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Concentrative nucleoside transporter 1 (hCNT1) promotes phenotypic changes relevant to tumor biology in a translocation-independent manner.

Pérez-Torras S, Vidal-Pla A, Cano-Soldado P, Huber-Ruano I, Mazo A, Pastor-Anglada M - Cell Death Dis (2013)

Bottom Line: We found that hCNT1 restoration in pancreatic cancer cells significantly altered cell-cycle progression and phosphorylation status of key signal-transducing kinases, promoted poly-(ADP-ribose) polymerase hyperactivation and cell death and reduced cell migration.Importantly, the translocation-defective transporter triggered these same effects on cell physiology.Moreover, this study also shows that restoration of hCNT1 expression is able to reduce tumor growth in a mouse model of pancreatic adenocarcinoma.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Barcelona and National Biomedical Research Institute of Liver and Gastrointestinal Diseases (CIBERehd), Barcelona, Spain. s.perez-torras@ub.edu

ABSTRACT
Nucleoside transporters (NTs) mediate the uptake of nucleosides and nucleobases across the plasma membrane, mostly for salvage purposes. The canonical NTs belong to two gene families, SLC29 and SLC28. The former encode equilibrative nucleoside transporter proteins (ENTs), which mediate the facilitative diffusion of natural nucleosides with broad selectivity, whereas the latter encode concentrative nucleoside transporters (CNTs), which are sodium-coupled and show high affinity for substrates with variable selectivity. These proteins are expressed in most cell types, exhibiting apparent functional redundancy. This might indicate that CNTs have specific roles in the physiology of the cell beyond nucleoside salvage. Here, we addressed this possibility using adenoviral vectors to restore tumor cell expression of hCNT1 or a polymorphic variant (hCNT1S546P) lacking nucleoside translocation ability. We found that hCNT1 restoration in pancreatic cancer cells significantly altered cell-cycle progression and phosphorylation status of key signal-transducing kinases, promoted poly-(ADP-ribose) polymerase hyperactivation and cell death and reduced cell migration. Importantly, the translocation-defective transporter triggered these same effects on cell physiology. Moreover, this study also shows that restoration of hCNT1 expression is able to reduce tumor growth in a mouse model of pancreatic adenocarcinoma. These data predict a novel role for a NT protein, hCNT1, which appears to be independent of its role as mediator of nucleoside uptake by cells. Thereby, hCNT1 fits the profile of a transceptor in a substrate translocation-independent manner and is likely to be relevant to tumor biology.

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Functional characterization of AdhCNT1. NP-9 and NP-29 cells were infected with AdhCNT1 or Adctrol at different MOIs, and all parameters were analysed 48 h post-infection. (a) hCNT1 sodium-dependent uptake of [3H]cytidine, calculated as uptake in NaCl medium minus uptake in choline chloride, measured in cells transduced with Adctrol (black bars) or AdhCNT1 (gray bars). Results are expressed as means±S.E.M. and correspond to a representative experiment (n=3). (b) Relative amounts of hCNT1 mRNA determined by RT-PCR. The graphics correspond to a representative experiment (n=3), and the error bars indicate the range of possible values define by the S.E.M. of the delta threshold cycles. (c) Western blot analysis of hCNT1 expression
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fig1: Functional characterization of AdhCNT1. NP-9 and NP-29 cells were infected with AdhCNT1 or Adctrol at different MOIs, and all parameters were analysed 48 h post-infection. (a) hCNT1 sodium-dependent uptake of [3H]cytidine, calculated as uptake in NaCl medium minus uptake in choline chloride, measured in cells transduced with Adctrol (black bars) or AdhCNT1 (gray bars). Results are expressed as means±S.E.M. and correspond to a representative experiment (n=3). (b) Relative amounts of hCNT1 mRNA determined by RT-PCR. The graphics correspond to a representative experiment (n=3), and the error bars indicate the range of possible values define by the S.E.M. of the delta threshold cycles. (c) Western blot analysis of hCNT1 expression

Mentions: The adenoviral vector AdhCNT1 was generated as a tool for overexpressing hCNT1 in a wide variety of cell lines. In order to examine its efficacy in inducing hCNT1-related function, we infected a panel of tumor cell lines with AdhCNT1 and the control adenovirus, Adctrol, at different multiplicities of infection (MOIs) and determined sodium-dependent cytidine uptake 48 h after infection. At the tested MOIs, cytidine uptake increased in a dose-dependent manner, whereas no hCNT1-related activity was observed after Adctrol infection of NP-9 cells, although NP-29 cells, when transduced with the empty vector did show some minimal Na+-coupled cytidine uptake (Figure 1a and Supplementary Figure S1). The pancreatic adenocarcinoma cell lines, NP-9 and NP-29, were chosen for further characterization of the effects of hCNT1 overexpression on several aspects of tumor cell biology. In both cell lines, infection with hCNT1 induced a dose-dependent increase in cytidine uptake (Figure 1a). hCNT1 mRNA, determined by quantitative reverse transcription-PCR (RT-PCR), was similarly increased in both cell lines in a dose-dependent manner 48 h after cDNA transduction (Figure 1b), resulting in a corresponding increase in the total amount of hCNT1 protein (Figure 1c). Under these conditions, no relevant changes in the mRNA levels of the endogenously expressed NTs, hENT1 and hENT2, were observed (Supplementary Figure S2).


Concentrative nucleoside transporter 1 (hCNT1) promotes phenotypic changes relevant to tumor biology in a translocation-independent manner.

Pérez-Torras S, Vidal-Pla A, Cano-Soldado P, Huber-Ruano I, Mazo A, Pastor-Anglada M - Cell Death Dis (2013)

Functional characterization of AdhCNT1. NP-9 and NP-29 cells were infected with AdhCNT1 or Adctrol at different MOIs, and all parameters were analysed 48 h post-infection. (a) hCNT1 sodium-dependent uptake of [3H]cytidine, calculated as uptake in NaCl medium minus uptake in choline chloride, measured in cells transduced with Adctrol (black bars) or AdhCNT1 (gray bars). Results are expressed as means±S.E.M. and correspond to a representative experiment (n=3). (b) Relative amounts of hCNT1 mRNA determined by RT-PCR. The graphics correspond to a representative experiment (n=3), and the error bars indicate the range of possible values define by the S.E.M. of the delta threshold cycles. (c) Western blot analysis of hCNT1 expression
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3674379&req=5

fig1: Functional characterization of AdhCNT1. NP-9 and NP-29 cells were infected with AdhCNT1 or Adctrol at different MOIs, and all parameters were analysed 48 h post-infection. (a) hCNT1 sodium-dependent uptake of [3H]cytidine, calculated as uptake in NaCl medium minus uptake in choline chloride, measured in cells transduced with Adctrol (black bars) or AdhCNT1 (gray bars). Results are expressed as means±S.E.M. and correspond to a representative experiment (n=3). (b) Relative amounts of hCNT1 mRNA determined by RT-PCR. The graphics correspond to a representative experiment (n=3), and the error bars indicate the range of possible values define by the S.E.M. of the delta threshold cycles. (c) Western blot analysis of hCNT1 expression
Mentions: The adenoviral vector AdhCNT1 was generated as a tool for overexpressing hCNT1 in a wide variety of cell lines. In order to examine its efficacy in inducing hCNT1-related function, we infected a panel of tumor cell lines with AdhCNT1 and the control adenovirus, Adctrol, at different multiplicities of infection (MOIs) and determined sodium-dependent cytidine uptake 48 h after infection. At the tested MOIs, cytidine uptake increased in a dose-dependent manner, whereas no hCNT1-related activity was observed after Adctrol infection of NP-9 cells, although NP-29 cells, when transduced with the empty vector did show some minimal Na+-coupled cytidine uptake (Figure 1a and Supplementary Figure S1). The pancreatic adenocarcinoma cell lines, NP-9 and NP-29, were chosen for further characterization of the effects of hCNT1 overexpression on several aspects of tumor cell biology. In both cell lines, infection with hCNT1 induced a dose-dependent increase in cytidine uptake (Figure 1a). hCNT1 mRNA, determined by quantitative reverse transcription-PCR (RT-PCR), was similarly increased in both cell lines in a dose-dependent manner 48 h after cDNA transduction (Figure 1b), resulting in a corresponding increase in the total amount of hCNT1 protein (Figure 1c). Under these conditions, no relevant changes in the mRNA levels of the endogenously expressed NTs, hENT1 and hENT2, were observed (Supplementary Figure S2).

Bottom Line: We found that hCNT1 restoration in pancreatic cancer cells significantly altered cell-cycle progression and phosphorylation status of key signal-transducing kinases, promoted poly-(ADP-ribose) polymerase hyperactivation and cell death and reduced cell migration.Importantly, the translocation-defective transporter triggered these same effects on cell physiology.Moreover, this study also shows that restoration of hCNT1 expression is able to reduce tumor growth in a mouse model of pancreatic adenocarcinoma.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Barcelona and National Biomedical Research Institute of Liver and Gastrointestinal Diseases (CIBERehd), Barcelona, Spain. s.perez-torras@ub.edu

ABSTRACT
Nucleoside transporters (NTs) mediate the uptake of nucleosides and nucleobases across the plasma membrane, mostly for salvage purposes. The canonical NTs belong to two gene families, SLC29 and SLC28. The former encode equilibrative nucleoside transporter proteins (ENTs), which mediate the facilitative diffusion of natural nucleosides with broad selectivity, whereas the latter encode concentrative nucleoside transporters (CNTs), which are sodium-coupled and show high affinity for substrates with variable selectivity. These proteins are expressed in most cell types, exhibiting apparent functional redundancy. This might indicate that CNTs have specific roles in the physiology of the cell beyond nucleoside salvage. Here, we addressed this possibility using adenoviral vectors to restore tumor cell expression of hCNT1 or a polymorphic variant (hCNT1S546P) lacking nucleoside translocation ability. We found that hCNT1 restoration in pancreatic cancer cells significantly altered cell-cycle progression and phosphorylation status of key signal-transducing kinases, promoted poly-(ADP-ribose) polymerase hyperactivation and cell death and reduced cell migration. Importantly, the translocation-defective transporter triggered these same effects on cell physiology. Moreover, this study also shows that restoration of hCNT1 expression is able to reduce tumor growth in a mouse model of pancreatic adenocarcinoma. These data predict a novel role for a NT protein, hCNT1, which appears to be independent of its role as mediator of nucleoside uptake by cells. Thereby, hCNT1 fits the profile of a transceptor in a substrate translocation-independent manner and is likely to be relevant to tumor biology.

Show MeSH
Related in: MedlinePlus