Cell Biology International

Ellis, I.R; Banyard, J.; Schor, S.L

doi: 10.1006/cbir.1999.0423pmid: 10728570

We have recently demonstrated that the three principal mammalian isoforms of transforming growth factor β (TGF‐β) exert distinct effects upon: (1) the migration of confluent adult fibroblasts into 3D gels of native type I collagen fibres (i.e. TGF‐β−1 and −2 had no apparent motogenic activity, whilst TGF‐β−3 induced a dose‐dependent stimulation of cell migration); and (2) the synthesis of hyaluronan (HA) by these cells is also affected by the TGF‐β isoforms in a manner which parallels their effect on cell migration. The objective of the present study is to elucidate the manner in which this differential activity of the TGF‐β−1, −2 and −3 may be modulated by experimental parameters. Data presented in this communication indicate that cytokine bioactivity is determined by a combination of cell density and the nature of the macromolecular substratum. Thus, we now report that all three TGF‐β isoforms inhibit the migration of subconfluent cells in the collagen gel assay. Our data confirm that the migration of confluent cells is stimulated by TGF‐β−3 and further indicate that this motogenic activity is completely abrogated by either TGF‐β−1 or −2 when these are co‐incubated with TGF‐β−3. In contrast to these results obtained using a native type I collagen substratum, all three isoforms stimulated adult fibroblast migration in the transmembrane assay (in which cells are adherent to a 2‐D porous polycarbonate substratum). The precise effect of TGF‐β isoforms on HA synthesis was also affected by cell density and the nature of the substratum in a manner which paralleled their diverse effects on cell migration (i.e. stimulation, inhibition or no effect). Streptomyces hyaluronidase completely neutralized the TGF‐β−3‐induced stimulation of confluent fibroblast migration, thus suggesting a mechanistic link between the cytokine‐induced cell migration and HA synthesis under these conditions. Taken together, these data indicate that: (1) the bioactivity of TGF‐β−1, −2 and −3 are determined by cell density, the macromolecular substratum and the presence of other cytokines; and (2) it is therefore necessary to define cytokine bioactivity within the context of a larger ‘tissue response unit’ which more fully defines the activity state of the target cell and its microenvironment.

Momchilova, A.; Markovska, T.; Pankov, R.

doi: 10.1006/cbir.1999.0430pmid: 10728571

Cultured NIH 3T3 fibroblasts were employed to investigate the changes in the phospholipid metabolism induced by Ha‐ ras transformation. All phospholipid fractions were reduced in ras ‐transformed fibroblasts except phosphatidylethanolamine (PE). The incorporation of labeled choline and ethanolamine into phosphatidylcholine (PC), PE and their corresponding metabolites were elevated in a similar manner in the transformed cells. The enhanced uptake of choline and ethanolamine correlated with the activation of choline kinase and ethanolamine kinase. Similarly, the uptake of arachidonic, oleic and palmitic acids by PC and PE was higher in ras ‐cells. Acyl‐CoA synthetases, which esterify fatty acid before their incorporation into lysophospholipids, were also activated. However, both CTP:phosphocholine‐cytidylyltransferase and CTP:phosphoethanolamine‐chytidyltransferase were inhibited in the transformed cells. This fact, taken together with the observed activation of choline‐ and ethanolamine kinases, led to accumulation of phosphocholine and phosphoethanolamine, which have been presumed to participate in the processes of tumor development. PC biosynthesis seemed to be carried out through the CDP‐choline pathway, which was stimulated in the oncogenic cells, whereas PE was more likely, a product of phosphatidylserine decarboxylation rather than the CDP‐ethanolamine pathway.

Louagie, H.; Schotte, P.; Vral, A.; Cornelissen, M.; Thierens, H.; Beyaert, R.; Ridder, L.; Philippe, J.

doi: 10.1006/cbir.1999.0429pmid: 10728572

Caspase 3 has been shown to be actively involved in the apoptotic process in thymocytes after γ‐irradiation. We examined caspase 3 activation in mature peripheral blood lymphocytes (PBL) after γ irradiation. Since the activation of caspase 3 is generally prceded by a decrease in mitochondrial membrane potential (ΔΨm) and cytochrome c release, these two parameters were also examined. Apoptosis in PBL after a 5‐Gy γ irradiation, is characterized by a decrease in ΔΨm, but surprisingly no release of cytochrome‐c and only a weak caspase 3 activation was noticed. In contrast, staurosporin treated PBL showed a decrease in ΔΨm with cytochrome‐c release and a clear caspase 3 activation. We were unable to block the decrease in ΔΨm with the caspase‐inhibitors zVAD‐fmk or zDEVD‐fmk after γ irradiation, but DNA fragmentation as measured by the TUNEL assay was partially inhibited. Therefore, in γ irradiated mature PBL, caspase‐dependent and ‐independent pathways, but not cytochrome c, seem to be involved in the apoptotic process.

Wolfe, Jason; Sasson, Shmuel Ben; Ron, Arie

doi: 10.1006/cbir.1999.0427pmid: 10728573

Homocysteine is causally associated with birth defects such as spina bifida, and with premature vascular disease. We have investigated the effects of homocysteine on a cell—cell interaction in a fundamental eukaryotic system, the free‐living ciliate Tetrahymena. Exogenously added homocysteine inhibits cell pairing in a dose‐dependent manner. These effects are exacerbated by adenosine, which by itself has little demonstrable influence on pairing. S ‐adenosylhomocysteine (SAH) is a product of the reaction between adenosine and homocysteine, and is an inhibitor of methyl transferases. We therefore predicted that protein methylation would be significantly inhibited by homocysteine. A direct test of that hypothesis involved a demonstration that incorporation of an isotopically labeled methyl group from methionine into proteins was significantly reduced by homocysteine. The undermethylated proteins are of low molecular weight, and might correspond to known methylatable signaling proteins. We show that vanadate, an inhibitor of protein phosphatase, also inhibits cell pairing, and that the effects of vanadate and homocysteine are additive. This is the first demonstration that methylation and possibly phosphorylation play a regulatory role in cell—cell interactions in ciliates.

Tzima, Eleni; POUJOL, Christel; Nurden, Paquita; Nurden, Alan T.; Orchard, Margaret A.; Walker, John H.

doi: 10.1006/cbir.1999.0426pmid: 10728574

We have previously shown biochemically that the physiological agonist thrombin can cause translocation of endogenous annexin V to a fraction containing all platelet membranes. This paper reports ultrastructural immunohistochemical data revealing that annexin V molecules localize with plasma membranes of blood platelets following thrombin activation. When ultrathin sections of resting platelets were examined by immunogold staining, annexin V was found to be cytosolic, having a generalized distribution throughout the platelet. After thrombin activation, annexin V became peripheral in location and plasmalemma association increased. Morphometric analysis of gold particles shows that annexin V relocates specifically to the plasma membrane and its underlying cytoskeleton following treatment with thrombin. In control platelets 6.1%±0.78 of annexin V is present at the plasma membrane and 15.0%±0.82 in the region corresponding to the membrane cytoskeleton (10–80nm); after stimulation with 0.5unit/ml thrombin for 2min this increased to 16.7%±0.22 and 40.4%±0.53, respectively.

Galleron, S.; Borderie, D.; Ponteziere, Ch; Lemarechal, H.; Jambou, M.; Roch‐Arveiller, M.; Ekindjian, O.G; Cals, M.J

doi: 10.1006/cbir.1999.0424pmid: 10728575

Reactive oxygen species (ROS) are released during the inflammation of the synovial membrane associated with cartilage degradation in osteoarthritis. In this work, we exposed synoviocytes to superoxide anions at concentrations that may cause either apoptosis or necrosis. We studied membrane organization, dehydrogenase mitochondrial activity and nuclear morphology and integrity, to determine the nature of the death process initiated by superoxide anions and tried to counteract ROS effects with α‐tocopherol. We found that oxidative stress caused synoviocytes to undergo a process of cell death of an apoptotic nature rather than necrotic. Mitochondrial injury occurred at an early stage, and the FITC‐annexin‐V‐positive/propidium iodide‐positive cells occurred later than the metabolic changes. DNA strand breaks were evident at 8h and nuclear condensation at 24h. No LDH activity was detected in culture supernatants. In our experimental conditions, α‐tocopherol had little effect on stress damage; the antioxidant properties of this molecule did not affect the apoptosis caused by superoxide anions.

Bačáková, L.; Mareš, V.; Lisá, V.

doi: 10.1006/cbir.1999.0417pmid: 10728576

In 1‐day cultures with 10% serum, the number of rat aortic smooth muscle cells (VSMC) adhering to the growth support was similar in cells from both sexes, whereas in 1% serum, the number of VSMC from male donors was lower. In 10% serum medium, the doubling time was significantly shorter and the number of (3H)thymidine‐labelled nuclei was higher in cells of high passage from male rats. In serum‐free medium, these differences increased and were also seen in cells of low passage number. Morphologically, the cells in male‐derived cultures at higher passage number were mainly spindle‐shaped, formed well‐developed ‘hills and valleys’ and possessed longitudinally oriented bundles of α‐actin‐containing microfilaments. Most cells from female rats were flat, polygonal, the multilayered ‘hills’ were less prominent, with α‐actin microfilaments forming a mesh‐like network.

Wheatley, Denys

doi: 10.1006/cbir.1999.0410pmid: N/A

Collins, Andrew

doi: 10.1006/cbir.1999.0447pmid: N/A

Saunders, Susan

doi: 10.1006/cbir.1999.0446pmid: N/A