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Oncogenomics: Piet Borst

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Piet Borst, Ph.D. professorGroup leader

About Piet Borst

Transporters, DNA base J

DNA base J
This project is an offshoot of our long-standing interest in the mechanisms of antigenic variation in African trypanosomes. Base J (β-glucosyl-hydroxymethyluracil), which we discovered in African trypanosomes in 1993 (Gommers-Ampt et al., Cell 1993; 75: 1129-1136), is a base present in kinetoplastid flagellates and ofEuglena. It replaces 1% of thymine in nuclear DNA and is predominantly located in repetitive sequences, such as telomeric repeats. We have shown that the initial step of base J synthesis, the conversion of a T-residue in DNA into hydroxymethyluracil, is catalysed by 2 enzymes belonging to the TET/JBP family of oxygenases (hydroxylases) that require Fe2+ and 2-oxoglutarate as cofactors. More recently we have shown that J is essential inLeishmaniafor the proper termination of transcription. Loss of J results in massive read-through of transcriptional stops and in death of the parasite. This project was discontinued in Amsterdam in 2012 and transferred to Peter Myler in Seattle. Borst remains involved as an adviser.

MULTIDRUG RESISTANCE OF CANCER CELLS
We are interested in mechanisms of drug resistance in cancer cells and have focussed on resistance caused by increased ATP-dependent transport of drug out of the cell, mediated by ATP-binding cassette (ABC) transporters. We have isolated genes for these transporters and characterized their substrate specificity and sensitivity to inhibitors in transfected cells. To study the physiological function in metabolism and defense of the body against drugs and xenotoxins of these transporters, we have inactivated genes for several drug transporters by targeted gene disruption in mice. Initially we looked at P-glycoproteins (ABCB1 and ABCB4); most recently we have studied the Multidrug Resistance-associated Protein (ABCC) family members MRP2, 3, 4, 5 and 6. MRPs are known to transport organic anions out of cells and these are often produced by conjugation of toxic compounds to hydrophilic organic anions, such as glucuronic acid. Although many substrates of MRPs are known, the list is incomplete. For some MRPs there is no idea yet of their physiological function.

Senior post-doc Koen van de Wetering has therefore initiated a systematic search for compounds conjugated to glucuronide or sulphate that are transported by MRPs by comparing the derivatives in plasma/urine of WT and KO mice using Mass Spectrometry. We have identified several glucuronidated and sulphated phyto-estrogens, derived from food, as novel substrates of MRPs (ABCC2), MRP3 (ABCC3) and BCRP (ABCG2). More recently we have studied MRP5 and MRP6 by this approach.

MRP5 (ABCC5)
In 2000 we generated a mouse KO of theMrp5gene. Although this gene is expressed in most mouse tissues, the KO mice had no phenotype (Wijnholds et al., PNAS, 97 (2000) 7476). MRP5 was found to transport some base and nucleotide analogs and later also some other drugs and cAMP and cGMP. A role for MRP5 in drug resistance or cyclic nucleotide metabolism in intact mice has not been demonstrated, however. We are therefore reinvestigating MRP5 and theMrp5KO mice using the metabolomics approach developed by Koen van de Wetering. Two new classes of substrates were recently identified in unpublished experiments: compounds related to neurotransmitters and a new class of compounds not previously known to exist in mammals.

MRP6 (ABCC6) and PXE
Pseudoxanthoma elasticum (PXE) is an autosomal recessive disease characterized by a progressive mineralization of connective tissue, resulting in skin, arterial and eye disease. Classical PXE is caused by mutations in the MRP6 (ABCC6) gene. Studies by Uitto et al. with Abcc6-/- mice have shown that the absence of ABCC6 in the liver is crucial for PXE and have confirmed the "metabolic disease hypothesis" for PXE, which states that tissue calcification is due to the absence of a plasma factor X secreted from the basolateral hepatocyte membrane. We are trying to find X by a concerted metabolomics approach, usingMrp6KO mice, cells transfected withMRP6constructs, vesicular transport assays and liver perfusates of WT and KO mice. MRP6 is closely similar to MRP1 and MRP3 and is able to (sluggishly) transport some organic anions. We therefore expect X to be an organic ion detectable by the methods available.

TRANSPORTOMICS
Finding endogenous substrates for MRP3 is time-consuming. If compound A is lower in the plasma of Mrp3 KO mice than of WT mice, one still has to demonstrate that this is not a secondary effect of MRP3 absence, but that MRP3 can transport A. As a short-cut we developed "transportomics". In this approach membrane vesicles containing MRP3 are incubated in body fluids and the compounds accumulating in the vesicles are analyzed by LC/MS. This method is fast; it can also be applied to human plasma/urine; no KO mice are required; and it is applicable to many transporters.

DRUG RESISTANCE IN "SPONTANEOUS" MOUSE TUMORS
In collaboration with Jos Jonkers (NKI-AVL), we have studied resistance mechanisms in "spontaneous" breast tumors arising in mice, conditionally defective in p53 and Brca1. This project will be continued by Sven Rottenberg, Borst remaining an adviser.

Co-workers

Experience

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Van Luenen, Henri

Henri van Luenen

Manager Research

Experience

I studied Biology at the Free University in Amsterdam.  As part of this study I did a rotation project in the lab of Piet Borst at the NKI and I stayed at the NKI ever since. Ronald Plasterk offered me a PhD student position at the NKI which I gladly accepted. After first studying adipogenesis in response to DNA hypomethylation, I switched to study the transposition mechanism of the Tc3 transposons in C. elegans. After my PhD I continued my research in the group of Piet Borst, first on the transferrin receptor in T. brucei and later on base J in Leishmania. This unusual base plays an important role in transcription termination in this parasite.

Besides my research I am also involved in the general operations of the NKI.

 

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Van de Wetering, Koen.jpg

Koen van de Wetering

Postdoctoral Fellow

Experience

After being trained as a veterinarian, I did my PhD at the Department of Biochemistry and Cell Biology at Utrecht University, where I worked on the innate immune system of the lung.
In 2003 I joined the group of Piet Borst to study the Multidrug Resistance Proteins (MRPs). We use metabolomics-based approaches to characterize the substrate spectrum of these efflux pumps. Our work currently focuses on MRP6, the absence of which results in the connective tissue disorder Pseudoxanthoma elasticum (PXE). We want to identify the physiological substrates of MRP6 to develop an effective treatment for this currently incurable disease.

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Key publications View All Publications

  • Transportomics: screening for substrates of ABC transporters in body fluids using vesicular transport assays

    FASEB J. 2012 Feb;26(2):738-47. Epub 2011 Oct 27

    Krumpochova P, Sapthu S, Brouwers J, De Haas M, De Vos R, Borst P, Van de Wetering K.

    link to PubMed
  • Glucosylated hydroxymethyluracil (DNA base J) prevents transcriptional read-through in Leishmania.

    Cell, 2012 Aug 31;150(5):909-21

    Van Luenen H, Farris C, Jan S, Genest PA, Tripathi P, Velds A, Kerkhoven RM, Nieuwland M, Haydock A, Ramasamy G, Vainio S, Heidebrecht et al.

    link to PubMed
 
 

Recent publications View All Publications

  • N-lactoyl-amino acids are ubiquitous metabolites that originate from CNDP2-mediated reverse proteolysis of lactate and amino acids

    Proc Natl Acad Sci U S A. 2015 May 26;112(21):6601-6

    Jansen RS, Addie R, Merkx R, Fish A, Mahakena S, Bleijerveld OB, Altelaar M4 IJlst L, Wanders RJ, Borst P, van de Wetering K

    link to PubMed
  • REV7 counteracts DNA double-strand break resection and affects PARP inhibition

    Nature. 2015 May 28;521(7553):541-4

    Xu G, Chapman JR, Brandsma I, Yuan J, Mistrik M, Bouwman P, Bartkova J, Gogola E, Warmerdam D, Barazas M, Jaspers JE, Watanabe K, Pieterse...

    link to PubMed
 

Contact

  • E-mail

    a.laan@nki.nl

  • Telephone Number

    +31 20 512 2015

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