Epigenetic modulation of tumor suppressor CCAAT/enhancer binding protein alpha activity in lung cancer.
BACKGROUND
Loss of tumor suppressor CCAAT/enhancer-binding protein-alpha (C/EBPalpha) expression is seen in several human malignancies, including acute myelogenous leukemia and lung cancer. We hypothesized that DNA methylation and histone acetylation of the C/EBPalpha promoter may modulate C/EBPalpha expression in lung cancer.


METHODS
We analyzed C/EBPalpha expression in 15 human lung cancer cell lines and in 122 human lung primary tumors by northern blotting, immunoblotting, and immunohistochemistry. C/EBPalpha promoter methylation was assessed using bisulfite sequencing, combined bisulfite restriction analysis, methylation-specific polymerase chain reaction, and Southern blotting. We examined the acetylation status of histones H3 and H4 at the C/EBPalpha promoter by chromatin immunoprecipitation. Binding of methyl-CpG-binding proteins MeCP2 and MBD2 and upstream stimulatory factor (USF) to the C/EBPalpha promoter was assayed in cell lines that were untreated or treated with histone deacetylase inhibitor trichostatin A and demethylating agent 5-aza-2'-deoxycytidine (5-aza-dC) by chromatin immunoprecipitation and by electrophoretic mobility shift assays.


RESULTS
DNA methylation and histone acetylation in the upstream region (-1422 to -896) of the C/EBPalpha promoter were associated with low or absent C/EBPalpha expression in 12 of 15 lung cancer cell lines and in 81 of 120 primary lung tumors. MeCP2 and MBD binding to the upstream C/EBPalpha promoter was detected in C/EBPalpha-nonexpressing cell lines; USF binding was detected in C/EBPalpha-expressing cell lines; however, in C/EBPalpha-nonexpressing cell lines USF binding was detected only after trichostatin A and 5-aza-dC treatment.


CONCLUSIONS
DNA hypermethylation of the upstream C/EBPalpha promoter region, not the core promoter region as previously reported, is critical in the regulation of C/EBPalpha expression in human lung cancer.
Division of Human Cancer Genetics, Department of Molecular Virology, Immunology, and Medical Genetics, The Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA.
Yasuhiro
Y
Tada
Romulo Martin
RM
Brena
Björn
B
Hackanson
Carl
C
Morrison
Gregory A
GA
Otterson
Christoph
C
Plass
D000107:Acetylation / Q000187:drug effects; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D015152:Blotting, Northern; D015139:Blotting, Southern; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000032:analysis* / Q000235:genetics; D002289:Carcinoma, Non-Small-Cell Lung / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D018899:CpG Islands; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004791:Enzyme Inhibitors / Q000494:pharmacology; D044127:Epigenesis, Genetic; D015972:Gene Expression Regulation, Neoplastic; D056572:Histone Deacetylase Inhibitors; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D015151:Immunoblotting; D007150:Immunohistochemistry; D008175:Lung Neoplasms / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D051783:Methyl-CpG-Binding Protein 2 / Q000378:metabolism; D016296:Mutagenesis; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D051720:Upstream Stimulatory Factors / Q000187:drug effects / Q000378:metabolism*
D022763:CCAAT-Enhancer-Binding Protein-alpha; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; C489325:MBD2 protein, human; C497392:MECP2 protein, human; D051783:Methyl-CpG-Binding Protein 2; D051720:Upstream Stimulatory Factors; C012589:trichostatin A; C041519:5-aza-2'-deoxycytidine-5'-monophosphate; D001374:Azacitidine