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The retinoid anticancer signal: mechanisms of target gene regulation.

Liu T, Bohlken A, Kuljaca S, Lee M, Nguyen T, Smith S, Cheung B, Norris MD, Haber M, Holloway AJ, Bowtell DD, Marshall GM - Br. J. Cancer (2005)

Bottom Line: In retinoid-sensitive neuroblastoma, lung and breast cancer cell lines, direct inhibition of retinoid-induced RARbeta2 expression blocked induction of only one of eight retinoid target genes (CYP26A1).Combined, rather than individual, inhibition of DUSP6 and RGS16 was required to block retinoid-induced growth inhibition in neuroblastoma cells, through phosphorylation of extracellular-signal-regulated kinase.In conclusion, sensitivity to the retinoid anticancer signal is determined in part by the transcriptional response of key retinoid-regulated target genes, such as RARbeta2, DUSP6, and RGS16.

View Article: PubMed Central - PubMed

Affiliation: Children's Cancer Institute Australia for Medical Research, Randwick NSW 2031, Australia.

ABSTRACT
Retinoids induce growth arrest, differentiation, and cell death in many cancer cell types. One factor determining the sensitivity or resistance to the retinoid anticancer signal is the transcriptional response of retinoid-regulated target genes in cancer cells. We used cDNA microarray to identify 31 retinoid-regulated target genes shared by two retinoid-sensitive neuroblastoma cell lines, and then sought to determine the relevance of the target gene responses to the retinoid anticancer signal. The pattern of retinoid responsiveness for six of 13 target genes (RARbeta2, CYP26A1, CRBP1, RGS16, DUSP6, EGR1) correlated with phenotypic retinoid sensitivity, across a panel of retinoid-sensitive or -resistant lung and breast cancer cell lines. Retinoid treatment of MYCN transgenic mice bearing neuroblastoma altered the expression of five of nine target genes examined (RARbeta2, CYP26A1, CRBP1, DUSP6, PLAT) in neuroblastoma tumour tissue in vivo. In retinoid-sensitive neuroblastoma, lung and breast cancer cell lines, direct inhibition of retinoid-induced RARbeta2 expression blocked induction of only one of eight retinoid target genes (CYP26A1). DNA demethylation, histone acetylation, and exogenous overexpression of RARbeta2 partially restored retinoid-responsive CYP26A1 expression in RA-resistant MDA-MB-231 breast, but not SK-MES-1 lung, cancer cells. Combined, rather than individual, inhibition of DUSP6 and RGS16 was required to block retinoid-induced growth inhibition in neuroblastoma cells, through phosphorylation of extracellular-signal-regulated kinase. In conclusion, sensitivity to the retinoid anticancer signal is determined in part by the transcriptional response of key retinoid-regulated target genes, such as RARbeta2, DUSP6, and RGS16.

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Induction of target gene expression by RA in neuroblastoma (SH-SY5Y, BE(2)-C), lung (SK-MES-1, Calu-6) and breast (MDA-MB-231, T47D) cancer cell lines, and neuroblastoma tissues. cDNA samples from cultured cells treated with 10 μM aRA (A and D), or 13-cis-RA (B) or solvent control at various time points, and duplicate cDNA samples from neuroblastoma arising in MYCN transgenic mice treated with 13-cis-RA or control (C) were subjected to independent competitive RT–PCR analyses using trans-intron PCR primers, together with housekeeping gene β2M primers. An equal aliquot of PCR product was then electrophoretically size-fractionated on a polyacrylamide gel as shown (A, B and C). Fold induction of a target gene by RA in RA-treated samples was calculated by ascribing the ratio between the level of expression of a target gene and that of β2M as 1.0 for control-treated samples (D).
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fig1: Induction of target gene expression by RA in neuroblastoma (SH-SY5Y, BE(2)-C), lung (SK-MES-1, Calu-6) and breast (MDA-MB-231, T47D) cancer cell lines, and neuroblastoma tissues. cDNA samples from cultured cells treated with 10 μM aRA (A and D), or 13-cis-RA (B) or solvent control at various time points, and duplicate cDNA samples from neuroblastoma arising in MYCN transgenic mice treated with 13-cis-RA or control (C) were subjected to independent competitive RT–PCR analyses using trans-intron PCR primers, together with housekeeping gene β2M primers. An equal aliquot of PCR product was then electrophoretically size-fractionated on a polyacrylamide gel as shown (A, B and C). Fold induction of a target gene by RA in RA-treated samples was calculated by ascribing the ratio between the level of expression of a target gene and that of β2M as 1.0 for control-treated samples (D).

Mentions: Microarray slides with 4500 cDNA clones, representing predominantly genes with known functions, were hybridised with cDNA from aRA- or control-treated cells. In total, 31 genes were up- or downregulated by ⩾2-fold by aRA in both cell lines, at one or more time points in triplicate (P<0.05) (listed in Table 2 according to the time course of changes in gene modification). This list of RA-regulated targets included genes coding for proteins known to be involved in: (i) retinoid binding and metabolism (RARβ2, cellular retinoid-binding protein 1 (CRBP1), and CYP26A1); (ii) the MAPK signalling pathway (regulator of G-protein signalling 16 (RGS16) and dual specificity phosphatase 6 (DUSP6)); (iii) cell structure and differentiation (filamin B (FLNB), c-RET proto-oncogene (RET), α polypeptide Cu2+ transporting ATPase (ATP7A), and δ-like 1 homolog (Drosophila) (DLK1)); and (iv) angiogenesis or cancer invasion (early growth response protein 1 (EGR1) and tissue plasminogen activator (PLAT)). Among the 31 genes, a classical RARE could be found in the promoter region of RARβ2, CYP26A1, CRBP1 and PLAT (Chambon, 1996; Sun and Lotan, 2002). While RGS16 expression was upregulated at 1 h after aRA (Table 2, Figure 1A), all other RA target genes showed a more gradual change in expression at 1, 3, and 7 days after commencement of aRA. Only four of 31 target genes demonstrated downregulated expression patterns after aRA treatment. In addition to those genes listed in Table 2, a further 21 genes exhibited ⩾2-fold change in expression in only one of the two neuroblastoma cell lines (data not shown). To validate the microarray data, semiquantitative, competitive RT–PCR was employed to assess expression of 11 selected genes shared by the two cell lines (Table 2). The results of RT–PCR were consistent with the microarray data in 10 out of 11 genes, with TIA1 as the only exception. Examples of the RT–PCR results were shown in Figure 1A.


The retinoid anticancer signal: mechanisms of target gene regulation.

Liu T, Bohlken A, Kuljaca S, Lee M, Nguyen T, Smith S, Cheung B, Norris MD, Haber M, Holloway AJ, Bowtell DD, Marshall GM - Br. J. Cancer (2005)

Induction of target gene expression by RA in neuroblastoma (SH-SY5Y, BE(2)-C), lung (SK-MES-1, Calu-6) and breast (MDA-MB-231, T47D) cancer cell lines, and neuroblastoma tissues. cDNA samples from cultured cells treated with 10 μM aRA (A and D), or 13-cis-RA (B) or solvent control at various time points, and duplicate cDNA samples from neuroblastoma arising in MYCN transgenic mice treated with 13-cis-RA or control (C) were subjected to independent competitive RT–PCR analyses using trans-intron PCR primers, together with housekeeping gene β2M primers. An equal aliquot of PCR product was then electrophoretically size-fractionated on a polyacrylamide gel as shown (A, B and C). Fold induction of a target gene by RA in RA-treated samples was calculated by ascribing the ratio between the level of expression of a target gene and that of β2M as 1.0 for control-treated samples (D).
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Related In: Results  -  Collection

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fig1: Induction of target gene expression by RA in neuroblastoma (SH-SY5Y, BE(2)-C), lung (SK-MES-1, Calu-6) and breast (MDA-MB-231, T47D) cancer cell lines, and neuroblastoma tissues. cDNA samples from cultured cells treated with 10 μM aRA (A and D), or 13-cis-RA (B) or solvent control at various time points, and duplicate cDNA samples from neuroblastoma arising in MYCN transgenic mice treated with 13-cis-RA or control (C) were subjected to independent competitive RT–PCR analyses using trans-intron PCR primers, together with housekeeping gene β2M primers. An equal aliquot of PCR product was then electrophoretically size-fractionated on a polyacrylamide gel as shown (A, B and C). Fold induction of a target gene by RA in RA-treated samples was calculated by ascribing the ratio between the level of expression of a target gene and that of β2M as 1.0 for control-treated samples (D).
Mentions: Microarray slides with 4500 cDNA clones, representing predominantly genes with known functions, were hybridised with cDNA from aRA- or control-treated cells. In total, 31 genes were up- or downregulated by ⩾2-fold by aRA in both cell lines, at one or more time points in triplicate (P<0.05) (listed in Table 2 according to the time course of changes in gene modification). This list of RA-regulated targets included genes coding for proteins known to be involved in: (i) retinoid binding and metabolism (RARβ2, cellular retinoid-binding protein 1 (CRBP1), and CYP26A1); (ii) the MAPK signalling pathway (regulator of G-protein signalling 16 (RGS16) and dual specificity phosphatase 6 (DUSP6)); (iii) cell structure and differentiation (filamin B (FLNB), c-RET proto-oncogene (RET), α polypeptide Cu2+ transporting ATPase (ATP7A), and δ-like 1 homolog (Drosophila) (DLK1)); and (iv) angiogenesis or cancer invasion (early growth response protein 1 (EGR1) and tissue plasminogen activator (PLAT)). Among the 31 genes, a classical RARE could be found in the promoter region of RARβ2, CYP26A1, CRBP1 and PLAT (Chambon, 1996; Sun and Lotan, 2002). While RGS16 expression was upregulated at 1 h after aRA (Table 2, Figure 1A), all other RA target genes showed a more gradual change in expression at 1, 3, and 7 days after commencement of aRA. Only four of 31 target genes demonstrated downregulated expression patterns after aRA treatment. In addition to those genes listed in Table 2, a further 21 genes exhibited ⩾2-fold change in expression in only one of the two neuroblastoma cell lines (data not shown). To validate the microarray data, semiquantitative, competitive RT–PCR was employed to assess expression of 11 selected genes shared by the two cell lines (Table 2). The results of RT–PCR were consistent with the microarray data in 10 out of 11 genes, with TIA1 as the only exception. Examples of the RT–PCR results were shown in Figure 1A.

Bottom Line: In retinoid-sensitive neuroblastoma, lung and breast cancer cell lines, direct inhibition of retinoid-induced RARbeta2 expression blocked induction of only one of eight retinoid target genes (CYP26A1).Combined, rather than individual, inhibition of DUSP6 and RGS16 was required to block retinoid-induced growth inhibition in neuroblastoma cells, through phosphorylation of extracellular-signal-regulated kinase.In conclusion, sensitivity to the retinoid anticancer signal is determined in part by the transcriptional response of key retinoid-regulated target genes, such as RARbeta2, DUSP6, and RGS16.

View Article: PubMed Central - PubMed

Affiliation: Children's Cancer Institute Australia for Medical Research, Randwick NSW 2031, Australia.

ABSTRACT
Retinoids induce growth arrest, differentiation, and cell death in many cancer cell types. One factor determining the sensitivity or resistance to the retinoid anticancer signal is the transcriptional response of retinoid-regulated target genes in cancer cells. We used cDNA microarray to identify 31 retinoid-regulated target genes shared by two retinoid-sensitive neuroblastoma cell lines, and then sought to determine the relevance of the target gene responses to the retinoid anticancer signal. The pattern of retinoid responsiveness for six of 13 target genes (RARbeta2, CYP26A1, CRBP1, RGS16, DUSP6, EGR1) correlated with phenotypic retinoid sensitivity, across a panel of retinoid-sensitive or -resistant lung and breast cancer cell lines. Retinoid treatment of MYCN transgenic mice bearing neuroblastoma altered the expression of five of nine target genes examined (RARbeta2, CYP26A1, CRBP1, DUSP6, PLAT) in neuroblastoma tumour tissue in vivo. In retinoid-sensitive neuroblastoma, lung and breast cancer cell lines, direct inhibition of retinoid-induced RARbeta2 expression blocked induction of only one of eight retinoid target genes (CYP26A1). DNA demethylation, histone acetylation, and exogenous overexpression of RARbeta2 partially restored retinoid-responsive CYP26A1 expression in RA-resistant MDA-MB-231 breast, but not SK-MES-1 lung, cancer cells. Combined, rather than individual, inhibition of DUSP6 and RGS16 was required to block retinoid-induced growth inhibition in neuroblastoma cells, through phosphorylation of extracellular-signal-regulated kinase. In conclusion, sensitivity to the retinoid anticancer signal is determined in part by the transcriptional response of key retinoid-regulated target genes, such as RARbeta2, DUSP6, and RGS16.

Show MeSH
Related in: MedlinePlus