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. 1999 Jul;45(1):51–57. doi: 10.1136/gut.45.1.51

Induction of retinoic acid receptor β mediates growth inhibition in retinoid resistant human colon carcinoma cells

B Nicke 1, E Riecken 1, S Rosewicz 1
PMCID: PMC1727569  PMID: 10369704

Abstract

BACKGROUND—The molecular mechanisms underlying the differential sensitivity of human colon carcinoma cells to retinoid mediated growth inhibition are poorly understood.
AIM—To identify the intracellular mechanisms responsible for resistance against retinoid mediated growth inhibition in human colon carcinoma cells.
METHODS—Anchorage independent growth of the human colon carcinoma cell lines HT29 and LoVo was determined by a human tumour clonogenic assay. Retinoid receptor expression was evaluated by reverse transcription polymerase chain reaction and northern blotting. Retinoid mediated transactivation was assessed by transient transfection of a pTK::βREx2-luc reporter construct. Retinoid receptor overexpression was achieved by selecting stably transfected cell clones.
RESULTS—Retinoid treatment resulted in profound dose dependent growth inhibition in HT29 cells, while LoVo cells were unaffected. The two cell lines express identical patterns of nuclear retinoid receptor mRNA transcripts. However, on retinoid treatment, retinoic acid receptor β gene expression was upregulated only in retinoid sensitive HT29 cells, but not in retinoid resistant LoVo cells. In accordance, stable overexpression of retinoic acid receptor β but not α or γ conferred retinoid mediated growth inhibition on LoVo cells.
CONCLUSION—Induction of retinoic acid receptor β expression is required and sufficent to confer retinoid mediated growth inhibition on human colon carcinoma cells.


Keywords: colon cancer; retinoic acid; retinoic acid receptor

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Figure 1  .

Figure 1  

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Differential effects of retinoid derivatives on anchorage independent growth. HT29 and LoVo cells were analysed using the human tumour clonogenic assay with the indicated final concentrations of each retinoid analogue (A, all-trans-retinoic acid; B, 13-cis-retinoic acid; C, 9-cis-retinoic acid). After 10 days the number of colonies was evaluated and expressed as percentage of vehicle treated controls. Values are mean (SEM) from three independent experiments, each performed in triplicate. *p<0.05 v controls.

Figure 2  .

Figure 2  

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Identical expression of nuclear retinoid receptors in HT29 and LoVo cells. Reverse transcription polymerase chain reaction (RT-PCR) analysis was performed on HT29 and LoVo cells using receptor specific oligonucleotide primers. The respective receptor subtype cDNA was used as a positive internal control. RT-PCR with (+) or without (−) prior reverse transcription (the latter served as a negative internal control) was performed. Duplicate aliquots from each RT-PCR product were electrophoresed through a 1% agarose gel and then subjected to Southern blot analysis using the respective cDNA probes for the retinoic acid receptors (RARs)/retinoid X receptors (RXRs) to ensure specificity of the amplification product. Size determination was performed using a DNA ladder. (A) RT-PCR for RARs; (B) RT-PCR for RXRs.

Figure 3  .

Figure 3  

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Differential autoregulation of retinoic acid receptor (RAR)β by retinoids. HT29 and LoVo cells were incubated with vehicle or 10 µM all-trans-retinoic acid (all-trans RA) for 24 hours. Then 25 µg total RNA was analysed by northern blotting using radioactively labelled cDNAs specific for the RAR subtypes. The blots were then stripped and rehybridised with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA to ensure equal RNA loading. The results shown are representative of three independent experiments.

Figure 4  .

Figure 4  

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Differential effects of all-trans-retinoic acid on transactivation. HT29 and LoVo cells were transiently transfected with the pTK::βREx2-luc reporter construct. After incubation with the indicated concentrations of all-trans RA for 24 hours, luciferase activity was determined and expressed as fold increase over basal (luciferase activity in the absence of retinoids). Values are mean (SEM) from six independent experiments, each performed in triplicate wells.

Figure 5  .

Figure 5  

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Overexpression of retinoic acid receptor (RAR)β restores retinoid sensitivity in LoVo cells. After stable transfection with the RARβ cDNA, overexpression of RARβ mRNA was confirmed by northern blotting (A). GAPDH, Glyceraldehyde-3-phosphate dehydrogenase. Transfected (pCMV:RARβ9, pCMV:RARβ12, pCMV:RARβ14), mock transfected (pCMV), and wild type (WT) LoVo cells were then analysed by human tumour clonogenic assay using the indicated concentrations of all-trans-retinoic acid. (B) After 10 days the number of colonies was evaluated and expressed as a percentage of vehicle treated contols. Results shown are mean (SEM) from three independent experiments, each performed in triplicate. *p<0.05 v controls.

Figure 6  .

Figure 6  

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Overexpression of retinoic acid receptor (RAR)α or γ does not restore retinoid sensitivity in LoVo cells. After stable transfection with the RARα and γ cDNA, overexpression of the respective RAR subtype mRNA was confirmed by northern blotting (A). Transfected (pCMV:RARα7, pCMV:RARα9, pCMV:RARγ23), mock transfected (pCMV), and wild type (WT) LoVo cells were then analysed by human tumour clonogenic assay using the indicated concentrations of all-trans-retinoic acid. (B) After 10 days the number of colonies was evaluated and expressed as a percentage of vehicle treated contols. Results shown are mean (SEM) from three independent experiments, each performed in triplicate.

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