J.
Cancer Mol. 3: 81-89, 2007
[Research Paper]
Induction of FADD Expression and Caspase Cascades Involved in RC-RNase-Induced
Apoptosis in Human Cervical Cancer Cells
Huey-Chung Huang, You-Di Liao, Li-Li Chen, and Tze-Sing Huang1
Institute of Biochemistry and Molecular Biology, College of Medicine,
National Taiwan University, Taipei, Taiwan [H.-C. Huang]; Institute of
Biomedical Sciences, Academia Sinica, Taipei, Taiwan [Y.-D. Liao];
National Institute of Cancer Research, National Health Research
Institutes, Taipei, Taiwan [L.-L. Chen, T.-S. Huang]
Abstract:
AIM:
RC-RNase is a novel member of RNase A superfamily from Rana
catesbeiana (bullfrog) oocytes. This study is to investigate the
cytotoxic effect of RC-RNase on cancer cells. The cytotoxic mechanisms
were investigated and discussed.
METHODS:
Cytotoxicity of RC-RNase to human cervical caner HeLa S3 cells was
determined by microculture tetrazolium test and trypan blue exclusion
assay. Apoptotic characteristics, such as chromatin condensation,
blebbing morphology, DNA ladder, phosphatidylserine translocation,
caspase activation, and
poly (ADP-ribose) polymerase (PARP) cleavage, were analyzed
by
generally described methods. Cell-cycle phases were analyzed by flow
cytometry, and cellular cyclin B1/CDC2 activity was detected by in
vitro histone H1 kinase assay. Western blot analyses were performed
to examine the levels of Fas, FADD, TNFR1, TRADD, cyclin B1, CDC2,
Wee-1, and the cleaved forms of Bid, PARP and caspases-3, 8 and 9.
RESULTS:
RC-RNase induced cell death with apoptotic characteristics in HeLa S3
cells. In vitro activity assays suggested that caspases-3, 8,
and 9 activities were all increased in RC-RNase-treated cells. The
results of Western blot analyses, including induction of FADD expression
and increases of the cleaved forms of Bid, PARP and caspases-3, 8 and 9,
confirmed that RC-RNase indeed induced the activation of caspases-3, 8,
and 9. Although RC-RNase did not significantly affect the cell-cycle
distribution, cyclin B1/CDC2 kinase activity was increased in RC-RNase-induced
apoptotic cells. Cellular Wee-1 level was reduced by RC-RNase
treatment, and CDC2 was kept in Tyr-15-dephosphorylated (active) form.
CONCLUSION:
RC-RNase was potent to induce FADD expression, caspase cascades, cyclin
B1/CDC2 activation, and ultimately apoptosis in HeLa S3 cells.
(Keywords:
RC-RNase; FADD; caspase; Wee-1; cyclin B1/CDC2)
Received
4/20/07; Revised 5/20/07; Accepted 5/23/07.
1Correspondence:
Dr. Tze-Sing Huang, National Institute of Cancer Research, National
Heath Research Institutes, 7F, No.161, Min-Chuan East Road Section 6,
Taipei 114, Taiwan, Republic of China. Phone: 886-2-26534401 ext 25138.
Fax: 886-2-27929654. E-mail:
tshuang@nhri.org.tw
2Abbreviations:
RNase, ribonuclease; MTT, microculture tetrazolium test; FITC,
fluorescein
isothiocyanate; PI,
propidium
iodide; TNFR1, tumor
necrosis factor-alpha receptor 1; PARP,
poly
(ADP-ribose) polymerase; tBid, truncated Bid; miRNA, microRNA; FADD, Fas-associating
death domain protein; TRADD, TNFR-associating death domain protein.
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