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Gene expression profiling by DNA microarray analysis in mouse embryonic fibroblasts transformed by rasV12 mutated protein and the E1A oncogene.

Vasseur S, Malicet C, Calvo EL, Labrie C, Berthezene P, Dagorn JC, Iovanna JL - Mol. Cancer (2003)

Bottom Line: Interestingly, most of the genes encoding structural proteins, secretory proteins, receptors, extracellular matrix components, 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.These data may explain, at least in part, the behavior of transformed cells in that 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.Yet, we also found very unexpected results.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre de Recherche INSERM EMI 0116, 163 Avenue de Luminy, BP172, 13009 Marseille, France. vasseur@marseille.inserm.fr

ABSTRACT

Background: Ras is an area of intensive biochemical and genetic studies and characterizing downstream components that relay ras-induced signals is clearly important. We used a systematic approach, based on DNA microarray technology to establish a first catalog of genes whose expression is altered by ras and, as such, potentially involved in the regulation of cell growth and transformation.

Results: We used DNA microarrays to analyze gene expression profiles of rasV12/E1A-transformed mouse embryonic fibroblasts. Among the approximately 12,000 genes and ESTs analyzed, 815 showed altered expression in rasV12/E1A-transformed fibroblasts, compared to control fibroblasts, of which 203 corresponded to ESTs. Among known genes, 202 were up-regulated and 410 were down-regulated. 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. Interestingly, most of the genes encoding structural proteins, secretory proteins, receptors, extracellular matrix components, 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. These data may explain, at least in part, the behavior of transformed cells in that 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. Yet, we also found very unexpected results. For example, proteases and inhibitors of proteases as well as all 8 angiogenic factors present on the array were down-regulated in transformed fibroblasts although they are generally up-regulated in cancers. This observation suggests that, in human cancers, proteases, protease inhibitors and angiogenic factors could be regulated through a mechanism disconnected from ras activation.

Conclusions: This study established a first catalog of genes whose expression is altered upon fibroblast transformation by rasV12/E1A. This catalog is representative of the genome but not exhaustive, because only one third of expressed genes was examined. In addition, contribution to ras signaling of post-transcriptional and post-translational modifications was not addressed. Yet, the information gathered should be quite useful to future investigations on the molecular mechanisms of oncogenic transformation.

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A. Expression of RAS was verified by immunoblot analysis in MEFs transduced with pBabe (control) or pBabe-rasV12/E1A (transformed) retroviruses. B. Morphological aspect of the pBabe and pBabe-rasV12/E1A transduced mouse embryonic fibroblats. C. Anchorage-independent growth of the rasV12/E1A transformed MEF. Fifty thousand cells were plated on 0.6% agar in DMEM-10% FCS and overlaid on 0.6% agar in the same medium. Photomicrographs were taken 10 days after plating. D. rasV12/E1A transformed MEF induce tumor formation. One million of pBabe and pBabe-rasV12/E1A transduced mouse embryonic fibroblast were injected in 200 μl PBS as xenografts in nude mice. Representative mice at day 18.
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Figure 1: A. Expression of RAS was verified by immunoblot analysis in MEFs transduced with pBabe (control) or pBabe-rasV12/E1A (transformed) retroviruses. B. Morphological aspect of the pBabe and pBabe-rasV12/E1A transduced mouse embryonic fibroblats. C. Anchorage-independent growth of the rasV12/E1A transformed MEF. Fifty thousand cells were plated on 0.6% agar in DMEM-10% FCS and overlaid on 0.6% agar in the same medium. Photomicrographs were taken 10 days after plating. D. rasV12/E1A transformed MEF induce tumor formation. One million of pBabe and pBabe-rasV12/E1A transduced mouse embryonic fibroblast were injected in 200 μl PBS as xenografts in nude mice. Representative mice at day 18.

Mentions: We used microarray analysis to compare expression profiles of ~12,000 genes in normal vs. rasV12/E1A-transformed fibroblasts. Figure 1 shows the phenotypic changes of the rasV12/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 rasV12/E1A-transformed fibroblasts relative to the empty retrovirus-transduced MEFs. We summarize the highlights below and present the full profile in Figure 2.


Gene expression profiling by DNA microarray analysis in mouse embryonic fibroblasts transformed by rasV12 mutated protein and the E1A oncogene.

Vasseur S, Malicet C, Calvo EL, Labrie C, Berthezene P, Dagorn JC, Iovanna JL - Mol. Cancer (2003)

A. Expression of RAS was verified by immunoblot analysis in MEFs transduced with pBabe (control) or pBabe-rasV12/E1A (transformed) retroviruses. B. Morphological aspect of the pBabe and pBabe-rasV12/E1A transduced mouse embryonic fibroblats. C. Anchorage-independent growth of the rasV12/E1A transformed MEF. Fifty thousand cells were plated on 0.6% agar in DMEM-10% FCS and overlaid on 0.6% agar in the same medium. Photomicrographs were taken 10 days after plating. D. rasV12/E1A transformed MEF induce tumor formation. One million of pBabe and pBabe-rasV12/E1A transduced mouse embryonic fibroblast were injected in 200 μl PBS as xenografts in nude mice. Representative mice at day 18.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC153489&req=5

Figure 1: A. Expression of RAS was verified by immunoblot analysis in MEFs transduced with pBabe (control) or pBabe-rasV12/E1A (transformed) retroviruses. B. Morphological aspect of the pBabe and pBabe-rasV12/E1A transduced mouse embryonic fibroblats. C. Anchorage-independent growth of the rasV12/E1A transformed MEF. Fifty thousand cells were plated on 0.6% agar in DMEM-10% FCS and overlaid on 0.6% agar in the same medium. Photomicrographs were taken 10 days after plating. D. rasV12/E1A transformed MEF induce tumor formation. One million of pBabe and pBabe-rasV12/E1A transduced mouse embryonic fibroblast were injected in 200 μl PBS as xenografts in nude mice. Representative mice at day 18.
Mentions: We used microarray analysis to compare expression profiles of ~12,000 genes in normal vs. rasV12/E1A-transformed fibroblasts. Figure 1 shows the phenotypic changes of the rasV12/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 rasV12/E1A-transformed fibroblasts relative to the empty retrovirus-transduced MEFs. We summarize the highlights below and present the full profile in Figure 2.

Bottom Line: Interestingly, most of the genes encoding structural proteins, secretory proteins, receptors, extracellular matrix components, 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.These data may explain, at least in part, the behavior of transformed cells in that 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.Yet, we also found very unexpected results.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre de Recherche INSERM EMI 0116, 163 Avenue de Luminy, BP172, 13009 Marseille, France. vasseur@marseille.inserm.fr

ABSTRACT

Background: Ras is an area of intensive biochemical and genetic studies and characterizing downstream components that relay ras-induced signals is clearly important. We used a systematic approach, based on DNA microarray technology to establish a first catalog of genes whose expression is altered by ras and, as such, potentially involved in the regulation of cell growth and transformation.

Results: We used DNA microarrays to analyze gene expression profiles of rasV12/E1A-transformed mouse embryonic fibroblasts. Among the approximately 12,000 genes and ESTs analyzed, 815 showed altered expression in rasV12/E1A-transformed fibroblasts, compared to control fibroblasts, of which 203 corresponded to ESTs. Among known genes, 202 were up-regulated and 410 were down-regulated. 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. Interestingly, most of the genes encoding structural proteins, secretory proteins, receptors, extracellular matrix components, 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. These data may explain, at least in part, the behavior of transformed cells in that 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. Yet, we also found very unexpected results. For example, proteases and inhibitors of proteases as well as all 8 angiogenic factors present on the array were down-regulated in transformed fibroblasts although they are generally up-regulated in cancers. This observation suggests that, in human cancers, proteases, protease inhibitors and angiogenic factors could be regulated through a mechanism disconnected from ras activation.

Conclusions: This study established a first catalog of genes whose expression is altered upon fibroblast transformation by rasV12/E1A. This catalog is representative of the genome but not exhaustive, because only one third of expressed genes was examined. In addition, contribution to ras signaling of post-transcriptional and post-translational modifications was not addressed. Yet, the information gathered should be quite useful to future investigations on the molecular mechanisms of oncogenic transformation.

Show MeSH
Related in: MedlinePlus