5-aza-2'-deoxycytidine induces retinoic acid receptor beta 2 demethylation, cell cycle arrest and growth inhibition in breast carcinoma cells.
Like all cancers, breast cancer is considered to result in part from the accumulation of multiple genetic alterations leading to oncogene overexpression and tumor suppressor loss. More recently, CpG island hypermethylation is known to be associated with gene silencing in cancer, and these silenced genes can be reactivated by 5-aza-2'-deoxycytidine (5-Aza-CdR). Retionoic acid receptor beta 2 gene is a tumor suppressor gene and the chemopreventive effects of retinoids are due to induction of RAR beta 2. In this study, the effect of 5-Aza-CdR RAR beta 2 restoration was investigated in the MRK-nu-1 human female breast cancer cell line. Changes of the RAR beta 2 methylation status were assessed by methylation-specific PCR. Reverse transcription PCR was used to evaluate RARb beta 2 restoration. Cell cycling and growth inhibition were studied using flow cytometric analysis of DNA content and CellTiter 96 AQueous non-radioactive cell proliferation assay, respectively. 5-Aza-CdR treatment resulted in complete demethylation of the RAR beta 2 gene. RAR beta 2 restoration was accompanied by cell cycle arrest (increase in the G0/G1- and decrease in the S- and G2/M-phases) and time-dependent growth inhibition. In conclusion, RAR beta 2 can be activated in vitro by 5-Aza-CdR, which may be one of the mechanisms for the tumor cell growth inhibition by 5-Aza-CdR.
Yang
Qifeng
Q
Department of Surgery, Affiliated Kihoku Hospital, Wakayama Medical University, Myoji 219, Katuragi-cho 649-7113, Wakayama, Japan. yang-qf@mail.wakayama-med.ac.jp
Shan
Liang
L
Yoshimura
Goro
G
Nakamura
Misa
M
Nakamura
Yasushi
Y
Suzuma
Takaomi
T
Umemura
Teiji
T
Mori
Ichiro
I
Sakurai
Takeo
T
Kakudo
Kennichi
K
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D001943:Breast Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology*; D002453:Cell Cycle / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D019175:DNA Methylation / Q000187:drug effects*; D000077209:Decitabine; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006801:Humans; D018168:Receptors, Retinoic Acid / Q000235:genetics*; D014407:Tumor Cells, Cultured
D000964:Antimetabolites, Antineoplastic; D004791:Enzyme Inhibitors; D018168:Receptors, Retinoic Acid; C082925:retinoic acid receptor beta; D000077209:Decitabine; D001374:Azacitidine