Spectrum of transforming sequences detected by tumorigenicity assay in a large series of human neoplasms

Abstract

We here summarize the analysis of 126 DNA samples from patients with hematopoietic neoplasias and solid tumors and from various tumor cell lines that were screened in the tumorigenicity assay. Thirty-eight samples were able to induce tumors after transfection in NIH/3T3 cells and injection into nude mice. Southern-blot analysis with a panel of onco-gene probes revealed human ras genes in the vast majority of cases (25 N-ras, 2 K-ras, 1 H-ras) but also activated FGF4, dbl, ret and mas genes respectively. DNA samples from the 6 remaining transfectants were cloned into EMBL-3 phages and screened with a human specific repetitive Alu probe. Direct hybridization of a transfectant cDNA library allowed cloning of the ufo oncogene. Application of the exon-trapping technique to alu-positive phage DNA from the other transfec-tants enabled us to isolate tre, cot, B-raf, p85/HUMORF8 and a novel oncogene. Int.

Activation ofgef-h1, a guanine nucleotide exchange factor for RhoA, by DNA transfection

“…None of the 28 oncogene specific probes available to us detected additional related sequences other than those con-stitutively present in mouse cells (NIH/3T3). 6 These data suggest the presence of a novel human oncogene in these mouse tumors.…”

“…Filters were exposed to Kodak X-Omat DS or XAR-5 film at Ϫ70°C with Ilford intensifier screens. 6 For hybridisation analysis, we used the following insert probes: 300 bp insert from the human repetitive specific Alu plasmid Blur-8, 31 1,162 bp fragment from the 3Ј end of gef-h1 (KIAA0651) and the complete human pask-cDNA (KIAA0135). Prior to blot hybridisation, a pre-reassociation of the COS-1 or COS-9 probe with an excess of competing, unlabeled human DNA was performed.…”

“…In order to identify the human oncogenic sequences present in U937T1e2 transfectants we performed a panel of investigations, 6 including 1) hybridisation of DNA from our transfectants with a collection of available oncogene probes, 2) hybridisation of DNA from Alu-positive cosmid clones (COS 1 and COS 9) to RNA or a cDNA library made of RNA from the U937T1e2 transfectant and 3) exon-trapping analyses of Alu-positive genomic clones (COS 1). 6 As our standard panel of investigations did not reveal the respective oncogene, we decided to sequence the 2 cosmid clones COS 1 and COS 9 completely. Along this line we applied a so called transposon-based method of sequencing.…”

“…2 However, improvements of the classical focus formation assay, such as the tumourigenicity assay, expression cDNA cloning and expression cloning by retroviral transfer of cDNA libraries led to the discovery of various other transforming genes. [3][4][5] Using the classical NIH/3T3 transformation assay and its different variants, more than 25 oncogenes have already been detected (references in Janssen et al, 1999). 6 Some of these oncogenes were present in an activated state in the original tumour material, whereas others became activated in vitro during the DNA-mediated gene transfer process.…”

Rearrangement and expression of myeov and hst in NIH/3T3 transfectants: A caveat for the interpretation of DNA transfection analyses

“…Using the tumorigenicity assay our group was able to identify and/or isolate a plethora of different oncogenes, such as mas, ufo, p85ß/humorf8, ret, cot, tre, hst (fgf4), dbl, B-raf, gef-h1 and myeov (1)(2)(3)(4)(5). This latter gene, myeov, was identified by the tumorigenicity assay using DNA from the tumour of a patient with gastric carcinoma and assigned to the chromosomal region 11q13.…”