Low-voltage shock impedance measurements: A false sense of security.

BACKGROUND: Implantable cardioverter defibrillators use low-voltage shock impedance measurements to monitor the lead integrity. However, previous case reports suggest that low-voltage shock impedance measurements may fail to detect insulation breaches that can cause life-threatening electrical short circuits. METHODS AND RESULTS: We report six cases of insulation breaches in transvenous defibrillation leads that were not obvious during standard interrogations and testing of the lead beforehand. In two cases, an electrical short circuit during commanded shock delivery for internal electrical cardioversion resulted in a total damage of the ICD generator. In one of these cases, commanded shock delivery induced ventricular fibrillation, which required external defibrillation. In two cases, a shock due to ventricular tachycardia was aborted as the shock impedance was less than 20 Ω. However, in both cases the tiny residual shock energy terminated the ventricular tachycardia. In contrast, in one case the residual energy of the aborted shock did not end ventricular fibrillation induced at defibrillator threshold testing. In one case, the ICD indicated an error code for a short circuit condition detected during an adequate shock delivery. CONCLUSIONS: This case series illustrates that low-voltage shock impedance measurements can fail to detect insulation breaches. These data suggest that in patients without a contraindication, traditional defibrillator threshold testing or high voltage synchronized shock at the time of device replacement should be considered.

Aldehyde dehydrogenase inhibitors: alpha,beta-acetylenic N-substituted aminothiolesters are reversible growth inhibitors of normal epithelial but irreversible apoptogens for cancer epithelial cells from human prostate in culture.

The pharmacomodulation of the N atom of alpha,beta-acetylenic aminothiolesters or the replacement of the thiolester moiety by more electrophilic groups did not permit any clear rationale to be established for improving the selective growth-inhibitory activity of this family of compounds over that of the previously synthesized alpha,beta-acetylenic aminothiolesters DIMATE and MATE [G. Quash, G. Fournet, J. Chantepie, J. Goré, C. Ardiet, D. Ardail, Y. Michal, U. Reichert, Biochem Pharmacol 64 (2002) 1279-92]. Hence DIMATE and MATE were investigated more thoroughly for selectivity and growth-inhibitory activity using human prostate epithelial normal cells (HPENC) on the one hand and human prostate epithelial cancer cells (DU145) on the other. Unequivocal evidence was obtained showing that both compounds were reversible growth inhibitors of HPENC but irreversible growth inhibitors of DU145. Growth-inhibition of DU145 was due to the induction of early apoptosis as revealed by the flow cytometric analytical profile of inhibitor-treated cells, of the decrease in the redox potential and increase in superoxide anion content of their mitochondria. Of the two intracellular enzymes: aldehyde dehydrogenases 1 and 3 (ALDH1 and ALDH3) targeted by DIMATE and MATE, ALDH3 was inhibited to the same extent by both compounds whereas ALDH1 was less susceptible to inhibition by MATE. As the induction of ALDH3 by xenobiotics is hormone-dependent, MATE, the more selective of the two inhibitors, is a useful tool not only for examining the role of the ALDH3 isoform in hormone-sensitive and resistant prostate cancer cells in culture but also for investigating if it can inhibit the growth of xenografts of prostate cancer in immunodeficient mice.

Cyclin B and E2F-1 expression in prostate carcinoma cells treated with the novel retinoid CD437 are regulated by the ubiquitin-mediated pathway.

E2F-1 and cyclin B are important regulators of the cell cycle, and their expressionand degradation are tightly regulated. Proteolysis of both molecules is mediated by the ubiquitin degradation pathway involving the activation of specific E3 ubiquitin ligases. Treatment of prostate carcinoma cells with the novel retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437/AHPN) results in the enhanced expression of E2F-1 and rapid degradation of cyclin B in the absence of the modulation of mRNA levels; this is accompanied by the S phase arrest of the cells and subsequent apoptosis. The elevated level of E2F-1 is because of the enhanced stability of the molecule, as indicated by pulse-labeling studies, demonstrating a prolonged half-life. The enhanced E2F-1 stability is associated with the concomitant acetylation of E2F-1, the disassociation of E2F-1 from the E2F-1 E3 ligase p45(SKP2), and decreased E2F-1 ubiquitination, suggesting CD437 inhibition of E-3 E2F-1 ligase activity. Exposure of the cells to CD437 also results in the enhanced association of the cyclin B E3 ligase APC with cyclin B and the rapid proteolysis of cyclin B. The CD437-enhanced proteolysis of cyclin B is blocked in the presence of the ubiquitin proteolysis inhibitor N-acetyl-leu-leu-norleu-al. Thus, CD437 modulates the expression of E2F-1 and cyclin B through the simultaneous stimulation and inhibition of the cyclin B and E2F-1 E3 ligases, respectively.

Ligation of RARgamma inhibits proliferation of phytohaemagglutinin-stimulated T-cells via down-regulating JAK3 protein levels.

The mechanisms whereby Vitamin A regulates the immune system are poorly understood. We have shown previously that retinoic acids, the Vitamin A derivatives, promote both apoptosis of neglected thymocytes and the activation-induced cell death of peripheral T-cells via ligating the nuclear retinoid receptor (RAR) gamma. In the present study, we found that human peripheral T-cells express RARalpha and gamma, but not RARbeta. Increasing concentrations of 9-cis RA inhibited phytohaemagglutinin (PHA)-induced proliferation of T-cells, an effect that could be mimicked only by addition of RARgamma agonists and could be inhibited by an RARgamma antagonist. Interleukin-2 (IL-2) produced is known to mediate PHA-induced proliferation of T lymphocytes. Ligation of RARgamma did not affect the PHA-induced high affinity IL-2 receptor expression, slightly reduced the PHA-induced IL-2 production, but interfered with the IL-2-mediated signal transduction resulting in inhibition of PHA-induced phosphorylation of retinoblastoma protein and of up-regulation of Bcl-2. Janus kinases JAK1 and JAK3 play a determinant role in IL-2-dependent signal transduction. Ligation of RARgamma did not affect the levels of JAK1, but prevented IL-2-induced expression of JAK3 resulting in inhibition of PHA-induced phosphorylation of Stat5 molecules. Our data suggest that the previously observed toxic effect of high concentrations of retinoids on the immune system might be mediated via formation of 9-cis RA, which via ligation of RARgamma not only induces cell death in immature thymocytes, but inhibits proliferation of T-cells as well.

Anaplerotic reactions in tumour proliferation and apoptosis.

Our aim in this commentary is to provide evidence that certain oxoacids formed in anaplerotic reactions control cell proliferation/apoptosis. In tumour cells with impaired Krebs cycle enzymes, some anaplerotic reactions do compensate for the deficit in oxoacids. One of these, oxaloacetate, derived from the transamination of asparagine but not of aspartate, is decarboxylated 4-fold more efficiently in polyoma-virus transformed cells than in their non-transformed counterparts. The deamidation of asparagine, in the cell culture medium, to aspartate by asparaginase decreases asparagine transamination and inhibits concomitantly the growth of asparaginase-sensitive lymphoma cells, suggesting a causal relationship between asparagine transamination and growth. Another oxoacid that can provide ATP when metabolised in mitochondria, but by the branched-chain oxoacid dehydrogenase complex (BCOADC), is 2-oxobutanoate. It has two origins: (a) deamination of threonine, and (b) cleavage of cystathionine, a metabolite derived from methionine. 2-Oxobutanoate in the presence of insulin promotes growth in G1/S arrested cells. But methionine also gives rise to another substrate of BCOADC, 4-methylthio-2-oxobutanoate (MTOB), which is synthesised exclusively from methylthioadenosine (MTA) by the action of MTA phosphorylase. In Met-dependent tumour cells with defective MTA phosphorylase, 2-oxobutanoate production would exceed that of MTOB. Further, BCOADC also has 3-fold greater affinity for 2-oxobutanoate than for MTOB; hence, the deficiency in 3-methylthio propionyl CoA, the final product of MTOB decarboxylation, would be exacerbated. Methional, the transient metabolic precursor in 3-methylthio propionyl CoA biosynthesis, is apoptogenic for both normal and bcl(2)-negative transformed cells in culture. Investigations of other causal relationships between the genes/enzymes mediating the homeostasis of anaplerotic oxoacids and cell growth/death may be worthwhile.

Novel competitive irreversible inhibitors of aldehyde dehydrogenase (ALDH1): restoration of chemosensitivity of L1210 cells overexpressing ALDH1 and induction of apoptosis in BAF(3) cells overexpressing bcl(2).

4-Amino-4-methyl-pent-2-ynthioc acid S-methyl ester (ampal thiolester: ATE) was used as a lead compound to synthesise new amino-substituted derivatives of alpha, beta acetylenic thiolester compounds as inhibitors of aldehyde dehydrogenase 1, (ALDH1). Of these compounds, the dimethyl derivative (DIMATE) was a competitive irreversible inhibitor (K(i) approximately 280 microM) of baker's yeast ALDH1 in vitro showing 80% inhibition at 400 microM when preincubated with the enzyme for 30min, whereas the trimethyl ammonium and the morpholine derivatives showed only 15% inhibition at 600 microM even after 60min preincubation. ATE inhibited ALDH1 activity in ALDH1-transfected L1210 T cells resistant to hydroperoxycyclophosphamide (HCPA) and inhibited growth synergistically in the presence of HCPA. In non-transfected L1210 counterparts ATE did not potentiate growth inhibition by HCPA. DIMATE was a 30-100-fold more effective growth inhibitor than ATE. Endogenous ALDH1 activities of BAF(3) cells over-expressing different levels of bcl(2) (0-100%) were similar (16-20mU/mg protein) and were all inhibited by DIMATE, reaching 20-30% at 4 microM. Up to 4 microM no apoptosis, as measured by DNA-fragmentation was observed, but at 8 and 10 microM DIMATE, DNA-fragmentation increased concomitantly with ALDH1 inhibition. No DNA-fragmentation was observed with ALDH1 irreversible inhibitors devoid of a thiolester group or with thiolesters which were not inhibitors of ALDH1. It was seen only with competitive irreversible inhibitors having the methanethiol and enzyme-inhibitory moieties. The methanethiol putatively released from DIMATE by ALDH1 esterase activity plays a role, albeit undefined, in lowering intramitochondrial glutathione levels which decreased by 47% as DNA-fragmentation increased.

Synthetic retinoid CD437 induces cell-dependent cycle arrest by differential regulation of cell cycle associated proteins.

BACKGROUND: The synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene (CD437) exhibits a wide spectrum antitumor activity through induction of cellular apoptosis and cell cycle arrest. We investigated the effects and mechanisms of CD437 on cell cycle arrest of gastric cancer cells. MATERIALS AND METHODS: The activities of CD437 on cell growth were analyzed by measuring total cellular DNA. The effects of CD437 on cell cycle phase distribution were analyzed using flow cytometry. The levels of cell cycle associated proteins were analyzed by Western blot. The activities of cyclin-dependent kinases (cdks) were analyzed by phosphorylation of the histone H1 protein. RESULTS: CD437 at concentrations between 0.1 and 10 microM profoundly suppressed the growth of all six gastric cancer cell lines. Growth suppression associated with induction of G0/G1 or G2/M arrest was cell-line-dependent. CD437 decreased levels of cdk inhibitor p21 in G2/M-arrested SC-M1 cells. However, CD437 increased p21 levels in G0/G1-arrested AGS cells. Total and activated cyclin-dependent kinases were differentially regulated by CD437 in AGS and SC-M1 cells. CD437 (1 microM) induced activity of cdk2- and p34cdc2-associated H1 kinase by 14.6- and 1.8-fold, respectively, in SC-M1 cells. In contrast, CD437 slightly increased (1.6-fold) the cdk2-associated H1 kinase activity in AGS cells. CONCLUSION: CD437 profoundly suppressed the growth of gastric cancer cells, which was associated with cell-dependent induction of G0/G1 or G2/M arrest. The differential regulation of p21 that leads to alteration in the activity of cdks may play a critical role in cell-line-dependent regulation of cell cycle arrest following treatment with CD437.

Retinoids induce Fas(CD95) ligand cell surface expression via RARgamma and nur77 in T cells.

Cells from the CD4+ murine T hybridoma line IP-12-7 enter the apoptotic suicide program via the Fas ligand (FasL)/Fas-mediated pathway upon TCR stimulation. This stimulus regulates the sensitization of the Fas death pathway and the cell surface appearance of preformed FasL. The apoptosis is dependent on new mRNA and protein synthesis and involves up-regulation of nur77. Two groups of nuclear receptors for retinoic acids (RA) have been identified: retinoic acid receptors (RAR) and retinoid X receptors. IP-12-7 cells express RARalpha and RARgamma. Here we show that,in the IP-12-7 T cells, RA also induced the expression and DNA binding of nur77, and the cell surface appearance of FasL. The induction was mediated via RARgamma. Despite the induced expression of cell surface FasL, only two structurally related RARgamma-selective compounds, CD437 and CD2325, initiated apoptosis in these cells. The lack of apoptosis induction by natural RA was related to the inability of RARgamma to sensitize the Fas death-pathway. Cell surface FasL, however, was able to induce cell death in Fas-bearing target cells. Natural RA also induced the expression of FasL in phytohemagglutinin-activated peripheral murine T cells. It is proposed that therapeutically administered RA might induce apoptosis in Fas-sensitive cells via induction of FasL expression in activated Tcells.

Ligation of retinoic acid receptor alpha regulates negative selection of thymocytes by inhibiting both DNA binding of nur77 and synthesis of bim.

Negative selection refers to the selective deletion of autoreactive thymocytes. Its molecular mechanisms have not been well defined. Previous studies in our laboratory have demonstrated that retinoic acids, physiological ligands for the nuclear retinoid receptors, selectively inhibit TCR-mediated death under in vitro conditions, and the inhibition is mediated via the retinoic acid receptor (RAR) alpha. The present studies were undertaken to investigate whether ligation of RARalpha leads to inhibition of TCR-mediated death in vivo and to identify the molecular mechanisms involved. Three models of TCR-mediated death were studied: anti-CD3-mediated death of thymocytes in wild-type mice, and Ag- and bacterial superantigen-driven thymocyte death in TCR-transgenic mice expressing a receptor specific for a fragment of pigeon cytochrome c in the context of the E(k) (class II MHC) molecule. Our data demonstrate that the molecular program of both anti-CD3- and Ag-driven, but not that of superantigen-mediated apoptosis involves up-regulation of nur77, an orphan nuclear receptor, and bim, a BH3-only member of the proapoptotic bcl-2 protein family, proteins previously implicated to participate in the negative selection. Ligation of RARalpha by the synthetic agonist CD336 inhibited apoptosis, DNA binding of nur77, and synthesis of bim induced by anti-CD3 or the specific Ag, but had no effect on the superantigen-driven cell death. Our data imply that retinoids are able to inhibit negative selection in vivo as well, and they interfere with multiple steps of the T cell selection signal pathway.

Identification and characterization of a cell cycle and apoptosis regulatory protein-1 as a novel mediator of apoptosis signaling by retinoid CD437.

CD437, a novel retinoid, causes cell cycle arrest and apoptosis in a number of cancer cells including human breast carcinoma (HBC) by utilizing an undefined retinoic acid receptor/retinoid X receptor-independent mechanism. To delineate mediators of CD437 signaling, we utilized a random antisense-dependent functional knockout genetic approach. We identified a cDNA that encodes approximately 130-kDa HBC cell perinuclear protein (termed CARP-1). Treatments with CD437 or chemotherapeutic agent adriamycin, as well as serum deprivation of HBC cells, stimulate CARP-1 expression. Reduced levels of CARP-1 result in inhibition of apoptosis by CD437 or adriamycin, whereas increased expression of CARP-1 causes elevated levels of cyclin-dependent kinase inhibitor p21WAF1/CIP1 and apoptosis. CARP-1 interacts with 14-3-3 protein as well as causes reduced expression of cell cycle regulatory genes including c-Myc and cyclin B1. Loss of c-Myc sensitizes cells to apoptosis by CARP-1, whereas expression of c-Myc or 14-3-3 inhibits CARP-1-dependent apoptosis. Thus, apoptosis induction by CARP-1 involves sequestration of 14-3-3 and CARP-1-mediated altered expression of multiple cell cycle regulatory genes. Identification of CARP-1 as a key mediator of signaling by CD437 or adriamycin allows for delineation of pathways that, in turn, may prove beneficial for design and targeting of novel antitumor agents.

Induction of apoptosis of human B-CLL and ALL cells by a novel retinoid and its nonretinoidal analog.

We have recently described a novel retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylic acid (CD437/AHPN) that induces apoptosis in a number of malignant cell types. We now describe our studies examining the effects of CD437 and a nonretinoidal analog (MM002) on the in vitro proliferation of the ALL-REH cell line, the in vitro and in vivo growth of a novel Epstein-Barr virus-negative (EBV(-)) B-cell chronic lymphocytic leukemia (B-CLL) cell line (WSU-CLL), and primary cultures of human B-CLL and acute lymphoblastic leukemia (ALL) cells. CD437 and MM002 induce apoptosis in both cell lines, as indicated by the activation of caspase-2 and caspase-3, cleavage of poly(adenosine diphosphate-ribose) (poly(ADP-ribose)) polymerase, increase in annexin V binding, and subsequent nuclear fragmentation. CD437-mediated apoptosis was not associated with the modulation of Bcl-2, Bax, or Mcl-1 levels, but was associated with the cleavage of the antiapoptotic protein Bcl-X(L) to a proapoptotic 18-kD form. This cleavage of Bcl-X(L) was dependent on caspase-3 activation since Bcl-X(L) cleavage and apoptosis were inhibited by the caspase-3 inhibitor Z-DVED-fmk. CD437 markedly inhibited the growth of WSU-CLL cells in severe combined immunodeficiency (SCID) mice. Tumor growth inhibition, growth delay, and log cell kill were 85.7%, 21 days, and 2.1, respectively, in the treated mice. Moreover, 1 of the 5 treated mice was tumor-free longer than 150 days and thus was considered cured. Exposure of primary cultures of both B-CLL and ALL cells obtained from patients to CD437 and MM002 resulted in their apoptosis. These results suggest that CD437 and MM002 analogs may have a potential role in the treatment of B-CLL and ALL.