Jeanette A. M. Maier

Purification of basic fibroblast growth factor from rat brain: Identification of a Mr 22,000 immunoreactive form

A 18,000-dalton protein which stimulates plasminogen activator (PA) activity in endothelial GM 7373 cells has been purified from rat brain by using heparin affinity chromatography and ion-exchange chromatography. The purified molecule stimulates PA activity in a dose-dependent manner between 1 and 30 ng/ml. It also stimulates proliferation of GM 7373 cells and DNA synthesis in NIH 3T3 cells in a similar concentration range. The molecule has been identified as a bFGF-like molecule on the basis of its biological activity, its affinity for heparin-Sepharose, and its cross-reactivity with anti-human bFGF antibodies. In the final preparation of the rat brain bFGF, trace amounts (less than 5%) of a contaminant were detectable. This contaminant has a molecular weight of 22,000 and cross reacts with several anti-human placental bFGF antibodies. On the basis of its affinity for heparin-Sepharose and its immunological characteristics, this protein appears to be an high molecular weight form of bFGF.

Characterization of a Mr 20,000 basic fibroblast growth factor-like protein secreted by normal and transformed fetal bovine aortic endothelial cells

A mitogenic and plasminogen activator (PA)-inducing activity for endothelial cells has been identified in serum-free culture medium of normal AG 7680 and transformed tumorigenic GM 7373 fetal bovine aortic endothelial (FBAE) cells. The activity binds to heparin-Sepharose and it is quenched by polyclonal anti-human placental basic fibroblast growth factor (bFGF) antibodies. In the serum-free conditioned medium of FBAE cells, the anti-bFGF antiserum recognizes an immunorective Mr 20,000 molecule which co-purifies with the mitogenic and PA-inducing activity on a heparin-Sepharose column. The partially purified Mr 20,000 bFGF-like molecule competes with the typical Mr 18,000 125I-bFGF form for the binding to high-affinity bFGF receptors in intact GM 7373 cells. Immunoprecipitation of biosynthetically labeled GM 7373 cells with anti-bFGF antiserum confirms the presence of a Mr 20,000 bFGF-like molecule in the conditioned medium of these cells and identifies the typical Mr 16,000 and Mr 18,000 bFGF forms and two high-molecular-weight immunoreactive Mr 22,000 and Mr 25,000 bFGF forms in their cell extract. Immunoreactive Mr 20,000 bFGF is detectable also in the conditioned medium of transformed nontumorigenic FBAE GM 7372 cells and of adult bovine aortic endothelial cells, but not in the culture medium of nonendothelial cell types, including rat and mouse fibroblasts, human hepatoma, and human endometrial adenocarcinoma cells. The results indicate that bovine endothelial cells secrete a Mr 20,000 bFGF-like molecule which shares several biological, biochemical, and immunological characteristics with the typical cell-associated Mr 18,000 bFGF.

Expression and alternative splicing of fibronectin mRNA in human diploid endothelial cells during aging in vitro.

Different mRNAs for fibronectin arise from the variable processing of a single primary transcript. We used ribonuclease protection assay to investigate the changes occurring in fibronectin expression and the alternative splicing of mRNA precursor during aging in vitro of human diploid endothelial cells. Senescent endothelial cells release more protein and contain 4-5-fold more fibronectin mRNA than young cells. The pattern of alternative splicing of fibronectin mRNA, with the EDA and the CS1 segments largely included (35% and 77%, respectively) and the EDB segment undetectable, correlates well with previous studies at the protein level both in vitro and in vivo. No changes in the splicing pattern of fibronectin mRNA precursor were detected during endothelial cellular senescence. The increased expression of fibronectin in senescent cells may be a result of the activity of interleukin-1 alpha, which is overexpressed in senescent endothelial cells. It could be also important in vivo during aging and in atherosclerotic lesions.

Induction of plasminogen activator activity by phorbol ester in transformed fetal bovine aortic endothelial cells. Possible independence from protein kinase C

12-O-tetradecanoyl phorbol 13-acetate (TPA) and 1,2-dioctanoyl-sn-glycerol (diC8) activate protein kinase C (PKC) in transformed fetal bovine aortic endothelial GM 7373 cells. Both molecules cause a similar increase in membrane-associated PKC activity and in the phosphorylation of a PKC-specific endogenous 87-kDa substrate in intact cells. Even though both TPA and diC8 exert a mitogenic activity in GM 7373 cells, only TPA induces also an increase in cell-associated plasminogen activator (PA) activity. Down-regulation of PKC which follows TPA-pretreatment completely abolishes the mitogenic activity of diC8 and the mitogenic and PA-inducing activity of TPA. However, both the PKC inhibitor H-7 and the down-regulation of PKC which follows a prolonged stimulation with diC8 do not abolish the PA-inducing activity of TPA. The PA-inducing activity of TPA is instead inhibited in cultures incubated in the presence of 1 mM EGTA or in a calcium-free medium. The data indicate that TPA and diC8 induce a different pattern of cellular activation in GM 7373 cells and that the PA-inducing activity of TPA might not be mediated by PKC.

Basic Fibroblast Growth Factor and Endothelial Cells: Receptor Interaction, Signal Transduction, Cellular Response-Dissociation of the Mitogenic Activity of bFGF from its Plasminogen Activator-Inducing Capacity

Basic fibroblast growth factor (bFGF) belongs to a class of heparin-binding growth factors which includes acidic FGF (aFGF) and five other related oncogene products (Burgess and Maciag, 1989). bFGF induces cell proliferation in a variety of cell types of mesodermal and neuro-ectodermal origin and appears to be a trophic factor for cultured neuronal cells. In vivo, bFGF may play a role in embryonic development (Kimelman and Kirschner, 1987) and induces neovascularization in different angiogenesis assays (Folkman and Klagsbrun, 1987). Also, anti-bFGF antibodies have been shown to inhibit angiogenesis in normal wound repair, indicating that bFGF is intrinsically involved in normal neovascularization (Broadley et al., 1989).

Liver DNA Damage by Chemical Carcinogens: Role of Thyroid Hormones

In 1948, Paschkis et al.14 demonstrated the protective effect of thiouracil on the induction of liver tumors by 2-acetylaminofluorene. Since then, several authors reported on the inhibition of hepatocarcinogenesis in hypothyroid rats1,2,8,12,19. These findings raise the point of the identification of the stage in chemical hepatocarcinogenesis which is inhibited by thyroid deficiency. Even though it has been demonstrated that thyroid digest increases the rate of growth of liver tumors in pituitary dwarf mice treated with aminofluorene3, therefore suggesting a role for thyroid hormones in the promotion and/or progression of liver tumors, several data indicate that thyroid activity might influence liver carcinogenesis also at the stage of initiation. Bielschowsky and Hall1, in fact, demonstrated that thyroidectomy, performed before, but not after, the administration of the carcinogen, prevents the development of liver tumors. These findings were confirmed by Goodall8 who demonstrated that in thyroidectomized animals the treatment with thyroid digest performed after aminofluorene administration does not restore the suscciptibility to the carcinogen. Moreover, thyroid hormones have been demonstrated to be necessary for the in vitro transformation of cultured cells by x-rays or chemical carcinogens9,10.