Background: The cyclin-dependent kinase inhibitor, p21, is well known for its role in cell cycle arrest. Novel anti-cancer agents that deplete iron pools demonstrate marked anti-tumor activity and are also active in regulating p21 expression. These agents induce p21 mRNA levels independently of the tumor suppressor, p53, and differentially regulate p21 protein expression depending on the cell-type. Several chelators, including an analogue of the potent anti-tumor agent, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), have entered clinical trials, and thus, their molecular mechanism of action is crucial to assess. Hence, this investigation examined how several iron chelators transcriptionally regulate p21.
Methods: Promoter-deletion constructs; luciferase assays; RT-PCR; western analysis; gene silencing; co-immunoprecipitation.
Results: The transcriptional regulation of the p21 promoter by iron chelators was demonstrated to be dependent on the chelator and cell-type examined. The potent anti-cancer chelator, Dp44mT, induced p21 promoter activity in SK-MEL-28 melanoma cells, but not in MCF-7 breast cancer cells. Further analysis of the p21 promoter identified a 50-bp region between -104 and -56-bp that was required for Dp44mT-induced activation in SK-MEL-28 cells. This region contained several Sp1-binding sites and mutational analysis of this region revealed the Sp1-3-binding site played a significant role in Dp44mT-induced activation of p21. Further, co-immunoprecipitation demonstrated that Dp44mT induced a marked increase in the interactions between Sp1 and the transcription factors, estrogen receptor-α and c-Jun.
Conclusions and general significance: Dp44mT-induced p21 promoter activation via the Sp1-3-binding site and increased Sp1/ER-α and Sp1/c-Jun complex formation in SK-MEL-28 cells, suggesting these complexes were involved in p21 promoter activation.
Keywords: Desferrioxamine; Dp44mT; Iron chelator; p21.
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