Intramitochondrial calcium regulation by the FHIT gene product sensitizes to apoptosis

Abstract

Despite the growing interest in the Fhit tumor suppressor protein, frequently deleted in human cancers, the mechanism of its powerful proapoptotic activity has remained elusive. We here demonstrate that Fhit sensitizes the low-affinity Ca(2+) transporters of mitochondria, enhancing Ca(2+) uptake into the organelle both in intact and in permabilized cells, and potentiating the effect of apoptotic agents. This effect can be attributed to the fraction of Fhit sorted to mitochondria, as a fully mitochondrial Fhit (a chimeric protein including a mitochondrial targeting sequence) retains the Ca(2+) signaling properties of Fhit and the proapoptotic activity of the native protein (whereas the effects on the cell cycle are lost). Thus, the partial sorting of Fhit to mitochondria allows to finely tune the sensitivity of the organelle to the highly pleiomorphic Ca(2+) signals, synergizing with apoptotic challenges. This concept, and the identification of the molecular machinery, may provide ways to act on apoptotic cell death and its derangement in cancer.

Fig. 1.

Subcellular localization of Fhit and effect on Ca²⁺⁻homeostasis. (A) Immunofluorescence labeling of Fhit (Left), mtGFP visualization (Middle), and merged image (Right) in HeLa cells. (I) Control, (II) Fhit-overexpressing cells. (B) Western blotting of Fhit and β tubulin as reference. Arrowheads denote the position of endogenous (lower) and His-6-tagged (higher) Fhit bands in mock-transfected (Mock) and Fhit-overexpressing (Fhit) HeLa cells. The level of overexpression is typical of the results obtained in all of the experiments of this paper. (C) Immunofluorescence labeling of overexpressed Fhit after plasma membrane permeabilization with digitonin. (D and E) [Ca²⁺] measurement in the ER (Left), cytosol (Middle), and mitochondria (Right) in controls (gray line) and Fhit-overexpressing cells upon 100 μM (D) and 10 μM (E) histamine. These and the following figures are representative of >10 experiments, giving similar results.

Fig. 2.

Assessment of mitochondrial Ca²⁺-uptake capacity in permeabilized and intact cells. (A) Mitochondrial Ca²⁺-uptake in permeabilized cells: Representative traces (Left) and average speed (Right). Where indicated (Ca²⁺ 1 μM) the medium was switched from IB/EGTA to IB/1 μM [Ca²⁺]. Gray trace, controls; black trace, Fhit-overexpressing HeLa cells. (B) Rapid imaging of the [Ca²⁺]_m increase elicited by 10 μM histamine, in control (mtRP/Control) and Fhit-overexpressing (mtRP/Fhit) HeLa cells. The images in the panel show the fluorescence changes of the mtRP probe, expressed in a pseudocolor scale (warmer colors revealing the [Ca²⁺]_m increases), as previously described (24). (C) Total number of generated mitochondrial Ca²⁺ hotspots, plotted through time after stimulation. (D) Mitochondrial [ATP] changes in control and Fhit-overexpressing HeLa cells. Cell luminescence was measured as previously described (26).

Fig. 3.

Subcellular localization and effect on Ca²⁺ signaling of mtFhit. (A) Map of the mtFhit cDNA and immunolocalization of the expressed protein. (B) [Ca²⁺] measurement in the mitochondria, ER (control 438 ± 26.90 μM vs. Fhit 411 ± 35.60 μM), and cytosol (control 2.48 ± 0.03 μM vs. Fhit 2.50 ± 0.03 μM) in controls (gray) and mtFhit-overexpressing cells (black) upon addition of 10 μM histamine.

Fig. 4.

The mitochondrial fraction of Fhit potentiates apoptotic challenges. (A) [Ca²⁺]_c and (B) [Ca²⁺]_m increases elicited by 1 mM menadione. (C and D) Assessment of cell viability via apoptotic counts (C) or Annexin V labeling (D) in controls, Fhit and mtFhit overexpressing cells, as indicated. In panel D, the percentage of cells over a common arbitrary level of fluorescence is indicated. (E) Distribution of HeLa cells in the cell cycle, as estimated by propidium iodide staining of DNA. The percentage of cells in G0/G1, S, and G2 is reported in each graph.