Reduced transcription of the RB2/p130 gene in human lung cancer.
Reduced expression of the retinoblastoma gene (RB)2/p130 protein, as well as mutation of exons 19, 20, 21, and 22 of the same gene, has been reported in primary lung cancer. However, it has been suggested by other investigators that mutational inactivation and loss of the RB2/p130 gene and protein, respectively, are rare events in lung cancer. In order to determine the contribution and mechanisms of RB2/p130 gene inactivation to lung cancer development and progression, we quantified RB2/p130 mRNA expression levels in a range of human lung cancer cell lines (n = 13) by real-time reverse transcription (RT)-polymerase chain reaction (PCR) analysis. In comparison to normal lung tissue, reduced transcription of the RB2/p130 gene was found in all small cell lung cancer cell lines examined, along with six out of the eight nonsmall cell lung cancers tested, most of which had inactivation of RB/p16 pathway. On the basis of Western blot analysis, the expression of RB2/p130 protein was consistent with RNA expression levels in all lung cancer cell lines examined. In addition, the mutational status of the RB2/p130 gene (specifically, exons 19, 20, 21, and 22) was determined in 30 primary lung cancers (from patients with distant metastasis) and 30 lung cancer cell lines by PCR-single strand conformation polymorphism (SSCP) analysis and direct DNA sequencing. There was no evidence of somatic mutations within the RB2/p130 gene in the 60 lung cancer samples (both cell lines and tumors) assessed, including the 11 lung cancer cell lines that displayed reduced expression of the gene. Furthermore, hypermethylation of the RB2/p130 promoter was not found in any of the above-mentioned 11 cell lines, as determined by a DNA methylation assay, combined bisulfite restriction analysis (COBRA). The results of the present study suggest that the reduced RB2/p130 expression seen in lung cancer may be in part transcriptionally mediated, albeit not likely via a mechanism involving hypermethylation of the RB2/p130 promoter. The observed reduction in RB2/p130 gene expression may be due to histone deacetylation, altered mRNA stability, and/or other forms of transcriptional regulation.
Fourth Department of Medicine, Nippon Medical School, Tokyo, Japan.
Hu
H
Xue Jun
Akihiko
A
Gemma
Yoko
Y
Hosoya
Kuniko
K
Matsuda
Michiya
M
Nara
Yukio
Y
Hosomi
Tetsuya
T
Okano
Futoshi
F
Kurimoto
Masahiro
M
Seike
Kiyoshi
K
Takenaka
Akinobu
A
Yoshimura
Minoru
M
Toyota
Shoji
S
Kudoh
D000230:Adenocarcinoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D018288:Carcinoma, Small Cell / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002294:Carcinoma, Squamous Cell / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D016022:Case-Control Studies; D019175:DNA Methylation; D004252:DNA Mutational Analysis; D004273:DNA, Neoplasm / Q000378:metabolism; D005091:Exons; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008168:Lung / Q000378:metabolism / Q000473:pathology; D008175:Lung Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D009154:Mutation; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D011506:Proteins; D016160:Retinoblastoma Protein / Q000235:genetics* / Q000378:metabolism; D050717:Retinoblastoma-Like Protein p130; D014158:Transcription, Genetic
D004273:DNA, Neoplasm; D010750:Phosphoproteins; D011506:Proteins; C493837:RBL2 protein, human; D016160:Retinoblastoma Protein; D050717:Retinoblastoma-Like Protein p130