Doxorubicin and 5-fluorouracil induced accumulation and transcriptional activity of p53 are independent of the phosphorylation at serine 15 in MCF-7 breast cancer cells

Abstract

The chemotherapeutic agents doxorubicin (dox) or 5-fluorouracil (5FU) are used to treat cancer cells as they cause irreparable DNA damage, inducing these aberrant cells to undergo cell death. The mediator of this process is presumed to be in part the tumor suppressor p53 which regulates genes involved in DNA repair and cell death. When MCF-7 breast cancer cells are treated with these drugs, we observed that the level of p53 and the p53 negative regulator, Mdm2, increased, as seen by others. But contrary to some reports, we observed minimal phosphorylation of p53 at serine 15 in MCF-7 cells after drug treatment. Interestingly, we determined that there was differential regulation of the kinases ATM and Chk2 with the drug treatments, likely the cause for the lack of phosphorylation of p53. We found a dramatic drop in p53 DNA binding affinity for p21 and other gene response elements (RE) after drug treatment. To determine if the p53 that accumulated in the drug treated cells was functionally active, we monitored changes in the protein products of two p53-regulated genes following drug treatment with and without the addition of a p53-specific siRNA. In response to 5FU, both p21 and Mdm2 proteins increased and that increase was alleviated if a p53-specific siRNA was added. This effect was not seen with the addition of dox. Thus, the phosphorylation at serine 15 is not necessary for the functional activation of this transcription factor. We propose a new model for the regulation of p53, Mdm2, and MdmX after drug treatment.

Keywords: :

• 5-fluorouracil
• ATM
• Chk2
• DNA binding
• doxorubicin
• MCF-7
• Mdm2
• MdmX
• p53
• posttranslational modification

Disclosure of Potential Conflicts of Interest

The authors declare no conflicts of interest.

Acknowledgments

The authors would like to acknowledge John Mallon for his assistance with protein extractions, DNA binding assays and thoughtful conversations; Diane Messina for assistance with maintenance of tissue culture; Mark Gerger for his help with statistical analyses; Amy Fedele for her assistance with the preparation of figures; and Sally Stem for her assistance with procurement of laboratory reagents. We would also like to acknowledge Sanofi Pasteur, Inc. for the use of laboratory supplies and facilities when needed.