Secreted proteins of normal andmyc-ras oncogene transformed rat embryo fibroblasts

Quiescent cultures of rat embryo fibroblasts synthesize and secrete several proteins in response to mitogenic stimulation. Two of these proteins have been characterized in this study and the effect of oncogenic transformation on these proteins was monitored. A serum induced 48,000 protein was shown to be related to plasminogen activator inhibitor while another serum-induced protein ofM _r 45,000 was found to be an inhibitor of DNA synthesis. Transformation of rat embryo fibroblasts with oncogenesmyc andras resulted in drastic reduction in the level of these proteins. The reduced levels of protease inhibitor may be responsible for the loss of anchorage dependence of the transformed cells. The DNA synthesis inhibitor protein may act as a negative growth regulator and reduced levels of this protein inmyc-ras transformed cells may accelerate the proliferation of these cells.     

Association of SV40 DNA sequences with nuclear matrix in SV40 transformed hamster fibroblasts

Nuclear matrices were isolated by the high-salt, non-ionic detergent method from SV40-transformed hamster fibroblasts (TSV5 cell line), and from hamster tumours derived from these cells. DNA isolated from matrices and total nuclei was hybridized with nick-translated SV40 DNA. The enrichment of matrix DNA with SV40 DNA sequences was observed in all five experiments with matrix DNA of TSV5 cells but only in five out of nine matrix DNA isolated from tumour cells.     

Viral and cellular oncogene expression during progressive malignant transformation of SV40 transformed human fibroblasts.

In vitro investigation of the multistep neoplastic progression which occurs during transformation of human cells has been hindered by resistance of human cells to both immortalization and tumorigenicity (Mut. Res. 199; 273, 1988). Previously our laboratory established a cell line, HSF4-T12, by transfection of normal human foreskin fibroblasts with the plasmid pSV3-neo which contains the early genes of simian virus 40 (SV40). A multistep progression in karyotypic alterations and transformed phenotype occurred resulting in a neoplastic cell line that was immortal, transformed, and tumorigenic. We have examined changes in the SV40 proteins, large T (T-antigen) and small t (t-antigen) antigens, and in the cellular protein, p53, during progressive transformation of these cells. Total viral protein expression relative to total cellular protein increased following immortalization of HSF4-T12 as did the ratio of T-antigen to t-antigen. Interestingly, no significant change in DNA content accompanied immortalization. However, during the progressive in vitro transformation of HSF4-T12 which occurred primarily post-immortalization, DNA index increased to 1.6 but only small additional increases in T-antigen expression were seen. No consistent or critical role for t-antigen in development of the tumorigenic phenotype was found in this system.     

Identification of conventional and novel endothelin receptors in sheep choroid plexus cells

Previous studies have demonstrated the presence of super-high affinity endothelin receptors with apparent K_d's on the order of pM in different brain tissues. This study was designed to characterize, in detail, the receptors present in SCP cells, a non-transformed sheep choroid plexus cell line. Competitive binding assays with receptor-selective ligands indicated the presence of at least three classes of binding sites: a conventional receptor of the ET_A subtype with a K_d = 0.4 nM that mediates an increase in intracellular levels of inositol 1,4,5-trisphosphate (IP₃) in response to ET-1 and two additional sites with much higher binding affinities. The latter two sites are not coupled to the common signal transduction pathways of IP₃, cAMP and cGMP. Northern blot analysis confirmed the presence of only the ET_A subtype mRNA in SCP cells. It remains to determined if the multiple binding sites are distinct gene products, multiple affinity states of a single receptor molecule or a result of cooperative association of one site with either the ligand or with other proteins.     

Increased activity of c-Src and Csk in fibroblasts transformed by v-src oncogene.

When c-Src and v-Src were immunoprecipitated together from hamster fibroblasts transformed by Rous sarcoma virus containing v-src oncogene, the total Src activity was almost threefold higher compared to c-Src activity in the control cells. The activity of v-Src immunoprecipitated separately, however, accounting for only 40% of the total Src activity, indicating that c-Src is activated upon transformation. An increased activity of Csk was also found in RSV-transformed cells. It decreased upon serum stimulation in parallel with an increase in Src kinase activity. In nontransformed cells, serum stimulation induced an enhanced Csk activity, but no changes in c-Src activity were observed. This may suggest that Csk may have more functions in hamster fibroblasts, in addition to its inhibitory effect on c-Src.     

A radioimmunocytological quantitative method for the rapid detection of ras oncogene p21 protein in mammalian cells

In order to provide a sensitive and quantitative detection method of ras p21 at the cytological level, the monoclonal antibody Y 13 259 and iodinated protein A were used to locate theras protein in various mammalian cell lines. The subsequent autoradiograph can be analysed by a computer-assisted system which showed in these reported experiments that the relative levels of p21 detected in these cells corresponded to results obtained earlier using conventional biochemical methods.To whom correspondence should be addressed.     

The induction of growth arrest in fibroblasts by SV40 T antigen

DNA tumor viruses such as SV40, Ras and papillomaviruses are the most commonly used agents in immortalization of non-hematopoietic cells, but the results are quite different. Some of them even lead instead to a senescence-like state. To verify the potential of SV40 T antigen-mediated immortalization or properties and functions of it to regulate cell growth, human dermal fibroblasts were cultured and then transfected with eukaryotic expressing plasmid psv3-neo which containing SV40 T DNA. We found that expression of oncogenic SV40 T in human dermal fibroblasts resulted in growth, arrest, earlier than the occurrence of control cell senescence, although telomerase was positive and cells grew faster than control ones in early stage following transfection. These observations suggest that SV40 T antigen can activate growth arrest in human dermal fibroblasts under normal growth condition instead of always prolonging the lifespan of fibroblasts. Moreover, high rate of cell division in early stage after transfection may be associated with the expression of telomerase activity.     

Neoplastic transformation and characterization of human fibroblasts by treatment with60Co gamma rays and the human c-Ha-ras oncogene

Summary Human fibroblasts (KMST-6) immortalized by treatment with⁶⁰Co gamma rays were further neoplastically transformed by transfection of the c-Ha-ras oncogene from human lung cancer. The ras-transfected cells formed undifferentiated fibrosarcoma in nude mice. One of the tumors was recultured and a neoplastic human fibroblast line, KMST-6/RAS, was established. To analyze multistep carcinogenesis of human cells, the cellular characteristics of these genetically matched immortalized (KMST-6) and neoplastic (KMST-6/RAS) cell lines were studied in detail. KMST-6/RAS cells showed an increased saturation density, colony formation on confluent monolayers of normal human fibroblasts, proliferation in neomycin-containing medium, anchorage-independent growth, and enhanced expression of the transfected c-Ha-ras oncogene, whereas the immortalized cells did not demonstate these characteristics. Unexpectedly, growth of KMST-6/RAS cells was serum-dependent, although they were neoplastic. Interestingly, the neoplastic cells did not show the criss-crossing or piling up growth pattern characteristic of transformed rodent fibroblasts.     

Expression of growth-regulated genes in normal and SV40 transformed hamster fibroblasts.

Transformation by the oncogenic virus SV40 has been shown to alter the expression of cellular genes at the level of RNA abundance. Many of these genes have yet to be identified. We have determined, by Northern blot analysis, the abundance levels of several growth-regulated genes in SV40-transformed cell lines to determine if their expression is altered and correlates with the ability of SV40 transformed cells to grow in low serum containing media. The mRNA abundance levels of the G1-specific genes 2A9/calcyclin, 2F1/translocase, and 4F1/vimentin were determined in the parental hamster fibroblast cell line, tk-ts13, and in two SV40 transformants, HR5 and HR8 cells, grown in medium containing 10% calf serum (normal medium) and in HR5 and HR8 cells adapted to passage in medium containing low serum. A spontaneous transformant of the parental line capable of growth in low serum in the absence of SV40 transformation (tk-ts13/1%), was also included in these studies. The low serum adapted SV40-transformed cells and the spontaneous tk-ts13 transformed cells grew more vigorously than their nonadapted counterparts in medium containing low serum. The low serum adapted cells also grew to higher saturation densities in low serum and to densities comparable to those in high serum, whereas the nonadapted cells grew to low saturation densities in low serum, but not as low as the untransformed parental.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of cholesterol in normal hamster fibroblasts and those transformed by the SV 40 virus. Effect of low density lipoproteins

The amount of cholesterol and the percentage of esterified cholesterol were increased in transformed cells. The cholesterol synthesis from [14C] sodium acetate was reduced and cholesteryl oleate uptake increased by 3 fold in transformed cells. The activity of acyl coenzyme A-cholesterol-acyltransferase, measured in situ was also increased in transformed cells. Studies with 125I-LDL pointed out an increase of binding, and especially of internalization of LDL by transformed cells. Finally, long term culture in a lipoprotein-deficient medium showed that transformed cells exhibited a higher ability (tested by growth rate and cholesterol synthesis) to adapt themselves to lipid depletion.     

A difference in the breakdown of phosphatidylinositol in normal and SV40 transformed mouse fibroblasts

Pulse chase experiments have shown that four reactions of phospholipid metabolism depend on cell population density in normal cells. Three of these reactions are influenced by population density also in tumor cells. Only the breakdown of phosphatidylinositol is not affected.     

Enhanced induction of SV40 replication from transformed mammalian cells by fusion with UV-irradiated untransformed cells

DNA-damaging agents such as ultraviolet (UV) light are known to cause stimulation of virus replication in SV40-transformed hamster and human cells. The dose-response curves of UV-induced SV40 replication in transformed hamster cells resemble that obtained for UV-enhanced reactivation (ER) and UV-enhanced mutagenesis (EM) of SV40 or herpes viruses in mammalian cells. We have investigated whether UV-enhanced production of SV40 from transformed hamster (THK) and human (NB-E) cells belongs to the same category of conditional responses as ER and EM. To answer this question we have made use of the phenomenon that fusion of the SV40-transformed cells with monkey cells that are permissive to SV40 results in a considerable increase in the production of SV40 virus. When THK or NB-E cells were fused with UV-irradiated CV-1 cells at various times after irradiation, induction of SV40 was further increased and reached a maximum value of 2--3-fold when fusion was delayed for 24-48 h after irradiation. The kinetics of enhanced SV40 induction resembled that of ER and EM, suggesting that the UV-stimulated part of the induction represents one of the pleiotropic responses that are transiently induced in mammalian cells by DNA-damaging agents. Evidence is presented, showing that this transient effect can be induced only in cells that are permissive to SV40 replication. This suggests that the enhanced induction observed after fusion with irradiated monkey cells may be attributed to a transient increase in the activity of "permissiveness' factors. No enhanced induction was found when the THK or NB-E cells were fused with irradiated rodent cells, that are not or only slightly permissive to SV40 replication.     

Expression of adenosine receptors in cardiac fibroblasts as a function of insulin and glucose level

Adenosine among other factors is known to regulate the growth and function of cardiac fibroblasts (CFs). Its action is mediated by cell-surface receptors linked to a variety of signaling systems. The goal of present work was to examine the effects of glucose and insulin on adenosine receptors (ARs) mRNA and protein level in primary culture of rat CFs by means of real-time PCR and Western blot. Elevated glucose level increased the expression of A(1)-AR, A(2A)-AR, decreased the expression of A(3)-AR, and had no effect on A(2B)-AR expression. On the other hand insulin suppressed the expression of A(1)-AR, and A(2B)-AR, and had no effect on A(2A)-AR and A(3)-AR expression. Our measurements showed that accumulation of cAMP in response to ARs agonists correlated well with the changes in receptors expression level. These results indicate that changes in glucose and insulin level independently and differentially regulate the ARs expression and functional state in CFs.     

Metabolically labeled cell membrane proteins in spontaneously and in SV40 virus transformed mouse fibroblasts.

A family of mouse fibroblast cell lines in exponential phase of growth were compared in protein constitution of their cell membranes. In preparations from these cells enriched in cell-surface membrane we observed one protein component (apparent molecular weight about 250 000) consistently to be reduced or absent in an SV40 virus transformed cell line, when compared with the normal cell line. No such compositional difference was observed in a spontaneously transformed tumorigenic clonal derivative cell line, or in subclones of such a derivative cell line, with or without SV40 virus infection. However, in metabolic labeling experiments with 14C-labeled mixed amino acids, a consistent decrease also was demonstrated in the biosynthesis of the same protein in the SV40 virus infected subclone, as compared to an uninfected sister subclone, during exponential growth. This specific difference in biosynthesis is apparently related to the presence and functioning of the SV40 gene, and correlates with the ability of these cells to grow in viscous medium, but not with cellular tumorigenicity.     

Prostaglandin synthetase activity and hormone responsiveness in normal and SV40 transformed WI-38 fibroblasts.

Prostaglandin (PG) synthetase activity and selective hormone responsiveness were examined in normal and SV40 transformed WI-38 fibroblasts (VA13-2RA). The transformed VA13-2RA cells have significantly reduced rates of PGE1, PGE2, PGF1alpha and PGF2alpha synthesis as compared to the normal WI-38 fibroblast. The transformed cell in contrast to the normal cell hyperresponds to stimulation by L-epinephrine (10 muM) and PGE1 (8.5 muM) but is unresponsive to bradykinin (BK) as measured by the accumulation of intracellular cyclic AMP. Indomethacin treatment does not significantly alter the PGE1 and L-epinephrine (EPI) responsiveness of the normal WI-38 fibroblast, however it abolishes the BK response in these cells. These results provide further evidence for the dependency of cell activation by bradykinin on the PG synthetase system. No experimental data was found to support the role of PGs as negative regulators of PGE1 and EPI responsiveness in the WI-38 fibroblast. Using the VA13-2RA cells, limited attempts to recover PG synthetase activity comparable to that found in normal WI-38 cells were unsuccessful. The VA13-2RA cell and its normal counterpart represent models for investigating the role of PGs in cell function and the mechanism of BK activation and its effect on cell metabolism.     

Mapping of the genes for rat endothelin receptor type A (ETAR) and type B (ETBR) to chromosomes 19 and 15 respectively

Chromosomal assignments of the genes for rat endothelin receptor type A (ET_AR) and type B (ET_BR) were performed by analysing somatic cell hybrid DNAs with the polymerase chain reaction (PCR) using primers specific for rat ET_AR and ET_BR genes. The genes for ET_AR and ET_BR were assigned to rat chromosomes 19 and 15 respectively.