Complexity, retinoid-responsive gene networks, and bladder carcinogenesis.

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 hRAR alpha nuclear 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 hRXR alpha regulate 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 hRAR alpha gene 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 microM all-trans-retinoic acid (aTRA) was measured for androgen and estrogen receptor; a set of genes involved with retinoid metabolism and action, hRAR alpha nd beta, hRXR alpha and beta 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 hRXR alpha and beta 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 hRAR alpha, 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 hRAR alpha 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. (ABSTRAC

Retinoid signaling in immortalized and carcinoma-derived human uroepithelial cells.

This paper investigates the presence and functionality of retinoid signaling pathways in human urinary bladder carcinoma and SV40-immortalized uroepithelial cell lines. Only two of eight cell lines were proliferation-inhibited by 10 microM of either all-trans or 13-cis-retinoic acid. Transactivation of the CAT gene under control of a retinoid-responsive element demonstrated functionality of the signaling pathway in both sensitive cell lines and four of six resistant cell lines. Relative RT-PCR analysis of a panel of retinoid-responsive and inducible genes demonstrated changes in expression levels of all the genes in response to-retinoic acid treatment together with numerous aberrations dysregulations. We conclude that retinoid signaling may be a target for inactivation during tumorigenesis by uncoupling gene expression, proliferation and differentiation. Therefore retinoids are more likely to be effective for chemoprevention than for treatment of bladder carcinomas.

Expression of retinoid-responsive genes occurs in colorectal carcinoma-derived cells irrespective of the presence of resistance to all-trans retinoic acid.

BACKGROUND AND OBJECTIVES: Retinoids are metabolized in human intestinal epithelial cells to all-trans retinoic acid; however, it is unknown whether these cells express retinoid receptors, and whether sensitivity or resistance to the hormone is associated with a particular pattern of expression of retinoid-responsive genes. METHODS: Northern blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR) were used to identify mRNAs for retinoid receptors. Both Relative RT-PCR and transfection of retinoid-inducible plasmid were applied to test functionality of the pathway in a model system for colorectal carcinoma progression (primary SW480, all-trans retinoic acid-sensitive cells vs. metastatic SW620, -insensitive cells). RESULTS: Three colorectal carcinoma-derived cell lines were inhibited by the hormone. Retinoic acid receptor type alpha (hRAR alpha) and retinoid X receptor type alpha (hRXR alpha) mRNAs were detected in normal enterocytes, colonocytes, and in all colorectal carcinoma-derived cells studied. Primary carcinomas and metastatic lesions expressed high amounts of hRAR alpha receptor protein, showing no simple correlation between the amounts of mRNA and receptor protein. No pattern of expression of the retinoid-responsive genes was associated with sensitivity or resistance to the retinoid. Expression of the genes occurred irrespective of resistance to the hormone or inactivity of the pathway. CONCLUSIONS: Colonocytes possess a molecular system for transduction of the retinoid signal. All-trans retinoic acid modifies gene expression and inhibits proliferation of these cells. Therefore, retinoids are likely to be effective in chemoprevention of colorectal carcinoma.

Retinoids and secosteroids induce gene expression in human colorectal carcinoma-derived cells but the macroscopic-scale effects remain unpredictable.

Expression of a number of retinoid-responsive genes (hRAR alpha, CRABP I, CRABP II, MK cytokine) and secosteroid-responsive genes (hVD3R, Calbindin) was studied in in vitro model of human colorectal carcinoma by Relative RT-PCR. MK cytokine mRNA has been identified in human colonocytes for the first time. Proliferation of SW480 cells was inhibited by 5 microM all-trans retinoic acid and 5 microM 1 alpha, 25-dihydroxycholecalciferol; however, SW620 cells were not inhibited by all-trans retinoic acid. Unexpectedly, SW620 cells were stimulated by nanomolar concentrations of 1 alpha, 25-dihydroxycholecalciferol. In the latter case, no induction of gene expression was seen. Gene expression was induced in both cell types, whether there was a responsive element in the promotor region or not, suggesting that signal transduction to cellular nucleus did occur. Also, the Scatchard analysis for hVD3R receptor protein confirmed that the amount of the protein was modified under the treatment with both hormones; however, non-linear relationship between the amount of the mRNA and the protein was observed. In general, the genes responded differently to the treatment than it had been predicted. While this variability could be ascribed to the genetic instability, we hypothesize that instability in the cellular network of genes, mRNAs, and proteins is responsible for the observed effects. Due to the complexity, a microscopic-scale phenomenon such as gene expression cannot determine a macroscopic-scale process such as proliferation. This study provides a molecular background for retinoid/secosteroid chemoprevention of colorectal carcinoma; however, these hormones should be applied early to control premalignant lesions rather than advanced carcinomas.

Expression of sex steroid receptor genes and comodulation with retinoid signaling in normal human uroepithelial cells and bladder cancer cell lines.

The expression of sex steroid receptor genes in human uroepithelial cells (UEC) and their role in bladder carcinogenesis is unknown. Expression of androgen receptor (hAR), estrogen receptor (hER), and vitamin d3 receptor (hVDR3) genes in normal human stromal cells (SC) and UEC, six bladder cancer cell lines, and two SV-40-immortalized cell lines (SVC) was determined by reverse transcriptase polymerase chain reaction (RT-PCR). Functionality was assessed indirectly by relative RT-PCR, which identified comodulation of mRNA expression between retinoic acid and sex steroid receptor genes. UEC and SC expressed hAR and hER mRNA constitutively at low levels, but only positive controls expressed hVDR3. Every cancer cell line and the SVC showed aberrant expression. Treatment of cells with all-trans-retinoic acid up-regulated hAR and hER expression, whereas treatment with sex steroids up-regulated retinoic acid receptor expression. Cell proliferation was not affected by sex steroids or by their inhibitors. Sex steroid signaling pathways are functional in UEC and appear to be altered during bladder tumorigenesis. The sex steroid receptors may play a role in normal differentiation.