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Restoration of p53 function in anaplastic Wilms' tumor
S J Delatte1, D J Hazen-Martin, G G Re
1Division of Pediatric Surgery, Department of Surgery, Medical University of South Carolina, Charleston, SC 29425, USA.
Background/Purpose:
Recent studies have reported a high incidence of p53 mutations in anaplastic Wilms' tumors (WT). Restoration of the normal p53 state by current gene therapy techniques is thus an attractive potential mode of therapy for this tumor, which is poorly responsive to standard therapy. The purpose of this study is to determine whether gene delivery of normal p53 is possible and to characterize the subsequent effect of restoring the wild-type p53 state.
Methods:
Anaplastic WT RM1 cells (mutant p53) were transduced with replication-deficient adenoviral vectors containing either the wild-type p53 gene (rAd-p53) or the gene encoding a green fluorescent protein (rAd-GFP). The transduction efficiency of adenovirus for RM1 cells was determined by flow cytometric analysis of rAd-GFP-transduced cells. The effect of p53 transduction on cell viability was evaluated using a colorimetric proliferation assay. Apoptosis was evaluated by labeling DNA breaks using a TUNEL assay (Apo-Direct kit).
Results:
Cells treated with increasing concentrations of viral particles relative to tumor cells (multiplicity of infection-MOI) showed a dose-dependent increase in the number of cells transduced. Twenty-four hours after viral treatment, the percentage of cells transduced for MOIs of 10, 50, 100, and 500 was 29.5, 60.9, 74.6, and 92.4, respectively; at 48 hours the percentage of cells transduced increased to 70.8, 90.7, 93. 7, and 96.3, respectively. Viral treatment at an MOI of 50 reduced cell proliferation by 10% at 17 hours and 97% at 5 days; at an MOI of 100, the relative reduction in proliferation was 15% and 99.8%, respectively. When assayed, 30% of cells became apoptotic at an MOI of 50, and 48% at an MOI of 100.
Conclusions:
Highly efficient delivery of the p53 tumor suppressor gene by adenoviral vector to anaplastic WT is possible. Subsequent restoration of the normal p53 state results in reduced viability and increased apoptosis. Gene replacement of p53 may represent a novel therapeutic agent for anaplastic Wilms' tumors.
Insights
Gene therapy can restore normal p53 in anaplastic Wilms' tumors (WT). Adenoviral delivery of the p53 gene effectively reduced tumor cell viability and increased apoptosis, offering a potential new treatment for WT.
Area of Science:
- Oncology
- Molecular Biology
- Gene Therapy
Background:
- Anaplastic Wilms' tumors (WT) frequently exhibit p53 mutations.
- WT is poorly responsive to conventional therapies.
- Restoring normal p53 function presents a potential therapeutic strategy.
Purpose of the Study:
- To assess the feasibility of gene delivery for normal p53 into anaplastic WT cells.
- To characterize the effects of restoring wild-type p53 status.
Main Methods:
- Anaplastic WT cells (RM1) were transduced using replication-deficient adenoviral vectors carrying either wild-type p53 (rAd-p53) or green fluorescent protein (rAd-GFP).
- Transduction efficiency was measured by flow cytometry.
- Cell viability was assessed using proliferation assays, and apoptosis was evaluated via TUNEL assay.
Main Results:
- Adenoviral transduction demonstrated a dose-dependent increase in efficiency, with over 90% transduction rates at high multiplicities of infection (MOI).
- Treatment with rAd-p53 significantly reduced cell proliferation (up to 99.8%) and induced apoptosis (up to 48%) in a dose-dependent manner.
- Higher MOIs correlated with greater reductions in proliferation and increased apoptosis.
Conclusions:
- Adenoviral vectors enable highly efficient delivery of the p53 gene to anaplastic WT.
- Restoring wild-type p53 function leads to decreased cell viability and enhanced apoptosis.
- p53 gene replacement therapy shows promise as a novel treatment for anaplastic Wilms' tumors.
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