Lipid peroxide-induced redox imbalance differentially mediates CaCo-2 cell proliferation and growth arrest

Dietary oxidants like lipid hydroperoxides (LOOH) can perturb cellular glutathione/glutathione disulphide (GSH/GSSG) status and disrupt mucosal turnover. This study examines the effect of LOOH on GSH/GSSG balance and phase transitions in the human colon cancer CaCo-2 cell. LOOH at 1 or 5 micro m were noncytotoxic, but disrupted cellular GSH/GSSG and stimulated proliferative activity at 6 h that paralleled increases in ornithine decarboxylase activity, thymidine incorporation, expression of cyclin D1/cyclin-dependent kinase 4, phosphorylation of retinoblastoma protein, and cell progression from G0/G1 to S. At 24 h, LOOH-induced sustained GSH/GSSG imbalance mediated growth arrest at G0/G1 that correlated with suppression of proliferative activity and enhanced oxidative DNA damage. LOOH-induced cell transitions were effectively blocked by N-acetylcysteine. Collectively, the study shows that subtoxic LOOH levels induce CaCo-2 GSH/GSSG imbalance that elicits time-dependent cell proliferation followed by growth arrest. These results provide insights into the mechanism of hydroperoxide-induced disruption of mucosal turnover with implications for understanding oxidant-mediated genesis of gut pathology.     

Human placental Hofbauer cells promote mitogen induced T cell proliferation

Hofbauer cells are a major cell type of the human placental villous core particularly numerous at the beginning of pregnancy. In the present study we have investigated whether or not Hofbauer cells could subserve the function of accessory cells for colony formation by phytohemagglutinin stimulated allogeneic T cells. Results showed that Hofbauer cells are capable to play an accessory role on T cell proliferation probably due to the release of interleukin 1-like soluble factors.     

Transcytosis of iota-toxin across polarized CaCo-2 cells

Iota-toxin from Clostridium perfringens type E is a binary toxin consisting of two independent proteins, an enzymatic Ia and binding Ib component. Ia catalyses ADP-ribosylation of actin monomers, thus disrupting the actin cytoskeleton. In this report, we show that Ia plus Ib applied apically or basolaterally induce a rapid decrease in the transepithelial resistance (TER) of CaCo-2 cell monolayers and disorganization of actin filaments as well as the tight and adherens junctions. Ib alone, on the apical or basolateral side, slowly decreased the TER without affecting the actin cytoskeleton, possibly via pore formation. Interestingly, the two iota-toxin components inoculated separately on each cell surface induced cytopathic effects and a TER decrease. Anti-Ib sera, raised against the whole molecule or the Ia docking domain and applied to the opposite cell side versus Ib, neutralized the TER decrease. In addition, radioactive Ib incubated in the basolateral compartment was detected on the apical side by selective cell surface biotinylation. This argues for a transcytotic routing of Ib to mediate internalization of Ia from the opposite cell surface. Bafilomycin A1 also prevented the cytopathic effects of Ia and Ib applied separately to each cell side, possibly by blocking translocation of Ia into the cytosol and/or the intracellular transport of Ib. Ib is either routed into the cell independently of Ia, trans-cytosed and permanently exposed on the opposite cell surface or continuously recycled between an endosomal compartment and the cell surface.     

On the cysteine and cystine content of proteins

Summary Analysis of published data on the cysteine and half-cystine content of proteins indicates that most intracellular proteins may be classified as sulfhydryl proteins (those containing cysteine but little or no half-cystine) and that such sulf-hydryl proteins have a low cysteine content. The mean cysteine content found for 32 intracellular mammalian proteins was 1.6 % and intracellular proteins of many bacteria have similar or lower values. Extracellular mammalian proteins are primarily disulfide proteins (those containing half-cystine but little or no cysteine) and have a high half-cystine content, the mean value found for some 34 extracellular mammalian proteins being 4.1 %. This is contrasted with many of the extracellular proteins from facultative bacteria which are cyst(e)ine-free proteins, being lacking in both cysteine and half-cystine. These and related observations are interpreted in terms of the evolution of life in a reducing atmosphere and the subsequent transition to an oxidizing environment. It is suggested that disulfide proteins evolved primarily after the accumulation of oxygen in the atmosphere.     

The intracellular ratio of cysteine and cystine in various tissues

1. The cysteine-cystine ratio was measured in rat kidney cortex, diaphragm, jejunum, liver and brain. 2. This ratio was determined by incubating these tissues in buffer containing [(35)S]cystine and then homogenizing the tissue in a buffered solution of N-ethylmaleimide. The products of this reaction were separated by high-voltage electrophoresis and the radioactivity in the cystine and 2-(l-2'-amino-2'-carboxyethylthio)-N-ethylsuccinimide regions was determined. 3. In these tissues cyst(e)ine was mainly present in the reduced form. 4. After incubation of [(35)S]cystine with rat jejunal segments it was found that 36% of the cystine in the medium has been reduced. 5. Anaerobiosis, Na(+)-free media, glucose and high concentrations of cystine and lysine were found not to affect significantly the cysteine-cystine ratio in rat kidney-cortex slices.     

Effect of eicosapentaenoic acid on triacylglycerol transport in CaCo-2 cells

The human intestinal cell line, CaCo-2, was used to study the effect of the n-3 fatty acid, eicosapentaenoic acid, on triacylglycerol secretion. In cells incubated with 250 microM eicosapentaenoic acid, the incorporation of [3H]glycerol into triacylglycerols secreted into the medium was decreased by 58% compared to cells incubated with 250 microM oleic acid. The incorporation of [3H]glycerol into cellular triacylglycerols was decreased 32% in cells incubated with eicosapentaenoic acid. In cells preincubated with [3H]glycerol to label existing triacylglycerols, the rates of secretion of preformed triacylglycerols were similar in response to the addition of either fatty acid. Initial uptake rates of the n-3 fatty acid were higher than for oleic acid. Both eicosapentaenoic acid and oleic acid were minimally oxidized to CO2. Oleic acid was predominantly incorporated into cellular triacylglycerols (62% vs. 47%), whereas more eicosapentaenoic acid was incorporated into cellular phospholipids (46% vs. 30%). Phospholipids of microsomes prepared from cells incubated with eicosapentaenoic acid were enriched in this fatty acid. The rate of synthesis of triacylglycerol and diacylglycerol acyltransferase activities were significantly less in microsomes prepared from cells incubated with eicosapentaenoic acid. Triacylglycerol mass secreted by CaCo-2 cells incubated with either fatty acid was similar. In CaCo-2 cells, eicosapentaenoic acid decreases the synthesis and secretion of newly synthesized triacylglycerol without decreasing the secretion of triacylglycerol mass. Modification of microsomal membrane phospholipid fatty acid composition is associated with a decrease in microsomal triacylglycerol synthesis and diacylglycerol acyltransferase activities.     

Phosphodiesterase in human colon carcinoma cell line CaCo-2 in culture

In a series of in vitro experiments we characterised the relationship between DNA distribution in the G1, S and G2/M phases of cell cycle and PDE and GST activity in CaCo-2 cells. The DNA distribution in CaCo-2 cells, was assessed by flow cytometry, with fluorescent dyes at different time points of culture. The exponential increase in cell number continued until day 10 when there was cell saturation. The effect of medium replacement on PDE activity was assayed in the first 10 h after medium replacement. The 6^th hour is the time at which PDE activity was found to be highest. We have assayed the PDE enzyme with cGMP and cAMP as substrates. Only cAMP was consumed from this enzyme. We found a very close correlation between the DNA distribution in the various phases of the cell cycle and the PDE activity. PDE activity was very high during the active replication phase, whereas GST activity was high after confluency.     

Morphometry of nuclear and nucleolar structures in a CaCo-2 cell line

The number of the nucleoli in a CaCo-2 cell nucleus does not generally depend on the quantity of DNA in the nucleus, but nucleolar DNA content is directly proportional to total nuclear DNA. However, in multinucleolar cells (three or more nucleoli), the nucleolar DNA content increases after 96 h incubation in culture without concomitant quantitative changes in nuclear DNA. The percentage of multinucleolar cells and the average number of nucleoli per nucleus increase with increasing incubation time. After 72 and 96 h in culture, multinucleolar cells show distinctive morphologies. The ratio of the sum of nucleolar perimeters to the nuclear perimeter increases linearly when the number of nucleoli in a nucleus increases, but there is no concomitant increase in total nucleolar area or DNA content, except in the 72 and 96 h populations. When the number of nucleoli in CaCo-2 cells increases after 48 and 60 h in culture, the amount of DNA per nucleolus decreases.     

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

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

Regulation of cholesterol esterification by micellar cholesterol in CaCo-2 cells

The regulation of acylcoenzyme A:cholesterol acyltransferase (ACAT) activity by cholesterol was studied in an established enterocyte cell line. CaCo-2 cells were grown in culture to confluency and dome formation. They were characterized morphologically by light and transmission electron microscopy. During the culture period, ACAT activity remained stable while the activities of the brush border enzymes sucrase and alkaline phosphatase progressively increased with time and plateaued 12 days after plating. As determined by the rate of incorporation of oleic acid into the individual lipid classes, the rate of triglyceride synthesis was twice that of phospholipid and 15 times that of cholesteryl ester synthesis in these cells. Incubating CaCo-2 cells with cholesterol solubilized in taurocholate micelles resulted in a significant increase in ACAT activity (149 +/- 5 pmol/dish per 2 hr vs. 366 +/- 5, (P less than 0.001) without changing the rates of triglyceride or phospholipid synthesis. The stimulation of ACAT activity by micellar cholesterol was rapid, occurring within 5 min and reaching a maximal effect by 2 hr. The regulation of ACAT activity by cholesterol was directly dependent upon the concentration of cholesterol solubilized in the micelle and was independent of protein synthesis. Incubating CaCo-2 cells with micellar cholesterol did not increase the esterification of, nor did the cholesterol enter the pool of, newly synthesized or performed cholesterol within 2 hr. The micellar cholesterol that was taken up by the cells was esterified within 5 min after starting the incubation. Progesterone, a known ACAT inhibitor, significantly decreased the rate of esterification of intracellular micellar cholesterol proving that the cholesterol taken up by CaCo-2 cells was indeed entering the ACAT pool. Despite increasing amounts of unesterified cholesterol entering the cells via micelles, the percent of cholesterol that was esterified at any one time remained constant at 1%. The results suggest that ACAT activity in CaCo-2 cells is stimulated by cholesterol delivered to the cells by way of taurocholate micelles. The rapid entry of this sterol into the ACAT substrate pool suggests that ACAT activity in CaCo-2 cells is regulated by the expansion of the cholesterol substrate pool that is being utilized by an unsaturated ACAT enzyme.     

CircTHBS1 targeting miR-211/CCND2 pathway to promote cell proliferation and migration potential in primary cystitis glandularis cells

The pathogenesis of cystitis glandular (CG) is unclear, but it is generally considered to be a neoplastic lesion of urothelial hyperplasia formed by long-term chronic stimulation. There is growing evidence that circRNAs play important roles in a variety of cellular processes. However, there are few reports on the role and molecular mechanism of circRNA in CG. In the present study, we first isolated primary cells from CG tissues and adjacent normal tissues. Further experiments showed that CircTHBS1 was up-regulated in primary CG cells (pCGs). The results of CCK-8 showed that the overexpression of CircTHBS1 promoted the viability of pCGs, while the deletion of CircTHBS1 reduced the cell viability. Knocking out CircTHBS1 also inhibited the migration of pCGs. In addition, we demonstrated that CircTHBS1 played a role in the adsorption of miR-211 by “sponge” in pCG. In turn, miR-211 can directly target CYCLIN D2 (CCND2) 3′UTR to perform its function. Finally, we confirmed the role and mechanism of CircTHBS1/miR-211/CCND2 regulation axis in pCGs. In summary, our study is the first to reveal the role and underlying mechanism of CircTHBS1 in CG, providing a potential biomarker and therapeutic target for human CG.     

SWI/SNF chromatin remodeling enzyme ATPases promote cell proliferation in normal mammary epithelial cells

The ATPase subunits of the SWI/SNF chromatin remodeling enzymes, Brahma (BRM) and Brahma‐related gene 1 (BRG1), can induce cell cycle arrest in BRM and BRG1 deficient tumor cell lines, and mice heterozygous for Brg1 are pre‐disposed to breast tumors, implicating loss of BRG1 as a mechanism for unregulated cell proliferation. To test the hypothesis that loss of BRG1 can contribute to breast cancer, we utilized RNA interference to reduce the amounts of BRM or BRG1 protein in the nonmalignant mammary epithelial cell line, MCF‐10A. When grown in reconstituted basement membrane (rBM), these cells develop into acini that resemble the lobes of normal breast tissue. Contrary to expectations, knockdown of either BRM or BRG1 resulted in an inhibition of cell proliferation in monolayer cultures. This inhibition was strikingly enhanced in three‐dimensional rBM culture, although some BRM‐depleted cells were later able to resume proliferation. Cells did not arrest in any specific stage of the cell cycle; instead, the cell cycle length increased by approximately 50%. Thus, SWI/SNF ATPases promote cell cycle progression in nonmalignant mammary epithelial cells. J. Cell. Physiol. 223:667–678, 2010. © 2010 Wiley‐Liss, Inc.