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Identification of p53 genetic suppressor elements which confer resistance to cisplatin
W M Gallagher1, M Cairney, B Schott
1CRC Department of Medical Oncology, CRC Beatson Laboratories, Glasgow, UK.
Abstract:
Loss of p53 function is associated with the acquisition of cisplatin resistance in the human ovarian adenocarcinoma A2780 cell line. Selection for cisplatin resistance of A2780 cells was used to isolate genetic suppressor elements (GSEs) from a retroviral library expressing random fragments of human or murine TP53 cDNA. Six GSEs were identified, encoding either dominant negative mutant peptides or antisense RNA molecules which corresponded to various regions within the TP53 gene. Both types of GSE induced cisplatin resistance when introduced individually into A2780 cells. Expression of antisense GSEs led to decreased intracellular levels of p53 protein. One sense GSE induced loss of p53-mediated activities such as DNA damage induced cell cycle arrest and apoptosis. A synthetic peptide, representing the predicted amino acid sequence of this GSE, conferred resistance to cisplatin when introduced into A2780 cells and inhibited the sequence specific DNA binding activity of p53 protein in vitro. Overall, these results directly indicate that inactivation of p53 function confers cisplatin resistance in these human ovarian tumour cells. We have identified short structural domains of p53 which are capable of independent functional interactions and highlighted the efficacy of this approach to discriminate biologically active GSEs from a random fragment library.
Insights
Loss of p53 protein function leads to cisplatin resistance in ovarian cancer cells. Researchers identified specific TP53 gene fragments that confer this resistance, offering new therapeutic targets.
Area of Science:
- Molecular Biology
- Cancer Research
- Genetics
Background:
- Cisplatin resistance is a major challenge in ovarian cancer treatment.
- The tumor suppressor protein p53 plays a critical role in cellular responses to DNA damage.
Purpose of the Study:
- To investigate the role of p53 function loss in cisplatin resistance.
- To identify specific TP53 gene domains that confer cisplatin resistance.
Main Methods:
- Used a retroviral library to express random TP53 cDNA fragments in cisplatin-resistant A2780 ovarian cancer cells.
- Isolated and characterized genetic suppressor elements (GSEs) encoding mutant peptides or antisense RNA.
- Assessed the effect of GSEs on p53 protein levels, DNA damage-induced cell cycle arrest, apoptosis, and cisplatin resistance.
Main Results:
- Six GSEs were identified, including dominant-negative mutant peptides and antisense RNA targeting TP53.
- Both types of GSEs induced cisplatin resistance in A2780 cells.
- Antisense GSEs reduced intracellular p53 levels, while one sense GSE impaired p53-mediated DNA damage responses.
- A synthetic peptide mimicking a GSE inhibited p53 DNA binding and conferred cisplatin resistance.
Conclusions:
- Inactivation of p53 function directly confers cisplatin resistance in human ovarian tumor cells.
- Identified functional domains within p53 capable of independent interactions.
- Demonstrated the utility of GSEs for identifying biologically active fragments within a library.