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Delivery of a Monomeric p53 Subdomain with Mitochondrial Targeting Signals from Pro-Apoptotic Bak or Bax

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Abstract

Purpose

p53 targeted to the mitochondria is the fastest and most direct pathway for executing p53 death signaling. The purpose of this work was to determine if mitochondrial targeting signals (MTSs) from pro-apoptotic Bak and Bax are capable of targeting p53 to the mitochondria and inducing rapid apoptosis.

Methods

p53 and its DNA-binding domain (DBD) were fused to MTSs from Bak (p53-BakMTS, DBD-BakMTS) or Bax (p53-BaxMTS, DBD-BaxMTS). Mitochondrial localization was tested via fluorescence microscopy in 1471.1 cells, and apoptosis was detected via 7-AAD in breast (T47D), non-small cell lung (H1373), ovarian (SKOV-3) and cervical (HeLa) cancer cells. To determine that apoptosis is via the intrinsic apoptotic pathway, TMRE and caspase-9 assays were conducted. Finally, the involvement of p53/Bak specific pathway was tested.

Results

MTSs from Bak and Bax are capable of targeting p53 to the mitochondria, and p53-BakMTS and p53-BaxMTS cause apoptosis through the intrinsic apoptotic pathway. Additionally, p53-BakMTS, DBD-BakMTS, p53-BaxMTS and DBD-BaxMTS caused apoptosis in T47D, H1373, SKOV-3 and HeLa cells. The apoptotic mechanism of p53-BakMTS and DBD-BakMTS was Bak dependent.

Conclusion

Our data demonstrates that p53-BakMTS (or BaxMTS) and DBD-BakMTS (or BaxMTS) cause apoptosis at the mitochondria and can be used as a potential gene therapeutic in cancer.

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Abbreviations

7-AAD:

7-Aminoactinomycin D

BakMTS:

MTS from Bak

BaxMTS:

MTS from Bax

CCO:

MTS from cytochrome c oxidase

CS:

C-segment

DBD:

DNA binding domain

E:

Nuclear export signal

MBD:

MDM2 binding domain

MDM2:

Murine double minute 2

MOMP:

Mitochondrial outer membrane permeabilization

MTS:

Mitochondrial targeting signal

NLS:

Nuclear localization signal

OTC:

MTS from ornithine transcarbamylase

PCC:

Pearson’s correlation coefficient

PRD:

Proline-rich domain

TA:

Transactivation domain

TD:

Tetramerization domain

TM:

Transmembrane

TMRE:

Tetramethylrhodamine, ethyl ester

TOM:

MTS from translocase of the outer membrane (TOM20)

XL:

MTS from Bcl-XL

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ACKNOWLEDGMENTS AND DISCLOSURES

Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under award number R01-CA151847. The authors would also like to acknowledge Christian Raab for his technical work. We also would like to thank Ben Bruno and Geoff Miller for reviewing the manuscript. We acknowledge the use of DNA/Peptide Core and Flow Cytometry Core (NCI Cancer Center Support Grant P30 CA042014, Huntsman Cancer Institute).

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Authors and Affiliations

  1. Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, Utah, USA

    Karina J. Matissek, Abood Okal, Mohanad Mossalam & Carol S. Lim

  2. Department of Pharmaceutics and Biopharmacy, Philipps-Universität, Marburg, Germany

    Karina J. Matissek

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  1. Karina J. Matissek
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  2. Abood Okal
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Correspondence to Carol S. Lim.

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Matissek, K.J., Okal, A., Mossalam, M. et al. Delivery of a Monomeric p53 Subdomain with Mitochondrial Targeting Signals from Pro-Apoptotic Bak or Bax. Pharm Res 31, 2503–2515 (2014). https://doi.org/10.1007/s11095-014-1346-y

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  • DOI: https://doi.org/10.1007/s11095-014-1346-y

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