Mitochondrial dysfunction is an essential step for killing of non-small cell lung carcinomas resistant to conventional treatment.

This corrects the article DOI: 10.1038/sj.onc.1205018.

Overcoming chemoresistance of non-small cell lung carcinoma through restoration of an AIF-dependent apoptotic pathway.

Non-small cell lung carcinomas (NSCLCs) are typically resistant against apoptosis induced by standard chemotherapy. We evaluated the effects of the two potential antitumor agents of the lamellarin class on a highly apoptosis-resistant NSCLC cell line. Both the marine alkaloid lamellarin-D and its synthetic amino derivative PM031379 induced the activation of Bax, the mitochondrial release of cytochrome c and apoptosis-inducing factor (AIF), as well as the activation of caspase-3. However, only PM031379 triggered cell death and sign of nuclear apoptosis coupled to the nuclear translocation of AIF. Depletion of AIF with small interfering RNA or microinjection of a neutralizing anti-AIF antibody largely prevented PM031379-induced cytotoxicity, underscoring the essential contribution of AIF to NSCLC killing. Using NSCLC cells lacking mitochondrial DNA, we showed that the generation of mitochondrial reactive oxygen species (ROS) was crucial for the PM031379-induced translocation of AIF to the nucleus and subsequently cell death. Pretreatment of NSCLC cells with menadione, a mitochondrial ROS generator, was able to restore the deficient chemotherapy-induced apoptosis of NSCLC cells. Altogether, these data suggest that mitochondrial ROS generation is crucial for overriding the chemoresistance of NSCLC cells. Moreover, this study delineates the unique mechanism of action of lamellarins as potential anticancer agents.

[Translational research and Cancer Plan]. / Recherche translationnelle et plan Cancer.

The French Cancer Plan 2003-2007 has made translational research central to its research programme, to ensure the care-research continuum and the quickest application possible for the most recent discoveries, for the patients' benefit. This is a new field of research, still little-known or ill-understood. A working group, composed of physicians and researchers from academic research and industrial research, sought to define translational research in cancerology and define the issues at stake in it. Translational research needs to develop in close connection with the patients in order to enable a bi-directional flow of knowledge from cognitive research toward medical applications and from observations made on patients toward cognitive research. Placed under the aegis of the French National Cancer Institute and Leem Research, the group has put forth a strategy for implementing translational research in cancerology in France to make it attractive, competitive and efficient and to foster the development of public-private partnerships.

[Activation of cardiac caspases: a double-edged weapon implicated in cardiac dysfunction]. / L'activation des caspases dans les cellules cardiaques: une arme a double tranchant impliquée dans la dysfonction cardiaque.

Low dead cell levels detected in various cardiac pathologies led to consider cardiac apoptosis as epiphenomena without repercussion on heart function. However, apoptosis prevention, and more specifically caspase inhibition, has been associated with major myocardial contractile performance improvement. This review describes specifically caspase involvement in myocardial dysfunction and highlights typical cardiomyocyte apoptosis signaling features. Thus caspase neutralization may represent a promising therapeutic strategy in heart disease treatments.

Hepatocyte nuclear factor 4 alpha ligand binding and F domains mediate interaction and transcriptional synergy with the pancreatic islet LIM HD transcription factor Isl1.

The orphan nuclear receptor HNF4alpha and the LIM homeodomain factor Isl1 are co-expressed in pancreatic beta-cells and are required for the differentiation and function of these endocrine cells. HNF4alpha activates numerous genes and mutations in its gene are associated with maturity onset diabetes of the young. Cofactors and transcription factors that interact with HNF4alpha are crucial to modulate its transcriptional activity, since the latter is not regulated by conventional ligands. These transcriptional partners interact mainly through the HNF4alpha AF-1 module and the ligand binding domain, which contains the AF-2 module. Here, we showed that Isl1 could enhance the HNF4alpha-mediated activation of transcription of the HNF1alpha, PPARalpha and insulin I promoters. Isl1 interacted with the HNF4alpha AF-2 but also required the HNF4alpha carboxy-terminal F domain for optimal interaction and transcriptional synergy. More specifically, we found that naturally occurring HNF4alpha isoforms, differing only in their F domain, exhibited different abilities to interact and synergize with Isl1, extending the crucial transcriptional modulatory role of the HNF4alpha F domain. HNF4alpha interacted with both the homeodomain and the first LIM domain of Isl1. We found that the transcriptional synergy between HNF4alpha and Isl1 involved an increase in HNF4alpha loading on promoter. The effect was more pronounced on the rat insulin I promoter containing binding sites for both HNF4alpha and Isl1 than on the human HNF1alpha promoter lacking an Isl1 binding site. Moreover, Isl1 could mediate the recruitment of the cofactor CLIM2 resulting in a further transcriptional enhancement of the HNF1alpha promoter activity.

Hepatocyte nuclear factor 4alpha enhances the hepatocyte nuclear factor 1alpha-mediated activation of transcription.

Hepatocyte Nuclear Factor 1alpha (HNF1alpha) and Hepatocyte Nuclear Factor 4alpha (HNF4alpha) are two liver-enriched transcription factors coexpressed in specific tissues where they play a crucial role through their involvement in a complex cross-regulatory network. HNF1alpha down regulates HNF4alpha-mediated activation of transcription via a direct protein-protein interaction. Here we show that HNF4alpha enhances the transcriptional activity of HNF1alpha in a DNA binding independent manner, thus indicating that it behaves as a HNF1alpha coactivator. Using mutations in the ligand binding domain (LBD) of HNF4alpha, we confirmed the involvement of the Activation Function 2 module and demonstrated the requirement of the integrity of the LBD for the interaction with HNF1alpha. Moreover, we show that HNF4alpha cooperates with p300 to achieve the highest HNF1alpha-mediated transcription rates. Our findings highlight a new way by which HNF4alpha can regulate gene expression and extend our knowledge of the complexity of the transcriptional network involving HNF4alpha and HNF1alpha.

[Micro-array based technologies to study the proteome: technological progress and applications]. / Nouvelle approche globale en protéomique: les biopuces à protéines. Techniques actuelles et applications.

Since these twenty last years, there is an increasing interest for large-scale analysis of biological function. In the field of transcriptome, the emergence of microarray-based technologies and the design of DNA biochips allow high-throughput studies of RNA expression in cell and tissue at a given moment. In the field of proteome, methods of reference are still the 2D electrophoresis followed by analysis with mass spectrometry. Technological progress makes it possible to apply microarray methods to proteomics study : they are protein biochips or protein arrays. Expression analysis of proteins in a cell or a tissue in simultaneous and highly parallel way give further information for large-scale studies of signaling pathway. Numerous applications of protein microarray-based assays are described in basic biological research and in medical research to identify diagnostic biomarkers of inflammatory and cancerous pathologies and to find out news drugs and new therapeutic targets. This review summarizes concrete applications of microarray-based technology in the field of proteome, describes fundamental technical stages in protein array development and highlights critical points which will be useful to improve this emerging proteomic method.

Hepatocyte nuclear factor 4 alpha isoforms originated from the P1 promoter are expressed in human pancreatic beta-cells and exhibit stronger transcriptional potentials than P2 promoter-driven isoforms.

The nuclear receptor hepatocyte nuclear factor (HNF) 4 alpha is involved in a transcriptional network and plays an important role in pancreatic beta-cells. Mutations in the HNF4 alpha gene are correlated with maturity-onset diabetes of the young 1. HNF4 alpha isoforms result from both alternative splicing and alternate usage of promoters P1 and P2. It has recently been reported that HNF4 alpha transcription is driven almost exclusively by the P2 promoter in pancreatic islets. We observed that transcripts from both P1 and P2 promoters were expressed in human pancreatic beta-cells and in the pancreatic beta-cell lines RIN m5F and HIT-T15. Expression of HNF4 alpha proteins originating from the P1 promoter was confirmed by immunodetection. Due to the presence of the activation function module AF-1, HNF4 alpha isoforms originating from the P1 promoter exhibit stronger transcriptional activities and recruit coactivators more efficiently than isoforms driven by the P2 promoter. Conversely, activities of isoforms produced by both promoters were similarly repressed by the corepressor small heterodimer partner. These behaviors were observed on the promoter of HNF1 alpha that is required for beta-cell function. Our results highlight that expression of P1 promoter-driven isoforms is important in the control of pancreatic beta-cell function.

[Are mitochondria targets of anticancer drugs responsible for apoptosis?]. / Mécanismes proapoptotiques des agents anticancéreux: la mitochondrie, une cible pharmacologique potentielle?

The vast majority of both chemical and physical anticancer treatments act through the induction of apoptotic cell death in vitro and in vivo. In numerous experimental systems, the apoptotic processes can be divided into three different phases. In the first one, multiple pro-apoptotic signal transduction pathways (e.g. P53, ROS production, etc.) are activated by various factors including anti cancer drugs. This first step is followed by an intermediate phase in which pro-apoptotic signals converge to mitochondria which in turn can finally trigger the last degradation phase of apoptosis. Consequently, mitochondria, play a pivotal role in the executive phase of apoptosis and could represent a novel attractive target for pro-apoptotic drugs. Indeed, unlike conventional anti tumour drugs which trigger pro-apoptotic signal transduction pathways upstream mitochondria, several compounds were shown to act directly on mitochondria to induce apoptosis. These drugs include betulinic acid, lonidamine, arsenic trioxide and two retinoids like CD437/AHPN and fenretinide/4-HPR. This review summarizes new data concerning these drugs targetted to mitochondria and highlights the new perspective they may offer in cancer therapy.

Progression of actinic keratosis to squamous cell carcinoma of the skin correlates with deletion of the 9p21 region encoding the p16(INK4a) tumor suppressor.

Actinic keratoses (AKs) are pre-neoplastic lesions that can develop into squamous cell carcinomas (SCCs) of the skin. Often AK and SCC have commonly altered p53. A status of another tumor suppressor, the p16(INK4a), was reported for SCC but not for AK. A comparative study of SCC and AK human samples by loss of heterozygosity (LOH) analysis determined that the p16(INK4a/ARF) locus is less frequently altered in AKs than in SCCs. These LOH data highly correlated with immunohistochemical findings demonstrating the presence of p16(INK4a) in the AK skin samples but its absence in SCC lesions. Our results imply that progression of AK into SCC may involve inactivation of p16(INK4a).

Real time RT-PCR shows correlation between retinoid-induced apoptosis and NGF-R mRNA levels.

Neurotrophins and retinoic acid have a critical role in the differentiation and the survival of neurons. All-trans-, 9-cis-retinoic acid (10(-6) M) or NGF (50-100 ng/ml) induced morphologic differentiation and inhibited cell growth in SH-SY5Y neuroblastoma cells after 7 days of culture. Continuous treatment of undifferentiated cells with all-trans- or 9-cis-retinoic (10(-6) M) did not induce apoptosis, whereas NGF-differentiated cells showed dramatic apoptosis after 2 to 4 days of retinoic acid treatment as evidenced by TUNEL reaction and flow cytometry analysis following propidium iodide staining. Addition of Ro41-5253 blocked all-trans-retinoic-induced apoptosis, suggesting that the apoptotic signaling pathway was mediated by RARs. The effects of all-trans- or 9-cis-retinoic acid on the expression of NGF receptors was evaluated using real-time fluorescence reverse transcription-PCR. A slight transient increase in the expression of p75(NGFR) mRNA was observed by 2 to 4 h after retinoid treatment of undifferentiated cells, whereas a larger increase in the expression of both TrkA and p75(NGFR) mRNA up to threefold the basal level, was observed by 2 to 6 h after retinoid treatment of NGF-differentiated cells. Our results suggest that NGF-differentiated cells may be more susceptible to retinoid-induced apoptosis than undifferentiated cells.

Maturity-onset diabetes of the young Type 1 (MODY1)-associated mutations R154X and E276Q in hepatocyte nuclear factor 4alpha (HNF4alpha) gene impair recruitment of p300, a key transcriptional co-activator.

Hepatocyte nuclear factor 4alpha (HNF4alpha) is a nuclear receptor involved in glucose homeostasis and is required for normal beta-cell function. Mutations in the HNF4alpha gene are associated with maturity-onset diabetes of the young type 1. E276Q and R154X mutations were previously shown to impair intrinsic transcriptional activity (without exogenously supplied co-activators) of HNF4alpha. Given that transcriptional partners of HNF4alpha modulate its intrinsic transcriptional activity and play crucial roles in HNF4alpha function, we investigated the effects of these mutations on potentiation of HNF4alpha activity by p300, a key co-activator for HNF4alpha. We show here that loss of HNF4alpha function by both mutations is increased through impaired physical interaction and functional cooperation between HNF4alpha and p300. Impairment of p300-mediated potentiation of HNF4alpha transcriptional activity is of particular importance for the E276Q mutant since its intrinsic transcriptional activity is moderately affected. Together with previous results obtained with chicken ovalbumin upstream promoter-transcription factor II, our results highlight that impairment of recruitment of transcriptional partners represents an important mechanism leading to abnormal HNF4alpha function resulting from the MODY1 E276Q mutation. The impaired potentiations of HNF4alpha activity were observed on the promoter of HNF1alpha, a transcription factor involved in a transcriptional network and required for beta-cell function. Given its involvement in a regulatory signaling cascade, loss of HNF4alpha function may cause reduced beta-cell function secondary to defective HNF1alpha expression. Our results also shed light on a better structure-function relationship of HNF4alpha and on p300 sequences involved in the interaction with HNF4alpha.

Defective caspase-3 relocalization in non-small cell lung carcinoma.

Many anticancer drugs exert their cytotoxicity through DNA damage and induction of apoptosis. Small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC) have different sensitivity to treatment with radiation and chemotherapeutic agents with SCLC being more sensitive than NSCLC both in vitro and in vivo. This difference might be related to the different susceptibility of small and non-small cell lung carcinoma to undergo apoptosis. The aim of this study was to investigate if deficiencies in the apoptotic pathways can explain the intrinsic resistance of NSCLC to anti-cancer treatment. Three different triggers were used to induce apoptosis. Etoposide and gamma-radiation, which are important parts of clinical lung cancer treatment, induce DNA-damage, whereas Fas ligation induces receptor-mediated apoptotic pathways. NSCLC cells were cross-resistant to all treatments, whereas SCLC cells, which do not express pro-caspase-8, were resistant to alphaFas-, but not to DNA-damage-induced apoptosis. Cytochrome c release, activation of caspase-9 and the executioner caspase-3 were observed in both types of lung cancer cells. However, cleavage of known nuclear substrates for caspase-3, such as PARP and DFF45/ICAD, was documented only in the sensitive SCLC cells but not in the resistant NSCLC cells. Moreover, relocalization of active caspase-3 from the cytosol into the nucleus upon treatment was observed only in the SCLC cell line. These results indicate that the inhibition of apoptosis in NSCLC occurs downstream of mitochondrial changes and caspase activation, and upstream of nuclear events.

Differential effects of caspase inhibitors on endotoxin-induced myocardial dysfunction and heart apoptosis.

Endotoxin is one of the major factors causing myocardial depression and death during sepsis in humans. Recently, it was reported that endotoxin may induce cardiomyocyte apoptosis. Also, multiple caspase activation has been implicated in endotoxin-induced apoptosis in several organ systems. In this study, we investigated whether endotoxin would increase myocardial caspase activities and evaluated the effects of in vivo administration (3 mg/kg) of the broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone(z-VAD.fmk), the caspase-3-like inhibitor benzyloxycarbonyl-Asp-Glu-Val-Asp-chloromethylketone (z-DEVD.cmk), and the caspase-1-like inhibitor acetyl-Tyr-Val-Ala-Asp-chloromethylketone (Ac-YVAD. fmk), on endotoxin-induced myocardial dysfunction and apoptosis. Endotoxin administration (10 mg/kg iv) induced myocardial contractile dysfunction that was associated with caspase activity increases and nuclear apoptosis. Broad-spectrum z-VAD.fmk and z-DEVD.cmk improved endotoxin-induced myocardial dysfunction and reduced caspase activation and nuclear apoptosis when given immediately and 2 h after endotoxin. In contrast, no effects of Ac-YVAD.fmk were observed on myocardial function and caspase-induced apoptosis. Administration of caspase inhibitors 4 h after endotoxin treatment was not able to protect the rat heart from myocardial dysfunction and nuclear apoptosis. These observations provide evidence that in our model, caspase activation plays a role in endotoxin-induced myocardial apoptosis. Caspase inhibition strategy may represent a therapeutic approach to endotoxin-induced myocardial dysfunction.

Control of retinoic acid receptor heterodimerization by ligand-induced structural transitions. A novel mechanism of action for retinoid antagonists.

Heterodimerization of retinoic acid receptors (RARs) with 9-cis-retinoic receptors (RXRs) is a prerequisite for binding of RXR.RAR dimers to DNA and for retinoic acid-induced gene regulation. Whether retinoids control RXR/RAR solution interaction remains a debated question, and we have used in vitro and in vivo protein interaction assays to investigate the role of ligand in modulating RXR/RAR interaction in the absence of DNA. Two-hybrid assay in mammalian cells demonstrated that only RAR agonists were able to increase significantly RAR interaction with RXR, whereas RAR antagonists inhibited RXR binding to RAR. Quantitative glutathione S-transferase pull-down assays established that there was a strict correlation between agonist binding affinity for the RAR monomer and the affinity of RXR for liganded RAR, but RAR antagonists were inactive in inducing RXR recruitment to RAR in vitro. Alteration of coactivator- or corepressor-binding interfaces of RXR or RAR did not alter ligand-enhanced dimerization. In contrast, preventing the formation of a stable holoreceptor structure upon agonist binding strongly altered RXR.RAR dimerization. Finally, we observed that RAR interaction with RXR silenced RXR ligand-dependent activation function. We propose that ligand-controlled dimerization of RAR with RXR is an important step in the RXR.RAR activation process. This interaction is dependent upon adequate remodeling of the AF-2 structure and amenable to pharmacological inhibition by structurally modified retinoids.

Caspase inhibition prevents cardiac dysfunction and heart apoptosis in a rat model of sepsis.

Despite intensive therapy, severe septic shock is commonly associated with myocardial dysfunction and death in humans. No new therapies have proven efficiency against cardiovascular alterations in sepsis. Here, we addressed the question of a beneficial effect of pharmacological inhibition of caspases on myocardial dysfunction following endotoxin treatment. Hearts from rats treated with endotoxin (10 mg/kg, intravenously) were isolated 4 h posttreatment for analysis. Assessment of myocardial contractility ex vivo and detection of apoptosis were performed. Hearts from endotoxin-treated rats displayed multiple caspase activities and also typical apoptosis pattern as detected by TUNEL, DNA fragmentation assays, and cytochrome c release as compared with control rats. z-VAD.fmk (3 mg/kg, intravenously), a broad spectrum caspase inhibitor (but not the irrelevant peptide z-FA.fmk), in coinjection with endotoxin, not only reduced caspase activities and nuclear apoptosis but also completely prevented endotoxin-induced myocardial dysfunction evaluated 4 h and even 14 h after endotoxin challenge. These data indicate that caspase activation plays an important role in myocardial cell dysfunction. Moreover, these results suggest that inhibitors of caspases may have important therapeutic applications in sepsis.

Functional properties of the R154X HNF-4alpha protein generated by a mutation associated with maturity-onset diabetes of the young, type 1.

Mutations in the hepatocyte nuclear factor 4alpha (HNF-4alpha) gene are associated with one form of maturity-onset diabetes of the young (MODY1). The R154X mutation generates a protein lacking the E-domain which is required for normal HNF-4alpha functions. Since pancreatic beta-cell dysfunction is a feature of MODY1 patients, we compared the functional properties of the R154X mutant in insulin-secreting pancreatic beta-cells and non-beta-cells. The R154X mutation did not affect nuclear localisation in beta-cells and non-beta-cells. However, it did lead to a greater impairment of HNF-4a function in beta-cells compared to non-beta-cells, including a complete loss of transactivation activity and a dominant-negative behaviour. .