Negative prognostic value of high levels of intracellular poly(ADP-ribose) in non-small cell lung cancer.

BACKGROUND: Cisplatin-resistant non-small cell lung cancer (NSCLC) cells are often characterized by alterations in vitamin B-related metabolic processes, including the overexpression and hyperactivation of poly(ADP-ribose) polymerase 1 (PARP1) and the downregulation of pyridoxal kinase (PDXK), correlating with elevated apoptosis resistance. Low PDXK expression is an established negative prognostic factor in NSCLC. PATIENTS AND METHODS: We determined by immunohistochemistry the expression of PARP1 and the level of its product, poly(ADP-ribose) (PAR), in two independent cohorts of patients with resected NSCLC. RESULTS: Intratumoral high levels (above median) of PAR (but not PARP1 protein levels) had a negative prognostic impact in both the training (92 stage I subjects) and validation (133 stage I and II subjects) cohorts, as determined by univariate and multivariate analyses. The simultaneous assessment of PAR and PDXK protein levels improved risk stratification. CONCLUSION: NSCLC patients with high intratumoral PARP1 activity (i.e. elevated PAR levels above median) and low PDXK expression (below median) had a dismal prognosis, while patients with low PARP1 activity and high PDXK expression had a favorable outcome. Altogether, these results underscore the clinical potential and possible therapeutic relevance of these biomarkers.

Systems biology of cisplatin resistance: past, present and future.

The platinum derivative cis-diamminedichloroplatinum(II), best known as cisplatin, is currently employed for the clinical management of patients affected by testicular, ovarian, head and neck, colorectal, bladder and lung cancers. For a long time, the antineoplastic effects of cisplatin have been fully ascribed to its ability to generate unrepairable DNA lesions, hence inducing either a permanent proliferative arrest known as cellular senescence or the mitochondrial pathway of apoptosis. Accumulating evidence now suggests that the cytostatic and cytotoxic activity of cisplatin involves both a nuclear and a cytoplasmic component. Despite the unresolved issues regarding its mechanism of action, the administration of cisplatin is generally associated with high rates of clinical responses. However, in the vast majority of cases, malignant cells exposed to cisplatin activate a multipronged adaptive response that renders them less susceptible to the antiproliferative and cytotoxic effects of the drug, and eventually resume proliferation. Thus, a large fraction of cisplatin-treated patients is destined to experience therapeutic failure and tumor recurrence. Throughout the last four decades great efforts have been devoted to the characterization of the molecular mechanisms whereby neoplastic cells progressively lose their sensitivity to cisplatin. The advent of high-content and high-throughput screening technologies has accelerated the discovery of cell-intrinsic and cell-extrinsic pathways that may be targeted to prevent or reverse cisplatin resistance in cancer patients. Still, the multifactorial and redundant nature of this phenomenon poses a significant barrier against the identification of effective chemosensitization strategies. Here, we discuss recent systems biology studies aimed at deconvoluting the complex circuitries that underpin cisplatin resistance, and how their findings might drive the development of rational approaches to tackle this clinically relevant problem.

Predictive biomarkers for cancer therapy with PARP inhibitors.

Poly(ADP-ribose) polymerase (PARP) inhibitors have raised high expectations for the treatment of multiple malignancies. PARP inhibitors, which can be used as monotherapies or in combination with DNA-damaging agents, are particularly efficient against tumors with defects in DNA repair mechanisms, in particular the homologous recombination pathway, for instance due to BRCA mutations. Thus, deficient DNA repair provides a framework for the success of PARP inhibitors in medical oncology. Here, we review encouraging results obtained in recent clinical trials investigating the safety and efficacy of PARP inhibitors as anticancer agents. We discuss emerging mechanisms of regulation of homologous recombination and how inhibition of DNA repair might be used in cancer therapy. We surmise that the identification of patients that are likely to benefit from PARP inhibition will improve the clinical use of PARP inhibitors in a defined target population. Thus, we will place special emphasis on biomarker discovery.

Characterization of novel MPS1 inhibitors with preclinical anticancer activity.

Monopolar spindle 1 (MPS1), a mitotic kinase that is overexpressed in several human cancers, contributes to the alignment of chromosomes to the metaphase plate as well as to the execution of the spindle assembly checkpoint (SAC). Here, we report the identification and functional characterization of three novel inhibitors of MPS1 of two independent structural classes, N-(4-{2-[(2-cyanophenyl)amino][1,2,4]triazolo[1,5-a]pyridin-6-yl}phenyl)-2-phenylacetamide (Mps-BAY1) (a triazolopyridine), N-cyclopropyl-4-{8-[(2-methylpropyl)amino]-6-(quinolin-5-yl)imidazo[1,2-a]pyrazin-3-yl}benzamide (Mps-BAY2a) and N-cyclopropyl-4-{8-(isobutylamino)imidazo[1,2-a]pyrazin-3-yl}benzamide (Mps-BAY2b) (two imidazopyrazines). By selectively inactivating MPS1, these small inhibitors can arrest the proliferation of cancer cells, causing their polyploidization and/or their demise. Cancer cells treated with Mps-BAY1 or Mps-BAY2a manifested multiple signs of mitotic perturbation including inefficient chromosomal congression during metaphase, unscheduled SAC inactivation and severe anaphase defects. Videomicroscopic cell fate profiling of histone 2B-green fluorescent protein-expressing cells revealed the capacity of MPS1 inhibitors to subvert the correct timing of mitosis as they induce a premature anaphase entry in the context of misaligned metaphase plates. Hence, in the presence of MPS1 inhibitors, cells either divided in a bipolar (but often asymmetric) manner or entered one or more rounds of abortive mitoses, generating gross aneuploidy and polyploidy, respectively. In both cases, cells ultimately succumbed to the mitotic catastrophe-induced activation of the mitochondrial pathway of apoptosis. Of note, low doses of MPS1 inhibitors and paclitaxel (a microtubular poison) synergized at increasing the frequency of chromosome misalignments and missegregations in the context of SAC inactivation. This resulted in massive polyploidization followed by the activation of mitotic catastrophe. A synergistic interaction between paclitaxel and MPS1 inhibitors could also be demonstrated in vivo, as the combination of these agents efficiently reduced the growth of tumor xenografts and exerted superior antineoplastic effects compared with either compound employed alone. Altogether, these results suggest that MPS1 inhibitors may exert robust anticancer activity, either as standalone therapeutic interventions or combined with microtubule-targeting chemicals.

Molecular mechanisms of cisplatin resistance.

Platinum-based drugs, and in particular cis-diamminedichloroplatinum(II) (best known as cisplatin), are employed for the treatment of a wide array of solid malignancies, including testicular, ovarian, head and neck, colorectal, bladder and lung cancers. Cisplatin exerts anticancer effects via multiple mechanisms, yet its most prominent (and best understood) mode of action involves the generation of DNA lesions followed by the activation of the DNA damage response and the induction of mitochondrial apoptosis. Despite a consistent rate of initial responses, cisplatin treatment often results in the development of chemoresistance, leading to therapeutic failure. An intense research has been conducted during the past 30 years and several mechanisms that account for the cisplatin-resistant phenotype of tumor cells have been described. Here, we provide a systematic discussion of these mechanism by classifying them in alterations (1) that involve steps preceding the binding of cisplatin to DNA (pre-target resistance), (2) that directly relate to DNA-cisplatin adducts (on-target resistance), (3) concerning the lethal signaling pathway(s) elicited by cisplatin-mediated DNA damage (post-target resistance) and (4) affecting molecular circuitries that do not present obvious links with cisplatin-elicited signals (off-target resistance). As in some clinical settings cisplatin constitutes the major therapeutic option, the development of chemosensitization strategies constitute a goal with important clinical implications.

Illicit survival of cancer cells during polyploidization and depolyploidization.

Tetraploidy and the depolyploidization of tetraploid cells may contribute to oncogenesis. Several mechanisms have evolved to avoid the generation, survival, proliferation and depolyploidization of tetraploids. Cells that illicitly survive these checkpoints are prone to chromosomal instability and aneuploidization. Along with their replication, tetraploids constantly undergo chromosomal rearrangements that eventually lead to pseudodiploidy by two non-exclusive mechanisms: (i) multipolar divisions and (ii) illicit bipolar divisions in the presence of improper microtubule-kinetochore attachments. Here, we describe the regulation and the molecular mechanisms that underlie such a 'polyploidization-depolyploidization' cascade, while focusing on the role of oncogenes and tumor suppressor genes in tetraploidy-driven tumorigenesis. We speculate that the identification of signaling/metabolic cascades that are required for the survival of tetraploid or aneuploid (but not diploid) cancer cells may pave the way for the development of novel broad-spectrum anticancer agents.

A novel source of tetraploid cancer cell precursors: telomere insufficiency links aging to oncogenesis.

Epithelial cancers of the elderly are caused by a combination of telomere dysfunction and the mutational invalidation of major tumor suppressors including p53. A recent article published in Cell by Davoli et al. shows that the simultaneous elimination of p53 and telomerase causes a state of chronic DNA damage that results in tetraploidization through endoreplication, that is, two consecutive S phases that are not separated by mitosis. As tetraploid cells represent a metastable intermediate between normal diploidy and cancer-associated aneuploidy, this novel route to tetraploidization may constitute (one of) the functional link(s) between aging and carcinogenesis.

An automated fluorescence videomicroscopy assay for the detection of mitotic catastrophe.

Mitotic catastrophe can be defined as a cell death mode that occurs during or shortly after a prolonged/aberrant mitosis, and can show apoptotic or necrotic features. However, conventional procedures for the detection of apoptosis or necrosis, including biochemical bulk assays and cytofluorometric techniques, cannot discriminate among pre-mitotic, mitotic and post-mitotic death, and hence are inappropriate to monitor mitotic catastrophe. To address this issue, we generated isogenic human colon carcinoma cell lines that differ in ploidy and p53 status, yet express similar amounts of fluorescent biosensors that allow for the visualization of chromatin (histone H2B coupled to green fluorescent protein (GFP)) and centrosomes (centrin coupled to the Discosoma striata red fluorescent protein (DsRed)). By combining high-resolution fluorescence videomicroscopy and automated image analysis, we established protocols and settings for the simultaneous assessment of ploidy, mitosis, centrosome number and cell death (which in our model system occurs mainly by apoptosis). Time-lapse videomicroscopy showed that this approach can be used for the high-throughput detection of mitotic catastrophe induced by three mechanistically distinct anti-mitotic agents (dimethylenastron (DIMEN), nocodazole (NDZ) and paclitaxel (PTX)), and - in this context - revealed an important role of p53 in the control of centrosome number.

Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes.

Cell death is essential for a plethora of physiological processes, and its deregulation characterizes numerous human diseases. Thus, the in-depth investigation of cell death and its mechanisms constitutes a formidable challenge for fundamental and applied biomedical research, and has tremendous implications for the development of novel therapeutic strategies. It is, therefore, of utmost importance to standardize the experimental procedures that identify dying and dead cells in cell cultures and/or in tissues, from model organisms and/or humans, in healthy and/or pathological scenarios. Thus far, dozens of methods have been proposed to quantify cell death-related parameters. However, no guidelines exist regarding their use and interpretation, and nobody has thoroughly annotated the experimental settings for which each of these techniques is most appropriate. Here, we provide a nonexhaustive comparison of methods to detect cell death with apoptotic or nonapoptotic morphologies, their advantages and pitfalls. These guidelines are intended for investigators who study cell death, as well as for reviewers who need to constructively critique scientific reports that deal with cellular demise. Given the difficulties in determining the exact number of cells that have passed the point-of-no-return of the signaling cascades leading to cell death, we emphasize the importance of performing multiple, methodologically unrelated assays to quantify dying and dead cells.

The inositol 1,4,5-trisphosphate receptor regulates autophagy through its interaction with Beclin 1.

The inositol 1,4,5-trisphosphate receptor (IP(3)R) is a major regulator of apoptotic signaling. Through interactions with members of the Bcl-2 family of proteins, it drives calcium (Ca(2+)) transients from the endoplasmic reticulum (ER) to mitochondria, thereby establishing a functional and physical link between these organelles. Importantly, the IP(3)R also regulates autophagy, and in particular, its inhibition/depletion strongly induces macroautophagy. Here, we show that the IP(3)R antagonist xestospongin B induces autophagy by disrupting a molecular complex formed by the IP(3)R and Beclin 1, an interaction that is increased or inhibited by overexpression or knockdown of Bcl-2, respectively. An effect of Beclin 1 on Ca(2+) homeostasis was discarded as siRNA-mediated knockdown of Beclin 1 did not affect cytosolic or luminal ER Ca(2+) levels. Xestospongin B- or starvation-induced autophagy was inhibited by overexpression of the IP(3)R ligand-binding domain, which coimmunoprecipitated with Beclin 1. These results identify IP(3)R as a new regulator of the Beclin 1 complex that may bridge signals converging on the ER and initial phagophore formation.

Enhancement of radiation response in p53-deficient cancer cells by the Aurora-B kinase inhibitor AZD1152.

Overexpression of the Aurora-B kinase correlates with oncogenic transformation and poor prognosis. We evaluated the effects of the bona fide Aurora-B kinase inhibitor AZD1152 on tumor responses to ionizing radiation (IR). When p53(wt) HCT116 and A549 cells were pretreated with AZD1152-HQPA prior to IR, additive effects were observed. Interestingly, more pronounced tumoricidal effects were observed in p53-deficient HCT116 and HT29 cells, as well as A549 cells treated with the p53 inhibitor cyclic pifithrin-alpha. In vivo studies on xenografted mice confirmed enhanced tumor growth delay after the combination of IR plus AZD1152-IR as compared to IR alone. Again, this effect was more pronounced with p53-/- HCT116 and p53-mutant xenografts. The AZD1152-mediated radiosensitization was mimicked by knockdown of Aurora-B with a short interference RNA or by inhibition of Aurora-B by transfection with an inducible kinase-dead Aurora-B. The radiosensitizing effect of AZD1152 was lost in CHK2-/- and 14-3-3-/- HCT116 cells. Altogether, these data indicate that AZD1152 can radiosensitize tumor cell lines in vitro and in vivo, the fact that these effects are exacerbated in p53-deficient cancer cells is of potential interest for further clinical development.

Caspase independence of radio-induced cell death.

Colon carcinoma cells subjected to gamma-irradiation (4 Gy) manifest signs of apoptosis (caspase activation, chromatin condensation, phosphatidylserine (PS) exposure on the cell surface, sub-diploid DNA content), correlating with their radiosensitivity, which is increased in cells lacking the 14-3-3sigma protein as compared to wild-type controls. Inhibition of caspases by addition of Z-Val-Ala-DL-Asp (OMe)-fluoromethylketone, by stable transfection with the Baculovirus gene coding for p35, or by Bax knockout reduced all signs of apoptosis, yet failed to suppress radio-induced micro- and multinucleation. Moreover, pharmacological caspase inhibition, p35 expression or Bax knockout had no effect on the clonogenic survival that was reduced by gamma-irradiation and caspase inhibition failed to abolish the increased radiosensitivity of 14-3-3sigma-deficient cells. Micro- and multinucleation was detectable among non-apoptotic cells lacking PS exposure, as well as among cells undergoing apoptosis. Moreover, a fraction of micro- or multinucleated cells manifested caspase activation, and videomicroscopic analyses revealed that such cells could succumb to caspase-dependent apoptosis. Altogether, these results suggest that genomic instability induced by gamma-irradiation can trigger apoptosis, although apoptosis is dispensable for radio-induced clonogenic death.

Mechanisms of apoptosis induction by the HIV-1 envelope.

The envelope glycoprotein complex (Env) of human immunodeficiency virus-1 (HIV-1) can induce apoptosis by a cornucopia of distinct mechanisms. A soluble Env derivative, gp120, can kill cells through signals that are transmitted by chemokine receptors such as CXCR4. Cell surface-bound Env (gp120/gp41), as present on the plasma membrane of HIV-1-infected cells, can kill uninfected bystander cells expressing CD4 and CXCR4 (or similar chemokine receptors, depending on the Env variant) by at least three different mechanisms. First, a transient interaction involving the exchange of lipids between the two interacting cells ('the kiss of death') may lead to the selective death of single CD4-expressing target cells. Second, fusion of the interacting cells may lead to the formation of syncytia which then succumb to apoptosis in a complex pathway involving the activation of several kinases (cyclin-dependent kinase-1, Cdk1; checkpoint kinase-2, Chk2; mammalian target of rapamycin, mTOR; p38 mitogen-activated protein kinase, p38 MAPK; inhibitor of NF-kappaB kinase, IKK), as well as the activation of several transcription factors (NF-kappaB, p53), finally resulting in the activation of the mitochondrial pathway of apoptosis. Third, if the Env-expressing cell is at an early stage of imminent apoptosis, its fusion with a CD4-expressing target cell can precipitate the death of both cells, through a process that may be considered as contagious apoptosis and which does not involve Cdk1, mTOR, p38 nor p53, yet does involve mitochondria. Activation of some of the above- mentioned lethal signal transducers have been detected in patients' tissues, suggesting that HIV-1 may indeed trigger apoptosis through molecules whose implication in Env-induced killing has initially been discovered in vitro.

Hepatitis crónica autoinmune / Chronic autoimmune hepatitis

La hepatitis autoinmune (HA) es una enfermedad inflamatoria crónica con destrucción progresiva del hígado produciendo necrosis, fibrosis y cirrosis. Diferentes estudios sugieren que es una enfermedad en la que existe una predisposición genética multifactorial que acoplada a algún factor desencadenante, gatilla una respuesta autoinmune dirigida contra los hepatocitos. La predisposición genética es un hecho reconocido en la HA llegándose a considerar la presencia de HLA-DR3 y DR4, como factores de riegos por si mismos para desarrollar la enfermedad. Se presenta el caso de una niña de 12 años cuyo cuadro clínico, hallazgos de laboratorio y de estudios de gabinete, fueron compatibles con hepatitis crónica autoinmune en fase de cirrosis e hipertensión portal.

The hepatitis autoinmune (HA) it is an illness inflammatory chronicle with progressive destruction of the liver producing necrosis, fibrosis and cirrhosis. Difíerent studies suggest that it is an illness in that it exists a bias genetic multifactori that coupled to some factor to unchail an answer managed autoinmune it is triggered against the hepatocell. The genetic bias is a fact recognized in there is her being ended up considering the presence of HLA-DR3 and DR4, as factors of waterings for if same to develop the illness.

Cyclin-dependent kinase-1: linking apoptosis to cell cycle and mitotic catastrophe.

The cyclin-dependent kinase 1 (Cdk1), formerly called Cdc2 (or p34(Cdc2)), interacts with cyclin B1 to form an active heterodimer. The activity of Cdk1 is subjected to a complex spatiotemporary regulation, required to guarantee its scheduled contribution to the mitotic prophase and metaphase. Moreover, the activation of Cdk1 may be required for apoptosis induction in some particular pathways of cell killing. This applies to several clinically important settings, for instance to paclitaxel-induced killing of breast cancer cells, in which the ErbB2 receptor kinase can mediate apoptosis inhibition through inactivation of Cdk1. The activation of Cdk1 participates also in HIV-1-induced apoptosis, upstream of the p53-dependent mitochondrial permeabilization step. An unscheduled Cdk1 activation may contribute to neuronal apoptosis occurring in neurodegenerative diseases. Finally, the premature activation of Cdk1 can lead to mitotic catastrophe, for instance after irradiation-induced DNA damage. Thus, a cell type-specific modulation of Cdk1 might be taken advantage of for the therapeutic correction of pathogenic imbalances in apoptosis control.

The C-terminal moiety of HIV-1 Vpr induces cell death via a caspase-independent mitochondrial pathway.

Previous biochemical studies suggested that HIV-1-encoded Vpr may kill cells through an effect on the adenine nucleotide translocase (ANT), thereby causing mitochondrial membrane permeabilization (MMP). Here, we show that Vpr fails to activate caspases in conditions in which it induces cell killing. The knock-out of essential caspase-activators (Apaf-1 or caspase-9) or the knock-out of a mitochondrial caspase-independent death effector (AIF) does not abolish Vpr-mediated killing. In contrast, the cytotoxic effects of Vpr are reduced by transfection-enforced overexpression of two MMP-inhibitors, namely the endogenous protein Bcl-2 or the cytomegalovirus-encoded ANT-targeted protein vMIA. Vpr, which can elicit MMP through a direct effect on mitochondria, and HIV-1-Env, which causes MMP through an indirect pathway, exhibit additive (but not synergic) cytotoxic effects. In conclusion, it appears that Vpr induces apoptosis through a caspase-independent mitochondrial pathway.