ABSTRACT
Aims-(1) To investigate the expression in human derived glioblastoma cell lines of two structurally related genes, novH (nephroblastoma overexpressed gene) and CTGF (connective tissue growth factor), which encode putative insulin-like growth factor binding proteins of a novel type. (2) To investigate whether the same transcription factors regulate CTGF and novH expression.Methods-Expression of novH and CTGF was analysed in 24 glioblastoma derived cell lines by northern blotting. The CTGF promoter region was characterised by nucleotide sequencing, RNase protection experiments, by transient transfections, and CAT assays.Results-CTGF and novH mRNA levels differed in the glioma cell lines studied. NovH and CTGF genes were not co-expressed in all cell lines. The CTGF promoter region was highly conserved compared with the corresponding region in the mouse (FISP12) and exhibited in vitro transcriptional activity.Conclusions-Although the coding regions of novH and CTGF are highly homologous, their promoter regions are substantially different, suggesting that these two genes may be regulated by different mechanisms. Considering that novH and CTGF are likely to be, respectively, negative and positive regulators of growth and that some glioma cell lines expressing novH are not tumorigenic, expression of these two genes might represent a key element in determining the stage of differentiation or the malignant potential, or both, of some tumour cell lines.
ABSTRACT
BA10-IR transformed cells, obtained by treating Syrian hamster embryo fibroblasts (HEF) with 7-methylbenz(a)anthracene and cultivated for a long period, are highly tumorigenic and grow in suspension as aggregates (spheroids) (Levy et al., 1976). They also grow in attached form or as spheroids in serum-free (S-) synthetic medium, without insulin and transferrin, and form anchorage-independent (AI) colonies in this same, but semi-solid, medium. This exceptional phenotype was acquired stepwise, after other transformation parameters, and appears to be related to the capacity of the transformed cells to respond to a mitogenic growth factor which they secrete. The response to this autocrine factor is amplified by insulin and transferrin. Untransformed HEF, at late and early passages, and also mouse and rat embryo fibroblasts, secrete factors equally active on BA10-IR cells; but HEF do not respond, in S- medium, to their factor, or that of BA10-IR cells. Rat FR3T3 fibroblasts transformed by Kirsten murine sarcoma virus (FR3T3-Ki cells) also form AI colonies in semi-solid S- medium, secrete an autocrine factor potentiated by insulin and transferrin, and respond to the factors active on BA10-IR cells. However, they form far fewer colonies without additives, and respond as well to the mitogenic factors only in the presence of insulin and transferrin. BA10-IR cells and FR3T3-Ki cells also release beta-TGF, or a related factor, in an active and a latent form, activable by acidification, and HEF latent, activable beta-TGF. However, the factors shed by BA10-IR cells or HEF which stimulate AI growth of BA10-IR and FR3T3-Ki cells are proteins which seem unrelated to known transforming growth factors. Two major cellular alterations characteristic of the transformed phenotype in vitro are the ability to grow in the absence of anchorage, in semi-solid medium, and reduced dependence on serum growth factors (Hanafusa, 1977; Tooze, 1980). These alterations are often expressed together, and anchorage independence also appears to be the in vitro transformation parameter which correlates best with the tumorigenicity of the transformed cells (Pollack et al., 1975; Shin et al., 1975; Cifone and Fidler, 1980). However, this correlation is not constant (cf., Tooze, 1980). The cellular changes which confer anchorage independence remain unknown, but the culture conditions which allow anchorage-independent (AI) growth are better known. This growth occurs in the same media which permit the growth of attached cells, but generally requires serum.(ABSTRACT TRUNCATED AT 400 WORDS)
