Differentiation-associated decrease in the proportion of fucosylated polylactosaminoglycans of CaCo-2 human colonic adenocarcinoma cells.

CaCo-2 cells are human colonic adenocarcinoma cells which can differentiate spontaneously into enterocytes when maintained confluent for extended periods of time. Cells kept in culture for 4 days (rapidly growing), 7-9 days (early confluence) and 19-22 days (late confluence) were incubated for 24 h with L-[5,6-3H]fucose or D-[6-3H]glucosamine in order to examine the changes in glycoprotein carbohydrate structure that occur during this differentiation. Labelled glycopeptides obtained by exhaustive Pronase digestion of the cell-surface and cell-pellet fractions were fractionated on Bio-Gel P-6. A high-Mr glycopeptide fraction which was excluded from Bio-Gel P-6 was present in all cases. These glycopeptides were then fractionated by affinity chromatography on Datura stramonium agglutinin-agarose. The glycopeptides which were specifically bound to the lectin column were largely degraded by endo-beta-galactosidase, thereby indicating that they consisted of fucosylated polylactosaminoglycans. The proportion of labelled polylactosaminoglycans decreased with increasing time in culture, whereas sucrase activity, which is characteristic of differentiated enterocytes, increased. These results demonstrate that a relatively large decrease in the proportion of fucosylated polylactosaminoglycans occurs with differentiation of CaCo-2 cells.     

Massive telomere loss is an early event of DNA damage-induced apoptosis

Chromosomal stability and cell viability require a proficient telomeric end-capping function. In particular, telomere dysfunction because of either critical telomere shortening or because of mutation of telomere-binding proteins results in increased apoptosis and/or cell arrest. Here, we show that, in turn, DNA damage-induced apoptosis results in a dramatic telomere loss. In particular, using flow cytometry for simultaneous detection of telomere length and apoptosis, we show that cells undergoing apoptosis upon DNA damage also exhibit a rapid and dramatic loss of telomeric sequences. This telomere loss occurs at early stages of apoptosis, because it does not require caspase-3 activation, and it is induced by loss of the mitochondrial membrane potential (Deltapsi(m)) and production of reactive oxygen species. These observations suggest a direct effect of mitochondrial dysfunction on telomeres.     

The expression of soluble and cell-bound alpha 2,6 sialyltransferase in human colonic carcinoma CaCo-2 cells correlates with the degree of enterocytic differentiation.

alpha 2,6 sialyltransferase towards the N-acetyllactosaminyl sequence (alpha 2,6 ST, E.C. 2.4.99.1) is one of the major sialyltransferases in human colonic cells; it strongly increases in human colorectal tumors and is largely expressed in fetal and neonatal rat colon. In this study we demonstrate that human colon carcinoma CaCo-2 cells, which differentiate spontaneously into enterocytes when maintained confluent for several days, exhibit a very high expression of alpha 2,6 ST both in the cell-bound and soluble form. When the CaCo-2 cells were cultured on porous membranes the soluble alpha 2,6 ST was mainly detected in the medium collected from the chamber corresponding to the basolateral face of the monolayer. The soluble alpha 2,6 ST could be concentrated and purified from the alpha 2,3 sialyltransferase by affinity chromatography on Blue Sepharose.     

Intrinsic disturbance of cellular redox balance enhances blood lymphocyte apoptosis in the spontaneously hypertensive rat

The spontaneously hypertensive rat (SHR) shows an altered cell apoptosis rate compared to normotensive controls by a mechanism that may involve redox imbalance. This study was designed to determine whether cellular oxidative stress and apoptosis in blood lymphocytes are enhanced in the SHR by intrinsic cellular abnormalities and/or by factors derived from selected organs (kidney, adrenals). We identified apoptosis and free radical production in isolated peripheral blood lymphocytes with flow cytometry before and after serum withdrawal, which exposes the cells to a pro-oxidative condition. Freshly drawn SHR lymphocytes showed higher levels of apoptosis and intracellular oxygen radicals than lymphocytes from normotensive Wistar-Kyoto rats (p < 0.05). Apoptosis and intracellular oxygen radicals were profoundly elevated after serum-free incubation in SHR cells and to a lesser extent in normotensive controls suspended in exactly the same medium (p < 0.01, SHR vs normotensives). Cell-permeable antioxidants, L-cysteine and Tempol, attenuated serum deprivation-induced apoptosis as well as cellular oxidative stress. Blood plasma and organ extracts from the SHR exerted pro- or antiapoptotic activity to the same degree as those from normotensives. The results suggest that blood lymphocyte apoptosis is enhanced in the SHR due to intrinsically disturbed cellular redox balance rather than altered activity in extracellular humoral factors.     

Activation of human T cells is associated with tyrosine phosphorylation of several cellular proteins

Human T lymphocytes are activated to proliferate after triggering the T Cell Antigen Receptor Complex. CD3-Ti, with either antigen, mitogenic lectins or monoclonal antibodies against its different subunits. Stimulation of Jurkat leukemic human T cells with anti-CD3 or anti-Ti monoclonal antibodies was found to induce, within 1 min, an increase in the phosphorylation of a set of cellular proteins that can be precipitated with anti-phosphotyrosine antibodies. Seven phosphotyrosine-containing proteins were separated with respective mol. wt of 21, 25, 38, 55, 70, 80 and 110 kDa, among which the 38 kDa species is predominant. Moreover, incubation of Jurkat T cells with sodium orthovanadate, a potent inhibitor of phosphotyrosine protein-phosphatases, was found to potentiate the effects of anti-CD3 mAb on tyrosine phosphorylation. In addition vanadate also induced IL-2 secretion in Jurkat cells when associated with the phorbol ester TPA, further demonstrating the importance of these phosphorylation reactions in the process of T cell activation. Our results therefore allow us to identify several protein substrates of a tyrosine kinase activity, whose stimulation appears to be an early event in human T cell activation through the antigen receptor pathway.     

A K(+)-selective channel in the colonic carcinoma cell line: CaCo-2 is activated with membrane stretch

CaCo-2 is a human colonic carcinoma cell line which becomes differentiated in culture to form a polarized epithelium exhibiting several of the functional characteristics of native colonic tissue. In the present study, CaCo-2 cells have been used for a patch-clamp study of colonic ion conductance pathways. A large, 120 pS K(+)-selective channel was found in cells forming subconfluent monolayers in culture. Unlike Maxi-K+ channels found in other epithelial cells, this channel was not activated with elevations in cytosolic Ca2+. Channel activity was stimulated with membrane depolarization and most markedly with membrane stretch. The application of negative pressure (20 mm-Hg) to both cell-attached and excised, inside-out membrane patches caused a burst of channel activity which disappeared within seconds of suction removal. Single-channel conductance of the pressure-activated channel was decreased when quinine (100 microM) was present in the patch pipette.     

Mitochondria hyperpolarization is an early event in oxidized low-density lipoprotein-induced apoptosis in Caco-2 intestinal cells

We investigated the mechanisms underlying the pro-apoptotic activity exerted by oxidized low-density lipoproteins (oxLDL) in Caco-2 intestinal cells, a cell line which retains many morphological and enzymatic features typical of normal human enterocytes. We found that: (i) oxLDL induced mitochondrial-mediated apoptosis by provoking first an increase in mitochondrial membrane potential, followed, later, by the typical apoptosis-associated depolarization (type II apoptosis); accordingly, (ii) caspase-9 inhibition significantly hindered apoptosis while caspase-8 inhibition did not; and finally (iii) dietary phenolic antioxidizing compounds exerted a significant protective antiapoptotic activity. These results point to mitochondrial hyperpolarization as 'sensitizing feature' in apoptotic proneness of Caco-2 intestinal cells to oxLDL exposure.     

Stimulation of colonic mucosal growth associated with oxidized redox status in rats

Limited data in animal models suggest that colonic mucosa undergoes adaptive growth following massive small bowel resection (SBR). In vitro data suggest that intestinal cell growth is regulated by reactive oxygen species and redox couples [e.g., glutathione (GSH)/glutathione disulfide (GSSG) and cysteine (Cys)/cystine (CySS) redox]. We investigated the effects of SBR and alterations in redox on colonic growth indexes in rats after either small bowel transection (TX) or 80% midjejunoileal resection (RX). Rats were pair fed +/- blockade of endogenous GSH synthesis with buthionine sulfoximine (BSO). Indexes of colonic growth, proliferation, and apoptosis and GSH/GSSG and Cys/CySS redox potentials (E(h)) were determined. RX significantly increased colonic crypt depth, number of cells per crypt, and epithelial cell proliferation [crypt cell bromodeoxyuridine (BrdU) incorporation]. Administration of BSO markedly decreased colonic mucosal GSH, GSSG, and Cys concentrations in both TX and RX groups, with a resultant oxidation of GSH/GSSG and Cys/CySS E(h). BSO did not alter colonic crypt cell apoptosis but significantly increased all colonic mucosal growth indexes (crypt depth, cells/crypt, and BrdU incorporation) in both TX and RX groups in a time- and dose-dependent manner. BSO significantly decreased plasma GSH and GSSG, oxidized GSH/GSSG E(h), and increased plasma Cys and CySS concentrations. Collectively, these data provide in vivo evidence indicating that oxidized colonic mucosal redox status stimulates colonic mucosal growth in rats. The data also suggest that GSH is required to maintain normal colonic and plasma Cys/CySS homeostasis in these animal models.     

Myocilin is associated with mitochondria in human trabecular meshwork cells.

The trabecular meshwork (TM) is a specialized tissue located at the chamber angle of the eye next to the cornea. This tissue is believed to be responsible for regulation of the aqueous humor outflow and control of the intraocular pressure (IOP). Alterations in functions of the TM may lead to IOP elevation and development of glaucoma, a major cause of blindness. The myocilin gene has recently been directly linked to open-angle glaucomas. The gene product was originally identified as a protein inducible in TM cells by treatment with glucocorticoids such as dexamethasone (DEX) and termed TIGR (TM inducible-glucocorticoid response). The exact nature and function of the myocilin protein so far still remain elusive. In this study, myocilin was localized to the perinuclear region of both DEX-treated and control TM cells. Its distribution overlapped considerably with that of mitochondria. Subcellular fractionation and Western blot analyses suggested a rather extensive association of myocilin with mitochondria. The DEX-treated TM cells were found to undergo apoptosis, when exposed to anti-Fas antibody, to a significantly higher degree than the untreated control cells. It appears that the TM cell integrity remains intact after DEX treatment. However, the induced myocilin or myocilin-mitochondria association seems to render the cells more susceptible to a second stress or challenge. This vulnerability may be the basis that ultimately leads to pathological consequences.     

Syncytia formation in HIV-1 infected cells is associated with an increase in cellular oleic acid

Infection of cells with the human immunodeficiency virus type-1 (HIV-1) usually results in the formation of giant multinuclear cells (syncytia) [(1986) Nature 322, 470-474; (1986) Nature 322, 725-728; (1985) Hum. Pathol. 18, 760-765; (1987) Arm. Neurol. 21, 490-496]. The appearance of syncytia is associated with an increase in the monounsaturated oleic acid content. This report describes experiments which compare the activity of known antiviral agents with that of saturated fatty acid derivatives in inhibiting oleic acid and syncytia formation. A concept is introduced which proposes that infection of cells with the human immunodeficiency virus causes a rise in cellular oleic acid which leads to increased membrane fluidity.     

Temporal expression of hypoxia-regulated genes is associated with early changes in redox status in irradiated lung

The development of normal lung tissue toxicity after radiation exposure results from multiple changes in cell signaling and communication initiated at the time of the ionizing event. The onset of gross pulmonary injury is preceded by tissue hypoxia and chronic oxidative stress. We have previously shown that development of debilitating lung injury can be mitigated or prevented by administration of AEOL10150, a potent catalytic antioxidant, 24h after radiation. This suggests that hypoxia-mediated signaling pathways may play a role in late radiation injury, but the exact mechanism remains unclear. The purpose of this study was to evaluate changes in the temporal expression of hypoxia-associated genes in irradiated mouse lung and determine whether AEOL10150 alters expression of these genes. A focused oligo array was used to establish a hypoxia-associated gene expression signature for lung tissue from sham-irradiated or irradiated mice treated with or without AEOL10150. Results were further verified by RT-PCR. Forty-four genes associated with metabolism, cell growth, apoptosis, inflammation, oxidative stress, and extracellular matrix synthesis were upregulated after radiation. Elevated expression of 31 of these genes was attenuated in animals treated with AEOL10150, suggesting that expression of a number of hypoxia-associated genes is regulated by early development of oxidative stress after radiation. Genes identified herein could provide insight into the role of hypoxic signaling in radiation lung injury, suggesting novel therapeutic targets, as well as clues to the mechanism by which AEOL10150 confers pulmonary radioprotection.     

Hepatocellular apoptosis in mice is associated with early upregulation of mitochondrial glucose metabolism

Hepatocyte death due to apoptosis is a hallmark of almost every liver disease. Manipulation of cell death regulatory steps during the apoptotic process is therefore an obvious goal of biomedical research. To clarify whether metabolic changes occur prior to the characteristic apoptotic events, we used ex vivo multinuclear NMR-spectroscopy to study metabolic pathways of [U-¹³C]glucose in mouse liver during Fas-induced apoptosis. We addressed whether these changes could be associated with protection against apoptosis afforded by Epidermal Growth Factor (EGF). Our results show that serum alanine and aspartate aminotransferase levels, caspase-3 activity, BID cleavage and changes in cellular energy stores were not observed before 3 h following anti-Fas injection. However, as early as 45 min after anti-Fas treatment, we observed upregulation of carbon entry (i.e. flux) from glucose into the Krebs-cycle via pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC) (up to 139% and 123% of controls, respectively, P < 0.001). This was associated with increased glutathione synthesis. EGF treatment significantly attenuated Fas-induced apoptosis, liver injury and the late decrease in energy stores, as well as the early fluxes through PDH and PC which were comparable to untreated controls. Using ex vivo multinuclear NMR-spectroscopic analysis, we have shown that Fas receptor activation in mouse liver time-dependently affects specific metabolic pathways of glucose. These early upregulations in glucose metabolic pathways occur prior to any visible signs of apoptosis and may have the potential to contribute to the initiation of apoptosis by maintaining mitochondrial energy production and cellular glutathione stores.