Mitochondria play an important role in energy production, Ca2+ homeostasis and cell death. In recent years, the role of the mitochondria in apoptotic and necrotic cell death has attracted much attention. In apoptosis and necrosis, the mitochondrial permeability transition (mPT), which leads to disruption of the mitochondrial membranes and mitochondrial dysfunction, is considered to be one of the key events, although its exact role in cell death remains elusive. We therefore created mice lacking cyclophilin D (CypD), a protein considered to be involved in the mPT, to analyse its role in cell death. CypD-deficient mice were developmentally normal and showed no apparent anomalies, but CypD-deficient mitochondria did not undergo the cyclosporin A-sensitive mPT. CypD-deficient cells died normally in response to various apoptotic stimuli, but showed resistance to necrotic cell death induced by reactive oxygen species and Ca2+ overload. In addition, CypD-deficient mice showed a high level of resistance to ischaemia/reperfusion-induced cardiac injury. Our results indicate that the CypD-dependent mPT regulates some forms of necrotic death, but not apoptotic death.

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Galectin-3 (Gal-3), a member of the ␤-galactoside binding protein family containing the NWGR antideath motif of the Bcl-2 protein family, is involved in various aspects of cancer progression. Previously, it has been shown that the antiapoptotic activity of Gal-3 is regulated by the phosphorylation at Ser 6 by casein kinase 1 (CK1). Here we questioned how phosphorylation at Ser 6 regulates Gal-3 function. We have generated serineto-alanine (S6A) and serine-to-glutamic acid (S6E) Gal-3 mutants and transfected them into the BT-549 human breast carcinoma cell line, which does not express Gal-3. BT-549 cell clones expressing wild-type (wt) and mutant Gal-3 were exposed to chemotherapeutic anticancer drugs. In response to the apoptotic insults, phosphorylated wt Gal-3 was exported from the nucleus to the cytoplasm and protected the BT-549 cells from drug-induced apoptosis while nonphosphorylated mutant Gal-3 neither was exported from the nucleus nor protected BT-549 cells from drug-induced apoptosis. Furthermore, leptomycin B, a nuclear export inhibitor, increased the cisplatin-induced apoptosis of Gal-3 expressing BT-549 cells. These results suggest that Ser 6 phosphoryaltion acts as a molecular switch for its cellular translocation from the nucleus to the cytoplasm and, as a result, regulates the antiapoptotic activity of Gal-3.

Galectin-3, a ␤-galactoside-binding protein, is implicated in cell growth, adhesion, differentiation, and tumor progression by interactions with its ligands. Recent studies have revealed that galectin-3 suppresses apoptosis and anoikis that contribute to cell survival during metastatic cascades. Previously, it has been shown that human galectin-3 undergoes post-translational signaling modification of Ser 6 phosphorylation that acts as an "on/off" switch for its sugar-binding capability. We questioned whether galectin-3 phosphorylation is required for its anti-apoptotic function. Serine to alanine (S6A) and serine to glutamic acid (S6E) mutations were produced at the casein kinase I phosphorylation site in galectin-3. The cDNAs were transfected into a breast carcinoma cell line BT-549 that innately expresses no galectin-3. Metabolic labeling revealed that only wild type galectin-3 undergoes phosphorylation in vivo. Expression of Ser 6 mutants of galectin-3 failed to protect cells from cisplatin-induced cell death and poly(ADPribose) polymerase from degradation when compared with wild type galectin-3. The non-phosphorylated galectin-3 mutants failed to protect cells from anoikis with G 1 arrest when cells were cultured in suspension. In response to a loss of cell-substrate interactions, only cells expressing wild type galectin-3 down-regulated cyclin A expression and up-regulated cyclin D 1 and cyclindependent kinase inhibitors, i.e. p21 WAF1/CIP1 and p27 KIP1 expression levels. These results demonstrate that galectin-3 phosphorylation regulates its anti-apoptotic signaling activity.

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