Fibroblast growth factor stimulates colony formation of differentiated chondrocytes in soft agar

The effect of fibroblast growth factor (FGF) on the growth of chondrocytes in soft agar was examined. FGF induced colony formation by chick embryo and rabbit chondrocytes. The colony-forming efficiency of FGF-exposed chondrocytes was similar to that of Rous sarcoma virus-transformed chondrocytes (15-20%). Other mitogenic agents tested, such as epidermal growth factor, insulin, insulin-like growth factor-l, and platelet-derived growth factor, induced very low levels of colony formation. The induction of growth in soft agar of chondrocytes by FGF was not due to cells' phenotypic transformation, because chondrocytes grown in soft agar with FGF retained the ability to synthesize cartilage-characteristic proteoglycan. FGF did not induce growth in soft agar of chondrocytes whose phenotypic expression was suppressed by retinoic acid or 5-bromodeoxyuridine. In addition, FGF did not induce growth in soft agar of primary fibroblasts and normal rat kidney (NRK) cells. These results suggest that FGF selectively stimulates growth of differentiated chondrocytes in soft agar.     

1 alpha,25-dihydroxyvitamin D3 stimulates colony formation of chick embryo chondrocytes in soft agar

The effect of vitamin D metabolites on the growth of chick embryo chondrocytes in soft agar was examined. 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] at 10(-8)-10(-7) M induced colony formation by chick embryo chondrocytes in soft agar in the presence of 10% fetal bovine serum. Furthermore, 1,25(OH)2D3 increased the number of colonies in the presence of a maximal dose of basic fibroblast growth factor, a potent mitogen for chondrocytes in soft agar. However, 24R,25 (OH)2D3 and other metabolites had little effect on the soft agar growth of chondrocytes in the presence or absence of basic fibroblast growth factor. These results suggest that 1,25(OH)2D3 is an active metabolite which may be involved in supporting cartilage growth.     

Mouse Balb/c3T3 cell mutant with low epidermal growth factor receptor activity: induction of stable anchorage-independent growth by transforming growth factor beta

A mutant clone (MO-5) was originally isolated as a clone resistant to Na+/K+ ionophoric antibiotic monensin from mouse Balb/c3T3 cells. MO-5 was found to show low receptor-endocytosis activity for epidermal growth factor (EGF): binding activity for EGF in MO-5 was less than one tenth of that in Balb/c3T3. Anchorage-independent growth of MO-5 was compared to that of Balb/c3T3 when assayed by colony formation capacity in soft agar. Coadministration of EGF and TGF-beta efficiently enhanced anchorage-independent growth of normal rat kidney (NRK) cells, but neither factor alone was competent to promote the anchorage-independent growth. The frequency of colonies appearing in soft agar of MO-5 or Balb/c3T3 was significantly enhanced by TGF-beta while EGF did not further enhance that of MO-5 or Balb/c3T3. Colonies of Balb/c3T3 formed in soft agar in the presence of TGF-beta showed low colony formation capacity in soft agar in the absence of TGF-beta. Colonies of MO-5 formed by TGF-beta in soft agar, however, showed high colony formation capacity in soft agar in the absence of TGF-beta. Pretreatment of MO-5 with TGF-beta induced secretion of TGF-beta-like activity from the cells, while the treatment of Balb/c3T3 did not induce the secretion of a significant amount of TGF-beta-like activity. The loss of EGF-receptor activity in the stable expression and maintenance of the "transformed" phenotype in MO-5 is discussed.     

Stimulation of cartilage-matrix proteoglycan synthesis by morphologically transformed chondrocytes grown in the presence of fibroblast growth factor and transforming growth factor-beta

The effects of transforming growth factor-beta (TGF-beta) on the synthesis of cartilage-matrix proteoglycan by cultured rabbit chondrocytes were examined. Rabbit chondrocytes were seeded at low density and exposed to a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with 0.5% fetal bovine serum, 1% bovine serum albumin, 50 micrograms/ml ascorbic acid, and 2 x 10(-7) M hydrocortisone (Medium A). Various combinations of TGF-beta, insulin-like growth factor-I (IGF-I), and fibroblast growth factor (FGF) were also added to Medium A, and the chondrocytes were grown to confluency. Chondrocytes grown with TGF-beta or FGF alone became flat or fibroblastic, those grown with FGF and TGF-beta became very elongated and formed distinct foci, and those grown with FGF and IGF-I showed the spherical configuration characteristic of overtly differentiated chondrocytes. Nevertheless, the incorporation of 3H with glucosamine into the large, chondroitin sulfate proteoglycan synthesized by cultures with FGF and TGF-beta was similar to that in cells grown with FGF and IGF-I and five times that in cells cultured with FGF alone. The increases in incorporation of 3H reflected real increases in proteoglycan synthesis, because chemical analyses showed an increase in the accumulation of macromolecules containing uronic acid in cultures with FGF and TGF-beta or with FGF and IGF-I. However, FGF in combination with either TGF-beta or IGF-I had little effect on the incorporation of 3H into small proteoglycans or hyaluronic acid. These results indicate that chondrocytes morphologically transformed with TGF-beta and FGF fully express the differentiated proteoglycan phenotype rather than the transformed glycosaminoglycan phenotype.     

Effect of transforming growth factor beta on cell proliferation and glycosaminoglycan synthesis by rabbit growth-plate chondrocytes in culture

The effects of the transforming growth factor beta (TGF-beta) on the growth and glycosaminoglycan synthesis of rabbit growth plate-chondrocytes in culture were studied. In serum-free medium, TGF-beta caused dose-dependent inhibition of DNA synthesis by chondrocytes, measured as [3H]thymidine incorporation (ED50 = 0.1-0.3 ng/ml). The inhibitory effect was maximal at a dose of 1 ng/ml, and extended for a duration of 16-42 h. In contrast, TGF-beta potentiated the synthesis of DNA stimulated by fetal calf serum (FCS). Addition of TGF-beta (1 ng/ml) to cultures containing 10% FCS increased [3H]thymidine incorporation to 1.6-times that in cultures with 10% FCS alone. Consistent with this finding, TGF-beta potentiated DNA synthesis stimulated by the purified growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and fibroblast growth factor (FGF). The maximal stimulation of DNA synthesis by FGF (0.4 ng/ml) was further potentiated dose dependently by TGF-beta (ED50 = 0.1 ng/ml, maximum at 1 ng/ml). When the cultures were treated with the optimal concentrations of TGF-beta (1 ng/ml) and FGF (0.4 ng/ml), [3H]thymidine incorporation was 3-times higher than that of cultures treated with FGF alone. This TGF-beta-induced potentiation of DNA synthesis was associated with replication of chondrocytes, as shown by a marked increase in the amount of DNA during treatment of sparse cultures of the cells with the growth factors for 5 days. In contrast, TGF-beta caused dose-dependent stimulation of glycosaminoglycan synthesis in confluent cultures of growth-plate chondrocytes (ED50 = 0.3 ng/ml, maximum at 1 ng/ml). This stimulatory effect of TGF-beta was greater than that of insulin-like growth factor I (IGF-I) or PDGF. Furthermore, TGF-beta stimulated glycosaminoglycan synthesis additively with IGF-I or PDGF. Recently, it has been suggested that bone and articular cartilage are rich sources of TGF-beta, whereas epiphyseal growth cartilage is not. Thus, the present data indicate that TGF-beta may be important in bone formation by modulating growth and phenotypic expression of chondrocytes in the growth plate, possibly via a paracrine mechanism.     

Co-ordination of TGF-beta and FGF signaling pathways in bone organ cultures

Transforming growth factor-beta (TGF-beta) is known to regulate chondrocyte proliferation and hypertrophic differentiation in embryonic bone cultures by a perichondrium dependent mechanism. To begin to determine which factors in the perichondrium mediate the effects of TGF-beta, we studied the effect of Insulin-like Growth Factor-1 (IGF-I) and Fibroblast Growth Factors-2 and -18 (FGF2, FGF18) on metatarsal organ cultures. An increase in chondrocyte proliferation and hypertrophic differentiation was observed after treatment with IGF-I. A similar effect was seen after the perichondrium was stripped from the metatarsals suggesting IGF-I acts directly on the chondrocytes. Treatment with FGF-2 or FGF-18 resulted in a decrease in bone elongation as well as hypertrophic differentiation. Treatment also resulted in a decrease in BrdU incorporation into chondrocytes and an increase in BrdU incorporation in perichondrial cells, similar to what is seen after treatment with TGF-beta1. A similar effect was seen with FGF2 after the perichondrium was stripped suggesting that, unlike TGF-beta, FGF2 acts directly on chondrocytes to regulate proliferation and hypertrophic differentiation. To test the hypothesis that TGF-beta regulates IGF or FGF signaling, activation of the receptors was characterized after treatment with TGF-beta. Activation was measured as the level of tyrosine phosphorylation on the receptor. Treatment with TGF-beta for 24h did not alter the level of IGFR-I tyrosine phosphorylation. In contrast, treatment with TGF-beta resulted in and increase in tyrosine phosphorylation on FGFR3 without alterations in total FGFR3 levels. TGF-beta also stimulated expression of FGF18 mRNA in the cultures and the effects of TGF-beta on metatarsal development were blocked or partially blocked by pretreatment with FGF signaling inhibitors. The results suggest a model in which FGF through FGFR3 mediates some of the effects of TGF-beta on embryonic bone formation.     

1α,25-Dihydroxyvitamin D3 stimulates colony formation of chick embryo chondrocytes in soft agar

The effect of vitamin D metabolites on the growth of chick embryo chondrocytes in soft agar was examined. 1,25-Dihydroxyvitamin D₃ [1,25(OH)₂D₃]at 10⁻⁸-10⁻⁷ M induced colony formation by chick embryo chondrocytes in soft agar in the presence of 10% fetal bovine serum. Furthermore, 1,25(OH)₂D₃ increased the number of colonies in the presence of a maximal dose of basic fibroblast growth factor, a potent mitogen for chondrocytes in soft agar. However, 24R,25 (OH)₂D₃ and other metabolites had little effect on the soft agar growth of chondrocytes in the presence or absence of basic fibroblast growth factor. These results suggest that 1,25(OH)₂D₃ is an active metabolite which may be involved in supporting cartilage growth.     

Transforming growth factor-beta: Its effect on phenotype reexpression by dedifferentiated chondrocytes in the presence and absence of osteogenin

Summary The single and combined actions of transforming growth factor (TGF)-beta and osteogenin were evaluated with regard to induction of colony formation and reexpression of the differentiated phenotype by dedifferentiated rabbit articular chondrocytes in soft agarose under serum-free conditions. TGF-beta alone did not promote colony formation and induced accumulation of proteoglycans and type II collagen at significantly lower levels than those induced by osteogenin. Although synergism between these two growth factors occurred with respect to the induction of colony formation, their joint action on reexpression of the differentiated phenotype was additive. Complex interactions between the two growth factors may explain the latter phenomenon.     

Autocrine growth stimulation by secreted Kaposi fibroblast growth factor but not by endogenous basic fibroblast growth factor

We studied the different potentials of a secreted and a nonsecreted member of the fibroblast growth factor (FGF) family to induce autocrine growth stimulation in human adrenal cortex carcinoma cells (SW-13). These epithelial cells express basic FGF (bFGF) cell surface receptors, and picomolar concentrations of bFGF suffice to induce anchorage-independent growth. The requirement for exogenously added bFGF contrasts with the intracellular storage of biologically active bFGF in SW-13 cells greater than 10,000-fold in excess of the concentration needed to stimulate anchorage independent growth. To study whether the expression of a secreted FGF would alter the growth phenotype of these cells, we transfected them with an expression vector coding for the Kaposi-fgf (K-fgf) oncogene. In contrast to controls, K-fgf-transfected cells secrete significant amounts of biologically active K-fgf protein into the growth media, show up to 50-fold increased colony formation in soft agar, and grow into rapidly progressing, highly vascularized tumors in athymic nude mice. A reversible inhibition of the autocrine growth stimulation in vitro is brought about by the polyanionic compound suramin. We conclude that FGF has to be released from SW-13 cells to function fully as a growth stimulator in vitro and in vivo.     

Growth factor regulation of human growth plate chondrocyte proliferation in vitro

Linear growth occurs as the result of growth plate chondrocytes undergoing proliferative and hypertrophic phases. Paracrine feedback loops that regulate the entry of chondrocytes into the hypertrophic phase have been shown and similar pathways likely exist for the proliferative phase. Human long-bone growth plate chondrocytes were cultured in vitro. The proliferative effects of a variety of factors were determined by [3H]thymidine uptake and the gene expression profile of these cells was determined by DNA microarray analysis. Serum, insulin-like growth factor (IGF)-I and -II, transforming growth factor-beta (TGF-beta, fibroblast growth factor (FGF)-1, -2, and -18, and platelet-derived growth factor (PDGF)-BB were potent stimulators of proliferation. FGF-10, testosterone, and bone morphogenetic proteins (BMP)-2, -4, and -6 inhibited proliferation. Microarray analysis showed that the genes for multiple members of the IGF-I, TGF-beta, FGF, and BMP pathways were expressed, suggesting the presence of autocrine/paracrine pathways that regulate the proliferative phase of growth plate-mediated growth.     

Fibroblast growth factor induces the soft agar growth of two non-transformed celllines

Summary Recent studies have determined that fibroblast growth factor (FGF) potentiates the soft agar growth responses of NRK-49F cells to several combinations of transforming growth factors (TGFs). In the current study, two other non-transformed cell lines, NR-6 and AKR-2B, which do not spontaneously form colonies in soft agar, were examined for their soft agar growth responses to FGF. Both the acidic form and basic form of FGF were found to induce the soft agar growth of these cells. In the case of NR-6 cells, the effects of TGF-β were also examined. TFG-β potentiated the soft agar growth response of NR-6 cells to FGF, but on its own did not induce soft agar growth. Attempts to identify other factors capable of modulating the response of these cells to FGF, led to the finding that both 12-0-tetra-decanoylphorbol-13-acetate and retinoic acid suppress FGF-induced soft agar growth of NR-6 cells and AKRR-2B cells. The finding that FGF induces the soft agar growth of both non-transformed cell lines, together with the findings of others that both forms of FGF are angiogenic, lends further support to the suggestion that FGF plays a significant role in the in vivo growth of some, and possibly many, tumors. This work was supported by grants from the Nebraska Department of Health (86-11R, 87-38), the National Institute of Child Health and Human Development (HD 19837, HD 21568) and the National Cancer Institute (Laboratory Cancer Research Center Support Grant CA 36727). Editor's Statement The last several years have seen extraordinary advances in the understanding of the biochemistry and physiology of heparin-binding growth factors. Among the activities of these peptides that may be of significance for neoplasia and wound healingin vivo is ability to promote anchorage independent growth of some cell types. In this study the interactions among several stimulatory and inhibitory factors are examined in a soft agar growth assay. An appreciation of these interactions is critical in attempts to relatein vitro effects to those in the intact organism.     

Purification and properties of a type beta transforming growth factor from bovine kidney

Type beta transforming growth factor (TGF-beta) has been purified 200 000-fold from bovine kidneys. This peptide is characterized by its ability to induce anchorage-dependent normal rat kidney cells to grow in soft agar in the presence of epidermal growth factor (EGF); TGF-beta is not mitogenic for cells grown in monolayer culture. Purified TGF-beta does not compete with EGF for binding to membrane receptors. The concentration of TGF-beta required to elicit a half-maximal response for formation of colonies greater than 3100 micron2 in the soft agar assay is 2-3 pM (55 pg/mL) when assayed in the presence of 0.8 nM EGF (5 ng/mL). The four-step purification procedure which includes chromatography of acid--ethanol tissue extracts on polyacrylamide sizing gels, cation exchange, and two steps of high-pressure liquid chromatography results in a 10% overall yield of colony-forming activity with a recovery of 3-4 micrograms/kg. Amino acid analysis of purified TGF-beta shows 16 half-cystine residues per mole. Analysis of the purified polypeptide by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels indicates that TGF-beta is composed of two closely related polypeptide chains cross-linked by disulfide bonds. In the absence of beta-mercaptoethanol, the colony-forming activity is associated with a single silver-staining band of molecular weight 25 000; in the presence of beta-mercaptoethanol, the TGF-beta is converted to an inactive species that migrates as a single band of molecular weight 12 500-13 000. Sequence analysis indicates that at least the first 15 N-terminal amino acids of the two TGF-beta subunits are identical.     

Behavior of transforming growth factors in serum-free media: an improved assay for transforming growth factors

Transforming growth factors (TGFs) are a relatively new category of factors that induce the anchorage-independent growth of non-transformed cells. These factors are usually detected by their ability to induce normal rat kidney (NRK) fibroblasts to grow in soft agar. Until now, this assay has been performed in serum-containing medium (SCM). Unfortunately, the background activity of this assay is variable and dependent on several factors, including passage number of the cells and the serum lot used. Furthermore, the addition of either EGF or TGF-beta alone results in the appearance of additional colonies, which decreases the sensitivity of the assay. To circumvent these problems, serum-free media have been developed that support the growth of the NRK cells at low density in both monolayer culture and soft agar. Long-term growth in monolayer cultures occurs in serum-free medium supplemented with laminin, insulin, transferrin, epidermal growth factor (EGF), fibroblast growth factor (FGF) and high density lipoprotein (HDL). Growth in soft agar occurs when TGFs are added to a serum-free medium, AIG medium, that contains insulin, transferrin, FGF and HDL. In contrast to the background activity observed when the assay is performed in SCM, no colonies form in the AIG medium unless TGFs are added and few, if any, colonies form if EGF or TGF-beta are added alone. Thus, the AIG medium provides an improved assay for TGFs. In addition, the AIG medium should prove useful for examining other factors, including serum factors, for TGF activity.     

Presence of transforming growth factor-beta-like activity in multiple fetal rat tissues

The presence and ontogeny of transforming growth factor-beta (TGF-beta)-like bioactivity in rat tissues was studied. Eight separate tissues were extracted in acetic acid and assayed for TGF-beta-like activity by NRK-49F cell colony formation in soft agar. Bioactivity was present in each tissue during late fetal life, being most abundant in skeletal muscle, liver and lung (7.6-87.3 ng equivalents TGF-beta/g tissue), but fell to barely detectable levels from 12 days after birth. Gel filtration (pH 2.5) on Sephadex G75 of extracted fetal skeletal muscle, or culture medium conditioned by isolated fetal myoblasts demonstrated bioactivity in the molecular weight range 25-40 Kd. The results show that TGF-beta-like activity is selectively present in multiple fetal and neonatal, but not adult, rat tissues. Expression may therefore be developmentally regulated.     

EGCG经AKT1-Mdm-2-p53途径抑制卵巢癌HO-891O细胞的增殖

初步探讨EGCG对卵巢癌HO-8910细胞增殖的抑制作用及其机制.方法:通过绘制细胞生长曲线、平皿克隆和软琼脂集落形成实验观察EGCG对HO-8910细胞增殖的抑制作用;Western-blotting检测AKT1、Mdm-2与p53蛋白的表达.结果:(1)细胞生长曲线、平皿克隆和软琼脂集落形成实验结果显示,EGCG可有效抑制HO-8910细胞的增殖(n=3,P＜0.05).(2)Westemblotting检测结果显示,EGCG处理后AKT1与Mdm-2蛋白表达均降低,而p53蛋白表达升高(P＜0.05).结论:EGCG通过抑制HO-8910细胞中AKT1与Mdm-2蛋白表达,促使p53蛋白表达而发挥其对细胞增殖的抑制作用.     

Effects of epidermal growth factor and transforming growth factor-beta 1 on rat heart endothelial cell anchorage-dependent and -independent growth

This report describes the effects of epidermal growth factor (EGF) and transforming growth factor-beta 1 (TGF-beta 1) on the anchorage-dependent and -independent growth of rat heart endothelial cells (RHE-1A). When RHE-1A cells were grown in monolayer culture with medium containing 10% fetal bovine serum (FBS) supplemented with epidermal growth factor (0.1-100 ng/ml), growth was stimulated fivefold when compared to that of cells grown in medium containing 10% FBS alone. The stimulatory effect of EGF on RHE-1A cell monolayer growth was dose-dependent and half-maximal at 5 ng/ml. The addition of TGF-beta 1 in the range 0.1-10 ng/ml had no effect on RHE-1A cell monolayer growth when added to medium containing 10% FBS alone or 10% FBS supplemented with EGF (50 ng/ml). RHE-1A cells failed to grow under anchorage-independent conditions in 0.3% agar medium containing 10% FBS. In the presence of EGF, however, colony formation increased dramatically. The stimulatory effect of EGF was dose-dependent in the range 0.1-100 ng/ml and was half-maximal at 5 ng/ml. In contrast to its effects under anchorage-dependent conditions, TGF-beta 1 (0.1-10 ng/ml) antagonized the stimulatory effects of EGF on RHE-1A cell anchorage-independent growth. The inhibitory effect of TGF-beta 1 was dose-dependent and half-maximal at 0.1 ng/ml. EGF-induced RHE-1A soft agar colonies were isolated and reinitiated in monolayer culture. They retained the cobblestone morphology and contact-inhibition characteristic of normal vascular endothelial cells. Each of the clones continued to express Factor VIII antigen. These findings suggest that TGF-beta may influence not only endothelial cell proliferation but also anchorage dependence. These effects may in turn be of relevance to endothelial cell growth and angiogenesis in vivo.     

Transforming growth factor-alpha expression is enhanced in human mammary epithelial cells transformed by an activated c-Ha-ras protooncogene but not by the c-neu protooncogene, and overexpression of the transforming growth factor-alpha complementary DNA leads to transformation

MCF-10A cells are a spontaneously immortalized normal human mammary epithelial cell line. MCF-10A cells were transfected with two expression vector plasmids containing either a human point-mutated c-Ha-ras protooncogene or the rat c-neu protooncogene. c-Ha-ras-transfected MCF-10A cells grow as colonies in soft agar, exhibit a 3- to 4-fold increase in their growth rate in serum-free medium, and show a reduced mitogenic response to exogenous epidermal growth factor (EGF) or transforming growth factor-alpha (TGF alpha) as compared to MCF-10A cells. c-Ha-ras-transfected MCF-10A cells express a 4- to 8-fold increase in TGF alpha mRNA levels and secrete 4- to 6-fold more TGF alpha protein as compared to MCF-10A cells. Addition of either an anti-TGF alpha neutralizing monoclonal antibody or an anti-EGF receptor blocking monoclonal antibody to the Ha-ras-transformed MCF-10A cells produces a 50 to 80% inhibition of colony formation of these cells in soft agar. c-neu-transfected MCF-10A cells grown in soft agar and exhibit an increase in their growth rate in serum-free medium at a level comparable to that observed in Ha-ras-transformed MCF-10A cells. Addition of an anti-c-erbB-2 monoclonal antibody inhibits the anchorage-independent growth of these cells in soft agar. However, c-neu-transformed MCF-10A cells show no increase in TGF alpha secretion and no change in their responsiveness to exogenous EGF or TGF alpha. A recombinant retroviral vector containing the human TGF alpha gene was also introduced into MCF-10A cells. TGF alpha-infected MCF-10A cells secrete 15- to 20-fold more TGF alpha protein than MCF-10A cells, form colonies in soft agar, exhibit an enhanced growth rate in serum-free medium, and show a decreased mitogenic response to exogenous EGF or TGF alpha at a level equivalent to Ha-ras-transformed MCF-10A cells. Growth of TGF alpha-infected MCF-10A cells in soft agar is completely inhibited by anti-TGF alpha neutralizing or anti-EGF receptor blocking monoclonal antibodies. These results suggest that TGF alpha is an intermediary in the transformation of human mammary epithelial cells by an activated c-Ha-ras gene, but not by the c-neu gene, and demonstrate that overexpression of this growth factor is able to transform immortalized human mammary epithelial cells which also express a sufficient complement of functional EGF receptors.     

Transforming growth factor-beta-like activity is secreted by rabbit renal cortical tubular cells in primary culture.

The activity of an autocrine growth factor in a medium conditioned by cultured rabbit renal cortical tubular cells was investigated. Little stimulatory growth activity for tubular cells was observed in the conditioned medium, and inhibitory activity was seen only in acidified conditioned medium. This factor stimulated the colony formation of NRK 49F cells in soft agar only with epidermal growth factor and inhibited the DNA synthesis of primary cultured rat hepatocytes, and its molecular weight was about 25 kDa. The factor was neutralized by the specific antibody to transforming growth factor (TGF)-beta 1. These results indicate that renal tubular epithelial cells can produce latent TGF-beta in primary culture.