Related Experiment Videos
Intercellular communication is reduced by TPA and Ki-ras p21 in quiescent, but not proliferating, NRK cells
A F Paulson1, R G Johnson, M M Atkinson
1Department of Genetics and Cell Biology, University of Minnesota, St. Paul 55108.
Abstract:
We investigated whether the growth state of NRK cells (proliferating or quiescent by serum deprivation) affected the ability of oncogenic Ki-ras p21 and the protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), to alter gap junctional communication. We evaluated gap junctional permeance by rate analysis of the transfer of a fluorescent dye, Lucifer Yellow, between cell pairs. We found that while the gap junctions of proliferating NRK cells were unresponsive to both TPA and to Ki-ras p21, junctional communication in quiescent cells was significantly inhibited by brief exposures to 100 ng/ml TPA. Furthermore, activity of Ki-ras p21 2 h prior to TPA exposure enhanced the inhibitory effect of TPA in quiescent cells. Junctional sensitivity to TPA was transient, with inhibition of junctional communication detected at 10 min and refractory after 60 min of continuous exposure. The suppression of junctional communication by TPA was completely prevented if the oncogenic p21 had been active for a longer period of time (48 h). The application of a phorbol ester derivative (4 alpha-PDD), which does not activate protein kinase C, did not affect the ability of quiescent cells to communicate. From these results we conclude that there is a cell-state dependence of junctional sensitivity to TPA in NRK cells and that ras p21 activity potentiates the junctional response to TPA. One interesting possibility is that this involved a cell-cycle effect.
Insights
Cell growth state impacts communication. Quiescent cells show inhibited gap junctional communication with 12-O-tetradecanoylphorbol-13-acetate (TPA), an effect potentiated by oncogenic Ki-ras p21 activity.
Area of Science:
- Cell Biology
- Molecular Oncology
Background:
- Gap junctional communication is vital for intercellular signaling.
- Oncogenic proteins like Ki-ras and signaling molecules such as TPA can modulate this communication.
- Cellular growth state may influence responses to these modulators.
Purpose of the Study:
- To determine if the growth state (proliferating vs. quiescent) of NRK cells affects their gap junctional communication in response to oncogenic Ki-ras p21 and TPA.
- To investigate the interaction between Ki-ras p21 activity and TPA in modulating gap junctional communication in different cell states.
Main Methods:
- NRK cells were induced into either proliferating or quiescent states via serum deprivation.
- Gap junctional communication was assessed by measuring Lucifer Yellow dye transfer between cell pairs.
- Cells were exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA) and/or activated oncogenic Ki-ras p21.
Main Results:
- Proliferating NRK cells showed no change in gap junctional communication with TPA or Ki-ras p21.
- Quiescent NRK cells exhibited significant inhibition of gap junctional communication upon brief TPA exposure.
- Pre-activation of Ki-ras p21 enhanced TPA's inhibitory effect in quiescent cells, but prolonged Ki-ras p21 activity (48h) prevented TPA-induced suppression.
- TPA-induced inhibition was transient, with communication returning to baseline after 60 minutes.
Conclusions:
- Gap junctional communication sensitivity to TPA is dependent on the cell's growth state in NRK cells.
- Oncogenic ras p21 activity potentiates the inhibitory response to TPA in quiescent cells, suggesting a role in cell-cycle regulation.
- These findings highlight the complex interplay between cellular state, oncogenic signaling, and intercellular communication.