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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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Related in: MedlinePlus

Expression levels of retinoic acid receptors and of transcriptional intermediary factors in HeLa cells. HeLa cells mRNA was extracted and analyzed by RT-PCR using specific primers to detect (A) hRARα, hRARβ, hRARγ, hRXRα, hRXRβ and hRXRγ transcripts; to confirm the lack of expression of hRARβ, hRARγ and hRXRγ by nested PCR (B) to characterize expression levels of nuclear coactivators (C) and nuclear corepressors (D). E) Western blot analysis of HeLa whole cell extracts. 100 μg of proteins were resolved by 8% SDS-PAGE and blotted onto a nitrocellulose membrane. This membrane was probed with antibodies specific for each indicated receptors, coactivators or corepressor. The left panel shows a silver-stained gel on which 10 μg of cell extract has been separated. Molecular masses are indicated in kDa.
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Figure 1: Expression levels of retinoic acid receptors and of transcriptional intermediary factors in HeLa cells. HeLa cells mRNA was extracted and analyzed by RT-PCR using specific primers to detect (A) hRARα, hRARβ, hRARγ, hRXRα, hRXRβ and hRXRγ transcripts; to confirm the lack of expression of hRARβ, hRARγ and hRXRγ by nested PCR (B) to characterize expression levels of nuclear coactivators (C) and nuclear corepressors (D). E) Western blot analysis of HeLa whole cell extracts. 100 μg of proteins were resolved by 8% SDS-PAGE and blotted onto a nitrocellulose membrane. This membrane was probed with antibodies specific for each indicated receptors, coactivators or corepressor. The left panel shows a silver-stained gel on which 10 μg of cell extract has been separated. Molecular masses are indicated in kDa.

Mentions: HeLa cells are known to express low levels of endogenous all-trans (RARs), 9-cis retinoic acid receptors (RXRs) and nuclear coactivators and corepressors. However, relative levels of expression of these proteins have not been monitored in this cell line and thus a comprehensive study was carried out to characterize mRNA levels coding for each protein. Using RT-PCR amplification of specific transcripts from total RNA, we observed that hRARα, hRXRα and hRXRβ were predominantly expressed in this cell line (Figure 1A). Using nested PCR primers, trace amounts of hRARβ were detected (Figure 1B), whereas hRARγ and hRXRγ were not detectable in these conditions (note that amplification products in the corresponding lanes are non specific amplification products and did not match the predicted size of amplified cDNA). We then assayed similarly expression levels for nuclear coactivators AIB1 [16], CBP [17], p300 [18], p/CIP [19], RAC3 [20], RIP140 [21], SRC1 [22], TIF1 [23], TIF2 [24] and TRIP1 [25]. As shown in Figure 1C, each coactivator mRNA was detectable in HeLa cells, and we also observed by Western blot analysis that members of the DRIP/TRAP coactivator family [26] are also expressed in this cell line (see panel E). Expression levels of nuclear corepressors N-CoR [27] and SMRT [28] were also assessed (Figure 1D). Among these two corepressors, only SMRT/TRAC2 was found to be significantly expressed. Two amplification products were characterized, reflecting the occurrence of two transcripts of SMRT as described previously [29].


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 levels of retinoic acid receptors and of transcriptional intermediary factors in HeLa cells. HeLa cells mRNA was extracted and analyzed by RT-PCR using specific primers to detect (A) hRARα, hRARβ, hRARγ, hRXRα, hRXRβ and hRXRγ transcripts; to confirm the lack of expression of hRARβ, hRARγ and hRXRγ by nested PCR (B) to characterize expression levels of nuclear coactivators (C) and nuclear corepressors (D). E) Western blot analysis of HeLa whole cell extracts. 100 μg of proteins were resolved by 8% SDS-PAGE and blotted onto a nitrocellulose membrane. This membrane was probed with antibodies specific for each indicated receptors, coactivators or corepressor. The left panel shows a silver-stained gel on which 10 μg of cell extract has been separated. Molecular masses are indicated in kDa.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC113761&req=5

Figure 1: Expression levels of retinoic acid receptors and of transcriptional intermediary factors in HeLa cells. HeLa cells mRNA was extracted and analyzed by RT-PCR using specific primers to detect (A) hRARα, hRARβ, hRARγ, hRXRα, hRXRβ and hRXRγ transcripts; to confirm the lack of expression of hRARβ, hRARγ and hRXRγ by nested PCR (B) to characterize expression levels of nuclear coactivators (C) and nuclear corepressors (D). E) Western blot analysis of HeLa whole cell extracts. 100 μg of proteins were resolved by 8% SDS-PAGE and blotted onto a nitrocellulose membrane. This membrane was probed with antibodies specific for each indicated receptors, coactivators or corepressor. The left panel shows a silver-stained gel on which 10 μg of cell extract has been separated. Molecular masses are indicated in kDa.
Mentions: HeLa cells are known to express low levels of endogenous all-trans (RARs), 9-cis retinoic acid receptors (RXRs) and nuclear coactivators and corepressors. However, relative levels of expression of these proteins have not been monitored in this cell line and thus a comprehensive study was carried out to characterize mRNA levels coding for each protein. Using RT-PCR amplification of specific transcripts from total RNA, we observed that hRARα, hRXRα and hRXRβ were predominantly expressed in this cell line (Figure 1A). Using nested PCR primers, trace amounts of hRARβ were detected (Figure 1B), whereas hRARγ and hRXRγ were not detectable in these conditions (note that amplification products in the corresponding lanes are non specific amplification products and did not match the predicted size of amplified cDNA). We then assayed similarly expression levels for nuclear coactivators AIB1 [16], CBP [17], p300 [18], p/CIP [19], RAC3 [20], RIP140 [21], SRC1 [22], TIF1 [23], TIF2 [24] and TRIP1 [25]. As shown in Figure 1C, each coactivator mRNA was detectable in HeLa cells, and we also observed by Western blot analysis that members of the DRIP/TRAP coactivator family [26] are also expressed in this cell line (see panel E). Expression levels of nuclear corepressors N-CoR [27] and SMRT [28] were also assessed (Figure 1D). Among these two corepressors, only SMRT/TRAC2 was found to be significantly expressed. Two amplification products were characterized, reflecting the occurrence of two transcripts of SMRT as described previously [29].

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