Chicken embryo fibroblasts exposed to weak,time-varying magnetic fields share cell proliferation,adenosine deaminase activity,and membrane characteristics of transformed cells

Chicken embryo fibroblasts (CEF) exposed to a sinusoidally varying magnetic field (SVMF) (100 Hz, 700 μT, for 24 h) showed a remarkable rise of segmental rotational relaxation rate of adenosine deaminase (ADA, EC 3.5.4.4) as determined by multifrequency phase fluorometry. Pyrene-labeled, small subunit ADA was applied to cultured (normal) CEF, which have available and abundant ADA complexing protein (ADCP) on their plasma membranes. Sine-wave-modulated fluorometry of the pyrene yielded a profile of phase angle vs. modulation frequency. In SVMF-treated cells and in Rous-sarcoma-virus (RSV) transformed cells the differential phase values at low modulation frequencies of the excitation are remarkably reduced. This effect is magnetic rather than thermal, because the temperature was carefully controlled and monitored; nevertheless to further check this matter we studied CEF, infected by the RSV-Ts68 temperature-sensitive mutant (36°C transformed, 41°C “revertant”). When grown at 36°C in the SVMF, cells did not show the slightest trend towards reversion, as would be expected had there been local heating. Concomitant with the increased segmental rotational relaxation rate of ADA, there was a decrease in fluorescence lifetime and a slight, yet significant, increase in membrane lipid “microfluidity.” These biophysical observations prompted us to examine the effect of SVMF on cell proliferation and ADA activity (a malignancy marker): higher rates of cell proliferation and reduced specific activity of ADA were observed. © 1993 Wiley-Liss, Inc.     

On the regulatory role of dipeptidyl peptidase IV (=CD=adenosine deaminase complexing protein) on adenosine deaminase activity

The molecular mechanism controlling the variable activity of the malignancy marker adenosine deaminase (ADA) is enigmatic. ADA activity was found to be modulated by the membrane-bound adenosine deaminase complexing protein (CP=DPPIV=CD26). The role of lipid-protein interactions in this modulation was sought. While direct solubilization of ADA in vesicles resulted in loss of ADA activity, the binding of ADA to CP reconstituted in vesicles restored the specific activity. The activity of ADA, free or bound to CP in solution, resulted in continuous linear Arrhenius plots. However, ADA bound to reconstituted CP exhibited two breaks associated with approximately 30% increased activity, at 25 and 13 degrees C, yielding three lines with similar apparent activation energies (E(a)). Continuum solvent model calculations of the free energy of transfer of the transmembrane helix of CP from the aqueous phase into membranes of various widths show that the most favorable orientations of the helix above and below the main phase transition may be different. We suggest that the 20% change in the thickness of the bilayer below and above the main phase transition may modify the orientation of CP in the membrane, thereby affecting substrate accessibility of ADA. This could account for ADA's reduced activity associated with increased membrane fluidity in transformed vs. normal fibroblasts.     

Immunohistochemical localization of adenosine deaminase complexing protein in intestinal mucosa and in colorectal adenocarcinoma as a marker for tumour cell heterogeneity

Summary Adenosine deaminase complexing protein (ADCP), a dimeric glycoprotein, has been reported to be decreased or deficient in transformed or cancer-derived cell lines, indicating its potential significance as an indicator of malignant transformation. A similar deficiency was reported in total homogenates of tumours of colon, kidney, lung and liver. In previous biochemical studies we failed to confirm the consistent reduction in ADCP concentration in cancer tissues. A possible explanation for our findings was thought to be intercellular heterogeneity in ADCP expression in individual tumour cells. To study ADCP expression in individual cells we developed an immunohistochemical method which was applied to tissue sections. Paraformaldehyde-lysine-periodate (PLP) solution was found to be a suitable fixative. Fixed tissue samples were paraffin-embedded, sectioned and stained for ADCP, using an indirect peroxidase-labelled antibody procedure. The protein was localized in normal colonic mucosa, mainly in the brush border region of the luminal epithelium and in cytoplasmic granules. Intense ADCP immunoreactivity was found also in the basal part of some cells. In cancer cells, three staining patterns were observed: membranous, diffuse cytoplasmic and granular cytoplasmic. The adenocarcinomas exhibited significant intratumour and intertumour heterogeneity in their staining types.Further studies on ADCP expression in colorectal cancer in relation to clinical and histopathological characteristics are warranted in order to fully evaluate the potential significance of ADCP as a cancer associated antigen.     

Distribution of adenosine deaminase complexing protein (ADCP) in human tissues

The normal distribution of adenosine deaminase complexing protein (ADCP) in the human body was investigated quantitatively by ADCP-specific radioimmunoassay (RIA) and qualitatively by immunohistochemistry. In these studies we used a specific rabbit anti-human ADCP antiserum. In all 19 investigated tissues, except erythrocytes, ADCP was found by RIA in the soluble and membrane fractions. From all tissues the membrane fractions contained more ADCP (expressed per mg protein) than the soluble fractions. High membrane ADCP concentrations were found in skin, renal cortex, gastrointestinal tract, and prostate. Immunoperoxidase staining confirmed the predominant membrane-associated localization of the protein. In serous sweat glands, convoluted tubules of renal cortex, bile canaliculi, gastrointestinal tract, lung, pancreas, prostate gland, salivary gland, gallbladder, mammary gland, and uterus, ADCP immunoreactivity was found confined to the luminal membranes of the epithelial cells. These data demonstrate that ADCP is present predominantly in exocrine glands and absorptive epithelia. The localization of ADCP at the secretory or absorptive apex of the cells suggests that the function of ADCP is related to the secretory and/or absorptive process.     

Immunological and structural properties of Rous sarcoma virus-transformed and tumor cells]

We compared the capacity of both normal and Rous sarcoma virus (RSV)-transformed chicken embryo fibroblast (CEF) cells as well as Rous sarcoma (RS) tumor cells to serve as targets in anti-tumor immunity assays. These studies showed that sera from tumor-bearing donors were able to stain transformed CEF more efficiently than RS cells as detected by immunofluorescence. In contrast, antiserum against the major viral glycoprotein, gp 85, stained a higher percentage of RS than transformed cells. Normal CEF cells, which served as controls, were essentially non-reactive with the immune system as judged by this type of assay. We observed that RS cells are considerably larger and contain far higher levels of protein than either normal or transformed CEF. Scanning electron microscopy revealed both the RS cells and transformed CEF to be rich in surface ruffles, blebs and microvilli as distinguished from the flattened, fusiform appearance of normal CEF cells.     

Transformation of chicken embryo fibroblast cells by avian retroviruses containing the human Fyn gene and its mutated genes.

The transforming activity of the human fyn protein, p59fyn, which is a kinase of the src family, was investigated by testing the effect of recombinant avian retrovirus (Fyn virus) expressing p59fyn on chickens or cultured chicken embryo fibroblast (CEF) cells. The Fyn virus did not induce transformed foci. After several passages of the virus stock on CEF cells, however, a few foci were detected in the presence of dimethyl sulfoxide. Chickens inoculated with Fyn virus at the stage of 12-day-old embryos developed fibrosarcomas 3 to 6 weeks after hatching. The viruses obtained from these foci and from one of the tumor tissues showed high transforming activity in the presence of dimethyl sulfoxide, suggesting that these viruses carry spontaneous mutations of the fyn gene. Four fyn genes from CEF DNAs infected with transforming viruses were molecularly cloned, and their products were confirmed to possess transforming activity. DNA sequence analysis of the fyn genes showed that two of the four mutants have Thr instead of Ile at position 338 in the kinase domain. The other two mutants carry deletions of 78 and 108 base pairs, respectively, which result in complete loss of region C of SH2. The overall level of proteins containing phosphotyrosine was significantly higher in transformed cells than in normal CEF cells. Our data indicate that when expressed at high levels in a retrovirus, normal p59fyn cannot cause cellular transformation, but that mutant p59fyn with either a single amino acid substitution in the kinase domain or a deletion including region C produces a transforming protein, perhaps due to enhanced tyrosine kinase activity. This is the first observation that deletion of region C can unmask the potential transforming activity of a src family kinase.     

Protein and lipid lateral diffusion in normal and Rous sarcoma virus transformed chick embryo fibroblasts.

We measured the lateral diffusion of the fluorescent lipid analogue dioctadecylindocarbocyanine iodide (DiI) and of membrane glycoproteins labeled with tetramethylrhodamine (TRITC) succinyl concanavalin A (SConA) via fluorescence photobleaching recovery (FPR) at selected times during a temperature downshift experiment on transformation-defective temperature-sensitive (td-ts) Rous sarcoma virus (RSV) NY68-transformed chicken embryo fibroblasts (CEF) and on identically treated CEF and RSV-transformed CEF. There were no significant differences in the lateral diffusion in DiI at any of the times measured. The lateral diffusion of TRITC-SConA on the RSV-transformed CEF, (1.32 +/- 0.12).10(-10) cm2 s-1, was approximately two times faster than that observed in normal CEF, (0.61 +/- 0.06).10(-10) cm2 s-1. In the cells undergoing RSV NY68-mediated transformation, TRITC-SConA diffusion increased over a 24-h period from a value comparable to that observed in normal CEF, (0.72 +/- 0.13).10(-10) cm2 s-1 to a value comparable to the RSV-CEF transformed cells, (1.74 +/- 0.20).10(-10) cm2 s-1. All diffusion measurements reported were made at the permissive temperature for RSV-NY68 (35 degrees C) unless stated otherwise. The changes in the lateral diffusion of TRITC-SConA occurred between the fifth and twelfth hour of the downshift course and could be associated with cytoskeletal disruption and/or fibronectin degradation, both known to occur at this time in RSV-transformed cells. To assess the contribution of extracellular matrix (ECM) degradation, SConA mobility was measured in normal and RSV-transformed cells treated with trypsin. This treatment increased SConA mobility approximately 4-fold in the normal cells relative to untreated controls and only 2-fold in the RSV-CEF transformed cells. No significant difference in SConA mobility between trypsinized spherical normal and transformed cells was apparent.     

Neutral protease activity of Rous sarcoma (RSV) transformed chick embryo fibroblasts.

The proteolytic activities of normal, Schmidt-Ruppin Rous sarcoma virus (SR-RSV) transformed, and infected (RAV) chick embryo fibroblasts (CEF) have been measured by a highly sensitive technique using 3H-acetylated haemoglobin as a substrate.When all 3 types of CEF cells were maintained in serumless media, no differences were detected in the amount of pH 3-4 protease activity released into the media over a 24-h period, and only negligible amounts of pH 7-6 proteolytic activity were found. When normal, transformed, and infected cells were maintained in serumless media and later incubated with 3H-acetylate haemoglobin, a significant proteolysis of the haemoglobin, a 6-fold increase compared to the normal CEF cells, was associated only with plates containing SR-RSV-CEF cells. A fluorescent assay for peptides confirmed that SR-RSV-CEF cells have increased cell-associated proteolytic activity. The net surface charge of the transformed CEF cells was unchanged by maintenance in serumless media but the net surface negativity of the normal and RAV-CEF cells was significantly increased by incubation in media minus serum for 24 h. This suggests that normal CEF cells, maintained in media plus serum, have a substance masking their surface charge which is absent from the surface of transformed cells, possibly because of proteolytic degradation.     

Effect of sinusoidally varying magnetic fields on cell proliferation and adenosine deaminase specific activity

The effect of sinusoidally varying magnetic fields (SVMF) on chick embryo fibroblasts (CEF) was examined by two independent methods: 1) measurement of cell proliferation at 0.06–0.7 mT (100, 60 and 50 Hz) using a colorimetric assay (MTT); 2) monitoring of specific activity of adenosine deaminase (ADA) at 0.3 and 0.7 mT, 60 Hz. Both increased cell proliferation and reduced ADA specific activity are associated with cell transformation. The MTT test showed an increase in cell proliferation of up to 64% after a 24 h exposure to SVMF at 100 Hz, 0.7 mT. Cell proliferation at constant frequency (100 Hz) depended on SVMF intensity. Cell proliferation at constant intensity (0.7 mT) increased with increasing field frequency. At 0.7 mT, 60 Hz cell proliferation increased by 31%, 28%, and 26% when measured by hemocytometry, ³H-thymidine incorporation, and the MTT assay, respectively. ADA specific activity in CEF decreased by circa 48% on exposure to SVMF at 60 Hz, 0.3 mT for 24 h; only a statistically insignificant trend was seen at 0.7 mT, 60 Hz. Our findings showed that CEF cell proliferation and ADA specific activity were modified by SVMF. Both methods, independently, qualitatively detect a magnetic field effect. Bioelectromagnetics 19:46–52, 1998. © 1998 Wiley-Liss, Inc.     

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.     

Effects of cocultivation with transformed cells on surface proteins of normal cells

Virally transformed fibroblasts do not have on their surface a major protein (large external transformation-sensitive, LETS) which is present in normal cells. Cocultivation of the transformation cells with normal cells whose surface proteins have been prelabelled induces an accelerated release of the LETS protein from the normal cells. We have investigated various conditions which affect this phenomenon. Our results show that alteration of cell surface proteins by cocultivation with the transformed cells is time and dose-dependent and requires cell contact. Serum was depleted at least 99% of plasminogen by affinity chromatography and used in the cocultivation experiments. It was found that activation of plasminogen was not required for the accelerated turnover of the LETS protein. Other diffusible proteases are also unlikely to be involved. The possibility that transformed cells have a membrane bound activity is discussed. The role of plasminogen activation was also tested for its relevance in transformation related proteolysis, growth and morphology of cells.     

Induction of two transformation-sensitive membrane polypeptides in normal fibroblasts by a block in glycoprotein synthesis or glucose deprivation.

Following transformation of chick embryo fibroblasts (CEF) by avian RNA tumor viruses, two membrane polypeptides with apparent molecular weights of 90,000 and 75,000 daltons have been found to be increased (Stone, Smith and Joklik, 1974). We find that this alteration in membrane proteins is not directly related to transformation.The 90,000 and 75,000 dalton proteins are present in increased amounts in a 3T3 fibroblast mutant (AD6) defective in glycoprotein synthesis. Feeding the mutant N-acetylglucosamine, a metabolite that bypasses the metabolic block, restores the amount of these two proteins to the levels found in normal cells. The 75,000 dalton protein is markedly reduced, and the 90,000 dalton protein disappears and is replaced by a fully glycosylated derivative with a molecular weight of 92,000 daltons.Two glucose derivatives, glucosamine and 2-deoxyglucose, are known to interfere with the glycosylation process. The addition of these substances to normal CEF and 3T3 cells specifically induces the accumulation of the 90,000 and 75,000 dalton membrane polypeptides.Finally, the deprivation of glucose for 24–48 hr also induces the synthesis of the 90,000 and 75,000 dalton polypeptides in normal fibroblasts. The induction of these two proteins by glucose starvation suggests that they have a role in glucose utilization.     

Basic defect in the expression of adenosine deaminase in ADA- SCID disease investigated through the cells of an obligate heterozygote

Summary The nature of the defect of a female baby who died of severe combined immunodeficiency (SCID) disease associated with adenosine deaminase deficiency (ADA^-) was investigated. Since tissue or tissue culture material was not available for subsequent studies, the expression of ADA in her cells was investigated in the somatic cell hybrid clones derived from a fusion between the lymphocytes from one of her two obligate heterozygote parents and thymidine kinase deficient Chinese hamster (a3) fibroblasts. The results of analyses of the human chromosomes and biochemical markers in 12 independent clones and 27 subclones indicated that the ADA deficiency in the patient is determined probably by a mutation in the structural gene for ADA in chromosome 20 leading either to the production of catalytically defective molecules or to the cessation of the production of ADA. Incidentally, the involvement of chromosome 2, which carries a gene for adenosine deaminase complexing protein (ADCP), in the causation of ADA deficiency was excluded. The in vitro approach through the cells from an obligate heterozygote described in this paper may have a general application in pursuing studies on other cases of inborn errors of metabolism whenever the material from the affected individuals (i.e., the homozygotes) is not available or not suitable for direct investigations.A part of this work was presented at the New York State Department of Health, Birth Defects Institute Symposium IX (Inborn Errors of Specific Immunity), Albany, October 16–18, 1978 and reported as an abstract in the proceedings of the Fifth International Conference on Human Gene Mapping, Edinburgh, July 1979. Cytogenet Cell Genet 22:164     

Altered signal transduction in erbB-transformed cells. Implication of enhanced inositol phospholipid metabolism in erbB-induced transformation

To clarify the signal transduction mechanism of the erbB gene (virus oncogene) products leading to cell growth and transformation, the alteration of signal transduction induced by enhanced inositol phospholipid metabolism was studied in chick embryo fibroblast cells (CEF cells) transformed by gag-fused erbB gene-carrying virus (GEV cells). The incorporations of 32P into phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate were markedly increased in GEV cells. In GEV cells, the activities of lipid kinases such as phosphatidylinositol (PI), PIP, and diacylglycerol (DG) kinases were also increased. The activities of other important enzymes involved in inositol phospholipid metabolism, such as CDP-DG:myo-inositol transferase and phospholipase C, were not changed in GEV cells. Increased inositol phospholipid metabolism might lead to the production of second messengers, such as 1,2-DG and inositol 1,4,5-trisphosphate. Indeed, the 1,2-DG content was also increased in GEV cells. Moreover, the activity of protein kinase C (the Ca2+/phospholipid-dependent enzyme), which should be stimulated by 1,2-DG, was elevated in GEV cells; the protein kinase C activity in the membrane fraction of GEV cells was especially high. When CEF cells were treated with tetradecanoylphorbol acetate, protein kinase C activator, plus Ca2+ ionophore, [3H]thymidine incorporation was markedly stimulated, and maximal stimulation was observed with 1 nM Ca2+ ionophore A23187 plus 100 nM TPA. On the other hand, when GEV cells were treated with TPA plus Ca2+ ionophore A23187, [3H]thymidine incorporation was consistently inhibited. Next, studies were made to determine whether the erbB gene product itself had kinase activity on PI, PIP, and DG after membranes were mildly solubilized with Triton X-100 to prevent inactivation of these kinases. Immunoprecipitates of a GEV cell lysate with antisera that reacted with the erbB gene product had PI kinase activity, whereas no activity was detected in those of lysates of uninfected CEF cells. However, the activity was very weak compared with the total cellular activity. No difference in the PIP and DG kinase activities of immunoprecipitates of cell lysates of uninfected CEF cells and GEV cells was observed. These results suggest that the erbB gene product enhances inositol phospholipid metabolism and subsequent signal transduction, but that the erbB gene product is not involved directly in lipid kinases, although it is closely associated with lipid kinase.     

Cell surface changes accompanying viral transformation: N-acetylneuraminic acid ectotransferase system activity.

The activity of the sialyl ectotransferase system of normal chick embryo fibroblasts (CEF) and chick embryo fibroblasts transformed with the Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV) have been compared. Neuraminidase treatment of the intact cells increased the sialyl ectotransferase system activity of control and transformed cells two to three times. The ectotransferase system activity increased as the pH was decreased from 7.8 to 6.0. The temperature optimum for both systems was 40 degrees C. Approximately 60% of the 14C-sialic acid incorporated at pH 6.5 or above could be removed with neuraminidase. The activity of the transformed cell system with or without neuraminidase treatment was more stimulated by addition of Mn2+ ions, particularly above pH 7.0. This difference in ion sensitivity indicates that a different cell surface phenomenon is being studied after transformation.     

Host range mutants of v-src: alterations in kinase activity and substrate interactions.

Host range mutants of Schmidt-Ruppin v-src that transform chicken embryo fibroblasts (CEF) but not Rat-2 cells were generated previously by linker insertion-deletion mutagenesis (J. E. DeClue and G. S. Martin, J. Virol. 63:542-554, 1989). One of these mutants, SRX5, in which Tyr-416 is substituted by the sequence Ser Arg Asp, retained high levels of kinase activity in vitro and in vivo, both in CEF and in Rat-2 cells. Phosphorylation of p36 (the calpactin I heavy chain) was drastically reduced in cells expressing SRX5 src, suggesting that the phenotype of SRX5 results from an alteration in substrate recognition by the src kinase. Three mutants, SPX1, SHX13, and XD6, containing linker insertions or small deletions within the src homology 2 (SH2) region, induced reduced levels of kinase activity in both CEF and Rat-2 cells. However, the residual levels of kinase activity in Rat-2 cells were above the threshold at which wild-type pp60v-src transforms Rat-2 cells, indicating that the reduction in kinase activity was not sufficient to account for the failure to transform. Cells infected by these mutants exhibited reduced levels of phosphorylation of 120- and 62-kDa proteins. We have reported elsewhere (M. F. Moran, C. A. Koch, D. Anderson, C. Ellis, L. England, G. S. Martin, and T. Pawson, Proc. Natl. Acad. Sci. USA 87:8622-8626, 1990) that ras GTPase-activating protein GAP and associated protein p62 are not tyrosine phosphorylated in Rat-2 cells expressing SHX13 or XD6. The transformation defect in Rat-2 cells may result from the failure to phosphorylate those proteins. The fifth mutant, XD4, contains a deletion which removes all of the src homology 3 (SH3) and most of the SH2 sequences of src. The protein encoded by XD4 is active as a kinase when expressed in CEF, indicating that in CEF the SH2 and SH3 regions of v-src are not necessary for kinase activity and transformation. The XD4 src product is not tyrosine phosphorylated and is inactive as a kinase when expressed in Rat-2 cells. Thus, host cell factors can affect the tyrosine phosphorylation and activity of the v-src kinase in the absence of the SH2 and SH3 regions. These results indicate that the host-dependent transformation phenotype results from alterations in src kinase activity and substrate specificity.     

Transformed cells have lost control of ribosome number through their growth cycle.

Previous studies on the synthesis and function of the protein synthetic machinery through the growth cycle of normal cultured hamster embryo fibroblasts (HA) were extended here to a series of four different clonal lines of polyoma virus-transformed HA cells. Under our culture conditions, these transformed cells could enter a stationary phase characterized by no mitotic cells, very low rates of DNA synthesis, and arrest in a post-mitotic pre-DNA synthetic state. Cellular viability was initially high in stationary phase but, unlike normal cells, transformed cells slowly lost viability. The rate of protein synthesis in the stationary phase of the transformed cells fell to 25-30% of the exponential rate. Though this reduction was similar to that seen in normal cells, it was accomplished by different means. The specific reduction in the ribosome complement per cell to values below that of any cycling cell seen in normal cells, was not seen in any of the transformed lines. This observation, which implies a loss of normal control of ribisome synthesis through the growth cycle after transformation, was confirmed in normal Chinese hamster embryo fibroblasts and transformed CHO cell lines. Normal control of ribosome synthesis was restored in L-73 and LR-73, growth control revertants of one of the transformed CHO lines. The transformed lines reduced their protein synthetic rates in stationary phase either by a greater reduction in the proportion of functioning ribosomes than that seen in normal cells or by a decrease in the elongation rate of functioning ribosomes; the latter effect was not seen in the normal cells. A model for growth control of normal cells and its derangement in transformed cells is presented.     

Basic fibroblast growth factor: production, mitogenic response, and post-receptor signal transduction in cultured normal and transformed fetal bovine aortic endothelial cells

Normal FBAE AG 7680 cells and chemically transformed FBAE GM 7373 cells were compared for their capacity to produce and to respond to bFGF. Normal FBAE cells showed higher levels of bFGF protein and of poly(A)+ bFGF mRNA than transformed GM 7373 cells, indicating that chemical transformation in FBAE cells is paralleled by a decrease of bFGF gene expression. Basic FGF induced cell proliferation in both normal and transformed FBAE cells. However, bFGF appeared to be much more potent in transformed than in normal cells. No differences in bFGF membrane receptors were observed between normal and transformed FBAE cells in terms of apparent molecular weight, number per cell, dissociation constant, and kinetic of downregulation. In respect to normal cells, however, transformed GM 7373 cells showed higher basal levels of PKC activity. This kinase is activated by bFGF and is involved in mediating the mitogenic activity of bFGF, as shown by the capacity of the PKC inhibitor H-7 to abolish the mitogenic activity of bFGF both in normal and transformed FBAE cells. Like bFGF, the PKC activators DAG and TPA exerted a stronger mitogenic activity in transformed than in normal FBAE cells. Thus, the different susceptibility of normal and transformed FBAE cells to bFGF appears to depend on differences in the post-receptor signal transduction mediated by PKC rather than on differences in bFGF receptors. The results indicate that chemical transformation causes significant modifications of bFGF physiology in FBAE cells. The relevance of these modifications to the genesis of tumors of vascular origin deserves further investigation.     

Different localization of the product of the v-rel oncogene in chicken fibroblasts and spleen cells correlates with transformation by REV-T

Reticuloendotheliosis virus strain T (REV-T) is a highly oncogenic avian retrovirus that transforms early lymphoid cells in vivo and in vitro, but REV-T does not transform chicken embryo fibroblasts (CEF). Using antisera to p59v-rel, the v-rel oncogene product of REV-T, we show that p59v-rel is expressed at equal levels and is a phosphoprotein in REV-T infected spleen cells and CEF. Biochemical fractionation and immunofluorescence of REV-T infected nontransformed CEF show that p59v-rel is loosely associated with the nucleus. However, in REV-T transformed spleen cells p59v-rel is primarily a cytoplasmic protein. MSB-1 cells, a Marek's disease virus transformed T cell leukemic line, and E26 virus transformed myeloid cells show nuclear staining of p59v-rel when they are infected by REV-T. Our results indicate that there is a correlation between a cytoplasmic localization of p59v-rel and transformation by REV-T, and they suggest that p59v-rel cannot transform cells in which it assumes solely a nuclear location.