Distinct transformation phenotypes induced by polyoma virus and simian virus 40 in rat fibroblasts and their control by an early viral gene function.

Several transformed cell lines established from Fisher rat cells (FR 3T3) infected with wild-type polyoma virus or simian virus 40 or early temperature-sensitive mutants (polyoma tsa and simian virus 40 tsA30) were studied for their transformation phenotypes. The distinct patterns which were obtained for polyoma and simian virus 40 transformants led to the conclusion that these two viruses express different transforming abilities in rat cells. The results obtained with temperature-sensitive mutant-derived transformants indicate that all of the transformation characteristics studied so far may be under the control of a viral function in polyoma tsa-transformed cells.     

Inhibition of a plasminogen activator from oncogenic virus-transformed mouse cells by rabbit antibodies against the enzyme

Antisera were raised in rabbits against an electrophoretically pure 48 000 dalton plasminogen activator from mouse cells transformed by an oncogenic virus. The IgG fraction of the antisera inhibited 48 000 dalton mouse plasminogen activators from a variety of sources (neoplastic and nonneoplastic), a 29 00) dalton plasminogen activator from mouse urine and a 48 000 dalton plasminogen activator from rat urine. No inhibition was observed of a 75 000 dalton plasminogen activator extracted from mouse lung, of mouse plasmin or of plasminogen activators from human urine and from oncogenic-virus transformed chicken cells. The IgG antibodies were stronger and more specific inhibitors of the 48 000 dalton mouse plasminogen activator than any previously tested compounds.     

Regulation of urokinase- and tissue-type plasminogen activator gene expression in the rat seminiferous epithelium

The secretion of plasminogen activator by seminiferous tubules at defined stages of the epithelial cycle is influenced both by neighboring spermatogenic cells and by hormones. We have used cRNA probes for urokinase-type (uPA) and tissue-type (tPA) plasminogen activators to analyze their mRNA levels in different stages of the epithelial cycle. Urokinase-type PA mRNA was most abundant in stages VII-VIII, while tPA mRNA levels showed smaller variations between the different stages. Both FSH and (Bu)2cAMP increased the steady-state level of tPA mRNA and tPA production without affecting those of uPA in stages VII-IX in vitro, whereas retinoic acid treatment selectively increased the concentration uPA mRNA and uPA production in stages II-VI. The results show that the expression of the uPA and tPA genes is differentially regulated in specific stages of the rat seminiferous epithelium.     

Modulation of urokinase plasminogen activator gene expression during the transition from quiescent to proliferative state in normal mouse cells.

We have investigated the regulation of urokinase (u-PA) mRNA in quiescent mouse fibroblasts and keratinocytes stimulated to divide by the addition of serum or epidermal growth factor (EGF), respectively. Serum stimulation of quiescent fibroblasts (BALB/c 3T3 or Swiss 3T3) results in an early and transient increase of u-PA mRNA level, which precedes by several hours the onset of DNA synthesis. A similar response is elicited by EGF stimulation of quiescent keratinocytes. The increase of u-PA mRNA parallels that of c-myc mRNA, does not require protein synthesis and is at least in part due to increase in template activity of the u-PA gene. Induction of terminal differentiation of mouse keratinocytes results in a decrease of u-PA mRNA which parallels the decrease of thymidine incorporation. In conclusion, variation in the level of u-PA mRNA is seen during G0/G1 transition and correlates with the proliferative state of these normal mouse cells.     

Purification and characterization of an inhibitor of plasminogen activator released by rat mammary adenocarcinoma cells

An inhibitor of plasminogen activator (PA) secreted by a tumorigenic, but non-metastatic, rat mammary adenocarcinoma cell line has been purified to apparent homogeneity and characterized. It strongly inhibited human urokinase, but was 100 times less potent in inhibiting bovine trypsin and had no effect on plasmin or thrombin. A secreted, urokinase-type PA (Mr 48 000) and a cell-associated PA from a metastatic rat adenocarcinoma cell line were also strongly inhibited. In contrast, a tissue-type PA (Mr 66 000), secreted by human melanoma cells, was only slightly inhibited. Purified inhibitor showed a band of Mr 66 000 in sodium dodecyl sulphate/polyacrylamide gel electrophoresis and an isoelectric point of 4.5 after chromatofocusing. The inhibition of human urokinase was non-competitive.     

Enhanced production and extracellular deposition of the endothelial- type plasminogen activator inhibitor in cultured human lung fibroblasts by transforming growth factor-beta

Cultured human embryonic lung fibroblasts were used as a model to study the effects of transforming growth factor-beta (TGF beta) on the plasminogen activator (PA) activity released by nontumorigenic cells into the culture medium. The cells were exposed to TGF beta under serum- free conditions, and the changes in PA activity and protein metabolism were analyzed by caseinolysis-in-agar assays, zymography, and polypeptide analysis. Treatment of the cells with TGF beta caused a significant decrease in the PA activity of the culture medium as analyzed by the caseinolysis-in-agar assays. The quantitatively most prominent effect of TGF beta on confluent cultures of cells was the induction of an Mr 47,000 protein, as detected by metabolic labeling. The Mr 47,000 protein was a PA inhibitor as judged by reverse zymography. It was antigenically related to a PA inhibitor secreted by HT-1080 tumor cells as demonstrated with monoclonal antibodies. The induced Mr 47,000 inhibitor was deposited into the growth substratum of the cells, as detected by metabolic labeling, immunoblotting analysis, and reverse zymography assays of extracellular matrix preparations. TGF beta also decreased the amounts of urokinase-type and tissue-type PAs accumulated in the conditioned medium, as detected by zymography. Epidermal growth factor antagonized the inhibitory effects of TGF beta by enhancing the amounts of the PAs. These results indicate that growth factors modulate the proteolytic balance of cultured cells by altering the amounts of PAs and their inhibitors.     

Regulation of the expression of type 1 plasminogen activator inhibitor in Hep G2 cells by epidermal growth factor

To identify factors potentially influencing expression of type 1 plasminogen activator inhibitor (PAI-1), we characterized the human tissue-specific distribution of PAI-1 mRNA and the influence of epidermal growth factor (EGF) on expression of steady state levels of PAI-1 mRNA and secretion of PAI-1 by Hep G2 cells. Two species of PAI-1 mRNA (3.2 and 2.2 kilobases) were detected, and the ratio of the two varied among tissues (3 to 5:1) in contrast to the 1:1 ratio detected in Hep G2 cells. Expression of PAI-1 mRNA was inversely related to the distribution of tissue-type plasminogen activator mRNA (2.3 kilobases). Nu-Serum, a growth media supplement, increased steady state levels of PAI-1 mRNA 5-fold within 3 h. Factors responsible were found to be trypsin-sensitive and dialysis-resistant. Antisera to EGF attenuated Nu-Serum-induced increases of PAI-1 mRNA by 57%, suggesting that EGF or EGF homologous peptides contributed to the response. EGF elicited increases of PAI-1 mRNA levels in a dose-dependent manner. Induction was rapid (7-fold at 3 h with 5 ng/ml) and complete within 10 h. The response was not attenuated by cycloheximide (25 micrograms/ml). Factor X and glyceraldehyde-3-phosphate dehydrogenase mRNA did not increase. Increased levels of PAI-1 antigen were detected in conditioned media of Hep G2 cells by 4 h and were maximal at 8 h (6-fold). We conclude that the expression of PAI-1 mRNA is tissue-specific and regulated by epidermal growth factor in Hep G2 cells.     

Unintegrated viral DNA is synthesized in the cytoplasm of avian sarcoma virus-transformed duck cells by viral DNA polymerase.

We have examined the location, structure, and mechanism of synthesis of unintegrated viral DNA present in fully transformed cultures of avian sarcoma virus-infected duck cells. De novo synthesis of the unintegrated forms several weeks after the initial infection was documented by labeling unintegrated DNA in both strands with 5-bromodeoxyuridine. The unintegrated DNA is synthesized in, and probably confined to, the cytoplasm, and it consists of duplexes of short "plus" strands (ca. 0.5 X 10(6) to 1.0 X 10(6) daltons) and "minus" strands the length of a subunit of the viral genome (ca. 2.5 X 10(6) to 3.0 X 10(6) daltons). The structure of the duplex and the mode of incorporation of density label support the hypothesis that the unintegrated DNA is synthesized from an RNA templated by virus-coded DNA polymerase.