Altered localisation of the copper efflux transporters ATP7A and ATP7B associated with cisplatin resistance in human ovarian carcinoma cells.
BACKGROUND
Copper homeostasis proteins ATP7A and ATP7B are assumed to be involved in the intracellular transport of cisplatin. The aim of the present study was to assess the relevance of sub cellular localisation of these transporters for acquired cisplatin resistance in vitro. For this purpose, localisation of ATP7A and ATP7B in A2780 human ovarian carcinoma cells and their cisplatin-resistant variant, A2780cis, was investigated.


METHODS
Sub cellular localisation of ATP7A and ATP7B in sensitive and resistant cells was investigated using confocal fluorescence microscopy after immunohistochemical staining. Co-localisation experiments with a cisplatin analogue modified with a carboxyfluorescein-diacetate residue were performed. Cytotoxicity of the fluorescent cisplatin analogue in A2780 and A2780cis cells was determined using an MTT-based assay. The significance of differences was analysed using Student's t test or Mann-Whitney test as appropriate, p values of < 0.05 were considered significant.


RESULTS
In the sensitive cells, both transporters are mainly localised in the trans-Golgi network, whereas they are sequestrated in more peripherally located vesicles in the resistant cells. Altered localisation of ATP7A and ATP7B in A2780cis cells is likely to be a consequence of major abnormalities in intracellular protein trafficking related to a reduced lysosomal compartment in this cell line. Changes in sub cellular localisation of ATP7A and ATP7B may facilitate sequestration of cisplatin in the vesicular structures of A2780cis cells, which may prevent drug binding to genomic DNA and thereby contribute to cisplatin resistance.


CONCLUSIONS
Our results indicate that alterations in sub cellular localisation of transport proteins may contribute to cisplatin resistance in vitro. Investigation of intracellular protein localisation in primary tumour cell cultures and tumour tissues may help to develop markers of clinically relevant cisplatin resistance. Detection of resistant tumours in patients may in turn enable individualization of the chemotherapy in the early stage of treatment.
Department of Clinical Pharmacy, Institute of Pharmacy, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany. akalayda@uni-bonn.de
Ganna V
GV
Kalayda
Christina H
CH
Wagner
Irina
I
Buss
Jan
J
Reedijk
Ulrich
U
Jaehde
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000493:pharmacokinetics / Q000494:pharmacology; D027682:Cation Transport Proteins / Q000378:metabolism*; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000493:pharmacokinetics / Q000494:pharmacology*; D003300:Copper / Q000378:metabolism*; D000073840:Copper-Transporting ATPases; D019008:Drug Resistance, Neoplasm; D011992:Endosomes / Q000378:metabolism; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D008247:Lysosomes / Q000378:metabolism; D018942:Macrolides / Q000494:pharmacology; D018613:Microscopy, Confocal; D010051:Ovarian Neoplasms / Q000188:drug therapy* / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism
D000970:Antineoplastic Agents; D027682:Cation Transport Proteins; D018942:Macrolides; C057620:bafilomycin A; D003300:Copper; D000251:Adenosine Triphosphatases; C081184:ATP7A protein, human; C000612464:ATP7B protein, human; D000073840:Copper-Transporting ATPases; D002945:Cisplatin