Abstract
Carcinogenesis involves inactivation or subversion of the normal controls of proliferation, differentiation, and apoptosis. However, these controls are robust, redundant, and interlinked at the gene expression levels, regulation of mRNA lifetimes, transcription, and recycling of proteins. One of the central systems of control of proliferation, differentiation and apoptosis is retinoid signaling. The hRARαnuclear receptor occupies a central position with respect to induction of gene transcription in that when bound to appropriate retinoid ligands, its homodimers and heterodimers with hRXRαregulate the transcription of a number of retinoid-responsive genes. These include genes in other signaling pathways, so that the whole forms a complex network. In this study we showed that simple, cause-effect interpretations in terms of hRARαgene transcription being the central regulatory event would not describe the retinoid-responsive gene network.
A set of cultured bladder-derived cells representing different stages of bladder tumorigenesis formed a model system. It consisted of 2 immortalized bladder cell lines (HUC-BC and HUC-PC), one squamous cell carcinoma cell line (SCaBER), one papilloma line (RT4), and 4 transitional cell carcinomas (TCC-Sup, 5637, T24, J82) of varying stages and grades. This set of cells were used to model the range of behaviors of bladder cancers. Relative gene expression before (constitutive) and after treatment with 10 µM all-trans- retinoic acid (aTRA) was measured for androgen and estrogen receptor; a set of genes involved with retinoid metabolism and action, hRAR α nd β, hRXR α and β CRBP, CRABP I and II; and for signaling genes that are known to be sensitive to retinoic acid, EGFR, cytokine MK, ICAM I and transglutaminase. The phenotype for inhibition of proliferation and for apoptotic response to both aTRA and the synthetic retinoid 4-HPR was determined. Transfection with a CAT-containing plasmid containing an aTRA-sensitive promoter was used to determine if the common retinoic acid responsive element (RARE)-dependent pathway for retinoid regulation of gene expression was active. Each of the genes selected is known from previous studies to react to aTRA in a certain way, either by up- or down-regulation of the message and protein.
A complex data set not readily interpretable by simple cause and effect was observed. While all cell lines expressed high levels of the mRNAs for hRXRαand β that were not altered by treatment with exogenous aTRA, constitutive and stimulated responses of the other genes varied widely among the cell lines. For example, CRABP I was not expressed by J82, T24, 5637 and RT4, but was expressed at low levels that did not change in SCaBER and at moderate levels that decreased, increased, or decreased sharply in HUC-BC, TCC-Sup and HUC-PC, respectively. The expression of hRARα, which governs the expression of many retinoid-sensitive genes, was expressed at moderate to high levels in all cell lines, but in some it was sharply upregulated (TCC-Sup, HUC-PC and J82), remained constant (5637 and HUC-BC), or was down-regulated (SCaBER, T24 and RT4). The phenotypes for inhibition of proliferation showed no obvious relationship to the expression of any single gene, but cell lines that were inhibited by aTRA (HUC-BC and TCC-Sup) were not sensitive to 4-HPR, and vice versa. One line (RT4) was insensitive to either retinoid. Transfection showed very little retinoid-stimulated transfection of the CAT reporter gene with RT4 or HUC-PC. About 2-fold enhancement transactivation was observed with SCaBER, HUC-BC, J82 and T24 cells and 3–8 fold with 5637, TCC-Sup cells. In HUC-BC, a G to T point mutation was found at position 606 of the hRARα gene. This mutation would substitute tyrosine for asparagine in a highly conserved domain.
These data indicate that retinoid signaling is probably a frequent target of inactivation in bladder carcinogenesis. However, it seems this inactivation is a random process, but the strongly interlinked nature of signaling pathways can lead to unexpected manifestations. The phenotype of a cell is probably determined more by a neural net type of calculation involving numerous inputs (risk factors) than it is by strict cause and effect, i.e. inactivation of a particular gene may lead to widespread alterations in the activities of genes linked to it. A novel perspective based upon complexity for the investigation of carcinogenesis is presented.