BCL2/BCL-X(L) inhibition induces apoptosis, disrupts cellular calcium homeostasis, and prevents platelet activation.

Apoptosis in megakaryocytes results in the formation of platelets. The role of apoptotic pathways in platelet turnover and in the apoptotic-like changes seen after platelet activation is poorly understood. ABT-263 (Navitoclax), a specific inhibitor of antiapoptotic BCL2 proteins, which is currently being evaluated in clinical trials for the treatment of leukemia and other malignancies, induces a dose-limiting thrombocytopenia. In this study, the relationship between BCL2/BCL-X(L) inhibition, apoptosis, and platelet activation was investigated. Exposure to ABT-263 induced apoptosis but repressed platelet activation by physiologic agonists. Notably, ABT-263 induced an immediate calcium response in platelets and the depletion of intracellular calcium stores, indicating that on BCL2/BCL-X(L) inhibition platelet activation is abrogated because of a diminished calcium signaling. By comparing the effects of ABT-263 and its analog ABT-737 on platelets and leukemia cells from the same donor, we show, for the first time, that these BCL2/BCL-X(L) inhibitors do not offer any selective toxicity but induce apoptosis at similar concentrations in leukemia cells and platelets. However, reticulated platelets are less sensitive to apoptosis, supporting the hypothesis that treatment with ABT-263 induces a selective loss of older platelets and providing an explanation for the transient thrombocytopenia observed on ABT-263 treatment.

Kv1.3 is the exclusive voltage-gated K+ channel of platelets and megakaryocytes: roles in membrane potential, Ca2+ signalling and platelet count.

A delayed rectifier voltage-gated K(+) channel (Kv) represents the largest ionic conductance of platelets and megakaryocytes, but is undefined at the molecular level. Quantitative RT-PCR of all known Kv alpha and ancillary subunits showed that only Kv1.3 (KCNA3) is substantially expressed in human platelets. Furthermore, megakaryocytes from Kv1.3(/) mice or from wild-type mice exposed to the Kv1.3 blocker margatoxin completely lacked Kv currents and displayed substantially depolarised resting membrane potentials. In human platelets, margatoxin reduced the P2X(1)- and thromboxaneA(2) receptor-evoked [Ca(2+)](i) increases and delayed the onset of store-operated Ca(2+) influx. Megakaryocyte development was normal in Kv1.3(/) mice, but the platelet count was increased, consistent with a role of Kv1.3 in apoptosis or decreased platelet activation. We conclude that Kv1.3 forms the Kv channel of the platelet and megakaryocyte, which sets the resting membrane potential, regulates agonist-evoked Ca(2+) increases and influences circulating platelet numbers.

Indanones and indenols from 2-alkylcinnamaldehydes via the intramolecular Friedel-Crafts reaction of geminal diacetates.

When treated with Ac2O at rt in the presence of 4-6 mol % FeCl3, 2-alkylcinnamaldehydes are converted to 2-alkyl-1H-inden-1-yl acetates through the intermediacy of gem-diacetates. Methanolysis of the indenyl acetates yields the corresponding indenols. Saponification yields 2-alkylindanones, providing, in effect, an intramolecular acylation employing catalytic levels of acid.

Thirty-three Years of Synthetic Organic Chemistry in Geneva: Reminiscences.

After thirty-three years of synthetic organic chemistry research in Geneva, three years as a postdoc at the Université de Genève and over thirty years working at Firmenich, a world-renowned flavour and fragrance company, Dr. Roger L. Snowden, Vice President Synthesis, Corporate R&D Division, Firmenich retraces his career back to 1975 when he arrived in Switzerland. A brief autobiographical sketch of this British national is presented, together with the reasons why Geneva turned out to be where he was to effect the major part of his scientific career.

Increased TNF expression in CD43++ murine blood monocytes.

Monocyte heterogeneity has been studied extensively in man but only recently tools have been developed to study blood monocyte populations in the mouse. We have used the MacGreen mouse model, which expresses the green fluorescent protein under the control of the promoter of the murine M-CSF receptor (CSF1 receptor, c-fms). Since both monocytes and granulocytes show GFP expression in this model the latter cells were excluded by staining with the Ly6G granulocyte marker. GFP+ Ly6G- blood monocytes were found to account for an average of 246+/-121cells/microl in these mice. These monocytes can be subdivided into CD43+ GR-1+ cells and CD43++ GR-1(-) cells, with the latter cells accounting for 140+/-77cells/mul, i.e. about 60% of all blood monocytes. After intraperitoneal injection of lipopolysaccharide (LPS) both blood monocyte subpopulations were depleted. The same was true after intranasal infection with Streptococcus pneumoniae but here the CD43++ subpopulation was preferentially reduced to 4cells/mul. For the study of TNF expression cells were stimulated in vitro with LPS from Salmonella abortus equi in the presence of Brefeldin A followed by intracellular staining and multicolor flow cytometry. Over a dose range of 10-100ng LPS/ml, TNF protein production was significantly higher in the CD43++ monocyte subset. At 1000ng LPS/ml 90% of all CD43++ monocytes stained positive for TNF and in terms of fluorescence intensity TNF was 5-fold higher compared to the CD43+ monocytes. These data indicate that the murine CD43++ monocyte subset exhibits features of pro-inflammatory monocytes and is functionally homologeous to the human CD14+CD16+ monocytes.

Cetalox and analogues: synthesis via acid-mediated polyene cyclizations.

Using a novel, acid-mediated cyclization methodology, a direct access to Cetalox ((+/-)-1; a commercially important ambergris-type odorant) and various structurally related didehydro (i.e., 19, 26, and 30) and tetradehydro (i.e., 28 and 37/38) analogues is described. Treatment of either (E,E)-14 or (E)-15 with an excess of FSO(3)H in 2-nitropropane at -90 degrees stereospecifically afforded (+/-)-1 in 40 and 42% yield, respectively. Under similar conditions, cyclization of (E)-18 or 20 furnished 19 in 60 and 64% yield, respectively. Analogously, using an excess of ClSO(3)H in CH(2)Cl(2) at -80 degrees, 26 is formed with high stereoselectivity by cyclization of either (E)-24 or (Z)-25 (52 and 31% yield, resp.); in the same manner, 28 was prepared from 27 (22% yield). The same principle was applied to the synthesis of racemic Superambrox (30), via cyclization of 35, but only with poor selectivity (22%) and low yield (7%). Another approach via cyclization of (E)-40 under solvolysis conditions (excess TFA in CH(2)Cl(2) at -10 degrees) gave a higher yield (15%) with improved selectivity (43%). Finally, cyclization of 34 (1:1 diastereoisomer mixture) afforded 37/38 (10:1) in 27% yield. The qualitative organoleptic properties of 19, 26, 28, 30, and 37/38 (10:1) are briefly discussed.

Conformational change and mitochondrial translocation of Bax accompany proteasome inhibitor-induced apoptosis of chronic lymphocytic leukemic cells.

Chemotherapy resistance remains a major clinical problem in patients with B-cell chronic lymphocytic leukemia (B-CLL). Proteasome inhibitors are able to induce apoptosis in chemotherapy-resistant B-CLL cells in vitro. Exposure of B-CLL cells to the proteasome inhibitors, MG132 and lactacystin, resulted in inhibition of proteasomal activity within 30 min of treatment and was accompanied by an increase in the level of ubiquitinated proteins. Proteasome inhibitors did not alter the levels of expression of the proapoptotic Bcl-2 family proteins, Bax and Bid, prior to the onset of apoptosis. Instead, proteasome inhibitors induced a caspase-independent conformational change in Bax (as shown by a conformation-specific Bax antibody) and its translocation to mitochondria, resulting in mitochondrial perturbation, as evidenced by loss of the mitochondrial membrane potential and cytochrome c release. Similar conformational change and subcellular localization of Bax were observed during apoptosis induced with fludarabine, chlorambucil and prednisolone. These data suggest that alteration of Bax conformation and its redistribution to mitochondria are common and early features of B-CLL apoptosis in response to proteasome inhibitors and other chemotherapeutic agents.

Mechanisms of resistance to TRAIL-induced apoptosis in primary B cell chronic lymphocytic leukaemia.

Primary B cells from B cell chronic lymphocytic leukaemia (B-CLL) were resistant to the novel selective cytotoxic agent, TNF-related apoptosis-inducing ligand (TRAIL). Low levels of the death-inducing TRAIL receptors, TRAIL-R1 and TRAIL-R2 but not the putative 'decoy' receptors, TRAIL-R3 and TRAIL-R4, were expressed on the surface of B-CLL cells. Resistance to TRAIL was upstream of caspase-8 activation, as little or no caspase-8 was processed in TRAIL-treated B-CLL cells. Low levels of a TRAIL death-inducing signalling complex (DISC) were formed in these cells, accompanied by the recruitment of endogenous FADD, caspase-8 and c-FLIP(L) but not c-FLIP(S). Both caspase-8 and c-FLIP(L) were cleaved to form two stable intermediates of approximately 43 kDa, which remained associated with the DISC. Caspase-8 was not further processed to its active heterotetramer. Thus the resistance of B-CLL cells to TRAIL may be due partly to low surface expression of the death receptors resulting in low levels of DISC formation and also to the high ratio of c-FLIP(L) to caspase-8 within the DISC, which would prevent further activation of caspase-8. Our results highlight the possibility of sensitising B-CLL cells to TRAIL by modulation of c-FLIP levels or by upregulation of surface expression of death receptors.

Low-penetrance genetic susceptibility and resistance loci implicated in the relative risk for radiation-induced acute myeloid leukemia in mice.

Inbred CBA/H mice are susceptible to radiation-induced acute myeloid leukemia (r-AML), and C57BL/6 mice are resistant. A genome-wide screen for linkage between genotype and phenotype (r-AML) of 67 affected (CBA/H x C57BL/6)F1 x CBA/H backcross mice has revealed at least 2 suggestive loci that contribute to the overall lifetime risk for r-AML. Neither is necessary or sufficient for r-AML, but relative risk is the net effect of susceptibility (distal chromosome 1) and resistance (chromosome 6) loci. An excess of chromosome 6 aberrations in mouse r-AML and bone marrow cells up to 6 months after irradiation in vivo suggests the locus confers a proliferative advantage during the leukemogenic process. The stem cell frequency regulator 1 (Scfr1) locus maps to distal chromosome 1 and determines the frequency of hemopoietic stem cells (HSCs) in inbred mice, suggesting that target size may be one factor in determining the relative susceptibility of inbred mice to r-AML.