Worm Breeder's Gazette 14(1): 74 (October 1, 1995)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

A multidrug resistance associated protein (MRP) confers resistance of C. elegans to heavy metals.

A. Broeks1, B. Gerrard.2, M. Dean2, R.H.A. Plasterk1

1 Division of Molecular Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
2 National Cancer Institute, Frederick Cancer Res. Dev. Fac., Frederick, MD 21702, USA.

Upon exposure to natural toxins tumor cells can become resistant to
structurally and functionally unrelated drugs (multidrug resistance or
MDR). The classical form of MDR is caused by a P-glycoprotein (P-gp)
encoded by the human MDR1 gene. Recently it has been found that MDR can
also be conferred by the Multidrug resistance associated protein (MRP),
a member of the ABC family of transporters. In C. elegans there is
besides the P-gp gene family also an MRP gene family. We investigate
whether the proteins encoded by these mrp genes are involved in the
protection of the nematode against natural occurring compounds.

The MRP gene has been first identified in a drug resistant small cell lung
cancer cell line. The protein is located in the plasma-membrane and
confers resistance by decreasing the intracellular drug concentration or
the intracellular distribution. The predicted protein consists of a
N-terminal half of eight transmembrane domains and an ATP-binding site
and a C-terminal half  of four transmembrane domains and an ATP binding
site. The MRP mRNA can be detected in all human tissues. The intracellular
distribution differs from that of the P- glycoproteins; P-glycoproteins
are predominantly expressed in the apical membranes of epithelial cells
whereas MRP is mainly found in intra-cellular vesicles.  Recent studies
suggest that also the mechanism of action of MRP differs from that of
P-gp. Mrp can also function as a transporter of glutathione conjugated
substrates. It is thought that MRP is involved in (acquired) resistance
against drugs beyond the classic MDR spectrum of drugs. The human MRP is
most related to the Leishmania tarentolae P-glycoprotein ltpgpA  and the
Yeast CF1 gene. Both can confer resistance to a number of heavy metal
ions. So far there are no clues for the natural physiological function of
the MRP protein.

In C. elegans four mrp gene family members have been identified; mrp-1,
-2, -3 and  -4. mrp-1 was first identified as a EST clone from Ventner and
Waterston's sequencing effort. The other three genes were identified as
cDNA clones from Y. Kohara's sequencing project. We started to analyze
mrp-1 and mrp-2. Both genes are localized on the X-chromosome next to
each other, the nucleotide sequence determination has almost been
completed. The predicted protein sequence of both genes resembles that
of the human MRP; they are both 71% similar at the amino acid level.
Using RT-PCR we found that mrp-1 in expressed in all developmental
stages. The highest expression was found in early larval stages. To
investigate the gene's function we isolated a Tc1 allele and a deletion
allele of mrp-1. In the mrp-1 deletion mutant 3 kb genomic DNA has been
deleted containing several transmembrane domains and the first
ATP-binding site. The mrp-1 knock-out was found to be sensitive to heavy
metals like cadmium and arsenite then wild-type animals (see fig 1).