5-aza-2 '-deoxycytidine leads to down-regulation of aberrant p16INK4A RNA transcripts and restores the functional retinoblastoma protein pathway in hepatocellular carcinoma cell lines
Si. Suh et al., 5-aza-2 '-deoxycytidine leads to down-regulation of aberrant p16INK4A RNA transcripts and restores the functional retinoblastoma protein pathway in hepatocellular carcinoma cell lines, CANCER LETT, 160(1), 2000, pp. 81-88
The inactivation of the cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor
p16INK4A may be caused by gene deletion, mutation or promoter hypermethylat
ion. We have previously reported that p16INK4A in hepatocellular carcinoma
(HCC) tissues and cell lines is inactivated predominantly by promoter hyper
methylation rather than genomic aberrations. In the present experiments, we
have studied the effects of the demethylating agent, 5-aza-2'-deoxycytidin
e (5-AZA/decitabine), on the expression of aberrant p16INK4A RNA transcript
s and the CDK-retinoblastoma gene pathway in HCC cell lines with p16INK4A p
romoter hypermethylation. The expression of aberrant p16INK4A RNA transcrip
ts was down-regulated and p16INK4A protein was strongly re-expressed in the
HCC cell lines, SNU 354, 398, 423 and 475 after 5-AZA/decitabine treatment
for 5 days. The re-expressed p16INK4A was functional, because it bound to
and inhibited CDK4 kinase activity, and increased the concentrations of the
hypophosphorylated form of retinoblastoma protein (pRB) in cells with a wi
ld type RE gene. Moreover, treatment with the demethylating agent led not o
nly to G1 cell cycle arrest, but also to the increased expression of the se
nescence-associated marker beta -galactosidase. This up-regulation of p16IN
K4A mRNA and protein correlated with demethylation of the p16INK4A promoter
. and with the down-regulation or disappearance of aberrant p161NK4A transc
ripts. The;e results suggest that the aberrant p16INK4A RNA transcript can
be transcribed from the methylated p16INK4A gene, and endogenous reactivati
on of functional p16INK4A mRNA by a demethylating agent can restore the pRB
pathway in HCC, and foster the terminal differentiation of the malignant c
ells. Therefore, demethylating agents, such as 5-AZA/decitabine, may have p
otential in the treatment of HCC. (C) 2000 Elsevier Science Ireland Ltd. Al
l rights reserved.