The expression of survivin and its related genes in adipocyte-derived stem cell by demethylation.
BACKGROUND
Survivin is thought to contribute to stem cell maintenance partly by a hypomethylation mechanism. This study attempted to elucidate the signal transduction pathway of adipocyte-derived stem cells (ASCs) by using a demethylating agent, 5-aza-2'-deoxycytidine (ADC), to analyze the survivin, MEK/ERK, c-Myc and p53 gene expression.


METHODS
Demethylation in the ASCs was induced by 1 microM ADC treatment. RT-PCR for survivin mRNA was preformed, before and 24, 48 and 72 hours (hr) after ADC treatment. Western blotting analysis was performed for p53, survivin, unphosphorylated and phosphorylated (p)-MEK, and p-ERK. Immunohistochemistry for ERK and survivin was done to evaluate the localization of the proteins.


RESULTS
ADC inhibited the population growth of the ASCs and it increased the number of apoptotic cells 24, 48, and 72 hr after treatment. ADC treatment slightly decreased the expression of survivin mRNA after 48 hr and its level was restored after 72 hr of treatment. Otherwise, the level of survivin protein gradually increased up to 48 hr and it was decreased at 72 hr. The levels of p-MEK and p53 were increased time-dependently. c-Myc and p-ERK were elevated after ADC treatment and their highest levels were seen 48 hr after treatment. The ADC treatment increased the nuclear expression of ERK and survivin in the ASCs.


CONCLUSIONS
The overexpression of p-MEK/ERK, p53, and c-Myc increased the survivin protein expression of the demethylated ASCs. These results suggest that demethylation could alter the expression of survivin, and p53, c-Myc and the MAPK (MEK/ERK) pathway might play a role in survivin's regulation in ASCs.
Yoon
Kwang
K
Department of Anesthesiology and Pain Medicine, Kosin University College of Medicine, Busan, Korea.
Lim
Young Soo
YS
Yu
Soo Bong
SB
Kim
Doo Sik
DS
Ryu
Sie Jeong
SJ
Kim
Kyung Han
KH
Jang
Tae Ho
TH
Kim
Se Hwan
SH