Determination Of DNA Synthesis Research Articles

Metformin: direct inhibition of rat ovarian theca-interstitial cell proliferation

To determine whether metformin has direct effects on ovarian theca-interstitial (T-I) cell proliferation through activation of adenosine monophosphate-activated protein kinase (AMPK). In vitro experimental study. Academic medical center laboratory. Immature Sprague-Dawley female rats. Ovarian T-I cells were isolated, purified, and cultured in the absence (control) or presence of insulin (1 μg/mL) with or without metformin or other activators/inhibitors of AMPK (AICAR, compound C). Proliferation assessed by determination of expression levels of proteins involved in cell cycle progression, cyclin D3, and cyclin-dependent kinase 4 (CDK4) with Western blot analysis, and determination of DNA synthesis with bromodeoxyuridine (BrdU) incorporation assay; activation of AMPK, Erk1/2, and S6K1 determined by Western blot analysis with the use of antibodies specific for the phosphorylated (activated) forms. Metformin inhibited insulin-induced ovarian T-I cell proliferation and the up-regulation of the cell cycle regulatory proteins, cyclin D3 and CDK4. Metformin independently activated AMPK in a dose-dependent manner. Treatment with metformin inhibited insulin-induced activation of Erk1/2 and S6K1. This effect was reversed with the addition of compound C, a known AMPK inhibitor. Metformin directly inhibits proliferation of ovarian T-I cells via an AMPK-dependent mechanism. These findings further validate the potential benefits of metformin in the treatment of conditions associated with hyperinsulinemia and excessive growth of ovarian T-I cells (such as polycystic ovary syndrome).

Statins Inhibit Growth of Human Theca-Interstitial Cells in PCOS and Non-PCOS Tissues Independently of Cholesterol Availability

Polycystic ovary syndrome (PCOS) is associated with ovarian enlargement, prominent theca-interstitial hyperplasia, and excessive androgen production. Recent clinical trials have demonstrated that statins, 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors, decrease androgen levels in women with PCOS. The present study evaluated the effect of statins on proliferation of human ovarian theca-interstitial cells. In vitro experiments were performed in the university research laboratory. Human theca-interstitial cells were isolated from ovaries of PCOS (n=4) and non-PCOS (n=4) patients. The cells were incubated for 48 h without additives (control) or with simvastatin (3-30 μm), mevastatin (3-30 μm), and/or the cell- and mitochondrion-permeable form of cholesterol (22-hydroxycholesterol; 10 μm). To determine whether the effects of statins could be affected by leukocytes, the experiment was carried out on cells not purified of leukocytes and cells purified using anti-CD-45 immunomagnetic beads. The effect of statins on proliferation was evaluated by determination of DNA synthesis using radiolabeled thymidine-incorporation assay and by quantification of viable cells using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenil)-2H-tetrazolium assay. Statins induced an inhibition of DNA synthesis in both the absence and the presence of 22-hydroxycholesterol; furthermore, 22-hydroxycholesterol alone also inhibited DNA synthesis. These effects of statins and 22-hydroxycholesterol were confirmed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenil)-2H-tetrazolium assay. Comparable inhibition of proliferation was observed in cells obtained from women with and without PCOS and in cell preparations treated and not treated with anti-CD-45 immunomagnetic beads. Statins inhibit proliferation of human theca-interstitial cells irrespective of the availability of cholesterol and independently of leukocytes both in normal and PCOS ovaries.

Identification of an Hexapeptide That Binds to a Surface Pocket in Cyclin A and Inhibits the Catalytic Activity of the Complex Cyclin-dependent Kinase 2-Cyclin A

The protein-protein complexes formed between different cyclins and cyclin-dependent kinases (CDKs) are central to cell cycle regulation. These complexes represent interesting points of chemical intervention for the development of antineoplastic molecules. Here we describe the identification of an all d-amino acid hexapeptide, termed NBI1, that inhibits the kinase activity of the cyclin-dependent kinase 2 (cdk2)-cyclin A complex through selective binding to cyclin A. The mechanism of inhibition is non-competitive for ATP and non-competitive for protein substrates. In contrast to the existing CDKs peptide inhibitors, the hexapeptide NBI1 interferes with the formation of the cdk2-cyclin A complex. Furthermore, a cell-permeable derivative of NBI1 induces apoptosis and inhibits proliferation of tumor cell lines. Thus, the NBI1-binding site on cyclin A may represent a new target site for the selective inhibition of activity cdk2-cyclin A complex.

Mevastatin inhibits proliferation of rat ovarian theca–interstitial cells by blocking the mitogen-activated protein kinase pathway

To evaluate mechanisms involved in mevastatin-induced inhibition of proliferation of ovarian theca-interstitial cells. In vitro study. Academic laboratory. Immature Sprague-Dawley female rats. Ovarian theca-interstitial cells were cultured without and with mevastatin in the presence and absence of serum, mevalonic acid, and/or insulin. Proliferation was assessed by determination of DNA synthesis by thymidine incorporation assay. Activation of extracellular signal-regulated kinase (Erk1/2) and of Akt/protein kinase B (PKB) was determined by ELISA. Mevastatin induced a concentration-dependent inhibition of theca-interstitial cell proliferation in the absence and in the presence of serum. Inhibitory effects of mevastatin were partly abrogated by mevalonic acid and by insulin. Mevastatin blocked basal and insulin-induced phosphorylation of ERK1/2. In contrast, mevastatin had no significant effect on either basal or insulin-induced phosphorylation of Akt/PKB. Mevastatin inhibits proliferation of theca-interstitial cells by a mechanism that involves depletion of mevalonic acid and selective inhibition of basal and insulin-induced activity of Erk1/2 pathway, but not Akt/PKB pathway. These effects of mevastatin may be a result of decreased isoprenylation of small GTPases.

Metabolic Stability of α-Methylated Polyamine Derivatives and Their Use as Substitutes for the Natural Polyamines

Metabolically stable polyamine derivatives may serve as useful surrogates for the natural polyamines in studies aimed to elucidate the functions of individual polyamines. Here we studied the metabolic stability of alpha-methylspermidine, alpha-methylspermine, and bis-alpha-methylspermine, which all have been reported to fulfill many of the putative physiological functions of the natural polyamines. In vivo studies were performed with the transgenic rats overexpressing spermidine/spermine N(1)-acetyltransferase. alpha-Methylspermidine effectively accumulated in the liver and did not appear to undergo any further metabolism. On the other hand, alpha-methylspermine was readily converted to alpha-methylspermidine and spermidine; similarly, bis-alpha-methylspermine was converted to alpha-methylspermidine to some extent, both conversions being inhibited by the polyamine oxidase inhibitor N(1), N(2)-bis(2,3-butadienyl)-1,4-butanediamine. Furthermore, we used recombinant polyamine oxidase, spermidine/spermine N(1)-acetyltransferase, and the recently discovered spermine oxidase in the kinetic studies. In vitro studies confirmed that methylation did not protect spermine analogs from degradation, whereas the spermidine analog was stable. Both alpha-methylspermidine and bis-alpha-methylspermine overcame the proliferative block of early liver regeneration in transgenic rats and reversed the cytostasis induced by an inhibition of ornithine decarboxylase in cultured fetal fibroblasts.

Mevastatin inhibits ovarian theca–interstitial cell proliferation and steroidogenesis

Statins reduce cardiovascular risks by improving hypercholesterolemia, reducing vascular smooth muscle proliferation, and ameliorating inflammation. Polycystic ovary syndrome (PCOS) is associated with increased cardiovascular risks and is characterized by ovarian theca-interstitial hyperplasia and hyperandrogenism. This study tested the hypothesis that mevastatin limits theca-interstitial proliferation and decreases steroidogenesis. In vitro study. Academic laboratory. None. Effects of mevastatin on cultured theca-interstitial cells. Proliferation was evaluated by determination of DNA synthesis using thymidine incorporation assay and by 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) assay. Production of P and T was determined by specific radioimmunoassays. Mevastatin induced a profound concentration-dependent inhibition of DNA synthesis. At the highest concentration (30 microM), mevastatin inhibited DNA synthesis by 92%. Similarly, in the MTT proliferation assay, mevastatin induced a concentration-dependent decrease in cell number. Mevastatin decreased production of P (by up to 49%) and T (by up to 52%); these effects remained significant when the effect on cell culture protein content was accounted for. Mevastatin inhibits proliferation of theca-interstitial cells; it also inhibits P and T production independently of the effects on cell growth. These findings provide a foundation for studies evaluating statins as potential therapeutic agents in the treatment of ovarian mesenchymal hyperplasia and hyperandrogenism characteristic of PCOS.

Proliferation of ovarian theca-interstitial cells is modulated by antioxidants and oxidative stress.

Maintenance of ovarian homeostasis requires precise regulation of proliferation of thecal- interstitial (T-I) cells. Recent evidence indicates that oxidative stress and antioxidants modulate proliferation of various tissues under both physiological and pathological conditions. This study evaluated the effects of oxidative stress and antioxidants on T-I proliferation. Rat T-I cells were cultured in serum-free medium and proliferation was assessed by determination of DNA synthesis using the thymidine incorporation assay, by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and by direct counting of steroidogenically active cells and steroidogenically inactive cells. Antioxidants and reactive oxygen scavengers induced a dose-dependent decrease of T-I proliferation. Vitamin E succinate was inhibitory at 10-100 micro mol/l, ebselen was inhibitory at 0.3-30 micro mol/l, and superoxide dismutase was inhibitory at 300-1000 IU/ml. In contrast, oxidative stress resulted in a biphasic effect. Modest oxidative stress induced by 1 mmol/l hypoxanthine and xanthine oxidase (3-30 micro U/ml) stimulated proliferation of T-I cells, while greater oxidative stress induced by xanthine oxidase (1 mU/ml) profoundly inhibited proliferation. Direct cell counting demonstrated comparable effects on steroidogenically active and inactive cells. Reactive oxygen species may play a role in the regulation of growth of ovarian mesenchyme. Under pathological conditions, such as those encountered in polycystic ovary syndrome, excessive oxidative stress and depletion of antioxidants may contribute to ovarian mesenchymal hyperplasia.

Genesis of hadacidin-induced cleft palate in hamster: morphogenesis, electron microscopy, and determination of DNA synthesis, cAMP, and enzyme acid phosphatase.

A morphological, electron microscopic, and biochemical study was undertaken to analyze the genesis of hadacidin-induced cleft palate in hamster fetuses. Gross and light microscopic observations indicated that hadacidin affected the growth of vertical palatal shelves to induce cleft palate. Electron microscopic observations showed that initial hadacidin-induced changes were seen in the mesenchymal cells. Within 12 hr of drug administration, the perinuclear space was swollen and a lysosomal response injury was evident in the mesenchymal cells. Subsequently, 24 hr after hadacidin treatment, lysosomes appeared in the epithelial cells; changes were also seen in the basal lamina which included separation of the lamina densa from the basal cells, duplication of lamina densa, and complete loss of basal lamina. Between 36 and 42 hr post-treatment, the cellular and basal lamina changes subsided, and the epithelium of vertical shelves underwent stratification. Biochemical determination of enzyme acid phosphatase indicated that the levels of enzyme activity in both the control and treated palatal tissues corresponded to the appearance of lysosomes. Measurement of cAMP levels suggested that the peak activity of cAMP corresponded to that of enzyme acid phosphatase and cell injury. The cAMP activity in hadacidin-injured cells, however, was significantly lower in comparison to that of the dying cells of control palates. Hadacidin treatment also affected DNA synthesis in the developing primordia of the palate. It was suggested that hadacidin injures the precursor cells of the palate prior to the appearance of the primordia, and subsequently affects their proliferative behavior, stunting the vertical growth of the palatal shelves and inducing a cleft palate.

Determination of DNA synthesis, estrogen receptors, and carcinoembryonic antigen in isolated cellular subpopulations of human breast cancer.

Primary breast adenocarcinomas obtained from ten patients were enzymatically digested using collagenase (1 mg/ml), hyaluronidase (1 mg/ml), elastase (0.1 mg/ml) and DNAse (0.2 mg/ml). The tumor cells were labeled with 3H-thymidine and, in some cases, with 3H-estradiol. The isolated cells were submitted successively to a Ficoll-Hypaque and a bovine serum albumin gradient, from which 12 fractions were obtained. In each fraction, several characteristics were determined: carcinoembryonic antigen (CEA), thymidine (dThd) incorporation, and estrogen receptors (ER). Three main cellular subpopulations were characterized: An intermediate density subpopulation (1.046-1.054 g/ml), in which the proliferating cells are concentrated. In this subpopulation a small number of CEA-positive cells are present, but ER containing cells are virtually absent. A high-density, small cell subpopulation that concentrates most of the ER-containing cells. This subpopulation lacks proliferating cells, but CEA-containing cells are abundant. A low-density subpopulation, lacking proliferating cells and with scarce ER-positive cells, although CEA-positive cells are frequent. These findings strongly suggest that proliferating cells lack ER.

Membrane Transport by Murine Lymphocytes

Abstract We have investigated membrane transport of thymidine and adenosine by bulk murine nonadherent spleen cells from animals treated with the lectin concanavalin A. Separation of cells from radioactive medium was achieved by means of the oil microfuge technique which permits incubation periods as short as 4 sec. We were able to distinguish between the membrane-linked function of transport and uptake which includes subsequent accumulation and metabolism of the transport solute. Previously we reported that cells derived from untreated mice could not be shown to translocate thymidine in a carrier-mediated fashion. In contrast, cells from concanavalin A-treated mice showed two membrane transport systems for thymidine, a high affinity system (Km = 160 μM) and one with low affinity (Km = 4 mM). The transport of thymidine conformed to the usual criteria for membrane carrier-linked function: increased uptake with time, substrate saturability and chemical specificity. In contrast to thymidine uptake, the transport of adenosine by cells from lectin-treated mice was similar to that shown by cells from untreated animals. Thus a specific membrane-linked activity is altered differentially in the course of mitogen-effected lymphocyte stimulation. We have found that determination of DNA synthesis by the standard method gave values which were as much as 700% too high, since the counts obtained by direct precipitation with TCA of cells incubated with radiolabeled thymidine exceed the cell-associated radiolabel obtained by the rapid sampling technique. With lectin-stimulated cells, the discrepancy observed was up to three times greater. Hence the validity of the standard assay for blastogenesis must be viewed with caution.

Autoradiography for determination of DNA synthesis in hamster bladder epithelium

Autoradiography for determination of DNA synthesis in hamster bladder epithelium

Formation of RNA in T4 phage-infected bacteria: Transcriptional regulation of early enzyme synthesis

The rates of synthesis were determined for those early mRNA species which code for early enzymes involved in phage-specific DNA synthesis. Evidence was found for a regulation mechanism which specifically restricts the transcription of early genes. The formation of early mRNA representing certain early genes was measured at different times after infection by phenotypic suppression by 5-fluorouracil of amber mutations in the early genes 1 (hydroxymethyl deoxycytidylate kinase), 42 (deoxycytidylate hydroxymethylase), and 56 (deoxycytidine triphosphatase), respectively. Suppression was measured by the determination of DNA synthesis and phage production. Since 5-fluorouracil suppression of amber mutations is effected by the incorporation of the analog into mRNA, the relative efficiency of suppression at different times after infection was interpreted to reflect the rate of mRNA formation at the time of fluorouracil addition. The regulation of early mRNA synthesis was also directly demonstrated by short-time labeling with uracil- 3H and fluorouracil- 3H after the infection of E. coli B with the mentioned phage mutants, which are DNA negative and produce no late mRNA. The uptake of 5-fluorouracil as an RNA precursor into the ribonucleotide pools of the infected cell was checked by electrophoretic analysis at different times after infection.