Short-term Monolayer Cultures Research Articles

Cell-to-Medium Concentration Ratio Overshoot in the Uptake of Statins by Human Hepatocytes in Suspension, but Not in Monolayer: Kinetic Analysis Suggesting a Partial Loss of Functional OATP1Bs

Suspended human hepatocytes (SHH) have long been used in assessing hepatic drug uptake, while plated human hepatocytes in short-term monolayer culture (PHH) have gained use in recent years. This study aimed to cross-evaluate SHH and PHH in measuring the hepatic uptake mediated by organic anion transporting polypeptide 1Bs (OATP1Bs). We compared the time courses of cell-to-medium (C/M) concentration ratios and initial uptake clearance values of the OATP1B substrates (pitavastatin, rosuvastatin, cerivastatin, pravastatin, dehydropravastatin, and SC-62807) between SHH and PHH. For all compounds except cerivastatin, the C/M ratios in SHH displayed an apparent overshoot (an initial increase followed by a decrease) during the 180-min uptake experiment, but not in PHH. Based on the literature evidence suggesting the possible internalization of OATP1Bs in primary hepatocytes, separate experiments measured the drug uptake after varying lengths of pre-incubation in the drug-free medium. The initial uptake clearances of pitavastatin and rosuvastatin declined in SHH beyond an apparent threshold time of 20-min drug-free pre-incubation, but not in PHH. Kinetic modeling quantitatively captured the decline in the active uptake clearance in SHH, and more than half of the active uptake clearances of pitavastatin and rosuvastatin were prone to loss during the 180-min uptake experiment. These results suggested a partial, time-delayed loss of the functional OATP1Bs in SHH upon prolonged incubation. Our results indicate that PHH is more appropriate for experiments where a prolonged incubation is required, such as estimation of unbound hepatocyte-to-medium concentration ratio (Kp,uu) at the steady-state.

Abstract 3911: High-throughput screening of patient-derived HCC organoids for drug discovery

Abstract Current in vitro models of primary liver cancer insufficiently recapitulates the tumor heterogeneity and pathophysiology of the original patient's tumor. This significantly hinders translational studies and often leads to the high rate of clinical trial attrition and poor patient response. Recent technological advances in organoid culture methods have been reported to overcome these limitations. Our group has successfully established 3 patient-derived HCC tumor organoids that closely recapitulate the patient's pathophysiology. Conventional short-term monolayer culture of the same 3 tumors as well as 3 non-tumoral adjacent liver and 3 normal liver organoids were established and compared with 5 HCC cell lines from ATCC for their drug response profiles. Subsequent drug screening was performed using a panel of 100 drugs, of which 70 are FDA-approved for either HCC or other cancers, as well as 30 pipeline compounds targeting frequently mutated pathways in HCC. We found that our screening pipeline could achieve robust z' scores and reproducible data points with technical and biological replicates. Principal component analysis of Total Area under curve (AUC) values show distinct and progressive changes between Normal Liver, Non-tumoral adjacent liver, and Tumor organoids. Upon comparison with HCC cell lines Hep3B, HepG2, Huh7, SNU398 and PLC5, we found HCC patient-derived short term cultures, particularly organoids have more diverse drug sensitivity profiles and may be a better indicator of patient heterogeneity. Interestingly, FDA-approved drugs for HCC such as Regorafenib show high sensitivity and tumor specificity to ATCC cell lines (ATCC vs Normal liver P=0.00017***), but this specificity was reduced and lost in patient-derived 2D cultures (P=0.045*) and organoids (P=0.895), respectively. This may explain the observed hepatotoxicity observed in HCC patients and warrant further investigation of more targeted ways of treating HCC. Citation Format: Alissa Michelle Go Wong, Winnie Cheung, Maggie Wong, Aikha Wong, Helena Lam, Zhe Zhang, Matthew Man, Hui Chen, David Close, Paul Johnston, Anthony Chan, Paul Lai, Joseph Kwong, Nathalie Wong. High-throughput screening of patient-derived HCC organoids for drug discovery [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3911.

Biological Effects of the Herbal Plant-Derived Phytoestrogen Bavachin in Primary Rat Chondrocytes.

The aim of this study was to examine the anabolic and anticatabolic functions of bavachin in primary rat chondrocytes. With bavachin treatment, chondrocytes survived for 21 d without cell proliferation, and the proteoglycan content and extracellular matrix increased. Short-term monolayer culture of chondrocytes showed that gene induction of both aggrecan and collagen type II, major extracellular matrix components, was significantly upregulated by bavachin. The expression and activities of cartilage-degrading enzymes such as matrix metalloproteinases and a disintegrin and metalloproteinase with thrombospondin motifs were inhibited significantly by bavachin, while tissue inhibitors of metalloprotease were significantly upregulated. Bavachin inhibits the expression of inducible nitric oxide synthase, a representative catabolic factor, and downregulated the expression of nitric oxide, cyclooxygenase-2, and prostaglandin E2 in a dose-dependent manner in chondrocytes. Our results suggest that the bavachin has anabolic and potent anticatabolic biological effects on chondrocytes, which may have considerable promise in treating articular cartilage degeneration in the future.

Protection of the differentiated Chondrocyte Phenotype during Monolayer expansion Cultures Using heat inactivated Fetal Calf Serum


 
 
 Introduction: autologous chondrocyte implantation (aci) is one successful treatment for osteoarthritic defects (1-4). However, chondrocytes undergo a conversion to a fibroblastic phenotype during their expansion in vitro. This process can lead to degradation and failure of the repaired tissue, and is termed “dedifferentiation” (5-9). we hypothesized that culturing chondrocytes in Heat inactivated Fetal calf serum (HiFcs) would lead to a slowing of the process of dedifferentiation. This study specifically examines how HiFcs affects gene expression and proliferation of bovine chondrocytes in short-term monolayer culture. Methods: we cultured primary chondrocytes extracted from bovine joints in Fcs or HiFcs for four passages (p0-p4). we then analysed the relative gene expression of aggrecan (agg), type ii-alpha1 collagen (col2a1), cartilage oligomeric matrix protein (comp), sry (sex determining region y)-box 9 (sox9), type i-alpha2 collagen (col1a2) and bcl-2-associated X protein (bax) using quantitative real-time polymerase chain reaction (qpcr). results: our results show significant down-regulation of col1a2 and up-regulations of agg, col2a1, comp and sox9 in HiFcs cultures when compared to those in Fcs cultures. discussion: Agg, col2a1 and comp are associated with the synthesis of cartilage matrix molecules while sox9 is associated with chondrogenesis (5-11). Col1a2 is associated with the presence of fibrous tissue which lacks the desired mechanical properties (5-9). Therefore, our results suggest that HiFcs can partially protect the chondrocytic phenotype from dedifferentiating to fibroblastic after one passage. To improve upon our results, more experiments are required to reveal the mechanism underlying the protection of chondrocytes offered by the heat inactivation of Fcs.
 
 

Inhibition of p38 MAPK signaling in chondrocyte cultures results in enhanced osteogenic differentiation of perichondral cells

Chondrocytes and osteoblasts originate from the same progenitor cell; however, both are characterized by distinct gene expression profiles once they are differentiated. Signals from differentiating chondrocytes, such as Indian hedgehog (Ihh), regulate the differentiation of osteoblast precursor cells. The MAPK pathways play important roles in controlling the differentiation of both chondrocytes and osteoblasts, with the p38 pathway being particularly relevant in skeletal cells. In the present study, we investigated the effects of p38 inhibition on osteoblastic marker gene expression in chondrocyte cultures. Using high-density micromass cultures of mesenchymal cells as well as chondrocytes that had differentiated in vivo and were maintained in short-term monolayer culture, we demonstrate elevated Runx2, Osterix and Osteocalcin transcript levels in chondrocyte cultures upon inhibition of p38 activity with the pharmacological inhibitor PD169316. Osteocalcin immunolocalization was restricted to perichondral/periosteal cells in micromass cultures, suggesting that inhibition of p38 results in increased periosteal osteogenesis. Coinciding with increased expression of these genes, we observed elevated levels of transcripts for Ihh and its target gene, Ptch, in response to p38 inhibition. Addition of recombinant hedgehog protein mimicked some effects of p38 inhibitors. We therefore suggest that p38 signaling regulates chondrocyte–perichondral cell communication during skeletal development, partially through increased Ihh signaling.

Region-specific constitutive gene expression in the adult porcine meniscus

The knee meniscus exhibits extensive spatial variations in native healing capacity, biochemical composition, and cell morphology that suggest the existence of distinct phenotypes for meniscus cells. Constitutive gene expression levels of appropriate extracellular matrix proteins may serve as useful molecular markers of cellular phenotypes; however, relatively little is known of variations in the gene expression for meniscus cells of different regions of the tissue. The objective of the present study was to evaluate constitutive differences between radial inner and outer regions in gene expression for extracellular matrix proteins relevant to the meniscus. A secondary objective was to determine if these region-specific differences in gene expression are maintained after periods of monolayer culture. The innermost regions of the meniscus were found to constitutively express higher mRNA levels for proteins highly expressed in articular cartilage, including aggrecan, type II collagen, and NOS2. In contrast, the outer meniscus was found to contain higher gene expression for proteins associated with fibrous tissues including type I collagen, and the proteases MMP2 and MMP3. Isolated inner and outer meniscus cells maintained these region-specific gene expression patterns for collagens and proteoglycans during short-term monolayer culture. The results provide new information that suggests the utility of constitutive gene expression levels as molecular markers to distinguish tissue and cells of the inner and outer meniscus.

Detection of rat Pneumocystis carinii proteinases and elastase and antipneumocystis activity of proteinase inhibitors in vitro.

Proteinases play an important role in survival of microorganisms and in pathogenicity of diseases. By using a modified SDS-gelatin-polyacrylamide gel system, proteinases of rat-P.carinii were detected as bands of proteolytic digestion after electrophoresis. P.carinii organisms obtained from dexamethasone immunosuppressed transtracheally infected rats were cultured in spinner flask suspension cultures to minimize host cell contamination. At pH 8.3, seven Pc-specific proteolytic bands were detected in three clusters of different molecular weights clearly different from host cell patterns. By using a range of pH, various preparations of organisms and both infected and uninfected culture media, proteolytic activities have been partially characterized. Elastase secretion has been assessed based on elastin digestion model. Proteinase inhibitors have been tested for their ability to inhibit P.carinii growth in HEL299 short-term monolayer cultures. Results indicate that proteolytic activities are involved in the proliferation of microorganisms since leupeptin exerted in vitro antipneumocystis activity while aprotinin enhanced P.carinii growth.

Ex vivo malignant glioma cells are sensitive to Fas (CD95/APO-1) ligand-mediated apoptosis

Fas (also known as CD95/APO-1) is a cell surface receptor and member of the tumor necrosis factor receptor superfamily which mediates apoptosis in sensitive cells upon oligomerization by specific antibodies or by its ligand (FasL). Recently, human glioma cell lines were found to be susceptible to Fas-mediated apoptosis triggered by α-Fas antibodies. However, whether the Fas system can also be targeted in ex vivo high grade gliomas is at present unknown. In the present investigation, α-Fas antibodies and FasL were tested in short-term monolayer cultures or in colony forming assays established from freshly resected tumors of patients with anaplastic astrocytomas (WHO grade III) and glioblastoma multiforme (WHO grade IV). Anti-Fas antibodies induced only moderate apoptosis in four of the 19 tested glioma cell cultures. This contrasts FasL which induced apoptosis in all of the 19 tumor cell cultures analyzed. Mean cytotoxicity of glioma cell cultures treated for 48 h with α-Fas antibodies or FasL was 9.6% and 44.3%, respectively. Irrespective of whether α-Fas antibodies or FasL were used, pretreatment with recombinant hu (rhu) IFN- γ and rhuTNF- α for 48 h did not sensitize glioma cells to Fas-mediated cytotoxicity. The long-term effect by FasL on tumor colony formation was more striking. FasL treatment resulted in more than 90% inhibition of clonal tumor cell growth of all the eight high grade gliomas analyzed. These results suggest that Fas targeting by FasL but not by α-Fas antibodies may provide a promising approach for locoregional glioma treatment.

Analysis of WT1 gene expression during mouse nephrogenesis in organ culture.

The temporal and spatial expression patterns of the Wilms tumor gene, WT1, were studied during the organogenesis of the mouse kidney in vitro. In situ hybridization and immunocytochemistry localized cellular expression of WT1 in whole kidney organ cultures to the induced metanephric mesenchyme and developing podocytes. Organ cultures were further characterized immunocytochemically with antibodies that specifically labeled the different tubular epithelial components and supporting mesenchyme of the developing nephrons. In organ cultures, the WT1 expression pattern could be visualized in induced metanephric mesenchyme and entire cell cohorts of differentiating podocytes. Expression of WT1 and cell specific markers were retained in short-term monolayer cultures of dissociated kidneys. The development of the metanephric kidney in vitro involves a highly restricted temporal and spatial cellular expression pattern of WT1 which closely follows that observed in tissue sections from gestational kidney isolated during organogenesis in the mouse.

Identification and Characterization of a Unique Chondrocyte Gene Involved in Transition to Hypertrophy

The character of differentiating chondrocytes in growing long bones has been defined by altered expression of a limited number of genes. To expand this set we have applied differential display to identify genes expressed in either mineralizing or nonmineralizing chondrocytes. One such gene,Band 17,has the following characteristics: (1)Band 17expression is predominantly found in cartilage destined for mineralization.Band 17mRNA is undetectable in articular cartilage and undetectable or weak in all other tissues tested. (2)Band 17expression is spatially restricted to the lower proliferative/upper hypertrophic zone of chondrocytes in the growth plate of long bones and embryonic vertebrae. (3) Induction of a hypertrophic phenotype in progenitor sternal chondrocytes by treatment with ascorbate increases expression ofBand 17.(4) Induction of hypertrophy in growth plate chondrocytes in short-term monolayer cultures correlates with a rapid but transient rise inBand 17message. Our interpretation of these findings is thatBand 17expression is associated with the transition to hypertrophy, not maintenance of the hypertrophic phenotype. Molecular analysis of the 3′ end ofBand 17cDNAs and genomic structure has shown thatBand 17is a single copy gene transcribed into four messages. Alternative splicing of these messages is predicted to result in two proteins that differ at the C-terminal by 131 amino acids. The longer protein contains a C-terminal consensus sequence that potentially targets this protein to the lumen of the endoplasmic reticulum. There is aBand 17homologue in humans, suggesting conservation ofBand 17function in mammals. In summary, the pattern of expression and the predicted primary structure identifyBand 17as unique among all previously known chondrocyte genes.

Effects of transforming growth factor-beta 1 and fibroblast growth factor on DNA synthesis in growth plate chondrocytes are enhanced by insulin-like growth factor-I.

The local tissue metabolism is controlled through the complex interaction between systemic and local growth factors. In recent years, an increasing number of autocrine or paracrine growth regulators have been identified in physeal cartilage. While these factors act to alter chondrocytes phenotypically and presumably are important mediators in the process of endochondral ossification, the manner in which they interact with the systemically regulated growth factor insulin-like growth factor-I is unknown. In the present study, the interactive effects of insulin-like growth factor-I with transforming growth factor-beta 1 or basic fibroblast growth factor were examined in short-term monolayer cultures of chick growth plate chondrocytes. [3H]thymidine incorporation was maximally stimulated 11-fold by fibroblast growth factor (10 ng/ml) and 3.5-fold by transforming growth factor-beta 1 following a 24-hour exposure in serum-containing cultures. The effects of transforming growth factor-beta 1 and fibroblast growth factor at both high and low concentrations were enhanced in a dose-dependent manner by insulin-like growth factor-I, with a 40-50% increase in DNA synthesis in the presence of 100 ng/ml of insulin-like growth factor-I. Since insulin-like growth factor-I increased [3H]thymidine incorporation after 48 hours (50% increase) but not after 24 hours of exposure, these observations represent a synergistic interaction. Total DNA in cultures treated for 5 days confirmed the modulating effect of insulin-like growth factor-I with transforming growth factor-beta 1 and fibroblast growth factor. The growth factors were further examined for their effects on markers of chondrocyte differentiation. While all three caused a dose-dependent inhibition of alkaline phosphatase activity, the effects of insulin-like growth factor-I were additive only to those of transforming growth factor-beta 1 and fibroblast growth factor. Similarly, insulin-like growth factor-I did not affect the sulfate incorporation stimulated by fibroblast growth factor or transforming growth factor-beta 1. Insulin-like growth factor-I had no effect on total protein synthesis after 24 hours and, although type-II collagen mRNA levels were stimulated, it had no effect on type-X collagen mRNA, as determined by quantitative in situ hybridization. Finally, insulin-like growth factor-I did not alter the dose-dependent stimulation of noncollagen protein synthesis and the inhibition of collagen synthesis caused by fibroblast growth factor and transforming growth factor-beta 1 in 24-hour cultures. Thus, the data suggest that insulin-like growth factor-I may have a role in augmenting the effects of other growth factors found in cartilage.(ABSTRACT TRUNCATED AT 400 WORDS)

Recombinant Bone Morphogenetic Protein-4, Transforming Growth Factor-β1, and Activin A Enhance the Cartilage Phenotype of Articular Chondrocytes in Vitro

Bone morphogenetic protein-4 (BMP-4) is a member of the transforming growth factor-β (TGF-β) supergene family and is characterized by its ability to induce singly de novo cartilage and bone in vivo. The influence of recombinant bone morphogenetic protein-4 and some related members, TGF-β1, activin A, and inhibin A, on articular chondrocyte metabolism in the presence and absence of extracellular matrix has been examined. BMP-4 and TGF-β1 stimulated [35S]-sulfate incorporation in a dose-dependent manner in short-term monolayer, micromass, and explant cultures. Activin A showed a slight but significant stimulation of proteoglycan synthesis while inhibin A decreased metabolic activity. The effects observed were most pronounced in the explant culture system. Although the relative influence of the growth factors was less apparent in chondrocytes isolated from adult cartilage, the qualitative responses were similar with cells obtained from young animals. The maintenance and enhancement of the cartilage phenotype was further investigated by Northern blot analysis. BMP-4 and TGF-β1 increased the levels of expression of type II collagen and proteoglycan aggrecan in short-term cultures, while activin A and inhibin A did not affect these parameters significantly when compared to serum-free control cultures. Binding experiments with 125I-BMP-4, revealed the presence of specific, high-affinity binding sites with an apparent dissociation constant of 110 pM and about 6000 receptors per cell. Chemical cross-linking showed the presence of three components (apparent size 200, 90, and 70 kDa), demonstrating the presence of functional receptors for BMP-4 on primary articular chondrocytes.

The Production of Transforming Growth Factor-β by Chick Growth Plate Chondrocytes in Short Term Monolayer Culture*

Transforming growth factor-beta (TGF beta) is capable of regulating the proliferation and phenotypic expression of growth plate chondrocytes in culture. Chondrocytes were isolated from the growth plates from the long bones of 3- to 5-week-old chicks. Conditioned medium was harvested from short term monolayer cultures for the assay of TGF beta production by these cells. A receptor competition assay using [125I]TGF beta was used to quantitate the amount of TGF beta in the conditioned medium. Acid-activated conditioned medium contained 5.0 +/- 0.4 ng/ml TGF beta, while conditioned medium that had not been exposed to acid had undetectable levels of the peptide by this assay. The initial cell plating density was inversely related to the amount of TGF beta produced on a per cell basis. Growth plate chondrocytes separated by countercurrent centrifugal elutriation into maturationally distinct subpopulations had different rates of TGF beta production; hypertropic chondrocytes produced significantly more TGF beta (4.5 ng/10(6) cells) than the smallest chondrocytes isolated (2.3 ng/10(6) cells). A variety of other growth mediators were tested for their ability to influence TGF beta production by chondrocytes, and it was found that only basic fibroblast growth factor could significantly influence TGF beta production, producing a 6-fold increase in TGF beta recovered in the conditioned medium. The production of TGF beta by growth plate chondrocytes implicates it as an important autocrine or paracrine regulator in the process of endochondral calcification.

Reversible mitochondrial swelling in cultured rat hepatocytes exposed to 1,2-dimethylhydrazine

The early structural changes of F344 rat hepatocytes exposed to the hepatocarcinogen 1,2-dimethylhydrazine (DMH) were characterized in short-term monolayer cultures. Continuous exposure of monolayers to DMH (2–16 m M) caused cytoplasmic vacuoles visible by phase-contrast microscopy in all hepatocytes within 6 hr of exposure. These changes preceded maximal release of lactate dehydrogenase (LDH) which occurred after 48 hr of continuous exposure to cytocidal concentrations of DMH (8–16 m M). Ultrastructurally, hepatocytes exposed to DMH (4 m M, 6 hr) showed a twofold increase in mitochondrial diameter from 340 ± 70 nm in control hepatocytes to 800 ± 140 nm in DMH-exposed cells. Hepatocyte monolayers exposed to DMH (4 m M, 6 hr) with subsequent removal of DMH attained normal phase-contrast appearance within 6 hr. Ultrastructural studies showed no significant differences when compared with control hepatocytes and mitochondrial diameters (330 ± 70 nm) were comparable with control hepatocytes. Pretreatment of hepatocytes with depletors of cellular reduced glutathione concentration, including 1,3-bis(2-chloroethyl)-1-nitrosourea (40 μ M) and diethyl maleate (160 μ M), did not potentiate hepatocellular vacuolation nor release of LDH from hepatocytes exposed to DMH (0–16 m M, 48 hr). These studies demonstrate a distinctive form of reversible high-amplitude mitochondrial swelling that can be monitored by phase-contrast microscopy of cultured hepatocytes in monolayers. Since DMH-induced mitochondrial swelling and its progression to irreversible injury are not potentiated by depletors of reduced thiols, this response appears distinct from prelethal mitochondrial swelling in hepatocytes subjected to oxyradical-mediated mechanisms of injury.

Use of monolayers of primary rat kidney cortex cells for nephrotoxicity studies

Kidney cells were isolated from rat kidney cortex and maintained in short-term monolayer cultures. A number of important parameters were studied in order to establish the usefulness of these cells for toxicity studies. Despite morphological differences between the cultured cells and similar cells in vivo, many relevant enzyme systems remained present and functional. Intracellular glutathione levels were stable up to 5 days in culture. The glutathione S-transferase activity during culture remained stable although at a lower level than in freshly isolated cells. Whereas rat kidney cytosol contained subunits 4, 7, 2 and 1, 3- and 5-day-old cultures contained glutathione transferase subunits 7, 2 and a small amount of subunit 1. Cytochrome P-450, although measurable in microsomes from freshly isolated cells, could not be determined after 1 day in culture. Organic anion transporters on the basolateral side and γ-glutamyl transpeptidase on the apical side were present. Through cytotoxicity studies, β-lyase activity could be demonstrated in the culture. Hence this monolayer culture system, which can be used in combination with filters, seems to be suitable for studying various mechanisms of nephrotoxicity.

Analysis of hemangiopericytic meningiomas by immunohistochemistry, electron microscopy and cell culture.

Intracranial hemangiopericytic meningiomas (HM) from seven patients were examined by immunostaining, transmission electron microscopy (TEM), scanning electron microscopy (SEM) and cell culture. Positive staining for Factor VIII-related antigen was restricted to capillary endothelial cells. There was no reaction with anti-glial fibrillary acidic protein (GFAP) serum or anti-S-100 protein serum. In these neoplasms TEM displayed extracellular basement membrane-like material (BMLM), cytoplasmic intermediate filaments (IF) associated with a dense body, dilated rough-surfaced endoplasmic reticulum containing BMLM, a small area of interdigitation of cell membranes, and a unique intercellular punctate or linear density. By SEM these tumors had intercellular shell-like or reticular structures and irregularly branched capillaries which were compressed by ellipsoidal- or carrot-shaped tumor cells. Short-term monolayer culture showed rapid and vigorous growth of tumor cells, and the formation of pseudolumens but not of whorls. The TEM of cultured cells also showed cytoplasmic IF associated with the dense body. By SEM the cultured cells were flat and had a discoid nucleus with conspicuous nucleolar hillocks. Our results show that HM are mainly poorly specialized mesenchyme-related tumors of the meninges; some possess a potential for aggressive growth and some for differentiation into smooth muscle cells. Further study is needed to determine their histogenesis.

Physiological concentrations of L-leucine control the release of prolactin from cultured rat pituitary cells.

In an attempt to further define the optimum experimental conditions for the in vitro study of PRL endocrinology, the effect of extracellular leucine concentration on PRL secretion was assessed. Rat pituitary cells, maintained in short term monolayer culture, were washed free of serum and placed in Minimum Essential Medium (MEM; 0.39 mM leucine) supplemented with nonessential amino acids and 0.1% gelatin. After the establishment of a constant rate of basal PRL release, the MEM was replaced with leucine-free MEM (supplemented as above), and the rate of PRL release was followed. PRL release from the cells in the leucine-free medium fell within 15 min to a rate 30% of that seen in the controls. Return to the control rate was achieved by the addition of L-leucine only. The D-isomer, isoleucine, valine, proline, leucyl-leucine, and leucine-agarose were ineffective. In the short term, the rate of PRL synthesis was found to be independent of extracellular leucine and the inhibition of release seen upon removal of leucine, and the subsequent recovery upon replacement of leucine was also observed when this experiment was conducted in the presence of 50 micrograms/ml cycloheximide. The L-leucine recovery was found to be dose dependent, with an increase in leucine concentration from 20 to 40 mg/liter causing a tripling of the PRL release rate. The addition of 10(-7) M TRH showed that leucine can be limiting in the response to this stimulant and allowed a response to as little as 10 mg/liter leucine to be discerned. From these experiments, it is concluded that physiological concentrations of leucine specifically and profoundly affect the release of PRL by a mechanism independent of protein synthesis. This leucine requirement for release has not been previously reported.

Monensin inhibits intracellular dissociation of asialoglycoproteins from their receptor.

Treatment of short-term monolayer cultures of rat hepatocytes with the proton ionophore, monensin, abolishes asialoglycoprotein degradation, despite little effect of the drug on either surface binding of ligand or internalization of prebound ligand. Centrifuging cell homogenates on Percoll density gradients indicates that, as a result of monensin treatment, ligand does not enter lysosomes but sediments instead in a lower density subcellular fraction that is likely an endocytic vesicle. Analyzing the degree of receptor association of intracellular ligand revealed that monensin prevents the dissociation of the receptor-ligand complex that normally occurs subsequent to endocytosis. The weak base, chloroquine, also blocks this intracellular dissociation. Evidence from sequential substitution experiments is presented, indicating that monensin and chloroquine act at the same point in the sequence of events leading to ligand dissociation. These data are discussed in terms of a pH-mediated dissociation of the receptor-ligand complex within a prelysosomal endocytic vesicle.

Insulin receptors of human term placental cells and choriocarcinoma (JEG-3) cells: characteristics and regulation.

Insulin has been implicated as a regulator of placental growth and function. In order to probe hormoneplacental interactions, an in vitro human placental cell culture model was employed in conjunction with the human choriocarcinoma cell line, JEG-3. Short term monolayer cultures of human term placentae were established from trypsinized chorionic villi and were presumed to be predominantly of syncytial origin on the basis of their electron microscopic morphology, their capacity to produce placental hormones, and their loss of replicative potential. Specific binding of [125I]iodoinsulin to both cell types was examined using whole cell radioreceptor assays, and was found to be a saturable process, time and temperature dependent, and proportional to cell protein and ligand concentration. At 4 C, a steady state was reached by 18 h and was maintained for 6 h. Specificity of the insulin receptor was evidenced by the inability of structurally unrelated hormones to inhibit [125I]iodoinsulin binding. Scatchard plot...

Cytological and functional analysis of inflammatory infiltrates in human malignant tumors: III. Further functional investigations using cultured autochthonous tumor cell lines and freeze-thawed infiltrating inflammatory cells

Short-term monolayer cultures, dominated by cells with malignant characteristics, were established from human tumors displaying an unusually strong host-inflammatory response. Upon repeated testings in the 51Cr release cytotoxicity assay, blood leukocytes were frequently cytotoxic (a) to autologous and allogeneic tumor cells, without any apparent restriction as to tumor origin or HLA type, and (b) to the so-called natural killer (NK) target cells. The anti-tumor cytotoxicity disappeared with time. The in situ inflammatory cells, freshly isolated or recovered from the deep freeze, did not display any type of cytotoxic activity. Nor were they notably suppressive to either type of blood leukocyte cytotoxic activity in the 51Cr release assay. Cytological analysis demonstrated that the “large granular lymphocytes” (LGL), known to be largely responsible for the NK activity in man, were prominent in the blood but not in the inflammatory infiltrate. These preliminary observations suggest that lack of cytotoxic activity in situ correlates with the absence of effector cells in the inflammatory infiltrate.