Piet Borst, Ph.D. professor, staff member and director emeritus
About Piet Borst
Experimental work in the Borst lab
The experimental work in my lab ended in 2015. Since the ongoing
projects were rather successful, they are being continued by former
collaborators. I remain involved as adviser and, on the side, I
remain active as reviewer, mentor, and lecturer on bio-ethics.
Mechanisms of anti-cancer drug resistance in mouse
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 was
started in my lab by post-doc Sven Rottenberg and is continued by
him (as PI) in Bern (Switzerland).
Recent developments in this project have led to two
- The Volume-Regulated Anion Channel (VRAC) acts as an entry port
of cisplatin and carboplatin into the cell. (In collaboration with
Thomas Jentsch, Berlin; and Thijn Brummelkamp, NKI). Loss of
functional VRAC leads to modest platinum-drug resistance.
- Continuing work on resistance to PARP inhibitors, led to the
discovery that loss of PAR glycohydrolase (PARG) can
cause PARG resistance. This mechanism can also occur in cells
deficient in BRCA2, which are unable to (partially) restore
homologous recombination, a resistance mechanism prevalent in
cells deficient in BRCA1.
Physiological functions of ABC
We are interested in mechanisms of drug resistance in cancer cells
and have focused 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 of these transporters in metabolism and defense of the
body against drugs and xenotoxins , 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 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 MRP2 (ABCC2), MRP3
(ABCC3) and BCRP (ABCG2). More recently we have studied MRP5 and
MRP6 by this approach.
In 2000 we generated a mouse KO of the Mrp5 gene.
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 have therefore reinvestigated
MRP5 and the Mrp5 KO mice using metabolomics. Two new classes of
substrates were identified: glutamate-conjugates, some of
which are 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.
Using a concerted metabolomics approach Robert Jansen and Koen
van de Wetering have shown that X is ATP. ATP is rapidly converted
in the circulation into pyrophosphate (PPi) and AMP. PPi is a known
inhibitor of tissue calcification and indeed PXE patients have only
about 40% of normal PPi levels in their plasma. This discovery has
inspired new treatment attempts for PXE. Our clinical colleagues in
Utrecht have tested bisphosphonates with modestly positive results.
As an independent PI in Philadelphia, Koen van de Wetering has
contributed to a study by our long-time collaborator Andras Varadi
(Budapest) showing that large doses of oral PPi can counteract
ectopic calcification in Abcc6 KO mice. Varadi has shown that oral
PPi is also taken up in humans and trials with oral PPi in PXE
patients are in progress.
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 (Cell 1993; 75:
1129-1136), is a base present in kinetoplastid flagellates and
Euglena. 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 in
Leishmania for the proper termination of transcription. Loss of J
results in massive read-through of transcriptional stops and in
death of the parasite (Cell 2012; 150: 909-921). This project was
discontinued in Amsterdam in 2012 and transferred to Peter Myler in
I remain involved as advisor in the structural work by
Anastassis Perrakis (NKI) on the enzymes involved in J
biosynthesis, J-binding proteins 1 and 2 (JBP1 and 2). We have
shown in recent years that JBP1 binds with high affinity to J in
DNA and that it can then hydroxylate a T 13 bp downstream in the
other strand. By a variety of structural approaches Perrakis is
trying to elucidate the structural features of JBP1 that allow this