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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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hCNT1 overexpression alters cell morphology and cell-cycle profile. All parameters were analysed in cells infected with Adctrol or AdhCNT1. (a) Morphology of pancreatic tumor cells after infection with Adctrol or AdhCNT1 at different MOIs. Pictures were taken on day 3 (NP-9) or 4 (NP-29) after infection. (b) G1 (gray), S (dark gray) and G2 (black) phases were quantified by flow cytometric analysis of propidium iodide-stained cells 48 h after infection. The graphic corresponds to a representative experiment (n=3). (c) Western blot analysis of cyclin E expression 48 h after infection at different MOIs
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fig2: hCNT1 overexpression alters cell morphology and cell-cycle profile. All parameters were analysed in cells infected with Adctrol or AdhCNT1. (a) Morphology of pancreatic tumor cells after infection with Adctrol or AdhCNT1 at different MOIs. Pictures were taken on day 3 (NP-9) or 4 (NP-29) after infection. (b) G1 (gray), S (dark gray) and G2 (black) phases were quantified by flow cytometric analysis of propidium iodide-stained cells 48 h after infection. The graphic corresponds to a representative experiment (n=3). (c) Western blot analysis of cyclin E expression 48 h after infection at different MOIs

Mentions: Unexpectedly, AdhCNT1 infection altered cell morphology and viability in both NP-9 and NP-29 cell lines, changes that were dose- and time-dependent (Figure 2a) and consistent with the induction of cell death. Interestingly, these changes were observed at the lowest MOI used (Figure 2a). These unexpected changes prompted us to analyze the cell-cycle profile 48 h after hCNT1 transduction. In both cell lines, overexpression of hCNT1 led to a dose-dependent accumulation of cells in S phase (Figure 2b). At the highest dose (25 MOI), the number of cells in S phase increased to >70% above control values in both cell lines (Figure 2b). Under the same conditions, no significant changes in cell-cycle profiles were observed in cells infected with Adctrol. Moreover, in both cell lines, cell-cycle alterations correlated with an increase in cyclin E expression (Figure 2c), which typically reached a peak at the G1–S transition.


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)

hCNT1 overexpression alters cell morphology and cell-cycle profile. All parameters were analysed in cells infected with Adctrol or AdhCNT1. (a) Morphology of pancreatic tumor cells after infection with Adctrol or AdhCNT1 at different MOIs. Pictures were taken on day 3 (NP-9) or 4 (NP-29) after infection. (b) G1 (gray), S (dark gray) and G2 (black) phases were quantified by flow cytometric analysis of propidium iodide-stained cells 48 h after infection. The graphic corresponds to a representative experiment (n=3). (c) Western blot analysis of cyclin E expression 48 h after infection at different MOIs
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: hCNT1 overexpression alters cell morphology and cell-cycle profile. All parameters were analysed in cells infected with Adctrol or AdhCNT1. (a) Morphology of pancreatic tumor cells after infection with Adctrol or AdhCNT1 at different MOIs. Pictures were taken on day 3 (NP-9) or 4 (NP-29) after infection. (b) G1 (gray), S (dark gray) and G2 (black) phases were quantified by flow cytometric analysis of propidium iodide-stained cells 48 h after infection. The graphic corresponds to a representative experiment (n=3). (c) Western blot analysis of cyclin E expression 48 h after infection at different MOIs
Mentions: Unexpectedly, AdhCNT1 infection altered cell morphology and viability in both NP-9 and NP-29 cell lines, changes that were dose- and time-dependent (Figure 2a) and consistent with the induction of cell death. Interestingly, these changes were observed at the lowest MOI used (Figure 2a). These unexpected changes prompted us to analyze the cell-cycle profile 48 h after hCNT1 transduction. In both cell lines, overexpression of hCNT1 led to a dose-dependent accumulation of cells in S phase (Figure 2b). At the highest dose (25 MOI), the number of cells in S phase increased to >70% above control values in both cell lines (Figure 2b). Under the same conditions, no significant changes in cell-cycle profiles were observed in cells infected with Adctrol. Moreover, in both cell lines, cell-cycle alterations correlated with an increase in cyclin E expression (Figure 2c), which typically reached a peak at the G1–S transition.

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