Enhanced invasive properties of rat embryo fibroblasts transformed by adenovirus E1A mutants with deletions in the carboxy-terminal exon.

E1A genes deficient in the carboxy-terminal exon can cooperate with activated ras oncogenes to induce transformation of rat embryo fibroblasts. However, the resulting transformed foci show a distinct appearance characterized by a decreased adhesion of the cells to the substrate. Here, we demonstrate that cell lines derived from foci showing the variant morphology are defective in down-regulation of stromelysin 1 metalloprotease expression and show an increased invasive propensity compared with cells transformed by wild-type E1A. The altered focus morphology, the high invasive propensity and the elevated stromelysin 1 expression were abrogated by glucocorticoid treatment. Our results show that E1A functions necessary for transformation and inhibition of invasive properties may be separated, and indicate that a 23 amino acid serine/threonine-rich region within the E1A carboxy-terminal exon is required for efficient repression of metalloprotease expression in transformed cells.

Lack of c-jun expression in a transformed cell line isolated by glucocorticoid promotion of ras-transfected rat embryo fibroblasts.

Rat embryo fibroblasts (REFs) are inefficiently transformed by the T24-ras oncogene. A contributing factor to cellular resistance to transformation is the limited tolerance to p21-ras oncoprotein expression. Here we present data suggesting that long-term glucocorticoid treatment of ras oncogene-transfected REFs results in increased tolerance to p21-ras oncoproteins, leading to expression of the transformed phenotype. Stably transformed cell lines that expressed high levels of H-ras and could be maintained in the absence of hormone were isolated. In three out of four lines studied, the AP-1-dependent collagenase gene was expressed at a low level. In one of these lines, low collagenase expression was paralleled by lack of c-jun mRNA. Immunochemical analysis revealed that progression to hormone independence was not paralleled by mutations in the p53 gene. We propose that a decreased expression of AP-1-driven genes may result in increased tolerance to p21-ras oncoprotein.

Repression of stromelysin metalloprotease expression in rat fibrosarcoma cells by dimethylsulfoxide.

Metalloproteases are implicated in conferring invasive properties to tumor cells. We show here that treatment of ras-oncogene-transformed rat fibroblasts with dimethylsulfoxide (DMSO) results in a reversible decrease in stromelysin mRNA. Furthermore, stromelysin expression was found to be repressed by DMSO, but not by glucocorticoid hormone, in a fibrosarcoma cell line showing low AP-1 (fos/jun) transcription factor activity. In two fibrosarcoma cell lines which express high levels of stromelysin and low levels of 68 kDa type IV collagenase, the DMSO-induced decrease in stromelysin expression was paralleled by a decreased invasive propensity.

Increases of stromelysin messenger-RNA expression in ras-transformed ref cells in-vivo.

Interstitial collagenase and stromelysin mRNA expression was examined in six rat cell lines isolated after transfection of embryo fibroblasts with polyomavirus large-T and T24-ras oncogenes. Increased levels of stromelysin mRNA were observed in tumors formed by cells with low levels of stromelysin expression before injection, whereas interstitial collagenase mRNA levels were not elevated in tumors. The lung colonizing - but not the chemoinvasive capacity - of two of these cell lines increased following growth in vivo. The increase in stromelysin expression in tumors was not necessarily paralleled by an increase in ras expression. These findings may be relevant to the phenomenon of clonal dominance of metastatic cells in primary tumors.

Limited invasive capacity of plt plus ras transformed rat fibrosarcoma cells effective in experimental metastasis.

Nine cell lines were isolated after cotransfection of rat embryo fibroblasts with polyomavirus large-T (plt) and T24-ras oncogenes. Five of these lines were highly tumorigenic following subcutaneous injection, but differed in their metastatic and in vitro invasive properties. Two cell lines, expressing low levels of ras mRNA, showed low capacity for experimental metastasis. Three cell lines, expressing high levels of ras mRNA, were tumorigenic and showed high capacity for experimental metastasis. High expression of interstitial collagenase, stromelysin and 92 kDa type IV collagenase was observed in the highly metastatic cell lines. Immunochemical analysis revealed that these cell lines expressed apparently wild-type p53 protein. Furthermore, the level of a 43 kDa/pI 5,44 polypeptide was elevated and the levels of a series of 41 to 43 kDa acidic polypeptides were decreased in the metastatic cells. Within this panel of transformed cell lines, high capacity for experimental metastasis did not correlate with high chemoinvasive capacity in the reconstituted basal membrane assay. The limited invasive propensity could not be attributed to low chemotactic or adhesive capacity. We conclude that in vitro invasion does not correlate with experimental metastasis in this model system.

Comparative-studies on the anti-invasive effects of retinoic Acid and staurosporine.

Retinoic acid and the protein kinase C inhibitor staurosporine have been reported to inhibit the invasiveness of tumor cells and may potentially be used to prevent metastatic disease. We report that retinoic acid reduced the invasiveness of 6 of 6 ras-transformed rat fibroblast cell lines and that inhibition did not require expression of the c-Jun component of AP-1. In contrast, staurosporine reduced the invasiveness of only 1 of 4 ras-transformed cell lines. The effect of staurosporine on the invasiveness of human tumor cell lines varied with cell type and length of treatment. We conclude that retinoic acid, but not necessarily staurosporine, decreases the invasiveness of ras-transformed fibroblasts.

Differential-effects of fibroblast coculture on mcf-7 and mda-231 breast-carcinoma cell invasion through matrigel.

The stimulative effect of conditioned media collected from fibroblasts or breast epithelial cells on breast carcinoma cell invasion through Matrigel was studied. Fibroblast conditioned medium was more efficient than media collected from breast epithelial cells in stimulating chemoinvasion of highly invasive MDA-MB-231 and MDA-MB-436 cells. In contrast, fibroblast conditioned medium was found to be less effective than breast epithelial cell conditioned medium in stimulating invasion of weakly metastatic MCF-7 cells. These data suggest that fibroblasts preferentially enhance the invasive capacity of highly malignant breast carcinoma cells.

Functions of the Golgi complex in cell division: formation of cell-matrix contacts and cell-cell communication channels in the terminal phase of cytokinesis.

The Golgi complex of mammalian cells is disorganized into dispersed vesicular and tubular elements during mitosis and is then reorganized into an interconnected system of cisternal stacks in each daughter cell during cytokinesis. Recent studies further indicate that the Golgi complex is typically relocated from the proximal to the distal side of the nucleus in the terminal phase of cytokinesis (as related to the intercellular bridge). Here, the functional role of this shift in position was approached using rat embryo fibroblasts synchronized with thymidine and nocodazole. Mitotic cells were collected by shaking and seeded in medium without or with brefeldin A (a fungal metabolite that inhibits protein secretion). They were fixed after one or two hours and stained for immunocytochemical demonstration of mannosidase II (a Golgi protein), fibronectin (an extracellular matrix protein), the fibronectin receptor (a member of the integrin family of proteins), and connexin 43 (a member of the connexin family of gap junction proteins). One hour after seeding, the cells had completed mitosis and progressed into cytokinesis. The Golgi complex was now usually located on the proximal side of the nucleus and overlapping fibrillar arrays of fibronectin and fibronectin receptors were observed in the contact zone between the daughter cells, while connexin 43 mainly occurred in fine dispersed spots. Two hours after seeding, the cells had spread out on the substrate and started to move apart. The Golgi complex was now usually located on the distal side of the nucleus. Moreover, fibronectin and fibronectin receptors were found to codistribute both in the contact zone between the daughter cells and in adhesive contacts beneath them, while connexin 43 was concentrated to plaques in the former zone. After treatment with brefeldin A, there was a diffuse cytoplasmic staining for mannosidase II and fibronectin and no distinct extracellular staining for fibronectin was noted. In addition, the connexin 43 positive plaques were reduced in size and number. Although the cells completed cytokinesis in the presence of the drug, they showed an increased tendency to detach from the substrate and locate on top of each other rather than to move apart normally. Taken together, the observations suggest that the change in position of the Golgi complex during cytokinesis serves the function to direct transport of secretory proteins as well as membrane constituents to different parts of the cell surface at different times.(ABSTRACT TRUNCATED AT 400 WORDS)

[3H]bumetanide binding and inhibition of Na+ + K+ + Cl- co-transport: demonstration of specificity by the use of MDCK cells deficient in co-transport activity.

Cultured renal MDCK cells possess a Na+ + K+ + Cl- co-transport system which is inhibited with high affinity by loop diuretics (K0.5 for bumetanide inhibition = 0.28 microM). By the use of 'mutant' cell lines deficient in co-transport flux the specific interaction of [3H]bumetanide with the co-transporter has been identified. [3H]Bumetanide uptake in parental MDCK-N cells in the range 0-1 microM comprises a non-saturable linear component, assessed by the inclusion of 100 microM-unlabelled bumetanide and a saturable component (K0.5 = 0.19 microM and Bmax = 0.55 pmol/10(6) cells). Though the magnitude of the linear non-specific component was little different between the parental MDCK-N cell line and two co-transport-deficient mutant cell lines (LKC1 and LKA3), the magnitude of the saturable component was markedly reduced in both co-transport-deficient mutants. In addition to the saturable component associated with flux inhibition a lower-affinity uptake displaceable by excess unlabelled bumetanide was evident in MDCK-N cells comprising 8 pmol/10(6) cells measured at 10 microM-[3H]bumetanide. This lower-affinity uptake was present in both co-transport-deficient mutant cell lines confirming its lack of association with inhibition of co-transport flux. In MDCK cells possessing the co-transporter, an estimate of the turnover number was made when co-transport flux and specific bumetanide uptake at 0.5 microM-[3H]bumetanide were measured in the same cell batch. At 37 degrees C this was 113 K+ ions site-1 s-1.

"Dome-curve"--three size classes of domes of MDCK epithelial monolayer.

Borders between monolayer of MDCK epithelial cells and domes were made visible by fixation and staining with 1% silver nitrate in 4% aqueous solution of mannitol. If the lengths of circumferences of domes are plotted against the frequency of their occurrence a "dome-curve" can be obtained. It was found that three size classes of domes exist in the MDCK monolayer: small with 0.0811 mm of circumference, medium with 0.1449 mm of circumference and large with 0.2439 mm of circumference.

Relationship between stimulation of sodium transport across frog skin and Ledakrin concentration.

Relationship between stimulation of sodium transport across frog skin and Ledakrin concentration. Acta Physiol. Pol., 1979, 30 (2): 261--265. Using the method of measurements of the short circuit current, introduced by Ussing, the relationship was investigated between stimulation of sodium ion transport across frog skin and the concentration of Ledakrin. It was found that the optimum concentration of the drug for investigating changes in ion transport is 10(-5) g per 1 ml of medium.

Early effect of ledakrin -- a nitro-derivative of acridine -- on cell membrane.

Ledakrin effect on the bioelectrical parameters of sodium ion transport (potential difference -- PD and short-circuit current -- SSC) and on Na22 transport across frog (R. esculenta) skin was studied in vitro. Ledakrin induced a biphasic action, after a phase of increase of PD, SCC and sodium flux the transport of sodium ions decreased gradually. The effect of this nitro-derivative of acridine was compared with that of Amiloride. When both these agents were given simultaneously only one phase developed, that is inhibition of sodium ion transport. In both situations there was a significant stimulation of outflux. The obtained results suggest that the external apical cell membrane of epithelial cells is the site of action of Ledakrin.

Cultivation of MDCK epithelial cells on chitosan membranes.

Deacetylated chitin upon evaporation from aqueous acetic acid solutions forms a thin, permeable and transparent porous membrane which can be successfully used as support of cell culture. An established MDCK cell line grown as monolayer on both chitosan membrane and millipore filter generates comparable bioelectrical properties when studied in a typical transporting chamber.

Elevated stromelysin-1 and reduced collagenase-IV expression in invasive rat embryo fibroblasts expressing E1A deletion mutants + T24-H-ras.

We have studied the in vitro invasive properties of 3 cell lines derived from the co-transfection of rat embryo fibroblasts (REF) with EIA genes deficient in exon 2 and T24-ras. All 3 cell lines showed invasive properties at passage 10 after isolation. Invasive cells expressed elevated levels of stromelysin-1 and reduced levels of 68-kDa type-IV collagenase compared with untransfected REF. In 2 cell lines the invasive capacity increased during in vitro propagation. The expression of stromelysin-1 increased during this process, whereas 68-kDa type-IV collagenase was persistently expressed at reduced levels. In the third clone analyzed, the invasive capacity decreased during culture, in parallel with decreased expression of stromelysin-1. The low level of stromelysin-1 expression observed in this cell line did not result from loss of AP-1-transcription-factor activity, and was not reversed by phorbol-ester treatment.