We used microarray analysis to compare expression profiles of ~12,000 genes in normal vs. ras^V12/E1A-transformed fibroblasts. Figure 1 shows the phenotypic changes of the ras^V12/E1A-transformed MEFs. With Affymetrix microarray technology, differential expression values greater than 1.7 are likely to be significant, based on internal quality control data. We present data which use a more stringent ratio, restricting our analysis to genes that are overexpressed or under-expressed at least 2.0 fold in ras^V12/E1A-transformed fibroblasts relative to the empty retrovirus-transduced MEFs. We summarize the highlights below and present the full profile in Figure 2.

Among the ~12,000 genes and ESTs analyzed, expression of 815 showed to be altered by at least 2.0 fold in the ras^V12/E1A-transformed fibroblasts, of which 203 corresponded to ESTs. Among known genes, 202 were up-regulated (Table 1)(see Additional file 1) whereas 410 were down-regulated (Table 2)(see Additional file 2) by ras^V12/E1A-transformation. It is interesting to note that about one half of genes encoding transcription factors, signaling proteins, membrane proteins, channels, or apoptosis-related proteins was up-regulated whereas the other half was down-regulated (Figure 2). However, after ras^V12/E1A-transformation most of genes encoding structural proteins, secretory proteins, receptors, proteases, protease inhibitors, extracellular matrix components, proteins involved in angiogenesis and cytosolic proteins, were down-regulated whereas genes encoding DNA-associated proteins (involved in DNA replication and reparation) and cell growth-related proteins were up-regulated (Figure 2). These data may explain, at least in part, the behavior of transformed cells. For example, down-regulation of structural proteins, extracellular matrix components, secretory proteins and receptors is consistent with reversion of the phenotype of transformed cells towards a less differentiated phenotype and up-regulation of cell growth-related proteins and DNA-associated proteins is consistent with their accelerated growth.

Primary embryo fibroblasts were isolated from 14.5 day-old SV129J mouse embryos following standard protocols 26. Cell were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal calf serum, 2 mM L-glutamine, 100 IU/ml penicilin G and 100 μg/ml streptomycin.

Oncogenic ras transforms most immortal rodent cells to a tumorigenic state, whereas transformation of mouse primary cells requires either a cooperating oncogene or the inactivation of a tumour suppressor gene. The adenovirus E1A oncogene cooperates with ras to transform primary mouse fibroblasts 7 and abrogates ras-induced senescence 27. Therefore, we transduced MEFs with the pBabe-ras^V12/E1A retroviral vector which expresses both the ras^V12 mutated protein and the E1A oncogene to obtain transformed fibroblasts. pBabe-ras^V12/E1A [described in ref. 27] and pBabe (as control) plasmids were obtained from S. Lowe. Bosc 23 ecotropic packaging (10⁶) cells were plated in a 6-well plate, incubated for 24 hr, and then transfected with PEI with 5 μg of retroviral plasmid. After 48 hr, the medium containing the virus was filtered (0.45 μm filter, Millipore) to obtain the first supernatant. MEFs were plated at 2 × 10⁵ cells per 35 mm dish and incubated overnight. For infections, the culture medium was replaced by an appropriate mix of the first supernatant and culture medium (V/V), supplemented with 4 μg/ml polybrene (Sigma), and cells were incubated at 37°C. As a control, we evaluated the ability of the retroviral vector to transduce MEFs by using a retroviral vector expressing the EGFP under control of the retroviral promoter located in the long terminal repeat. About 30% of MEFs expressed high levels of EGFP fluorescence 48 h after transduction (data not shown), indicating that retroviral vectors are well adapted to our experimental set-up. Retrovirus-infected cells were selected with puromycin (0.7 μg/ml). Transformation of MEFs by the pBabe-ras^V12/E1A retroviral vector was evaluated by examining changes in their morphological aspect, by quantifying expression of the RAS protein by western blot, by monitoring cell proliferation, colony formation in soft-agar and tumors in nude mice. In soft-agar assays, pBabe-ras^V12/E1A transformed cells formed colonies at high frequency (Figure 1). Similarly, transformed cells produced tumors in all (3/3) athymic nude mice when injected subcutaneously, whereas control MEFs did not (0/3) (Figure 1).

One hundred μg of total protein extracted from cells was separated with standard procedures on 12.5% SDS-PAGE using the Mini Protean System (Bio-Rad) and transferred to a nitrocellulose membrane (Sigma). The intracellular level of RAS was estimated by Western blot using the H-ras (C-20) polyclonal antibody (1:200) purchased from Santa Cruz Biotechnology, Inc.

Total RNA was isolated by Trizol (Gibco-BRL by Invitrogen). Twenty μg of total RNA was converted to cDNA with SuperScript reverse transcriptase (Gibco-BRL by Invitrogen), using T7-oligo-d(T)₂₄ as a primer. Second-strand synthesis was performed using T4 DNA polymerase and E. Coli DNA ligase followed by blunt ending by T4 polynucleotide kinase. cDNA was isolated by phenol-chloroform extraction using phase lock gels (Brinkmann). cDNA was in vitro transcribed using the T7 BioArray High Yield RNA Transcript Labeling Kit (Enzo Biochem, New York, N.Y.) to produce biotinylated cRNA. Labelled cRNA was isolated using an RNeasy Mini Kit column (Qiagen). Purified cRNA was fragmented to 200–300 mer cRNA using a fragmentation buffer (100 mM potassium acetate-30 mM magnesium acetate-40 mM Tris-acetate, pH 8.1), for 35 min at 94°C. The quality of total RNA, cDNA synthesis, cRNA amplification and cRNA fragmentation was monitored by micro-capillary electrophoresis (Bioanalizer 2100 by Bioanalyser 2100, Agilent Technologies). The cRNA probes were hybridized to an MGu74Av2 Genechip (Affymetrix, Santa Clara, CA). The MGu74Av2 Genechip represents ~6,000 sequences of mouse Unigene that have been functionally characterized and ~6,000 sequences ESTs clusters. Each sequence in the chip is represented by 32 probes : 16 "perfect match" (PM) probes that are complementary to the mRNA sequence and 16 "mismatch" (MM) probes that only differ by a single nucleotide at the central base (more detailed information about the MGu74Av2 Genechip can be obtained in the web site http://www.affymetrix.com. Fifteen micrograms of fragmented cRNA was hybridized for 16 h at 45°C with constant rotation (60 rpm). Microarrays were processed in an Affymetrix GeneChip Fluidic Station 400. Staining was made with streptavidin-conjugated phycoerythrin (SAPE) followed by amplification with a biotinylated anti-streptavidin antibody and a second round of SAPE, and then scanned using an Agilent GeneArray Scanner (Agilent Technologies). Expression value (signal) is calculated using Affymetrix Genechip software MAS 5.0 (for fully description of the statistical algorithms see http://affymetrix.com/support/technical/whitepapers/sadd_whitepaper.pdf. Briefly, signal is calculated as follow : First, probe cell intensities are processed for global background. Then, MM value is calculated and subtracted to adjust the PM intensity in order to incorporate some measure of non-specific cross-hybridization to mismatch probes. Then, this value is log-transformed to stabilize the variance. Signal is output as the antilog of the resulting value. The 20 probe pairs representing each gene are consolidated into a single expression level. Finally, software scales the average intensity of all genes on each array within a data set. Final value of signal is considered representative of the amount of transcript in solution.

Housekeeping controls β-actin and GAPDH genes serve as endogenous controls and are useful for monitoring the quality of the target. Their respective probe sets are designed to be specific to the 5', middle, or 3' portion of the transcript. The 3'/5' signal ratio from these probe sets is informative about the reverse transcription and in vitro transcription steps in the sample preparation. Then, an ideal target in which all transcripts was full-length transcribed would have an identical amount of signal 3' and 5' and the ratio would be equal to 1. Differences greater than three fold between signal at 3' and 5' for these housekeeping genes indicate that RNA was incompletely transcribed or target may be degraded. Ratio of fluorescent intensities for the 5' and 3' ends of these housekeeping genes was <2.

Hybridization experiments were repeated twice using independent cRNA probes synthezised with RNA from two independent sets of MEF-infected cells. Genes were considered as differentially expressed when both hybridizations showed >2 folds change. Data presented in this work represent the average of both hybridizations. The list of unchanged genes should be obtained from authors upon request.

Synthesis of probes: One microgram of total RNA from MEF cells was subjected to PCR with reverse transcription using the One Step RT-PCR kit (Gibco-BRL) according to the manufacturer's protocol to synthezise specific cDNA probes. PCR were carried out for 32 cycles, each cycle consisting in a denaturing step for 1 min at 94°C, an annealing step for 2 min at 56°C, and a polymerization step for 2 min at 72°C. Selected RNA species were amplified using the following primers: p8, sense, 5'-ggagagagcagactaggcata-3' and antisense, 5'-gttgctgccacccaagggcat-3'; transgelin, sense, 5'-ccagccagctctgcagatggg-3'and antisense, 5'-gcaggcagatttctgagttc-3'; serum amyloid A3, sense, 5'-ggatgaagccttccattgcc-3' and antisense, 5'-gaagagctacaccgccactc-3'; lysyl oxidase, sense, 5'-taaaacgactgtccccaacc-3' and antisense, 5-tcacggccgttgttagtgta-3'; thrombospondin 2, sense, 5'-aagcccagtcgggcttacgg-3' and antisense, 5-tgctggagctggagccctgc-3'; extracellular superoxide dismutase, sense, 5'-ccttagttaacccagaaatct-3' and antisense 5'-gtacctcaaaggtgctcactgg-3'; biglycan, sense, 5'-ggctgctttctgcttcacagg-3' and antisense 5'-gcaactgaccatcacctccta-3'; myb proto-oncogene, sense, 5'-ctaaaccatttcatgaggag-3' and antisense, 5-aacaaatgcaaaattcaccc-3'; cytokeratin, sense, 5'-ctggtctcagcagattgagg-3' and antisense, 5-ggtaggtggcaatctctgcc-3' ; high mobility group protein 2, sense, 5'-cgtctgccttctgcctgttttg-3' and antisense 5'-gcccttgacacggtatgcagc-3' and ezrin, sense, 5'-caacgaggagaagcggatca-3' and antisense 5'-gtgtgacacctgcctgcagtg-3'. Specificity of the PCR products was confirmed by direct DNA sequencing.

Northern blot hybridization: RNA samples (10 μg) were submitted to electrophoresis on a 1% agarose gel and vacuum blotted onto Hybond-N membranes (Amersham). The filters were hybridized with the ³²P-labeled probes for 16 h at 65°C in 5X SSPE (1X SSPE is 180 mM NaCl, 1 mM EDTA, 10 mM NaH₂PO₄, pH 7.5), 5X Denhardt solution, 0.5% SDS and 100 μg/ml single stranded herring sperm DNA. Filters were then washed four times for 5 min at room temperature in 2X SSC, 0.2% SDS, twice for 15 min at 50°C in 0.2X SSC, 0.2% SDS, and once for 30 min in 0.1X SSC at 50°C before autoradiography exposure on Kodak X-Omat films at -80°C from 8 hr to 4 days.