Abstract
Purpose
Members of paraneoplastic Ma (PNMA) family have been identified as onconeuronal antigens, which aberrant expressions in cancer cells of patients with paraneoplastic disorder (PND) are closely linked to manifestation of auto-immunity, neuro-degeneration, and cancer. The purpose of present study was to determine the role of PNMA5 and its functional relationship to MOAP-1 (PNMA4) in human cancer cells.
Methods
PNMA5 mutants were generated through deletion or site-directed mutagenesis and transiently expressed in human cancer cell lines to investigate their role in apoptosis, subcellular localization, and potential interaction with MOAP-1 through apoptosis assays, fluorescence microscopy, and co-immunoprecipitation studies, respectively.
Results
Over-expressed human PNMA5 exhibited nuclear localization pattern in both MCF-7 and HeLa cells. Deletion mapping and mutagenesis studies showed that C-terminus of PNMA5 is responsible for nuclear localization, while the amino acid residues (391KRRR) within the C-terminus of PNMA5 are required for nuclear targeting. Deletion mapping and co-immunoprecipitation studies showed that PNMA5 interacts with MOAP-1 and N-terminal domain of PNMA5 is required for interaction with MOAP-1. Furthermore, co-expression of PNMA5 and MOAP-1 in MCF-7 cells significantly enhanced chemo-sensitivity of MCF-7 to Etoposide treatment, indicating that PNMA5 and MOAP-1 interact synergistically to promote apoptotic signaling in MCF-7 cells.
Conclusions
Our results show that PNMA5 promotes apoptosis signaling in HeLa and MCF-7 cells and interacts synergistically with MOAP-1 through its N-terminal domain to promote apoptosis and chemo-sensitivity in human cancer cells. The C-terminal domain of PNMA5 is required for nuclear localization; however, both N-and C-terminal domains of PNMA5 appear to be required for pro-apoptotic function.
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Abbreviations
- KDa:
-
KiloDalton
- FBS:
-
Fetal Bovine Serum
- CHAPS:
-
3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate
- FITC:
-
Fluorescein Isothiocyanate
- EDTA:
-
Ethylenediaminetetraacetic Acid
- Triton X-100:
-
Octylphenol Decaethylene Glycol Ether
- fLuc:
-
Firefly luciferase
- β-Gal:
-
β-Galactosidase
- GFP:
-
Green Fluorescent Protein
- SDS-PAGE:
-
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
- MOAP-1:
-
Modulator of Apoptosis 1
- PBS:
-
Phosphate-Buffered Saline
- OD:
-
Optical Density
- DAPI:
-
4′,6-Diamidino-2-phenylindole
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Acknowledgments
The research was supported by Sunway University Research Grant (INT-FST-BIOS-2013-07, INT-FST-BIOS-0115-03) and FRGS (Ministry of Higher Education, Malaysia) Research Grant (SG05/SYUC/02/1) awarded to K.O. Tan. In addition, S.W. Pang was partially supported by INT-RRO-2014-017 (Sunway University). Authors would like to express their appreciations to Drs. Victor Yu, Lau BC (National University of Singapore, NUS) for generous donation of plasmid clones or research materials, and Dr. Shing-Leng Chan (Cancer Science Institute, NUS, Singapore) for helpful comments and feedback on some of the data presented in this manuscript.
Authors’ contributions
Y. H. L., S. W. P., and K. O. T designed research. Y. H. L. and S.W. P. performed experiments, Y. H. L., S. W. P., C. L. P., and K. O. T. analyzed the data, C. L. P edited the manuscript and K. O. T. wrote the paper.
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Lee, Y.H., Pang, S.W., Poh, C.L. et al. Distinct functional domains of PNMA5 mediate protein–protein interaction, nuclear localization, and apoptosis signaling in human cancer cells. J Cancer Res Clin Oncol 142, 1967–1977 (2016). https://doi.org/10.1007/s00432-016-2205-5
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DOI: https://doi.org/10.1007/s00432-016-2205-5