Hyperproduction of human interferon gamma by rat cells maintained in low-serum medium using the fibronectin gene promoter.

An expression vector, pF1900M, which expresses a cloned gene at a high level in quiescent mammalian cells was constructed using the rat fibronectin (FN) promoter. Human interferon gamma (HuIFN-gamma) cDNA inserted downstream of the FN promoter in pF1900M was introduced into rat 3Y1 cells and several IFN-producing cell lines were established. These cells secreted a low level of IFN when they were growing but secreted at a high level after they had reached confluence. The level was further increased when the confluent cells were maintained in low-serum medium and a cell line, I7, produced 4 x 10(5) IU/ml of IFN, comparable to that produced by genetically engineered Escherichia coli in 2 days. The IFN-producing ability of I7 cells could be maintained by successive replacements of low-serum medium for at least 2 weeks. HuIFN-gamma secreted into the medium had a molecular weight range of 22,000 to 25,000, similar to that of IFN-gamma produced by human lymphocytes. The N-linked glycosylation of HuIFN-gamma seemed to occur properly, since treatment of the IFN with N-glycanase resulted in a reduction of molecular weight to 17,000, which corresponds to that calculated from the deduced amino acid sequence of HuIFN-gamma.     

Intracellular and secreted proteins of density-inhibited, serum-arrested and proliferating mouse embryo cells

Short-term labelling of secondary cultures of mouse embryo fibroblasts with [14-C] aminoacids enabled the identification and quantitation of proteins specific for quiescent and proliferative stages. Intracellular and secreted proteins of cells maintained under different growth conditions were resolved in high resolution SDS-polyacrylamide gradient gels. Two proteins, identified as fibronectin and procollagens and a 34 000 D polypeptide were found to be secreted by all three types (density-arrested, serum arrested and proliferating) of cells. Both types of arrested cells exclusively secreted a 375 000 D protein while the proliferating cells specifically secreted a 48 000 D polypeptide. During progression of cells from quiescence to proliferation, two intracellular proteins showed major variations. A 205 000 D intracellular protein was found to be synthesized in higher amounts by proliferating cells than by arrested cells. Another protein, identified as actin, showed a marked increase in synthesis following the release of cells from serum arrest. The arrested cells showed reduced levels of actin synthesis and the turning-off process in the synthesis of actin was found to be relatively slow as the cells entered into quiescence.     

Alteration of the chemotactic response of NIH/3T3 cells to PDGF by growth factors, transformation, and tumor promoters

The platelet-derived growth factor (PDGF) is a potent chemoattractant for cells that respond to PDGF as a mitogen. The chemotactic response of these cells to PDGF is inversely related to their rate of proliferation, with quiescent cells exhibiting a 25-fold greater chemotactic response than exponentially growing cells. Factors that stimulate the growth of quiescent cells (EGF, FGF, PDGF, and serum) decrease the cells' migratory response to PDGF but not to fibronectin, suggesting that the decreased migration is not due to a general paralysis of cell motility. Transformed lines of NIH/3T3 cells lose their ability to respond to PDGF as a chemoattractant but can still migrate in response to fibronectin. Similarly, after treatment of 3T3 cells with the tumor-promoter phorbol myristate acetate, which induces a transformation-like phenotype, the cells no longer respond to PDGF as a chemoattractant but retain their migratory response to fibronectin. Thus it appears that the growth state of the cells can alter their migratory response to PDGF. These data suggest that growth factors, transformation, and tumor promoters specifically alter the cells' ability to respond to the PDGF-mediated chemotactic signal. It appears that both transformation and tumor promoters accomplish this by altering PDGF-binding to the cell surface.     

Plasma fibronectin is synthesized and secreted by hepatocytes

Primary cultures of hepatocytes of rats and hamsters were established and examined for the synthesis and secretion of fibronectin. Hepatocytes of both species secreted fibronectin as a soluble dimeric protein which could be purified by its affinity for gelatin and using specific antisera. Plasma and cellular fibronectins could be clearly resolved on two-dimensional gels. In both species, the majority of the fibronectin secreted by hepatocytes was of the plasma type, as shown by analyses on one- and two-dimensional gels. The secretion of plasma fibronectin increased with time in culture, both in absolute terms and relative to the secretion of albumin. Even during the first day of culture, the secretion of fibronectin relative to that of albumin appeared to be sufficient to account for the relative levels of these two proteins in plasma. Hepatocytes of both species secreted preferentially the chain of plasma fibronectin with higher apparent molecular weight, although the faster migrating chain was also secreted. In addition, hamster hepatocytes cultured for 2 or more days appeared to secrete a cellular form of fibronectin. Possible origins for the different chain types of cellular and plasma fibronectins are discussed.     

A 63 kDa protein is secreted from BALB/c-3T3 cells entering the G1 phase from the G0 state

A 63 kDa protein is detectable in the culture fluid of mouse BALB/c-3T3 cells traversing from the G0 state to the G1 phase, whereas it is undetectable in the culture fluid of quiescent or growing BALB/c-3T3 cells. Secretion of the protein is maximal at 10 h after serum addition. G0-specific ts mutant cells (rat tsJT60) also secrete the 63 kDa protein only when the quiescent cells are stimulated by serum addition at permissive temperature. These facts indicate that the 63 kDa protein is secreted only from cells traversing from the G0 state to the G1 phase.     

Relation between the regulation of DNA synthesis and the production of two secreted glycoproteins by 12-O-tetradecanoylphorbol-13-acetate in 3T3 cells and in phorbol ester nonresponsive 3T3 variants

12-O-tetradecanoylphorbol-13-acetate (TPA), a potent tumor promoter, acts similarly to growth factors by selectively increasing the rate of production of the secreted proteins, mitogen regulated protein (MRP) and major excreted protein (MEP) by murine 3T3 cells. MRP, a 34 kilodalton (kDa) glycoprotein, is a member of the prolactin-growth hormone family of proteins. MEP, a 39 kDa glycoprotein, is a lysosomal thiol protease that is also secreted. The aim of our investigation was to determine the relation between increases in MRP and MEP production and the initiation of DNA synthesis in response to mitogens. The TNR-9 cell line is a variant of 3T3 cells in which growth factors, but not TPA and teleocidin, stimulate DNA synthesis and cell division. Using [35S]methionine to metabolically label proteins and SDS polyacrylamide gel electrophoresis to resolve the proteins, we found that growing cultures of 3T3 and TNR-9 cells responded equally well to TPA and teleocidin with increased rates of production of MRP and MEP. By contrast, the responses of quiescent TNR-9 cells to these tumor promoters in the increased production of MRP and MEP was greatly diminished compared with quiescent 3T3 cells. The changes in production of MRP in response to tumor promoters in quiescent and growing cells paralleled similar changes in the level of MRP mRNA. In summary, the ability to TPA and teleocidin to increase the rate of production of MRP and MEP correlated with the ability of these tumor promoters to stimulate DNA synthesis in quiescent 3T3 and TNR-9 cells. Evidently the biochemical condition that distinguishes TNR-9 from 3T3 cells and that limits the ability of tumor promoters to stimulate the production of MEP and MRP, and perhaps also DNA synthesis in TNR-9 cells occurs only when the cells are quiescent.     

Correlation between a specific molecular weight form of plasminogen activator and metabolic activity of 3T3 cells

In quiescent cultures of 3T3 cells, plasminogen activator (PA) is found predominantly as a 75,000 dalton species. When quiescent cells are exposed to mitogenic agents such as phorbol myristate acetate, Ca++, or 25% serum, the absolute levels of PA in cell lysates may either increase or decrease. However, a consistent observation is that in the stimulated cultures PA is found predominantly as a 49,000 dalton species. This also is the predominant form of PA in growing and transformed cells. Concomitant with the mitogen-induced stimulation of the 49,000 dalton PA in quiescent cultures is a change in morphology to one that is characteristic of growing and transformed cells. The data suggest that PA is not operative in causing the morphological change that occurs with activation; however, the 49,000 dalton PA in particular is closely related to the pleiotypic response accompanying growth stimulation and transformation.     

H1 variant synthesis in proliferating and quiescent human cells

The synthesis of histone H1 isoprotein species in human cells of several different types and in several different physiological states was studied. Up to five H1 and two H1 degrees isoprotein species could be resolved by two-dimensional electrophoresis. All five H1 isoprotein species were synthesized in exponentially growing cultures of IMR-90 human fibroblasts; in quiescent IMR-90 cells the synthesis of three H1 isoprotein species was greatly decreased while the synthesis of two others was much less affected. When DNA synthesis in exponentially growing cultures of IMR-90 was inhibited, the pattern of H1 isoprotein synthesis became similar to that found in quiescent cultures. Other human cells, isolated from blood, yielded similar results. These results suggest that the pattern of H1 synthesis is the same for cells in non-S phases of the cell cycle and in quiescent cells. Thus for histone H1 in human cells the relationship of the variant synthesis pattern to the growth state and DNA replication is similar to that of the core histone H3 but not that of H2A.     

The activities of MMP‐9 and total gelatinase respond differently to substrate coating and cyclic mechanical stretching in fibroblasts and myoblasts

The current study was designed to investigate whether the activities of TGC (total gelatinase and collagenase) as well as MMP‐9 (matrix metalloproteinase‐9, gelatinase B) secreted by the cultured fibroblasts and myoblasts were influenced by the specific extracellular substrates and by cyclic mechanical strain. Fibroblasts (Rat 2) and myoblasts (C2C12) were cultured with either fibronectin, laminin or collagen type I for 24 h and applied with or without a biaxial deformation at 1 Hz using the Flexcell FX‐4000 system. MMP‐9 activity was increased in fibroblasts when the cells were in contact with fibronectin and laminin, while in myoblasts, enhanced activity of the secreted enzyme was only observed when collagen was present. TGC activity expressed from myoblasts was increased in cells growing on all three types of extracellular proteins in response to the mechanical stimulation, but in fibroblasts, such an increase was only observed in cells grown on the laminin coating. In summary, our data demonstrate that the activities of MMP‐9 synthesized by fibroblasts tend to be regulated by the specific extracellular protein the cells are in contact with, whereas the gelatinolytic actions of proteases produced by myoblasts are more responsive to the mechanical deformation.     

Biosynthesis and secretion of fibronectin in human melanoma cells

The biosynthesis and secretion of cellular fibronectin from human melanoma cells have been investigated by pulse-chase/immunoprecipitation analysis. Melanoma cells synthesize endoglycosidase H (Endo H)-sensitive glycoprotein precursors of fibronectin glycoproteins which chase to an Endo H-resistant monomer with an apparent Mr of 240,000 (240 K). This molecule, which has a significantly higher molecular weight than normal plasma or cellular fibronectin, is rapidly secreted by melanoma cells, resulting in the secretion of 80% of newly synthesized fibronectin in 120 min, following a 10-min biosynthetic pulse. This active secretory process can be inhibited by brief exposure of melanoma cells to sodium monensin (10(-7) M), which also results in a modified fibronectin of lower apparent Mr. Monosaccharide-incorporation studies of melanoma fibronectin reveal that monensin significantly inhibits galactose and fucose incorporation into this glycoprotein, correlating with reported effects of monensin on Golgi apparatus functions. These studies indicate that this tumor-associated and biosynthetically altered cellular fibronectin is a rapidly secreted major N-linked glycoprotein of metastatic human melanoma cells.     

Differential repair of potentially lethal damage in exponentially growing and quiescent 9L cells

The alteration of potentially lethal damage repair by postirradiation treatment with hypertonic saline (0.5 M PBS) was investigated in exponentially growing and quiescent 9L cells in vitro. A single dose of X rays (8.5 Gy) immediately followed by a 30-min treatment with hypertonic PBS at 37 degrees C reduced the survival of exponentially growing 9L cells by a factor of 13-18 compared to survival of irradiated immediately and delayed-plated cells, while the survival of quiescent cells was reduced by only a factor of 5-8. Survival curves confirmed the relative resistance of the quiescent 9L cells versus exponentially growing 9L cells to X rays plus hypertonic treatment. Both the slope and the shoulder of the survival curve were reduced to a greater extent in exponentially growing cells than in the quiescent cells by hypertonic treatment. The response of quiescent cells cannot be explained by either the duration of hypertonic treatment or the redistribution of the cells into G1 phase. We show that quiescent 9L cells can recover from hypertonically induced potentially lethal damage when incubated under conditions which have been found to delay progression through the cell cycle, and postulate that an altered chromatin structure or an enhanced repair capacity of quiescent 9L cells may be responsible for their resistance.     

Cloning of a growth arrest-specific and transforming growth factor beta-regulated gene, TI 1, from an epithelial cell line.

By cDNA cloning and differential screening, five genes that are regulated by transforming growth factor beta (TGF beta) in mink lung epithelial cells were identified. A novel membrane protein gene, TI 1, was identified which was downregulated by TGF beta and serum in quiescent cells. In actively growing cells, the TI 1 gene is rapidly and transiently induced by TGF beta, and it is overexpressed in the presence of protein synthesis inhibitors. It appears to be related to a family of transmembrane glycoproteins that are expressed on lymphocytes and tumor cells. The four other genes were all induced by TGF beta and correspond to the genes of collagen alpha type I, fibronectin, plasminogen activator inhibitor 1, and the monocyte chemotactic cell-activating factor (JE gene) previously shown to be TGF beta regulated.     

Senescent and quiescent cell inhibitors of DNA synthesis : Membrane-associated proteins

Cytoplasts derived from senescent and quiescent human diploid cells inhibit DNA synthesis initiation when fused with cells capable of proliferation. When the cytoplasts were subjected to a variety of conditions (trypsin and cycloheximide treatment and growth on fibronectin), this inhibitory activity was lost, suggesting that the inhibitors involved were proteins associated with the surface membranes of the cells. We have studied the quiescent cell inhibitor in greater detail and determined that surface membrane-enriched preparations isolated from quiescent cells and proteins extracted from these membrane preparations have DNA synthesis-inhibitory activity.     

An immunological approach to quantifying the saprotrophic growth dynamics of Trichoderma species during antagonistic interactions with Rhizoctonia solani in a soil-less mix

Studies of the saprotrophic growth dynamics of Trichoderma species and their fungal hosts during antagonistic interactions are severely hampered by the absence of methods that allow the unambiguous identification and quantification of individual genera in complex environments such as soil or compost containing mixed populations of fungi. Furthermore, methods are required that allow discrimination between active hyphal growth and other components of fungal biomass such as quiescent spores that are produced in large numbers by Trichoderma species. This study details the use of monoclonal antibodies to quantify the saprotrophic growth dynamics of the soil-borne plant pathogen Rhizoctonia solani and biological control strains of Trichoderma asperellum and Trichoderma harzianum during antagonistic interactions in peat-based microcosms. Quantification was based on the immunological detection of constitutive, extracellular antigens that are secreted from the growing tip of Rhizoctonia and Trichoderma mycelium and, in the case of Trichoderma harzianum, from quiescent phialoconidia also. The Trichoderma-specific monoclonal antibody (MF2) binds to a protein epitope of the enzyme glucoamylase, which was shown by immunofluorescence and immunogold electron gold microscopy studies of Trichoderma virens in vitro to be produced at the origin of germ tube emergence in phialoconidia and from the growing tip of germ tubes. In addition, a non-destructive immunoblotting technique showed that the enzyme was secreted during active growth of Trichoderma asperellum mycelium in peat. The Rhizoctonia solani-specific monoclonal antibody (EH2) similarly binds to a protein epitope of a glycoprotein that is secreted during active mycelial growth. Extracts derived from lyophilized mycelium were used as a quantifiable and repeatable source of antigens for construction of calibration curves. These curves were used to convert the absorbance values obtained in ELISA tests of peat extracts to biomass equivalents, which allowed comparisons of the saprotrophic growth dynamics of the pathogen and antagonists to be made in single or mixed species microcosms. Trichoderma species were able to compete successfully with R. solani for nutrients and to prevent saprotrophic growth of the pathogen. Specificity of the Trichoderma quantitative assay was tested in non-sterile soil-based microcosms artificially inoculated with T. asperellum. The assay was highly specific and only detected T. asperellum population dynamics. No cross-reactivity was found with extracts from soil samples containing contaminant fungi.     

Transformation of rat liver cell line by Rous sarcoma virus causes loss of cell surface fibronectin, accompanied with secretion of metallo-proteinase that preferentially digests the fibronectin

An epithelial cell line derived from the liver of a normal Buffalo rat (BRL) was transformed by Rous sarcoma virus (RSV). The RSV-transformed cells were separated into five clones (RSV-BRL1 through 5), which were morphologically different. RSV-BRL cells exhibited the following characteristics distinct from those of BRL cells: tumorigenicity, irregular cell arrangement, loose intercellular junction, growth in soft agar (anchorage-independent growth) except for RSV-BRL3 and 5, and loss of cell surface fibronectin. When BRL cells were cultured in the standard medium supplemented with the serum-free conditioned medium of RSV-BRL cells, the amount of the cell surface fibronectin decreased significantly. It was found that RSV-BRL cells secreted a proteinase capable of hydrolyzing the fibronectin, whereas BRL cells secreted hardly any of this proteinase. The fibronectin-hydrolyzing proteinase (FNase) could also hydrolyze plasma fibronectin added as an exogenous substrate. The hydrolysis of plasma fibronectin was inhibited by ethylenediamine tetraacetate, but stimulated by rho-chloromercuribenzoate and calcium ion. This indicates that FNase is a metallo-enzyme, but not a serine or thiol enzyme. In addition to the proteinase, RSV-BRL cells secreted plasminogen activator and a proteinase inhibitor which inhibited the activity of plasmin but not FNase.     

Quiescence in 9L cells and correlation with radiosensitivity and PLD repair

The onset of quiescence, changes in X-ray sensitivity, and changes in capacity for potentially lethal damage (PLD) repair of unfed plateau-phase 9L44 cell cultures have been systematically investigated. The quiescent plateau phase in 9L cells was the result of nutrient deprivation and was not a cell contact effect. Eighty-five to 90% of the plateau-phase cells had a G1 DNA content and a growth fraction less than or equal to 0.15. The cell kinetic shifts in the population were temporally correlated with a developing radioresistance, which was characterized by a larger shoulder in the survival curve of the quiescent cells (Dq = 5.71 Gy) versus exponentially growing cells (Dq = 4.48 Gy). When the quiescent plateau-phase cells were refed, an increase in radiosensitivity resulted which approached that of exponentially growing 9L cells. Delayed plating experiments after irradiation of exponentially growing cells, quiescent plateau-phase cells, and synchronized early to mid-G1-phase cells indicated that while significant PLD repair was evident in all three populations, the quiescent 9L cells had a higher PLD repair capacity. Although data for immediate plating indicated that 9L cells may enter quiescence in the relatively radioresistant mid-G1 phase, the enhanced PLD repair capacity of quiescent cells cannot be explained by redistribution into G1 phase. When the unfed quiescent plateau-phase 9L cells were stimulated to reenter the cell cycle by replating into fresh medium, the first G1 was extended by 6 h compared with the G1 of exponentially growing or refed plateau-phase 9L cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of protein secretion in Chinese hamster ovary cells by cell cycle position and cell density : Plasminogen activator, procollagen and fibronectin

To study the relationship between cell growth control, cell contact, and protein secretion, we examined the production of plasminogen activator, procollagen, and fibronectin by Chinese hamster ovary (CHO) fibroblasts, both as a function of position in the cell cycle and as a function of cell density. CHO fibroblasts that were synchronized at hourly intervals throughout the cell cycle by mitotic selection in an automated roller bottle apparatus secreted plasminogen activator only during the G2 and M phases of the cell cycle (10–14 h after mitotic selection). Cell-associated plasminogen activator activity was variable during G1 and S, but was greatly reduced during G2 and M. In contrast, secretion of the connective tissue matrix proteins, procollagen and fibronectin, was controlled by cell density rather than by cell cycle position. Type III procollagen and fibronectin were secreted throughout the cell cycle with no pronounced variations. Type I procollagen was not secreted by cycling cells and was observed in confluent cultures only after 24–48 h. To correlate these changes in protein secretion patterns with cell shape and contact, we used scanning electron microscopy (SEM) to study the appearance of CHO cells after mitotic selection. Actively dividing cells retained a high proportion of rounded, ruffled, and blebbed cells during all phases of the cell cycle. Only with increased cell density in contact-inhibited confluent cultures did most cells begin to flatten and spread. Thus, secretion of and attachment to extracellular matrix did not occur in rapidly dividing cells, but appeared to require the increased cell-cell contact and spreading that accompanies contact inhibition of growth. On the other hand, increased secretion of plasminogen activator was directly related to cell division and may be part of a sequence of events that allows cells growing in culture to loosen extracellular attachments in preparation for rounding and cytokinesis.