Colin S.Cooper, Philip R.Tempest, M.Patricia Beckman',. Carl-Henrik Heldin2 and Peter Brookes. Chemical Carcinogenesis Section, Institute of Cancer ...
The EMBO Journal vol.5 no. 10 pp.2623 -2628, 1986
Amplification and overexpression of the met gene in spontaneously transformed NIH3T3 mouse fibroblasts
Colin S.Cooper, Philip R.Tempest, M.Patricia Beckman', Carl-Henrik Heldin2 and Peter Brookes Chemical Carcinogenesis Section, Institute of Cancer Research, Chester Beatty Laboratories, Fulham Road, London SW3 6JB, UK, 'Department of Medical and Physiological Chemistry, University of Uppsala, and 2Ludwig Institute for Cancer Research (Uppsala Branch), Box 595, Biomedical Center, S-751 23 Uppsala, Sweden Communicated by R.A.Weiss
We have identified a class of transformed NIH3T3 mouse fibroblasts that arise at low frequencies in transfection experiments with DNA from both neoplastic and non-neoplastic cells and that may result from a low level of spontaneous transformation of NIH3T3 cells. DNA from the transformed cells was unable to transform NIH3T3 cells in a second cycle of transfection and, where examined, the cells showed no evidence for the uptake of the transfected DNA sequences. The results of Southern analyses demonstrate that a mouse homologue of the human met oncogene is amplified 4- to 8-fold in 7 of 10 lines of these transformed NIH3T3 mouse fibroblasts. The cells containing the amplified gene also exhibit at least a 20-fold overexpression of an 8.5-kb mRNA that is homologous to met. To test the hypothesis that met encodes a growth factor receptor, we examined the binding of plateletderived growth factor, epidermal growth factor, insulin-like growth factor I and gastrin-releasing peptide to transformed and non-transformed NIH3T3 cells. The results show that there is no significant elevation of the binding of these growth factors to cells containing amplification and overexpression of met. Key words: gene amplification/met oncogene/mouse fibroblast transformation/growth factor binding Introduction Activated oncogenes that are present in some primary tumours and transformed cell lines can be detected by their ability to efficiently induce foci of morphologically transformed NIH3T3 mouse fibroblasts in DNA transfection experiments. (Shih et al., 1979; Cooper and Neiman, 1980; Perucho et al., 1981; Marshall et al., 1982; Blair et al., 1982; Pulciani et al., 1982; Balmain and Pragnell, 1983). In these experiments, transformation of the NIH3T3 cells results from the uptake and stable incorporation of the activated oncogenes, and DNA from the transformed cells can be used to transform NIH3T3 cells in a second round of transfection. Most of the oncogenes identified in these studies are members of the ras family of oncogenes, which are activated by point mutations in the codons for amino acids 12 or 61 (for a review, see Marshall, 1985). Other genes including B-lym, mel, neu and met have, however, also been identified. (Diamond et al., 1983; Cooper et al., 1984b; Padua et al., 1984; Schechter et al., 1984). The met gene was originally identified by transfection of DNA from the N-methyl-N'-nitro-N-nitrosoguanidine transformed human cell line called MNNG-HOS (Cooper et al., 1984a, 1984b) and in this particular cell line is activated IRL Press Limited, Oxford, England
by gene rearrangement, the 5' end of the activated met gene is derived from chromosome 1 while the 3' end is derived from chromosome 7 (Park et al., 1986). Additional studies have demonstrated that the region of activated met derived from chromosome 7 is homologous to a family of genes that encode protein kinases (Dean et al., 1985). DNA transfection into NIH3T3 cells has also been used to demonstrate the transforming potential of genes from normal cells. Cooper et al. (1980) have demonstrated that sonicated fragments of DNA from normal chicken and mouse cells can transform NIH3T3 cells with low efficiencies. In contrast, in a second cycle of transfection, high mol. wt DNA from the transformed NIH3T3 cells efficiently transformed NIH3T3 cells indicating that normal cellular genes had been activated by rearrangement of normal chicken and mouse DNAs during the original transfection. A low frequency of another class of transformed NIH3T3 cells has been observed in transfection assays with high mol. wt DNAs from both normal and neoplastic cells (Cooper et al., 1980; Perucho et al., 1981). DNAs from these transformed NIH3T3 cells are unable to transform NIH3T3 cells in a second cycle of transfection and they have been referred to as spontaneous transformants, although the precise mechanism of transformation has not been defined. Amplification and overexpression of N-myc and of cellular homologues of genes such as c-myc, rasK and erbB have been detected in many types of tumour and it has been suggested that overexpression of these genes may contribute to tumourigenesis (Collins and Groudine, 1982; Dalla Favera et al., 1982; Kohl et al., 1983; Schwab et al., 1983a, 1983b; Libermann et al., 1985). This hypothesis is supported by the high frequency with which amplification of particular oncogenes is observed in some tumour types and by the observation that, in some cases, the amplification may be associated with particular stages in tumour progression. For example the amplification of N-myc is detected in 20-30% of advanced stages of human neuroblastomas (stages III and IV) but not in less advanced stages of this disease (stages I and H) (Brodeur et al., 1984). In the present study we have examined the role of oncogene amplification and overexpression in the generation of spontaneously transformed NIH3T3 cells. Our results show that a high proportion of the transformed NIH3T3 cells have a 4- to 8-fold amplification and at least a 20-fold overexpression of a mouse gene that is homologous to met. The binding of growth factors to cell lines containing amplified met has also been examined.
Results Detection of transformed NIH3T3 mouse fibroblasts In our transfection experiments we detected a class of morphologically transformed NIH3T3 cells that arose at low frequencies in transfections with DNA from both neoplastic (8 foci/197 recipient cultures) and non-neoplastic (2 foci/ 104 recipient cultures) cells. The foci consisted of cells that were spindle shaped and refractile when observed using phase contrast microscopy and had similar morphologies to foci induced by activated ras 2623
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Table I. Properties of transformed NIH3T3 mouse cells Cell lines and tissues
Relative no. Efficiency of of mouse met transformation gene copies of NIH3T3 cells by DNA (foci/recipient cultures)
Tumours in Human DNA mice (no. of sequencesb tumours/sites injected)a
Controls Mouse thymus 1 NIH3T3 cells 1 Spontaneously transformed NIH3T3 cellsc Line 1 6 Line 2 4 Line 3 8 Line 4 6 Line 5 8 Line 6 8 Line 7 8 Line 8 1 Line 9 1 Line 10 1 NIH3T3 cells transformed by activated oncogened N-PAI-I 1 1 N-PA1-2 N-MNNG-HOS-1 1 N-MNNG-HOS-2 1
0/12 0/12
NT 0/4
NA NA
0/8 0/12 0/7 0/8 1/8 1/8 0/8 0/12 0/8 0/8
4/4 6/6 6/6 6/6 6/6 6/6 6/6 NT 6/6 6/6
NA NA NA NA
85/4 14/8 31/4 33/4
NT 4/4 5/5 5/5
NA NA
+ + + +
aTumorigenicity of NIH3T3 cells was assessed by injecting 5 x 105 cells s.c. into nude mice. All of the tumours arose in
