Related Experiment Videos
Thapsigargin-resistant intracellular calcium pumps. Role in calcium pool function and growth of
R T Waldron1, A D Short, D L Gill
1Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore 21201, USA.
Abstract:
Exposure of cells to the intracellular Ca2+ pump blocker, thapsigargin (TG), results in emptying of Ca2+ pools and termination of cell proliferation (Short, A. D., Bian, J., Ghosh, T. K., Waldron, R. T., Rybak, S. L., and Gill, D. L. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 4986-4990). DC-3F Chinese hamster lung cells were made resistant to TG by long-term stepwise exposure to increasing TG concentrations in culture (Gutheil, J. C., Hart, S. R., Belani, C. P., Melera, P. W., and Hussain, A. (1994) J. Biol. Chem. 269, 7976-7981). Since these cells (DC-3F/TG2) grow in the presence of TG, it was important to ascertain what Ca2+ pool function they retain. TG-resistant DC-3F/TG2 cells cultured with 2 microM TG had a doubling time (24 h) not significantly different from the parent DC-3F cells without TG. Analysis of TG-induced inhibition of 45Ca2+ uptake into permeabilized parent DC-3F cells revealed two distinct Ca2+ pump activities with 20,000-fold different sensitivities to TG; the IC50 values for TG were 200 pM and 4 microM, representing 80% and 20% of total pumping activity, respectively. Total pump activity in parent DC-3F and resistant DC-3F/TG2 cells was similar (0.23 +/- 0.10 and 0.18 +/- 0.08 nmol of Ca2+/10(6) cells, respectively). In DC-3F/TG2 cells, up to 100 nM TG had no effect on Ca2+ pumping; however, almost all pumping was blocked at higher TG concentrations with an IC50 of 5 microM. In both cell types, each Ca2+ pump activity (regardless of TG sensitivity) had high Ca2+ affinity (Km values congruent to 0.1 microM) and similar ATP dependence and vanadate sensitivity. In DC-3F cells, the TG-sensitive Ca2+ pool was releasable with inositol 1,4,5-trisphosphate (InsP3) or GTP and was oxalate-permeable; the TG-insensitive pool in these cells was not InsP3-releasable. GTP-induced Ca2+ uptake in the presence of oxalate indicated Ca2+ transfer between distinct pools in the DC-3F cells. In resistant DC-3F/TG2 cells, almost 50% of total TG-insensitive Ca2+ accumulation was releasable with InsP3; unlike the parent cells, this pool was not oxalate-permeable, and GTP induced no Ca2+ transfer between pools in the presence of oxalate. Thus, whereas InsP3 releases Ca2+ only from the high TG sensitivity Ca2+ pumping pool in parent DC-3F cells, in resistant DC-3F/TG2 cells the TG-resistant Ca2+ pumping pool now contains functional InsP3 receptors.(ABSTRACT TRUNCATED AT 400 WORDS)
Insights
Thapsigargin-resistant cells retain calcium (Ca2+) pool function, with InsP3 receptors now present in the TG-resistant Ca2+ pumping pool. This study investigates Ca2+ pump activity and InsP3-mediated release in resistant cells.
Area of Science:
- Cell Biology
- Molecular Pharmacology
Background:
- Thapsigargin (TG) blocks intracellular Ca2+ pumps, emptying Ca2+ pools and halting cell proliferation.
- TG-resistant DC-3F/TG2 cells were developed to investigate retained Ca2+ pool functions.
- Understanding Ca2+ handling in resistant cells is crucial for cell proliferation studies.
Purpose of the Study:
- To determine the Ca2+ pool function retained by TG-resistant DC-3F/TG2 cells.
- To analyze the characteristics of Ca2+ pump activities and their sensitivities to TG.
- To investigate the role of InsP3 and GTP in Ca2+ release and transfer in both parent and resistant cells.
Main Methods:
- Development of TG-resistant DC-3F/TG2 cells through stepwise TG exposure.
- Analysis of 45Ca2+ uptake inhibition by TG in permeabilized cells to determine IC50 values.
- Assessment of Ca2+ pool releasability using inositol 1,4,5-trisphosphate (InsP3) and GTP, with and without oxalate.
Main Results:
- Two distinct Ca2+ pump activities with differing TG sensitivities were identified in parent DC-3F cells.
- DC-3F/TG2 cells exhibited resistance to TG, with altered InsP3-mediated Ca2+ release from the TG-insensitive pool.
- GTP-induced Ca2+ transfer between pools was observed in parent cells but not in resistant cells.
Conclusions:
- TG-resistant cells maintain Ca2+ pump activity, but the regulation of Ca2+ pools is altered.
- In resistant cells, the TG-insensitive Ca2+ pool gains InsP3 responsiveness.
- These findings highlight the adaptive mechanisms of Ca2+ handling in drug-resistant cells.