[Effect of inhibitor-1 of protein phosphatase I on apoptosis of human cervical carcinoma cell line HeLa].
OBJECTIVE
Inhibitor-1 of protein phosphatase I(PPI 1) is one of the regulatory subunits of protein phosphatase 1 (PP1). When phosphorylated by protein kinase A (PKA) at Thr-35, PPI 1 inhibits the activity of PP1. This study was to observe the effects of wild type and activated mutant of PPI 1 (PPI 1/wt and PPI 1/sI 1) on the apoptosis of human cervical carcinoma cell line HeLa, and to explore its possible mechanism.


METHODS
Plasmids containing PPI 1/wt or PPI 1/sI 1 were transfected into HeLa cells. Stable cell clones expressing PPI 1 protein (HeLa-PPI 1/wt and HeLa-PPI 1/sI 1) were selected by G418. Untransfected and mock-transfected HeLa cells were used as control. The expression of PPI 1/wt and PPI 1/sI 1 were confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Cell clonogenic ability was detected by colony formation assay. The morphologic alterations of the cells were observed with Hoechst 33258 staining. The apoptosis before and after stimulation of tumor-necrosis factor (TNF) was detected by flow cytometry. The expression of apoptosis-related proteins in the cells was detected by Western blot.


RESULTS
PPI 1/wt and PPI 1/sI 1 were efficiently expressed in HeLa cells. The clonogenic ability of HeLa-PPI 1/sI 1 cells was inhibited. The apoptosis of HeLa-PPI 1/sI 1 cells were enhanced. The expression of P53, P27 and BAK were up-regulated, while the expression of Bcl-2 and Bcl-x(L) were down-regulated. After TNF stimulation, the apoptosis rate was 19.06% for HeLa-PPI 1/sI 1 cells, 2.67% for HeLa-mock cells, and 1.89% for untransfected HeLa cells. PPI 1/sI 1 enhanced the phosphorylation of JNK, and induced the activation of P38 at 30 min after TNF stimulation.


CONCLUSIONS
PPI 1/sI 1 could induce apoptosis of HeLa cells, and enhance the sensitivity of HeLa cells to TNF stimulation, which might enhance the activation of JNK and p38 signal transduction.
Zeng
Qi-Yan
QY
Department of Biochemistry and Molecular Biology, Guangxi Medical University, Nanning, Guangxi, 530021, PR China. zengnn6@hotmail.com
Qin
Xue
X
Zhang
Hong
H
D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D048670:MAP Kinase Kinase 4 / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
D051017:Apoptosis Regulatory Proteins; D047908:Intracellular Signaling Peptides and Proteins; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; C021042:protein phosphatase inhibitor-1; D048051:p38 Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4