Evaluation of bioactive sphingolipids in 4-HPR-resistant leukemia cells

Background -- N-(4-hydroxyphenyl)retinamide (4-HPR, fenretinide) is a synthetic retinoid with potent pro-apoptotic activity against several types of cancer, but little is known regarding mechanisms leading to chemoresistance. Ceramide and, more recently, other sphingolipid species (e.g., dihydroceramide and dihydrosphingosine) have been implicated in 4-HPR-mediated tumor cell death. Because sphingolipid metabolism has been reported to be altered in drug-resistant tumor cells, we studied the implication of sphingolipids in acquired resistance to 4-HPR based on an acute lymphoblastic leukemia model. Methods -- CCRF-CEM cell lines resistant to 4-HPR were obtained by gradual selection. Endogenous sphingolipid profiles and in situ enzymatic activities were determined by LC/MS, and resistance to 4-HPR or to alternative treatments was measured using the XTT viability assay and annexin V-FITC/propidium iodide labeling. Results -- No major crossresistance was observed against other antitumoral compounds (i.e. paclitaxel, cisplatin, doxorubicin hydrochloride) or agents (i.e. ultra violet C, hydrogen peroxide) also described as sphingolipid modulators. CCRF-CEM cell lines resistant to 4-HPR exhibited a distinctive endogenous sphingolipid profile that correlated with inhibition of dihydroceramide desaturase. Cells maintained acquired resistance to 4-HPR after the removal of 4-HPR though the sphingolipid profile returned to control levels. On the other hand, combined treatment with sphingosine kinase inhibitors (unnatural (dihydro)sphingosines ((dh)Sph)) and glucosylceramide synthase inhibitor (PPMP) in the presence or absence of 4-HPR increased cellular (dh)Sph (but not ceramide) levels and were highly toxic for both parental and resistant cells. Conclusions -- In the leukemia model, acquired resistance to 4-HPR is selective and persists in the absence of sphingolipid profile alteration. Therapeutically, the data demonstrate that alternative sphingolipid-modulating antitumoral strategies are suitable for both 4-HPR-resistant and sensitive leukemia cells. Thus, whereas sphingolipids may not be critical for maintaining resistance to 4-HPR, manipulation of cytotoxic sphingolipids should be considered a viable approach for overcoming resistance.

Efeito da 1,10-fenantrolina e seus derivados complexados em metal na atividade proteolítica de Leishmania braziliensis

Leishmanioses são um grupo de doenças com um largo espectro de manifestações clínicas, as quais variam desde lesões cutâneas até o envolvimento visceral severo, podendo levar ao óbito. A leishmaniose é, ainda hoje, uma doença negligenciada, estando entre os agravos prioritários do programa de pesquisa sobre doenças da pobreza da Organização Mundial da Saúde (OMS). Além de não haver vacinas disponíveis, a terapia é baseada em medicamentos injetáveis que causam sérios efeitos colaterais, tornando o tratamento inviável para muitos países endêmicos. Drogas derivadas de metal representam um novo arsenal terapêutico antimicrobiano e anti-câncer. Os inibidores de peptidase/agentes quelantes tais como 1,10-fenantrolina e seus derivados, no estado livre de metal ou como ligantes com metais de transição, interferem com a função de vários sistemas biológicos. Em trabalhos anteriores, nosso grupo descreveu que o parasito L. braziliensis produziu moléculas gp63 sensíveis a 1,10-fenantrolina. No presente trabalho, demonstramos a distribuição celular da molécula gp63 em uma cepa virulenta de L. braziliensis por meio de análises bioquímicas e imuno-histoquímica. Depois disso, relatamos os efeitos inibitórios de três compostos derivados da 1,10-fenantrolina, 1,10-fenantrolina-5,6-diona (phendio), [Cu(phendio)2] e [Ag(phendio)2], nas atividades metalopeptidases celulares e extracelulares produzidas por promastigotas de L. braziliensis, bem como as suas ações sobre a viabilidade do parasita e na interação com as células de macrófagos murinos. As moléculas gp63 foram detectadas em compartimentos de parasitos, incluindo membrana citoplasmatica e bolsa flagelar. O tratamento de promastigotas de L. braziliensis durante 1 hora com 1,10-fenantrolina e seus derivados resultou numa inibição significativa da viabilidade celular e mostrou um mecanismo de ação irreversível. Estes inibidores de metalopeptidases induziram apoptose em promastigotas de L. braziliensis, demonstrada através da marcação com anexina/iodeto de propídio e ensaio TUNEL. O pré-tratamento de promastigotas com os inibidores de metalopeptidases induziram uma diminuição na expressão de moléculas de superfície gp63, assim como uma redução significativa no índice de associação com macrófagos. Em paralelo, macrófagos infectados com L. braziliensis e tratados com 1,10-fenantrolina e seus derivados promoveram uma potente redução sobre o número de amastigotas intracelulares. O tratamento de macrófagos com 1,10-fenantrolina e seus derivados não induziram o aumento de óxido nítrico. A ação combinatória sobre a capacidade de crescimento entre os compostos derivados da 1,10-fenantrolina e Glucantime, quando ambos foram utilizados em concentracões sub-inibidoras, também foi observada. In vivo os compostos derivados da 1,10-fenantrolina e seus drivados foram capazes de controlar o tamanho das lesões a partir da terceira semana de tratamento em relação ao controle não tratado em hamsters infectados quando administrado por via intraperitoneal. Os animais tratados com os compostos apresentaram maior resposta intradérmica (DTH) aos antígenos de L. braziliensis. Coletivamente, a 1,10-fenantrolina e seus derivados metálicos apresentam uma nova perspectiva de estudos para o desenvolvimento de novos fármacos anti-L. braziliensis

Apoptose induzida por estreptococos do grupo B em células epiteliais respiratórias A549

Streptococcus agalactiae, ou Streptococcus do grupo B (GBS), é um importante patógeno oportunista que causa pneumonia, sepse e meningite em recém-nascidos e infecções em adultos imunocomprometidos. O pulmão aparentemente é o portal de entrada para o EGB na corrente sanguínea o que pode evoluir para uma septicemia. Os mecanismos de virulência relevantes envolve a habilidade do EGB em penetrar e sobreviver intracelularmente em células hospedeiras. Neste trabalho, foram analisados os mecanismos moleculares da apoptose epitelial induzida pelo EGB, e a produção de óxido nítrico (NO) e espécies reativas de oxigênio (ROS) em células epiteliais respiratórias A549 durante a infecção por EGB. Todas as amostras de EGB exibiram a capacidade de aderir e invadir células A549. A sobrevivência intracelular do EGB em células A549 ocorreu durante 24 h de incubação sem replicação do patógeno. No entanto, a amsotra 88641-V isolada de vagina não sobreviveu após 0,5 h de interação. O EGB promoveu a perda de viabilidade do epitélio durante a infecção. As alterações morfológicas em células A549 infectadas com o EGB incluem arredondamento celular, condensação nuclear, encolhimento celular e perda de contato célula-célula e célula-substrato. A dupla marcação AV/IP revelou que amostras de EGB sorotipo III induziram apoptose enquanto amostras do sorotipo V induziram morte celular semelhante a necrose em células A549. Caspase-3 foi ativada durante a apoptose induzida por EGB em células epiteliais. No entanto, a ativação de caspases-8 e -9 foi detectada apenas para a amostra 88641-V e as amostras EGB do sorotipo III, respectivamente. Experimentos comparativos de Immunoblotting revelaram que o EGB induziu um aumento da expressão Bim, uma proteína pró-apoptótica e diminuiu a expressão de Bcl-2 e Bcl-xL, proteínas anti-apoptóticas. As células A549 apresentaram perda de potencial de membrana mitocondrial Δψm e co-localização com o Bax. Ensaio de espectrometria de massa identificou a proteína PI-2a, uma proteína estrutural de pili, que exibe atividade carboxipepdidase. Descobrimos que os dois sorotipos (III e V) induziram a produção ROS e NO em células A549. Em conclusão, a apoptose induzida pelo EGB em células A549 é um mecanismo importante de virulência, resultando na destruição de tecidos, escape do sistema imune do hospedeiro com espalhamento bacteriano e, em consequência, a doença invasiva ou uma infecção sistémica.

Avaliação da ação antitumoral in vitro da pterocarpanoquinona LQB 118 e estudo de alguns mecanismos de ação

Tem sido descrito que o acúmulo de mutações em proto-oncogenes e genes supressores de tumor contribui para o direcionamento da célula à carcinogênese. Na maioria dos casos de câncer, as células apresentam proliferação descontrolada devido a alterações na expressão e/ou mutações de ciclinas, quinases dependentes de ciclinas e/ou inibidores do ciclo celular. Os tumores sólidos figuram entre o tipo de câncer mais incidente no mundo, sendo a quimioterapia e/ou hormônio-terapia, radioterapia e cirurgia os tratamentos mais indicados para estes tipos de tumores. Entretanto, o tratamento quimioterápico apresenta diversos efeitos colaterais e muitas vezes é ineficaz. Portanto, a busca por novas moléculas capazes de conter a proliferação destas células e com baixa toxicidade para o organismo se faz necessário. Este trabalho teve por objetivo avaliar a ação antitumoral in vitro de um novo composto sintético, a pterocarpanoquinona LQB118, sobre algumas linhagens tumorais humanas de alta prevalência e estudar alguns dos seus mecanismos de ação. As linhagens tumorais estudadas neste trabalho foram os adenocarcinomas de mama (MCF7) e próstata (PC-3), e carcinoma de pulmão (A549). A citotoxicidade foi avaliada pelo ensaio do MTT e a proliferação celular pela contagem de células vivas (exclusão do corante azul de tripan) e análise do ciclo celular (citometria de fluxo). A expressão gênica foi avaliada por RT-PCR e a apoptose foi avaliada por condensação da cromatina (microscopia de fluorescência-DAPI), fragmentação de DNA (eletroforese) e marcação com anexina V (citometria de fluxo). Das linhagens tumorais testadas, a de próstata (PC3) foi a que se mostrou mais sensível ao LQB 118, e em função deste resultado, os demais experimentos foram realizados com esta linhagem tumoral. O efeito citotóxico do LQB 118 se mostrou tempo e concentração dependente. Esta substância inibiu a proliferação celular e prejudicou a progressão do ciclo celular, acumulando células nas fases S e G2/M. Buscando esclarecer os mecanismos desta ação antitumoral, demonstrou-se que o LQB 118 inibe a expressão do mRNA do fator de transcrição c-Myc e das ciclinas D1 e B1, e induz a apoptose de tais células tumorais. Em suma, o LQB 118 é capaz de inibir a proliferação das células tumorais de próstata, alterando a expressão do mRNA de alguns genes reguladores do ciclo celular, resultando em interrupção do ciclo celular e indução de apoptose, indicando este composto como um potencial candidato a futuro medicamento no tratamento do câncer de próstata.

Modulação das vias de sinalização intracelulares pelo sistema NAD(P)H oxidase em melanoma humano

Evidências têm mostrado que as espécies reativas de oxigênio (ROS) geradas pela NAD(P)H oxidase são importantes moduladores de diversas funções celulares como migração, crescimento, proliferação e sobrevivência. Estudos recentes demonstraram o envolvimento da atividade da NAD(P)H oxidase no crescimento e sobrevivência de células de melanoma. Neste trabalho, investigamos o efeito da inibição da NAD(P)H oxidase por difenileneiodônio (DPI) sobre o crescimento das células de melanoma humano MV3 e observamos que este composto reduziu o crescimento destas células em aproximadamente 50%. A inibição da NAD(P)H oxidase induziu mudanças no formato celular, com arredondamento, diminuição do espraiamento e descolamento celular. Esta redução foi acompanhada por um rearranjo do citoesqueleto de actina, diminuição da fosforilação no resíduo Tyr397 da quinase de adesão focal (FAK) e redução na associação de FAK com actina e com a tirosina quinase c-Src. Isto indica que a inibição da geração de ROS está modulando negativamente vias de sinalização ativadas por integrinas, o que freqüentemente conduz a um tipo particular de morte celular conhecida por anoikis. Comprovando a ocorrência deste fenômeno, observamos que a inibição da atividade da NAD(P)H oxidase aumentou a apoptose das células de melanoma e induziu a ativação da caspase-3. Nossos resultados mostram ainda que a inibição da viabilidade celular por DPI foi revertida com o pré-tratamento das células MV3 com um inibidor de tirosina fosfatases (ortovanadato de sódio). Em resumo, este estudo mostra que a geração de ROS por NAD(P)H oxidase está envolvida nos mecanismos de sobrevivência em células de melanoma, uma vez que afetam as vias de sinalização dependentes de FAK-Src, através da inibição da atividade de proteína tirosina fosfatases.

Enhanced apoptotic action of trichosanthin in HIV-1 infected cells

Trichosanthin (TCS) is a type 1 ribosome-inactivating protein (RIP) effective against HIV-1 replication. The mechanism is not clear. Present results suggested that the antiviral action tray be partly mediated through enhanced apoptosis on infected cells. TCS induced apoptosis in normal H9 cells and this action was more potent in those infected with HIV-1. In flow cytometry study, TCS induced larger population of apoptotic H9 cells chronically infected with HIV-1 in a dose-dependent manner. At TCS concentration of 25 mu g/ml. 8.4% of normal H9 cells were found to be apoptotic whereas the same concentration induced 24.5% in HIV-1 chronically infected cells. Such difference was not found in the control experiments without TCS treatment. Two other studies supported this action. Cytotoxic study showed that cell viability was always lower in HIV-1 infected cells after TCS treatment, and DNA fragmentation studs confirmed more laddering in infected cells. The mechanism of TCS induced apoptosis in normal or infected H9 cells is not clear. Results in this study demonstrated that TCS is snore effective in inducing apoptosis in HIV-1 infected cells. This may explain in part the antiviral action of TCS. (c) 2005 Elsevier Inc. All rights reserved.

Characterization and expression analysis of Paralichthys olivaceus voltage-dependent anion channel (VDAC) gene in response to virus infection

Voltage-dependent anion channel (VDAC, also known as mitochondrial porin) is acknowledged to play an important role in stress-induced mammalian apoptosis. In this study, Paralichthys olivaceus VDAC (PoVDAC) gene was identified as a virally induced gene from Scophthalmus Maximus Rhabdovirus (SMRV)-infected flounder embryonic cells (FEC). The full length of PoVDAC cDNA is 1380 bp with an open reading frame of 852 bp encoding a 283 amino acid protein. The deduced PoVDAC contains one alpha-helix, 13 transmembrane beta-strands and one eukaryotic mitochondrial porin signature motif. Constitutive expression of PoVDAC was confirmed in all tested tissues by real-time PCR. Further expression analysis revealed PoVDAC mRNA was upregulated by viral infection. We prepared fish antiserum against recombinant VDAC proteins and detected the PoVDAC in heart lysates from flounder as a 32 kDa band on western blot. Overexpression of PoVDAC in fish cells induced apoptosis. Immunofluoresence localization indicated that the significant distribution changes of PoVDAC have occurred in virus-induced apoptotic cells. This is the first report on the inductive expression of VDAC by viral infection, suggesting that PoVDAC might be mediated flounder antiviral immune response through induction of apoptosis. (c) 2007 Elsevier Ltd. All rights reserved.

Toxicity of microcystins in the isolated hepatocytes of common carp (Cyprinus carpio L.)

The toxicity of hepatotoxic microcystins produced mainly by Microcystis aeruginosa in mammals and fishes was well studied in recent years. However, there were scarcely reports in toxic effects of microcystins on isolated hepatocytes of fishes, especially investigation of microcystin-induced apoptosis and/or necrosis in carp hepatocytes. In the present study, the isolated hepatocytes of common carp were exposed to various concentrations of microcystins (0.01, 0.1, 1, 10, 100, 1000 mu g L-1) for 2, 4, 8, 16 and 24 h, respectively, and cytotoxicity of microcystins in the toxin-treated cells was determined. Results of this study showed that cytotoxicity of microcystins on carp hepatocytes was time and dose-dependent, and the approximate LC50 of microcystins in carp hepatocytes was 169.2 mu g L-1. The morphological changes typical of apoptosis, such as blebbing of cell membrane, condensation and fragmentation of cell nucleus were observed in the hepatocytes exposed to microcystins (1, 10 and 100 mu g L-1) using fluorescence and differential interference contrast microscopy. Agarose gel electrophoresis of DNA demonstrated a typical apoptotic "ladder pattern" in microcystin-treated hepatocytes after 16 h of exposure. Results of the present study indicated that the form of cell death in microcystin-treated hepatocytes depend on the exposure dose of toxin. When lower concentration of microcystins (10 and 100 mu g L-1) was used for exposure, carp hepatocytes died in apoptosis while, when higher one used (1000 mu g L-1), they died in the form of necrosis. (C) 2006 Elsevier Inc. All rights reserved.

Targeting aberrant kinase activity in myeloid leukaemias

Acute myeloid leukaemia (AML) is the most common form of acute leukaemia in adults. Its treatment has remained largely unchanged for the past 30 years. Chronic myeloid leukaemia (CML) represents a tremendous success story in the era of targeted therapy but significant challenges remain including the development of drug resistance and disease persistence due to presence of CML stem cells. The Aurora family of kinases is essential for cell cycle regulation and their aberrant expression in cancer prompted the development of small molecules that selectively inhibit their activity. Chapter 2 of this thesis outlines the efficacy and mechanism of action of alisertib, a novel inhibitor of Aurora A kinase, in preclinical models of CML. Alisertib possessed equipotent activity against CML cells expressing unmutated and mutated forms of BCR-ABL. Notably, this agent retained high activity against the T315I and E255K BCR-ABL mutations, which confer the greatest degree of resistance to standard CML therapy. Chapter 3 explores the activity of alisertib in preclinical models of AML. Alisertib disrupted cell viability, diminished clonogenic survival, induced expression of the forkhead box O3 (FOXO3a) targets p27 and BCL-2 interacting mediator (BIM), and triggered apoptosis. A link between Aurora A expression and sensitivity to ara-C was established. Chapter 4 outlines the role of the proto-oncogene serine/threonine-protein (PIM) kinases in resistance to ara-C in AML. We report that the novel small molecule PIM kinase inhibitor SGI-1776 disrupted cell viability and induced apoptosis in AML. We establish a link between ara-C resistance and PIM over-expression. Finally, chapter 5 explores how the preclinical work outlined in this thesis may be translated into clinical studies that may lead to novel therapeutic approaches for patients with refractory myeloid leukaemia.

Ubiquitylation of p53 by the APC/C inhibitor Trim39.

Tripartite motif 39 (Trim39) is a RING domain-containing E3 ubiquitin ligase able to inhibit the anaphase-promoting complex (APC/C) directly. Through analysis of Trim39 function in p53-positive and p53-negative cells, we have found, surprisingly, that p53-positive cells lacking Trim39 could not traverse the G1/S transition. This effect did not result from disinhibition of the APC/C. Moreover, although Trim39 loss inhibited etoposide-induced apoptosis in p53-negative cells, apoptosis was enhanced by Trim39 knockdown in p53-positive cells. Furthermore, we show here that the Trim39 can directly bind and ubiquitylate p53 in vitro and in vivo, leading to p53 degradation. Depletion of Trim39 significantly increased p53 protein levels and cell growth retardation in multiple cell lines. We found that the relative importance of Trim39 and the well-characterized p53-directed E3 ligase, murine double minute 2 (MDM2), varied between cell types. In cells that were relatively insensitive to the MDM2 inhibitor, nutlin-3a, apoptosis could be markedly enhanced by siRNA directed against Trim39. As such, Trim39 may serve as a potential therapeutic target in tumors with WT p53 when MDM2 inhibition is insufficient to elevate p53 levels and apoptosis.

CARBON MONOXIDE AND PREGNANCY: A SEARCH FOR A POSSIBLE THERAPEUTIC IN THE TREATMENT OF PRE-ECLAMPSIA

Pre-eclampsia (PE) is a pregnancy disorder that affects roughly 5-7% of all pregnancies and is a leading cause of both maternal and fetal/neonatal morbidity and mortality. With no present cure for the disease, researchers are interested in the lower incidence of PE observed among the cigarette smoking pregnant population. However, women who use smokeless tobacco do not experience the same decreased incidence of PE, leading to hypothesis of protection against PE from the largest combustible product of cigarette smoke, carbon monoxide (CO). Studies evaluated levels of CO in PE women and found that they were statistically lower than those of healthy pregnancy. Researchers have found CO to possess many cytoprotective and regulatory properties and specifically within the placenta, it has been found to increase perfusion pressure, decrease oxidative stress, decreases ischemia/reperfusion induced apoptosis and maintain endothelial functioning. The idea for use of CO as a possible therapeutic for PE has thus become a real possibility. This study determined CO levels in pregnant women ± smoking as well as in PE women±smoking, as to discover a possible therapeutic range for future treatments. The best correlated automated CO measurement device with blood CO levels was determined, for use in future clinical studies. This thesis also sought a possible CO delivery concentration, in order to achieve the CO levels observed in the human correlation study. A threshold level of maternal CO exposure in a murine animal model was found, for which fetal and maternal negative toxicities were not observed. The results of this thesis lend a few more pieces to the complicated puzzle involving CO and PE and offer another step toward the possibility of a therapeutic treatment/prevention using this gaseous molecule.

Chemotherapy and TRAIL-mediated colon cancer cell death: the roles of p53, TRAIL receptors, and c-FLIP.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has recently attracted attention as a potential therapeutic agent in the treatment of cancer. We assessed the roles of p53, TRAIL receptors, and cellular Fas-associated death domain-like interleukin-1beta-converting enzyme inhibitory protein (c-FLIP) in regulating the cytotoxic effects of recombinant TRAIL (rTRAIL) alone and in combination with chemotherapy [5-fluorouracil (5-FU), oxaliplatin, and irinotecan] in a panel of colon cancer cell lines. Using clonogenic survival and flow cytometric analyses, we showed that chemotherapy sensitized p53 wild-type, mutant, and null cell lines to TRAIL-mediated apoptosis. Although chemotherapy treatment did not modulate mRNA or cell surface expression of the TRAIL receptors death receptor 4, death receptor 5, decoy receptor 1, or decoy receptor 2, it was found to down-regulate expression of the caspase-8 inhibitor, c-FLIP. Stable overexpression of the long c-FLIP splice form but not the short form was found to inhibit chemotherapy/rTRAIL-induced apoptosis. Furthermore, siRNA-mediated down-regulation of c-FLIP, particularly the long form, was found to sensitize colon cancer cells to rTRAIL-induced apoptosis. In addition, treatment of a 5-FU-resistant cell line with 5-FU down-regulated c-FLIP expression and sensitized the chemotherapy-resistant cell line to rTRAIL. We conclude that TRAIL-targeted therapies may be used to enhance conventional chemotherapy regimens in colon cancer regardless of tumor p53 status. Furthermore, inhibition of c-FLIP may be a vital accessory strategy for the optimal use of TRAIL-targeted therapies.

c-FLIP: A Key Regulator of Colorectal Cancer Cell Death

c-FLIP is an inhibitor of apoptosis mediated by the death receptors Fas, DR4 and DR5 and is expressed as long (c-FLIPL) and short (c-FLIPS) splice forms. We found that siRNA-mediated silencing of c-FLIP induced spontaneous apoptosis in a panel of p53 wild-type, mutant and null colorectal cancer (CRC) cell lines and that this apoptosis was mediated by caspase 8 and FADD. Further analyses indicated the involvement of DR5 and/or Fas (but not DR4) in regulating apoptosis induced by c-FLIP siRNA. Interestingly, these effects were not dependent on activation of DR5 or Fas by their ligands TRAIL and FasL. Overexpression of c-FLIPL, but not c-FLIPS, significantly decreased spontaneous and chemotherapy-induced apoptosis in HCT116 cells. Further analyses with splice form-specific siRNAs indicated that c-FLIPL was the more important splice form in regulating apoptosis in HCT116, H630 and LoVo cells, although specific knock down of c-FLIPS induced more apoptosis in the HT29 cell line. Importantly, intra-tumoral delivery of c-FLIP-targeted siRNA duplexes induced apoptosis and inhibited the growth of HCT116 xenografts in Balb/c SCID mice. In addition, the growth of c-FLIPL overexpressing CRC xenografts was more rapid than control xenografts, an effect that was significantly enhanced in the presence of chemotherapy. These results indicate that c-FLIP inhibits spontaneous death ligand-independent, death receptor-mediated apoptosis in CRC cells and that targeting c-FLIP may have therapeutic potential for the treatment of colorectal cancer.

Chk1 Suppresses a Caspase-2 Apoptotic Response to DNA Damage that Bypasses p53, Bcl-2, and Caspase-3

Evasion of DNA damage-induced cell death, via mutation of the p53 tumor suppressor or overexpression of prosurvival Bcl-2 family proteins, is a key step toward malignant transformation and therapeutic resistance. We report that depletion or acute inhibition of checkpoint kinase 1 (Chk1) is sufficient to restore ?-radiation-induced apoptosis in p53 mutant zebrafish embryos. Surprisingly, caspase-3 is not activated prior to DNA fragmentation, in contrast to classical intrinsic or extrinsic apoptosis. Rather, an alternative apoptotic program is engaged that cell autonomously requires atm (ataxia telangiectasia mutated), atr (ATM and Rad3-related) and caspase-2, and is not affected by p53 loss or overexpression of bcl-2/xl. Similarly, Chk1 inhibitor-treated human tumor cells hyperactivate ATM, ATR, and caspase-2 after ?-radiation and trigger a caspase-2-dependent apoptotic program that bypasses p53 deficiency and excess Bcl-2. The evolutionarily conserved "Chk1-suppressed" pathway defines a novel apoptotic process, whose responsiveness to Chk1 inhibitors and insensitivity to p53 and BCL2 alterations have important implications for cancer therapy. © 2008 Elsevier Inc. All rights reserved.

Anti-apoptotic effects of Z alpha-1 antitrypsin in human bronchial epithelial cells.

α1-antitrypsin (α1-AT) deficiency is a genetic disease which manifests as early-onset emphysema or liver disease. Although the majority of α1-AT is produced by the liver, it is also produced by bronchial epithelial cells, amongst others, in the lung. Herein, we investigate the effects of mutant Z α1-AT (ZAAT) expression on apoptosis in a human bronchial epithelial cell line (16HBE14o-) and delineate the mechanisms involved.

Control, M variant α1-AT (MAAT)- or ZAAT-expressing cells were assessed for apoptosis, caspase-3 activity, cell viability, phosphorylation of Bad, nuclear factor (NF)-κB activation and induced expression of a selection of pro- and anti-apoptotic genes.

Expression of ZAAT in 16HBE14o- cells, like MAAT, inhibited basal and agonist-induced apoptosis. ZAAT expression also inhibited caspase-3 activity by 57% compared with control cells (p = 0.05) and was a more potent inhibitor than MAAT. Whilst ZAAT had no effect on the activity of Bad, its expression activated NF-κB-dependent gene expression above control or MAAT-expressing cells. In 16HBE14o- cells but not HEK293 cells, ZAAT upregulated expression of cIAP-1, an upstream regulator of NF-κB. cIAP1 expression was increased in ZAAT versus MAAT bronchial biopsies.

The data suggest a novel mechanism by which ZAAT may promote human bronchial epithelial cell survival.