Molecular Analysis of Rat Embryo Cell Transformants Induced by α-particles

Abstract

An immortal cell line was established by transfecting a myc oncogene into rat embryo cells (REC:myc)¹. This cell line was diploid, contact inhibited and grew well in culture. Exposure to a single 200 cGy dose of 6 MeV α-particles transformed these cells with a frequency of focus formation of ∼ 3·6 × 10⁻⁴ compared with a transformation frequency of < 7·8 × 10⁻⁶ for primary cultures of REC. Isolates of α-particle-induced REC:myc (REC:myc:α) foci displayed anchorage-independent growth in soft agar and were tumourigenic in nude mice. Molecular studies demonstrated no alteration of gene structure or expression of the transfected or of the endogenous cmyc genes. Similarly, there was no alteration of the structure of Ha-ras, Ki-ras, or N-ras. The expression of Ha-ras, Ki-ras, N-ras and raf was not altered significantly. Assay for dominant oncogenes via DNA-mediated gene transfer into NIH3T3 cells was positive for nine of 13 REC:myc:α transformants. All NIH3T3 isolates contained bands hybridizing to rat repetitive DNA. NIH3T3 transformants from a tertiary round of transfection were analysed by Southern blot analysis for the presence of Ki-ras, N-ras, raf, trk, abl, fms, src, mos, fos, sis, fps, erbA, erbB or neu oncogenes of REC origin, and none were detected. Tertiary NIH3T3 transformants from three REC:myc:α transformants contained bands corresponding to Ha-ras but no point mutations were identified at the known hotspots of exons 1 or 2 of the donor REC:myc:α transformants. The inactivation of the tumour suppressor genes Rb, and p53, and the antimetastasis gene, nm23, was evaluated by Southern and Northern hybridization analysis. Southern blots demonstrated that at least one allele of Rbp53 and nm23 was present and no large scale structural changes were detected. No expression of Rb or p53 was detected in REC:myc or the α-particle-induced REC:myc transformants. The expression of nm23 was not altered in the transformed cell lines. While the analysis of the role of tumour suppressor gene inactivation in radiation-induced cell transformation is only in the initial stages, the results of DNA-mediated gene transfer into NIH3T3 cells suggest that unidentified dominant oncogenes are associated with α-particle-induced transformation in vitro.