The molecular mode of action of lonidamine, a therapeutic agent employed in cancer chemotherapy, has been elusive. Here we provide evidence that lonidamine (LND) acts on mitochondria to induce apoptosis. LND provokes a disruption of the mitochondrial transmembrane potential which precedes signs of nuclear apoptosis and cytolysis. The mitochondrial and cytocidal eects of LND are not prevented by inhibitors of caspases or of mRNA or protein synthesis. However, they are prevented by transfection-enforced overexpression of Bcl-2, an oncoprotein which inhibits apoptosis by stabilizing the mitochondrial membrane barrier function. Accordingly, the cell death-inducing eect of LND is ampli®ed by simultaneous addition of PK11195, an isoquinoline ligand of the peripheral benzodiazepine receptor which antagonizes the cytoprotective eect of Bcl-2. When added to isolated nuclei, LND fails to provoke DNA degradation unless mitochondria are added simultaneously. In isolated mitochondria, LND causes the dissipation of the mitochondrial inner transmembrane potential and the release of apoptogenic factors capable of inducing nuclear apoptosis in vitro. Thus the mitochondrion is the subcellular target of LND. All eects of LND on isolated mitochondria are counteracted by cyclosporin A, an inhibitor of the mitochondrial PT pore. We therefore tested the eect of LND on the puri®ed PT pore reconstituted into liposomes. LND permeabilizes liposomal membranes containing the PT pore. This eect is prevented by addition of recombinant Bcl-2 protein but not by a mutant Bcl-2 protein that has lost its apoptosisinhibitory function. Altogether these data indicate that LND represents a novel type of anti-cancer agent which induces apoptosis via a direct eect on the mitochondrial PT pore.

Myelodysplastic syndrome (MDS) is a preneoplastic condition that frequently develops into overt acute myeloid leukemia (AML). The P39 MDS/AML cell line manifested constitutive NF-kappaB activation. In this cell line, NF-kappaB inhibition by small interfering RNAs specific for p65 or chemical inhibitors including bortezomib resulted in the down-regulation of apoptosis-inhibitory NF-kappaB target genes and subsequent cell death accompanied by loss of mitochondrial transmembrane potential as well as by the mitochondrial release of the caspase activator cytochrome c and the caspase-independent death effectors endonuclease G and apoptosis-inducing factor (AIF). Bone marrow cells from high-risk MDS patients also exhibited constitutive NF-kappaB activation similar to bone marrow samples from MDS/AML patients. Purified hematopoietic stem cells (CD34+) and immature myeloid cells (CD33+) from high-risk MDS patients demonstrated the nuclear translocation of the p65 NF-kappaB subunit. The frequency of cells with nuclear p65 correlated with blast counts, apoptosis suppression, and disease progression. NF-kappaB activation was confined to those cells that carried MDS-associated cytogenetic alterations. Since NF-kappaB inhibition induced rapid apoptosis of bone marrow cells from high-risk MDS patients, we postulate that NF-kappaB activation is responsible for the progressive suppression of apoptosis affecting differentiating MDS cells and thus contributes to malignant transformation. NF-kappaB inhibition may constitute a novel therapeutic strategy if apoptosis induction of MDS stem cells is the goal.

Mercuric chloride induces an autoimmune glomerulonephritis in Brown-Norway (BN) but not in Lewis (LEW) rats. Injection of HGCl2 into BN rats regularly produced a transient appearance of plaque-forming cells (PFC) of anti-2,4,6-trinitrophenyl and anti-sheep red blood cell specificity and circulating anti-single-stranded DNA antibodies. Addition of HgCl2 to spleen cell cultures from BN rats induced an increase in anti-trinitrophenyl PFC and reverse PFC. This effect was no longer observed when nylon wool column-depleted or anti-Thy-1 antiserum-treated spleen cells were cultured in the presence of HgCl1. These data suggest that HgCl2 acts as a polyclonal activator on spleen cells in BN rats, but not on isolated B lymphocytes. In contrast, no effect of HgCl2 on immunoglobulin production was observed in LEW rats. Since polyclonal activation and immune-type nephritis are both seen in BN but not in LEW rats, polyclonal activation may participate in the pathogenesis of the HgCl2-induced autoimmune disease of BN rats.

HLA-G is involved in regulating T cell responses. Various mechanisms have been proposed to explain the inhibition of T cell proliferation. In this context, the possible role of HLA-G in cell cycle regulation remains to be explored. Using stably transfected M8 cells expressing the secreted isoform (HLA-G5) of HLA-G, we investigated the role of HLA-G in inducing apoptosis and in controlling the cell cycle of activated T cells. Soluble HLA-G (HLA-G5) inhibited both CD4 and CD8 T cell proliferation. However, HLA-G5 did not induce T cell apoptosis, as determined by 3,3′-diethyloxacarbocyanine and propidium iodine labeling. It induced accumulation of the retinoblastoma protein, but not its phosphorylated and active form. Treatment of activated T cells with HLA-G5 also reduced the amounts of cyclin D2, E, A, and B by >80%. In contrast, it induced an accumulation of p27kip, but not p21cip, in activated T cells. HLA-G does not induce apoptosis of alloreactive T cells, but induces p27kip1 and inhibits cell cycle progression.