The effect of amphotericin b-deoxycholate on proliferation and protein synthesis in human skin fibroblast cultures

Summary Cultures of adult human skin fibroblasts were grown in the presence of the recommended antifungal dose (3 μg per ml) of amphotericin B-deoxycholate. A reduction in cell culture growth, measured as DNA content and protein content per culture, was observed. However, radioisotope incorporation into noncollagen protein and, to a lesser extent, collagen protein was enhanced. These effects were due to amphotericin B, not to deoxycholate. These observations were made under several growth conditions and indicate that cell proliferation or isotope-labeling studies in fibroblasts in the presence of amphotericin B-deoxycholate are susceptible to errors in interpretation. Supported by PHS Grants AM-02456, AM-15312 and AM-17047, by the Kroc Foundation, and by the American Diabetes Association, Washington Affiliate. Recipient of Research Career Development Award AM-47142 from NIAMDD, and to whom requests for reprints should be addressed.     

Evaluation of growth rate in adhering cell cultures using a simple colorimetric method

In this paper we present a simple colorimetric method for evaluating cell growth in adhering cell cultures. This technique is based on the staining of basophilic cellular compounds (mainly nucleic acids) with methylene blue. To check its reliability, we have compared the quantity of dye fixed by the cells with the number of cells released from the culture substratum by trypsinization, and with the total cellular protein content determined by the Lowry's method. In these experiments we have used human skin fibroblasts and human epithelial cells derived from the epithelium of the full-term umbilical cord. Like other alternative methods, the methylene blue colorimetric technique can be used in 96-well microplates and allows the rapid collection of large amounts of data. As an illustration, we report on the selection of optimal composition of culture medium for human skin fibroblasts.     

Simultaneous Rates of Ribonucleic Acid and Deoxyribonucleic Acid Syntheses for Estimating Growth and Cell Division of Aquatic Microbial Communities

A method for measuring rates of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) syntheses using a single radioactive precursor has been devised and tested using bacterial cultures and natural assemblages of marine and freshwater microorganisms. The procedure is based upon the uptake and incorporation of exogenous [³H]adenine into cellular adenosine triphosphate and deoxyadenosine triphosphate pools which serve as the immediate precursors for the adenine incorporated into RNA and DNA, respectively. It is proposed that the DNA/RNA rate ratio is correlated with the specific growth rate of microorganisms and can be used as an index for estimating and comparing the productivities of microbial assemblages in nature. This technique can also be used to detect discontinuous growth and cell division processes which frequently occur in surface plankton populations. The DNA/RNA rate ratios measured in a variety of aquatic ecosystems ranged from 3.3 to 31.8% without significant correlation to total microbial biomass.     

Homogeneous cultivation of animal cells for the production of virus and virus products

Homogeneous technique facilitates the cultivation of large quantities of cells, reduces the risk of contamination by eliminating many manipulations, and makes practical the control of conditions such as pH and oxygen tension. Although most animal cells will not multiply in free suspension, certain cell lines have lost the requirement of being attached to a solid surface. These cells can be subcultured indefinitely but have some resemblance to cancer cells such as their abnormal karyotype. Certain cell linen developed from human embryonic tissue maintain their diploid character after repeated subculture and would seem to be ideal for the production of vaccines. However, strict regulations exist for viral products for human injection in that only cells taken from normal tissue and subcultured but once may be used. A microcarrier method in which cells adhere to DEAE-Sephadex beads permits a suspension culture which may be termed quasihomogeneous. The attached cells may be retained by sedimentation or by screening as the medium is replaced. Cell debirs from the original tissue is difficult to remove from microcarrier cultures; modifications of the trypsinization technique have alleviated but not solved this problem. Conditions for virus replication can be less critical than those for cell growth in that oxygen tension seems to have little influence on virus production. In cases where rate of virus production increases with specific growth rate of cells, homogeneous culture would have a advantage in maintaining a high cell mogeneous culture would have a valuble advantage in maintaining a high cell growth rate for a longer time. Some virus infections destroy cells, but others cause little change in cellular mteabolism except that virus is continually produced. The latter type can be conducted with a microcarrier in continuous culture with a virus titer exceeding 10⁷ plaque forming units per milliliter for over 50 days with Rubella-infected BHK cells.     

Establishment of a suite of assays that support the discovery of proteasome stimulators

BackgroundThe proteasome catalyzes the degradation of many mis-folded proteins, which are otherwise cytotoxic. There is interest in the discovery of proteasome agonists, but previous efforts to do so have been disappointing.MethodsThe cleavage of small fluorogenic peptides is used routinely as an assay to screen for proteasome modulators. We have developed follow-on assays that employ more physiologically relevant substrates.ResultsTo demonstrate the efficacy of this workflow, the NIH Clinical Collection (NCC) was screened. While many compounds stimulated proteasome-mediated proteolysis of the pro-fluorogenic peptide substrates, most failed to evince activity in assays with larger peptide or protein substrates. We also show that two molecules claimed previously to be proteasome agonists, oleuropein and betulinic acid, indeed accelerate hydrolysis of the fluorogenic substrate, but have no effect on the turnover of a mis-folded protein in vitro or in cellulo. However, two small molecules from the NCC, MK-866 and AM-404, stimulate the proteasome-mediated turnover of a mis-folded protein in living cells by 3- to 4-fold.ConclusionAssays that monitor the proteasome-mediated degradation of larger peptides and proteins can distinguish bona fide agonists from compounds only able to stimulate the cleavage of short, non-physiologically relevant peptides.General significanceA suite of assays has been established that allows the discovery of bona fide proteasome agonists. AM-404 and MK-866 can be useful tools for cell culture experiments, and can serve as scaffolds to generate more potent 20S stimulators.     

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins ^{1, 2}. Maintenance of proteasome activity was implicated in many key cellular processes, like cell''s stress response ³, cell cycle regulation and cellular differentiation ⁴ or in immune system response ⁵. The dysfunction of the ubiquitin-proteasome system has been related to the development of tumors and neurodegenerative diseases ^{4, 6}. Additionally, a decrease in proteasome activity was found as a feature of cellular senescence and organismal aging ^{7, 8, 9, 10}. Here, we present a method to measure ubiquitin-proteasome activity in living cells using a GFP-dgn fusion protein. To be able to monitor ubiquitin-proteasome activity in living primary cells, complementary DNA constructs coding for a green fluorescent protein (GFP)–dgn fusion protein (GFP–dgn, unstable) and a variant carrying a frameshift mutation (GFP–dgnFS, stable ¹¹) are inserted in lentiviral expression vectors. We prefer this technique over traditional transfection techniques because it guarantees a very high transfection efficiency independent of the cell type or the age of the donor. The difference between fluorescence displayed by the GFP–dgnFS (stable) protein and the destabilized protein (GFP-dgn) in the absence or presence of proteasome inhibitor can be used to estimate ubiquitin-proteasome activity in each particular cell strain. These differences can be monitored by epifluorescence microscopy or can be measured by flow cytometry.     

Selective and nonselective isolation of temperature-sensitive mutants of mouse L-cells and their characterization

Mutants of mouse L-cells which are temperature-sensitive for growth have been obtained by using both selective and nonselective isolation procedures on populations treated with the mutagen nitrosoguanidine. Selective isolation was carried out by utilizing a five-day treatment with ³H-TdR and ara-C as selective agents at the nonpermissive temperature. Nonselective isolation was performed by isolating 1400 clones in the absence of selective agents and then testing them for temperature-sensitivity. From this experiment we obtained a minimum estimate of 6 × 10^?3 for the frequency of mutants in the mutagentreated population. The mutants were characterized by their plating efficiencies, growth in suspension culture, and uptake of isotopic precursors of DNA, RNA, and protein. A range in phenotypes was observed, and there appeared to be some differences between the mutants obtained by the two types of isolation procedures. In uptake experiments the most marked reductions in the rates of precursor incorporation were seen with ³H-TdR, rather than ³H-UR or ³H-Leu. Different mutant lines showed considerable variation in the rate of cessation of DNA synthesis as well as the time required for termination of cell division. These experiments suggest that both types of isolation procedures are feasible for obtaining temperature-sensitive mutants having a range of phenotypes.     

A damaged DNA binding protein 2 mutation disrupting interaction with proliferating-cell nuclear antigen affects DNA repair and confers proliferation advantage

In mammalian cells, Nucleotide Excision Repair (NER) plays a role in removing DNA damage induced by UV radiation. In Global Genome-NER subpathway, DDB2 protein forms a complex with DDB1 (UV-DDB), recognizing photolesions. During DNA repair, DDB2 interacts directly with PCNA through a conserved region in N-terminal tail and this interaction is important for DDB2 degradation. In this work, we sought to investigate the role of DDB2-PCNA association in DNA repair and cell proliferation after UV-induced DNA damage. To this end, stable clones expressing DDB2^Wt and DDB2^PCNA- were used. We have found that cells expressing a mutant DDB2 show inefficient photolesions removal, and a concomitant lack of binding to damaged DNA in vitro. Unexpected cellular behaviour after DNA damage, such as UV-resistance, increased cell growth and motility were found in DDB2^PCNA- stable cell clones, in which the most significant defects in cell cycle checkpoint were observed, suggesting a role in the new cellular phenotype. Based on these findings, we propose that DDB2-PCNA interaction may contribute to a correct DNA damage response for maintaining genome integrity.     

Differential effects of cAMP and cGMP on in vitro epidermal cell growth.

Primary keratinocyte cultures free of dermal fibroblasts were used to investigate the effect of varying cyclic AMP (cAMP) concentrations on epidermal cell function. Addition of 10^?3, 10^?4 or 10^?5 M dibutyryl cAMP to plated cells (day 1) results by day 5 in a dose dependent increase of [³H]TdR incorporation into DNA as determined by increases in both the labeling index and incorporation of ³H label into an isolated DNA fraction. 8-Bromo cAMP, another cAMP analogue, likewise induced keratinocyte proliferation. The proliferative response was dose and time dependent, and 5- to 6-fold increases in ³H label incorporated into DNA were seen at day 6, 8 and up until day 15 of culture. Moreover, elevation of cellular cAMP by addition of cholera toxin, an irreversible stimulator of adenylate cyclase, also demonstrated a time dependent stimulation of [³H]TdR uptake into DNA and increased the labeling index. Specific histochemical staining for keratinaceous protein (Kreyberg technique) demonstrated that elevated cAMP levels also enhance the production of specialized (differentiated) epidermal cells. Determination of the level of cAMP and cyclic GMP (cGMP) by RIA of partially purified fractions of the cultures revealed that addition of 8-bromo cAMP or cholera toxin to the cultures increased the levels of cAMP but not of cGMP. Addition of 8-bromo cGMP to the keratinocytes on day 1 at concentrations of 10^?6, 10^?7 or 10^?8 M had no effect on culture proliferation on days 4, 6 and 8, although qualitative changes in the electron microscopic pattern of the culture stratification and specialization were observed. The results indicate (1) both large and moderate increases in cellular cAMP levels induce keratinocyte culture proliferation and specialization in the absence of fibroblasts or dermal influences, (2) the quantitative enhancement of keratinocyte growth and specialization occurs without apparent participation of cGMP, (3) cGMP may be a qualitative effector of epidermal cell differentiation.     

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

Few naturally-occurring plasmids are maintained in mammalian cells. Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus (EBV) and Kaposi''s Sarcoma-associated herpesvirus (KSHV), which cause multiple human malignancies ^1-3. These two genomes are replicated in a licensed manner, each using a single viral protein and cellular replication machinery, and are passed to daughter cells during cell division despite their lacking traditional centromeres ^4-8.Much work has been done to characterize the replications of these plasmid genomes using methods such as Southern blotting and fluorescence in situ hybridization (FISH). These methods are limited, though. Quantitative PCR and Southern blots provide information about the average number of plasmids per cell in a population of cells. FISH is a single-cell assay that reveals both the average number and the distribution of plasmids per cell in the population of cells but is static, allowing no information about the parent or progeny of the examined cell.Here, we describe a method for visualizing plasmids in live cells. This method is based on the binding of a fluorescently tagged lactose repressor protein to multiple sites in the plasmid of interest ⁹. The DNA of interest is engineered to include approximately 250 tandem repeats of the lactose operator (LacO) sequence. LacO is specifically bound by the lactose repressor protein (LacI), which can be fused to a fluorescent protein. The fusion protein can either be expressed from the engineered plasmid or introduced by a retroviral vector. In this way, the DNA molecules are fluorescently tagged and therefore become visible via fluorescence microscopy. The fusion protein is blocked from binding the plasmid DNA by culturing cells in the presence of IPTG until the plasmids are ready to be viewed.This system allows the plasmids to be monitored in living cells through several generations, revealing properties of their synthesis and partitioning to daughter cells. Ideal cells are adherent, easily transfected, and have large nuclei. This technique has been used to determine that 84% of EBV-derived plasmids are synthesized each generation and 88% of the newly synthesized plasmids partition faithfully to daughter cells in HeLa cells. Pairs of these EBV plasmids were seen to be tethered to or associated with sister chromatids after their synthesis in S-phase until they were seen to separate as the sister chromatids separated in Anaphase¹⁰. The method is currently being used to study replication of KSHV genomes in HeLa cells and SLK cells. HeLa cells are immortalized human epithelial cells, and SLK cells are immortalized human endothelial cells. Though SLK cells were originally derived from a KSHV lesion, neither the HeLa nor SLK cell line naturally harbors KSHV genomes¹¹. In addition to studying viral replication, this visualization technique can be used to investigate the effects of the addition, removal, or mutation of various DNA sequence elements on synthesis, localization, and partitioning of other recombinant plasmid DNAs.     

Stimulation of guanylate cyclase and RNA polymerase II activities in HeLa cells and fibroblasts by biotin

HeLa cells cultured in a biotin-deficient medium showed reduced rates of protein synthesis, DNA synthesis and growth. Continuous synthesis is required for the increase in DNA synthesis observed upon addition of biotin to cells cultured in biotin-deficient medium. The addition of biotin to the biotin-deficient culture medium increased the activity of guanylate cyclase in both HeLa cells and fibroblasts. Both cell types cultured in biotin deficient medium showed reduced activity of RNA Polymerase II. The exogenous addition of biotin to the biotin-deficient cell cultures also resulted in increased activity of RNA Polymerase II in HeLa cells and fibroblasts. The maximal response was observed in 4 hours. Significant increase in enzyme activity was observed at 10^–8 M biotin in the culture medium. The growth promoting effect of biotin seems to involve stimulations of cellular guanylate cyclase and RNA Polymerase II activity.     

Unbalanced growth as a normal feature of development of Bdellovibrio bacteriovorus

In this study we have investigated the rates and spatial patterns of chromosome replication and cell elongation during the growth phase of wild-type and facultatively prey-independent mutant strains of Bdellovibrio bacteriovorus. For the facultatively prey-independent mutants, the total DNA content of synchronously growing cultures was found to increase exponentially, as the multiple chromosomes within each filamentous cell replicated simultaneously. Cell mass, measured as total cellular protein, also increased exponentially during this period, apparently by means of multiple elongation sites along the filament wall. The relative rates of DNA and protein synthesis were unbalanced during growth, however, with the cellular concentration of DNA increasing slightly faster than that of protein. The original cellular DNA: protein ratio was restored in the progeny cells by continued protein synthesis during the septation period that follows the termination of DNA replication. Because of technical problems, these experiments could not be conducted on the wild-type cells, but similar results are assumed. This unusual pattern of unbalanced growth may represent an adaptation by bdellovibrios to maximize their progeny yield from the determinate amount of substrate available within a given prey cell.     

Analysis of chain length,substitution patterns,and unsaturation of AM-404 derivatives as 20S proteasome stimulators

Proteasome-mediated degradation of proteins is a vital cellular process and is performed by the ubiquitin-dependent proteasome system (UPS) and the ubiquitin-independent proteasome system (UIPS). While both systems are necessary to maintain healthy cell function, many disease states are characterized by reduced activity of the UPS, and the UIPS cannot by itself maintain proper protein levels. It has been suggested that the 20S core particle (20S CP), the isoform of the proteasome in the UIPS that can degrade proteins without a ubiquitin tag, can be stimulated with a small molecule to assist the 20S CP to accept and hydrolyze substrates more rapidly. Several small molecule stimulators of the 20S CP have since been discovered, including AM-404, an arachidonic acid derivative. AM-404 has previously been shown to inhibit fatty acid amide hydrolase activity. We wished to evaluate what structural components of AM-404 are required to stimulate the 20S CP with the long-term goal of using this information to design a stimulator with better drug-like qualities. We synthesized numerous derivatives of AM-404, varying the chain length, substitutions, and degree of unsaturation. Through this endeavor, we obtained several molecules capable of stimulating the 20S CP to various degrees. We discovered that though chain length is important, the presence of a cis-alkene in a specific location in the aliphatic chain has the greatest impact on the ability to stimulate the 20S CP. Two of the derivatives maintain modest stimulatory activity, and have improved toxicity over AM-404.     

Mechanism of action of benzo(a)pyrene and nicotine on hormone production by rat pituitary tumor cells

The effects of the cyclic aromatic hydrocarbon, benzo(a)pyrene (BaP) and that of the tobacco alkaloid, nicotine, on prolactin (PRL) and growth hormone (GH) synthesis by rat pituitary tumor cells in culture (GH cells) have been studied. Treatment of GH cells with nicotine (0.1–300 μg/ml) neither affected the growth, nor significantly altered the general pattern of hormone production in these cells. BaP at concentrations greater than 5 μg/ml irreversively inhibited the growth of these cells. The sublethal concentrations of BaP, which did not affect either 1) cell growth, or 2) amino acid transport or 3) total protein synthesis or degradation, did however inhibit specifically, hormone synthesis by these cells. More interestingly concentrations of nicotine which did not affect either cell growth or hormone synthesis, modulated both of these cellular processes in the presence of BaP. A concentration dependent stimulation of microsomal BaP monooxygenase activity was observed in nicotine or BaP treated cells. The effects of these drugs on stimulation of BaP monooxygenase activity seems to be additive. Nicotine also enhanced the association of radioactivity (presumably [³H] BaP metabolites) with DNA in [³H] BaP treated cells. It is concluded that nicotine by itself did not demonstrate any cytotoxic effect nor influence hormone synthesis in GH cells. However, this constituent of tobacco smoke stimulated BaP monooxygenase activity and the interaction of [³H] BaP metabolites with cellular DNA and also modulated BaP induced inhibition of hormone synthesis in GH cells.     

Vascular growth factor binding kinetics to the endothelial cell basement membrane, with a kinetics-based correction for substrate binding

Vascular growth factors, including vascular endothelial growth factor and fibroblast growth factor-2, bind to heparan sulfate proteoglycans in the basement membrane. While this binding, storage, and release system provides a critical model for controlled drug release devices, basement membrane—growth factor binding kinetics have not been fully established. We modified endothelial cell—growth factor binding kinetics protocols for the basement membrane. The basement membrane showed low affinity for fibroblast growth factor-2 (K _d = 185.8 nM), with a slow off rate (k _off = 0.00338 min^?1). However, results were confounded by growth factor binding to tissue culture polystyrene in a manner strikingly similar to basement membrane. Since substrate binding could not be blocked, a binding kinetics based correction technique was developed to account for polystyrene growth factor binding. This method was validated by conducting binding kinetics experiments on bacteriologic plates that exhibit little growth factor binding. This novel method will improve our understanding of cell and protein interaction with the basement membrane in health and disease. They can also further be applied to develop biomimetic drug delivery systems.     

Control of normal differentiation of myeloid leukemic cells. XI. Induction of a specific requirement for cell viability and growth during the differentiation of myeloid leukemic cells

Normal hematopoietic cells require the presence of a protein (MGI) in the appropriate conditioned medium (CM) for cell viability and growth and for differentiation to mature macrophages and granulocytes. Clones of myeloid leukemic cells have been established in culture (D⁺ clones) which require CM with this protein for differentiation, but not for cell viability and growth. It has been shown that these leukemic cells can be induced by CM to again require, like normal cells, the presence of CM for cell viability and growth. Induction of this requirement, which will be referred to as RVG, occurred before the D⁺ cells differentiated to mature granulocytes. Clones of myeloid leukemic cells (D^? clones) that could not be induced to differentiate to mature cells, did not show the induction of RVG. The steroid hormones prednisolone and dexamethasone can induce some, but not all the changes associated with differentiation of D⁺ cells. Incubation with these steroids did not result in the induction of a requirement for these steroids for cell growth and viability. Studies with CM from different sources have shown, that all batches that induced RVG also induced differentiation of D⁺ cells and that both activities were inhibited after treating the CM with trypsin. It is suggested that the same protein (MGI) may be involved in both activities. Incubation of D⁺ cells with CM resulted in an increase in agglutinability by concanavalin A and this increase was maintained even in the absence of CM. This suggests, that the induction of RVG in D⁺ myeloid leukemic cells is associated with a change in the cell surface membrane.     

Assimilatory Sulfur Metabolism in Marine Microorganisms: Sulfur Metabolism, Protein Synthesis, and Growth of Alteromonas luteo-violaceus and Pseudomonas halodurans During Perturbed Batch Growth

The antibiotic protein synthesis inhibitor chloramphenicol specifically blocked the incorporation of [³⁵S]sulfate into the residue protein of two marine bacteria, Pseudomonas halodurans and Alteromonas luteo-violaceus. Simultaneous inhibition of total protein synthesis occurred, but incorporation of ³⁵S into low-molecular-weight organic compounds continued. A. luteo-violaceus rapidly autolyzed, with similar reduction in cell counts, total culture protein and cellular sulfur, whereas P. halodurans remained viable. Treatment with chloramphenicol, growth during nitrogen and carbon limitation, and the carbon and energy sources used for growth did not alter the sulfur content of P. halodurans protein. The mean value (1.09%, by weight), representing a wide variety of environmentally relevant growth conditions, was in agreement with model protein composition. The variability of cellular composition of P. halodurans and A. luteo-violaceus is discussed with respect to the measurement of bacterial growth in natural environments. Total carbon and nitrogen per cell varied greatly (coefficient of variation, ca. 100%) depending on growth conditions. Variation in total sulfur and protein per cell was much less (coefficient of variation, <50%), but the least variation was found for sulfate incorporation into residue protein (coefficient of variation, ca. 15%). Thus, sulfate incorporation into residue protein can be used as an accurate measurement of de novo protein synthesis in these bacteria.     

Cell cycle regulation by growth factors and nutrients in normal and transformed cells

Summary SV3T3 cells, originally responsive to epidermal growth factor (EGF) and displaying density-dependent inhibition of growth, lose responsiveness to the growth factor after several passages and then proliferate without restriction, but continue to display EGF receptor sites at the cell surface. Proliferation of primary fetal rat hepatocytes is not stimulated by EGF, but cells bind it to an extent comparable to that of responsive 3T3 cells. Therefore presence of EGF receptors does not imply that cells are responsive to the growth factor. The relevance of some growth-factor-induced events for DNA synthesis initiation is dicussed. In various primary and secondary cell cultures, Ca⁺⁺-levels appear to be involved in controlling cell proliferation. In contrast, in 3T3-4a cells, levels of Ca⁺⁺ ions are not tightly coupled to DNA synthesis initiation; effects of growth factors are not mediated by extracellular Ca⁺⁺ ions, but cells have a Ca⁺⁺-sensitive restriction, point in G₁. In various cell types in primary or secondary culture or in 3T3-4a cells, polyamine, levels are not tightly coupled to induction of proliferation. Therefore growth-factor-induced ornithine decarboxylase is not an event essential for DNA synthesis initiation. Normal but not transformed cells have a spermidine/spermine-sensitive restriction point in G₁. Although rRNA synthesis appears to be necessary for induction of proliferation, preliminary data obtained by double-beam flow microfluorometry suggest that cellular RNA levels might not affect rate of entry into S phase and, furthermore, that 3T3-4a cells can enter S without accumulating RNA above levels present in quiescent cells. It appears that none of the events induced during the prereplicative phase that have been studied in 3T3 cells are essential for DNA synthesis initiation under normal culture conditions. Presented in the Opening Symposium on Nutritional Factors and Differentiation at the 28th Annual Meeting of the Tissue Culture Association, New Orleans, Louisiana, June 6–9, 1977. This work was supported by Research Grants GM 20101, CA 15087, CA 14195, CA 12227 and CA 11176 from the USPHS, and Grant BC-30D from the American Cancer Society.