ALBERT

Notes: [Auszug] Apo2L/TRAIL stimulates cancer cell death through the proapoptotic receptors DR4 and DR5, but the determinants of tumor susceptibility to this ligand are not fully defined. mRNA expression of the peptidyl O-glycosyltransferase GALNT14 correlated with Apo2L/TRAIL sensitivity in pancreatic carcinoma, ...

Notes: [Auszug] Fas ligand (FasL) is produced by activated T cells and natural killer cells and it induces apoptosis (programmed cell death) in target cells through the death receptor Fas/Apo1/CD95 (ref. 1). One important role of FasL and Fas is to mediate immune-cytotoxic killing of cells that are ...

Notes: In order to clone the glutamate synthase gene (glt) from salt-tolerant Bradyrhizobium spp. strain WR1001, a genomic cosmid library was constructed using the vector pHC79. The library was mass-conjugated into Escherichia coli ET1194 (glt−) in a triparental mating, using pRK2013 as a mobilizing plasmid. Transconjugants of ET1194 which grew on minimal medium and had Glt+ phenotype were selected for the isolation of recombinant cosmids. Plasmid pHL27, which contained a 40-kb insert of WR1001 DNA and complemented ET1194 to Glt+ phenotype, was chosen to delimit the glutamate synthase gene. The region that encoded glt in cosmid pHL27 was determined by Southern blot analysis using the cloned glt gene of E. coli DH1 as a probe. An 11-kb HindIII DNA fragment from pHL27 contained the fully functional glt gene. Partial PstI and EcoRI restriction enzyme digests of cosmid pHL27 were subcloned into the vectors pBS+ and pUC8 and transformed into ET1194 to test for Glt+ phenotype. The glt coding region was defined by aligning the physical maps of the subclones

Notes: Abstract 1. We have investigated the role of reactive oxygen species (ROS) in cell death induced by ischemia or application of the excitatory amino acid agonist, N-methyl-D-aspartate (NMDA) or kainate (KA), in acutely isolated rat cerebellar granule cell neurons, studied by flow cytometry. Various fluorescent dyes were used to monitor intracellular calcium concentration, ROS concentration, membrane potential, and viability in acutely dissociated neurons subjected to ischemia and reoxygenation alone, NMDA or kainate alone, and ischemia and reoxygenation plus NMDA or kainate. 2. With ischemia followed by reoxygenation, ROS concentrations rose slightly and there was only a modest increase in cell death after 60 min. 3. When NMDA or kainate alone was applied to the cells there was a large increase in ROS and in intracellular calcium concentration but only a small loss of cellular viability. However, when NMDA or kainate was applied during the reoxygenation period there was a large loss of viability, accompanied by membrane depolarization, but the elevations of ROS and intracellular calcium concentration were not greater than seen with the excitatory amino acids alone. 4. These observations indicate that other factors beyond ROS and intracellular calcium concentration contribute to cell death in cerebellar granule cell neurons.

Notes: Normal chicken, mouse, and human embryo fibroblasts release into their culture media transforming growth factors (TGFs) in a latent form. Their soft agar colony-forming activity on two widely used target cells, rat NRK-49F and mouse AKR-2B, is essentially revealed only after prior acidification of cell-conditioned media. These TGFs are EGF-dependent when assayed on NRK-49F cells and EGF-independent on AKR-2B cells. The TGF activity from the chicken source is released in three (apparent) molecular weight forms of 500 kd, 125 kd, and 20 kd.

Notes: Transformation of rat NRK-49F cells (49F) by Kirsten murine sarcoma virus (Ki-MSV) renders these cells (Ki-49F cells) capable of autonomous anchorage independent (Al) growth. As compared to nontransformed 49F cells, the transformation by Ki-MSV does not modify the cell response to transforming growth factor-β (TGF-β) in monolayer conditions, but alters it in A I growth conditions. The growth of nontransformed or Ki-MSV-transformed adherent 49F cells is slowed down by porcine TGF-β, and this effect is reversed by epidermal growth factor (EGF). This decrease in the cell growth rate, induced by TGF-β, does not affect the cloning efficiency of untransformed and transformed adherent 49F cells. Contrarily, porcine TGF-β decreases the A I cloning efficiency of Ki-49F cells in agar-gelled medium; this effect is only partly reversed by EGF, which does not synergise with TGF-β to enhance the A I growth as in the case of untransformed 49F cells. Media conditioned by 49F cells, Ki-49F cells, and chicken embryo fibroblasts contain a latent TGF-β whose capacity to promote the A I growth of 49F cells and to inhibit that of Ki-49F cells is unmasked by acidification. The same situation exists concerning TGF-β from human platelets. Neutral extracts are inefficient in both tests of promotion and inhibition of A I growth and contain an acid-activable component with an apparent molecular weight of 600 kd. In acid extracts, a 5-9 kd apparent molecular weight component is responsible for the A I growth enhancement of 49F cells and the A I growth inhibition of Ki-49F cells. Further purification by reverse phase chromatography shows that both activities strictly coelute at the same point (32%) of an acetonitrile gradient. These results indicate that TGF-β is present in physiological conditions as a latent form which requires activation for inhibiting the A I growth of transformed cells as well as for enhancing that of 49F cells.

Notes: Conditioned media from Rous sarcoma virus transformed chicken embryo fibroblasts stimulate the uptake of 2-deoxyglucose in normal chicken fibroblasts. The factor responsible for this effect, which is also shed in very low amount by non-transformed fibroblasts, is destroyed by trypsin and not linked to the protease and plasminogen activator activities present in the media. Its apparent molecular weight, determined by gel filtration, is about 20.000 daltons. The factor released by transformed cells might be related to the monomeric form of a family of glucose binding and transport proteins recently reported by Lee and Lipmann ('78) to be detached by detergents from normal and transformed cells.

Notes: Chicken embryo fibroblasts and hamster BHK cells transformed by Rous sarcoma virus (RSV) release in their culture media growth factors which enhance markedly anchorage-independent colony formation in gelified medium, at the restrictive temperature (41°5 C), of chicken embryo fibroblasts (CEF) infected by RSV mutants with a ts mutation of the src gene. This action is not observed with uninfected CEF, and, therefore, appears to require some expression of the viral src gene in the target cells. The enhancing factors are proteins related to the family of the transforming growth factors (TGFs) by their molecular weight (about 20 kd), their heat and acid resistance, and their sensitivity to dithiothreitol. They do not compete with 125I EGF for binding on the EGF receptors of the membrane of A431 cells. As chicken embryo fibroblasts are devoid of EGF receptors, their activity is not potentiated by EGF.

Notes: BA 10-IR transformed cells, obtained by treating Syrian hamster embryo fibroblasts (HEF) with 7-methylbenz(a)anthracene and cultivated for a long period, are highly tumorigenic and grow in suspension as aggregates (spheroids) (Levy et al., 1976). They also grow in attached form or as spheroids in serumfree (S-) synthetic medium, without insulin and transferrin, and form anchorage-independent (AI) colonies in this same, but semi-solid, medium. This exceptional phenotype was acquired stepwise, after other transformation parameters, and appears to be related to the capacity of the transformed cells to respond to a mitogenic growth factor which they secrete. The response to this autocrine factor is amplified by insulin and transferrin. Untransformed HEF, at late and early passages, and also mouse and rat embryo fibroblasts, secrete factors equally active on BA 10-IR cells; but HEF do not respond, in S- medium, to their factor, or that of BA10-IR cells. Rat FR3T3 fibroblasts transformed by Kirsten murine sarcoma virus (FR3T3-Ki cells) also form AI colonies in semi-solid S- medium, secrete an autocrine factor potentiated by insulin and transferrin, and respond to the factors active on BA10-IR cells. However, they form far fewer colonies without additives, and respond as well to the mitogenic factors only in the presence of insulin and transferrin.BA10-IR cells and FR3T3-Ki cells also release β-TGF, or a related factor, in an active and a latent form, activable by acidification, and HEF latent, activable β-TGF. However, the factors shed by BA10-IR cells or HEF which stimulate AI growth of BA10-IR and FR3T3-Ki cells are proteins which seem unrelated to known transforming growth factors.Two major cellular alterations characteristic of the transformed phenotype in vitro are the ability to grow in the absence of anchorage, in semi-solid medium, and reduced dependence on serum growth factors (Hanafusa, 1977; Tooze, 1980). These alterations are often expressed together, and anchorage independence also appears to be the in vitro transformation parameter which correlates best with the tumorigenicity of the transformed cells (Pollack et al., 1975; Shin et al., 1975; Cifone and Fidler, 1980). However, this correlation is not constant (cf., Tooze, 1980).The cellular changes which confer anchorage independence remain unknown, but the culture conditions which allow anchorage-independent (AI) growth are better known. This growth occurs in the same media which permit the growth of attached cells, but generally requires serum. The action of serum is presumably the result of the cooperation between various serum mitogens, such as PDGF and EGF (Heldin and Westermark, 1984), and growth factors which synergize with them, such as insulin and transferrin, which are required for the growth of most cells (cf., Barnes and Sato, 1980).In a number of cases, the capacity to form AI colonies has been related to the secretion by the transformed cells of proteins, operationally called transforming growth factors (or TGF), which reversibly confer the capacity to grow without anchorage in serum-containing medium and also other parameters of transformation to certain established, but untransformed, fibroblastic cells (Roberts et al., 1983; Brown and Blakeley, 1984; Lawrence, 1985). These TGFs fall into at least two distinct classes:(1)α-TGF, which binds to the EFG receptors and is structurally and functionally related to EFG; and (2) β-TGF, which is unrelated to EFG and acts synergistically with α-TGF or EGF to promote the Al growth of rat NRK (49F clone) indicator cells (Anzano et al., 1982). However, β-TGF antagonizes the mitogenic activity of EGF on attached NRK cells and inhibits the Al growth of many neoplastic cells. It can also either stimulate or inhibit this growth, depending on the mitogenic factors in the medium (Roberts et al., 1985). Furthermore, it is found in the culture medium or extracts of both untransformed and transformed cells (Roberts et al., 1983; Lawrence, 1985), and is shed by these cells in a high molecular weight latent form, activated by acidification (Lawrence et al., 1984; Lawrence, Pircher, and Jullien, 1985; Pircher et al., 1984; Krycève-Martinerie et al., 1985).The frequent correlation observed between anchorage-independence, reduced serum-dependence, and TGF production has led to the suggestion that transformed cells displaying these phenotypic characters may be autocrine systems stimulated by their TGFs (Sporn and Todaro, 1980; Sporn and Roberts, 1985). In line with this model, it has been shown that the Al growth of virally or chemically transformed cells, or explanted cancer cells, was stimulated by α-TGF (De Larco and Todaro, 1978; Ozanne, Fulton, and Kaplan, 1980; Todaro, Fryling, and De Larco, 1980), or β- or unclassified TGF (Krycève-Martinerie et al., 1982; Kaplan and Ozanne, 1982; Tucker et al., 1983) shed by these cells. The TGFs (or accompanying factors) shed by some of these cells were also shown to stimulate their growth, in monolayer culture, in serum-free (S-) medium containing insulin and/or transferrin (Kaplan, Anderson, and Ozanne, 1982; Kaplan and Ozanne, 1982). McClure (1983) further observed that mouse BALB/c3T3 cells transformed by simian virus 40 were able to form Al colonies in semi-solid S- medium containing insulin and transferrin, in response to an autocrine growth factor which was not characterized. More recently, Sauvaigo et al. (1986) observed the autonomous proliferation of a human melanoma cell line and a metastatic variant of this line in S- medium without additives, also in response to an uncharacterized autocrine factor.We report here data obtained in the study of a line of chemically tranformed hamster embryo fibroblasts (HEF), called BA 10-IR (Leavy et al., 1976), which grow in S- medium without insulin and transferrin, both as attached cells and unattached aggregates (spheroids). These cells form Al colonies in semi-solid S- medium without additives, although this capacity is enhanced by insulin and transferrin. This phenotype is accompained by the secretion of an autocrine factor which stimulates Al growth of BA10-IR cells, in S- medium, and is potentiated by insulin and transferrin. Untransformed HEF and rat and mouse embryo fibroblasts secrete factors which display a similar activity on BA10-IR cells, but not on themselves. We have further observed that rat FR3T3 fibroblasts transformed by Kristen murine sarcoma virus (Ki-MSV) also form Al colonies in semi-solid S- medium, shed an autocrine factor potentiated by insulin and transferrin, and respond to the factors active on BA10-IR cells. But their colning efficiency without additives is much lower. BA10-IR cells and Ki-MSV-transformed FR3T3 cells also secrete active as well as latent β-TGF activated by acidification, and HEF latent, activable β-TGF. However, the factors stimulating Al growth in S- medium appear to be unrelated to known TGFs.