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Nucleoside Transporter Profiles in Human Pancreatic Cancer Cells

Role of hCNT1 in 2',2'-Difluorodeoxycytidine- Induced Cytotoxicity

Departament de Bioquímica i Biologia Molecular Universitat de Barcelona, Barcelona, E-08071 Spain

Purpose: Concentrative nucleoside transporter (CNT) 1, CNT3, equilibrative nucleoside transporter (ENT) 1, and, to a lesser extent, ENT2, appear to be the transporters responsible for 2',2'-difluorodeoxycytidine (gemcitabine; Gemzar) uptake into cells. Gemcitabine is used currently in the treatment of pancreatic cancer, but the role of specific nucleoside carrier proteins in gemcitabine cytotoxicity has not been elucidated. Indeed, it is not known which nucleoside transporters are expressed in human pancreas.

Experimental Design: In this study we have used four cell lines [pancreatic neoplasia (NP)9, NP18, NP29, and NP31] derived from human pancreatic adenocarcinomas to monitor the pattern of nucleoside transporter expression, and we have heterologously expressed the high-affinity gemcitabine transporter human orthologue (h) CNT1 to monitor its role in drug responsiveness.

Results: All of the cell lines take up gemcitabine mostly via the hENT1 transporter, which is expressed at high levels. Reverse transcription-PCR analysis of the other four cloned plasma membrane transporter mRNAs revealed very different expression patterns among NP cell lines, with apparent selective loss or decrease of hCNT mRNAs. NP cells transiently express hCNT1-type Na⁺-dependent nucleoside transport activity at low/medium cell density but not in confluent cultures. Cells expressing hCNT1 in a more constitutive manner were cloned after stable transfection of hCNT1. Despite high constitutive hENT1 activity, this increased sensitivity to gemcitabine.

Conclusion: In summary, human pancreatic adenocarcinoma cells overexpress hENT1, although they retain the ability to express a functional hCNT1 transporter, an isoform that confers sensitivity to gemcitabine.