Stimulation of DNA synthesis by heated human serum in primary cultured normal adult rat hepatocytes

In primary culture of normal adult rat hepatocytes, human serum heated at 56°C for 30 min stimulated dose-dependently [³H]thymidine incorporation into trichloroacetic acid insoluble fraction of the cells, most of which was solubilized into hot trichloroacetic acid solution. The solubilized fraction was reduced when hydroxyurea was added to the culture. The heated serum also increased dose-dependently protein synthesis and cell viability determined from morphological findings. These results suggest that human serum has heat-stable factors stimulating DNA synthesis and maintaining cell viability of cultured rat hepatocytes.     

Stimulation of growth of primary cultured adult rat hepatocytes without growth factors by coculture with nonparenchymal liver cells

DNA synthesis of adult rat parenchymal hepatocytes alone in primary culture can be stimulated only by the addition of humoral growth factors to the culture medium. However, when parenchymal hepatocytes were cocultured with nonparenchymal liver cells from adult rats, their DNA synthesis was markedly stimulated in the absence of added growth factors or calf serum. DNA synthesis of parenchymal hepatocytes was not stimulated by conditioned medium from nonparenchymal liver cells and was greatest when the parenchymal cells were plated on 24-h cultures of nonparenchymal liver cells. A dead feeder layer of nonparenchymal cells was almost as effective as a feeder layer of viable nonparenchymal cells. These results suggest that the stimulation of DNA synthesis in parenchymal hepatocytes was not due to some soluble factors secreted by nonparenchymal liver cells but to an insoluble material(s) produced by the nonparenchymal liver cells. This insoluble material(s) was collagenase- and acid-sensitive, suggesting that it was a protein containing collagen. The effect of nonparenchymal liver cells was specific: coculture with hepatoma cells, liver epithelial cells, or Swiss 3T3 cells did not stimulate DNA synthesis in parenchymal hepatocytes. Added insulin and epidermal growth factor showed additive effects with nonparenchymal cells in the cocultures. These results suggest that DNA synthesis in parenchymal hepatocytes is stimulated not only by various humoral growth factors but also by cell-cell interaction between parenchymal and nonparenchymal hepatocytes, possibly endothelial cells. This cell-cell interaction may be important in repair of liver damage and liver regeneration.     

Stimulation of cyclic GMP synthesis in human cultured glomerular cells by atrial natriuretic peptide

Recently a stimulatory effect of atrial natriuretic peptide (ANP) on the particulate guanylate cyclase system has been reported in the glomeruli from different species. Using cultures of homogeneous human glomerular cell lines, we found that rat and human ANP stimulated markedly cGMP formation in epithelial cells with a threshold dose of 1 nM. A 20-fold increase was obtained at 5 microM. Stimulation was also present but less substantial (2-fold at 5 microM) in mesangial cells. cGMP was formed rapidly and released in the medium. ANP and sodium nitroprusside, an activator of soluble guanylate cyclase, had additive effects on cGMP formation. ANP did not inhibit cAMP formation in both cell lines. These results demonstrate that, at least in the human species, epithelial cells represent the main target of ANP in the glomerulus. Synthesis of cGMP in the glomerular epithelial cells in response to ANP also suggests that the excess of urinary cGMP produced by the kidney which is observed after ANP administration is of glomerular rather than of tubular origin.     

Stimulation by insulin of DNA synthesis in cultured mouse fibroblasts]

With the aid of autoradiography, the effect of insulin on entering S- from G1-period of the mitotic cycle and on the rate of DNA synthesis of the mouse fibroblasts (L), was studied,--in the cells incubated for 24 hr in serum-free medium. In these conditions the cells were temporarily blocked in G1-period. Insulin (100 mcU/ml) increased by 1.5-fold the amount of cells in S-period as well as caused a marked stimulation of DNA synthesis.     

Stimulation of cellular DNA synthesis by human cytomegalovirus

Human cytomegalovirus (CMV) is able to induce cellular DNA synthesis in both permissive (human embryonic lung) and nonpermissive (Vero) cells. The induction of cell DNA synthesis was assayed by the incorporation of [methyl-³H]thymidine into macromolecules having the buoyant density characteristics of cell DNA. The DNA synthesis induced by CMV infection appears to represent normal semiconservative replication as opposed to repair synthesis. Both strains of CMV tested were capable of inducing cell DNA synthesis. Virus exposed to heat or UV light prior to infection lost the ability to induce DNA synthesis, indicating that a virus-coded function expressed after infection is responsible for stimulation of cell DNA synthesis.     

Cytogenetic response to asbestos fibers in cultured human primary mesothelial cells from 10 different donors

The ability of amosite asbestos fibers to induce chromosomal aberrations in human primary mesothelial cells obtained from pleural effusions of 10 noncancerous patients was investigated. The glutathione S-transferase M1 (GSTM1) genotypes of the patients were determined, since the GSTM1 null genotype has been associated with increased susceptibility to lung cancer and chemically induced cytogenetic damage. Four of the patients represented the GSTM1 null genotype, and six the GSTM1 positive genotype. Successful chromosome aberration analyses were obtained from six cases, three of them with the GSTM1 null genotype. The level of aberrant cells in unexposed cultures ranged from 2.0% to 7.5%. Statistically significant increases (2.3–3.0-fold compared to controls) in the number of aberrant cells were observed in two cases only: in one case treated with 1 μg/cm² of amosite, and in another treated with 2 μg/cm² of amosite. Cell cultures from four individuals showed minor or no increases in the numbers of aberrant cells in the doses tested (1 and 2 μg/cm²). Chromosome breaks were the major type of aberration. The amosite exposed cells with significantly increased aberrations were from patients with GSTM1 positive genotypes. Two cases that showed no cytogenetic response to asbestos fibers were of the GSTM1 null genotype. Thus, our results suggest that the lack of the GSTM1 gene does not render human mesothelial cells more susceptible to chromosomal damage induced by asbestos. GSTM1 null cells appeared, however, to be more sensitive to the growth inhibitory effects of asbestos than did GSTM1 positive cells. Variation in the cytogenetic response of human primary mesothelial cells to asbestos fibers was observed to exist, but the fibers do not appear to be potent inducers of structural chromosomal aberrations in these cells. It remains to be established whether individual sensitivity to asbestos fibers, due to specific genetic traits, exists.     

Stimulation of ribosomal RNA synthesis during hypertrophic growth of cultured heart cells by phorbol ester

Primary cultures of neonatal cardiac myocytes were used to determine the effects of tumor-promoting phorbol esters on ribosomal RNA (rRNA) synthesis during myocyte growth. Treatment of myocytes with phorbol-12,13-dibutyrate (PDBu) increased protein accumulation by 25% and RNA content by 20%. Rates of rRNA synthesis were measured to assess the mechanism by which rRNA accumulated during myocyte growth. Rates of rRNA synthesis were determined from the incorporation of [³H]uridine into UMP of purified rRNA and the specific radioactivity of the cellular UTP pool. After 24h of PDBu treatment, cellular rates of 18S and 28S rRNA synthesis were accelerated by 67% and 64%, respectively. The increased rate of rRNA synthesis accounted for the net increase in myocyte rRNA content after PDBu treatment.     

DNA damage and growth inhibition in cultured human cells by bleomycin congeners

Bleomycin is hypothesized to cause cell growth inhibition and cell death via DNA cleavage. We have attempted to determine if net DNA cleavage is directly related to growth inhibition by measuring whether both parameters vary in parallel. Of primary importance to these studies was use of several bleomycin congeners. We have shown that these congeners vary in their abilities both to inhibit cell growth and to cause DNA damage. Bleomycin B2, tallysomycin, and phleomycin were the most potent growth inhibitors, and bleomycin B2 caused the most DNA damage. N-Acetylbleomycin A2 was inactive in both assays. The net amount of DNA damage measured at two levels of growth inhibition was compared for each congener and was found to vary widely among the congeners. Similarly, the degree of growth inhibition at a given level of submaximal DNA damage was found to vary widely when individual congeners were compared to each other. Hence, growth inhibition and net DNA damage due to bleomycin are not directly correlated with each other when individual congeners are compared to each other.     

Translesion DNA synthesis across non-DNA segments in cultured human cells

DNA lesions that have escaped DNA repair are tolerated via translesion DNA synthesis (TLS), carried out by specialized error-prone DNA polymerases. To evaluate the robustness of the TLS system in human cells, we examined its ability to cope with foreign non-DNA stretches of 3 or 12 methylene residues, using a gap-lesion plasmid assay system. We found that both the trimethylene and dodecamethylene inserts were bypassed with significant efficiencies in human cells, using both misinsertion and misalignment mechanisms. TLS across these non-DNA segments was aphidicolin-sensitive, and did not require poleta. In vitro primer extension assays showed that purified poleta, polkappa and poliota were each capable of inserting each of the four nucleotides opposite the trimethylene chain, but only poleta and polkappa could fully bypass it. Poleta and poliota, but not polkappa, could also insert each of the four nucleotides opposite the dodecamethylene chain, but all three polymerases were severely blocked by this lesion. The ability of TLS polymerases to insert nucleotides opposite a hydrocarbon chain, despite the lack of any similarity to DNA, suggests that they may act via a mode of transient and local template-independent polymerase activity, and highlights the robustness of the TLS system in human cells.     

Stimulation of DNA synthesis in human fibroblasts by thrombin.

Earlier work showed that thrombin stimulates proliferation of human fibroblasts in serumfree medium. This work demonstrates (1) that thrombin has to be prensent during most or all of the G1 period to ensure maximal DNA synthesis, (2) that DNA synthesis increases about three hours later after thrombin than after serum treatment, (3) that both thrombin and serum activate transport of uridine, D--2-deoxy-glucose and putrescine, (4) that thrombin is able to increase 3H-thymidine incorporation also in SV40 transformed human fibroblasts, in HeLa cells and in two continuous monkey cell lines.     

Stimulation by thyroid hormone of phosphate transport in primary cultured renal cells

The regulation by thyroid hormone of phosphate transport in primary cultured chick renal cells was examined. The more physiologically active L-analogs of triiodothyronine and thyroxine, but not the D-analogs of the hormones, stimulated the Na+-dependent phosphate uptake system. Na+-independent phosphate uptake and Na+-dependent uptakes of alpha-methylglucoside and L-proline were unaffected. The increase in Na+-dependent phosphate uptake was concentration dependent, exhibited an induction period, and was blocked by inhibitors of RNA and protein synthesis. The stimulation of phosphate uptake by triiodothyronine was due to an increased Vmax rather than to an altered affinity for phosphate. These findings demonstrate that thyroid hormone acts directly on renal cells to modulate phosphate transport and suggest that the renal cell system may serve as a model to examine the mechanism by which thyroid hormone controls gene expression and regulates plasma membrane transport function.     

Stimulation of DNA repair in human cells damaged by UV and ionizing radiation with soluble growth factors of photomodified blood

Exposure of a small skin area of volunteers to UV light in 1 minimal erythemal dose is accompanied by rapid appearance in the circulating blood of soluble factors able to restore proliferation of X-ray-damaged autologous lymphocytes, to decrease frequency of chromosome breaks, and to stimulate unscheduled DNA synthesis. The appearance of such an activity in the blood can be also induced without skin irradiation. For this, one volume of a directly UV-irradiated blood is to be mixed in vitro with 10-fold volumes of intact blood, thus modeling the in vivo situation, when a small amount of transcutaneously UV-irradiated blood mixes with intact blood in the circulation. It has been found that the platelet-derived growth factor and epidermal growth factor, added at physiological concentrations to the culture medium, decrease chromosome break frequency in X-damaged cells.     

Stimulation of catecholamine synthesis in cultured bovine adrenal medullary cells by leptin

Recently, we characterized leptin receptors in bovine adrenal medullary cells (Yanagihara et al. 2000). Here we report the stimulatory effect of leptin on catecholamine synthesis in the cells. Incubating cells with leptin (10 nM) for 20 min increased the synthesis of 14C-catecholamines from [14C]tyrosine, but not from L-3,4-dihydroxyphenyl [3-14C]alanine. The stimulation of catecholamine synthesis in the cells by leptin was associated with the phosphorylation and activation of tyrosine hydroxylase, the rate-limiting enzyme of catecholamine biosynthesis. The incubation of cells with leptin resulted in a rapid activation of the mitogen-activated protein kinases (MAPKs). An inhibitor of MAPK kinase, U0126, nullified the stimulatory effect of leptin on the synthesis of 14C-catecholamines. Leptin potentiated the stimulatory effect of acetylcholine on 14C-catecholamine synthesis, whereas leptin failed to enhance the phosphorylation and activation of tyrosine hydroxylase induced by acetylcholine. These findings suggest that leptin stimulates catecholamine synthesis via the activation of tyrosine hydroxylase by two different mechanisms, i.e., one is dependent on tyrosine hydroxylase phosphorylation mediated through the MAPK pathway and the second is independent of enzyme phosphorylation.     

Stimulation of protein synthesis in cultured heart muscle cells by glucose.

Glucose stimulated the rate of incorporation of [3H]leucine into HCLO4-insoluble fraction of cultured rat heart muscle cells under both aerobic and anaerobic conditions. In the aerobic system the incorporation proceeded at a constant rate during 3h of incubation with and without glucose whereas in the anaeorbic system the incorporation ceased after approx. 60 min and could be renewed only by the addition of glucose. No correlation was found to exist between the above effect of glucose on protein synthesis and glucose-dependent changes in the intracellular ATP concentration. The extent of the stimulation of protein synthesis was related to the concentration of glucose. The effect of glucose was suppressed by cycloheximide but was not affected by actinomycin D. Glucose had no effect on the rate of transport of alpha-aminoisobutyric acid. Mannose also stimulated [3H]leucine incorporation. Substances that did not produce lactate were ineffective. Iodoacetate inhibited the stimulatory effect of glucose, but pyruvate, which by itself had no apprecialbe stimulatory action, relieved the inhibition induced by iodoacetate. There was no concomitant change in the concentration of ATP when iodoacetate inhibition was reversed by pyruvate. L-Lactate or other intermediates of energy metabolism could not relieve the inhibitory effect of iodoacetate.     

Stimulation of DNA synthesis in quiescent rabbit chondrocytes in culture by limited exposure to somatomedin-like growth factors

Cartilage-derived factor (CDF), extracted from fetal bovine cartilage, and multiplication-stimulating activity (MSA) stimulated DNA synthesis in quiescent rabbit costal chondrocytes in culture under serum-free conditions. As described previously, when added in the presence of fibroblast growth factor (FGF) or epidermal growth factor (EGF) a somatomedin-like growth factor, CDF or MSA, synergistically stimulated DNA synthesis in the cultured chondrocytes. The present study showed that exposure of the cells to MSA or CDF for only the initial 5 h was sufficient for transmission of their full stimulatory effect. Furthermore, the limited exposure did not alter the time course of stimulation of DNA synthesis: [3H]thymidine incorporation into DNA began to increase after 16 h and reached a maximum after 24 h. In contrast to the somatomedin-like growth factors, FGF and EGF were required continuously in the culture medium during traverse of the entire G1 phase for stimulation of DNA synthesis, and the mitogenic effects of FGF and EGF in cultured chondrocytes were stronger than those of CDF and MSA. Synergistic stimulation of DNA synthesis by CDF or MSA in the presence of FGF or EGF could be observed as long as FGF or EGF was continuously present, even when CDF or MSA was withdrawn after the first 5 h of culture. These findings suggest that, in contrast to FGF and EGF, somatomedin-like growth factors affect an early distinct stage in the G1 phase of chondrocytes.     

Stimulation of DNA synthesis in cultured mouse peritoneal macrophages after fusion injection of macrophage growth factor

Macrophage growth factor (MGF), when injected into cultivated mouse peritoneal macrophages by Sendai virus-fusion with loaded human erythrocyte ghosts, induced macrophage DNA synthesis within 40 h. DNA synthesis was observed in approx. 10% of the injected cells. Fusion products between macrophages and red cell ghosts were identified by co-injection of ¹²⁵I-labelled human serum albumin. These studies suggest that the mechanism of the physiological effect of MGF may depend on the endocytosis of at least part of the MGF molecule.     

Effect of growth factors on hyaluronan synthesis in cultured human fibroblasts.

The effect of various growth factors on the synthesis of hyaluronan in human fibroblasts was investigated. When tested in medium containing 0.5% fetal calf serum, platelet-derived growth factor (PDGF)-BB was found to stimulate hyaluronan synthesis; the maximal response was equal to or higher than that obtained with 10% fetal calf serum. PDGF-AA gave only a limited effect, indicating that the stimulatory effect of PDGF on hyaluronan synthesis was mainly transduced via the B-type PDGF receptor. Epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and transforming growth factor (TGF)-beta 1 also stimulated hyaluronan synthesis; their effects were less than that of PDGF-BB, but combinations of factors produced potent stimulatory effects on hyaluronan synthesis. All factors stimulated hyaluronan synthesis in sparse as well as dense cultures. The effects of the factors on hyaluronan synthesis did not correlate with their mitogenic activities; PDGF-BB, EGF and bFGF are equipotent mitogens, but PDGF-BB had a much more potent effect on hyaluronan synthesis, and TGF-beta actually inhibits the growth of fibroblasts under the conditions of the assay.     

Stimulation of DNA synthesis in skin fibroblasts by human hepatocyte growth factor/scatter factor.

The effect of hepatocyte growth factor/scatter factor (HGF/SF) on the proliferation of human skin fibroblasts was examined. At concentrations above 1.0 ng/ml, both native and recombinant human HGF/SF stimulated the DNA synthesis determined by [³H]thymidine incorporation, which was completely inhibited by an anti-human HGF/SF monoclonal antibody. The maximal DNA synthesis in the treated cells was nearly twice that in untreated cells. HGF/SF also caused an increase in the labelling index, DNA content and cell number. The effect of HGF/SF was more than additive to the maximal effect of insulin and epidermal growth factor, other mitogens for the fibroblasts. These results indicate that human skin fibroblasts are sensitive to the mitogenic action of HGF/SF.