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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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Differential expression of mRNA species in HeLa cells treated by retinoids. Differential display RT-PCR analysis of HeLa transcripts obtained from cells treated with 1μM of the indicated retinoid for 4 hours. Total RNA was extracted and purified from HeLa cells and reverse-transcribed with the H-T11G (AAGCT11G, left panel), H-T11A (AAGCT11A, middle panel) or H-T11C (AAGCT11C, right panel) primers. PCR amplification of cDNAs was carried out using the same 3' primer and the H-AP3 primer (AAGCTTTGGTCAG), the H-AP6 primer (AAGCTTGCACCAT), the H-AP12 primer (AAGCTTGAGTGCT) and the H-AP15 primer (AAGCTTACGCAAC) (from left to right) in the presence of α-[33P] dATP. Amplified cDNA fragments were analyzed on 6% sequencing gels and visualized by autoradiography. Typical lanes are shown, with size markers appearing on the left. Selectively regulated mRNAs are indicated by dots. These materials were extracted from the gel, re-amplified by PCR with the same set of primers, cloned into the pCR-TRAP vector (GenHunter) and sequenced.
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Figure 2: Differential expression of mRNA species in HeLa cells treated by retinoids. Differential display RT-PCR analysis of HeLa transcripts obtained from cells treated with 1μM of the indicated retinoid for 4 hours. Total RNA was extracted and purified from HeLa cells and reverse-transcribed with the H-T11G (AAGCT11G, left panel), H-T11A (AAGCT11A, middle panel) or H-T11C (AAGCT11C, right panel) primers. PCR amplification of cDNAs was carried out using the same 3' primer and the H-AP3 primer (AAGCTTTGGTCAG), the H-AP6 primer (AAGCTTGCACCAT), the H-AP12 primer (AAGCTTGAGTGCT) and the H-AP15 primer (AAGCTTACGCAAC) (from left to right) in the presence of α-[33P] dATP. Amplified cDNA fragments were analyzed on 6% sequencing gels and visualized by autoradiography. Typical lanes are shown, with size markers appearing on the left. Selectively regulated mRNAs are indicated by dots. These materials were extracted from the gel, re-amplified by PCR with the same set of primers, cloned into the pCR-TRAP vector (GenHunter) and sequenced.

Mentions: Total RNA was then extracted from HeLa cells treated for 4 hours with 1μM atRA, our reference compound, 1μM CD3106/AGN 193109, a RAR-specific antagonist [31], 1μM TTNPB and 20 nM Am580, two synthetic RARα-specific agonists, 1μM CD367, a RAR-specific agonist [32] and 1μM CD2425, a RXR-specific retinoid. mRNAs were then randomly amplified using various combinations of oligodT anchored primers (see legend to figure 2 for more details) and arbitrary chosen primers in the presence of α-[33P]-labelled dATP. Products were visualized by autoradiography of high resolution sequencing gels. Examination of autoradiographies allowed the identification of several mRNAs species that were specifically induced upon treatment with some of the retinoids described above. Typical results are shown in Figure 2 for four different sets of primers which allowed the amplification of differentially expressed genes. This differential expression was noted for about 60% of the primer sets, while others did not show any significant variations. Based on visual examination of 40 sequencing gels, cDNAs were extracted from the gel, reamplified by PCR using the same set of primers than that used in the initial RT-PCR reaction, cloned in the PCR-Trap vector by T/A cloning, and sequenced. 140 cDNAs were identified (hereafter noted synthetic retinoid-induced genes or SRIG) following this procedure and could be classified into three categories: (i) sequences with no homology with any known human genes; (ii) sequences overlapping with previously identified ESTs and (iii) sequences homologous to genes with known function(s). These results are summarized in Table 1 and Table 2. 28 new ESTs were identifed and sequences were deposited in GenBank.


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)

Differential expression of mRNA species in HeLa cells treated by retinoids. Differential display RT-PCR analysis of HeLa transcripts obtained from cells treated with 1μM of the indicated retinoid for 4 hours. Total RNA was extracted and purified from HeLa cells and reverse-transcribed with the H-T11G (AAGCT11G, left panel), H-T11A (AAGCT11A, middle panel) or H-T11C (AAGCT11C, right panel) primers. PCR amplification of cDNAs was carried out using the same 3' primer and the H-AP3 primer (AAGCTTTGGTCAG), the H-AP6 primer (AAGCTTGCACCAT), the H-AP12 primer (AAGCTTGAGTGCT) and the H-AP15 primer (AAGCTTACGCAAC) (from left to right) in the presence of α-[33P] dATP. Amplified cDNA fragments were analyzed on 6% sequencing gels and visualized by autoradiography. Typical lanes are shown, with size markers appearing on the left. Selectively regulated mRNAs are indicated by dots. These materials were extracted from the gel, re-amplified by PCR with the same set of primers, cloned into the pCR-TRAP vector (GenHunter) and sequenced.
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Related In: Results  -  Collection

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Figure 2: Differential expression of mRNA species in HeLa cells treated by retinoids. Differential display RT-PCR analysis of HeLa transcripts obtained from cells treated with 1μM of the indicated retinoid for 4 hours. Total RNA was extracted and purified from HeLa cells and reverse-transcribed with the H-T11G (AAGCT11G, left panel), H-T11A (AAGCT11A, middle panel) or H-T11C (AAGCT11C, right panel) primers. PCR amplification of cDNAs was carried out using the same 3' primer and the H-AP3 primer (AAGCTTTGGTCAG), the H-AP6 primer (AAGCTTGCACCAT), the H-AP12 primer (AAGCTTGAGTGCT) and the H-AP15 primer (AAGCTTACGCAAC) (from left to right) in the presence of α-[33P] dATP. Amplified cDNA fragments were analyzed on 6% sequencing gels and visualized by autoradiography. Typical lanes are shown, with size markers appearing on the left. Selectively regulated mRNAs are indicated by dots. These materials were extracted from the gel, re-amplified by PCR with the same set of primers, cloned into the pCR-TRAP vector (GenHunter) and sequenced.
Mentions: Total RNA was then extracted from HeLa cells treated for 4 hours with 1μM atRA, our reference compound, 1μM CD3106/AGN 193109, a RAR-specific antagonist [31], 1μM TTNPB and 20 nM Am580, two synthetic RARα-specific agonists, 1μM CD367, a RAR-specific agonist [32] and 1μM CD2425, a RXR-specific retinoid. mRNAs were then randomly amplified using various combinations of oligodT anchored primers (see legend to figure 2 for more details) and arbitrary chosen primers in the presence of α-[33P]-labelled dATP. Products were visualized by autoradiography of high resolution sequencing gels. Examination of autoradiographies allowed the identification of several mRNAs species that were specifically induced upon treatment with some of the retinoids described above. Typical results are shown in Figure 2 for four different sets of primers which allowed the amplification of differentially expressed genes. This differential expression was noted for about 60% of the primer sets, while others did not show any significant variations. Based on visual examination of 40 sequencing gels, cDNAs were extracted from the gel, reamplified by PCR using the same set of primers than that used in the initial RT-PCR reaction, cloned in the PCR-Trap vector by T/A cloning, and sequenced. 140 cDNAs were identified (hereafter noted synthetic retinoid-induced genes or SRIG) following this procedure and could be classified into three categories: (i) sequences with no homology with any known human genes; (ii) sequences overlapping with previously identified ESTs and (iii) sequences homologous to genes with known function(s). These results are summarized in Table 1 and Table 2. 28 new ESTs were identifed and sequences were deposited in GenBank.

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