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TPCK-induced apoptosis and labelling of the largest subunit of RNA polymerase II in Jurkat cells

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Abstract

N-α-Tosyl-l-phenylalanyl chloromethyl ketone (TPCK) is an affinity label for chymotrypsin-like proteases and has been extensively used as an experimental tool in apoptosis research to probe the role of proteases in cell death. While TPCK blocks some apoptotic changes and induces others, the cellular target or targets for TPCK have not been identified. Here we investigated for the first time the cellular targets for TPCK using a polyclonal anti-tosyl antibody. We have found that TPCK rapidly and irreversibly labels numerous intracellular proteins and have identified one as RPB1, the largest subunit of RNA polymerase II. We show that TPCK inhibits DNA binding by RNA polymerase and that TPCK inhibits transcription. Inhibition of transcription is known to induce apoptosis and while TPCK may trigger death through interaction with multiple targets, our data suggests that the pro-apoptotic effects of TPCK may be explained in part by the inhibition of RNA polymerase II activity.

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Funding

We are indebted to The Irish Cancer Society for a project grant (CRI05FER) that supported this research.

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Authors and Affiliations

  1. Pharmacology and Therapeutics, School of Medicine and National Centre for Biomedical Engineering Science, School of Science, Room 213, Orbsen Building, NUI Galway, Galway, Ireland

    Z. Fabian, P. O’Brien & H. O. Fearnhead

  2. Department of Medical Biology, University of Pécs, Pecs, Hungary

    Z. Fabian

  3. Science Foundation Ireland Undergraduate Research Experience and Knowledge Award Programme, Dublin, Ireland

    K. Pajęcka

Authors
  1. Z. Fabian
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  2. P. O’Brien
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  3. K. Pajęcka
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  4. H. O. Fearnhead
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Corresponding author

Correspondence to H. O. Fearnhead.

Additional information

Z. Fabian and P. O’Brien contributed equally to this work.

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Fabian, Z., O’Brien, P., Pajęcka, K. et al. TPCK-induced apoptosis and labelling of the largest subunit of RNA polymerase II in Jurkat cells. Apoptosis 14, 1154–1164 (2009). https://doi.org/10.1007/s10495-009-0386-y

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