Calpain has long been implicated in the regulation of cell cycle, mostly based on studies with inhibitors that lack strict specificity toward the enzyme. Further, previous work has primarily focused on one particular point, the G1 checkpoint, and made no attempt at dissecting the full cycle in terms of calpain action. To extend and complement these findings, we tested the effect of a specific inhibitor, PD 150606, on granulocyte-macrophage-colony stimulating factor (GM-CSF)-stimulated human TF-1 cells by flow cytometry following single- and double labelling by propidium iodide and bromodeoxyuridine. Using a new algorithm of analysis, we determined the time-dependence of the absolute number of cells leaving G1, S and G2M phases following the application of the inhibitor. Our results point to the simultaneous involvement of calpain activity in promoting the cycle at the G1 checkpoint and somewhere in the G2M compartment. Furthermore, the inhibitor significantly impedes the progress of cells through the S phase, indicating calpain activity in S phase checkpoint signalling. Overall, our analysis suggests that calpain regulates the cell cycle at more points than previously thought.
- Cyclin-dependent kinase
- Dimethyl sulfoxide
- Fluorescence-activated cell sorting
- Granulocyte-macrophage-colony stimulating factor
- Normal goat serum
- Phosphate-buffered saline
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