The human urokinase-plasminogen activator gene and its promoter.

The urokinase type of plasminogen activator (uPA) is subject to regulation by hormones, phorbol esters and oncogenic transformation. This enzyme has been suggested to play a key role in processes involving cell migration and tissue remodeling, and to be essential for tumor metastasis. In order to study these processes, we have isolated the human uPA gene, and have determined its entire nucleotide sequence. The gene is organized in 11 exons and is 6.4 kb long. The 5' end of uPA mRNA has been determined by both S1 mapping and primer extension experiments. A fragment of 800 bp containing the entire 5' flanking region shows promoter activity when introduced upstream of a bacterial chloramphenicol acetyltransferase gene and introduced into human cells. The hexanucleotide sequence GGCGGG, previously found at similar regions in several viral and eukaryotic promoters and shown to be essential for promoter activity (McKnight et al. (1984) Cell, 37, 253-262), is repeated three times between the CAAT and the TATA boxes.     

Identification of cells responsible for urokinase-type plasminogen activator synthesis and secretion in human diploid kidney cell cultures

Human urokinase-type plasminogen activator (uPA) is a serine protease that converts plasminogen to plasmin. It is produced and secreted by a variety of different human cells in vivo and in vitro. We have studied human diploid kidney cell (HKC) cultures prepared from neonatal kidney tissue and cultures of purified populations of HKC to determine which cells synthesize and secrete uPA into the culture medium. Antibodies against cell specific antigens and uPA were used to correlate specific kidney cell types with uPA synthesis. In addition, secretion of uPA activity into growth and uPA production media was determined for each cell type and cultures containing a mixture of cell types. The results of these studies demonstrated that glomerular visceral epithelial and kidney tubular epithelial cells synthesize and secrete uPA into the culture medium.     

The 'transition probability model' and the regulation of proliferation of human diploid cell cultures during aging.

Genealogies derived from time-lapse cinemicrophotographic studies of aging human diploid cell cultures were analysed in terms of the 'transition probability' model. It was found that the distribution of intermitotic times obtained from middle passage cells deviated only slightly from that predicted by the model. In contrast, the plot for late passage cultures did not fit the predicted pattern and appeared to be composed of multiple curves. These changes are discussed in reference to cellular senescence as expressed by normal human diploid cells in vitro.     

Changes in the cell surface of human diploid fibroblasts during cellular aging.

The electrophoretic mobility of 13 human diploid cell strains, TIG-1, TIG-2, TIG-3, TIG-7, WI-38, IMR-90, MRC-5, MRC-9, TIG-1H, TIG-1L, TIG-2M, TIG-2B, and TIG-3S, which were established from different tissues of human embryos, was studied at different passages. The net negative surface charge of the cells was characteristic for each cell strain and decreased significantly during the in vitro aging of the cells. The decrease in the net negative charge of the cells correlated well with the decrease in cell density throughout the life span of the cells. A strict linear correlation between the electrophoretic mobility and the number of cells harvested at each passage was obtained for all the human diploid cell strains. Moreover, almost the same linear regression coefficient of the cells was obtained among these cell strains. Therefore, the net negative surface charge of human diploid cell strains could serve as a cell surface marker for in vitro cellular aging.     

The fate of the primary diploid population during spontaneous transformation of growth factor-supplemented murine cell cultures

Primary cultures derived from lung and renal tissue of the newborn harvest mouse (Micromys minutus) were serially passaged in media supplemented with epidermal growth factor, hydrocortisone, transferrin, insulin, and triiodothyronine. Although these growth factor supplements eliminated the growth crisis commonly encountered during the initial stages of murine primary cultures, the original diploid cell fraction clearly underwent such a "crisis"; the truly diploid cells invariably disappeared as these cultures reached 20 to 40 population doublings. They were replaced, either gradually or precipitously, by various heteroploid cell fractions. In three of four independent cultures, these "established" cells were hypotetraploid and appeared to be derived from a small number of progenitors already present during the very early (precrisis) culture stages. In contrast to rather frequent DNA changes displayed by clones and subclones derived from the various heteroploid cell lineages, the predominant components of the established mass cultures displayed a highly constant DNA fluorescence pattern. Our results suggest that primary murine cell cultures develop heteroploid cell lineages even if the initial growth crisis is mitigated by growth factor supplements. These heteroploid cells appear to respond more efficiently to stimulation by various growth factors than the primary diploid cell population.     

Alteration of ceramide monohexoside in human diploid fetal lung fibroblasts during cell aging

Serially cultured human diploid fibroblasts have a finite lifetime in vitro, and this phenomenon was postulated as cellular aging. Neutral glycosphingolipids (GSLs) from human fetal lung-derived diploid fibroblast cell lines, WI-38 and TIG-1 cells, were studied during cellular aging. Both cell lines had at least four types of neutral GSLs. It was found that neutral GSLs changed with aging, the most conspicuous alteration being a 2-4-fold increase in the content of ceramide monohexoside. This change was invariably observed in either WI-38 or TIG-1 cells.     

Plasminogen activator from human embryonic kidney cell cultures: Evidence for a proactivator

The nature of the trypsin-activatable plasminogen activator produced by kidney cell cultures (Bernik, M.B. (1973), J. Clin. Invest. 52, 823–834) was investigated using human embryonic kidney (HEK) cell cultures in serum-free medium. Plaminogen activator activity ratios (trypsin-activated/ untreated controls) in HEK cell-conditioned media were maximal (up to 3) during the first week of culture and remained nearly constant at approximately 2 for the next 3–5 weeks, while the total plasminogen activator titer increased in a nearly linear manner. Therefore, coincident with progressive cell dengeration and death, the ratios decreased to near unity due to “spontaneous” activation of the enzyme, which was inhibited in cell-free conditioned media by the pancreatic trypsin inhibitor Kunitz and benzamidine. Since the activator is not inhibited by the trypsin inhibitor, it is concluded that a protease other than the plasminogen activator is responsible for the activation. Increases in the plasminogen activator titers (about 2-fold) were similarly obtained by culturing the cells in medium containing low concentrations (0.05–0.10 μg/ml) of trypsin for up to about 6 weeks. The presence of the trypsin inhibitor in HEK cell cultures decreased the rate of activation, resulting in higher activity ratios (up to 6), and the total plasminogen activator activity was reduced only minimally (<20%), if at all, by the highest concentration of the trypsin inhibitor (100 μg/ml) tested.Affinity chromatography of conditioned media with activity ratios of 1.6–2 separated the plasminogen activator into an active fraction and a fraction which was activated a minimum of 200-fold by trypsin and contained no measurable activity prior to activation. Gel filtration of crude conditioned media or partially purified activator separated the plasminogen activator activity into two peaks; both were trypsin-activatable, and their relative proportions varied with the isolation conditions. The results indicate the occurrence of a proenzyme form of the plasminogen activator in the culture media.     

A new cell surface marker of aging in human diploid fibroblasts

The relationship of cell surface changes to proliferative decline of human diploid fibroblasts was investigated using the concanavalin A-mediated red blood cell adsorption assay. The amount of the red blood cells adsorbed to human diploid fibroblasts via concanavalin A increased continuously from the early phases of cell passage up through cell senescence, while the amount of 3H-concanavalin A binding did not change to a significant extent. The red blood cell adsorption is not a function of cell cycle phase and time spent in culture. Cocultivation of young cells with old cells also did not affect the adsorption capacity of respective cells. Thus, the concanavalin A-mediated red blood cell adsorption can be expected to serve as a new cell surface marker for aging in vitro. Using this marker, it was revealed that transient cell size or 3H-thymidine incorporating capacity di not have a direct relationship with the division age of a cell. Small rapidly dividing cells in old populations resemble large slowly dividing or nondividing cells of the same populations and differ from small rapidly dividing cells in young populations, in terms of cell surface properties.     

Transcription factor activity during cellular aging of human diploid fibroblasts.

Human diploid fibroblasts display a limited proliferative life span in vitro, which is directly correlated to the age of the donor from which the cells were explanted. In an effort to identify molecular events that may underlie the loss of proliferative potential in aging fibroblasts, we have determined, at the protein level, the abundance of several cell-cycle-regulated proteins and the activity of the two major members of the activator protein-1 (AP-1) DNA binding complex. We find that cyclin A and p34cdc2 expression is decreased by two- to four-fold in old fibroblasts, but that Fos expression and binding activity are reduced by as much as 95% in old, as opposed to young cells, despite equivalent amounts of p105Rb and Jun proteins being expressed. We have further determined that the composition of the protein complex which binds a consensus (-TGACTCA-) AP-1 site changes dramatically during in vitro aging. Since we have shown previously that AP-1 activity is required for progression through the cell cycle, we propose that the quantitative and qualitative changes seen in AP-1 may play a direct role in the gradual loss of proliferative ability seen as cells approach senescence.     

Purification and characterization of single-chain urokinase-type plasminogen activator from human cell cultures

A urokinase-type plasminogen activator was purified from conditioned media of several human cell cultures, but preferably from the human lung adenocarcinoma line CALU-3 (ATCC, HTB-55), using a combination of chromatography on zinc chelate-Sepharose, SP-Sephadex C-50, and Sephadex G-100. Final yields of 65-100 micrograms/liter of starting material were obtained with a 290-fold purification factor and a recovery of 30%. The purified plasminogen activator consists of a single polypeptide chain with Mr 54,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and is very similar or identical to single-chain urokinase-type plasminogen activator on the basis of immunodiffusion, amino acid composition, and the lack of specific binding to fibrin. It has very low amidolytic activity on Pyroglu-Gly-Arg-rho-nitroanilide and is converted to two-chain urokinase by limited exposure to plasmin. It has a specific activity of 60,000 IU/mg on fibrin plates and directly activates plasminogen following Michaelis-Menten kinetics with Km = 1.1 microM and kappa cat = 0.0026 S-1. It is concluded that the plasminogen activator purified from CALU-3-conditioned media is physically and kinetically identical to single-chain urokinase-type plasminogen activator. With the present straightforward purification method and a readily available source, sufficient amounts of single-chain urokinase-type plasminogen activator can be obtained for more detailed investigations of its biochemical, biological, and thrombolytic properties.     

Characterization of the cell cycle of cultured human diploid cells: Effects of aging and hydrocortisone

Age-related changes in the cytokinetics of human diploid cells in vitro have been compared in normal cultures and in cultures in which lifespan has been prolonged by the addition of hydrocortisone to the medium. For both cultures, with advancing age the fraction of cells in the actively proliferating pool decreased and the intercellular variation in cell cycle times increased. The average cell cycle time was prolonged during aging due almost entirely to changes in the duration of G₁. The duration of S remained constant, while a small delay in G₂ was observed in late passage cells near the end of their lifespan. Although the same pattern of change in proliferative parameters occurred in both control and hydrocortisone-treated cultures, the changes were somewhat delayed in the presence of the steroid. The results are interpreted in terms of several cell cycle models and suggest that the events controlling cell proliferation are sensitive to hydrocortisone modulation during the G₁ and possibly the G₂ periods.     

Expression of proliferationg cell nuclear antigen during the cell cycle of human diploid fibroblasts

Summary Proliferating cell nuclear antigen mRNA levels were determined in human diploid fibroblasts as they progressed through the cell cycle. PCNA message levels were low at G0, gradually increased following entrance into G1, peaked at G1/S, and declined during S phase. PCNA mRNA was determined to have a half life of 12 hours when cells were blocked at the G1/S interface. PCNA protein levels increased two- to three-fold as cells moved from G0 to S phase.