Regulation of proliferation of UM-SCV-1A and UM-SCV-6 vulvar carcinoma cells by cytokines

 The biology and pathogenesis of vulvar carcinoma are poorly understood at present. In order to understand this disease better, we have used recently developed squamous cell carcinoma lines of the vulva as models. Two cell lines originating from two individuals (UM-SCV-1A and UM-SCV-6) were cultured in vitro in 10% fetal calf serum. The effects of interleukins 10 and 13, interferons α and γ, granulocyte/macrophage-growth-stimulating factor (GM-CSF), tumor necrosis factor α (TNFα), and transforming growth factor β (TGFβ) on the proliferation of the cells was investigated by using radioactively labelled uridine as tracer. In addition, an investigation on the molecular structure of extracted cellular DNA was carried out to investigate whether programmed cell death (apoptosis) would be inducible by any of the factors. In UM-SCV-1A cells, interleukin-10 (IL-10) and interleukin-13 (IL-13) caused an approximately 12-fold decrease in DNA synthesis in cells cultured for 72 h (P<0.001), while GM-CSF had no significant effect. TGFβ showed a significant inhibitory effect on deoxyuridine incorporation (P<0.001), which was 2.0- and 4.2-fold at 48 h and 72 h, respectively. TFGα showed a 1.2-fold inhibitory effect on DNA synthesis at 48 h (P<0.01) and a 1.5-fold inhibition at 72 h (P<0.05). Interferon γ (IFNγ) showed an inhibitory effect on DNA synthesis (1.3-fold; P<0.01). In UM-SCV-6 cells, both IL-10 and IL-13 showed inhibitory effects on deoxyuridine incorporation (1.3- and 1.4-fold at 48 h, respectively; P<0.001) that were even more pronounced at 72 h (2.4- and 2.5-fold respectively; P<0.001). IFNγ caused a 3.6-fold inhibition of DNA synthesis by UM-SCV-6 cells at 72 h (P<0.001). Both TFGβ and TNFα inhibited uridine incorporation (3.0- and 1.6-fold at 48 h, respectively; 2.7-fold at 72 h for both factors). GM-CSF inihibited DNA synthesis by UM-SCV-6 cells 1.3- 2.0-fold at 48 h and 72 h, respectively. In dose/response analyses, the effect of INFα on DNA synthesis was inhibitory in both cell lines at 48 h, while stimulatory effects were observed at 72 h. Electrophoretic analyses of DNA isolated from cells cultured in the presence or absence of different factors did not reveal DNA fragmentation. All cytokines, with the exception of IFNα, showed inhibitory effects on DNA synthesis by vulvar carcinoma cells. Of the factors studied, the recently described interleukins 10 and 13 showed potent inhibition of cell growth, encouraging further investigation on the molecular mechanisms of the observed inhibition. Apoptosis does not seem to be induced in the two vulvar carcinoma cell lines by any of the cytokines studied. Received: 26 March 1996 / Accepted: 5 December 1996     

DNA synthesis in the imaginal wing discs of the American bollwormHelicoverpa armigera (Hübner)

The effect of two insect growth regulators of plant origin viz. plumbagin and azadirachtin and the ecdysteroids 20-hydroxyecdysone, makisterone A and a phytoecdysteroid on DNA synthesis in imaginal wing discs of day 4 final instarHelicoverpa armigera larvae was studied. DNA synthesis increased with increase in time of incubation up to 8 h and decreased later without the addition of moulting hormone. Addition of 20-hydroxyecdysone supported long term acquisition of competence for DNA synthesis in the wing discs. Both DNA synthesis and protein content were drastically reduced in plumbagin and azadirachtin-treated insects. Underin vitro conditions, plumbagin had a more pronounced inhibitory effect than azadirachtin. All the ecdysteroids tested, viz. makisterone A, 20-hydroxyecdysone and the ecdysteroidal fraction from the silver fernCheilanthes farinosa enhanced DNA synthesis     

Mechanism of Action of Showdomycin

Among the syntheses of DNA, RNA and protein in Escherichia coli cells, the DNA synthesis was found to be preferentially inhibited at lower concentrations of showdomycin. At such lower concentrations of this antibiotic, serious decreases in the synthesis of deoxycytidine phosphates and in de novo synthesis of deoxythymidine phosphates were found in parallel with the decrease in the synthesis of DNA, although the syntheses of other pyrimidine nucleotides were not significantly diminished. The salvage synthesis of deoxythymidine phosphates was very resistant to this antibiotic. The inhibitory action of this antibiotic on DNA synthesis could be reversed by the concomitant addition of a thiol compound or a nucleoside. When a nucleoside was added after the completion of the inhibition by showdomycin, the recovery of the DNA synthesis from the inhibition was detected only after the recovery of the syntheses of pyrimidine ribotides, pyrimidine deoxyribotides and RNA have become distinct.     

Studies on the inhibition of bacterial macromolecule synthesis by roquefortine,a mycotoxin from Penicillium roqueforti

Summary The inhibitory effect of roquefortine, a secondary metabolite of Penicillium roqueforti, on bacterial protein, RNA and DNA synthesis was studied. Similar results were obtained in colorimetric measurements and in studying the incorporation of radioactive precursors. They show that RNA synthesis was most significantly affected by roquefortine. Inhibition of protein and DNA synthesis was less pronounced and might be a result of primary inhibition of RNA synthesis.Abbreviations RNA ribonucleic acid - DNA desoxyribonucleic acid - DMSO dimethylsulfoxide - Cpm counts per min - ATCC American Type Culture Collection     

Mechanism of Inhibition of DNA Synthesis by 1-β-D-Arabinofuranosyl-E-5-(2-Bromovinyl)uracil

The mechanism of the inhibitory action of 1-β-D -arabinofuranosyl-E-5-(2-bromovinyl) uracil triphosphate (BV-araUTP) on DNA synthesis by Escherichia coli DNA polymerase I Klenow fragment was studied. Acting as a chain terminator, BV-araUTP inhibited DNA synthesis by Klenow fragment more effectively than 2′, 3′-dideoxythymidine triphosphate (ddTTP). However, the incorporation sites of BV-araU monophosphate were restricted at consecutive dTMP sequence whereas ddTMP was incorporated at every dTMP site.     

Regulation of cell proliferation inhibitory and stimulatory factors diffused by 3T3 cultured cells

The growth rate of normal cells multiplied in vitro decreases as the cell density of the culture increases. Previous results suggested that this density-dependent inhibition of growth in nontransformed cells was due to the diffusion of growth inhibitory substances in the medium of dense cultures. In this paper, we demonstrate that dense cultures of 3T3 cells secrete inhibitory and stimulatory factors. Macromolecules of conditioned medium were fractionated on Biogel P150 and the different fractions were tested on quiescent cultures of 3T3 cells stimulated or not to proliferate by addition of alpha globulin. When target cells were not stimulated to proliferate by addition of exocrine growth factors, we observed the inhibitory activity of a large molecular weight inhibitor (IDF45) and the stimulatory activity of autocrine growth factors (fraction about 35 and 10 K molecular weight), on the incorporation of 14C inosine into nucleotide pool and RNA. However, DNA synthesis was significantly stimulated with fraction 10 K only. This discrepancy between the stimulation of RNA and DNA synthesis may be explained by the presence, simultaneously, of inhibitory and stimulatory factors in fraction 35 and 10 K molecular weight. The presence of inhibitory factor was demonstrated when the fractions were tested on target cells stimulated to proliferate by alpha globulin addition and labeled with 14C thymidine. In these conditions, the stimulatory activity of autocrine growth factors was not observable, and only the inhibitory activity on DNA synthesis of fractions 35 and 10 K appeared. It is tempting to assume that the regulation of in vitro cell proliferation is determined by the balance between these antagonist stimulatory and inhibitory autocrine growth factors.     

High temperature enhances cytotoxicity of mercury (HgCl2) on Hela S3 cells

The combined effect of mercury (HgCl₂) and high temperature on the growth and synthesis of nucleic acid and protein, and on the cell cycle of HeLa S₃ cells was investigated. The subsequent growth of the cells was dose-dependently inhibited by mercury at 37.2° and 41.2°C. The inhibitory effect of mercury on subsequent growth was enhanced at the higher temperature. IC₅₀ values for DNA and RNA synthesis but not protein synthesis, at 41.2°C, were significantly lower than those at 37.2°C (P<0.05,P<0.01, respectively). Flow cytometric analysis using synchronous cells indicated the possibility of blocking of cell cycle progression in the early part of S phase by the combined treatment. These results suggest that the cytotoxicity of mercury to cell growth was enhanced at the higher temperature and that this enhancement is related to the increased inhibitory effect of mercury on DNA and RNA synthesis and on the cell cycle at high temperatures.     

DNA synthesis in rat hepatocytes: Inhibition by a platelet factor and stimulation by an endogenous factor

Primary monolyer cultures of adult rat hepatocytes can be induced to undergo DNA synthesis in serum-free medium in the presence of insulin, glucagon, and epidermal growth factor (three factors). We have found that hepatocyte DNA synthesis is affected not only by an endogenous stimulant produced by the hepatocytes and released into the culture medium. Serum has a strong inhibitory effect on hepatocyte DNA synthesis. Partially purified human platelet extract (“platelet inhibitor”) inhibits the three-factor-induced DNA synthesis in a concenration-dependent manner. Pure βTGF at 0.5 ng/ml as well as HPLC-purified PDGF at 10 ng/ml completely inhibit the three-factor-induced DNA synthesis. Determination of the time required for the presence of the three factors and the platelet inhibitor to exert their effects indicated that the inhibition of DNA synthesis is caused not by competition of the platelet inhibitor with any of the three factors but through an independent pathway. Hepatocyte DNA synthesis is density-dependent and is greater if medium is not changed during the course of an experiment than if medium is changed daily. Hepatocyte-conditioned medium is also affective in stimulating DNA synthesis beyond the level induced by the three factors. These results suggest that an endogenous stimulant for hepatocyte DNA synthesis is produced by the hepatocytes themselves. Our studies demonstrate that hepatocyte DNA synthesis is subject to both stimulatory and inhibitory controls. Unlike the three factors, the endogenous stimulant can overcome the inhibition by the platelet inhibitor, suggesting the importance of these factors in the physiological control of hepatocyte DNA synthesis.     

Modulation by the src oncogene of the effect of inhibitory diffusible factor IDF45

Density-dependent inhibition of growth has been assumed to be under the control of inhibitory molecules diffusing from dense cell cultures. Growth inhibitory factors have been fractionated or purified from medium conditioned by different cell types. In the present work, it was shown that IDF45 (inhibitory factor diffusing from 3T3 cells) decreased DNA synthesis in chick embryo fibroblasts (CEF) and was an inhibitor of CEF growth; this inhibition was reversible. Since similitudes between oncogene products and growth factors have been observed, it was of interest to compare the inhibitory effect of IDF45 upon the stimulation of DNA synthesis induced either by serum or by pp60-src. CEF infected by Ny68 virus (a mutant of Rous sarcoma virus ts for the expression of transformation) were density-inhibited at 41 degrees C, but were stimulated at this temperature by addition of 1% serum. This stimulation was 94% inhibited by IDF45. The same Ny68-infected cells could also be stimulated by transfer to 37 degrees C, the permissive temperature (in the absence of serum). The stimulation of DNA synthesis by src expression was poorly inhibited by IDF45. From our results, it appears that oncogene expression in CEF induces a loss in their sensitivity to IDF45. This would explain why transformed cells escape DDI of growth.     

Changes induced by expression of the v-src gene in the regulation of cell proliferation. Hypothesis and preliminary results

Similarities between the mode of action of growth factors and the oncogene product (pp 60 src protein) of Rous Sarcoma virus have been described. However, a major difference is that addition of growth factors does not induce a malignant transformation of cells. The present work proposes a hypothesis concerning this difference. Various data suggest that density-dependent inhibition (DDI) of growth in non-transformed cells is due to the diffusion of growth inhibitory molecules. Inhibitory factors of 45 K (IDF 45) and 12 K have been fractionated. We assume that the stimulation of DNA synthesis induced by growth factor addition to dense quiescent cultures of non-transformed cells leads to an increase in the activity of autocrine inhibitory molecules in such a manner that the growth factor stimulatory effect is only transient, and cells re-enter the Go phase. On the contrary, the stimulation of DNA synthesis by v-src transformation would not be counterbalanced by inhibitory diffusing factors and cells would not enter Go phase. We present preliminary results which support this assumption. Dense quiescent cultures of chick embryo fibroblasts infected by Ny 68 virus (ts mutant for transformation of Rous Sarcoma virus) were stimulated to proliferate either by addition of growth factors in cultures maintained at 41 degrees C or by expression of transformation (by the cell transfer from 41 to 37 degrees C, the permissive temperature for expression of transformation). Stimulation of DNA synthesis by growth factors was totally inhibited by the inhibitory diffusing factors of 45 K (IDF45) whereas the stimulation of DNA synthesis produced by transformation was reproducibly not decreased by IDF45.     

Influence of sodium selenite and selenomethionine on DNA/RNA synthesis and BaP binding to spleen lymphocytes in culture

In the present study, an attempt was made to provide some information regarding the effects of organic and inorganic selenium compounds on DNA/RNA synthesis and benzo(a)pyrene uptake in cultured lymphocytes from mice spleen. It was clear from the results that there was a significant inhibition of DNA/RNA synthesis with increasing concentration of either form of selenium from 0.1ΜM to 1 mM in culture medium. However, when used at the same level as selenite with respect to selenium content, selenomethionine exerted more DNA/RNA synthesis inhibitory effect than selenite. Benzo(a)pyrene uptake by proliferating lymphocytes was also significantly reduced with increasing selenium concentration (0.1 ΜM to 1 mM). However, both forms of selenium at the same selenium concentration showed almost the same inhibitory effect on the cellular uptake of benzo(a)pyrene, which indicated that some factor(s) other than the DNA synthesis are also involved in the interaction between benzo(a)pyrene and cells. Involvement of the changes in the carcinogen metabolism and glutathione level has been discussed. Present studies show that organic selenium as a source of selenium is a more potent chemopreventive compared to the inorganic one. This information may have a useful therapeutic potential.     

DNA polymerase activity in phytohemagglutinin-stimulated and non-stimulated human lymphocytes

Requirements and optimal conditions have been studied for the activity of DNA polymerase from phytohemagglutinin-stimulated and non-stimulated human lymphocytes. Differences were found in thermal stability and inhibitory effect of KC1 and p-chloromercuribenzoate. The relationship was determined between DNA polymerase activity, cellular pools of dATP, dTTP and incorporation of deoxythymidine into DNA during transformation. The increase in polymerase activity was paralleled by a similar increase in the pools of dATP and dTTP. The enzyme activity and the pool sizes of both nucleotides reached a maximum simultaneously with the peak of deoxythymidine incorporation into DNA. Studies in which protein synthesis was limited by cycloheximide showed that both the DNA polymerase activity and the rise in the pool sizes of both nucleotides were abolished. This implies that the de novo synthesis is required for the enzymes involved.     

dam methylation and the initiation of DNA replication on oriC plasmids

Summary Plasmid DNA containing the replication origin of the Escherichia coli chromosome (oriC) has been shown to be inefficient as a template for DNA synthesis in vitro when isolated from dam mutants. here, we extend this study to hemimethylated oriC plasmids and to replication in dam-3 mutant enzyme extracts. The results show that: (1) hemimethylated oriC plasmids replicate with the same low efficiency as nonmethylated DNA; (2) DNA synthesis starts at oriC regardless of the methylated state of the template; (3) replication in dam-3 enzyme extracts is inefficient because this strain is deficient in DnaA protein; and (4) consistent with this observation, the copy number of the oriC plasmid pFH271 is reduced in the dam-3 mutant. However, we have found that low DnaA protein levels in dam-3 mutants are not sufficient to explain the reduced transformation efficiency of oriC plasmids. We suggest that there must exist in vivo inhibitory factors not present or present in low quantities in vitro which specifically recognize the hemimethylated or nonmethylated forms of the oric region.     

DNA replication initiation by 6-DMAP treatment in maturing oocytes and dividing embryos from marine invertebrates

6-dimethylaminopurine (6-DMAP), a potent protein kinase inhibitor, drives most cells into an interphasic stage. Experiments were undertaken with oocytes from three marine invertebrate species, i.e., Mytilus edulis, Spisula solidissima, and Strongylocentrotus droebachiensis, wherein oocytes were arrested at different phases of meiosis. 6-DMAP induced a continuous DNA synthesis in meiotic cells, whereas it allowed a single round of DNA replication in treated mitotic cells, regardless of species considered. The effects of 6-DMAP were accompanied in all cases by rephosphorylation on tyrosine of the p34^cdc2 homolog, the M-phase promoting factor (MPF) catalytic subunit. The fact that 6-DMAP overcomes the inhibitory control of replication during meiosis suggests that this process depends upon protein phosphorylation, while DNA synthesis regulation in mitotic cells relies on 6-DMAP-insensitive events. © 1996 Wiley-Liss, Inc.     

Inhibition of β-galactosidase synthesis inEscherichia coli after infection with different DNA and RNA phages

Infection ofEscherichia coli with T1, T2r⁺, T3 and T4 phages leads to an immediate inhibition of β-galactosidase synthesis. Similar results were obtained with the virulent mutant of phage lambda. The degree of inhibition of β-galactosidase synthesis depends on the time delay between the addition of the inducer and the phage particles, and on the amount of phage DNA, which has penetrated into the host cell. RNA phage MS2 exhibited no inhibitory effect on enzyme synthesis.     

Inorganic phosphate is necessary for the stimulation of DNA synthesis in swiss 3T3 cells by pure mitogenic agents

We compared the ability of dialysed fetal bovine serum and of combinations of purified growth-promoting factors such as insulin, epidermal growth factor (EGF), vasopressin, fibroblast-derived growth factor and antitubulin agents to stimulate DNA synthesis in 3T3 cells maintained in the absence or presence of inorganic phosphate (P_i). When DNA synthesis was stimulated by serum in the absence of P_i the level induced was 70% of that observed in P_i-containing medium. In contrast, combinations of growth-promoting factors in the absence of P_i stimulated less than 8% of the DNA synthesis which they induced in complete medium. Addition of as little as 50 μM P_i fully restored the ability of the factors to stimulate DNA synthesis. Cells stimulated by purified mitogens in the absence of P_i became blocked in early G1, and for up to 48 h the block was reversible by readdition of p_i. The effectiveness of dialysed serum to stimulate DNA in the absence of P_i suggest that dialysed serum might contain a component capable of supplying P_i to support DNA synthesis. Indeed, delipidization of serum by solvent extraction resulted in loss of ability to stimulate DNA synthesis in the absence of p_i, but delipidized serum stimulated DNA synthesis virtually, as well as dialysed serum in the presence of P_i. Previous conclusions suggesting that P_i is not essential for DNA synthesis appear to require re-evaluation.     

Inhibition of nucleic acids synthesis in Chlorella by 2,4-dinitrophenol a unique concentration effect on DNA synthesis

Nucleic acids and protein synthesis in synchronously growing Chlorella cells were inhibited by 2,4-dinitrophenol. RNA and protein synthesis decreased gradually from about 100% at 0.1 mM to almost 0% at 10 mM dinitrophenol. DNA synthesis was strongly inhibited at 0.5 mM but less at 1 mM concentration of the inhibitor. Beyond 1 mM the inhibitory effect increased again. A transient exposure to 0.5 and 10 mM dinitrophenol was fully reversible and cell division after the inhibition proceeded normally except for a slight delay.Abbreviation DNP 2,4-dinitrophenol     

THYMUS DERIVED INHIBITOR OF LYMPHOCYTE PROLIFERATION I. ISOLATION AND ASSESSMENT OF TISSUE SPECIFICITY

Preparations from bovine thymus tissue were analyzed for their inhibitory effects during in vitro lymphocyte proliferation. The results indicate that these preparations strongly inhibit DNA synthesis in stimulated human peripheral lymphocytes, bone marrow cells, thymocytes and lymphoblastoid cells. This inhibition was dose-dependent and not due to cytotoxicity of the preparations. No inhibition was found of the spontaneous proliferation of HeLa cells and human fibroblasts indicating that the inhibitory effect was specific for proliferating lymphocytes. Control preparations from bovine liver or kidney did not show any suppression in the test systems used.     

Inhibition by Retinoic Acid of Albumin and DNA Synthesis in Adult Rat Hepatocytes

RA down-regulated albumin and DNA synthesis of adult rat hepatocytes in a dose-dependent manner at dose ranges of 10^?9–10^?5 m. 10^?5 m of RA decreased albumin synthesis as well as growth factors EGF, aFGF, bFGF, and HGF-induced DNA synthesis to half of the control. These inhibitory effects of RA were not a consequence of the toxicity of RA to hepatocytes.