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Selective sensitization to DNA-damaging agents in a human rhabdomyosarcoma cell line with inducible wild-type p53
A A Gibson1, F G Harwood, D M Tillman
1Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
Abstract:
Drug-induced cytotoxicity or apoptosis may be influenced by the expression of the p53 tumor suppressor gene and by the specific oncogene expressed, which may dictate the threshold at which a cytotoxic response may by induced. The objective of the study was to elucidate how DNA-damaging agents with different mechanisms of action were sensitized in the context of expression of the Pax3/FKHR fusion protein, a transformation event unique to alveolar rhabdomyosarcomas (ARMSs), and wild-type p53 (wtp53). A wtp53 cDNA was subcloned into the pGRE5-2/EBV vector with dexamethasone-inducible overexpression and transfected into Rh30 ARMS cells that express Pax3/FKHR and a mutant p53 phenotype. Following dexamethasone induction of wtp53 overexpression in a derived clone (Cl.#27), growth was slowed, and cells accumulated in G1. Functional wtp53 activity was demonstrated by selective transactivation of p50-2, a wtp53 chloramphenicol acetyltransferase reporter construct, and by up-regulated expression of endogenous p21Waf1. Data demonstrated p53-dependent sensitization (> or = 4-fold) to bleomycin, actinomycin D, and 5-fluorouracil and considerably less p53-dependence (< or = 2-fold) for doxorubicin, topotecan, etoposide, and cisplatin in Cl.#27 compared to an equivalent clone containing the pGRE5-EBV vector alone (VC#3). Data demonstrate that ARMS cells show a selective sensitization to DNA-damaging agents when wtp53 is overexpressed. The cytotoxic activity of agents that are not potentiated substantially must, therefore, depend upon p53-independent factors that relate to the mechanism of drug action.
Insights
Overexpressing wild-type p53 (wtp53) selectively sensitizes alveolar rhabdomyosarcoma cells to certain DNA-damaging drugs. This finding highlights p53
Area of Science:
- Oncology
- Molecular Biology
- Cancer Genetics
Background:
- Drug-induced cytotoxicity is influenced by p53 tumor suppressor gene expression and oncogenes.
- Alveolar rhabdomyosarcomas (ARMS) are characterized by the Pax3/FKHR fusion protein and often a mutant p53 phenotype.
- Understanding drug response in ARMS requires investigating the role of wild-type p53 (wtp53) in conjunction with Pax3/FKHR.
Purpose of the Study:
- To determine how DNA-damaging agents with varying mechanisms are sensitized by the expression of Pax3/FKHR and wtp53 in ARMS cells.
- To elucidate the role of wtp53 in modulating the sensitivity of ARMS cells to different chemotherapeutic agents.
Main Methods:
- Subcloning of wtp53 cDNA into a dexamethasone-inducible vector.
- Transfection into Rh30 ARMS cells expressing Pax3/FKHR and mutant p53.
- Induction of wtp53 overexpression and assessment of cell growth, cell cycle (G1 arrest), and functional p53 activity (reporter assays, p21Waf1 expression).
- Comparative drug sensitivity assays using bleomycin, actinomycin D, 5-fluorouracil, doxorubicin, topotecan, etoposide, and cisplatin.
Main Results:
- Dexamethasone induction of wtp53 overexpression in ARMS cells (Cl.#27) led to slowed growth and G1 cell cycle arrest.
- Functional wtp53 activity was confirmed by reporter gene transactivation and increased p21Waf1 expression.
- Significant p53-dependent sensitization (≥4-fold) was observed for bleomycin, actinomycin D, and 5-fluorouracil.
- Lower p53-dependence (≤2-fold) was noted for doxorubicin, topotecan, etoposide, and cisplatin, suggesting p53-independent mechanisms for these agents.
Conclusions:
- Overexpression of wtp53 in ARMS cells results in selective sensitization to specific DNA-damaging agents.
- The degree of drug sensitization is dependent on both the drug's mechanism of action and the p53 status.
- Agents with substantial un-potentiated cytotoxic activity likely rely on p53-independent pathways in this cellular context.