Augmented DNA Synthesis Research Articles

Accelerated DNA replication fork speed due to loss of R-loops in myelodysplastic syndromes with SF3B1 mutation

Myelodysplastic syndromes (MDS) with mutated SF3B1 gene present features including a favourable outcome distinct from MDS with mutations in other splicing factor genes SRSF2 or U2AF1. Molecular bases of these divergences are poorly understood. Here we find that SF3B1-mutated MDS show reduced R-loop formation predominating in gene bodies associated with intron retention reduction, not found in U2AF1- or SRSF2-mutated MDS. Compared to erythroblasts from SRSF2- or U2AF1-mutated patients, SF3B1-mutated erythroblasts exhibit augmented DNA synthesis, accelerated replication forks, and single-stranded DNA exposure upon differentiation. Importantly, histone deacetylase inhibition using vorinostat restores R-loop formation, slows down DNA replication forks and improves SF3B1-mutated erythroblast differentiation. In conclusion, loss of R-loops with associated DNA replication stress represents a hallmark of SF3B1-mutated MDS ineffective erythropoiesis, which could be used as a therapeutic target.

Translin restricts the growth of pubertal mammary epithelial cells estrogen-independently in mice

Translin, a ubiquitous RNA/DNA-binding protein that forms a hetero-octamer together with Translin-associated factor X (TRAX), possesses endoribonuclease activity and plays a physiological role in restricting the size and differentiation of mesenchymal precursor cells. However, the precise role of Translin in epithelial cells remains unclear. Here, we show evidence that Translin restricts the growth of pubertal mammary epithelial cells. The mammary epithelia of Translin-null females exhibited retarded growth before puberty, but highly enhanced growth and DNA synthesis with increased ramification after the onset of puberty. Primary cultures of Translin-null mammary epithelial cells showed augmented DNA synthesis in a ligand-independent and ligand-enhanced manner. Translin-null ovariectomized mice implanted with slow-release estrogen pellets showed enhanced length and ramification of the mammary glands. Mammary epithelial growth was also observed in ovariectomized Translin-null mice implanted with placebo pellets. Luciferase reporter assays using embryonic fibroblasts from Translin-null mice showed unaltered estrogen receptor α function. These results indicate that Translin plays a physiological role in restricting intrinsic growth, beyond mesenchymal cells, of pubertal mammary epithelial cells.

Src homology 2 (SH2) domain containing protein tyrosine phosphatase-1 (SHP-1) dephosphorylates VEGF Receptor-2 and attenuates endothelial DNA synthesis, but not migration

BackgroundVascular endothelial growth factor receptor-2 (VEGFR-2, KDR), a receptor tyrosine kinase, regulates mitogenic, chemotactic, hyperpermeability, and survival signals in vascular endothelial cells in response to its ligand vascular permeability factor/ vascular endothelial growth factor (VPF/VEGF). SHP-1 is a protein tyrosine phosphatase known to negatively regulate signaling from receptors such as EGF receptor, IL3 receptor, erythropoietin receptor and also KDR. However, the mechanism by which SHP-1 executes KDR dephosphorylation, the targeted tyrosine residue(s) of KDR and also overall downstream signaling or phenotypic change(s) caused, is not defined.ResultsHere, we have demonstrated that KDR and SHP-1 are constitutively associated and upon VEGF treatment, the phosphatase activity of SHP-1 is stimulated in a c-Src kinase dependent manner. Knockdown of SHP-1 by siRNA or inhibition of c-Src by an inhibitor, results in augmented DNA synthesis perhaps due to increased phosphorylation of at least three tyrosine residues of KDR 996, 1059 and 1175. On the other hand, neither tyrosine residue 951 of KDR nor VEGF-mediated migration is affected by modulation of SHP-1 function.ConclusionTaken together our results define the tyrosine residues of KDR that are regulated by SHP-1 and also elucidates a novel feed back loop where SHP-1 is activated upon VEGF treatment through c-Src and controls KDR induced DNA synthesis, eventually leading to controlled angiogenesis.

The SPARC-related Factor SMOC-2 Promotes Growth Factor-induced Cyclin D1 Expression and DNA Synthesis via Integrin-linked Kinase

Secreted modular calcium-binding protein-2 (SMOC-2) is a recently-identified SPARC-related protein of unknown function. In mRNA profiling experiments we, found that SMOC-2 expression was elevated in quiescent (G0) mouse fibroblasts and repressed after mitogenic stimulation with serum. The G0-specific expression of SMOC-2 was similar to that of platelet-derived growth factor-beta receptor (PDGFbetaR), a major mitogenic receptor. Therefore, we tested a possible role for SMOC-2 in growth factor-induced cell cycle progression. SMOC-2 overexpression augmented DNA synthesis induced by serum and fibroblast mitogens (including PDGF-BB and basic fibroblast growth factor). Conversely, SMOC-2 ablation by using small interfering RNA attenuated DNA synthesis in response to PDGF-BB and other growth factors. Mitogen-induced expression of cyclin D1 was attenuated in SMOC-2-ablated cells, and cyclin D1-overexpressing cells were resistant to inhibition of mitogenesis after SMOC-2 ablation. Therefore, cyclin D1 is limiting for G1 progression in SMOC-2-deficient cells. SMOC-2 ablation did not inhibit PDGF-induced PDGFbetaR autophosphorylation or PDGF-BB-dependent activation of mitogen-activated protein kinase and Akt kinases, suggesting that SMOC-2 is dispensable for growth factor receptor activation. However, integrin-linked kinase (ILK) activity was reduced in SMOC-2-ablated cells. Ectopic expression of hyperactive ILK corrected the defective mitogenic response of SMOC-2-deficient cells. Therefore, SMOC-2 contributes to cell cycle progression by maintaining ILK activity during G1. These results identify a novel role for SMOC-2 in cell cycle control.

Effects of Ca-Containing Phosphate Glasses on T Lymphocytes In Vitro

Calcium phosphate-based glasses (PG) are of interest as both scaffold and delivery materials for tissue rebuilding because of their chemical similarity to bone. Since it is essential that these materials exhibit local and systemic biocompatibility and do not adversely affect host tissues, the present study was undertaken to examine the effects of PG containing different amounts of Ca on human T lymphocytes in vitro. This was carried out by measuring the effects of extracts of the PG on the direct and mitogen-induced activation of T cells from human peripheral blood, as well as assessing CD4 and CD8, surface antigens which define T-helper and T-suppressor cells, respectively. The results showed that DNA synthesis by resting T lymphocytes was unaffected by all the PG. However, extracts of the PG containing 24 mol% of Ca caused a very marked inhibition of mitogen-induced T cell activation. This PG also reduced both the resting CD4+ and CD8+ T cells, as well as activated CD8+ cells. In contrast, high Ca-PG significantly augmented DNA synthesis by mitogen-activated T cells. These experiments show that PG containing differing levels of Ca can have pronounced and differential effects on the activation and function of T lymphocytes in vitro.

Induction of Cell Cycle Arrest and Apoptosis by Dephosphorylation of ERK, Akt, and Bcl-2 and Phosphorylation of JNK by LY293111 in Human Anaplastic Large Cell Lymphoma (ALCL).

The prognosis of patients with anaplastic large cell lymphoma (ALCL) treated with standard chemotherapy remains poor. Leukotriene B4 (LTB4) receptor has been reported to exhibit a growth-promoting activity in lymphoma, since it augments DNA synthesis and increases cell proliferation of lymphocytes. LY293111 (2-[2-propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-hydroxyphenoxy]-propoxy]-phenoxy] benzoic acid sodium salt) is a Leukotriene B4 receptor antagonist, which was found to be safe and tolerable in Phase I clinical trials. LY293111 inhibits pancreatic cancer cell growth, induces apoptosis, and reduces growth of colon cancer in vivo. In the present study, we investigated the potential therapeutic effects and mechanisms of action of LY293111 in an ALCL cell line. We utilized the Sup/M2 cell line derived from human anaplastic large cell lymphoma as a model. Dose-response studies demonstrated that the IC50 of LY293111 was 4μM after 5 days of treatment. LY293111 caused retardation of Sup/M2 cell entry into S phase in a dose-dependent fashion as measured by BrdU/propidium iodide flow cytometry. LY293111 at 2.5μM induced complete G1-S cell cycle arrest in cells synchronized by serum starvation. LY293111 upregulated p21 and p27 protein expression and reduced cyclin E mRNA and protein. Pre-treatment with LY293111 for 4 hours resulted in profound inhibition of serum-induced phosphorylation of ERK1/2, Akt, and Bcl-2. Concomitantly, phosphorylation of stress-activated kinase JNK dramatically increased following LY293111 treatment. LY293111 induced pronounced apoptosis of Sup/M2 cells and caspase inhibition by Benzyloxycarbonyl -Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK) abrogated this effect. LY293111 induced cleavage of the caspase-9, -3, PARP and XIAP, but had no effect on caspase-8, suggesting activation of the intrinsic apoptotic pathway. Accordingly, early loss of mitochondrial membrane potential was observed in a dose-dependent fashion. At 72 hrs, LY293111 decreased Bcl-2 expression levels. These findings provide the first evidence that LY293111 inhibits ALCL proliferation by arresting cells in G1 phase of the cell cycle, as a result of cyclin E downregulation and induction of cell-cycle inhibitory proteins p21 and p27. LY293111 induces apoptosis in Sup/M2 lymphoma cells, with activation of the mitochondrial apoptosis pathway, including cleavage of caspase 9, caspase 3, PARP, XIAP, and Bcl-2. Furthermore, LY293111 treatment markedly shifts signaling toward the JNK stress-related pathway and away from cytoprotective MEK/ERK and AKT signaling. These results suggest that LY293111 may have a utility in the management of aggressive non-Hodgkin's lymphomas.

The roles of IGF-I and IGFBP-3 in the regulation of proximal tubule, and renal cell carcinoma cell proliferation

Insulin-like growth factor I (IGF-I), a potent proximal tubule cell (PTC) mitogen, has been implicated in the progression of many human cancers. Our previous work on human renal tissues has suggested that IGF-I and several of its binding proteins (IGFBP-3 and -6) are up-regulated in clear cell renal cell carcinoma (RCC). To further elucidate the role of IGF-I and IGFBPs in RCC growth, immunohistochemistry, thymidine incorporation, and Western analysis were performed in primary cultures of normal PTC (priPTC) and clear-cell RCC (priRCC), as well as in SN12K1 cells (a cell line derived from metastatic RCC). By immunohistochemistry, IGFBP-3 and IGF-I were prominently expressed in SN12K1 cells, and weakly expressed in priPTC and priRCC. Incubation with 100 ng/mL IGF-I significantly augmented DNA synthesis by priPTC (mean +/- SD 120.7%+/- 19.7% of controls, P < 0.05), priRCC (238.7%+/- 279.9% of controls, P < 0.01), and SN12K1(120.0%+/- 22.9% of controls, P < 0.05). Neutralizing antibodies to IGF-I and IGF-I receptor significantly suppressed SN12K1 growth (81.9%+/- 13.5% of control, P < 0.01 and 87.4%+/- 16.2% of control, P < 0.05, respectively). Removal of endogenous IGFBP-3 by an anti-IGFBP-3 increased SN12K1 DNA synthesis (243.9%+/- 35.3% of control, P < 0.001), which was partially abrogated by coincubation with exogenous IGFBP-3 (135.97%+/- 5.9% of controls, P < 0.001). Using Western analysis, IGFBP-3 expression was enhanced in IGF-I-stimulated SN12K1 cells exposed to exogenous IGF-I. Coincubation with anti-IGFBP-3 further enhanced IGF-I-induced DNA synthesis. RCC cells express IGF-I and IGFBP-3, and are responsive to exogenous IGF-I stimulation. Moreover, in SN12K1 cells (derived from metastatic RCC), autocrine IGF-I and IGFBP-3 actions, respectively, stimulated and inhibited growth. These results suggest that IGF-I and IGFBP-3 may be potential candidates for therapeutic manipulation in patients with advanced RCC.

Amiloride kills malignant glioma cells independent of its inhibition of the sodium-hydrogen exchanger.

Previously, we demonstrated that malignant glioma cell lines have increased intracellular pH (pHi) as a result of increased activities of the type I sodium/hydrogen exchanger (NHE1). This alkalotic pHi of 7.2 to 7.4 is favorable for augmented glycolysis, DNA synthesis, and cell cycle progression. Conversely, reductions in pHi have been associated with reduced rates of proliferation in transformed cell types. The effects of reducing pHi directly and by NHE1 inhibition on human malignant glioma cells were systematically compared with those on primary rat astrocytes. Neither cariporide, nor direct acidification to pHi 6.9 altered the proliferative rates or viabilities of human U87 or U118 malignant glioma cell lines. However, amiloride significantly impaired glioma cell proliferation and viability while not affecting astrocytes at concentrations (500 microM) that exceeded its inhibition of NHE1 in glioma cells (IC50 = 17 microM). Preventing a reduction of pHi did not alter the drug's antiproliferative and cytotoxic effects on glioma cells. These findings indicated that amiloride's cytotoxic effects on glioma cells are independent of its ability to inhibit NHE1 or to reduce intracellular pHi. The amiloride derivative 2,4 dichlorobenzamil (DCB) inhibits the sodium-calcium exchanger (NCX) and was both antiproliferative and cytotoxic to glioma cells at low doses (20 microM). By contrast, KB-R7943 [(2-[2-[4-nitrobenzyloxy]phenyl]ethyl)-isothioureamethanesulfonate] preferentially blocks sodium-dependent calcium influx by NCX (reverse mode) and was nontoxic to glioma cells. It is proposed that DCB (20 microM) and amiloride (500 microM) impair calcium efflux by NCX, leading to elevations of intracellular calcium that initiate a morphologically necrotic, predominantly caspase-independent glioma cell death.

Fibrin monomer and fibrinopeptide B act additively to increase DNA synthesis in smooth muscle cells cultured from human saphenous vein

Purpose: We investigated the hypothesis that fibrinogen increased DNA synthesis (and cell proliferation) of smooth muscle cells (SMCs) cultured from human saphenous vein and that the increased DNA synthesis was attenuated when cells were cultured on polymeric collagen. Methods: SMCs were cultured from human saphenous vein on plastic, fibronectin, monomeric, and polymeric collagen. Fibrinogen products were prepared by proteolytic digestion. DNA synthesis was measured by bromodeoxyuridine incorporation into DNA, cell proliferation by cell counting, cyclic adenosine monophosphate by enzyme-linked immunosorbent assay, and fibrinopeptide B labeled with iodine 125 used for binding studies. Results: Fibrin monomer (0.003-0.1 μmol/L) stimulated a concentration-dependent increase in DNA synthesis of up to 10-fold, which could be inhibited by the peptide Bβ15-42. The stimulation of DNA synthesis was highest for cells cultured on plastic and lowest for cells cultured on type I collagen polymer. Much higher concentrations of fibrinogen (0.3-1 μmol/L) were required to effect similar increases in DNA synthesis. Fibrinogen had a particular effect to augment DNA synthesis, up to 14-fold, when cells were cultured on monomeric type I collagen. This augmented DNA synthesis was inhibited by a neutralizing antibody to urokinase-type plasminogen activator. Incubation of cells cultured on collagen monomer with fibrinogen resulted in production of fibrinopeptide B. Fibrinopeptide B (5 μmol/L) increased DNA synthesis by fourfold and had additive effects with fibrin monomer to increase DNA synthesis. Iodinated tyrosine fibrinopeptide B bound to SMCs (dissociation constant 0.6 μmol/L). Conclusion: Cultured human saphenous vein SMCs appear to have high-affinity receptors for fibrin monomer and fibrinopeptide B, the engagement of which stimulates DNA synthesis. These mechanisms may be pertinent to the association between fibrinogen and vein graft stenosis in vivo. (J Vasc Surg 2001;33:847-53.)

Effects of mechanical vibration on DNA and proteoglycan syntheses in cultured articular chondrocytes

The objective of this study was to determine the effects of mechanical vibration loading on DNA and proteoglycan syntheses in cultured rabbit articular chondrocytes. Chondrocyte culture plates were placed in a vibratory apparatus and subjected to a mechanical vibratory load at various frequencies and periods during culture. Mechanical vibration was applied at a sinusoidal waveform of 1.4 G-acceleration with frequencies of 200, 300, 400, 800, and 1600 Hz. 3H-thymidine and H235SO4 incorporation were used to detect radiolabeled DNA and proteoglycan syntheses, respectively. A frequency of 300 Hz showed a time-dependent augmentation of DNA synthesis and gave a maximal increase on day 3 with periodic vibration (8 h per day), and at 72 h or longer with continuous vibration. It also promoted proteoglycan synthesis in long-term cul-ture (from 3 to 15 days) by periodic vibration. However, frequencies above 400 Hz suppressed biosynthesis. These results suggest that mechanical vibration at certain frequencies may modulate the biosynthetic response of articular chondrocytes.

Complement (C5b-9) induces DNA synthesis in rat mesangial cells in vitro

The membrane attack complex C5b-9 causes injury in many forms of immune-mediated glomerular diseases characterized by mesangial cell (MC) proliferation and inhibiting C5b-9 decreases MC proliferation in vivo. Membrane insertion of sublytic quantities of the membrane attack complex of complement (C5b-9) is a potent stimulus for cell activation and the production of a variety of cytokines, growth factors, oxidants, matrix components, and other nephritogenic molecules. In vivo, a common response of MC to C5b-9--mediated injury is cell proliferation, an event closely linked to matrix expansion and sclerosis. In this study, we tested the hypothesis that C5b-9 might also serve as a mitogenic stimulus for MCs. Rat MCs in vitro were exposed anti-Thy1 antibody and 2% normal PVG serum (a complement source) to induce sublytic C5b-9 attack and DNA synthesis and cell number were measured. Control MCs were exposed to antibody and C6-deficient PVG serum. Sublytic C5b-9--induced injury to MCs is sufficient to induce DNA synthesis. Furthermore, C5b-9 augmented DNA synthesis induced by platelet-derived growth factor (PDGF) and 5% fetal calf serum. C5b-9--induced DNA synthesis was reduced by inhibiting reactive oxygen species (ROS) with superoxide dismutase and catalase, but not by neutralizing the mitogenic growth factors PDGF and basic fibroblast growth factor (bFGF). This study demonstrates that C5b-9 may directly increase DNA synthesis in cultured MCs, which are mediated in part by the release of ROS, and that C5b-9 also augments DNA synthesis induced in MCs by other known mitogens.

Mouse B1 cell line responds to lipopolysaccharide via membrane-bound CD14

The role of membrane-bound CD14 in the response of mouse B1 cell lines to lipopolysaccharide (LPS) was studied. The surface profile of mouse TH2.52 B cells was positive for CD5, IgM, B220, CD11b and F4/80, suggesting that TH2.52 cells carried the typical phenotype of B1 cells. Furthermore, TH2.52 B1 cells were found to express membrane-bound CD14, which plays a critical role in LPS recognition. TH2.52 B1 cells responded to a very low concentration of LPS and exhibited: (i) augmentation of DNA synthesis; (ii) activation of nuclear factor (NF)-kappaB; and (iii) phosphorylation of extracellular signal regulated kinase 1/2 (Erk1/2). They were markedly inhibited by anti-CD14 antibody. Therefore, the expression of membrane-bound CD14 was suggested to provide high sensitivity to LPS for TH2.52 B1 cells.

Dual Effect of Adenosine on Vascular Smooth Muscle [3H]-Thymidine DNA Labeling: Receptor-Mediated Modulation of DNA Synthesis and Inhibition of Thymidine Uptake

This study examined the contribution of cAMP signaling to the modulation of vascular smooth muscle cell (VSMC) proliferation by adenosine. At a concentration of 1 mM, adenosine inhibited [³H]-thymidine uptake, measured as the initial rate of isotope influx, by 10-fold. Diminution of [³H]-thymidine uptake by adenosine was independent of the presence of A₁- and A₂-receptor antagonists, indicating that adenosine competes with thymidine for plasma membrane transporter-binding sites. Considering these results, in order to estimate [³H]-thymidine DNA labeling, VSMCs were preincubated with adenosine for 48 h, and adenosine was then omitted during the subsequent 2 h of incubation in [³H]-thymidine-containing medium. In serum-depleted VSMCs, preincubation with 100 µM or 1,000 µM adenosine augmented DNA synthesis by approximately 6- and 3-fold, respectively, whereas the increment of DNA synthesis triggered by serum was decreased in the presence of adenosine by 20–30%. Both cAMP production and inhibition of DNA synthesis by adenosine in serum-supplied cells were independent of the presence of the A₁-antagonist 1,2-dipropyl-8-cyclopentylxanthine (DPCPX), but were abolished by the A₂-antagonist 1,3-dimethyl-7-propylxanthine (DMPX). In contrast, the activation of DNA synthesis in serum-depleted cells by adenosine was decreased in the presence of DPCPX and DMPX by approximately 30 and 40%, respectively. Both in serum-supplied and -depleted VSMCs, dose-dependent elevation of cAMP production with an adenylate cyclase activator, forskolin, reduced DNA synthesis by up to 40–60%. Thus, our results show that in addition to suppressing thymidine uptake, adenosine depresses the DNA synthesis triggered by serum-derived growth factors and stimulates DNA synthesis in serum-depleted cells. These data also suggest that the inhibition of DNA synthesis is mediated by cAMP production where the activation of DNA synthesis is independent of cAMP signaling.

Physiological concentrations of insulin augment pancreatic cancer cell proliferation and glucose utilization by activating MAP kinase, PI3 kinase and enhancing GLUT-1 expression.

Pancreatic carcinoma is characterized by poor prognosis and lack of response to conventional therapy for reasons that are not clear. Because of the structural relationship between the exocrine and endocrine pancreas and high concentrations of islet hormones bathing pancreatic tissue, we hypothesized that pancreatic cancer cell proliferation and glucose utilization are regulated by pancreatic islet hormones, particularly insulin. Based on this, the effect of islet hormones on pancreatic cancer cells in vitro was investigated. Five pancreatic cancer cell lines, CD11, CD18, HPAF, PANC-1, and MiaPaCa2 were used to investigate the effect of islet hormones on cell proliferation, glucose utilization, and GLUT-1 expression. Insulin, but not somatostatin and glucagon, induced pancreatic cancer cell growth in a concentration- and time-dependent manner. At concentrations within the range of those in the intrapancreatic vasculature, insulin (10(-10)-10(-8) mol/L) markedly increased [3H]-thymidine incorporation. Insulin significantly enhanced glucose utilization of pancreatic cancer cells before it enhanced cell proliferation. The MAPK kinase inhibitor PD 098059 abolished insulin-stimulated DNA synthesis and partially reduced insulin-stimulated glucose uptake. In contrast, the PI3 kinase inhibitor wortmannin substantially inhibited insulin-induced glucose uptake and partially blocked thymidine incorporation. Furthermore, after 24-hour treatment with insulin, GLUT-I expression in pancreatic cancer cells was markedly increased, indicating that insulin enhances glucose utilization partly through increasing glucose transport. These findings suggest that insulin stimulates proliferation and glucose utilization in pancreatic cancer cells by two distinct pathways. Insulin augments DNA synthesis mainly by MAP kinase activation and glucose uptake mainly by PI3 kinase activation and enhancement of GLUT-I expression. High intrapancreatic concentrations of insulin are likely to play an important role in stimulating pancreatic cancer growth indirectly by increasing substrate availability as well as by direct action as a trophic factor.

Physical interaction between CDK9 and B-Myb results in suppression of B-Myb gene autoregulation.

B-Myb is a transcription factor belonging to the myb family, whose activity has been associated with augmented DNA synthesis and cell cycle progression. We showed recently that B-Myb autoregulates its own expression through promoter transactivation. We report in this study that CDK9, the cyclin T associated kinase, which phosphorylates and activates RNA-Polymerase II, suppresses B-Myb autoregulation through direct interaction with the carboxyl-terminus of the B-Myb protein. Down-regulation of the transactivating ability of B-Myb is independent of the kinase activity of CDK9, because a kinase deficient mutant (dn-CDK9) also represses B-myb gene autoregulation. Overexpression of CDK9 did not result in suppression of p53-dependent transactivation or inhibition of the basal activity of the promoters tested so far, demonstrating that CDK9 is a B-Myb-specific repressor. Rather, transfection of the dominant negative dn-CDK9 construct inhibited the basal activity of the reporter genes, confirming an essential role for CDK9 in gene transcription. In addition, Cyclin T1 restores B-Myb transactivating activity when co-transfected along with CDK9, suggesting that the down-regulatory effect observed on B-Myb is specifically due to CDK9 alone. Thus, our data suggest that CDK9 is involved in the negative regulation of activated transcription mediated by certain transcription factors, such as B-Myb. This may indicate the existence of a feedback loop, mediated by the different activities of CDK9, which links basal with activated transcription.

Modulation of T-lymphocyte proliferation by exogenous natural ceramides and sphingosylphosphorylcholine.

Sphingolipids such as ceramide and sphingosine are abundantly present in the stratum corneum of epidermis. In atopic stratum corneum, sphingosylphosphorylcholine (SPC) is present in association with a reduction in the amount of ceramides. We have previously shown that the cellular kinetics of T cells are affected by exogenous addition of sphingosine and synthetic ceramides, raising the possibility that sphingolipids diffusing from the stratum corneum modulate skin-infiltrating T cells. By using two natural ceramides and murine T cells, this study further clarified the conditions under which exogenous ceramides enhance the proliferation of T cells. KLH-specific T cell clones 28-4 and 24-2 proliferated in response to natural ceramides when cultured for 44-48 h in the presence of concanavalin A at 1 microg per ml. Elongation of culture periods adversely inhibited the T cell proliferation, suggesting the existence of an optimal exposure time. Augmentation of DNA synthesis by natural ceramides was more pronounced in tumor necrosis factor alpha (TNFalpha)-sensitive 28-4 cells than in less sensitive 24-2 cells, and TNFalpha-induced proliferation of 28-4 cells was suppressed by the concomitant addition of natural ceramides. Similar to ceramides, SPC augmented the proliferation of resting spleen cells. Our study suggests that ceramide modulation of T cell proliferation depends on the TNFalpha sensitivity and activation level of T cells and that SPC also has a mitogenic potential for T cells.

Sequential morphological and biological changes in the glandular stomach induced by oral administration of catechol to male F344 rats.

Histogenesis and mechanisms of catechol-induced rat glandular stomach carcinogenesis were investigated in male F344 rats. Groups of 5 or 6 rats were treated with dietary catechol at doses of 1, 0.5, 0.1, and 0.01% for 12 hr or for 1, 2, 3, or 7 days or at a dose of 0.8% for 1, 2, 4, 12, and 24 wk; rats were then euthanatized. The initial morphological changes were edema of the gastric wall, inflammatory-cell infiltration, erosion in the pyloric region close to the duodenum, and considerable increase in apoptosis at 12 hr; later, changes included augmented DNA synthesis and cell proliferation, as evaluated by bromodeoxyuridine labeling index and thickness of mucosa, respectively, on day 1. Downward hyperplasia due to excess regeneration appeared at edges of ulceration at week 2. This lesion disappeared, and then submucosal hyperplasia appeared in the course of adenoma development. Only slight expression of c-myc or c-fos was apparent after 30-min oral administration or 1-, 3-, and 6-hr oral administration of catechol. No increase in lipid peroxide levels was evident in gastric epithelium fed catechol for 1 wk. The amount of catechol distributed in the glandular stomach and forestomach epithelium, which is not a target for carcinogenesis, did not differ 1, 3, 6, and 24 hr after a single intragastric dose of 75 mg/kg body weight. Amounts of catechol bound to tissue protein were also not specifically high in the glandular stomach. These results indicate that regenerative cell proliferation due to toxicity plays an important role in catechol-induced glandular stomach carcinogenesis. Protein binding and free radicals may not be largely responsible for the toxicity.

Low-density lipoprotein-induced formation of nitric oxide by cultured vascular smooth muscle cells of the rat.

There is evidence that low-density lipoprotein (LDL) plays a crucial role in atherogenesis. On the cellular level, LDL has been shown to activate a number of mechanisms involved in atherogenesis and vasoconstriction. Local immoderate vasoconstriction is physiologically antagonized by nitric oxide, which is released from the endothelium. To find out whether LDL also influences the synthesis of nitric oxide in vascular smooth muscle cells, both the conversion of arginine to citrulline and the production of nitrite were determined as a measure of nitric oxide formation. After incubation of rat vascular smooth muscle cells with native LDL (25 micrograms mL-1) for 24 h, the production of both L-[14C]-citrulline [39600 (3600) cpm mg-1 cell protein] and nitric oxide [2.95 (0.56) mumol L-1] were about twice and 1.5-fold the amount of the corresponding values in untreated cells (mean +/- SD, P < 0.05, n = 4). Oxidized LDL was less effective than the native form. The presence of the arginine analogue NG-methyl-L-arginine reduced citrulline production dose-dependently but augmented DNA synthesis, both induced by LDL. In addition, the lipoprotein caused a 1.6-fold increase in cyclic GMP production following a 24-h incubation [control = 10.9 (3.8) pmol mg-1 cell protein, P = 0.016]. The results suggest that native LDL might partly impair its atherogenic potential on the vasculature by stimulating the production by smooth muscle cells of both nitric oxide and cyclic GMP.

Role of CD44 in the reaction of vascular smooth muscle cells to arterial wall injury.

CD44, the principal receptor for hyaluronic acid, is a widely distributed cell surface proteoglycan involved in cellular activation, proliferation, and migration. These processes are also central to the vascular smooth muscle cell's response to arterial wall injury. We evaluated the expression of CD44 and its isoform, CD44-V6, on vascular smooth muscle cells in vitro and in vivo and assessed the role of CD44 in DNA synthesis. Cultured vascular smooth muscle cells expressed CD44 and CD44-V6 at levels equal to or higher than those of the beta 1 and beta 2 integrins. In a rat carotid artery balloon injury model, CD44 and CD44-V6 mRNAs were unregulated in vascular smooth muscle cells after injury, and CD44 protein expression was greatest at the luminal edge of the growing neointima. CD44-expressing smooth muscle cells proliferated actively, and hyaluronic acid expression increased after injury in a temporal pattern similar to that of CD44. Through binding to hyaluronic acid, CD44 augmented DNA synthesis in cultured human and rat smooth muscle cells by 48 +/- 7.8 and 100 +/- 12.5%, respectively, an effect inhibited by an anti-CD44 antibody that blocked hyaluronate binding. These observations support a role for CD44 in the reaction of vascular smooth muscle cells to arterial wall injury.

Stimulation of macrophage DNA synthesis by polyanionic substances through binding to the macrophage scavenger receptor.

We previously demonstrated that ligands of macrophage scavenger receptors such as acetylated low density lipoprotein (LDL), oxidized LDL and advanced glycation-end products (AGE) of the Maillard reaction induce the growth of peritoneal exudate macrophages, and that the activity of AGE is inhibited by the presence of an antibody for granulocyte/macrophage colony-stimulating factor (GM-CSF). To evaluate the suggested role of the scavenger receptor in the induction of macrophage growth, we compared the effect of various polyanionic compounds which were reported to either have or not to have competent activity for the binding of acetylated LDL to scavenger receptors on macrophage DNA synthesis. Among the polyanions exhibiting such activity, polyguanilic acid (poly G) and dextran sulfate strongly augmented macrophage DNA synthesis, although they did not increase macrophage cell number. On the other hand, polyanions which are not ligands for the scavenger receptors did not show a significant augmenting effect, suggesting that the binding of polyanions to the scavenger receptor is important but not, by itself, sufficient. The augmentation of DNA synthesis in macrophages cultured with dextran sulfate or poly G was inhibited by the co-presence of anti-GM-CSF antibody, suggesting that the reaction is mediated by GM-CSF. However, dextran sulfate did not augment the production of GM-CSF in macrophages. Therefore, GM-CSF spontaneously present in macrophages might be a prerequisite for the induction of DNA synthesis.