Arachidonic Acid Metabolism In Murine Fibrosarcoma Cells With Differing In Vivo And In Vitro Characteristics

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/144314/1/ijc1985363383.pd

Use of recombinant and synthetic peptides as attachment factors for cells on microcarriers

Polystyrene culture dishes and polystyrene microcarriers were coated with Pronectin-F and poly- l -lysine (polylysine), either alone or in combination. Pronectin-F is a recombinant peptide containing repeats of the RGD cell-attachment sequence from fibronectin. Polylysine is a polymer of l -lysine. Pronectin-F supported attachment of Madin-Darby Canine Kidney (MDCK) cells at concentrations as low as 0.025 μg/cm 2 of surface area. The cells rapidly spread after attachment. Polylysine at concentrations of 0.05–0.5 μg/cm 2 also supported cell attachment but cells did not rapidly spread after attachment to this substrate. Higher concentrations of polylysine could not be used because of toxicity. When the two peptides were used in conjunction, MDCK cells attached and spread at lower peptide concentrations than they did when either substrate was used alone. These findings suggest that recombinant Pronectin-F alone or in conjunction with a cationic polymer could be a useful replacement for materials such as gelatin or collagen which are currently used as microcarrier surfaces.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42614/1/10616_2004_Article_BF00749935.pd

Phorbol ester binding and phorol ester-induced arachidonic acid metabolism in a highly responsive murine fibrosarcoma cell line and in a less-responsive variant

Phorbol ester binding was examined in two lines of murine fibrosarcoma cells. The two cell lines were isolated from the same parent tumor but respond differentially to stimulation with phorbol esters. In one of the lines, these agents stimulate a rapid attachment and spreading response and induce directional migration. The other cell line does not migrate in response to stimulation with phorbol esters and the attachment and spreading response is slow. The cell line which responds actively to phorbol ester stimulation is highly malignant when injected into syngeneic animals while the other line is of low tumorigenicity and is virtually non-metastatic. In spite of these differences, both lines were found in the present study to bind [ 3 H]4β-phorbol-12β, 13α-dibutyrate in a receptor-mediated fashion. The characteristics of binding were virtually identical between the two cell lines. In additional studies, arachidonic acid metabolism was examined in the same two lines. In the highly responsive line, PMA stimulated a rapid release of [ 3 H]arachidonic acid and its conversion into cyclooxygenase and lipoxygenase products. In the less-responsive line, PMA stimulated a slower release of [ 3 H]arachidonic acid from prelabeled cells. The quantity of arachidonic acid metabolites produced was also much less. These studies suggest that the disparity between the two cell lines in their response to phorbol ester stimulation is not the result of differences in the initial interaction between the cells and ligand but may result from alterations in their signal transductance mechanism. This may be the result of inherent differences in capacity for arachidonic acid metabolism.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42593/1/10585_2004_Article_BF00053473.pd

Substrate-dependent differences in production of extracellular matrix molecules by squamous carcinoma cells and diploid fibroblasts

Two human squamous carcinoma cell lines and human diploid fibroblasts were examined for the production of extracellular matrix (ECM) molecules including fibronectin (FN), laminin (LN), and thrombospondin (TSP) when grown on a number of different substrates. The substrates used included glass, plastic, collagen (gelatin), and DEAE-dextran. Levels of TSP as indicated by enzyme-linked immunosorbent assay did not vary significantly as a function of substrate. In contrast, LN levels in the culture medium were significantly decreased when the cells were grown on DEAE-dextran or collagen-linked dextran as compared to the other substrates. FN levels were slightly lower in the culture medium of the cells grown on DEAE-dextran. Biosynthetic labeling followed by immunoprecipitation indicated that the reduction in LN was due, in part, to decreased biosynthesis. Previous studies have indicated that LN influences the behavior of epithelial cells in culture and that the cells, themselves, are a major source of the LN. The differences in LN production noted here indicate that the production of this ECM component is influenced by the substratum on which the cells are grown. These differences could contribute to alterations in biological properties that are known to be influenced by the substratum.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/37897/1/260331003_ftp.pd

Characterization of thrombospondin synthesis, secretion and cell surface expression by human tumor cells

Previous studies have shown that thrombospondin (TSP) is an adhesion factor for some human tumor cells. The previous studies have shown further that tumor cells which utilize TSP as an adhesion factor also synthesize it. This study continues the effort to understand how TSP production and expression are regulated in human tumor cells and the consequences of this for the cells. It is shown that differences among cell lines in their capacity to biosynthesize TSP are associated with differences in TSP specific mRNA levels. This indicates that biosynthesis is regulated at the transcriptional level. There is also a direct relationship between TSP biosynthesis and secretion into the culture medium and expression at the cell surface. The cells which are the most biosynthetically active secrete amounts of TSP into the culture medium that are sufficient to elicit a detectable response in the cell-substrate adhesion assay. The kinetics of TSP secretion by these cells are in accord with the kinetics of attachment and spreading of the same cells in the absence of exogenous adhesion factors. These data are consistent with the idea that endogenously produced TSP promotes the adhesion of the cells which synthesize it in an autocrine manner.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42595/1/10585_2005_Article_BF01753679.pd

all-trans -Retinoic acid preserves viability of fibroblasts and keratinocytes in full-thickness human skin and fibroblasts in isolated dermis in organ culture

Human dermal fibroblast and human epidermal keratinocyte survival was examined under various conditions in organ culture. Using cell recovery from organ-cultured tissue as the criterion, it was observed that no keratinocytes and few fibroblasts survived incubation for 10–12 days in serum-free basal medium containing a low level (0.15 m M ) of extracellular Ca 2+ . Increasing the extracellular Ca 2+ concentration to 1.4 m M or treating the tissue with 3 Μ M retinoic acid (RA) under low Ca 2+ conditions resulted in increased keratinocyte and fibroblast survival; the two treatments together were more effective than either treatment alone. The same treatments preserved fibroblast survival when pieces of isolated dermal tissue were incubated in organ culture and also supported fibroblast survival in monolayer culture. These findings indicate that recovery of keratinocytes and fibroblasts from skin after maintenance in organ culture provides a simple but definitive measure of the viability of the major cellular elements present in the tissue. These findings suggest that RA treatment enhances survival of both fibroblasts and keratinocytes and that these effects of RA can be seen at physiological Ca 2+ concentrations as well as at suboptimal levels of extracellular Ca 2+ . Finally, these results indicate that the dermis is a direct target of RA.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47248/1/403_2004_Article_BF00371569.pd

Retinoid Toxicity for Fibroblasts and Epithelial Cells Is Separable From Growth Promoting Activity

Three different retinoids with widely varying capacity to stimulate skin repair in vivo and stimulate fibroblast and epithelial cell growth in vitro were examined for capacity to lyse the same cells at high concentrations. These included all-trans retinoic acid (RA), tetrahydro tetramethyl napthalenyl benzoic acid (TTNPB), and its biologically inactive structural analogue, meta-carboxy TTNPB. Despite their differing capacities to stimulate skin repair and cell growth, all of the agents were cytotoxic for fibroblasts and epithelial cells over the same range of concentrations (0.6 – 3 × 10-5 M). Cytotoxicity for both fibroblasts and epithelial cells was blocked by addition of phosphatidylcholine to the cells along with the retinoid. In the presence of high concentrations of RA (0.75 – 3 × 10-5 M) and phosphatidylcholine, proliferation was observed. The proliferative response was greater under these conditions than in the presence of an optimal concentration of RA (0.75 – 3 × 10-6 M) without phos-phatidylcholine. These data suggest that toxicity of retinoids can be separated, at least partially, from their growth-promoting activities