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Cytotoxic effects of Ad5CMV-p53 expression in two human nasopharyngeal carcinoma cell lines
1Departments of Radiation Oncology and Research, Princess Margaret Hospital/Ontario Cancer Institute, Toronto, Ontario, M5G 2M9 Canada.
Abstract:
Nasopharyngeal carcinoma (NPC) is a malignant disease of the head/neck region with a 5-year survival level of approximately 65%. To explore novel therapeutic strategies in the management of this disease, the potential of Ad5CMV-p53-mediated gene transfer to NPC cells was investigated in vitro. Two NPC cell lines, CNE-1 and CNE-2Z, were infected with either Ad5CMV-p53 or Ad5CMV-beta-galactosidase and evaluated for transduction efficiency and cytotoxicity. At a multiplicity of infection of 50 plaque-forming units (pfu)/cell, Ad5CMV-beta-galactosidase infection and beta-galactosidase expression were detected in almost 100% of treated NPC cells. High levels of recombinant p53 protein expression were also observed in the NPC cell lines when treated with Ad5CMV-p53 at 50 pfu/cell. Expression of recombinant p53 was dose and time dependent, with peak levels observed at 24 h. A marked increase in WAF1/CIP1 expression was also observed in NPC cells after Ad5CMV-p53 infection. Expression of bcl-2 and bax were minimally detectable at baseline; infection with Ad5CMV-p53 induced no changes in the protein levels in the NPC cells. Growth of NPC cells treated with Ad5CMV-p53 was observed to be significantly inhibited when determined by either the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide or clonogenic assay. Infection with Ad5CMV-p53 at 25 pfu/cell resulted in survival levels of 0.35 and 11% in CNE-1 and CNE-2Z cells, respectively. Chromatin condensation and DNA fragmentation were also observed, demonstrating that these cells were undergoing apoptosis. However, when GM38 (normal human fibroblasts) were subjected to identical treatments, they demonstrated significantly lower infection efficiency and transgene expression and were resistant to Ad5CMV-p53-mediated cytotoxicity. These data demonstrate the efficacy of Ad5CMV-p53-mediated gene therapy in human NPC, thus warranting additional investigations of this therapeutic strategy.
Insights
Gene therapy using Ad5CMV-p53 effectively targets nasopharyngeal carcinoma (NPC) cells, significantly inhibiting tumor growth and inducing apoptosis. This novel approach shows promise for treating NPC, with minimal impact on normal cells.
Area of Science:
- Oncology
- Gene Therapy
- Molecular Biology
Background:
- Nasopharyngeal carcinoma (NPC) is a head and neck malignancy with a moderate 5-year survival rate.
- Novel therapeutic strategies are crucial for improving NPC patient outcomes.
- Gene transfer offers a potential avenue for targeted cancer treatment.
Purpose of the Study:
- To investigate the efficacy of Ad5CMV-p53-mediated gene transfer in nasopharyngeal carcinoma (NPC) cells in vitro.
- To evaluate the transduction efficiency and cytotoxicity of Ad5CMV-p53 in NPC cell lines.
- To assess the therapeutic potential of p53 gene therapy for NPC.
Main Methods:
- Two human NPC cell lines (CNE-1, CNE-2Z) were infected with Ad5CMV-p53 or Ad5CMV-beta-galactosidase.
- Transduction efficiency and transgene expression (p53, beta-galactosidase) were quantified.
- Cytotoxicity was assessed using MTT and clonogenic assays.
- Apoptosis markers (chromatin condensation, DNA fragmentation) and related gene expression (WAF1/CIP1, bcl-2, bax) were analyzed.
- Normal human fibroblasts (GM38) were used as a control.
Main Results:
- Ad5CMV-p53 achieved high transduction efficiency and recombinant p53 expression in NPC cells, dependent on dose and time.
- Significant inhibition of NPC cell growth and induction of apoptosis (chromatin condensation, DNA fragmentation) were observed.
- NPC cells treated with Ad5CMV-p53 showed drastically reduced survival rates (0.35% for CNE-1, 11% for CNE-2Z).
- Normal fibroblasts (GM38) exhibited lower infection efficiency and resistance to Ad5CMV-p53-mediated cytotoxicity.
Conclusions:
- Ad5CMV-p53-mediated gene therapy demonstrates significant efficacy against human nasopharyngeal carcinoma cells in vitro.
- The treatment effectively inhibits NPC cell proliferation and induces apoptosis.
- The selective toxicity towards NPC cells with relative safety for normal fibroblasts supports further investigation of this therapeutic strategy for NPC.