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Modulation of fibroblast response to maitotoxin along the cell division cycle
J Berreur-Bonnenfant1, M Ammar, A Dubreuil
1ECMR, CNRS URA 1449, Université P.M. Curie, Paris, France.
Abstract:
Maitotoxin (MTX) induces an increase of [Ca2+]i and of phosphoinositide breakdown in various cell types. The [Ca2+]i increase followed with fluorescent probes on cell suspensions has been described as slow and lasting, in contrast to the "signal" induced by calcium ionophores such as ionomycin. MTX effects have been studied on two fibroblastic cell lines, BHK21 C13 and FR 3T3, synchronized by serum deprivation treatment performed in an isoleucine-free medium for BHK21 C13 cells. In BHK21 C13 cells, flow cytometry analysis showed that two stages, G1/S and G2/M, were particularly susceptible to MTX treatment. Scanning laser cytometry demonstrated that calcium response of FR 3T3 fibroblasts followed with Indo-1 varied during the cell division cycle. The [Ca2+]i increase was almost always vertical, but its delay after MTX addition lasted from zero (S and G2/M transition) to 10-20 min (G1) or more (G2). No [Ca2+]i change could be detected during mitosis. The [Ca2+]i response at the S phase was biphasic. These observations suggest that (1) the lasting response described in the literature represents a global cell population effect, and (2) cells are more sensitive to MTX at specific stages of the cell division cycle, which could correspond to periods when calcium signals have been detected in different cell types.
Insights
Maitotoxin (MTX) triggers calcium increases in cells, with responses varying by cell cycle stage. This suggests MTX sensitivity is cell-cycle dependent, impacting calcium signaling research.
Area of Science:
- Cell Biology
- Toxicology
- Biochemistry
Background:
- Maitotoxin (MTX) is known to elevate intracellular calcium ([Ca2+]i) and induce phosphoinositide breakdown in diverse cell types.
- MTX-induced [Ca2+]i increases are characterized as slow and sustained, differing from rapid signals evoked by calcium ionophores like ionomycin.
Purpose of the Study:
- To investigate the impact of Maitotoxin (MTX) on intracellular calcium ([Ca2+]i) dynamics in synchronized fibroblastic cell lines.
- To determine if MTX sensitivity and calcium response vary across different phases of the cell division cycle.
Main Methods:
- Synchronization of BHK21 C13 and FR 3T3 fibroblasts using serum deprivation in isoleucine-free medium.
- Flow cytometry and scanning laser cytometry with Indo-1 to analyze intracellular calcium ([Ca2+]i) changes in response to MTX.
- Assessment of MTX effects on cell cycle progression, specifically at G1/S and G2/M transitions.
Main Results:
- BHK21 C13 cells showed heightened susceptibility to MTX at the G1/S and G2/M stages of the cell cycle.
- In FR 3T3 cells, the delay in [Ca2+]i increase after MTX addition varied significantly with cell cycle phase (0 min in S/G2/M transition, 10-20 min in G1).
- No significant [Ca2+]i changes were observed during mitosis; the S phase exhibited a biphasic calcium response.
Conclusions:
- The observed sustained calcium responses to MTX in cell populations likely represent an averaged effect across different cell cycle stages.
- Cells exhibit distinct sensitivities to MTX at specific points in the cell division cycle, correlating with periods of endogenous calcium signaling.