antibodies in an immunotherapy protocol, methods must be identified for the optimum expansion, activation, and retargeting of lymphocyte-effector populations from cancer patients. In this study, we have compared the proliferative responses of different normal and renal cell carcinoma (RCC) patient lymphocyte preparations (PBL, tumor-infiltrating lymphocytes) stimulated in vitro for periods up to 12 days with a variety of growth factor combinations (anti-CD3, rIL-2, rIL-4). These activated lymphocyte preparations were then tested in vitro for their ability to kill RCC tumor cells and tumor cell lines in the presence of HC preparations (anti-CD3 mAb covalently linked to mAb reactive to different RCC tumor-associated Ag). RCC patient PBL cultured with anti-CD3 plus rIL-2 for 12 days resulted in a 3- to 160-fold expansion of effector cells. These cells, as well as tumor infiltrating lymphocytes, when retargeted with appropriate HC antibodies were capable of mediating high levels of killing of autologous tumor cells. No constitutive autologous anti-tumor cell response was detected in the absence of added HC antibodies. Of the five anti-RCC mAb tested (A6H, K29, K20, UR07, and URO 3), HC containing URO 3 x anti-CD3 and K20 x anti-CD3 elicited the highest level of tumor cell lysis by the activated lymphocyte effector cells. Together these results demonstrate that HC antibodies may be a useful imunotherapeutic reagent for directing the killing of RCC tumor cells by autologous lymphocytes.     

Intracellular microelectrode measurements in small cells evaluated with the patch clamp technique.

Microelectrode penetration of small cells leads to a sustained depolarization of the resting membrane potential due to a transmembrane shunt resistance (Rs) introduced by the microelectrode. This has led to underestimation of the resting membrane potential of various cell types. However, measurement of the fast potential transient occurring within the first few milliseconds after microelectrode penetration can provide information about pre-impalement membrane electrophysiological properties. We have analyzed an equivalent circuit of a microelectrode measurement to establish the conditions under which the peak of the impalement transients (Ep) approaches the pre-impalement resting membrane potential (Em) of small cells most closely. The simulation studies showed that this is the case when the capacitance of the microelectrode is low and the membrane capacitance of the cell high. In experiments performed to assess the reliability of Ep as a measure of Em, whole-cell patch clamp measurements were performed in the current clamp mode to monitor, free from the effects of Rs, Em in cultured human monocytes. Microelectrode impalement of such patch clamped cells and measurement of Ep made it possible to detect correlation between Ep and Em and showed that for small cells such as human monocytes Ep is on average 6 mV less negative than the resting membrane potential.     

The role of increased proteolysis in the atrophy and arrest of proliferation in serum-deprived fibroblasts

When cultured fibroblasts are deprived of serum, the degradation of long-lived proteins and RNA increases, the cells stop proliferating, and they decrease in size. To determine the role of the increased protein catabolism in these responses, we studied the effects of inhibitors of intralysosomal proteolysis in Balb/c 3T3 cells. When these cells were placed in serum-deficient medium (0.5% serum), the rate of degradation of long-lived proteins increased about twofold within 30 min. This increase was reduced by 50-70% with inhibitors of lysosomal thiol proteases (Ep475 and leupeptin) or agents that raise intralysosomal pH (chloroquine and NH4Cl). By contrast, these compounds had little or no effect on protein degradation in cells growing in 10% serum. Thus, in accord with prior studies, lysosomes appear to be the site of the increased proteolysis after serum deprivation. When 3T3 cells were deprived of serum for 24-48 hours, the rate of protein synthesis and the content of protein and RNA and cell volume decreased two- to fourfold. The protease inhibitor, Ep475, reduced this decrease in the rate of protein synthesis and the loss of cell protein and RNA. Cells deprived of serum and treated with Ep475 for 24-48 hours had about twice the rate of protein synthesis and two- to fourfold higher levels of protein and RNA than control cells deprived of serum. The Ep475-treated cells were also about 30% larger than the untreated cells. Thus, the protease-inhibitor prevented much of the atrophy induced by serum deprivation. The serum-deprived fibroblasts also stopped proliferating and accumulated in the G1 phase of the cell cycle. The cells treated with Ep475 accumulated in G1 in a manner identical to untreated serum-deprived cells. Other agents which inhibited protein breakdown in serum-deprived cells also did not prevent the arrest of cell proliferation. Thus the enhancement of proteolysis during serum deprivation appears necessary for the decrease in size and protein synthesis, but probably not for the cessation of cell proliferation. When cells deprived of serum in the presence or absence of Ep475 were stimulated to proliferate by the readdition of serum, the larger Ep475-treated cells began DNA synthesis 1-2 hours later than the smaller untreated cells. Thus, after treatment with Ep475, the rate of cell cycle transit following serum stimulation was not proportional to the cell's size, protein, or RNA content, or rate of protein synthesis.     

Adherence of fimbriate and non-fimbriate strains of Yersinia enterocolitica to human epithelial cells

Strains of Yersinia enterocolitica from different O serogroups were tested for their ability to adhere to cultured epithelial cells (H.Ep 2) after growth of bacteria in broth at 22 C or 37 C. Strains that adhered to H.Ep 2 cells generally did so better after growth at 22 C than at 37 C. The data indicate that the ability to produce either of the mannose-resistant (MR) hemagglutinins, MR/Y or K1, each associated with fimbriae, does not correlate with ability to adhere to epithelial cells in this particular in vitro model.     

Cultivation of cottontail rabbit epidermal (Sf1Ep) cells on microcarrier beads and their use for suspension cultivation of Treponema pallidum subsp. pallidum

In vitro propagation of Treponema pallidum can be achieved by cocultivation with Sf1Ep cells. This study had two objectives: (i) to achieve suspension cultivation of Sf1Ep cells and (ii) to develop procedures for achieving the replication of T. pallidum in those cell cultures. Seven suspension cultures of Sf1Ep cells yielded an average of 7.2 x 10(8) T. pallidum (36-fold increase) after 12 days.     

人肾细胞癌细胞阳离子脂质体的转染效率

以ＭＴＳ染色法测定实验剂量的Ｌｉｐｏｆｅｃｔｉｎ对细胞的毒性作用,以β－半乳糖苷酶基因为报告基因,通过Ｌｉｐｏｆｅｃｔｉｎ而转染,用Ｘ－ｇａｌ染色法,测定转染效率,结果表明实验剂量（１０μｇ／ｍｌ）的Ｌｉｐｏｆｅｃｔｉｎ对细胞生长无明显毒性。Ｌｉｐｏｆｅｃｔｉｎ对多数肾细胞癌细胞的转染是有效的,且转染效率随Ｌｉｐｏｆｅｃｔｉｎ 度的增高（２．５－１０μｇ／ｍｌ）而增高,说明Ｌｉｐｏｆｅｃｔｉｎ可安     

Cultivation of cottontail rabbit epidermal (Sf1Ep) cells on microcarrier beads and their use for suspension cultivation of Treponema pallidum subsp. pallidum.

In vitro propagation of Treponema pallidum can be achieved by cocultivation with Sf1Ep cells. This study had two objectives: (i) to achieve suspension cultivation of Sf1Ep cells and (ii) to develop procedures for achieving the replication of T. pallidum in those cell cultures. Seven suspension cultures of Sf1Ep cells yielded an average of 7.2 x 10(8) T. pallidum (36-fold increase) after 12 days.     

Modulation of erythropoietin concentrations by manipulation of hypercarbia.

The present studies were done to determine whether preventing the respiratory alkalosis, which is known to occur with acute "hypoxic" stimuli, would lead to alterations in plasma concentrations of erythropoietin (Ep). Rats were subjected to two acute stresses, hypoxia and blood loss, separately and in combination, with and without the added stress of hypercarbia. Hypercarbia in all experimental groups was associated with a decrease in plasma concentrations of Ep. This reduction in plasma Ep with hypercarbia could not be fully explained by the higher arterial pO2S or p50S of the hypercarbic rats. Hypercarbia may have indirectly suppressed Ep production by increasing blood flow to the site of Ep production. Alternatively, the cell of origin of Ep could be sensitive to changes in pH and/or PCO2. It was further demonstrated that neither the onset nor the degree of reticulocytosis could be predicted by the plasma Ep concentrations. It is likely that the removal of red blood cells led to a decrease in marrow transit time with the early emergence of reticulocytes after acute blood loss.     

Inhibition of bone resrption by selctive inactivators of cysteine proteinases

Incativators of cystein proteinases (CPs) were tested as inhibitors of bone resorption in vitro and in vivo. The following four CP inactivators were tested: Ep453, the membrane-permeant produrg of Ep475, a compound with low membrane pereability which inhibits cathepsins B, L, S, H, and calpain; Ep453, the membrane-permeant prodrug of Ep475; CA074, a compound with low membrane permeability which selectivly inactives cathepsin B; and CA07Me, the membrane-permanent prodrug of CA074. The test systems consisted of (1) monitoring the release of radioisotope from prelabelled mousecalvarial explants and (2) assessing the extene of bone resorption in and isolated osteoclast assay using confocal laser microscopy. Ep453, Ep475, and CA074Me inhebited both stimulated and basal bone resorption in vitro while CA074 WASA without effect; The inhibition was reversible and dose dependent. None of the inhibitors affected protein synthesis, DNA synthesis, the PTH-enhanced secretion of β-glucuronised, and N-acetyle-β-glucosaminidase, or the spontaneous release of lactate dehyrogenese. Ep453, Ep475, and CA074Me does-dependently inhibited the resorption activity of isolated areat osteoclassts cultured on bone slices with a maximal effect at 50 μM. The munber of resorption pits and their mean volume was reduced, whilest the mean administration subcutaneously at a dose of 60 μg/g body weight inhibited bone resorption in vivo as measured by an in vivo/in vitro assay, by about 20%. This study demonestration that cathepsins B,L, and/or S are involved in bone resorpotion in vitro and in vivo. Whilest cathepsin L and/or S act extracellularly, and possibly intractually, cathepsin B mediate its effects intracellularly perpheps through the activation of other proteinases involved in subsosteoclastic collagen degradition.     

Effects of prostaglandins on erythropoiesis

A model has been presented for the role of the kidney in the physiologic and pathophysiologic control of erythropoiesis. It is postulated that an oxygen deficit created by anemia or hypobaric hypoxia results in the release of prostacyclin and its metabolite 6-keto PGE1, and the release of PGE2 with ischemic hypoxia. Prostacyclin, 6-keto-PGE1, or PGE2 activation of adenylate cyclase, an increase in cyclic AMP, activation of a protein kinase and the phosphorylation of hydrolases, which have been released from lysosomes by hypoxia, lead to increased biosynthesis of erythropoietin (Ep). The mechanism of labilization of lysosomes and the release of hydrolases from these cell organelles is postulated to be related to increases in cyclic GMP levels in a renal cell. An Ep-producing human renal carcinoma cell line grown in tissue culture has been demonstrated to produce significant amounts of PGE2. Meclofenamate, an inhibitor of prostaglandins synthesis, was found to inhibit in vitro production of PGE2, Ep, and dome formation in these renal carcinoma cells, giving support to our hypothesis that pathophysiologic production of Ep tumor cells depends upon prostaglandins production. An Ep-producing clone from this renal carcinoma cell line has been developed that contains low electron density (LED) cells after the cells reach confluency, which show a cytoplasm, with abundant and widely dilated endoplasmic reticulum, an oval nucleus, dispersed chromatin, and prominent nucleoli. These are the cells responsible for dome formation and Ep production. Non-EP-producing clones have also been produced from this renal carcinoma cell line, which did not produce domes even at high cell density and had a distinctly different cell type than the Ep-producing clone. Thus, it is postulated that prostacyclin (PGI2) and its metabolite 6-keto PGE1 play a significant role in hypoxic hypoxia stimulation of Ep production and PGE2 is involved in ischemic hypoxia and renal carcinoma cell production of Ep. A modulating effect of PGE2 and PGD2, the two primary bone marrow prostaglandins, has been proposed in Ep stimulation of the erythroid progenitor cell compartment (CFU-E and BFU-E).     

Interleukin-22 suppresses the growth of A498 renal cell carcinoma cells via regulation of STAT1 pathway

Background

Renal cell carcinoma (RCC) is one of the most common kidney cancers and is highly resistant to chemotherapy. Accumulating evidence suggests that interleukin-22 (IL-22) may mediate host defense against varietal pathogens as a proinflammatory and anti-inflammatory cytokine. The purpose of this study is to assess the inhibitory effects of IL-22 on human RCC cell line A498 and to investigate the possible mechanisms underlying the anti-tumor effects of this cytokine.

Methodology

A498 cells, a RCC cell line, were used to assess the inhibitory growth effects of IL-22 using the MTT assay and flow cytometric analysis in vitro. BALB/C nude mice bearing A498 cell xenografts were used to examine the antitumor efficacy of IL-22 in vivo. Western blotting assay was performed to detect the regulation of the intracellular signaling pathway of IL-22.

Principal Findings

We found that IL-22 suppressed the growth of A498 cells in a dose-dependent manner and inhibited the growth of A498 xenografts. We also observed that IL-22 produced a dose-dependent inhibitory effect on A498 cells that involved the induction of G2/M cell cycle arrest without cell apoptosis. Moreover, we showed that the phosphorylation of STAT1 was increased and the phosphorylation of ERK1/2 was attenuated in A498 cells exposed to IL-22. The growth inhibition of A498 cells was partially revised after IL-22 treatment as the expression of STAT1 was knocked down. And inflammatory cytokines, interferon-α and tumor necrosis factor-α (TNF-α) were barely involved in the suppression of A498 cell xenografts treated with IL-22.

Conclusions

IL-22 dose-dependently suppresses RCC cell line A498 cells in vitro and induces growth inhibition of A498 cell-bearing mouse xenografts. These results suggest that the anti-RCC effects of IL-22 are at least partially mediated through regulation of STAT1 signaling pathways and G2/M cell cycle arrest, rather than by inducing apoptosis and inflammatory cytokines.