A transcriptome map of cellular transformation by the fos oncogene

Abstract

Background: The c-fos gene was originally identified as the cellular homolog of the oncogene v-fos carried by the Finkel-Biskis-Jenkins and Finkel-Biskis-Reilly murine osteogenic sarcoma retroviruses. Sustained expression of fos is sufficient to induce cellular transformation in vitro and tumorigenesis in vivo. Fos functions as a component of the AP-1 transcription factor complex to regulate gene transcription and several differentially expressed genes have been identified in cells transformed by fos. We have extended these studies by constructing a cellular system for conditional transformation by v-fos. Using Affymetrix-based DNA microarray technology, we analyzed transcriptional changes over the course of transformation and reversion in an inducible v-fos system.

Results: Microarray analyses of temporal gene expression during the process of v-fos mediated cellular transformation and morphological reversion revealed a remarkably dynamic transcriptome. Of the more than 8000 genes analyzed in this study, 3766 genes were categorized into 18 gene-expression patterns by using self-organizing map analysis. By combining the analysis of gene expression profiles in stably transformed cells with the analysis of sequential expression patterns during conditional transformation, we identified a relatively small cohort of genes implicated in v-fos mediated cellular transformation.

Conclusion: This approach defines a general conditional cell transformation system that can be used to study the endogenous transcription regulatory mechanisms involved in transformation and tumorigenesis. In addition, this study is the first reported analysis of dynamic changes in gene expression throughout experimentally controlled morphological transformation mediated by v-fos.

Figure 1

Expression of v-Fos and cell morphology rearrangement in LacIv-fos system during conditional transformation and reversion. (A) Expression of v-Fos protein expression during conditional morphological transformation of LacIv-fos cells. At time zero (t = 0), Fos protein was not detectable in cells. 24 hours after removal of IPTG, Fos protein expression was induced and the immunoreactivity increased over the 72 hour transformation period. After re-addition of IPTG, Fos protein levels rapidly decreased with no detectable signal observed after 72 hours of reversion. (B) Induction of v-Fos expression resulted in dramatic cytoskeletal changes in LacIv-fos cells. Cells were stained with anti-vinculin antibody (green) to detect focal adhesion contact sites and phalloidin (red) was used to track alterations in the actin cytoskeleton. In the presence of IPTG (t = 0), cells have well established focal adhesion sites and coordinated F-actin staining. Seventy-two hours after removal of IPTG (t = 72), cells lacked defined focal adhesion contacts and displayed disorganized F-actin staining

Figure 2

Self-organizing map (SOM) analysis of microarray expression data in the LacIv-fos system during morphological transformation and reversion. (A) Results of SOM analysis of genes depicting co-regulated clusters of genes across the seven time points throughout v-Fos mediated morphological transformation and reversion. Genes whose expression did not change significantly across time points were eliminated by using a variance filter (see Methods). The 3766 probe sets (out of a possible 8799) that passed the variation filter were grouped into 18 clustered patterns. Blue bars at the top of each graph represent the relative number of probe sets included within the SOM bin. (B) A gene-expression heat map of genes within group 8. This bin represents genes dramatically upregulated specifically during the 72 hour v-Fos transformation period (-IPTG). (C) Gene-expression heat map of genes within group 11. This bin represents genes displaying sustained downregulation during the 72 hr v-Fos transformation period (+ITPG). Red signal represents upregulated genes and green represents downregulated genes. (D-E) Normalized expression values of all genes within group 8 (D) or group 11 (E) are plotted to demonstrate the reliability of the representative SOM patterns shown in panel A.

Figure 3

Expression of representative genes conditionally regulated in LacIv-fos cells and differentially expressed in both CMVc-fos cells and FBJ/R cells. Expression patterns of selected genes upregulated (A) or downregulated (B) specifically during conditional transformation, as well as in cells stably transformed by either c-Fos or v-Fos. Gene names are indicated above each pair of graphs. Line graphs indicate raw signal values obtained from the two independent LacIv-fos time course experiments. Time points at 24, 48 and 72 hours in the absence of IPTG (transforming) and at 24, 48 and 72 hours after the re-addition of IPTG (reverting) are indicated by arrows below each line graph. Bar graphs indicate raw signal values obtained in microarray analyses of the parental 208F fibroblast cell line (white), the stably transformed c-fos cell line (CMVc-fos; grey) and the stably transformed v-fos cell line (FBJ/R; black).

Figure 4

Expression of representative genes conditionally regulated in LacIv-fos cells and stably regulated in FBJ/R cells, but not CMVc-fos cells. Expression patterns of selected genes upregulated (A) or downregulated (B) specifically during conditional transformation, as well as in cells stably transformed by v-Fos. Gene names are indicated above each pair of graphs. Line graphs indicate raw signal values obtained from the two independent LacIv-fos time course experiments. Time points at 24, 48 and 72 hours in the absence of IPTG (transforming) and at 24, 48 and 72 hours after the re-addition of IPTG (reverting) are indicated by arrows below each line graph. Bar graphs indicate raw signal values obtained in microarray analyses of the parental 208F fibroblast cell line (white), the stably transformed c-Fos cell line (CMVc-fos; grey) and the stably transformed v-Fos cell line (FBJ/R; black).