The effect of the tumor promoter, phorbol myristate acetate (PMA), on hyaluronic acid (HA) synthesis by chicken embryo fibroblasts

Treatment of chicken embryo frbroblasts (CEF) with the tumor promoter, phorbol myristate accetate (PMA), resulted in a rapid increase in their ability to synthesize the glycosaminoglycan, hyaluronic acid (HA), whereas the parent compound, phorbol, had no effect. CEF cultures incubated with PMA (100 ng/ml) for 6 h resulted in a 15-fold increase in HA synthetase activity compared with phorbol-treated control cultures. The incorporation of [³H]acetate into HA and chemical determination of this polymer also demonstrated increased synthesis of HA by cells treated with PMA. We have previously shown that CEF infected with a temperature-sensitive mutant of Rous sarcoma virus, LA24, exhibit increased synthesis of HA upon transformation. PMA treatment of cells transformed with RSV-LA24 results in a further 1.5-fold stimulation of HA synthesis. These data indicate that PMA causes an increased synthesis of HA in CEF which is analogous to the increased synthesis of HA found in virally transformed CEF.     

Plasminogen activator in chick embryo muscle cells: induction of enzyme by RSV, PMA and retinoic acid.

To explore the generality of the effects of sarcoma viruses, tumor-promoting phorbol esters and retinoic acid, we have studied plasminogen activator production in differentiating chick myogenic cultures. Although slightly higher than in chick fibroblast cultures, the level of spontaneously synthesized enzyme is low; it reaches a peak shortly after maximum cell fusion has been completed and then declines. Rous sarcoma virus (RSV) transformation of differentiating myotubes was accomplished by infecting myoblasts with a temperature-sensitive mutant, maintaining cultures at the nonpermissive temperature until completion of fusion and shifting to permissive temperatures at selected times thereafter. RSV transformation, phorbol myristate acetate (PMA) and retinoic acid all induced high levels of plasminogen activator production by differentiating myotubes in the absence of DNA synthesis. In comparison with fibroblasts, virus-induced enzyme synthesis by myogenic cultures proceeded more slowly but ultimately reached comparably high levels. Whereas cAMP strongly repressed RSV- and PMA-induced plasminogen activator production by chick fibroblasts, it weakly stimulated enzyme synthesis by myotubes. This suggests that enzyme induction by RSV and PMA is not mediated primarily through effects on cAMP metabolism.     

Phorbol ester-induced morphological changes in transformed chick fibroblasts: evidence for direct catalytic involvement of plasminogen activator.

The tumor promoter phorbol myristate acetate (PMA) induces the production of the serine protease plasminogen activator (PA) in cultures of normal chick embryo fibroblasts (CEF) and synergistically enhances PA production in Rous sarcoma virus-transformed chick embryo fibroblasts (RSVCEF). Following PMA treatment of serum-free RSVCEF cultures, PA induction is accompanied by distinct morphological changes, including enhanced cell clustering and the formation of dense cellular aggregates. These alterations in the morphology of the PMA-treated transformed cells are inhibited by several protease inhibitors, including leupeptin, NPGB, SBTI, benzamidine and DFP, the specific inhibitor of serine enzymes. A number of protease inhibitors are ineffective in preventing the PMA-induced morphological changes; these include inhibitors of trypsin, chymotrypsin, elastase, thrombin and, most importantly, plasmin. The use of a fluorescent substrate to assay PA directly demonstrated that the pattern of inhibiton of PA activity correlates exactly with the inhibition of morphological changes. The of 3H-DFP to label and characterize serine zymes in the culture fluid from PMA-treated cells further indicated that PA is the serine protease responsible for the morphological changes. Thus PA itself can catalytically alter cellular behavior in culture independent of plasminogen, until not its only known natural substrate.     

Acetylcholine receptor: effects of proteolysis on receptor metabolism

Previous studies (Miskin, R., T. G. Easton, and E. Reich, 1970, Cell. 15:1301-1312) have shown that sarcoma virus transformation and tumor promoters reduced the cell surface concentration of acetylcholine receptors (AChR) in differentiating chick embryo myogenic cultures. Both of these agents also induced high rates of plasminogen activator (PA) synthesis in myogenic cultures (Miskin, R., T. G. Easton, A. Maelicke, and E. Reich, 1978, Cell. 15:1287-1300), and the present work was performed to establish whether proteolysis might significantly affect receptor metabolism. Proteolysis in myogenic cultures was modulated by one or more of the following: stimulation of PA synthesis, direct addition of plasmin, removal of plasminogen, or addition of plasmin inhibitors. The results were: (a) When the rates of proteolysis were raised either by addition of plasmin or by stimulating PA synthesis in the presence of plasminogen, both the steady-state concentration and the half-life of surface AChR decreased, but the rate of receptor synthesis was unaffected. (b) The magnitude of these effects, and their dependence on added plasminogen, indicated that proteolysis initiated by plasminogen activation could account almost entirely for the reduction in receptor half-life produced by sarcoma virus transformation and phorbol ester. (c) The rate of receptor synthesis, which is also reduced by viral transformation and tumor promoters, was not modified by proteolysis; hence plasmin action may be responsible for a large part, but not all of the change in surface receptor under these conditions. (d) The plasmin catalysed changes in receptor parameters appear to occur in response to modified membrane metabolism resulting from proteolysis of surface components other than AChR itself.     

Modulation by the src oncogene of the effect of inhibitory diffusible factor IDF45

Density-dependent inhibition of growth has been assumed to be under the control of inhibitory molecules diffusing from dense cell cultures. Growth inhibitory factors have been fractionated or purified from medium conditioned by different cell types. In the present work, it was shown that IDF45 (inhibitory factor diffusing from 3T3 cells) decreased DNA synthesis in chick embryo fibroblasts (CEF) and was an inhibitor of CEF growth; this inhibition was reversible. Since similitudes between oncogene products and growth factors have been observed, it was of interest to compare the inhibitory effect of IDF45 upon the stimulation of DNA synthesis induced either by serum or by pp60-src. CEF infected by Ny68 virus (a mutant of Rous sarcoma virus ts for the expression of transformation) were density-inhibited at 41 degrees C, but were stimulated at this temperature by addition of 1% serum. This stimulation was 94% inhibited by IDF45. The same Ny68-infected cells could also be stimulated by transfer to 37 degrees C, the permissive temperature (in the absence of serum). The stimulation of DNA synthesis by src expression was poorly inhibited by IDF45. From our results, it appears that oncogene expression in CEF induces a loss in their sensitivity to IDF45. This would explain why transformed cells escape DDI of growth.     

Effects of retinoic acid and other retinoids on the growth and differentiation of 3T3 supported human keratinocytes

Using mitomycin C treated 3T3-Swiss fibroblasts as feeder cells, human keratinocytes derived from infant foreskins were subcultured in the presence of trans-retinoic acid (RA) and other retinoids. At 1 microgram/ml (3 x 10(-6) M) and higher RA concentrations plating efficiency was markedly reduced. Addition of retinoids to 1 microgram/ml after colonies were established produced no change in the rate of cell production, but caused differentiated cells to be sloughed earlier than in control cultures. Electron microscopy showed wider extra cellular spaces the contained numerous villi, increased numbers of microvilli at the surfaces of the outermost cells, and decreased number of cell layers all of which were consistent with the observed desquamatory effects of RA. Retinoic acid also extended the time during which cell population increased so that RA treated cultures produced more cells than control cultures over their respective lifetimes. Keratin polypeptides represented a smaller percentage of the total solubilizable protein and more keratin was present as acid soluble prekeratin in postconfluent RA treated cultures. This may be a consequence of early desquamation rather than a decrease in keratin synthesis. No unusual proteins were visible in RA treated cultures by simple sodium dodecylsulfate electrophoresis, nor was there increase in specific activities of three lysosomal enzymes.     

Metabolism of acetylcholine receptor in chick embryo muscle cells: effects of RSV and PMA.

We have investigated some aspects of the metabolism of the integral membrane protein acetylcholine receptor (AChR) in normal and transformed cultures of chick embryo muscle cells. Turnover of AChR in control muscle cell cultures was compared with turnover in cultures infected and transformed by a temperature-sensitive mutant of Rous sarcoma virus (RSV) and with cultures treated with the tumor promoter phorbol myristate acetate (PMA). The parameters of AChR metabolism were estimated using 125I-alpha-bungarotoxin as a stoichiometric high affinity ligand for the AChR. We found that both RSV transformation and PMA increased the rate of degradation and decreased the rate of synthesis of AChR. The consequent reduction in steady state receptor levels suggests that oncogenic transformation and tumor promoter significantly alter the metabolism of cell surface membranes. We also observed that parameters of AChR metabolism in control cultures changed systematically in a pattern which depended upon the age of the culture as well as the use of embryo extract or fetal bovine serum as medium supplements. The muscle cell system allows quantitative measurement of an integral membrane protein and its metabolism, and may serve as a more general model for alterations in membrane and surface receptor metabolism associated with the transformed state.     

Modulation of synovial fibroblast plasminogen activator and plasminogen activator inhibitor production by protein kinase C.

Phorbol myristate acetate (PMA) added to human synovial fibroblast cultures caused a dose-dependent increase in the production of plasminogen activator inhibitor-type 1 (PAI-1). In addition, PMA inhibited endogenous and interleukin-1 (IL-1) induced plasminogen activator (PA) activity, while increasing mRNA PAI-1 levels. Other protein kinase C (PKC) activators, mezerein and teleocidin B4, caused similar effects. The simultaneous addition of the PKC antagonists, H-7 or staurosporine, prevented the inhibition of PA activity by PMA. This study shows that activation of PKC inhibits PA and stimulates PAI production in human synovial fibroblasts. These results suggest that activation of PKC may play an important role in regulating increased PA production associated with joint destruction in rheumatoid arthritis (RA).     

Replication of the Reticuloendotheliosis Virus (Strain T) in Chicken Embryo Cell Culture

Investigations were conducted on the in vitro replication of the reticuloendotheliosis (RE) virus (strain T) in specific-pathogen-free chicken embryo fibroblast (CEF) cultures. Active virus production was detected in the tissue culture fluid 24 hr after infection. When injected into chickens, samples taken 42 hr after infection of the cell cultures killed approximately 50% of the birds at a 1:100 dilution. The RE virus titer remained at this level for 5 days before declining. Cell-free virus preparations from tissue cultures rarely resulted in 100% mortality of the assay birds. The level of cell-associated virus was very low. Evidence that the reticuloendotheliosis was not induced by a mycoplasma was indicated by failure to isolate an organism on PPLO Agar (Difco) and failure of kanamycin or amphotericin B to inhibit multiplication of RE virus in vitro. RE virus appeared to be unrelated to members of the avian leukosis and sarcoma complex. It did not induce resistance in CEF cultures to sarcoma viruses of the A or B subgroup of this complex. Similarly, preinfection of cell cultures with leukosis viruses of the A or B subgroup did not inhibit or reduce the replication of RE virus.     

Transformation-sensitive isoactin in passaged chick embryo fibroblasts transformed by Rous sarcoma virus

Transformation by Rous sarcoma virus (RSV) has been reported to block the expression of differentiated cell products in chicken cells. The expression of these proteins may or may not be suppressed when temperature-sensitive mutants are shifted from the nonpermissive to the permissive temperature. A general characteristic of cellular transformation is the disruption of the microfilament system. In passaged chick embryo fibroblast cultures (CEF), this system is principally composed of isomeric forms of actin designated alpha, beta, and gamma by their isoelectric focusing and when subjected to SDS-PAGE behavior. We present evidence that an alpha-actin in CEF cultures, identified by its electrofocusing behavior, retention in the cytoskeleton, and DNase 1 binding properties, is selectively and dramatically reduced in amount upon transformation by RSV. Little or no reduction is observed in the beta- and gamma-isoactins. The reduction of alpha-actin is shown to be reversible and transformation related by use of a temperature-sensitive mutant, tsNY68. The decrease in this transformation-sensitive isoactin is apparently due to a decrease in synthesis, though other possibilities are discussed. A specific decrease in a particular isoactin after transformation may give insight into the mechanism by which the microfilaments are normally maintained.     

Enhancement of melanotic expression in cultured mouse melanoma cells by retinoids

Retinoic acid (RA), which reduces the rate of cell proliferation in S91 mouse melanoma clone C2 cells, was found to stimulate the expression of their melanotic phenotype. RA treatment also induced the extension of long cellular processes. The RA effects on melanogenesis included stimulation of tyrosinase activity and augmentation of cellular melanin content to levels 3- to 4-fold higher than in untreated cultures at similar cell densities. These effects became apparent after 48 hours of exposure to 10(-5) M RA and increased thereafter. Half-maximal stimulation in cells treated for 6 days occurred at 5 X 10(-7) M RA. Although the degrees of melanogenesis enhancement by RA (10(-5) M) and by alpha-melanocyte stimulatory hormone (2 X 10(-7) M) were similar, the former did not alter the intracellular cAMP level, whereas the latter induced a transient 4-fold increase. In high-passage (p28) cells, as well as in low-passage cells (less than p10) treated with tyrosinase inhibitor phenylthiocarbamate, melanin synthesis was suppressed in the absence and presence of RA, yet the ability of RA to inhibit cell proliferation was not compromised. In the presence of the tumor promotor phorbol myristate acetate (greater than 5 X 10(-9) M) melanin synthesis in control as well as in cells exposed to RA was dramatically inhibited. Phorbol which is not active in tumor promotion had no effect on melanogenesis. In addition to RA, other retinoids, such as 13-cis-retinoic acid, retinyl acetate, the TMMP analog of RA and the phenyl analog of RA, but not the pyridyl analog of RA or retinyl palmitate, also inhibited cell growth and enhanced melanin synthesis.     

Enhancing effects of retinoic acid on monoclonal antibody production of human-human hybridomas

The effects of retinoids on the production of monoclonal antibody of human-human hybridomas were examined. IgG antibody secretion of a hybridoma CLNH11 was enhanced up to about two- to fourfold by retinoic acid (RA) at concentrations ranging from 10(-9) to 10(-5) M, where RA had little effect on the growth rate and saturation density of the cell. Among other retinoids, retinol magnified the antibody production as well as RA. Retinal and retinyl acetate had weak effects. Retinyl palmitate showed no effect. RA also enhanced the production of monoclonal antibodies from other human-human hybridomas: SLNF10, IgG-producing; CoLNE10, IgA-producing; TOS/H8, IgM-producing. RA and human hybridomas provide a defined system to study the effects of retinoids on immune responses at a molecular level.     

Role of the mitogenic property and kinase activity of p60src in tumor formation by Rous sarcoma virus

Expression of the src gene of Rous sarcoma virus in chicken embryo neuroretinal cells results in morphological transformation and sustained proliferation of this normally resting cell population. PA101 and PA104 are two mutants of Rous sarcoma virus which induce neuroretinal cell proliferation in the absence of morphological transformation. Their mitogenic property is temperature sensitive, and they both encode p60src proteins with low kinase activity. To study the role of the mitogenic function and protein kinase activity of p60src in tumorigenesis, we investigated the oncogenicity of PA101 and PA104. Both mutants were less tumorigenic than wild-type virus when injected into chicks. Tumorigenicity was further assayed by inoculating infected chicken embryo fibroblasts and neuroretinal cells onto the chorioallantoid membrane of embryonated duck eggs. This system provides a nonpermissive and immunodeficient environment for xenogenic cell grafting and allows the study of cell tumorigenicity within a temperature range of 37 to 39.5 degrees C. Chicken embryo fibroblasts and neuroretinal cells infected with PA101 were as tumorigenic as wild type-infected cells at 37 degrees C, but tumor development was significantly reduced at 39.5 degrees C. In contrast, both cell types infected with PA104 displayed sharply reduced tumorigenicity. Cell cultures derived from PA101 tumors induced on the chorioallantoid membrane were similar to the corresponding cells maintained in vitro in terms of morphology, production of plasminogen activator, relative amounts of phosphotyrosine in total cellular proteins, and phosphorylation of 34,000-molecular-weight protein. These results indicate that the expression of the mitogenic function of src does not account per se for cell tumorigenicity and that tumor formation is compatible with low levels of p60src protein kinase activity.     

In vitro effects of retinoids on murine thymus-dependent and thymus-independent mitogenesis

The effects of three retinoids: all-trans-retinoic acid (RA), 13-cis-retinoic acid (13-cis-RA), and N-(4-hydroxyphenyl)retinamide (4-HPR) on murine splenic lymphocyte proliferative responses to mitogens were evaluated. The responses to T-cell mitogens, PHA and Con A, and a T-cell-dependent B-cell mitogen, PWM were significantly potentiated by these retinoids. However proliferative responses to a B-cell mitogen, Escherichia coli LPS were unaffected or inhibited. All three retinoids at concentrations ranging from 10(-6) to 10(-15) M significantly potentiated Con A-induced proliferative responses. In response to PWM, 10(-13) M RA, 10(-12) M 13-cis RA, and 10(-11) M 4-HPR were the lowest concentrations producing significant potentiation. Endpoint concentrations of retinoids significantly potentiating responses to PHA were; 10(-9) M RA, 10(-8) M 13-cis RA, and 10(-6) M 4-HPR. These responses were independent of retinol contained in fetal calf serum supplemented medium since responses were reproduced in serum-free medium devoid of retinol. Optimal potentiation by retinoids of responses to these T-cell-dependent mitogens were found at superoptimal concentrations of mitogen suggesting a selective inhibition of T-suppressor cells. Thus, potentiation of T-cell-dependent mitogen responses provides the most sensitive biological assay yet described for detection of retinoid activity and is a reproducible system to explore the cellular and molecular mechanisms of retinoid-mediated immunopotentiation.     

Studies on the synthesis and structure of mitochondrial DNA in cells infected by rous sarcoma viruses and on the occurrence of intramitochondrial virus-like particles in certain RSV-induced tumor cells

1. The synthesis of mitochondrial DNA in CEF in vivo at 3,4 and 6 days after infection with RSV (Schmidt-Ruppin, subgroup A) was progressively stimulated 2 to 4-fold as compared with that in uninfected CEF cells grown in parallel. 2. The stimulation of mtDNA synthesis in vivo upon transformation was found to be temperature dependent when the thermosensitive mutant of RSV, T5, was used to infect the cells. 3. In contrast to mtDNA synthesis, nuclear DNA synthesis did not differ in transformed and uninfected cells, nor did it change significantly upon temperature shifts. 4. MtDNA (monomeric and catenated dimeric forms) in transformed and uninfected CEF replicate by displacement synthesis. Various replication intermediates are described. 5. The restriction endonuclease EcoRI cleaves closed circular mtDNA from CEF at one specific site. 6. Heteroduplex molecules formed between nicked circular and/or EcoRI cleaved mt DNA molecules from uninfected and transformed CEF revealed, with a few exceptions, no detectable base sequence heterogeneity in at least 98% of cases. 7. Intramitochondrial virus like particles (IMV) are described in hamster tumor cells. The evidence suggests both engulfment of cytoplasmic particles by mitochondria and the presence of intramitochondrial incomplete forms of particles. Bromodeoxyuridine was found to enhance the frequency of IMV.     

Differential effects of retinol and retinoic acid on cell proliferation: a role for reactive species and redox-dependent mechanisms in retinol supplementation

While some authors suggest that retinoids are potential anti-proliferative and antioxidant agents, evidence has suggested those present pro-oxidant properties, which might lead to malignant proliferation. These discordances stimulated one to investigate the proliferative/anti-proliferative properties of two major retinoids, retinol (ROH) and retinoic acid (RA). In Sertoli cells, ROH increased proliferation while RA was anti-proliferative. ROH increased DNA synthesis, decreased p21 levels and induced cell cycle progression. ROH increased reactive species (RS) production and stimulated p38, JNK1/2 and ERK1/2 MAPKs activation. Antioxidant treatment with Trolox blocked ROH-induced RS production, MAPKs activation and proliferation; MAPKs inhibition blocked proliferation. The potential sites of RS indicate that ROH-induced RS is promoted via mitochondria and xanthine oxidase. In contrast, RA induced neither RS production nor MAPKs activation. RA decreased DNA synthesis and increased p21 leading to cell arrest. Overall, data show that ROH, but not RA, is able to induce proliferation through non-classical and redox-dependent mechanisms.     

Differential effects of retinol and retinoic acid on cell proliferation: A role for reactive species and redox-dependent mechanisms in retinol supplementation

While some authors suggest that retinoids are potential anti-proliferative and antioxidant agents, evidence has suggested those present pro-oxidant properties, which might lead to malignant proliferation. These discordances stimulated one to investigate the proliferative/anti-proliferative properties of two major retinoids, retinol (ROH) and retinoic acid (RA). In Sertoli cells, ROH increased proliferation while RA was anti-proliferative. ROH increased DNA synthesis, decreased p21 levels and induced cell cycle progression. ROH increased reactive species (RS) production and stimulated p38, JNK1/2 and ERK1/2 MAPKs activation. Antioxidant treatment with Trolox blocked ROH-induced RS production, MAPKs activation and proliferation; MAPKs inhibition blocked proliferation. The potential sites of RS indicate that ROH-induced RS is promoted via mitochondria and xanthine oxidase. In contrast, RA induced neither RS production nor MAPKs activation. RA decreased DNA synthesis and increased p21 leading to cell arrest. Overall, data show that ROH, but not RA, is able to induce proliferation through non-classical and redox-dependent mechanisms.     

Early release of the density-dependent inhibition of phosphate uptake and ATP synthesis after src gene expression in chick embryo fibroblasts

Our results showed that the expression of the src gene in chick embryo fibroblasts (CEF) released the density-dependent inhibition (DDI) of phosphate metabolism (phosphate uptake and phosphorylation of small organic compounds). With increasing cell density, phosphate metabolism decreased by 58% in normal CEF and, in contrast, increased by 20% in Rous sarcoma virus (RSV)-transformed CEF. The same change in the DDI was observed in CEF infected by NY68 (a ts mutant for transformation of RSV) and maintained at the permissive temperature (37 degrees C) instead of the restrictive temperature (41.5 degrees C) for the expression of transformation. An interesting feature was that the release of the DDI of phosphate metabolism was an early event in the process of transformation, since it was almost concomitant with the stimulation of the pp60 src kinase activity following the shift from 41.5 to 37 degrees C of NY68 CEF. The phosphorylation of small organic compounds (Po) was more strongly increased by the change in temperature than was 32Pi accumulation. Furthermore, the percentage increases of Po and adenosine triphosphate (ATP) labelling with 32P were similar, suggesting that the expression of src gene enhanced ATP synthesis. In glucose-free medium, the stimulation of Po-labelling was still observed but was decreased. Therefore the activation of glycolytic activity is not an absolute requirement, but is necessary for the maximum effect of transformation on the release of DDI of phosphate metabolism. Oligomycin added in complete medium did not prevent the increase in Po-labelling. From these results, we assumed that ATP turnover was stimulated as a consequence of enhanced ATP degradation. We verified that the stimulation of Po phosphorylation was not a consequence of increased ATP utilization for RNA or protein synthesis. The stimulation of Po labelling was specifically abolished by quercetin. This drug inhibited the transformed cells more strongly than the non-transformed cells.     

Immune response and resistance to Rous sarcoma virus challenge of chickens immunized with cell-associated glycoproteins provided with a recombinant avian leukosis virus.

The Rous-associated virus 1 env gene, which encodes the envelope gp85 and gp37 glycoproteins, was isolated and inserted in place of the v-erbB oncogene into an avian erythroblastosis virus-based vector, carrying the neo resistance gene substituted for the v-erbA oncogene, to generate the pNEA recombinant vector. A helper-free virus stock of the pNEA vector was produced on an avian transcomplementing cell line and used to infect primary chicken embryo fibroblasts (CEFs) or quail QT6 cells. These infected cells, selected with G418 (CEF/NEA and QT6/NEA, respectively) were found to be resistant to superinfections with subgroup A retroviruses. The CEF/NEA preparations were used as a cell-associated antigen to inoculate adult chickens by the intravenous route compared with direct inoculations of NEA recombinant helper-free virus used as a cell-free antigen. Chickens injected with the cell-associated antigen (CEF/NEA) exhibited an immune response demonstrated by induction of high titers of neutralizing antibodies and were found to be protected against tumor production after Rous sarcoma virus A challenge. Conversely, no immune response and no protection against Rous sarcoma virus A challenge were observed in chickens directly inoculated with cell-free NEA recombinant virus or in sham-inoculated chickens.