Inhibition of protease activity in cultures of rous sarcoma virus-transformed cells: effect on the transformed phenotype.

We have examined the role of proteolytic activity in the genesis and maintenance of the transformed phenotype by growing cultures of chick embryo fibroblasts transfromed by Rous sarcoma virus either in medium containing plasminogen-free serum or in medium to which protease inhibitors were added. Alterations in morphology, adhesiveness, and hexose transport were used as markers for the transformed state. Addition of the trypsin inhibitors NPGB or Soy Bean Trypsin Inhibitor at concentrations which inhibited transformation-associated fibrinolysis restored adhesiveness and morphology to near normal, but did not affect the rate of hexose transport. Growth of Rous-infected cells in plasminogen-free medium blocked the appearance of morphological and adhesive alterations, but allowed the rate of hexose transport to increase to the transformed level. Thus we were able to separate the appearance of transformation-specific changes in morphology and adhesiveness (which apparently require fibrinolytic activity) from the increased rate of hexose transport (which is independent of fibrinolytic activity). Another trypsin inhibitor, TLCK, although it did not inhibit fibrinolysis, was very effective at restoring adhesiveness and morphology as well as hexose transport to normal. This raises the possibility that there is another, perhaps earlier, protease involved in the genesis of the transformed phenotype.     

Control of the uptake of amino acids by serum in chick embryo cells,untransformed or transformed with rous sarcoma virus

Summary Forty to fifty minutes after removal of serum, the net total uptake of amino acids in growing secondary cultures of normal or virus-transformed chick embryo cells, stopped or proceeded only at a highly reduced rate. In both normal and transformed cells, theinitial (0–40 min) rate of the above uptake was the same in the absence of serum as in its presence. The initial rate of the total uptake of amino acids in growing transformed cells was about the same as in growing normal cells. Neither in the normal nor in the transformed cells was the rate of the total uptake of amino acids reduced by cell confluence alone. In highly dense, hyperconfluent cultures of normal cells in which cell growth was arrested, the rate of uptake in the absence or in the presence of serum was four- to fivefold lower than the rate obtained in growing normal cells under similar conditions; in the absence of serum, the net uptake stopped after 40 min in the hyperconfluent cultures as well. It appears that cells growing in tissue culture require a serum factor for maintenance of the required high rates of uptake of amino acids and that the inhibition of growth at high cell densities is a result of depletion of this factor from serum, or the inability of the cells in a dense culture to respond to the factor. A serum factor is apparently also required for maintenance of the reduced rates of uptake of amino acids observed in hyperconfluent cultures.     

Dynamic mechanical testing of human skin ‘in vivo’

The problems involved in measurement of the dynamic mechanical properties of human skin in vivo are examined. On the basis of these observations, the design of a rotary position servo for in vivo testing is outlined. Using this device, certain hitherto unidentified characteristics of the mechanical behaviour of human skin are illustrated.     

Interactions between cell populations influence expression of the transformed phenotype in irradiated rat tracheal epithelial cells

A combined in vivo-in vitro model has been utilized to evaluate the influence of cell-cell interactions on expression of radiation-induced transformation in irradiated rat tracheal epithelial cells. Two types of cell interactions are evaluated. One type of interaction appears to involve direct cell-cell contact as occurs within the intact tissue. The other type of communication appears to be mediated via a diffusible factor, most likely transforming growth factor type-beta. The data suggest the need to be cautious when extrapolating from transformation data obtained following irradiation of low-density rapidly proliferating cell cultures to the in vivo model.     

Granulated ‘marginal cell layer’ in the rat anterior pituitary gland

A granulated ‘marginal layer cell’ was observed in the lining of Rathke's residual pouch of 5 and 10 day-old rat anterior pituitary glands. Immunohistochemistry was not employed to identify the precise function of these cells. However, the cytological characteristics of nearly all of the cells indicated that they resembled GH-secreting cells, with a few displaying morphological features of corticotrophs. In pituitary glands of 5–20 day-old rats, both ends of Rathke's residual pouch extended into the pars distalis at the site of transitional zone of this lobe and of the pars intermedia. The cells within the ‘invading’ residual pouch contained numerous microvilli. In the middle portion of the residual pouch, cavities lined by ‘marginal layer cells’ had numerous microvilli and were adjoined by junctional complexes. In the adult rat pituitary gland, there were no granulated cells in the ‘marginal cell layer’ and no invasion of the residual pouch into the anterior lobe. From these data the possible source of the follicle and of the folliculo-stellate cells in the anterior pituitary of the rat is proposed.     

Quantitation of the viral DNA present in somatic cell hybrids between mouse and SV40-transformed human cells

Recent studies of somatic cell hybrids between mouse cells and SV40-transformed human cells have demonstrated a correlation between the expression of SV40 T-antigen and the presence of human chromosome 7. We have used two types of nucleic acid hybridization procedures to detect and quantitate the presence of viral DNA sequences in the DNA of the hybrid cell clones. Results of reassociation kinetics as well as hybridization with a single-strand probe indicate that SV40 DNA is present only in those hybrid clones which both contain human chromosome 7 and express the SV40 T-antigen. SV40 DNA was not detectable either in the clones which had lost human chromosome 7, or in the rare clones which retain human chromosome 7 but which do not express T-antigen. We have thus extended the correlation between human chromosome 7 and the SV40 T-antigen to the presence of integrated SV40 DNA in somatic cell hybrid clones.     

Conditions required for activation of the mouse albumin or α-fetoprotein gene in hybrids between mouse lymphoblastoma and rat hepatoma cells

Activation of two previously silent mouse hepatic genes has been investigated in hybrid cells between pseudodiploid mouse lymphoblastoma cells and hyperdiploid or hypertetraploid rat hepatoma cells. In this material, activation of the mouse albumin gene is a frequent event, whereas activation of mouse alpha-fetoprotein (AFP) occurs only in those cells that produce large amounts of albumin. Quantitative tests of hybrid populations for the activated proteins and their mRNAs revealed the expected sizes and structures: moreover, as in hepatoma cells, the amount of both rat and mouse albumin produced was directly proportional to the intracellular concentration of the corresponding mRNA. The cellular environment required for activation of the liver-specific genes was investigated by cell-by-cell analysis of each hybrid clone. Immunostaining for the presence of rat and mouse albumin and mouse AFP revealed unexpected heterogeneity in the phenotypes of the hybrid populations, which were found to contain cells that: (a) failed to express either of the proteins; (b) produced all three; (c) produced both rat and mouse albumin; or (d) produced rat albumin only. Karyotypic analysis indicated that the hybrid-cell phenotype depended on parental chromosome ratios rather than absolute numbers of chromosomes. It was found for albumin and mouse AFP that the fraction of immunostained cells was equal to the fraction of metaphases that contained a minimal rat-to-mouse chromosome ratio of 2.5 and 9, respectively. It is concluded that in those hybrids, expression of liver-specific genes is regulated by extinguishers, but in a dose-dependent fashion, suggesting the intervention of antagonistic activators from the rat hepatoma chromosomes.     

Developmental characteristics of somatic cell hybrids between totipotent mouse teratocarcinoma and rat intestinal villus cells

Hybrids between mouse PCC4-azal teratocarcinoma cells and rat epithelial intestinal villus cells (PCI hybrids) are phenotypically teratocarcinoma cells. They express several teratocarcinoma-specific traits but do not express functions specific for differentiated cells. Tumour formation is partially or completely suppressed. Some of the hybrids show more extensive differentiation both in vitro and in vivo than the PCC4-azal parental line. The hybrids are capable of endoderm formation in monolayer cultures and of the formation of embryoid bodies in suspension cultures. Two of the tumour-forming hybrids generate derivatives of all three germ layers, whereas differentiation in the PCC4-azal tumours is restricted to the formation of primitive neuronal tissues.     

Reduced synthesis of pp60src and expression of the transformation-related phenotype in interferon-treated Rous sarcoma virus-transformed rat cells.

Treatment of Rous sarcoma virus-transformed rat cells with rat interferon-alpha (specific activity, 10(6) U/mg of protein) for 24 h caused a 50% reduction in intracellular pp60src-associated protein kinase activity. Staphylococcus aureus V8 protease digestion of pp60src, derived from 32P-labeled monolayer cultures incubated with or without interferon, revealed no differences either in the phosphopeptide pattern or in the phosphoserine-phosphotyrosine ratio. However, [3H]leucine pulse-labeling experiments showed that the synthesis of pp60src was reduced by 42 to 48%, relative to the level of bulk protein synthesis, in the interferon-treated cultures. Rat interferon-alpha also reduced the growth rate of Rous sarcoma virus-transformed rat cells in a dose-dependent manner over a 72-h period. The decrease in growth rate was accompanied by increases in the thickness and number of actin fibers per cell and by a decline in intracellular tyrosine phosphorylation by pp60src. The results suggest that interferon can inhibit the expression of the transformation-related phenotype by selectively reducing the synthesis of the Rous sarcoma virus transforming gene product. However, the interferon effects on the cytoskeletal organization and proliferation of Rous sarcoma virus-transformed cells may be due at least in part to the predominance of interferon-induced phenotypic changes over those caused by pp60src.     

Expression of the Rous sarcoma virus src gene in avian macrophages fails to elicit transformed cell phenotype.

Infection of avian macrophages with Rous sarcoma virus does not induce any changes in the morphology, growth behavior, or expression of macrophage-specific proteins. The absence of cellular transformation does not result from a block in the synthesis of viral proteins, since infectious viruses are released from a majority of cells in the culture. In this report, we examine the synthesis, processing, and functional activity of pp60src in Rous sarcoma virus-infected macrophages to determine whether the absence of transformation is due to an alteration in the functional expression of pp60src. Although the absolute level of pp60src was reduced compared with fibroblasts, the protein exhibited the same phosphorylation pattern and subcellular distribution and was able to phosphorylate immunoglobulin in the immune complex-protein kinase assay. These results imply that the failure of Rous sarcoma virus to transform macrophage may be due to a restriction in the cellular response to a functional src protein, perhaps due to the absence of cellular products which are essential for mediating pp60src-induced transformation.     

Expression of viral p21ras during acquisition of a transformed phenotype by rat adrenal cortex cells infected with Kirsten murine sarcoma virus.

Rat adrenal cortex cells infected with Kirsten murine sarcoma virus acquire a transformed phenotype in a progressive fashion. The expression of the viral p21ras does not appear to correlate with the degree of transformation of the adrenocortical cells but rather is produced at similar levels as the culture becomes transformed. This indicates that the expression of an oncogenic form of p21ras is not of itself sufficient to completely transform rat adrenal cortex cells.     

‘Cross-talk’ between phospholipase C and adenylyl cyclase involves regulation of G-protein levels in GH3 rat pituitary cells

We have investigated the possibility that adenylyl cyclase (AC) activity and membrane protein levels of the α-subunits of the stimulatory and inhibitory G-proteins of AC (G_sα and G_i−2α) in cultured prolactin-producing rat pituitary adenoma cells (GH₃ cells) are modulated by phospholipase C (PLC)-generated second messengers. Pretreatment of cells (6–48 h) with ionomycin (1 μM) or 1-oleoyl-2-acetylglycerol (OAG; 1μM) showed that ionomycin regulated G_sα levels in a time-dependent, biphasic manner; a two-fold increase followed a 40% initial reduction, while OAG lowered G_sα levels by more than 50% at all time-points. G_i−2α levels remained unchanged by both pretreatments. OAG, but not ionomycin, increased basal AC activity without increasing enzyme protein levels. Alterations in AC responsiveness to peptide hormones (e.g. thyroliberin and vasoactive intestinal peptide) correlated to membrane G_s protein α-subunit content. These results demonstrate the involvement of G-protein translation regulation as one mechanism of ‘cross-talk’ between the PLC- and AC-dependent signalling pathways.     

Regulation of expression of tyrosine aminotransferase in somatic cell hybrids between rat hepatoma cells and mouse fibroblasts

Somatic cell hybrids between rat hepatoma cells and mouse 3T3 fibroblasts fail to produce the liver-specific enzyme tyrosine aminotransferase. A novel approach using gamma-irradiation to induce chromosome loss from the non-expressing parent cell was applied to dissect genetically the factors in 3T3 cells that interact with the regulation of expression of tyrosine aminotransferase in these hybrids. Suppression of basal and steroid-inducible tyrosine aminotransferase activities was progressively relieved with increasing dose of radiation. The wide range in degree of reexpression suggests a complex of regulatory mechanisms. Suppression of steroid-inducibility was not linked to the mouse X-chromosome. Nor did the mouse genome affect the modulation of enzyme activity induced by insulin and by serum.     

Dissociation between transformed and differentiated phenotype in rat thyroid epithelial cells after transformation with a temperature-sensitive mutant of the Kirsten murine sarcoma virus.

Differentiated rat thyroid epithelial cells, infected in vitro with a temperature-sensitive mutant of the Kirsten murine sarcoma virus, expressed at the permissive temperature (33 degrees C) some phenotypic properties typical of transformed cells, including morphological features, colony formation in agar, and induction of tumors in newborn animals. Specific functional markers of these differentiated cells, i.e., synthesis/secretion of thyroglobulin, synthesis of thyroglobulin mRNA and iodide uptake, were blocked during growth at 33 degrees C. Normal morphology, failure to grow in agar, and the requirement of hormones for optimal growth were all restored after shifting to the temperature nonpermissive for transformation (39 degrees C), though the typical differentiated functions remained blocked. Infection with a leukemia helper virus clone (Moloney or Kirsten murine leukemia virus) did not lead to the loss of the differentiated phenotype of rat epithelial thyroid cells, thus demonstrating that the loss of the differentiated phenotype is caused by the sarcoma virus component. These results indicate that the expression of some of the phenotypic properties of transformed differentiated rat thyroid epithelial cells is under the direct control of the p21 thermosensitive activity, whereas the block in the expression of two typical differentiation markers of thyroid epithelial cells is irreversible and probably controlled by different mechanisms.