Pulse-labeled Cultures Research Articles

Rapid Kinetic Labeling of Arabidopsis Cell Suspension Cultures: Implications for Models of Lipid Export from Plastids

Cell cultures allow rapid kinetic labeling experiments that can provide information on precursor-product relationships and intermediate pools. T-87 suspension cells are increasingly used in Arabidopsis (Arabidopsis thaliana) research, but there are no reports describing their lipid composition or biosynthesis. To facilitate application of T-87 cells for analysis of glycerolipid metabolism, including tests of gene functions, we determined composition and accumulation of lipids of light- and dark-grown cultures. Fatty acid synthesis in T-87 cells was 7- to 8-fold higher than in leaves. Similar to other plant tissues, phosphatidylcholine (PC) and phosphatidylethanolamine were major phospholipids, but galactolipid levels were 3- to 4-fold lower than Arabidopsis leaves. Triacylglycerol represented 10% of total acyl chains, a greater percentage than in most nonseed tissues. The initial steps in T-87 cell lipid assembly were evaluated by pulse labeling cultures with [(14)C]acetate and [(14)C]glycerol. [(14)C]acetate was very rapidly incorporated into PC, preferentially at sn-2 and without an apparent precursor-product relationship to diacylglycerol (DAG). By contrast, [(14)C]glycerol most rapidly labeled DAG. These results indicate that acyl editing of PC is the major pathway for initial incorporation of fatty acids into glycerolipids of cells derived from a 16:3 plant. A very short lag time (5.4 s) for [(14)C]acetate labeling of PC implied channeled incorporation of acyl chains without mixing with the bulk acyl-CoA pool. Subcellular fractionation of pea (Pisum sativum) leaf protoplasts indicated that 30% of lysophosphatidylcholine acyltransferase activity colocalized with chloroplasts. Together, these data support a model in which PC participates in trafficking of newly synthesized acyl chains from plastids to the endoplasmic reticulum.

Transforming growth factor beta 1 stimulates type V collagen expression in bovine vascular smooth muscle cells

Vascular smooth muscle cells (SMCs) produce the bulk of the connective tissue of major arteries, including collagen types I, III, and V. Recently, we have shown, they also have the capacity to synthesize the alpha 1 chain of type XI, a collagen related to type V (Brown, K., Lawrence, R., and Sonenshein, G. (1991) J. Biol. Chem. 266, 23268-23273). Furthermore, expression of types V and XI collagen were coordinately regulated with respect to serum deprivation and cell density in a fashion distinct from that for types I and III. To begin to determine the factors that influence vascular SMC production of types V/XI collagen, we have examined the effects of transforming growth factor (TGF)-beta 1, a major modulator of connective tissue expression. In serum-deprived confluent cultures of bovine pulmonary artery SMCs, TGF-beta 1 treatment increased the steady-state levels of the mRNAs of collagen types V and XI, as well as of types I and III, elastin and fibronectin. The largest increase was seen for alpha 2(V) procollagen. The increase in alpha 2(V) mRNA was detectable by 12 h after addition of 2 ng/ml TGF-beta 1, and concentrations as little as 0.5 ng/ml were effective. A similar increase in alpha 2(V) mRNA levels was observed with SMCs derived from the aortic arch and carotid artery. Type V collagen protein was found to be elevated by TGF-beta 1 treatment in both the conditioned media and the cell layer associated fraction of pulse-labeled cultures. A slight decrease in SMC proliferation as judged by DNA content, [3H]thymidine incorporation, and steady-state levels of histone H3.2 mRNA resulted from TGF-beta 1 treatment. These results suggest that the elevated levels of TGF-beta 1 in the vessel wall during atherosclerosis may be, in part, responsible for the increase in type V collagen that typifies advanced fibrotic lesions.

Biosynthesis of procalcitonin in small cell carcinoma of the lung

Immunoreactive calcitonin (CT) secreted by DMS 53, a cell line derived from human small cell carcinoma of the lung, consists almost entirely of molecular species larger than the mature hormone (Mr 3,420). Messenger RNA isolated from DMS 53 cells and nude mouse tumors was translated in wheat germ systems, and the products were precipitated with CT-specific antisera. Analyses of the translation products by electrophoresis on 15% polyacrylamide-sodium dodecyl sulfate gels indicated synthesis of a Mr 16,500 preprohormone that was reduced to Mr 14,500 by cotranslation with microsomal membranes. Immunoprecipitation of CT from media from pulse-labeled cultures revealed two major products (Mr 16,500 and Mr 14,500) and up to three minor secreted polypeptides (Mr 9,400, 8,400, and 6,800). Intracellular CT from cell homogenates appeared almost entirely as a single major product (Mr 14,500) and possibly 3-4 minor components (Mr 16,500; 9,200, 8,400, and 6,800). No glycosylated forms of CT were demonstrable by lectin binding methods or labeling attempts with tritiated sugars. The presence of multiple CT species in DMS 53 cells suggests significant post-translational processing of the larger precursor molecules and the accumulation and secretion by small cell carcinoma of the lung of several intermediate immunoreactive forms via a glycosylation-independent secretory pathway.

Heterogeneity in the replicating population of cultured human epidermal keratinocytes

We studied the replication of keratinocytes in stratified squamous epithelia. Other studies have revealed functional and morphological heterogeneity in the replicating population of such cells. To examine possible kinetic heterogeneity, we determined the cell-cycle lengths of replicating cells in cultures of human epidermal keratinocytes. A double-label assay was developed, which measures the time between two successive cycles of DNA synthesis. The first cycle of DNA synthesis was marked by pulse labeling cultures for a brief period with 14C-thymidine (dThd), and the second cycle was detected by labeling at a later time with bromodeoxyuridine (BrdUrd). The time taken for the 14C-labeled DNA to become doubly labeled with BrdUrd was shown to correspond to the length of the cell cycle. In subconfluent cultures in which the cell number increased at an exponential rate, the average cell-cycle time was 21.5 h. In confluent cultures in which desquamation was balanced by cell renewal, the average cell cycle was 31.5 h. However, in confluent cultures, three populations of replicating cells were evident, these having cycle times of 22, 33, and 40 h. In subconfluent cultures, there was no clear evidence for cell-cycle heterogeneity of the replicating cells, although the most rapidly cycling cells in these cultures had a cycle time (16 h) considerably less than the most rapidly cycling cells in the confluent cultures (21 h). It is possible that the rapidly cycling cells seen in the subconfluent cultures were stem cells. The heterogeneity seen in the replicating population is entirely consistent with the epidermal proliferation unit model of renewal, and the three populations of replicating cells probably represent cells undergoing amplification divisions.

Interval between the synthesis and assembly of cytoskeletal proteins in cultured neurons

We have used pulse-chase experiments to study the time interval between the synthesis and assembly of tubulin and neurofilament proteins (NFP) in sympathetic neurons grown in tissue culture. After varying pulse-chase times, cultures were extracted with Triton X-100 such that polymerized tubulin and NFP were insoluble, while unassembled tubulin and NFP were quantitatively solubilized. The partitioning of labeled tubulin and NFP between Triton X-100-soluble and insoluble, or cytoskeletal, fractions was determined with an isoelectric focusing X SDS gel electrophoresis assay. Labeled tubulin and NFP in cultures pulse-labeled for 5-10 min partitions primarily with the soluble fraction. When pulse-labeled cultures were chased for increasing periods of time, relatively more of the total labeled tubulin and NFP partitioned with the cytoskeleton, attaining maximal values after chase times of 60-120 and 15-30 min, respectively. The maximal values for the relative levels of labeled tubulin and NFP in polymer were 70-75 and greater than 90%, respectively. The levels of labeled tubulin and NFP synthesized during a short pulse-label remained constant for at least 2 hr, indicating that selective turnover of soluble tubulin and NFP does not detectably contribute to the changes in solubility properties of these proteins observed in the pulse-chase experiments. These results indicate that newly synthesized tubulin and NFP are rapidly assembled from soluble precursors. The lag between the synthesis and assembly of the 145,000-molecular-weight NFP is not related to its phosphorylation because its initial incorporation into the cytoskeleton occurs prior to its phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Cerebral microvascular smooth muscle in tissue culture.

Cerebral endothelium is being studied rather extensively in tissue culture, but no reports are available describing the tissue culture of cerebral microvascular smooth muscle. The present paper describes for the first time the isolation and culture of non-neoplastic mouse cerebral vascular smooth muscle. Microvessels from a dounce homogenate of mouse brain are plated onto plastic culture dishes in Dulbecco's modified Eagle media plus 20% fetal bovine serum and treated briefly with collagenase. Cells migrate from vessels and proliferate sufficiently to be transferred out of primary culture in 2 to 3 wk. Light microscopy reveals generally broad, polygonal cells that grow collectively in a "hill and valley" pattern. By transmission electron microscopy the cells possess many characteristics of smooth muscle: basal laminas, clusters of pinocytotic vesicles, and bundles of thin filaments. Several ill-defined cell-to-cell junctions are also present. Isoelectric focusing and sodium dodecyl sulfate-electrophoresis of cellular proteins on polyacrylamide gels after pulse labeling cultures with [S-35]methionine demonstrate that these cells actively synthesize a smooth-muscle-specific isoactin, alpha-actin. The identity of alpha-actin is confirmed by analysis of NH2-terminal peptides after actin digestion with trypsin and subsequent peptide cleavage with thermolysin. Both their morphology and active synthesis of alpha-actin strongly suggest that these cells are of smooth-muscle origin. Future studies of their metabolism and interactions with endothelium and astrocytes should provide a better understanding of the cerebral microcirculation.

Sequential study on the influence of adriamycin on cell proliferation and ultrastructure of cultured mammary tumor cells.

The time-dependent cytocidal and growth inhibitory effects of Adriamycin (ADM) on monolayer cultures of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumor cells were analyzed. The inhibitory effect on cell proliferation was assessed by colony formation in soft agar. Growth inhibition and [3H]thymidine labeling indices clearly demonstrate a dose-dependent antimitotic and cytotoxic effect of the drug. At low concentrations (10(-9)-10(-8) M), 90-100% of cells survived 24-hr exposure. At a higher concentration (10(-5) M), 75-80% of cells survived after 8-hr exposure; by 72 hr only 20-30% of the cells remained. Autoradiographic examination of the pulse-labeled cultures demonstrated no change in the proportion of cells in S-phase during the first 4 hr of treatment. Subsequently DNA synthesis was completely abolished and remained inhibited for the duration of the experiment (72 hr). Clonogenic assay revealed a complete arrest of growth in cells exposed to 10(-5) M ADM and greater than 60% inhibition of cell proliferation at 10(-7) M. Ultrastructural changes were not observed in cells during the first 4 hr of treatment; however, after 8 hr most surviving cells exhibited alterations in nuclear chromatin. The surviving cells showed mitochondrial degeneration, myelin body formation, and vacuolization of the endoplasmic reticulum. This study shows the potential usefulness of the primary culture system in drug evaluation. In addition, serial observation of the effects of ADM revealed a cell subpopulation of the primary culture with differential sensitivity to the drug.

Modulation of proliferation in epidermal keratinocyte cultures by lowered oxygen tension

The modulation of proliferation and differentiation in primary epidermal keratinocyte cultures by lowered gas phase oxygen tensions was studied. Neonatal mouse epidermal keratinocyte cultures were grown in an Heraeus type B 5060 EK/O2 incubator in oxygen tensions between 5% and 15% (within the physiologic range); the oxygen tension of ambient air being 21%. Cell morphology was studied using histochemical stains and electron microscopy. Differentiation was assessed using autoradiography of SDS PAGE gels of six serially extracted cell protein fractions with [3H]leucine as a marker. Autoradiographs using [14C]glucosamine and 32Pi as markers were also assessed as a measure of other cell functions. Proliferation was studied using autoradiography of [3H]thymidine ([3H]TdR) pulse-labeled cultures and [3H]TdR incorporation into isolated DNA fractions. The results of these studies showed that lowering the oxygen tension in the gas phase reversibly inhibited cell proliferation. There was a direct arithmetic relationship between the proliferative rate of the cultures and the oxygen tension. No change in differentiation as defined by [3H]leucine indexing of protein synthesis was seen. Other markers of cell function, such as [14C]glucosamine glycosylation and [32P] phosphorylation of proteins were also unchanged. These results suggest that oxygen tension regulates only proliferation in epidermal keratinocytes. This epidermal response is well adapted to its role in the healing wound, and is an example of a tissue-specific modification of a regulatory function.

Stimulation of albumin synthesis in mouse hepatoma cells by Bt 2cAMP: Cell cycle specificity

Addition of 1 m m dibutyryl cyclic AMP (Bt 2cAMP) to cultures of mouse hepatoma cells, Hepa, specifically stimulates the synthesis of serum proteins including albumin. This stimulation is accompanied by an inhibition of cell proliferation. We have investigated these phenomena in synchronous cultures of Hepa. Proliferation of Hepa was arrested by isoleucine starvation. Synchronous growth was initiated by addition of complete growth medium or complete growth medium supplemented with 1 m m Bt 2cAMP. S phase and mitosis were estimated by determinations of [ 3H]thymidine incorporation and by cell numbers. The rate of albumin synthesis relative to total protein synthesis was measured by pulse labeling cultures for 30 min with [ 3H]leucine and comparing amounts of immunoprecipitable label with trichloroacetic acid-precipitable label. Treatment of synchronous cultures with Bt 2cAMP did not alter the duration of S phase or the onset of mitosis. The relative rate of albumin synthesis in Bt 2cAMP-treated culture began increasing after mitosis. The timing of the Bt 2cAMP stimulation of albumin synthesis was further investigated by adding Bt 2cAMP to cultures of Hepa at various times after the initiation of synchronous growth. The relative rate of albumin synthesis was then measured at a fixed postmitotic time. An increased relative rate of albumin synthesis was observed only in cultures exposed to Bt 2cAMP before or during S phase. Thus the postmitotic increase in the synthesis of albumin requires the presence of Bt 2cAMP during S phase.

Replication of viral DNA sequences integrated within the chromatin of SV40-transformed chinese hamster lung cells

To determine when during S phase integrated viral DNA sequences in several tsA SV40-transformed Chinese hamster cell clones replicate, we pulse-labeled cultures with BrdUrd and subsequently collected mitotic cells during sequential time intervals. Restriction endonuclease mapping indicated that each of the three SV40-transformed Chinese hamster lung cell clones contained a single viral DNA sequence at a different, but in each case unique, chromosomal site. DNA was extracted from each population of mitotic cells and then was resolved into dense, BrdUrd-containing and light, unsubstituted DNA fractions by cesium chloride gradient centrifugation. In each pair of samples obtained, we measured viral DNA sequences by solution hybridization using single-stranded SV40 32P-labeled DNA probes. Our results support the conclusions that specific genes within a mammalian DNA are programmed to replicate at particular times during S phase, and that the SV40 A gene product, large T antigen, programs integrated viral DNA sequences to replicate very early in S phase. The fractions of viral DNA replicated early in S phase appeared to correlate with each clone's content of functional large T antigen at permissive and nonpermissive culture temperatures.

Asynchrony in late replication between homologous autosomes in primary cultures of Chinese hamster fibroblasts.

Asynchronies in late replication of the autosomal chromosome pair No. 5, and to some extent of pair No. 4, were found after thymidine pulse labeling cultures of partially synchronized Chinese hamster lung fibroblasts from nine to nine and a half hours and from nine and a half to ten hours after block removal. In contrast to this, no asynchrony could be detected in the replication of homologous autosomes after continuous labeling for the last two hours of the S-phase. - G-banding and C-banding revealed no differences between the homologous autosomes. - These findings indicate that besides the known form of asynchronous replication in mammalian cells during S-phase on the chromosomal level, there also exists an asynchronous replication between homologous autosomes of the same complement.

Synthesis of developmental proteins in morphogenetic mutants of Polysphondylium pallidum

Changes in labeling of 9 polypeptides were followed during development of 17 morphogenetic mutants using sodium dodecyl sulfate-polyacrylamide gel electrophoresis of pulse-labeled cultures. In most cases the change in labeling of a particular polypeptide either occurred normally or failed to occur altogether in a particular mutant, and cases where the change was partial or of a pattern different from the wild type were rare. From a table showing labeling changes in polypeptides during development of the several mutants a diagram was constructed which implies causal relations among the labeling events.

A ribonucleoprotein precursor of both the 30S and 50S ribosomal sunbunits of Escherichia coli.

A ribonucleoprotein particle (46S) has been isolated from [3H]uridine pulse-labeled cultures of E. Coli AB301/105. Evidence from pulse chase experiments and from protein analysis suggested that this particle may give rise to both the 30S and 50S ribosomal subunits. Direct deproteinization of the particle yielded 30S RNA, while deproteinization after treatment with a crude RNase III preparation yielded products similar to 23S and 16S RNA. This result is consistent with the idea that the 46S ribonucleoprotein is the in vivo counterpart of 30S RNA, which is the in vitro product obtained after phenol extraction.

The effect of bacteriophage T2 infection on the synthesis of transfer RNA in Escherichia coli

During the initial 5 minutes of T2 bacteriophage infection of Escherichia coli, there is substantial incorporation of labeled guanine into the transfer RNA (tRNA) fraction, strongly suggesting that new tRNA is synthesized early in infection. In this 5-minute period the amount of labeled guanine incorporated into the tRNA fraction of T2-infected bacteria is 23% of that in uninfected bacteria during an equivalent period. The tRNA fractions from T2-infected bacteria and uninfected bacteria were compared by pulse-labeling cultures of each for 5 minutes with 3H and 14C isotopes of guanine and partially resolving a mixture of the doubly labeled tRNA species by reversed phase chromatography. The chromatographic pattern of the labeled tRNA from uninfected cultures of E. coli was very similar to that of nonlabeled carrier tRNA from uninfected cultures. tRNA prepared from pulse-labeled T2-infected cultures of E. coli had a chromatographic pattern that was radically different from that of tRNA prepared from uninfected cultures. These results indicate either that the tRNA synthesized after T2 infection of E. coli represents species of tRNA entirely different from those made in uninfected bacteria, or that relative proportions of the different species made are greatly altered after phage infection, some being made preferentially and others in reduced amounts. Evidence is presented that a substantial amount of the material in the tRNA fraction made during T2 infection can accept amino acids.

Collagen synthesis by isolated bone cells: Stimulation by ascorbic acid in vitro

Bone cells isolated from rat calvaria were cultured on a flat surface, and collagen synthesis was determined by the incorporation of radioactive proline into peptide hydroxyproline. Addition of ascorbic acid to the incubation medium markedly increased the radioactivity of hydroxyproline in cold water-soluble and -insoluble protein fractions, without increasing the radioactivity of peptide-bound proline or the DNA content of the cell cultures. Stimulation was demonstrated with low concentrations of ascorbic acid (3 μM) and was directly proportional to the starting concentration. Hydroxylation of radioactive proline that had previously been incorporated into cell protein was stimulated despite puromycin- or cycloheximide-induced inhibition of protein synthesis. These results indicate that ascorbic acid promotes collagen synthesis in isolated bone cells by directly stimulating the hydroxylation of a proline-rich peptide. Inhibitors of protein synthesis consistently increased the radioactivity of peptide hydroxyproline when added to pulse-labeled cultures in the absence of ascorbic acid. The data suggest that they prevented the dilution of radioactive, unhydroxylated collagen precursor with non-radioactive precursor, thus providing substrate of higher specific activity for hydroxylation.