Growth properties of RSV-transformed rat sarcoma (XC) cells

RSV-transformed rat sarcoma (XC) cells were characterized as to their ability to anchorage-dependent and anchorage-independent growth in various culture conditions. The proliferation of XC cells was dependent on serum concentration and initial cell density. The relationship between seeding density and the growth rate of XC cells indicated that the investigated cells produce factors with growth stimulating and growth inhibiting activity. The anchorage-independent growth inhibiting activity was found in ultrafiltrate (500 Mr 10,000) of XC cells conditioned medium. The obtained results suggests that anchorage-independent growth of XC cells may be regulated by two types of autocrine factors, which are antagonistic in their biological affects.     

Mammalian oocyte growth in vitro is stimulated by soluble factor(s) produced by preantral granulosa cells and by Sertoli cells

We have evaluated the possibility that mouse oocyte growth in vitro could be achieved under the influence of soluble compound(s) released by different somatic cell types. For this purpose, zona-free denuded oocytes from 12-day-old mice were cultured on monolayers of NIH-3T3 fibroblasts, which are able to establish gap junctional communications with them, in the presence or absence of media conditioned by preantral granulosa cells or by Sertoli cells, plated at increasing concentrations from 0.3–1 × 10⁶ ml⁻¹ cells. After 3 days, no increase in vitellus diameter was recorded from fibroblast-coupled oocytes maintained in culture medium or in the presence of media conditioned by 0.3 × 10⁶ ml⁻¹ Sertoli cells. By contrast, increasing proportions of coupled oocytes grew, provided the continuous presence of media conditioned by 0.5 or 1 × 10⁶ ml⁻¹ Sertoli cells, or by 0.3, 0.5, and 1 × 10⁶ ml⁻¹ preantral granulosa cells. Since the ligand of c-kit, the growth factor KL, promotes the growth in vitro of oocytes cultured in follicles from 8-day-old mice, an antibody against mouse KL was used to evaluate whether in our culture conditions KL might also be responsible for the growth of oocytes from 12-day-old mice. No inhibition of growth was evident in oocytes cultured directly on preantral granulosa or Sertoli-cell monolayers. Furthermore, the growth of fibroblast-coupled oocytes cultured in media conditioned by preantral granulosa cells was not significantly affected by the presence of this antibody during culture. By contrast, a high percentage of oocytes cultured on fibroblasts in the presence of media conditioned by Sertoli cells showed a significant inhibition of growth and no metabolic cooperativity. It was concluded that, besides KL, other bioactive factor(s) released by either preantral granulosa or Sertoli cells can induce a significant stimulation of mouse oocyte growth in vitro. © 1996 Wiley-Liss, Inc.     

Reduced binding of epidermal growth factor by avian sarcoma virus-transformed rat cells

Rat cells transformed by Rous sarcoma virus and Fujinami sarcoma virus bound 5-10% of the amount of epidermal growth factor (EGF) bound by normal cells. Scatchard plot analysis indicated that the reduction in binding by transformed cells was due to a decreased number of receptors rather than to altered binding affinity. In experiments with temperature sensitive mutants of Rous sarcoma virus and Fujinami sarcoma virus significant loss of EGF binding occurred within one hour of shift from non-permissive to permissive temperature. Conditioned media from various normal and transformed cell lines were examined for the ability to inhibit EGF binding to normal cells or to cause "down regulation" of EGF receptors. No activity of either type was found. EGF-dependent phosphorylation in isolated membrane preparations was also examined. Membranes from normal cells displayed EGF-dependent phosphorylation of a Mr 180,000 protein presumed to be the EGF receptor. This activity was absent in membranes from transformed cells. The data suggest a close correlation between activation of avian sarcoma virus transforming gene products and modulation of the EGF growth regulatory system.     

Anchorage-independent growth of RSV-transformed rat (GCA, W 12 and XC) cells

Three RSV-transformed rat cell lines: GCA, W12 and XC were characterized as to their ability to anchorage-independent growth in comparison to normal rat kidney (NRK-49F) cells. Differences in the threshold density (TD) and colony forming efficiency (CFE) of the investigated cells are described. The ability of virally transformed cells to stimulation of soft agar colony formation of NRK cells in coculture assay was presented. The production of TGFs-like factors by GCA, W12 and XC cells was suggested.     

3H-thymidine incorporation method versus colony forming assay in the determination of anchorage-independent cell growth of rat sarcoma (XC) and Morris hepatoma 7777 (MH) cells.

The modification of 3H-thymidine incorporation method of Tanigawa was used to the estimation of anchorage-independent growth of virally and chemically transformed rat cells. The relationship between colony forming assay and 3H-TdR incorporation test was determined, depends on the composition of culture medium and the period of incubation of rat sarcoma (XC) cells with thymidine. The influence of exogenous mitogens (RFG, TGF beta 1 and insulin) and autocrine factor (at different step of purification) on the growth of Morris hepatoma 7777 (MH) cells was estimated by both methods. Regression analysis comparing the results of colony counting and thymidine incorporation revealed good correlation between the two methods. The modification can be used the determination of growth stimulating or growth inhibiting activity and in multistep purification procedure of autocrine growth factors.     

Growth control of androgen-dependent Shionogi carcinoma cells by growth factor(s) produced from androgen-independent Shionogi carcinoma cells in a paracrine mechanism

Androgen-dependent (SC3) and -independent (CADO21) cloned cell lines were established from androgen-dependent mouse mammary tumor (Shionogi carcinoma 115). The effects of conditioned medium (CM) collected from SC3 and CADO21 cells on the anchorage-independent growth of SC3 cells in soft agar were studied. CM prepared from SC3 cells in the absence of testosterone was unable to stimulate the growth of SC3 cells, whereas CM prepared from SC3 cells in the presence of 10(-8) M testosterone stimulated the growth of SC3 cells in a concentration-dependent manner (21 colonies at 10% and 48 colonies at 20%) and this growth-stimulatory effect was not inhibited by 10(-6) M cyproterone acetate. CM prepared from CADO21 cells in the absence of testosterone was also able to stimulate the SC3 cell growth in a concentration-dependent manner (9 colonies at 10% and 19 colonies at 20%). These results suggest that the growth of androgen-dependent SC3 cells is stimulated by androgen-induced growth factor(s) produced from the same cells (autocrine mechanism) and is also regulated by autonomous growth factor(s) produced from androgen-independent cancer cells formed from the dependent cancer cells (paracrine mechanism). A suggested possible mechanism of the progression from androgen-dependent to -independent growth of cancer cells is also discussed.     

Contact-activated migration of melanoma B16 and sarcoma XC cells.

During migration, tumour cells interact with neighbouring neoplastic and normal host cells, and such interaction may influence their motile activity. We investigated the effect of homotypic collisions on the motile activity of two tumour cell lines, mouse melanoma B16 and rat sarcoma XC, and nontransformed human skin fibroblasts. It was found that the tumour cells show only limited motile activity when moving as single cells without contact with neighbours. At a higher density of the culture (and also at a greater number of cell to cell contacts) the activation of motility of investigated tumour cells was observed. On the other hand, the normal human skin fibroblasts showed a typical reaction of density-dependent inhibition of motility. The motile activity of tumour cells was not affected by conditioned media and was visibly dependent on a direct physical contact among colliding cells. The activation of cell movement was observed about 40-50 min after the initial contact between tumour cells. Contact-activated migration of neoplastic cells was inhibited by 50 microM verapamil (a selective voltage-gated calcium channel inhibitor) and 10 microM gadolinium chloride (a nonspecific blocker of mechanosensitive ion channels) but not by pertussis toxin. The observation that homotypic collisions among tumour cells strongly increase their motile activity suggests that contact-activated migration may play a significant role in tumour invasion and metastasis.     

Induction of P-glycoprotein functional activity and multidrug resistance by a soluble factor(s) produced by some mammalian cells.

In the previous study we have found that Djungarian hamster fibroblasts with high levels of multidrug resistance (MDR) (colchicine-resistance index RI of 1000 to 42000) produce soluble factor(s) communicating MDR to the drug-sensitive cells of the same species by elevating the functional activity of P-glycoprotein (Pgp). Here we have shown that these cells can influence human tumor cells in the same fashion. Rat hepatoma McA RH7777 cells and their colchicine-resistant derivatives are shown to produce a factor with similar effects (induction of MDR and Pgp functional activity in the drug-sensitive cells). These effects seem to depend on the drug resistance level of the donor cells. Our results show that induction of the Pgp-mediated MDR is not species-specific and the tumor cells with intrinsic MDR (arising from the tissue with a high level of Pgp expression) can produce a factor(s) communicating this type of drug resistance to the sensitive cells.     

Transforming growth factors (TGFs) produced by R-XC cells

Chemical fractionation as a first step of purification of TGFs from R-XC cells and culture medium was described. Physicochemical treatment indicated that the crude preparation of RGF's contains a heat and acid stable and dithiothreitol and trypsin sensitive polypeptides related with the alpha and/or beta TGFs. The influence of partially purified TGFs on REF cell colony formation in soft agar was documented.     

Characterization of high molecular weight transforming growth factor alpha produced by rat hepatocellular carcinoma cells

In addition to the mature 50 amino acid transforming growth factor alpha (TGF alpha), some transformed cells appear to produce multiple higher molecular weight forms. The structure and derivation of most of these larger soluble TGF alpha species remain to be established. We previously reported that a chemically induced rat hepatocellular carcinoma cell line, JM1, secreted acid-stable proteins which bind to epidermal growth factor receptors and stimulate DNA synthesis in primary cultures of normal adult rat hepatocytes. Purification and characterization of these hepatoma-derived growth factors have indicated their relationship to TGF alpha. Two EGF-competing activities of apparent Mr 30K and 10K were separated by gel filtration of concentrated JM1-conditioned medium and further purified by ion-exchange chromatography and reverse-phase HPLC. Both growth factors were detected by a radioimmunoassay specific for TGF alpha. Western blotting with antibodies to the 50 amino acid TGF alpha revealed that the lower molecular weight factor comigrated with the synthetic 6-kDa rat TGF alpha. The higher molecular weight TGF alpha appeared on immunoblots as a diffuse band of 18-21 kDa, which converted to the mature 6-kDa form upon digestion with elastase, confirming a precursor-product relationship. However, the 18-21-kDa proteins did not react with antibodies directed against the carboxy-terminal cytoplasmic segment of the transmembrane TGF alpha precursor. Enzymatic deglycosylation of the 18-21-kDa TGF alpha species by sequential removal of sialic acids and O- and N-linked carbohydrate reduced the molecular weight to 11K. The size and soluble nature of this polypeptide suggest that it represents the extracellular domain of the transmembrane TGF alpha precursor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Type beta transforming growth factor from feline sarcoma virus-transformed rat cells. Isolation and biological properties

We have isolated a strongly mitogenic, type beta transforming growth factor (beta TGF) released by Snyder-Theilen feline sarcoma virus-transformed rat embryo (FeSV-Fre) cells that induces phenotypic transformation of normal NRK cells when they are concomitantly stimulated by analogues of epidermal growth factor (EGF). Molecule filtration chromatography separates beta TGF from an EGF-like TGF (eTGF) which is also present in acid extracts from medium conditioned by FeSV-Fre cells (J. Massagué, (1983) J. Biol. Chem. 258, 13606-13613). Final purification of beta TGF is achieved by reverse phase high pressure liquid chromatography (HPLC) on octadecyl support, molecular filtration HPLC, and nonreducing dodecyl sulfate-polyacrylamide gel electrophoresis steps, yielding a 300,000-fold purified polypeptide with a final recovery of 21%. The purified rat beta TGF consists of two Mr = 11,000-12,000 polypeptide chains disulfide-linked as a Mr = 23,000 dimer. Induction of anchorage-independent proliferation of NRK cells by rat beta TGF depends on the simultaneous presence of eTGF or EGF. In the presence of a saturating (300 pM) concentration of either rat eTGF or mouse EGF, half-maximal anchorage-independent proliferation of NRK cells is obtained with 4-6 pM rat beta TGF. In the presence of a saturating (20 pM) concentration of rat beta TGF, half-maximal anchorage-independent proliferation of NRK cells is obtained with either rat eTGF or mouse EGF at a 50-70 pM concentration. Rat beta TGF is also able to induce DNA synthesis and cell proliferation on growth-arrested NRK, human lung, and Swiss mouse 3T3 fibroblast monolayers, this effect being half-maximal at 2-3 pM beta TGF for NRK cells. These results identify eTGF and beta TGF as the two synergistically acting factors responsible for the transforming action of culture fluids from FeSV-Fre cells.     

Platelet derived growth factor(s) for a hormone-responsive rat mammary tumor cell line.

Mammary tumor cell growth factor(s) has been identified in extracts of platelets from both male and female rats, as well as in extracts prepared from pooled outdated human platelets. When assayed by the growth promotion of MTW9/PL rat mammary tumor cells in culture, platelet extracts alone were able to support growth 50--75% as well as whole serum. The mitogenic activity from crude human platelet lysates was shown to be trypsin sensitive, relatively stable to extremes of pH, labile to heat treatment at 70 degrees, non-dialysable, ammonium sulfate precipitable, not removed by 56 degrees charcoal treatment, and of apparent molecular weight of 30,000 to 50,000 daltons as estimated by G-100 Sephadex chromatography. The platelet derived mammary growth factor activity was not replaced or potentiated by thrombin or known hormones and growth factors such as prolactin, insulin, 17-beta-estradiol, progesterone, hydrocortisone, L-thyroxine, and mouse epidermal growth factor. The experimental report demonstrates that platelets are a rich source growth factor activity for rat epithelial mammary tumor cells, and that the activity appears to be a polypeptide(s) different from other mitogenic activities known to influence growth of mammary tissue.     

Ionic responses and growth stimulation induced by nerve growth factor and epidermal growth factor in rat pheochromocytoma (PC12) cells

Rat pheochromocytoma cells (clone PC12) respond to nerve growth factor (NGF) by the acquirement of a phenotype resembling neuronal cells. In an earlier study we showed that NGF causes an increase in Na+,K+ pump activity, as monitored by ouabain-sensitive Rb+ influx. Here we show that addition of epidermal growth factor (EGF) to PC12 cells resulted in a stimulation of Na+,K+ pump activity as well. The increase of Na+,K+ pump activity by NGF or EGF was due to increased Na+ influx. This increased Na+ influx was sensitive to amiloride, an inhibitor of Na+,H+ exchange. Furthermore, no changes in membrane potential were observed upon addition of NGF or EGF. Amiloride-sensitive Na+,H+ exchange in PC12 cells was demonstrated by H+ efflux measurements and the effects of weak acids on Na+ influx. These observations suggest that both NGF and EGF activate an amiloride-sensitive, electroneutral Na+,H+ exchange mechanism in PC12 cells. These findings were surprising in view of the opposite ultimate biological effects of NGF and EGF, e.g., growth arrest vs. growth stimulation. However, within 24 h after addition, NGF was found to stimulate growth of PC12 cells, comparable to EGF. In the presence of amiloride, this stimulated growth by NGF and EGF was abolished. In contrast, amiloride did not affect NGF-induced neurite outgrowth of PC12 cells. From these observations it is concluded that in PC12 cells: (a) NGF has an initial growth stimulating effect; (b) neurite outgrowth is independent of increased amiloride-sensitive Na+ influx; and (c) growth stimulation by NGF and EGF is associated with increased amiloride-sensitive Na+ influx.     

A homologue of platelet-derived growth factor produced by rat alveolar macrophages

Rat alveolar macrophages secrete a growth factor that renders rat lung fibroblasts competent to initiate DNA synthesis in vitro in the presence of platelet-poor plasma. This biological activity resembles that of platelet-derived growth factor (PDGF). After separation from putative associated binding proteins by chromatography under acidic conditions, the macrophage-derived factor exhibited a relative molecular weight similar to that of highly purified human PDGF. The factor bound to a monospecific antibody to human PDGF and thus could be quantitated in an enzyme immunoassay for PDGF. It competed with radiolabeled human PDGF for receptor sites for PDGF on rat lung fibroblasts, and binding to these receptor sites could be specifically inhibited by anti-PDGF. These data strongly support the view that the factor derived from rat alveolar macrophages is homologous to human PDGF and is similar to human macrophage-derived PDGF-like growth factor. Furthermore, we have demonstrated that the lung contains both an effector cell (pulmonary macrophage) and a potential target cell (interstitial fibroblast) for this cytokine. Therefore the rat appears to be an appropriate animal model in which to study macrophage-derived PDGF-like growth factors as mediators of proliferation in pulmonary fibrogenesis.     

Stimulatory effect of thymic factor(s) on steroidogenesis in cultured rat granulosa cells.

Thymic cells from immature female rats were isolated and used for production of thymic cell culture conditioned medium (TCM). Granulosa cells were obtained from immature diethylstilbestrol (DES)-treated rats. TCM stimulated basal progesterone and estradiol secretion from the granulosa cells in a dose and time dependent manner. Maximal stimulation of progesterone production occurred at 48 hours of incubation, during which period TCM caused approximately 5 times more progesterone secretion than heart cell conditioned medium (HCM) or mock extract (ME). The maximum progesterone secretion by granulosa cells occurred when they were exposed to 48% TCM causing 7 times more progesterone secretion than controls. Under the same maximum stimulatory conditions, however, TCM only approximately doubled estradiol secretion compared to concentrations secreted in the presence of HCM or ME. Thus, the effect of TCM on progesterone secretion was more prominent than its effect on estradiol secretion. The stimulatory action of TCM was not mimicked by HCM, thymosin-alpha 1 or thymulin. Furthermore, the stimulatory action of TCM on steroidogenesis did not appear to be mediated by the cAMP system. The stimulatory factor(s) in TCM were heat, acid and acetone labile, but could not be sedimented by activated charcoal. Thus, the present study demonstrates that the secretory product(s) of thymic epithelial cells can stimulate steroidogenesis in cultured rat granulosa cells. Our data imply that thymic factor(s) may have a direct effect on ovarian function.     

Epidermal growth factor-like transforming growth factor. I. Isolation, chemical characterization, and potentiation by other transforming factors from feline sarcoma virus-transformed rat cells

An acid-stable transforming growth factor (TGF) that interacts with epidermal growth factor (EGF) receptors and is structurally related to EGF was isolated from serum-free culture fluids of Snyder-Theilen feline sarcoma virus-transformed rat embryo (FeSV-Fre) cells. Purification of this EGF-like TGF (eTGF) was achieved by molecular filtration chromatography and successive reverse-phase high pressure liquid chromatography steps on octadecyl support eluted with acetonitrile and 1-propanol gradients, respectively. Rat eTGF consists of a 7.4-kD single polypeptide chain that co-migrates with biological activity in dodecyl sulfate-polyacrylamide electrophoresis gels. Like preparations of a related TGF from human melanoma cells (Marquardt, H., and Todaro, G.J. (1982) J. Biol. Chem. 257, 5220-5225), but unlike EGF from rat, human, or mouse, rat eTGF has phenylalanine and lacks methionine. However, the sequence of the first 30 amino acid residues in rat eTGF is H2N-Val-Val-Ser-His-Phe-Asn-Lys-Cys-Pro-Asp-Ser-His-Thr-Gln-Tyr-Cys-Phe-His-Gly - Thr-(x)-Arg-Phe-Leu-Val-Gln-Glu-Glu-(Lys)-(Lys)-, which is significantly (20% and 28%) homologous to the NH2-terminal region of mouse EGF and human EGF, respectively. In addition to eTGF, molecular filtration chromatography of acid-soluble extracts from medium conditioned by FeSV-Fre cells resolved a 14-kD transforming factor(s) apparently devoid of intrinsic mitogenic activity but able to elicit a strong anchorage-independent growth response in the presence of eTGF or EGF. These results show that: 1) a 7.4-kDa TGF structurally and functionally related to EGF has been isolated from FeSV-Fre cells and 2) the full anchorage-independent growth-promoting activity of medium conditioned by FeSV-Fre cells is due to the coordinate action of at least two types of factors, the 7.4-kDa eTGF and a second 14-kDa transforming factor(s).     

AIDS-associated Kaposi's sarcoma cells in culture express vascular endothelial growth factor.

PCR cloning and cDNA sequencing have been used to identify mRNAs of two splice products of the vascular endothelial growth factor (VEGF) gene, VEGF121 and VEGF165, in cells isolated from Kaposi's sarcomas (KS) of AIDS patients (AIDS-KS). As demonstrated by Northern blot analysis, AIDS-KS cells as well as tumor cells show a high expression level of the VEGF gene as compared to primary human vascular cells like smooth muscle cells or endothelial cells. In addition to the lower expression of the gene, vascular cells express a 3.9 kb band together with a 3.2 kb band instead of a 3.9 kb and a 4.3 kb band in AIDS-KS cells. Our data suggest that the angiogenic properties of AIDS-KS cells might be mediated by the secretion of this growth factor and that this factor alone or in combination with other endothelial mitogens may be involved in endothelial proliferation associated with Kaposi's sarcoma.