Interaction of circulating cell-derived and plasma growth factors in stimulating cultured smooth muscle cell replication

Multiple growth factors that circulate in plasma have been shown to stimulate cellular growth in vitro. The plasma growth factors appear to stimulate DNA synthesis in cultured fibroblasts only after prior exposure of cell growth factors derived from circulating cell types, such as platelets and macrophages. The purpose of these studies was to investigate the role of the plasma growth factors in stimulating smooth muscle cell replication following exposure to platelet-derived growth factor (PDGF). Following transient exposure to PDGF, insulin stimulated smooth muscle cell replication but only when supraphysiologic concentrations were used (i.e., greater than 1.0 μg/ml). Somatomedin-C (Sm-C), in contrast, was found to stimulate a 320% increase in [³H]thymidine incorporation when concentrations that are present in extracellular fluids were used (i.e., 0.5–10 ng/ml). Epidermal growth factor (EGF), an important mitogen for multiple cell types, caused a 70% increase in [³H]thymidine incorporation when added to quiescent cells following PDGF exposure, and EGF caused a substantial increase in the absolute level of [³H]thymidine incorporation when coincubated with Sm-C. When EGF (1 ng/ml) was incubated simultaneously with concentrations of Sm-C between 1 and 10 ng/ml plus Sm-C-deficient plasma, maximal [³H]thymidine incorporation was 2.1-fold greater in the presence of EGF. In contrast, insulin (20 ng/ml), when coincubated with Sm-C under similar conditions, had no enhancing effect on the cellular response to Sm-C. None of the plasma factors tested was an effective stimultant of replication when incubated either in serum-free medium or in the presence of Sm-C-deficient plasma without prior PDGF exposure. Hydrocortisone was shown to inhibit smooth muscle cell replication in concentrations between 10^?7 and 10^?5M. In summary, multiple plasma growth factors can stimulate the smooth muscle cell replication, and Sm-C appears to be most effective of those tested. Insulin and EGF appear to work by different mechanisms; that is, EGF can facilitate the cellular response to Sm-C, whereas insulin is effective only at supraphysiologic concentrations at which it will directly bind to Sm-C receptors.     

The proliferative effects of retinoic acid on primary cultures of adult rat type II pneumocytes depend upon cell density

Retinoic acid (RA) is important for maintaining integrity of alveolar epithelial cells, but the mechanism has not been defined. We cultured type II pneumocytes at confluent, high cell density (10⁴ cells/mm²) and found that RA (10⁻⁶ M) inhibited thymidine incorporation to 60% of control, despite a dose-dependent increase in epidermal growth factor receptor (EGFR) levels. However, at lower, subconfluent density (10² cells/mm²), RA stimulated thymidine incorporation to 280% of control. EGF increased thymidine incorporation at concentrations as low as 0.1 ng/mL, but no further increase was observed at higher concentrations up to 100 ng/mL. In subconfluent cells co-treated with EGF (100 ng/mL) and increasing concentrations of RA (10⁻⁸ M–10⁻⁵ M RA), thymidine incorporation was significantly greater at all concentrations than RA alone, with greatest increases observed at 10⁻⁷ (422% of control) and 10⁻⁶ (470% of control) M RA. In summary, the effects of RA on thymidine incorporation are sensitive to changes in cell density. RA inhibits thymidine incorporation at high cell density and stimulates thymidine incorporation at low density. RA increases EGFRs in cultured type II pneumocytes, and EGF stimulates thymidine incorporation independent of the cultured cell density. These data may help to explain how RA mediates lung repair in vivo.     

Alterations of the incorporation of [3H]thymidine in cells in culture regulated by nucleoplasmic extract

Porcine skin nucleoplasmic extract (PSNE) was shown to alter the incorporation of [³H]thymidine into DNA of selected porcine, bovine, and human cell populations in culture. PSNE stimulated incorporation of [³H]thymidine into DNA of porcine and bovine dermal cells an average of 300 and 200% of control value, respectively. When porcine and bovine epidermal cells were exposed to PSNE the treatment inhibited [³H]thymidine incorporation into DNA by an average of 48 and 45%, respectively. Similar inhibitions were observed for porcine and bovine kidney, porcine lung, and human KB cells. Thus, the effect of PSNE on the incorporation of [³H]thymidine into DNA of various cultured cells was either stimulatory to dermal cells or inhibitory to a variety of other cell types, including skin epidermal cells. The stimulatory and inhibitory effects of PSNE were abolished by heating PSNE for 5 min in boiling water before its addition to cell cultures. This suggests that macromolecular structure is important in the action of PSNE. This project was supported by a grant from the Research Advisory Board, University of Nevada, Reno, NV.     

Direct cell culture assay of thin-layer-chromatographed material without prior substance elution

A method enabling the direct assay in a cell culture system of material separated by thinlayer chromatography (TLC) without prior elution of active material from the TLC sheet is described. After chromatographic development on a plastic cellulose sheet, small disks were punched out from the sheet and transferred to microwells used for cell culture. Lymphoid cells in culture medium were incubated with the disks and the DNA synthesis was measured by [³H]thymidine incorporation. The reliability of the method was tested with l-alanine and thymidine, which are known to enhance the DNA synthesis of lymphoid cells and to interfere with the uptake of labeled thymidine, respectively. The incorporation of [³H]thymidine was stimulated with disks containing l-alanine and inhibited with disks containing unlabeled thymidine, completely in agreement with the expected results. Elution experiments showed that more than 75% of l-alanine and thymidine on the TLC disks was eluted into the culture medium within 1 h. Elution of material of higher molecular weight was also demonstrated. The method was used for the separation and assay of a thymocyte-specific growth factor isolated from calf thymus and greatly facilitated the detection of the active material after TLC.     

Adaptation of thymidine utilization to changing rates of DNA synthesis in the cell cycle

Summary In synchronous cultures of P-815 murine mastocytoma and of Chinese hamster ovary (CHO) cells, the relative contribution of exogenous thymidine to DNA synthesis was studied by comparing rates of (³H)thymidine incorporation with the rate of DNA synthesis as derived from incorporation of (³H)thymidine (10^–5 m) in the presence of amethopterin. In synchronous P-815 cultures, time-dependent variations of DNA synthesis rates were in close agreement with those of (³H)thymidine incorporation rates at concentrations of the precursor ranging from 5 × 10^–8 to 10^–5 m. Similarly, in synchronous CHO cell cultures prepared by two different methods, time-dependent changes in DNA synthesis rate were almost identical with those of the rate of incorporation of (³H)thymidine supplied at 5 × 10^–8 m. Thus, at a given thymidine concentration in the medium, the proportion of thymine residues in DNA that were derived from exogenous thymidine remained nearly constant, even though rates of cellular DNA synthesis underwent pronounced changes. This indicates that in the synchronous culture systems used, utilization of exogenous thymidine is efficiently adapted to changing rates of DNA synthesis.In partial fulfillment of the requirements for the degree of Ph.D. by G.G.M.     

Effect of exogenous gangliosides on human neural cell division

Human neural cells in exponential growth phase were transferred to a serum-free medium and maintained for 72 hr without any detectable loss in viability. The two normal fetal cell lines (CH_I and CH_II) showed a serum-dependent cell proliferation, but the glioblastoma multiforme cells (12–18) were able to continue proliferating in this totally synthetic medium. The incorporation of [³H] thymidine into the acid-precipitable fraction of both normal and neoplastic human neural cells was assayed in the presence and the absence of exogenous gangliosides by a convenient new method. In serum-free medium, gangliosides (50 μM) inhibited the thymidine incorporation into the normal fetal cells within 24 hr and, in serum containing medium, reduced their proliferation within 48 hr. No such effects were detectable in the glioma cells. The inhibition of thymidine incorporation in the normal cells was reversible upon removal of the gangliosides. These results indicate a role of gangliosides in the postmitotic phase of normal human neural cells resulting in the regulation of cell proliferation.     

Differential patterns of mitochondrial,chloroplastic and nuclear DNA synthesis in the synchronous cell cycle of Chlamydomonas reinhardtii

Nuclear DNA (ncDNA) synthesis in Chlamydomonas reinhardtii was measured by both ³²P[or-thophosphoric acid] (³²P) and [¹⁴C]adenine incorporation and found to be highly synchronous. Ca. 85% of incorporation was confined to the first 6 h of the dark period of a synchronized regime consisting of an alternating light-dark period of 12 h each. In contrast, no such synchronous incorporation pattern was found for chloroplast (cp) and mitochondrial (mt) DNAs in the same cell population. These two organellar DNAs also exhibited different ³²P-incorporation patterns in the cell cycle. Considerable amounts of ³²P were incorporated into cpDNA throughout the light-dark synchronous cycle under both mixo- and phototrophic growth conditions, although the second 6-h light period under phototrophy showed an increase not apparent under mixotrophy. This change in growth conditions did not affect ³²P incorporation into mtDNA, which was found throughout the cell cycle, with a modest peak in the first 6-h of the dark period. The pattern of [³H]thymidine incorporation into cpDNA was also determined. Under synchronous phototrophic conditions, this pattern was quite different from that obtained with ³²P. Most [³H]thymidine incorporation occurred during the light period of the synchronous cycle; this period had been shown previously by density transfer experiments to be the time of cpDNA duplication. Such preferential [³H]thymidine incorporation into cpDNA in the light period was not observed under mixotrophic synchronous growth conditions; in these, [³H]thymidine incorporation was detected throughout the cell cycle. This lack of coincidence between the patterns of ³²P- and of [³H]thymidine incorporation into cpDNA during the synchronous cell cycle indicates that in addition to replication, the considerably reiterated organelle-DNA molecules may also regularly undergo an extensive repair process during each cell cycle.     

Lack of effect of butyrate on S phase DNA synthesis despite presence of histone hyperacetylation

The effects of sodium butyrate on [³H]thymidine incorporation and cell growth characteristics in randomly growing and synchronized HeLa S₃ cells have been examined in an attempt to determine what effects, if any, butyrate has on S phase cells. Whereas 5 mM sodium butyrate rapidly inhibits [⁵H]thymidine incorporation in a randomly growing cell populations, it has no effect on incorporation during the S phase in cells synchronized by double thymidine block techniques. This lack of effect does not result from an impaired ability of the S phase cells to take up butyrate, since butyrate administration during this period leads to histone hyperacetylation that is identical with that seen with butyrate treatment of randomly growing cells. Furthermore, the ability to induce such hyperacetylation with butyrate during an apparently normal progression through S phase indicates that histone hyperacetylation probably has no effect on the overall process of DNA replication. Temporal patterns of [³H]thymidine incorporation and cell growth following release from a 24-h exposure to butyrate confirm blockage of cell growth in the G1 phase of the cell cycle. Thus, the inhibition by butyrate of [³H]thymidine incorporation in randomly growing HeLa S₃ cell populations can be accounted for solely on the basis of a G1 phase block, with no inhibitory effects on cells already engaged in DNA synthesis or cells beyond the G1 phase block at the time of butyrate administration.     

Incorporation of fucose into HeLa cell plasma membranes during the cell cycle

The metabolism of HeLa cell plasma membranes during the cell cycle was studied by following the incorporation of radioactive precursor l-[³H]fucose into plasma membranes of synchronized cells. Maximal incorporation of the radioactive precursor was observed in late S phase of the cell cycle. This discrete period of increased incorporation of precursor into the plasma membranes implies the existence of a distinct control mechanism which may relate cell surface phenomena to the cell cycle.     

Role of oxidized human plasma low density lipoproteins in atherosclerosis: effects on smooth muscle cell proliferation

The effects of oxidized human plasma low density lipoproteins (Ox-LDL) on the proliferation of cultured aortic smooth muscle cells was studied, employing viable cell counting, [³H] thymidine incorporation into DNA, and the release of lactate dehydrogenase (LDH) into the medium. Oxidized LDL (prepared by incubation of LDL with copper sulfate) exerted a concentration-dependent stimulation (2 fold, compared to control) of aortic smooth muscle cell proliferation at low concentrations (0.1 µg – 10 µg/ml medium). On the other hand, at high concentrations (25–200 µg/ml), Ox-LDL produced a pronounced decrease in viable cells, a decrease in the incorporation of [³H] thymidine into DNA, and an increase in the release of LDH in the medium. In this report, the previously postulated biological roles of oxidized-LDL in atherosclerosis are discussed in view of these findings.Abbreviations Ox-LDL Oxidized human plasma Low Density Lipoproteins - SMC Smooth Muscle Cells - LDH Lactate Dehydrogenase - LPC Lysophosphatidycholine - PC Phosphatidylcholine - TNF Tumor Necrosis Factor     

Membranes of animal cells. V. Biosynthesis of the surface membrane during the cell cycle

KB cells grown in suspension culture were synchronized by using a double thymidine block. At various times throughout the life cycle aliquots of cells were pulsed with ¹⁴C-L-leucine, ¹⁴C-D-glucosamine and ¹⁴C-choline for one hour periods. Surface membranes, cell particulates and soluble proteins were isolated and their ¹⁴C specific activities were determined. It was found that there was a marked increase in the rate of incorporation into surface membrane just after division. The pattern of incorporation was the same for all three isotopic precursors. The rate of incorporation of isotopic precursors into soluble proteins was constant throughout the cycle. Some increase in rate of incorporation of isotope into the particulate fraction was observed during division.     

Comparison of effects of epidermal and insulin-like growth factors,gastrin releasing peptide and retinoic acid on fetal lung cell growth and maturation in vitro

The role in cell multiplication and maturation of several factors present in the late fetal lung was explored on isolated fetal rat pulmonary fibroblasts and alveolar epithelial type II cells cultivated in serum-free medium. The low degree of reciprocal contamination of each cell population was assessed by immunocytochemistry. Epidermal Growth Factor (EGF) stimulated thymidine incorporation and DNA accumulation in both cell types. In type II cells, it increased labeled-choline incorporation into surfactant phosphatidylcholine (PC), consistently with previous data obtained with lung explant cultures, but not into non-surfactant PC. Insulin-like growth factor (IGF)-I slightly stimulated DNA accumulation in fibroblasts although it did not significantly stimulate thymidine incorporation, contrary to IGF-II which presented a dose-dependent stimulating activity of thymidine incorporation. Neither IGF-I nor IGF-II stimulated type II cell growth. IGFs thus appear to primarily control the growth of lung mesenchyme. In type II cells, they stimulated the most non-surfactant PC biosynthesis. Gastrin releasing peptide (GRP) which was recently reported to promote fetal lung growth in vivo and to stimulate surfactant biosynthesis in lung organ culture revealed as a growth factor for type II cells only, at concentrations below 10 ⁻⁹ M. At concentration 10 ⁻⁸ M, although it did not affect DNA synthesis, GRP tended to increase surfactant and non-surfactant-PC biosynthesis. Retinoic acid inhibited thymidine incorporation into type II cells on a dose-dependent manner but nevertheless enhanced surfactant-PC biosynthesis to a similar extent as EGF. It is suggested that retinoic acid may represent a differentiation or maturation factor for the alveolar epithelium.     

Rates of incorporation of radioactive molecules during the cell cycle

We report measurements of the incorporation of radioactive molecules during short labeling periods, as a function of cell-cycle stage, using a cell-sorter-based technique that does not require cell synchronization. We have determined: (1) tritiated thymidine (³H-TdR) incorporation throughout S-phase in Lewis lung tumor cells in vitro both before and after treatment with cytosine arabinoside; (2) ³H-TdR incorporation throughout S-phase in KHT tumor cells in vitro and in vivo; (3) ³H-TdR incorporation throughout S-phase in Chinese hamster ovary cells and compared it with DNA synthesis throughout S-phase; (4) a mathematical expression describing ³H-TdR incorporation throughout S-phase in Chinese hamster M3-1 cells; and (5) the simultaneous incorporation of ³H-TdR and ³⁵S-methionine as they are related to cell size and DNA content in S49 mouse lymphoma cells. In asynchronously growing cells in vitro and in vivo, ³HH-TdR incorporation was generally low in early and late S-phase and highest in mid-S-phase. However, in Lewis lung tumor cells treated with cytosine arabinoside ³H-TdR incorporation was highest in early and late S-phase and lowest in mid-S-phase. Incorporation of ³⁵S-methionine increased continuously with cell size and DNA content. Incorporation of ³H-TdR in CHO cells was proportional to DNA synthesis.     

Approach to the mechanism of Zajdela's hepatoma cell growth inhibition induced by concanavalin A and phytohaemagglutinin

Cell growth of tumour ascites cell was inhibited by concanavalin A, phytohaemagglutinin and Ricinus lectin at 2–100 μg/ml. As expected, the Ricinus lectin inhibited the protein synthesis estimated by leucine incorporation and decreased thymidine incorporation, whereas concanavalin A and phytohaemagglutinin stimulate the uptake and the incorporation of both leucine and thymidine, and thus, synthesis of protein and DNA. Theses results suggest that different mechanisms are involved in the hepatoma cell growth inhibition by the lectins. This difference was not related to the kinetic characteristics of the lectin interactions with the cells whihc represent a first and necessary step. It was showed that concanavalin A and phytohaemagglutinin as well as chloroquine inhibited the ¹⁴C-labelled asialofetuin degradation. We can conclude that Ricinus lectic present a toxic effect whereas both concanavalin A and phytohaemagglutinin show an anti-protease activity.     

Electron microscopic autoradiographic and electron microscopic immunohistochemical studies on the anti-HPO antibody-producing cells

The secondary immune responses in mouse popliteal lymph nodes to horseradish peroxidase (HPO) were studied by a combination of electron microscopic autoradiography and electron microscopic immunohistochemistry in order to clarify the relationship between antibody-producing and DNA-synthesizing capacities of the plasmacytic series. The anti-HPO antibody-containing cells, which increased in number 72 h after the secondary antigenic stimulation, were mainly immunoblasts and immature plasma cells. Immunoblasts containing anti-HPO antibody incorporated [³H]thymidine more actively than did immature plasma cells containing anti-HPO antibody. In 144 h after the secondary antigenic stimulation, antibody containing cells consisted mainly of mature plasma cells and immature plasma cells. Immature plasma cells containing the anti-HPO antibody incorporated a little [³H]thymidine, but mature plasma cells containing anti-HPO antibody did not incorporate any [³H]thymidine.     

Stimulation of DNA synthesis,cell multiplication,and ornithine decarboxylase in 3T3 cells by multiplication stimulating activity (MSA)

Multiplication stimulating activity (MSA) has been purified from the conditioned media of rat liver cells in culture by a modification of the procedure of Dulak and Temin. Purified MSA stimulates [³H] thymidine incorporation into DNA in subconfluent, serum starved 3T3 cells. Cell cycle analysis by the flow microfluorometer shows that the [³H] thymidine incorporation data reflects DNA synthesis. MSA also stimulates the multiplication of serum starved subconfluent 3T3 cells. MSA is approximately 10-fold less active in 3T3 cells than in chick embryo fibroblasts in stimulating [³H] thymidine incorporation into DNA. MSA causes a 2–10-fold increase in ornithine decarboxylase (ODC) activity in 3T3 cells and the dose response curve parallels the dose response curve for [³H] thymidine incorporation into DNA. The Km of ODC for ornithine is 0.12 mM. There is a 30% decrease in the activity of ornithine transaminase (OTA) during the time period in which MSA causes an increase in ODC activity. Insulin also stimulates [³H] thymidine incorporation into DNA, cell multiplication and ODC activity over the same concentration range as shown for MSA, however, the extent of stimulation by insulin is less than that observed following MSA addition.     

A stromal cell line from rainbow trout spleen, RTS34st, that supports the growth of rainbow trout macrophages and produces conditioned medium with mitogenic effects of leukocytes

Summary A rainbow trout spleen cell line, RTS34, was developed from a long-term hemopoietic culture. This cell line consisted of a mixed stromal cell layer with an associated cell population of macrophage-like cells that formed proliferative foci and released nonadherent progeny cells into the culture medium. A stromal cell line, RTS34st, was isolated from the RTS34 cell line. RTS34st cultures contained cells with fibroblast-like and epithelial-like morphologies and showed enhanced [³H]thymidine incorporation in response to either FBS or rainbow trout serum. The combination of FBS and trout serum was synergistic. Conditioned medium from RTS34st stimulated thymidine incorporation by peripheral blood and head kidney leukocytes, but not by leukocytes from the spleen. In addition, RTS34st provided a hemopoietic inductive microenvironment for immature precursor cells, selectively supporting the growth of macrophage-like cells. Therefore, RTS34st appears useful for studying the different roles of the stroma in regulating hemopoiesis in fish.     

A comparative kinetic analysis of proliferation in vitro of Con-A-treated splenocytes and syngeneic leukaemia cells

Abstract. The growth kinetics of Con-A-treated mouse splenocytes and syngeneic leukaemia cells cultured in vitro were compared with respect to (i) the total cell number, (ii) the rate of [¹⁴C]thymidine incorporation (measured by pulse-labelling the cells at various times of incubation), and (iii) the labelling index of the cell populations. By correlating the thymidine incorporation, labelling index and cell number data, it has been established that, for both types of cells, the rate of [¹⁴C]thymidine incorporation is directly proportional to the number of cells synthesizing DNA. A new approach to cytokinetic analysis has been developed, showing that important information can be obtained by determining the cumulative kinetics of [¹⁴C]thymidine incorporation. The latter has been calculated by integrating the area underneath the time course of the rate of thymidine incorporation, and was directly proportional to the overall growth of both leukaemia cells and Con-A-stimulated splenocytes. Based on this proportionality, an estimate of the average duration of the S phase for both types of cells was calculated, suggesting that normal and neoplastic blasts maintain this parameter at a constant value (7.6 and 5.9 hr, respectively) throughout different stages of growth. The percentage of Con-A-responsive cells within the initial splenocyte population and their overall proliferation in vitro have been determined by a procedure which measures the cumulative kinetics of thymidine incorporation and the kinetics of cell total number in the presence or in the absence of the lectin, as well as in the presence of Con-A plus colcemid. A minor fraction (11%) of the initial splenocytes is recruited into cycle by Con-A, proliferating with similar kinetics to that of leukaemia cells in the same conditions. The great majority of the initial splenocyte population is unaffected by Con-A, decaying exponentially throughout the incubation with the same half-life (28 hr), both in the presence or in the absence of the lectin.     

Activity of an Attached and Free-Living Vibrio sp. as Measured by Thymidine Incorporation, p-Iodonitrotetrazolium Reduction, and ATP/DNA Ratios

Three independent techniques, [³H]thymidine incorporation, the reduction rate of p-iodonitrotetrazolium violet (INT) to INT formazan normalized to DNA, and the ratio of ATP to DNA, were adapted to measure the activity of attached and unattached estuarine bacteria. In experiments employing the estuarine isolate Vibrio proteolytica, nutrient concentrations were manipulated by varying the concentration of peptone-yeast extract. In the presence of exogenous nutrients, the activity of free-living cells was greater than that of attached cells as measured by [³H]thymidine incorporation and ATP/DNA ratios. In the absence of peptone-yeast extract, however, the activity of attached cells surpassed that of free-living cells as determined by [³H]thymidine incorporation and INT formazan normalized to DNA. Of the three techniques, [³H]thymidine incorporation was deemed most sensitive for detecting changes in activity resulting from slight differences in nutrient concentration. By this technique, attached cells were much less sensitive to changing nutrient concentrations than were free-living cells. Below a threshold concentration, attached cell activity remained constant, while the activity of unattached cells decreased as a function of decreasing nutrient concentration. The results suggest that loss of cell surface area available for substrate uptake due to attachment may be an important factor in determining the relative activities of attached and free-living cells.     

DETERMINATION OF RATES OF DNA SYNTHESIS IN CULTURED MAMMALIAN CELL POPULATIONS

In cultures of a murine mastocytoma, endogenous synthesis of thymidine phosphates, as determined by the incorporation of [³H]deoxyuridine into DNA, was reduced within 15 min to less than 3% of control values by the addition of amethopterin (10 µM) in combination with hypoxanthine and glycine. If [³H]thymidine and unlabeled thymidine were added simultaneously with amethopterin, the increase with time of radioactivity in cellular DNA was linear at least between 30 and 90 min, while radioactivity in the acid-soluble nucleotide fraction remained constant during this time interval, indicating that intracellular thymidine nucleotides had the same specific activity as exogenously supplied [³H]thymidine. This permitted calculation of the amount of thymidine incorporated per hour into DNA of 10⁶ cells. In conjunction with the base composition of mouse DNA, these results were used to calculate rates of DNA synthesis. Cell proliferation rate, cell cycle time, and the duration of the S period were not affected to any appreciable extent by the addition of amethopterin and thymidine. Rates of DNA synthesis, as derived from thymidine incorporation rates, were in good agreement with those derived from the measured mean DNA content of exponentially multiplying cells and rates of cell proliferation.