Some effects of trypsin dissociation on the inhibition of reaggregation among embryonic chicken neural retina cells by cycloheximide

The effects of cycloheximide on the reaggregation of trypsin-dispersed, embryonic chicken, neural retina cells were investigated. The cells were used either immediately after isolation (F-cells), or after 24 hr prior culture under conditions not permitting reaggregation (V-cells). The parameters of aggregation used were the size of aggregates formed in gyrotatory shaker cultures, and the concentration of single cells in these cultures. At both 24 and 48 hr, following treatment with cycloheximide, the mean diameters of the aggregates of F-cells showed a greater reduction than the V-cells, when compared with their corresponding controls. In addition, cycloheximide resulted in a higher concentration of single cells, that is less cell/cell adhesion, than in comparable controls. A higher proportion of single cells were present in the F-cell cultures in the presence of cycloheximide than in the V-cell cultures. Thus, the degree of inhibition of cell adhesion by one inhibitor of protein synthesis differed in F-cells and V-cells. These experiments may serve to focus attention on some secondary effects of tryptic dissociation, which is an often overlooked factor in subsequent studies of reaggregating embryonic cells, particularly in those experiments involving the use of metabolic inhibitors.     

Contact-mediated changes in cycloheximide-induced agglutinability of BHK cells

Suspension cultures of BHK cells grow in MEM supplemented with 10% fetal calf serum at about 50% the rate of corresponding monolayer cultures. If the serum supplement is reduced to 2% no increase in cell number is observed. When 10% serum is used small spheroids comprising 3–4 cells form within a 24 h period, but in 2 % serum the cells remain single over the same period. The addition of cycloheximide to contact-inhibited monolayer cultures induces high levels of ConA agglutinability within 6 h, yet growing non-confluent cells are rendered only about half as agglutinable by the same treatment. Cycloheximide treatment of suspension cultures causes growing cells to become agglutinable, but non-growing cells, which do not form spheroids, remain non-agglutinable even after 24 h of treatment. This suggests that the pronounced effect of cycloheximide on the agglutinability of contact-inhibited cells in monolayer culture reflects their confluence rather than suspended growth, and that the turnover rate of surface molecules determining the agglutinability state of cells is enhanced as cell-to-cell contact increases.     

Induction of an oligodendroglial enzyme in C-6 glioma cells maintained at high density or in serum-free medium.

The relationship between cell density and the activity of 2':3'-cyclic nucleotide 3'-phosphohydrolase (CNP), an enzyme believed to be specific to oligodendroglial cells and myelin in the brain, has been studied in cultured C-6 glioma cells. Over a 12-day period, the specific activity of CNP underwent a 4-fold increase in conjunction with an increase in the cell density (total protein/flask) and a decline in the growth rate of the cultures. In contrast, the specific activity of Na+,K+-ATPase was not influenced by cell density. Experiments with cultures seeded at different initial densities indicated that the increase in CNP activity coincided with the attainment of a specific cell density rather than with the length of time that the cells were maintained in culture. Arrest of cell proliferation in non-confluent C-6 cells by means of thymidine blockade was not sufficient to cause an increase in the activity of CNP; however, removal of serum from the culture medium resulted in a 3-fold induction of the enzyme in the absence of a high degree of cell contact. The induction of CNP in cells maintained in serum-free medium paralleled the development of a series of distinct morphological changes reminiscent of glial differentiation, which occurred within 48 hours after removal of the serum. Inhibition of protein synthesis by cycloheximide prevented the induction of CNP in serum-free cultures. The demonstration that an enhancement of an oligodendroglial characteristic in C-6 glioma cells can be obtained by growing the cells to high density or by removing serum from the medium, provides further support for the suggestion that these cells may be analogous to the glial stem cells present in the developing brain.     

Deoxyribonucleic Acid Replication in Simian Virus 40-Infected Cells IV. Two Different Requirements for Protein Synthesis During Simian Virus 40 Deoxyribonucleic Acid Replication

The replication of simian virus 40 (SV40) deoxyribonucleic acid (DNA) was inhibited by 99% 2 hr after the addition of cycloheximide to SV40-infected primary African green monkey kidney cells. The levels of 25S (replicating) and 21S (mature) SV40 DNA synthesized after cycloheximide treatment were always lower than those observed in an infected untreated control culture. This is consistent with a requirement for a protein(s) or for protein synthesis at the initiation step in SV40 DNA replication. The relative proportion of 25S DNA as compared with 21S viral DNA increased with increasing time after cycloheximide treatment. Removal of cycloheximide from inhibited cultures allowed the recovery of viral DNA synthesis to normal levels within 3 hr. During the recovery period, the ratio of 25S DNA to 21S DNA was 10 times higher than that observed after a 30-min pulse with (3)H-thymidine with an infected untreated control culture. The accumulation of 25S replicating SV40 DNA during cycloheximide inhibition or shortly after its removal is interpreted to mean that a protein(s) or protein synthesis is required to convert the 25S replicating DNA to 21S mature viral DNA. Further evidence of a requirement for protein synthesis in the 25S to 21S conversion was obtained by comparing the rate of this conversion in growing and resting cells. The conversion of 25S DNA to 21S DNA took place at a faster rate in infected growing cells than in infected confluent monolayer cultures. A temperature-sensitive SV40 coat protein mutation (large-plaque SV40) had no effect on the replication of SV40 DNA at the nonpermissive temperature.     

Antiviral factor production by infected chick embryo fibroblasts]

The secretion of antiviral factor (AF) by infected cell cultures was examined. Activity of AF depended on the cell culture used. AF produced by infected chick embryo fibroblasts had maximal activity. No activity was registered in BHK-21 cells, whereas human embryo fibroblasts and cell line Vero produced a low level of activity. Actinomycin D and cycloheximide prevented the production of AF. The results indicate that VEE virus-infected chick embryo fibroblasts produce AF which may be attributed to nonspecific factors of cell defense.     

Cycloheximide resistance in carrot culture: a differentiated function

Cultured carrot cells grow as unorganized callus tissue in medium containing auxin. Upon removal of the auxin from the medium, they grow in an organized manner and differentiate into embryos. In the normal cell line, W001C, the callus growth can be inhibited by cycloheximide, but the embryonic growth cannot. A variant cell line, WCH105, whose callus growth is resistant to cycloheximide, was isolated. The mechanism of cycloheximide resistance in embryos of both lines and in WCH105 callus was found to be cycloheximide inactivation. In addition to auxin, bromodeoxyuridine can also promote callus growth in carrot culture. Callus cultures maintained by bromodeoxyuridine behave the same as do those maintained by auxin. WCH105 callus is resistant, whereas W001C callus is sensitive to cycloheximide inhibition. Except for the onset of embryogenesis, cycloheximide inactivation is expressed throughout the embryo developmental stages up to the plantlets. These results suggest that cycloheximide inactivation is a function expressed in the differentiated, but not in the undifferentiated, tissues.     

Localization of insulin-like immunoreactivity in the neurons from primary cultures of rat brain

Brains from 1-day-old rats were dissociated with trypsin and the cells were maintained in culture for 3-4 weeks. These primary cultures contained insulin-like immunoreactivity in the limited populations of neurons. Typically, the fluorescent staining pattern observed in the soma was homogeneous and varicosity-like structures were observed on the neurites of the majority of insulin-like immunoreactive neurons. Serum deprivation of brain cell cultures did not reduce immunoreactivity, whereas cycloheximide caused approx. 80% decrease in the number of insulin-like immunoreactive neurons. Incubation of these cultures with [3H]valine resulted in the incorporation of radioactivity into immunoprecipitable insulin. These results suggest that insulin-like immunoreactivity present in the Central Nervous System (CNS) neurons may be synthesized by brain cell cultures.     

Nitric oxide synthase induction in glial cells: Effect on neuronal survival

In primary rat cortical glial cell cultures lipopolysaccharide (LPS) induced a dose- and time-dependent increase of intracellular cyclic GMP concentration associated with a release of nitrite. The LPS-induced cyclic GMP and nitrite increase was enhanced by interferon-γ and was prevented by L-N^G- nitroarginine, dexamethasone and cycloheximide. Thus indicates that LPS effect occured via the production of nitric oxide (NO) and involved new protein synthesis suggesting the induction of NO syntahse in these cells. Furthermore this induction was Ca²⁺-independent and was blocked by an inhibitor of the synthesis of tetrahydrobiopterin. The inducible NO synthase was also expressed by C6 glioma cells. In primary mixed cultures containing both neuronal and glial cells, the effects of LPS were less important than in primary glial cell cultures suggesting that glial cells rather than neurons expressed the inducible form of NO synthase. On the other hand no change on neuronal viability was observed after NO synthase induction by LPS in this culture type. This study indicates that glial cells are able to induce NO synthase without affecting neuronal survival.     

Cell density-dependent stimulation of glutamine synthetase activity in cultured mouse teratoma cells

A density dependent stimulation of glutamine synthetase (GS) activity has been observed in cultures of mouse teratoma cells. GS specific activity increased as cultures approached confluency to a level greater than 2-fold over the basal level found in sparse cultures. After confluency the GS specific activity returned to the basal level found in sparse cultures. The enzyme increase could not be attributed to age of cultures, medium or glutamine depletion, cell leakage of GS, or change in the amount of cellular protein. Dibutyryl cyclic AMP (db-cAMP) plus theophylline lowered GS specific activity both in cultured teratoma and in teratoma obtained from ascites grown tumors. The enzyme increase observed in cultured teratoma cells could be prevented by cycloheximide, and enhanced by hydrocortisone or actinomycin D.     

Interaction of Chlamydia trachomatis with human genital epithelium in culture

Primary cultures of human endometrial and ectocervical epithelial cells were examined as a new model system to study genital infection by Chlamydia trachomatis. Initial studies demonstrated that these cells were indeed susceptible to chlamydial infection. Inocula, adjusted to produce inclusions in 50 to 80% of equivalent numbers of standard McCoy cells, resulted in infection rates of approximately 15 to 30% for the columnar cells of the endometrium and 5 to 10% for the squamous cells of the ectocervix. Exposure of cultures to DEAE-dextran and centrifugation-assisted inoculation, manipulations reported to enhance infection of HeLa and McCoy cells, did not alter the number of inclusion-positive genital cells. Addition of cycloheximide to the post-inoculation culture medium slightly increased numbers of inclusion-bearing cells while growth of genital cells in hormone-supplemented medium resulted in a variable effect on inclusion development and a significant reduction in the association of radiolabelled organisms with these cells. The basis for the different levels of infection in McCoy versus genital cell cultures was revealed by immunofluorescence analysis of chlamydial association with host cells immediately after inoculation. Chlamydiae failed to adhere to many cells in the genital cell cultures while adherence to McCoy cells was uniform. In addition, the association of radiolabelled C. trachomatis was significantly lower with genital cells than with McCoy cells. Finally, culture conditions were defined which markedly inhibited inclusion development without an immediate loss of chlamydial growth potential. This investigation indicates that primary genital cell cultures are susceptible to chlamydial infection and will be valuable for studies on the nature of C. trachomatis interactions with natural human target cells.     

Characterization of factor(s) in culture supernatants affecting cell social behavior.

Treatment of embryonic chick heart fibroblast cultures with 0.2 M urea reversibly increases cellular overlap. The increase in cellular overlapping over that in control cultures may be quantitated by the overlap ratio (R), the ratio of the number of superimposed nuclei observed, to the number expected to occur when cells are assumed to be distributed randomly over the culture substratum (R = observed/expected overlaps). Reversal of the urea-induced increase in R is blocked by 0.2 micrograms/ml cycloheximide. In the presence of cycloheximide, normal (low) overlap ratios are restored to urea-treated cultures by adding non-dialyzable material recovered by washing fibroblast monolayers with serum-free medium. The overlap ratio assay revealed no effect of supernatant material added either to urea-treated cultures in the continued presence of urea, or to untreated cultures. Although unfiltered supernatants were shown by SDS-polyacrylamide gel electrophoresis to contain fibronectin (CSP; LETS; MWappar. = 220,000 d) and smaller proteins, the ability to reverse the urea-induced increase in overlap ratio was present in Diaflo and Millipore filtrates of culture supernatants in which fibronectin was greatly depleted or absent. In contrast, purified fibronectin preparations failed to lower urea-induced increases in overlap-ratio. Partially purified, biologically active supernatants, prepared from 14C-leucine or 125I-labeled cultures, contained several macromolecules smaller than fibronectin that were labeled by both radioisotopes. In particular, one band (MWappar. = 58--60,000 d) was present in polyacrylamide gels of active supernatant and also depleted in gels of homogenates from urea-treated cultures. These results indicate that external macromolecules other than fibronectin are synthesized by cultured fibroblasts and can affect cell social behavior or culture morphology.     

Inhibition of cell-substratum attachment of cultured rat heart cells by protein synthesis inhibitors

Addition of cycloheximide to growth medium of neonatal rat heart cell cultures prevented cell-substratum attachment. Even concentrations of cycloheximide which inhibited only 50% of normal protein synthesis prevented some cells from attaching. Cells which required the longest time to attach were most dependent on protein synthesis. The kinetics of cell-substratum adhesion in the presence of various concentrations of cycloheximide supported the hypothesis that repair of damaged cell membranes was required prior to attachment. An alternate hypothesis that protein synthesis was required for substratum attachment either to synthesize new unique proteins or higher concentrations of existing proteins not damaged by enzymes was not supported by experimentally obtained data. If the second hypothesis were true, no cells would have attached when protein synthesis was completely inhibited (greater than 95%) and all cells should have been equally affected by protein synthesis inhibition; such was not the case. Inhibition of mRNA formation by actinomycin D also should have inhibited attachement completely and this was not observed. Since attachment was minimally affected by actinomycin D, protein synthesis on long-lived mRNA was apparently sufficient for cell-substratum adhesion.     

Inhibition of cell-substratum attachment of cultured rat heart cells by protein synthesis inhibitors.

Addition of cycloheximide to growth medium of neonatal rat heart cell cultures prevented cell-substratum attachment. Even concentrations of cycloheximide which inhibited only 50% of normal protein synthesis prevented some cells from attaching. Cells which required the longest time to attach were not dependent on protein synthesis. The kinetics of cell-substratum adhesion in the presence of various concentrations of cycloheximide supported the hypothesis that repair of damaged cell membranes was required prior to attachment. An alternate hypothesis that protein synthesis was required for substratum attachment either to synthesize new unique proteins or higher concentrations of existing proteins not damaged by enzymes was not supported by experimentally obtained data. If the second hypothesis were true, no cells would have attached when protein synthesis was completely inhibited (greater than 95%) and all cells should have been equally affected by protein synthesis inhibition; such was not the case. Inhibition of mRNA formation by actinomycin D also should have inhibited attachment completely and this was not observed. Since attachment was minimally affected by actinomycin D, protein synthesis on long-lived mRNA was apparently sufficient for cell-substratum adhesion.     

Pineal gland cells in culture : Morphology, biochemistry, differentiation, and co-culture with sympathetic neurons

We present here the initial report of a method for reproducibly obtaining primary cell cultures from pineal glands of 2-day-old rats. During culture, the putative pinealocytes became associated with each other in “nests”. Cells in these nests displayed vesicle-crowned rodlets and cilia, which are fine structural features in vivo of pinealocytes from neonatal rats. Treatment of the cultured cells with either norepinephrine or dibutyryl-cyclic AMP (db-cAMP) resulted in an increase in the activity of serotonin N-acetyltransferase, a marker activity for pineal function. This stimulation could be blocked by either cycloheximide or actinomycin D, and norepinephrine stimulation was also blocked by -propranolol. Further, the pineal cell cultures were able to support the growth of dispersed cells of rat superior cervical ganglia and to allow neurite outgrowth in these co-cultures, though the presence of nerve growth factor (NGF) in the medium of these cultures could not be detected.     

A novel time resolved fluorometric assay of anoikis using Europium-labelled Annexin V in cultured adherent cells

Background: Adherent cells undergo apoptosis when detached from their home ground, a process called anoikis (homelessness).Methods: We developed a new and sensitive method to analyse apoptosis and anoikis of adherent cell types using a time resolved fluorometric assay with Europium-labelled Annexin V. Anoikis was induced with tumor necrosis factor- /cycloheximide and three cell fractions of the cell cultures were prepared and analysed. Fraction 1 consisted of adherent cells, analysed while growing on their support (without detachment by trypsinisation). Fraction 2 contained detached cells due to anoikis (floating cells) and fraction 3 contained apoptotic bodies. Both fractions 2 and 3 were present in the culture medium and were isolated by differential centrifugation.Results: TNF- treatment of three different types of adherent cell cultures induced a significant increase of the amount of floating cells (anoikis) and apoptotic bodies compared to control cell cultures. Also in the adherent cell fractions a small amount of apoptosis was observed.Conclusions: The novel time resolved assay provides the ability to analyse the cell death cascade in adherent cell cultures of the same sample at the same time in a sensitive and reproducible way.     

Ligninase production in agitated conditions by Phanerochaete chrysosporium

Abstract Extracellular H₂O₂-dependent ligninase activity of Phanerochaete chrysosporium was produced in agitated culture conditions when veratryl alcohol or veratraldehyde were added to the cultures. The enzyme production was suppressed by cycloheximide indicating that true protein synthesis occurred. The activated cultures were also able to degrade synthetic lignin. Reduction of veratraldehyde to corresponding alcohol during secondary metabolism was a good indicator of the effect of agitation on cell metabolism. Too high agitation speed led to complete inhibition of both the reduction reaction and the ligninolytic activity.     

Cycloheximide elicits in human fibroblasts a response characteristic for initiation of cell proliferation

Earlier I found that a variety of stimuli to proliferation of cultured human fibroblasts caused an increase in the rate of putrescine transport into the cells. This paper reports the effects of cycloheximide on putrescine transport in stationary and growing cultures. Cycloheximide in concentrations that inhibited protein synthesis caused increased putrescine transport in serumstarved and density-inhibited cultures. Similar effects were found with pactamycin, also an inhibitor of protein synthesis. Actinomycin D in concentrations that suppressed messenger RNA (mRNA) synthesis, did not cause increased putrescine transport. When both serum and cycloheximide were added to serum-starved cultures, the increase in putrescine transport was greater than when serum alone was added. However, cycloheximide had an inhibitory effect when added 1–2 h after addition of serum. These results suggest that one or more rapidly metabolizing proteins may be important in the regulation of putrescine transport and initiation of cell growth.     

Study on the turnover of the receptor for the third component of complement on human lymphoid cells.

The in vitro turnover of the receptor for the third component of complement (C3) was studied in normal peripheral blood lymphocytes (PBL) and in lymphoblastoid cells from established cell cultures of both "normal" and "malignant" origin. The turnover was evaluated by studying i) the disappearance rate of the C3-receptor in cells in which the protein synthesis was blocked by cycloheximide and puromycin, ii) the reexpression rate of the C3-receptor after treatment of the cells with either rabbit antiserum against B lymphocytes or mouse C activated through the alternative pathway by inulin. The results show that the C3-receptor of all the lymphoid cells has roughly a half-life of about 3 to 4 hr. However, the cultured lymphoblastoid cells were less sensitive than normal PBL to inhibition by cycloheximide and showed a faster reexpression rate of the C3-receptor. A spontaneous release of the receptor was found to occur, since a receptor-like activity was detected in the spent culture medium of long-term cultured lymphoid cells.     

The relation of protein synthesis to chondroitin sulphate biosynthesis in cultured bovine cartilage.

The effect of cycloheximide on chondroitin sulphate biosynthesis was studied in bovine articular cartilage maintained in culture. Addition of 0.4 mM-cycloheximide to the culture medium was followed, over the next 4h, by a first-order decrease in the rate of incorporation of [35S]sulphate into glycosaminoglycan (half-life, t 1/2 = 32 min), which is consistent with the depletion of a pool of proteoglycan core protein. Addition of 1.0 mM-benzyl beta-D-xyloside increased the rate of incorporation of [35S]sulphate and [3H]acetate into glycosaminoglycan, but this elevated rate was also diminished by cycloheximide. It was concluded that cycloheximide exerted two effects on the tissue; not only did it inhibit the synthesis of the core protein, but it also lowered the tissue's capacity for chondroitin sulphate chain synthesis. Similar results were obtained with chick chondrocytes grown in high-density cultures. Although the exact mechanism of this secondary effect of cycloheximide is not known, it was shown that there was no detectable change in cellular ATP concentration or in the amount of three glycosyltransferases (galactosyltransferase-I, N-acetylgalactosaminyltransferase and glucuronosyltransferase-II) involved in chondroitin sulphate chain synthesis. The sizes of the glycosaminoglycan chains formed in the presence of cycloheximide were larger than those formed in control cultures, whereas those synthesized in the presence of benzyl beta-D-xyloside were consistently smaller, irrespective of the presence of cycloheximide. These results suggest that beta-D-xylosides must be used with caution to study chondroitin sulphate biosynthesis as an event entirely independent of proteoglycan core-protein synthesis, and they also indicate a possible involvement of the core protein in the activation of the enzymes of chondroitin sulphate synthesis.     

Regulation of glycogen metabolism in primary cultures of rat hepatocytes. Restoration of acute effects of glucose in cells from diabetic rats involves protein synthesis

Defective acute regulation of hepatic glycogen synthase by glucose and insulin, caused by severe insulin deficiency, can be corrected in adult rat hepatocytes in primary culture by inclusion of insulin, triiodothyronine, and cortisol in a chemically defined serum-free culture medium over a 3-day period (Miller, T. B., Jr., Garnache, A. K., Cruz, J., McPherson, R. K., and Wolleben, C. (1986) J. Biol. Chem. 261, 785-790). Using primary cultures of hepatocytes isolated from normal and diabetic rats in the same serum-free chemically defined medium, the present study addresses the effects of cycloheximide and actinomycin D on the chronic actions of insulin, triiodothyronine, and cortisol to facilitate the direct effects of glucose on the short-term activation of glycogen synthase. The short-term presence (1 h) of the protein synthesis blockers had no effect on acute activation of glycogen synthase by glucose in primary hepatocyte cultures from normal rats. Normal cells maintained in the presence of cycloheximide or actinomycin D for 2 and 3 days exhibited unimpaired responsiveness to glucose activation of synthase. The protein synthesis inhibitors were effective at blocking the restoration of glucose activation of synthase in diabetic cells in media which restored the activation in their absence. Restoration of glycogen synthase phosphatase activity by insulin, triiodothyronine, and cortisol in primary cultures of diabetic hepatocytes was also blocked by cycloheximide or actinomycin D. These data clearly demonstrate that restoration of acute glycogen synthase activation by glucose and restoration of glycogen synthase phosphatase activity in primary cultures of hepatocytes from adult diabetic rats are dependent upon the synthesis of new protein.