Abstract
The aryl hydrocarbon receptor (AhR), a ligand-activated member of the basic helix-loop-helix (bHLH)/PER-ARNT-SIM (PAS) transcription superfamily, is known to regulate the toxicity of polyaromatic halogenated hydrocarbon environmental chemicals, most notably dioxin. However, the AhR has also been implicated in multiple stages of tumorigenesis. Medulloblastoma (MB), a primary cerebellar brain tumor arising in infants and children, is thought to originate from abnormally proliferating cerebellar granule neuron precursors (GNPs). GNPs express high levels of the AhR in the external germinal layer of the developing cerebellum. Moreover, our laboratory has previously reported that either abnormal activation or deletion of the AhR leads to dysregulation of GNP cell cycle activity and maturation. These observations led to the hypothesis that the AhR promotes the growth of MB. Therefore, this study evaluated whether the AhR serves a pro-proliferative role in an immortalized MB tumor cell line (DAOY). We produced a stable AhR knockdown DAOY cell line [AhR short hairpin RNA (shRNA)], which exhibited a 70% reduction in AhR protein levels. Compared with wild-type DAOY cells, AhR shRNA DAOY cells displayed an impaired G1-to-S cell cycle transition, decreased DNA synthesis, and reduced proliferation. Furthermore, these cell cycle perturbations were correlated with decreased levels of the pro-proliferative gene Hes1 and increased levels of the cell cycle inhibitor p27kip1. Supplementation experiments with human AhR restored the proliferative activity in AhR shRNA DAOY cells. Taken together, our data show that the AhR promotes proliferation of MB cells, suggesting that this pathway should be considered as a potential therapeutic target for MB treatment.
Footnotes
This research was supported by the National Institutes of Health National Institute of Environmental Health Sciences [Grants R01-ES016357, P30-ES01247, T32-ES07026] and The Bristol-Myers Squibb Pharmaceutical Research Institute.
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS:
- AhR
- aryl hydrocarbon receptor
- bHLH
- basic helix-loop-helix
- TCDD
- 2,3,7,8-tetrachlorodibenzo-p-dioxin
- MB
- medulloblastoma
- GNP
- cerebellar granule neuron precursors
- EGL
- external germinal layer
- DMSO
- dimethyl sulfoxide
- BSA
- bovine serum albumin
- DMEM
- Dulbecco's modified Eagle's medium
- PBS
- phosphate-buffered saline
- GFP
- green fluorescent protein
- shRNA
- short hairpin RNA
- hAhR
- human aryl hydrocarbon receptor
- EGFP
- enhanced green fluorescent protein
- FACS
- fluorescence-activated cell sorting
- CFSE
- carboxyfluorescein diacetate succinimidyl ester
- DAPI
- 4,6-diamidino-2-phenylindole
- PCR
- polymerase chain reaction
- Q-PCR
- quantitative real-time PCR
- GAPDH
- glyceraldehyde-3-phosphate dehydrogenase
- PI
- propidium iodide.
- Received December 19, 2011.
- Accepted February 6, 2012.
- Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
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