Effect of prostaglandins on protein, RNA, DNA and collagen synthesis in experimental wounds.

The effect of exogenous prostaglandins E1, E2 and F2alpha (PGE1, PGE2 and PGF2alpha) on 3H-leucine, 3H-uridine, 3H-thymidine and 3H-proline incorporation in experimental cutaneous wounds has been studied in rats. Prostaglandins E1 and E2 markedly stimulate the incorporation of these tritiated precursors, into protein, RNA, DNA and collagen synthesis, whereas F2 inhibits it. All tested prostaglandins exhibit their maximum effect within the first hours following administration. Most active is PGE1. These observations indicate that application of prostaglandins significantly stimulate incorporation with protein, RNA, DNA and collagen synthesis in the skin of wounded rats and thus, may play a role in epidermal cell growth and division as well as in scar-forming tissue.     

Effects of calcitonin and calcitonin gene-related peptide on 3H-thymidine incorporation into DNA of rat thyroid lobes in vitro.

The effects of a 4 h incubation of rat thyroid lobes, in the presence of calcitonin (CT) and calcitonin gene-related peptide (CGRP) on the incorporation of 3H-thymidine into DNA, were investigated. In other groups the thyroid lobes were incubated during exposure to CT and thyrotropin (TSH), and to CGRP together with TSH. All concentrations of CT (10(-6)-10(-8) M) revealed a tendency towards lowering 3H-thymidine uptake, but the effect was not statistically significant. The influence of CGRP was dose-dependent; the lowest concentration of CGRP (10(-9) M) significantly enhanced DNA synthesis in the incubated rat thyroids; an intermediate dose of the peptide (10(-8) M) had no effect, while the highest concentration of CGRP (10(-7) M) decreased 3H-thymidine incorporation. Calcitonin (10(-7) M), as well as CGRP (10(-8) M), suppressed the stimulatory effect of TSH on 3H-thymidine incorporation.     

AN AUTORADIOGRAPHIC ANALYSIS OF THE ROOT EPIDERMIS OF THE SWITCH GRASS (PANICUM VIRGATUM)

Analyses of ³H-uridine, ³H-thymidine, and ³H-lysine incorporation in the root epidermis of Panicum virgatum were undertaken. Highly significant differences between the mean incorporation of ³H-uridine and ³H-lysine in epidermal and adjacent cortical cells were observed. While the cortex exhibited a steady decrease of precursor incorporation with distance from the apex, the epidermal cells exhibited differential incorporation. These results were regarded as further evidence for the hypothesis that cells of two maturation potentials exist in the epidermis of this panicoid grass. Treatment with 20.0 μg/ml of actinomycin D resulted in a differential inhibition in the epidermal-cortical incorporation of ³H-uridine. The possibility of endopolyploidy in the epidermis was suggested by the observation that root hairs, hair initials, and some epidermal cells incorporated two to four times more ³H-thymidine than meristematic cells. Neither puromycin nor actinomycin D treatment affected the protein-positive particles present in the cytoplasm of epidermal cells in this grass. Similarly, RNase did not affect their structural integrity. Attempts to clarify the significance of these inclusions and their possible role, if any, in the differentiation of the epidermis are now in progress.     

Effects of growth and differentiation inducing factors on protein kinase-C of cultured intestinal crypt cells

To assess the role of protein kinase-C (PK-C) in the growth and differentiation of small intestinal enterocytes, IEC-6 cells (a cell line derived from the crypts of rat small intestine) were incubated with factors known to induce growth (insulin, epidermal growth factor, gastrin, somatostatin and transferrin) or differentiation (transforming growth factor-beta, retinoic acid and phorbol 12-myristate 13-acetate (PMA)). Cell proliferation (3H-thymidine incorporation) and PK-C activity (Ca++/phospholipid dependent) were measured. Among growth promoting factors only epidermal growth factor, insulin and transferrin were associated with increased 3H-thymidine incorporation, and none of these agents induced PK-C activation as measured by its translocation from cytosol to membrane fraction. Of the differentiation inducing factors, only PMA translocated PK-C from cytosol to membrane. PMA also inhibited 3H-thymidine incorporation in a dose dependent manner. These results suggest that growth and proliferation of enterocytes occur independent of PK-C signal transduction.     

Further studies on the enhancing factor and its possible mechanism of action

In this study the nature of binding of enhancing factor (EF) and its mode of action are examined. EF binds to A431 cells through its own receptor, which is distinct from the receptor for epidermal growth factor (EGF). EF binds to the cell membrane and in turn provides a binding site for EGF. Data analyzed from Scatchard plots show that prior treatment of formalin-fixed A431 cells with EF for 30 minutes results in an increase in the number of binding sites for 125I-EGF. 3H-Thymidine incorporation studies, using the EGF-receptorless cell line NR-6, indicate that neither EF nor EGF alone stimulates the cells to synthesise DNA, but when both are added together the cells show 3H-thymidine incorporation. The role of EF may be to trap EGF and make it available to the cells through its own receptors even in the absence of EGF receptors. EF also induces anchorage-independent growth of normal fibroblasts in soft agar only in the presence of EGF.     

Observatons on the growth and metabolic functions of cultured cells derived from human adipose tissue.

The growth and metabolic activity of cultured cells derived from human adipose tissue (CAT cells) were studied and compared to cultured skin fibroblasts. The morphological appearance of the CAT cells was distinctly different from that of fibroblasts. The growth rate of CAT cells as measured by 3H-thymidine incorporation was much slower than the fibroblast growth rate. Cultured CAT cells synthesized significantly 14C-glucose, while fibroblast cultures had a higher metabolic rate as measured by CO2 production. Insulin stimulated 3H-thymidine incorporation in both CAT and fibroblast cultures. The CAT cells did not show a consistent insulin response of lipid or CO2 production, but this may be a reflection of donor age or nutritional status. Even though the CAT cell may be a type of stromal cell peculiar to adipose tissue rather than a preadipocyte or adipocyte, it may prove useful in studies of human obesity.     

INCORPORATION OF TRITIATED THYMIDINE DURING VARIOUS STAGES OF LEAF DEVELOPMENT OF XANTHIUM PENNSYLVANICUM

³H-thymidine was incorporated into leaf tissue of Xanthium pennsylvanicum during the stage of active cell division, during cellular differentiation, and into mature cells. Incorporation into nuclear DNA was high in the early stages of development. No nuclear incorporation was found after cessation of cell division. However, significant incorporation could be demonstrated in cytoplasm of differentiating and mature cells. Depending upon the time of growth in the radioisotope and the time of growth after treatment, ³H-thymidine, or its metabolized fraction, was incorporated into the secondary wall depositions of epidermal cells, mesophyll parenchyma cells, xylem cells, and chloroplasts. Autoradiographic technique and liquid scintillation spectrometry were used in these studies. The significance of ³H-thymidine incorporation into various organelles is discussed in relation to cell metabolism and its regulation during leaf development.     

The autocrine regulation of proliferation in cell cultures. I. A study of the heparin-binding factors with growth-stimulating activity contained in media conditioned with transformed rat fibroblasts]

A study was made of the medium conditioned by spontaneously transformed rat embryo fibroblasts of line Rec1-sf, which are capable of unlimited reproduction in medium free of serum and of other endogenous growth factors (c-medium). Addition of c-medium to stationary cultures of nontransformed rat embryo fibroblasts (REF), spontaneously transformed REF (line Rec1), and cells of Rec1-sf stimulated the incorporation of 3H-thymidine by 1.5-6 times. SDS-polyacrilamide-gel electrophoresis of proteins, marked by 35S-metionine of c-medium of the cell line Rec 1-sf, demonstrated that this medium had proteins with molecular mass from 10 to 110 kDa. The fractionating divisible by 100 ultra-concentrates of c-medium with utilization of heparin-sepharose allowed to isolate two types of heparin-binding proteins. The proteins of the first type took about 5% of all the proteins of c-medium; they were eluted with 1.1 M NaCl and stimulated the incorporation of 3H-thymidine in REF, Rec1 and Rec1-sf cultures by 1.3-1.9 times. The second type proteins took about 1% of all the proteins of c-medium and were eluted with 2M NaCl, and, like the main endogenic basic growth factor of fibroblasts, stimulated the incorporation of 3H-thymidine into REF and Rec1-sf, but not into the culture of Rec1 line cells. The results obtained are discussed in terms of a hypothesis of autocrine regulation of cell proliferation.     

Effects of EGF and PMA on the growth and proliferation of IEC-6 cells

Proliferation of an epithelial line (IEC-6) derived from the crypts of rat jejunum was induced with epidermal growth factor (EGF). EGF enhanced synthesis of protein, RNA, and DNA in a dose-dependent manner. Protein synthesis increased within 6-12 hours of exposure to EGF and remained elevated for 72 hours. Maximal 3H-thymidine incorporation occurred 48 hours after addition of EGF. The stimulatory effect of EGF on 3H-thymidine incorporation was two-fold greater in serum-free media than in media containing fetal calf serum (FCS). In contrast to EGF, phorbol-12-myristate-13-acetate (PMA) decreased 3H-thymidine uptake by IEC-6 cells and had no effect on either protein synthesis or RNA synthesis. EGF did not alter protein kinase-C activity in IEC-6 cells whereas PMA induced enzyme activity: activity was translocated from cytosol to membrane. Moreover, the EGF-associated increase in 3H-thymidine uptake was not altered by amiloride. These data suggest protein kinase-C activation may not be involved in the proliferation of IEC-6 cells.     

DNA replication in mammalian cells after the action of physical, chemical or biological factors. VI. The recovery of DNA synthesis in a culture of irradiated cells

The kinetics of DNA synthesis restoration in cultured HeLa cells and in L-929 mouse fibroblasts irradiated by gamma-rays of 60Co with a dose of 10 Gy was studied. Early after irradiation the rate of DNA synthesis in HeLa cells measured with 3H-thymidine incorporation was seen to decrease. Two hours later the incorporation starts to increase to reach the control level 4 hours after irradiation and then becomes even higher than this level. The distribution of cells among phases of the cell cycle measured with flow cytometry undergoes changes. 4-6 hours after irradiation part of S-phase cells increased contributing presumably to the elevating of 3H-thymidine incorporation observed at this time. The restoration of the incorporation was suppressed by inhibitors of protein and RNA synthesis--cycloheximide and actinomycin D. It is suggested that the processes of restoration of DNA synthesis in irradiated cells can be of inducible nature. In irradiated HeLa and L-929 cells the restoration of DNA synthesis is resistant to novobiocin, an inhibitor of DNA replication.     

Ethanol inhibits hormone stimulated hepatocyte DNA synthesis

Insulin, glucagon, and epidermal growth factor (EGF) addition stimulated DNA synthesis in primary hepatocyte cell cultures prepared from adult rat liver. The addition of ethanol (20-200mM) to the culture medium resulted in a substantial reduction in DNA synthesis as measured by 3H-thymidine incorporation and autoradiography. This effect was specific for differentiated hepatocytes compared to fibroblasts and two other human hepatoma cell lines. These studies demonstrate in a cell culture system that one of the major properties of ethanol is the inhibition of hepatocyte DNA synthesis.     

Bovine thecal cells secrete factor(s) that promote granulosa cell proliferation

To determine if soluble factors (other than steroids) secreted by bovine thecal cells may be involved in local regulation of follicular development, we examined the effects of thecal cell secretory products on the growth of granulosa cells obtained from the same follicles. DNA synthesis (assessed by the incorporation of 3H-thymidine) by granulosa cells plated on coverslips and cocultured with, but not directly in contact with, thecal cells in organ culture dishes in a serum-free medium was 5-fold greater than controls. The effect of the thecal cell-secreted products on DNA synthesis by granulosa cells was significantly higher than the maximum response produced by epidermal growth factor (EGF). Thecal cell-conditioned medium stimulated 3H-thymidine incorporation into the DNA of granulosa cells and a normal rat kidney cell line in a dose-dependent manner. The increases in 3H-thymidine incorporation into granulosa cell DNA subsequently lead to an increase in cell number. Preliminary characterization studies using ultrafiltration membranes indicated that the mitogenic factor was retained in the greater than 10,000 molecular weight fraction. The activity was stable to heating at 90 degrees C for 5 min and was not extracted in ether. The thecal cell-generated growth factor may act as a paracrine regulator of granulosa cell growth, thus providing the dominant follicle with autonomy over other follicles in the cohort.     

Proliferation of Buffalo rat liver cells in serum-free medium does not depend upon multiplication-stimulating activity (MSA).

A line of Buffalo rat liver cells (BRL 3A) that multiplies in the absence of serum produces a family of polypeptides termed MSA that can partially satisfy the serum requirement for growth of chick embryo fibroblasts. Temin, Pierson and Dulak (1972) proposed that BRL cells multiply in serum-free medium because they produce MSA. This does not appear to be the case. We have studied three BRL cell lines: 3A2 and 3A have diverged from the same original isolate from normal liver; 61t is a spontaneous transformant of a different isolate. All three cell lines showed a 10 fold increase in cell number during 5 days in serum-free medium. However, 3A-conditioned medium stimulated 3H-thymidine incorporation into DNA in chick embryo fibroblasts and human skin fibroblasts; 3A2- and 61t-conditioned media did not. After ion-exchange chromatography or gel filtration of the conditioned media and measurement of MSA by 3H-thymidine incorporation or radioreceptor assay, MSA again was found in the 3A medium but not in the 3A2 or 61t media. The absence of MSA in the 3A2 and 61t media was not due to inactivation of MSA by these two cell lines. Addition of partially purified MSA to 3A2 cells did not increase their multiplication rate in serum-free medium. We conclude that the ability of the BRL cells to multiply in serum-free medium is independent of the level of MSA in the medium.     

Reduced cell sensitivity to glucocorticoid hormones in hypercholesterolemia

The number of 3H-dexamethasone binding sites in lymphocytes of subjects with hypercholesterolemia (HCS) was found to be decreased as compared to the receptor level in normolipidemic patients (N). In HCS-lymphocytes, the dexamethasone-induced inhibition of 3H-thymidine and 14C-acetate incorporation was less pronounced (by 20% and 22%, respectively) than in control cells, which is suggestive of the decreased sensitivity of HCS-lymphocytes to the hormone. An addition of 5-25% HCS blood sera to human skin fibroblast cultures caused a 10-50% decrease in the number of 3H-dexamethasone binding sites and diminished the Kd values 2-3 times. Lipid-depleted HCS-sera had no effect on the glucocorticoid reception in fibroblasts, whereas very low (VLDL) and low (LDL) density lipoproteins inhibited the 3H-dexamethasone binding to the cells. The most potent inhibiting effect was exerted by VLDL (both N-VLDL and HCS-VLDL). HCS-VLDL were more effective than N-LDL. HCS-HDL and N-HDL did not effect the 3H-dexamethasone binding to fibroblasts. In cells preincubated with VLDL dexamethasone inhibited the incorporation of 3H-thymidine and 14C-acetate less intensively (by 27% and 20%, respectively) than in control fibroblasts. The experimental results are suggestive of a decreased sensitivity of peripheral HCS-cells to glucocorticoids, which may shed some light on the mechanism of hypercholesterolemia realization into coronary heart disease and atherosclerosis.     

Mechanism of nonspecific macrophage-mediated cytotoxicity: evidence for lack of dependence upon oxygen.

Peritoneal macrophages elicited in C3H/HJ mice by the i.p. injection of Corynebacterium parvum were cytotoxic to allogeneic virus-transformed fibroblasts in vitro. Cytotoxicity was demonstrated in a morphologic (plaque) assay, and quantitated by measuring macrophage-mediated inhibition of incorporation of 3H-thymidine by the target cells. The cytotoxic effect was well established by 6 hr of macrophage-fibroblast interaction, and was retained in cultures from which the supernatant was removed before the addition of 3H-thymidine. Cytotoxic activity of macrophages diminished rapidly after 22 hr of cultivation in vitro. Maximal cytotoxic effect could be prolonged by addition of C. parvum, 50 microgram/ml to macrophage monolayers preincubated in vitro for 22 hr. It could neither be retained nor regenerated when C. parvum was added to monolayers greater than 22-hr old. C. parvum-activated macrophages, grown under anaerobic conditions for 8 hr, retained the ability to phagocytize heat-killed Candida albicans and to exclude trypan blue dye. There was a small but significant reduction in the ability of macrophages to inhibit 3H-thymidine incorporation by target fibroblasts under anaerobic conditions. The cytotoxic effect of activated macrophages in air was not altered by the presence of catalase and was enhanced by enzymatically active superoxide dismutase. We conclude that the processes involved in macrophage-mediated cytotoxicity against allogeneic fibroblasts in this system are largely independent of oxygen.     

1,25-Dihydroxycholecalciferol modulates 3H-thymidine incorporation in FRTL5 cells.

1,25-dihydroxycholecalciferol (1,25(OH)2D3) possesses proliferation and differentiation modulating effects in many cell types in vitro. We studied the effect of 1,25(OH)2D3 on 3H-thymidine incorporation in FRTL5 cells, a cultured rat thyroid follicular cell line. 1,25(OH)2D3 alone at 10(-11) and 10(-9) M exerted no effect on 3H-thymidine incorporation. However, at 10(-7) M, 1,25(OH)2D3 slightly enhanced 3H-thymidine incorporation. In the presence of 5% calf serum, 1,25(OH)2D3 increased 3H-thymidine incorporation induced by calf serum in a dose-dependent manner. 1,25(OH)2D3 also enhanced 3H-thymidine incorporation induced by PMA, an extrinsic stimulator of protein kinase C, without directly affecting PMA-induced protein kinase C translocation. In contrast to the stimulatory effects of 1,25(OH)2D3 on the calf serum and PMA-induced 3H-thymidine incorporation, 1,25(OH)2D3 inhibited the increase in 3H-thymidine incorporation induced by TSH in a dose-dependent manner. This effect of 1,25(OH)2D3 on TSH-induced 3H-thymidine incorporation may be, in part, due to post-cAMP pathways since 1,25(OH)2D3 also inhibited the increase in 3H-thymidine incorporation induced by Bu2cAMP without affecting the TSH-induced increase in cAMP. The stimulatory effect of insulin on 3H-thymidine incorporation, a cAMP-independent process, was also inhibited by 1,25(OH)2D3. We conclude that 1,25(OH)2D3 affects 3H-thymidine incorporation in FRTL5 cells raising the possibility of a physiologic role for 1,25(OH)2D3 in the growth and function of thyroid follicular cells.     

The effect of transforming growth factor beta on the intensity of cellular DNA synthesis in relation to the type and conditions of cultivation

A study was made of the influence of transforming growth factor beta (0.05-50.00 ng/ml) on the intensity of 3H-thymidine incorporation in the DNAs of pseudonormal mouse fibroblasts of NIH 3T3 line, of cells of NRK-49F line from normal rat kidney, and of cells of A-549 line from human lung adenocarcinoma. The experiments were carried out in the absence and in the presence of epidermal growth factor (5 ng/ml), and(or) insulin (1 micrograms/ml), as well as in the presence of different concentrations of fetal calf serum, and while using different time of incubation of cells with the above mentioned growth factors. It was shown that depending on the culture conditions the transforming growth factor beta exerted stimulatory, inhibitory or no action on the intensity of DNA synthesis in the cells of the same type. An attempt was made to analyse the reasons, which may significantly determine the direction of regulatory influence of the transforming growth factor beta on DNA replication in the cells.     

Human epidermal growth factor and the proliferation of human fibroblasts

The effect of human epidermal growth factor (hEGF), a 5,400 molecular weight polypeptide isolated from human urine, on the growth of human foreskin fibroblasts (HF cells) was studied by measuring cell numbers and the incorporation of labeled thymidine. The addition of hEGF to HF cells growing in a medium containing 10% calf serum resulted in a 4-fold increase in the final density. The presence of hEGF also promoted the growth of HF cells in media containing either 1% calf serum or 10% gamma globulin-free serum. The addition of hEGF to quiescent confluent monolayers of HF cells, maintained in a medium with 1% calf serum for 48 hours, resulted in a 10- to 20-fold increase in the amount of ³H-thymidine incorporation after 20–24 hours. The stimulation of thymidine incorporation was maximal at an hEGF concentration of 2 ng/ml, was dependent on the presence of serum, and was enhanced by the addition of ascorbic acid. In confluent cultures of HF cells, subject to density dependent inhibition of growth, hEGF was able to stimulate DNA synthesis more effectively than fresh calf serum. Human EGF stimulated DNA synthesis in quiescent cultures, however, regardless of cell density. The addition of rabbit anti-hEGF inhibited all effects of this growth factor on HF cells.     

Reduced uptake and incorporation of 3H-thymidine in fanconi anemia fibroblasts

Summary Uptake of ³H-thymidine and its incorporation into DNA was studied in fibroblastic cell lines derived from normal individuals, patients with Fanconi anemia, and those heterozygous for this genetic trait. Uptake and incorporation for the normal cells were about five and seven times higher, respectively, than for Fanconi anemia fibroblasts; mean values for heterozygotes were intermediate. This effect was dependent on the duration of cell exposure to ³H-thymidine and was not observed with other labeled compounds. Thus, a genetically-determined metabolic defect may exist in Fanconi anemia patients which can be readily studied at the cellular level. This finding may be relevant to the observed clinical, cytogenetic, biochemical, and biologic properties related to expression of the Fanconi anemia gene.     

Quantitative historadioautographic analysis of the cellular proliferation characteristics of the aortic wall in rabbits with experimental atherosclerosis

Aortic intima cell proliferation has been studied in 49 rabbits with progressing experimental hypercholesterolemia, using 3H-thymidine. It has been shown that focal cell proliferation is related to infiltration sites of apo-B lipoproteins and can be observed already at pre-lipid stage of atherogenesis. 4 types of proliferating cells involved in the formation of lipid plaques have been identified: endothelial cells, smooth muscle cells, fibroblasts and macrophages, with the latter predominating in the atherosclerotic plaque cover.