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Amiloride added together with bumetanide completely blocks mouse 3T3-cell exit from G0/G1-phase and entry into
Abstract:
In this study we tested the hypothesis that stimulation of univalent-cation fluxes which follow the addition of growth factors are required for cell transition through the G1-phase of the cell cycle. The effect of two drugs, amiloride and bumetanide, were tested on exit of BALB/c 3T3 cells from G0/G1-phase and entry into S-phase (DNA synthesis). Amiloride, an inhibitor of the Na+/H+ antiport, only partially inhibited DNA synthesis induced by serum. Bumetanide, an inhibitor of the Na+/K+ co-transport, only slightly suppressed DNA synthesis by itself, but when added together with amiloride completely blocked cell transition through G1 and entry into S-phase. Similar inhibitory effects of the two drugs were found on the induction of ornithine decarboxylase (ODC) (a marker of mid-G1-phase) in synchronized cells stimulated by either partially purified fibroblast growth factor (FGF) or serum. To test this hypothesis further, cells arrested in G0/G1 were stimulated by serum, insulin or FGF. All induced similar elevations of cellular K+ content during the early G1-phase of the cell cycle. However, serum and FGF, but not insulin, released the cells from the G0/G1 arrest, as measured by ODC enzyme induction. This result implies that the increase in cellular K+ content may be necessary but not sufficient for induction of early events during the G1-phase. The synergistic inhibitory effects of amiloride and bumetanide on the two activities stimulated by serum growth factors, namely ODC induction (mid-G1) and thymidine incorporation into DNA (S-phase), suggested that the amiloride-sensitive Na+/H+ antiport system together with the bumetanide-sensitive Na+/K+ transporter play a role in the mitogenic signal.
Insights
Cell cycle progression requires ion flux. Amiloride and bumetanide drugs blocked cell transition through G1 and S-phase, indicating ion transporters are crucial for growth factor signaling.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Cell cycle progression is regulated by growth factors.
- Univalent-cation fluxes are implicated in cell cycle transitions.
Purpose of the Study:
- To investigate the role of univalent-cation fluxes in G1-phase cell cycle progression.
- To determine if Na+/H+ antiport and Na+/K+ co-transport are required for growth factor-induced cell cycle entry.
Main Methods:
- BALB/c 3T3 cells were synchronized in G0/G1 phase.
- Cells were treated with amiloride (Na+/H+ inhibitor) and bumetanide (Na+/K+ inhibitor).
- DNA synthesis and ornithine decarboxylase (ODC) induction were measured as markers of cell cycle progression.
Main Results:
- Amiloride partially inhibited serum-induced DNA synthesis.
- Bumetanide alone had minimal effect, but combined with amiloride, it completely blocked G1 to S-phase transition.
- Both drugs inhibited ODC induction by fibroblast growth factor (FGF) and serum.
- Increased cellular K+ content was observed with serum, insulin, and FGF, but only serum and FGF induced ODC, suggesting K+ increase is necessary but not sufficient.
Conclusions:
- The Na+/H+ antiport and Na+/K+ transporter play a synergistic role in mitogenic signaling.
- These ion transport systems are essential for cell transition through G1 and entry into S-phase.
- While increased intracellular K+ is necessary, it is not sufficient for initiating early G1 events.