[Re-expression of p16 gene in myeloma cell line U266 by arsenic trioxide].
OBJECTIVE
DNA methylation status regulates gene expression and is associated with oncogenesis. Demethylation of DNA has been proposed as a possible new strategy for cancer prophylaxis and treatment. S-adenosylmethionine is required as methyl donor for both arsenic metabolism and DNA methylation. The present study was designed to explore the possibility and mechanism of re-expression of the silenced p16 gene in human myeloma cell line U266 cells by arsenic trioxide (As2O3).


METHODS
The U266 cell line in which the p16 gene is silenced due to hypermethylation was treated with different concentration of As2O3. The PCR technique combined with HpaII and its isoschizomer MspI was used to assess p16 gene methylation status. The mRNA expression levels of p16 and DNA methyltransferases (DNMTs) genes were determined with RT-PCR technique. Western blot was performed for P16 protein expression analysis.


RESULTS
(1)The characteristic hypermethylation with losing expression of p16 gene in U266 cells was confirmed. After agarose gel electrophoresis, the genomic DNA digested with MspI showed a "smear" pattern, however the HpaII digested DNA showed a strong single band. There was no PCR amplified product of p16 gene when MspI digested DNA was used as the templates, whereas a 340 bp p16 gene product was found in HpaII digested DNA sample as the undigested DNA was amplified. Also, neither p16 mRNA nor P16 protein was detectable when assessed with RT-PCR and Western blot, respectively. (2)When the genomic DNA from cells treated with (0.5-2.0) micromol/L As2O3 was digested with HpaII and then analyzed by electrophoresis, a "smear" pattern was observed. The 340 bp product could not be amplified if such digested DNA was used as PCR templates. Detectable expression of both p16 mRNA and P16 protein was found in the cells treated with 1.0 micromol/L and 2.0 micromol/L As2O3. (3)Expression levels of DNMT 3A and 3B mRNA were increased in the treated cells and were dependent on As2O3 concentration, however no significant difference was found between each two groups (P >0.05).


CONCLUSIONS
As2O3 could induce p16 gene re-expression in human myeloma cell line U266 through DNA demethylation.
Li
Jin-Qing
JQ
Department of Hematology,Peking University First Hospital, Beijing 100034, PR China.
Li
Yuan
Y
Shi
Yong-Jin
YJ
Wu
Shu-Lan
SL
D000970:Antineoplastic Agents / Q000494:pharmacology*; D000077237:Arsenic Trioxide; D001152:Arsenicals / Q000494:pharmacology*; D045744:Cell Line, Tumor; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000096:biosynthesis* / Q000235:genetics; D019175:DNA Methylation / Q000187:drug effects; D000090205:DNA Methyltransferase 3A; D004273:DNA, Neoplasm / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D019942:Genes, p16; D006801:Humans; D009101:Multiple Myeloma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D010087:Oxides / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics
D000970:Antineoplastic Agents; D001152:Arsenicals; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004273:DNA, Neoplasm; C000717967:DNMT3A protein, human; D010087:Oxides; D012333:RNA, Messenger; D000090205:DNA Methyltransferase 3A; D000077237:Arsenic Trioxide