p53-Dependent activation of a molecular beacon in tumor cells following exposure to doxorubicin chemotherapy.
In an effort to begin developing a non-invasive strategy for in-vivo detection of the cellular DNA damage response, we engineered a molecular beacon to detect expression of p21(WAF1/CIP1), a gene whose transcription is directly activated by the p53 tumor suppressor protein. Introduction of a phosphorothioate-modified p21-beacon by transfection in human tumor cells led to a slight background signal that increased in a dose dependent manner between 50 and 400 nM beacon. Strong nuclear signal was observed following treatment of wild-type p53-expressing human H460 lung cancer cells for 8 hours with the chemotherapeutic agent doxorubicin (adriamycin). Similar induction was observed in wild-type p53-expressing HCT116 cells. Interestingly, following doxorubicin exposure, there was activation of the p21-beacon in p21-null HCT116 cells, which was not observed in p53-null HCT116, or mutant p53-expressing DLD1 cells that are either wild-type or p21-null. Increased signal from the phosphorothioate-modified p21-beacon in doxorubicin-treated cells likely resulted from sequence-specific hybridization as well as sequence-independent cleavage that may occur due to p53-dependent activation of endonucleases during apoptosis. We conclude that activation of p53 by chemotherapy leads to a strong signal from a p21-beacon that may be useful in further testing both in vitro and in vivo. Strategies need to be developed to optimize the gene or damage specificity as well as the sensitivity to therapeutic response of this non-invasive imaging approach.
Shah
Rishita
R
Laboratory of Molecular Oncology and Cell Cycle Regulation, Department of Medicine, Abramson Cancer Center, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104, USA.
El-Deiry
Wafik S
WS
D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis; D014408:Biomarkers, Tumor / Q000032:analysis*; D018797:Cell Cycle Proteins / Q000032:analysis / Q000096:biosynthesis*; D002467:Cell Nucleus; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000494:pharmacology*; D004720:Endonucleases / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000032:analysis; D006801:Humans; D008175:Lung Neoplasms / Q000473:pathology; D014162:Transfection; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000494:pharmacology*
D000903:Antibiotics, Antineoplastic; D014408:Biomarkers, Tumor; C493956:CDKN1A protein, human; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004791:Enzyme Inhibitors; D016159:Tumor Suppressor Protein p53; D004317:Doxorubicin; D004720:Endonucleases