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Selective alteration of gene expression in response to natural and synthetic retinoids.

Brand C, Ségard P, Plouvier P, Formstecher P, Danzé PM, Lefebvre P - BMC Pharmacol. (2002)

Bottom Line: This suggested that gene-selective modulation could be achieved by structurally distinct retinoids.Furthermore, this differential ability to regulate promoter activities was also observed in murine P19 cells for the RARbeta2 and CRABPII gene, showing conclusively that retinoid structure has a dramatic impact on the regulation of endogenous genes.Our findings therefore show that some degree of selective induction or repression of gene expression may be achieved when using appropriately designed ligands for retinoic acid receptors, extending the concept of selective modulators from estrogen and peroxisome proliferator activated receptors to the class of retinoid receptors.

View Article: PubMed Central - HTML - PubMed

Affiliation: INSERM U 459 and Ligue nationale contre le Cancer, Faculté de Médecine Henri Warembourg, 1, place de Verdun, 59045 Lille cedex, France. brand@lille.inserm.fr

ABSTRACT

Background: Retinoids are very potent inducers of cellular differentiation and apoptosis, and are efficient anti-tumoral agents. Synthetic retinoids are designed to restrict their toxicity and side effects, mostly by increasing their selectivity toward each isotype of retinoic acids receptors (RARalpha,beta, gamma and RXRalpha, beta, gamma). We however previously showed that retinoids displayed very different abilities to activate retinoid-inducible reporter genes, and that these differential properties were correlated to the ability of a given ligand to promote SRC-1 recruitment by DNA-bound RXR:RAR heterodimers. This suggested that gene-selective modulation could be achieved by structurally distinct retinoids.

Results: Using the differential display mRNA technique, we identified several genes on the basis of their differential induction by natural or synthetic retinoids in human cervix adenocarcinoma cells. Furthermore, this differential ability to regulate promoter activities was also observed in murine P19 cells for the RARbeta2 and CRABPII gene, showing conclusively that retinoid structure has a dramatic impact on the regulation of endogenous genes.

Conclusions: Our findings therefore show that some degree of selective induction or repression of gene expression may be achieved when using appropriately designed ligands for retinoic acid receptors, extending the concept of selective modulators from estrogen and peroxisome proliferator activated receptors to the class of retinoid receptors.

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Expression of ferritin H mRNA in HeLa cells treated with different ligands of the retinoic acid receptor. HeLa cells were treated with the different ligands for four hours. Total RNA (20 μg) was probed sequentially with fluorescein-labeled partial human ferritin H cDNA and 18S rRNA probes. Blots were quantified using a Storm™ apparatus. Values for the ferritin H transcript were normalized to the 18S rRNA level. A) Upper panel: ferritin H transcript in HeLa cells treated with various retinoids. Lower panel: 18S rRNA. B) Quantification of ferritin H expression. Results are presented as the mean +/- S.E.M. of three different experiments. Cells were treated with 25nM atRA, 30nM CD3106, 80nM TTNPB, 20 nM Am580, 10nM CD367 and 100nM CD2425 for 4 hours.
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Figure 3: Expression of ferritin H mRNA in HeLa cells treated with different ligands of the retinoic acid receptor. HeLa cells were treated with the different ligands for four hours. Total RNA (20 μg) was probed sequentially with fluorescein-labeled partial human ferritin H cDNA and 18S rRNA probes. Blots were quantified using a Storm™ apparatus. Values for the ferritin H transcript were normalized to the 18S rRNA level. A) Upper panel: ferritin H transcript in HeLa cells treated with various retinoids. Lower panel: 18S rRNA. B) Quantification of ferritin H expression. Results are presented as the mean +/- S.E.M. of three different experiments. Cells were treated with 25nM atRA, 30nM CD3106, 80nM TTNPB, 20 nM Am580, 10nM CD367 and 100nM CD2425 for 4 hours.

Mentions: To further validate our initial screening, probes ranging in size from 200 to 350 bp were obtained by PCR using primers flanking the insertion site of the cDNA. 96 probes were thus synthesized, spotted onto a nylon membrane to generate cDNAs arrays. Total RNAs extracted from HeLa cells treated for 4 hours by the indicated retinoid (see Figure 3) was reverse-transcribed in the presence of α-[32P] dCTP and hybridized to membranes. Twenty genes showed marked differential regulation in this secondary screening, exhibiting various pharmacological profiles (data not shown). Of interest were genes found to be induced upon treatment with the RAR antagonist CD3106 (SRIG 61, 62 and 150). Two genes were repressed upon atRA treatment, but induced in the presence of other synthetic agonists (SRIG 146 and 177) and three were activated to various extent by retinoid agonists (SRIG 126, 148, 169). Other genes followed a more complex pattern which does not match a simple relationship between RAR transcriptional activation, pharmacological properties of retinoids and gene expression levels, reflecting a likely involvement of multiple, retinoid-induced steps in gene regulation.


Selective alteration of gene expression in response to natural and synthetic retinoids.

Brand C, Ségard P, Plouvier P, Formstecher P, Danzé PM, Lefebvre P - BMC Pharmacol. (2002)

Expression of ferritin H mRNA in HeLa cells treated with different ligands of the retinoic acid receptor. HeLa cells were treated with the different ligands for four hours. Total RNA (20 μg) was probed sequentially with fluorescein-labeled partial human ferritin H cDNA and 18S rRNA probes. Blots were quantified using a Storm™ apparatus. Values for the ferritin H transcript were normalized to the 18S rRNA level. A) Upper panel: ferritin H transcript in HeLa cells treated with various retinoids. Lower panel: 18S rRNA. B) Quantification of ferritin H expression. Results are presented as the mean +/- S.E.M. of three different experiments. Cells were treated with 25nM atRA, 30nM CD3106, 80nM TTNPB, 20 nM Am580, 10nM CD367 and 100nM CD2425 for 4 hours.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC113761&req=5

Figure 3: Expression of ferritin H mRNA in HeLa cells treated with different ligands of the retinoic acid receptor. HeLa cells were treated with the different ligands for four hours. Total RNA (20 μg) was probed sequentially with fluorescein-labeled partial human ferritin H cDNA and 18S rRNA probes. Blots were quantified using a Storm™ apparatus. Values for the ferritin H transcript were normalized to the 18S rRNA level. A) Upper panel: ferritin H transcript in HeLa cells treated with various retinoids. Lower panel: 18S rRNA. B) Quantification of ferritin H expression. Results are presented as the mean +/- S.E.M. of three different experiments. Cells were treated with 25nM atRA, 30nM CD3106, 80nM TTNPB, 20 nM Am580, 10nM CD367 and 100nM CD2425 for 4 hours.
Mentions: To further validate our initial screening, probes ranging in size from 200 to 350 bp were obtained by PCR using primers flanking the insertion site of the cDNA. 96 probes were thus synthesized, spotted onto a nylon membrane to generate cDNAs arrays. Total RNAs extracted from HeLa cells treated for 4 hours by the indicated retinoid (see Figure 3) was reverse-transcribed in the presence of α-[32P] dCTP and hybridized to membranes. Twenty genes showed marked differential regulation in this secondary screening, exhibiting various pharmacological profiles (data not shown). Of interest were genes found to be induced upon treatment with the RAR antagonist CD3106 (SRIG 61, 62 and 150). Two genes were repressed upon atRA treatment, but induced in the presence of other synthetic agonists (SRIG 146 and 177) and three were activated to various extent by retinoid agonists (SRIG 126, 148, 169). Other genes followed a more complex pattern which does not match a simple relationship between RAR transcriptional activation, pharmacological properties of retinoids and gene expression levels, reflecting a likely involvement of multiple, retinoid-induced steps in gene regulation.

Bottom Line: This suggested that gene-selective modulation could be achieved by structurally distinct retinoids.Furthermore, this differential ability to regulate promoter activities was also observed in murine P19 cells for the RARbeta2 and CRABPII gene, showing conclusively that retinoid structure has a dramatic impact on the regulation of endogenous genes.Our findings therefore show that some degree of selective induction or repression of gene expression may be achieved when using appropriately designed ligands for retinoic acid receptors, extending the concept of selective modulators from estrogen and peroxisome proliferator activated receptors to the class of retinoid receptors.

View Article: PubMed Central - HTML - PubMed

Affiliation: INSERM U 459 and Ligue nationale contre le Cancer, Faculté de Médecine Henri Warembourg, 1, place de Verdun, 59045 Lille cedex, France. brand@lille.inserm.fr

ABSTRACT

Background: Retinoids are very potent inducers of cellular differentiation and apoptosis, and are efficient anti-tumoral agents. Synthetic retinoids are designed to restrict their toxicity and side effects, mostly by increasing their selectivity toward each isotype of retinoic acids receptors (RARalpha,beta, gamma and RXRalpha, beta, gamma). We however previously showed that retinoids displayed very different abilities to activate retinoid-inducible reporter genes, and that these differential properties were correlated to the ability of a given ligand to promote SRC-1 recruitment by DNA-bound RXR:RAR heterodimers. This suggested that gene-selective modulation could be achieved by structurally distinct retinoids.

Results: Using the differential display mRNA technique, we identified several genes on the basis of their differential induction by natural or synthetic retinoids in human cervix adenocarcinoma cells. Furthermore, this differential ability to regulate promoter activities was also observed in murine P19 cells for the RARbeta2 and CRABPII gene, showing conclusively that retinoid structure has a dramatic impact on the regulation of endogenous genes.

Conclusions: Our findings therefore show that some degree of selective induction or repression of gene expression may be achieved when using appropriately designed ligands for retinoic acid receptors, extending the concept of selective modulators from estrogen and peroxisome proliferator activated receptors to the class of retinoid receptors.

Show MeSH
Related in: MedlinePlus