Abstract
AIM: To investigate the apoptosis-promotive action of the P53 protein C-terminus (P53c) agsinst tumors with p53 gene mutation. METHODS: Protein transduction domain of human immunodeficiency virus TAT protein (TAT) was used to deliver P53c into tumor cells, and the oxygen-dependent degradation domain (ODD) was used to control the stability of P53c in tissue. TAT-p53c (TPc) gene was constructed by PCR, and cloned into the pGEX4T vector. The fusion proteins of glutathione-S- transferase (GST)-TAT-ODD-P53c (GST-TOPc) and GST-TAT-P53c (GST-TPc), etc, were expressed in BL21(DE3) and purified by affinity column. Immunohistochemistry analysis was used to determine the transduction of TAT fusion protein into the SW480 cells. The viability of SW480 cells treated with the fusion proteins were measured by the MTT method, and the flow cytometry was employed to analyze the apoptosis of SW480 cells caused by the fusion proteins. RESULTS: A series of pGEX vectors were constructed, and the soluble proteins were expressed in E. coli. The result of Western blot showed that the fusion proteins well immunoreacted with the mouse anti-Schistosoma japonica GST monoclonal antibody. Immunohistochemistry analysis showed that GST-TPc, GST-TOPcm and GST-TOPc could all permeate into the SW480 cells. GST-TPc obviously decreased the viability of SW480 cells and led to the cell apoptosis in vitro. The fusion proteins bearing the ODD domain exhibits mild apoptosis-promoting action on the tumor cells under the aerobic condition. CONCLUSION: TAT mediates the fusion proteins accross the cell membrane. GST-TPc induces the apoptosis of the P53 mutated-tumor cells.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 28-33 |
| Number of pages | 6 |
| Journal | Chinese Journal of Pharmacology and Toxicology |
| Volume | 21 |
| Issue number | 1 |
| State | Published - Feb 2007 |
| Externally published | Yes |
Bibliographical note
Funding Information:This work was funded by the British Columbia Blueberry Council, the Natural Sciences and Engineering Research Council of Canada – Agriculture Canada Research Partnerships Program, the British Columbia Investment Agriculture Foundation, and Agriculture and Agri-Food Canada Matching Investment Initiative. We wish to acknowledge the numerous blueberry growers, consultants, and field scouts who submitted plant samples for analysis and allowed research to be conducted in their fields. We thank Mark Sweeney, Jim Rahe, Elizabeth Hudgins, Vippen Joshi, Andrea Buonassisi, Dave Raworth, Debbie Henderson, Tom Lowery, Pete Bristow, Colleen Harlton, Bob Costello, Ken Sojonky, Julie Bidulka, Heather Carriere, Naomi Van Huizen, Rhonda MacCoy, Rudra P. Singh, and Brad Hill-man for providing advice and information during various aspects of this study.
Keywords
- Apoptosis
- Oxygen-dependent degradation domain
- Protein P53
- Protein transduction domain
