Research interest
DNA base J
This project is an offshoot of our long-standing interest in the mechanisms of antigenic variation in African trypanosomes. b-glucosyl-hydroxymethyluracil (base J), 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 in Euglena. It replaces 1% of thymine in DNA and is predominantly located in repetitive sequences, such as telomeric repeats. We have characterized a J-binding protein (JBP1) that binds with high specificity to J-containing duplex DNA (Cross et al. EMBO J 1999; 18: 6573-6581). We have shown that JBP1 is a thymidine hydroxylase that catalyses the first step of J biosynthesis, the conversion of T in DNA into hydroxymethyluracil. JBP1 appears to belong to the family of Fe2+ and 2-oxoglutarate-requiring dioxygenases, as does a second putative hydroxylase, JBP2. In the kinetoplastid Leishmania, a JBP1 KO is lethal. In contrast, JBP2 is dispensable in Leishmania under normal growth conditions, but JBP2 KO strains are hypersensitive to bromodeoxyuridine (BrdU). During growth in BrdU, Leishmania loses its J, which is located for > 98% in telomeric repeats in this organism. How J loss leads to cell death is unclear. We do not find alterations in DNA integrity or cell cycle blocks. A. recent breakthrough came form the discovery by R. Sabatini (University of Georgia, US) that trypanosomes have J marking transcriptional stop sites. Using immuno-precipitation of J-DNA and deep sequencing, we have also found the 1% of non-telomeric DNA in Leishmania in specific chromosome-internal positions, partly at transcriptional stops. We have also found an interesting interaction between J and histone H3/H3V (H3 variant). We think that the tools are now on board to solve the elusive function of base J. We are also continuing attempts to set up assays for hydroxylase activity and for the putative glucosyl transferase catalyzing the second step in J biosynthesis. With Anastassis Perrakis (NKI-AVL) we are trying to determine the structure of JBP1-J-DNA complexes by crystallography; with Paul Wentworth (Scripps, California), we are looking for inhibitors of the binding of JBP1 to DNA and of its hydroxylase function.
MULTIDRUG RESISTANCE OF CANCER CELLS
We are interested in mechanisms of drug resistance in cancer cells and focus 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 are characterizing 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 family); most recently we have studied the Multidrug Resistance Protein (ABCC) family members MRP2, 3, 4, 5 and 6.
MRP3 (ABCC3) is an organic anion transporter contributing to the cellular export of endogenous or exogenous (toxic) compounds, conjugated to glutathione, sulphate or glucuronate. Using KO mice, we have shown that murine MRP3 is important for basolateral export of glucuronated compounds from the gut epithelium into blood. We have 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 MRP3 substrates by this approach and anticipate that it may also be helpful to find substrates of other MRPs and BCRP (ABCG2).
In collaboration with Sören Moestrup (University of Arhus, Denmark) we have shown that the ABC transporter MRP1 (ABCC1) is able to transport vitamin B12 (cobalamin) out of cells. Using our Mrp1 KO mice, we have shown that Mrp1 has a physiological role in cobalamine homeostasis.
MRP6 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. Recent studies by Uitto et al. on Abcc6-/- mice show that the absence of ABCC6 in the liver is crucial for PXE and confirm the “metabolic disease hypothesis” for PXE, which states that tissue calcification is due to the absence of a plasma factor secreted from the basolateral hepatocyte membrane. We have proposed that this plasma factor is vitamin K (precursor). We think that vitamin K (precursor) is secreted by ABCC6 from the liver as a glutathione-, glucuronide-, or sulphate conjugate and that this supplements the vitamin K need of peripheral tissues that receive insufficient vitamin from the diet, because dietary vitamin K is effectively extracted from blood by the liver. Peripheral tissue vitamin K is needed for the gamma-carboxylation of glutamate residues in proteins known to be required for counteracting calcification of connective tissue throughout the body.
We have started an ambitious program to test this hypothesis in collaboration with Arthur Bergen (AMC), who generated an Abcc6-/- mouse. We have made conjugates of menadione (Vit. K3) and are testing these in vesicular transport assays with MRP6-vesicles.
DRUG RESISTANCE IN “SPONTANEOUS” MOUSE TUMORS
In collaboration with Jos Jonkers (NKI-AVL), we are studying resistance mechanisms in “spontaneous” tumors arising in mice, conditionally defective in p53 and Brca1. These mice contain floxed alleles of these two genes and a Cre recombinase gene driven by a Keratin14 promotor, active only in epithelial cells, resulting in breast (and skin) cancer. When treated with the maximum tolerable dose of doxorubicin, docetaxel or topotecan, the breast tumors initially respond but eventually always develop resistance. Resistance is often associated with upregulation of the Mdr1a and Mdr1b genes (Abcb1), which encode drug-transporting P-glycoproteins and we have shown with specific inhibitors that remarkably low levels of Abcb1 upregulation (5-fold the levels in sensitive tumors) suffice to make the tumor multidrug resistant. We are also using this mouse model to test new anticancer drugs and drug combinations. Impressive tumor regression has been obtained with a new inhibition of Poly-ADP-ribose polymerase I (PARPI) in collaboration with KuDOS/AstraZeneca. We have crossed disrupted alleles for the Abcb1 and Abcg2 genes into our mouse model to further test the importance of these transporters in drug resistance and to uncover other forms of resistance not mediated by these transporters. Upregulation of Abcg2 is important for topotecan resistance in this model, but even in Abcg2-/- mice tumors eventually become resistant, often by downregulation of the topotecan target topoisomerase I.
In contrast to the results obtained with MDR drugs, we have been unable to obtain cisplatin resistance in this tumor model. The tumors respond to each new treatment with cisplatin, but are never fully eradicated. Although we have identified a tumor-initiating cell (“stem cell”) in this tumor model characterized by high surface expression of CD24 and CD49f, this fraction does not appear to be enriched in the “remnants” from which the tumors regrow after chemotherapy. We are testing the hypothesis that the resistance of “remnants” is not due to specific biochemical defense mechanisms of the putative tumor stem cells, but to the ability of a sub-fraction of the cells to go into “hibernation”, i.e. stop cell cycle progression until the drug is gone and the DNA damage has been repaired. We have succeeded in isolating tumor cell lines in low O2 that resemble the original tumor. These cultured cells have allowed us to study the fraction of cells surviving cisplatin. Remarkably, this fraction does not contain G2/M cells, but appears to stall in G1/G0. We are studying how these cells escape cisplatin-induced death and what needs to be done to kill them.
Key publications
Beedholm-Ebsen R, Van de Wetering K, Hardlei T, Nexo E, Borst P, Moestrup SK. Identification of multidrug resistance protein 1 (MRP1/ABCC1) as a molecular gate for cellular export of cobalamin. Blood 2009; 115:1632-39
Borst P, Van de Wetering K, Schlingemann R. Does the absence of ABCC6 (multidrug resistance protein 6) in patients with Pseudoxanthoma elasticum prevent the liver from providing sufficient vitamin K to the periphery? Cell Cycle 2008;7:1575-9
Borst P, Rottenberg S, Jonkers J. How do real tumors become resistant to cisplatin? Cell Cycle 2008;7:1353-9
Borst P, Sabatini R. Base J: discovery, biosynthesis and possible functions. Ann Rev Microbiol 2008;62:235-51
Pajic M, Iyer JK, Kersbergen A, Van der Burg E, Nygren AO, Jonkers J, Borst P, Rottenberg S. Moderate increase in Mdr1a/1b expression causes in vivo resistance to doxorubicin in a mouse model for hereditary breast cancer. Cancer Res 2009;69:6396-404
Rottenberg S, Kersbergen A, Van der Burg E, Nygren AOH, Zander S, Derksen PW, de Bruin M, Zevenhoven J, Lau A, Boulter R, Cranston A, O'Connor MJ, Martin NMB, Borst P, Jonkers J. High sensitivity of BRCA1-deficient mammary tumors to the PARP inhibitor AZD2281 alone and in combination with platinum drugs. Proc Natl Acad Sci U S A 2008;105:17079-84
Rottenberg S, Nygren AOH, Pajic M, Van Leeuwen FWB, Van der Heijden I, Van de Wetering K, Liu X, de Visser KE, Gilhuijs KG, Van Tellingen O, Schouten JP, Jonkers J, Borst P. Selective induction of chemotherapy resistance of mammary tumors in a conditional mouse model for hereditary breast cancer. Proc Natl Acad Sci U S A 2007;104:12117-22
Vainio S, Genest PA, ter Riet B, van Luenen H, Borst P. Evidence that J-binding protein 2 is a thymidine hydroxylase catalyzing the first step in the biosynthesis of DNA base J. Mol Biochem Parasitol 2009;164:157-61
Van de Wetering K, Burkon A, Feddema W, Bot A, de Jonge H, Somoza V, Borst P. Intestinal breast cancer resistance protein (BCRP)/Bcrp1 and multidrug resistance protein 3 (MRP3)/Mrp3 are involved in the pharmacokinetics of resveratrol. Mol Pharmacol 2009;75:876-85
Van de Wetering K, Feddema W, Helms JB, Brouwers JF, Borst P. Targeted metabolomics identifies glucuronides of dietary phytoestrogens as a major class of MRP3 substrates in vivo. Gastroenterol 2009;137:1725-35
Yu Z, Genest PA, ter Riet B, Sweeney K, DiPaolo C, Kieft R, Christodoulou E, Perrakis A, Simmons JM, Hausinger RP, Van Luenen HGAM, Rigden DJ, Sabatini R, Borst P. The protein that binds to DNA base J in trypanosomatids has features of a thymidine hydroxylase. Nucleic Acids Res 2007;35:2107-15
Zander S, Kersbergen A, Van der Burg E, De Water N, Van Tellingen O, Gunnarsdottir S, Jaspers J, Nygren AOH, Jonkers J, Borst P, Rottenberg S. Sensitivity and acquired resistance of BRCA1; -53-deficient mouse mammary tumors to the topoisomerase I inhibitor topotecan. Cancer Res 2010;70:1700-10
More publications by Piet Borst on PubMed
Biographic sketch
Piet Borst studied medicine in Amsterdam, where he obtained a PhD in biochemistry with Bill Slater, studying the properties of mitochondria in cancer cells. He then spent 2 years in New York University with Severo Ochoa, where he studied replication of RNA bacteriophages in E. coli with fellow-postdoc Charles Weissmann. He returned to Amsterdam to become Professor of Biochemistry and Molecular Biology in Amsterdam University, where he worked on the biogenesis of mitochondria and other cell organelles in a variety of organisms. In 1983 he became director of research of The Netherlands Cancer Institute, where he continued his research on mechanisms of antigenic variation in trypanosomes and started a new project on mechanisms of drug resistance in cancer cells. In 1999 he stepped down as director, but he continues his work in the cancer institute as fulltime staff member.
Piet Borst is a foreign associate of the National Academy of Sciences (US); a foreign honorary member of the American Academy of Arts and Sciences (US) and a foreign member of the Royal Society (GB). He is also a member of the Innovation Platform, a small thinktank chaired by the Dutch Prime Minister.
Co-workers
Henri Van Luenen PhD Academic staff
Charlotte Guyader PhD post-doc
Sven Rottenberg DVM PhD Dipl ECVP Senior post-doc
Pankaj Tripathi PhD post-doc
Koen Van de Wetering DVM PhD Senior post-doc
Nikola Banishki MSc PhD student
Petra Krumpochova MSc PhD student
Guotai Xu MSc PhD student
Serge Zander DVM MSc
Marcel De Haas Technician
Liesbeth Van Deemter Technician
Sabrina Jan Technician
Ariena Kersbergen Technician
Sunny Sapthu Technician
Wendy Sol Technician
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