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.
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.
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
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