Reduced occurrence of programmed cell death and gliosis in the retinas of juvenile rabbits after short-term treatment with intravitreous bevacizumab

OBJECTIVE: Bevacizumab has been widely used as a vascular endothelial growth factor antagonist in the treatment of retinal vasoproliferative disorders in adults and, more recently, in infants with retinopathy of prematurity. Recently, it has been proposed that vascular endothelial growth factor acts as a protective factor for neurons and glial cells, particularly in developing nervous tissue. The purpose of this study was to investigate the effects of bevacizumab on the developing retinas of juvenile rabbits. METHODS: Juvenile rabbits received bevacizumab intravitreously in one eye; the other eye acted as an untreated control. Slit-lamp and fundoscopic examinations were performed both prior to and seven days after treatment. At the same time, retina samples were analyzed using immunohistochemistry to detect autophagy and apoptosis as well as proliferation and glial reactivity. Morphometric analyses were performed, and the data were analyzed using the Mann-Whitney U test. RESULTS: No clinical abnormalities were observed in either treated or untreated eyes. However, immunohistochemical analyses revealed a reduction in the occurrence of programmed cell death and increases in both proliferation and reactivity in the bevacizumab-treated group compared with the untreated group. CONCLUSIONS: Bevacizumab appears to alter programmed cell death patterns and promote gliosis in the developing retinas of rabbits; therefore, it should be used with caution in developing eyes.

Mechanism and Efficiency of Cell Death of Type II Photosensitizers: Effect of Zinc Chelation

A series of meso-substituted tetra-cationic porphyrins, which have methyl and octyl substituents, was studied in order to understand the effect of zinc chelation and photosensitizer subcellular localization in the mechanism of cell death. Zinc chelation does not change the photophysical properties of the photosensitizers (all molecules studied are type II photosensitizers) but affects considerably the interaction of the porphyrins with membranes, reducing mitochondrial accumulation. The total amount of intracellular reactive species induced by treating cells with photosensitizer and light is similar for zinc-chelated and free-base porphyrins that have the same alkyl substituent. Zinc-chelated porphyrins, which are poorly accumulated in mitochondria, show higher efficiency of cell death with features of apoptosis (higher MTT response compared with trypan blue staining, specific acridine orange/ethidium bromide staining, loss of mitochondrial transmembrane potential, stronger cytochrome c release and larger sub-G1 cell population), whereas nonchelated porphyrins, which are considerably more concentrated in mitochondria, triggered mainly necrotic cell death. We hypothesized that zinc-chelation protects the photoinduced properties of the porphyrins in the mitochondrial environment.

Proteasome inhibition and ROS generation by 4-nerolidylcatechol induces melanoma cell death

Induction of apoptotic cell death in response to chemotherapy and other external stimuli has proved extremely difficult in melanoma, leading to tumor progression, metastasis formation and resistance to therapy. A promising approach for cancer chemotherapy is the inhibition of proteasomal activity, as the half-life of the majority of cellular proteins is under proteasomal control and inhibitors have been shown to induce cell death programs in a wide variety of tumor cell types. 4-Nerolidylcatechol (4-NC) is a potent antioxidant whose cytotoxic potential has already been demonstrated in melanoma tumor cell lines. Furthermore, 4-NC was able to induce the accumulation of ubiquitinated proteins, including classic targets of this process such as Mcl-1. As shown for other proteasomal inhibitors in melanoma, the cytotoxic action of 4-NC is time-dependent upon the pro-apoptotic protein Noxa, which is able to bind and neutralize Mcl-1. We demonstrate the role of 4-NC as a potent inducer of ROS and p53. The use of an artificial skin model containing melanoma also provided evidence that 4-NC prevented melanoma proliferation in a 3D model that more closely resembles normal human skin.

Fhos encodes a Drosophila Formin-Like Protein participating in autophagic programmed cell death

Larval tissues undergo programmed cell death (PCD) during Drosophila metamorphosis. PCD is triggered in a stage and tissue-specific fashion in response to ecdysone pulses. The understanding of how ecdysone induces the stage and tissue-specificity of cell death remains obscure. Several steroid-regulated primary response genes have been shown to act as key regulators of cellular responses to ecdysone by inducing a cascade of transcriptional regulation of late responsive genes. In this article, the authors identify Fhos as a gene that is required for Drosophila larval salivary gland destruction. Animals with a P-element mutation in Fhos possess persistent larval salivary glands, and precise excisions of this P-element insertion resulted in reversion of this salivary gland mutant phenotype. Fhos encodes the Drosophila homolog of mammalian Formin Fhos. Fhos is differentially transcribed during development and responds to ecdysone in a method that is similar to other cell death genes. Similarly to what has been shown for its mammalian counterpart, FHOS protein is translocated to the nucleus at later stages of cell death. Fhos mutants posses disrupted actin cytoskeleton dynamics in persistent salivary glands. Together, our data indicate that Fhos is a new ecdysone-regulated gene that is crucial for changes in the actin cytoskeleton during salivary gland elimination in Drosophila. genesis 50:672684, 2012. (c) 2012 Wiley Periodicals, Inc.

Carvedilol protects against apoptotic cell death induced by cisplatin in renal tubular epithelial cells

Cisplatin is a highly effective chemotherapeutic drug; however, its use is limited by nephrotoxicity. Studies showed that the renal injury produced by cisplatin involves oxidative stress and cell death mediated by apoptosis and necrosis in proximal tubular cells. The use of antioxidants to decrease cisplatin-induced renal cell death was suggested as a potential therapeutic measure. In this study the possible protective effects of carvedilol, a beta blocker with antioxidant activity, was examined against cisplatin-induced apoptosis in HK-2 human kidney proximal tubular cells. The mitochondrial events involved in this protection were also investigated. Four groups were used: controls (C), cisplatin alone at 25 mu M (CIS), cisplatin 25 mu M plus carvedilol 50 mu M (CV + CIS), and carvedilol alone 50 mu M (CV). Cell viability, apoptosis, caspase-9, and caspase-3 were determined. Data demonstrated that carvedilol effectively increased cell viability and minimized caspase activation and apoptosis in HK-2 cells, indicating this may be a promising drug to reduce nephrotoxicity induced by cisplatin.

Nuclear Transfer with Apoptotic Bovine Fibroblasts: Can Programmed Cell Death Be Reprogrammed?

Cell death by apoptosis is considered to be irreversible. However, reports have indicated that its reversibility is possible if the cells have not yet reached the "point of no return.'' In order to add new information about this topic, we used cells at different moments of apoptotic process as nuclear donors in somatic cell nuclear transfer (SCNT) in order to test if programmed cell death can be reversed. Adult bovine fibroblasts were treated with 10 mu M of staurosporine (STP) for 3 h and analyzed for phosphatidylserine externalization (Annexin assay) and presence of active caspase-9. Annexin-positive (Anx +) and Caspase-9-positive (Casp-9 +) cells were isolated by FACS and immediately transferred into enucleated in vitro matured bovine oocytes. After STP treatment, 89.9% of cells were Anx + (4.6% in control cells; p < 0.01) and 24.9% were Casp-9 + (2.4% in control cells; p < 0.01). Fusion and cleavage were not affected by the use apoptotic cells (p > 0.05). Also, the use of Anx + cells did not affect blastocyst production compared to control (26.4% vs. 22.9%, respectively; p > 0.05). However, blastocyst formation was affected by the use of Casp-9 + cells (12.3%; p < 0.05). These findings contribute to the idea of that apoptosis is reversible only at early stages. Additionally, we hypothesize that the "point of no return'' for apoptosis may be located around activation of Caspase-9.

Cocaine induces cell death and activates the transcription nuclear factor kappa-b in pc12 cells

Cocaine is a worldwide used drug and its abuse is associated with physical, psychiatric and social problems. The mechanism by which cocaine causes neurological damage is very complex and involves several neurotransmitter systems. For example, cocaine increases extracellular levels of dopamine and free radicals, and modulates several transcription factors. NF-κB is a transcription factor that regulates gene expression involved in cellular death. Our aim was to investigate the toxicity and modulation of NF-κB activity by cocaine in PC 12 cells. Treatment with cocaine (1 mM) for 24 hours induced DNA fragmentation, cellular membrane rupture and reduction of mitochondrial activity. A decrease in Bcl-2 protein and mRNA levels, and an increase in caspase 3 activity and cleavage were also observed. In addition, cocaine (after 6 hours treatment) activated the p50/p65 subunit of NF-κB complex and the pretreatment of the cells with SCH 23390, a D1 receptor antagonist, attenuated the NF-κB activation. Inhibition of NF-κB activity by using PDTC and Sodium Salicilate increased cell death caused by cocaine. These results suggest that cocaine induces cell death (apoptosis and necrosis) and activates NF-κB in PC12 cells. This activation occurs, at least partially, due to activation of D1 receptors and seems to have an anti-apoptotic effect on these cells.

Reduced occurrence of programmed cell death and gliosis in the retinas of juvenile rabbits after shortterm treatment with intravitreous bevacizumab

OBJECTIVE: Bevacizumab has been widely used as a vascular endothelial growth factor antagonist in the treatment of retinal vasoproliferative disorders in adults and, more recently, in infants with retinopathy of prematurity. Recently, it has been proposed that vascular endothelial growth factor acts as a protective factor for neurons and glial cells, particularly in developing nervous tissue. The purpose of this study was to investigate the effects of bevacizumab on the developing retinas of juvenile rabbits. METHODS: Juvenile rabbits received bevacizumab intravitreously in one eye; the other eye acted as an untreated control. Slit-lamp and fundoscopic examinations were performed both prior to and seven days after treatment. At the same time, retina samples were analyzed using immunohistochemistry to detect autophagy and apoptosis as well as proliferation and glial reactivity. Morphometric analyses were performed, and the data were analyzed using the Mann-Whitney U test. RESULTS: No clinical abnormalities were observed in either treated or untreated eyes. However, immunohistochemical analyses revealed a reduction in the occurrence of programmed cell death and increases in both proliferation and reactivity in the bevacizumab-treated group compared with the untreated group. CONCLUSIONS: Bevacizumab appears to alter programmed cell death patterns and promote gliosis in the developing retinas of rabbits; therefore, it should be used with caution in developing eyes

Retinal cell death induced by TRPV1 activation involves NMDA signaling and upregulation of nitric oxide synthases

The activation of the transient receptor potential vanilloid type 1 channel (TRPV1) has been correlated with oxidative and nitrosative stress and cell death in the nervous system. Our previous results indicate that TRPV1 activation in the adult retina can lead to constitutive and inducible nitric oxide synthase-dependent protein nitration and apoptosis. In this report, we have investigated the potential effects of TRPV1 channel activation on nitric oxide synthase (NOS) expression and function, and the putative participation of ionotropic glutamate receptors in retinal TRPV1-induced protein nitration, lipid peroxidation, and DNA fragmentation. Intravitreal injections of the classical TRPV1 agonist capsaicin up-regulated the protein expression of the inducible and endothelial NOS isoforms. Using 4,5-diaminofluorescein diacetate for nitric oxide (NO) imaging, we found that capsaicin also increased the production of NO in retinal blood vessels. Processes and perikarya of TRPV1-expressing neurons in the inner nuclear layer of the retina were found in the vicinity of nNOS-positive neurons, but those two proteins did not colocalize. Retinal explants exposed to capsaicin presented high protein nitration, lipid peroxidation, and cell death, which were observed in the inner nuclear and plexiform layers and in ganglion cells. This effect was partially blocked by AP-5, a NMDA glutamate receptor antagonist, but not by CNQX, an AMPA/kainate receptor antagonist. These data support a potential role for TRPV1 channels in physiopathological retinal processes mediated by NO, which at least in part involve glutamate release.

UVB-induced cell death signaling is associated with G1-S progression and transcription inhibition in primary human fibroblasts

DNA damage induced by ultraviolet (UV) radiation can be removed by nucleotide excision repair through two sub-pathways, one general (GGR) and the other specific for transcribed DNA (TCR), and the processing of unrepaired lesions trigger signals that may lead to cell death. These signals involve the tumor suppressor p53 protein, a central regulator of cell responses to DNA damage, and the E3 ubiquitin ligase Mdm2, that forms a feedback regulatory loop with p53. The involvement of cell cycle and transcription on the signaling to apoptosis was investigated in UVB-irradiated synchronized, DNA repair proficient, CS-B (TCR-deficient) and XP-C (GGR-deficient) primary human fibroblasts. Cells were irradiated in the G1 phase of the cell cycle, with two doses with equivalent levels of apoptosis (low and high), defined for each cell line. In the three cell lines, the low doses of UVB caused only a transient delay in progression to the S phase, whereas the high doses induced permanent cell cycle arrest. However, while accumulation of Mdm2 correlated well with the recovery from transcription inhibition at the low doses for normal and CS-B fibroblasts, for XP-C cells this protein was shown to be accumulated even at UVB doses that induced high levels of apoptosis. Thus, UVB-induced accumulation of Mdm2 is critical for counteracting p53 activation and apoptosis avoidance, but its effect is limited due to transcription inhibition. However, in the case of XP-C cells, an excess of unrepaired DNA damage would be sufficient to block S phase progression, which would signal to apoptosis, independent of Mdm2 accumulation. The data clearly discriminate DNA damage signals that lead to cell death, depending on the presence of UVB-induced DNA damage in replicating or transcribing regions.

Autophagic programmed cell death in the suspensor and endosperm of Vicia faba : An ultrastructural study

Programmed cell death (PCD) is a widely spread phenomenon among multi-cellular organisms. Without the deletion of cells no longer needed, the organism will not be able to develop in a predicted way. It is now belived that all cells have the capacity to self-destruct and that the survival of the cells is depending on the repression of this suicidal programme. PCD has turned out to show similarities in many different species and there are strong indications that the mechanisms running the programme might, at least in some parts, be evolutionarily conserced. PCD is a generic term for different programmes of cell destruction, such as apoptosis and autophagic PCD. An important tool to determine if a cell is undergoing PCD is the transmitting electron microscope. The aims of my study were to find out if, and in what way, the suspensor and endosperm in Vicia faba (Broad bean), which are short-lived structures, undergoes PCD. The endosperm degradation preceed the suspensor cell death and they differ to some extent ultrastructurally. The cell death occurs in both tissues about 13-14 days after pollination when the embryo proper is mature enough to support itself. It was found that both tissues are committed to autophagic PCD, a cell death characteristic of conspicuous formations of autophagic vacuoles. It was shown by histochemical staining that acid phosphatases are accumulated in these vacuoles but are also present in the cytoplasm. These vacuoles are similar to autophagic vacuoles formed in rat liver cells, indicating that autophagy is a widely spread phenomenon. DNA fragmentation is the first visible sign of PCD in both tissues and it is demonstrated by a labelling technique (TUNEL). In the endosperm nuclei the heterochromatin subsequently appears in the form of a network, while in the suspensor it is more conspicuous, with heterochromatin that forms large electron dense aggregates located close to the nuclear envelope. In the suspensor, the plastids develop into chromoplasts with lycopene crystals at the same time or shortly after DNA fragmentation. This is probably due to the fact that the suspensor plastids function as hormone producing organelles and support the embryo proper with indispensable growth factors. Later the embryo will be able to produce its own growth factors and the synthesis of these, in particular gibberelines, might be suppressed in the suspensor. The precursors can then be used for synthesis of lycopene instead. Both the suspensor and endosperm are going through autophagic PCD, but the process differs in some respects. This is probably due the the different function of the two tissues, and that the signals that trigger the process presumably are different. The embryo proper is probably the source of the death signal affecting the suspensor. The endosperm, which has a different origin and function, might be controlling the death signal within its own cell. The death might in this case be related to the age of the cell.

Hox genes and the regulation of programmed cell death in the embryonic central nervous system of Drosophila melanogaster

This study deals with the function and regulation of programmed cell death, or apoptosis, in the development of the embryonic central nervous system of Drosophila melanogaster. The first part provides a description of apoptosis-deficient embryos, which showed that preventing apoptosis does not cause gross morphological defects in the CNS, as it appears well organized despite the presence of too many cells. An analysis of the incidence and pattern of apoptosis over the course of development discloses a partly very orderly pattern suggesting tight spatio-temporal control, but also reveals random apoptotic cells, which suggests a certain amount of plasticity in the embryo. This analysis also allowed precise identification of some of the dying neural cells in the embryo, and establishment of single cell models for studying regulation of segment-specific apoptosis in the embryonic CNS. In the second part of the work, further investigations into mechanisms controlling segment-specific apoptosis revealed the involvement of two Hox genes, Antennapedia (Antp) and Ultrabithorax (Ubx), in this process. Hox genes control the formation of segment-specific structures in their domains of expression, but also regulate organ and tissue morphogenesis. The study presented here shows that Antp and Ubx play antagonistic roles in motoneuron survival in the embryo. Ubx expression in the CNS is strongly upregulated at a late point in development, when most cells have begun to differentiate. This upregulation shortly precedes Ubx-dependent, segment-specific apoptosis of two differentiated motoneurons. It could further be demonstrated that Antp is required for proper development of the NB7-3 lineage and for survival of the NB7-3 motoneuron in the anterior thoracic segments. In segments where Antp and Ubx expression overlaps, Ubx counteracts the anti-apoptotic function of Antp, resulting in cell death. Thus, these two Hox genes play opposing roles in the survival of differentiated neurons in the late developing nervous system. They thereby contribute to establishment of correct connections between outward-projecting neurons and their targets, which is crucial for the assembly of functional neural circuits, as these have to fulfill region-specific locomotion and sensory requirements along the antero-posterior body axis.

Mechanistic study of cell death induction by O 6-methylating agents, focusing on the role of homologous recombination as a molecular target for sensitization of tumor cells to chemotherapeutic alkylating agents

Chemotherapeutic SN1‑methylating agents are important anticancer drugs. They induce several covalent modifications in the DNA, from which O6‑methylguanine (O6MeG) is the main toxic lesion. In this work, different hypotheses that have been proposed to explain the mechanism of O6MeG‑triggered cell death were tested. The results of this work support the abortive processing model, which states that abortive post‑replicative processing of O6MeG‑driven mispairs by the DNA mismatch repair (MMR) machinery results in single‑strand gaps in the DNA that, upon a 2nd round of DNA replication, leads to DNA double‑strand break (DSB) formation, checkpoint activation and cell death. In this work, it was shown that O6MeG induces an accumulation of cells in the 2nd G2/M‑phase after treatment. This was accompanied by an increase in DSB formation in the 2nd S/G2/M‑phase, and paralleled by activation of the checkpoint kinases ATR and CHK1. Apoptosis was activated in the 2nd cell cycle. A portion of cells continue proliferating past the 2nd cell cycle, and triggers apoptosis in the subsequent generations. An extension to the original model is proposed, where the persistence of O6MeG in the DNA causes new abortive MMR processing in the 2nd and subsequent generations, where new DSB are produced triggering cell death. Interestingly, removal of O6MeG beyond the 2nd generation lead to a significant, but not complete, reduction in apoptosis, pointing to the involvement of additional mechanisms as a cause of apoptosis. We therefore propose that an increase in genomic instability resulting from accumulation of mis‑repaired DNA damage plays a role in cell death induction. Given the central role of DSB formation in toxicity triggered by chemotherapeutic SN1‑alkylating agents, it was aimed in the second part of this thesis to determine whether inhibition of DSB repair by homologous recombination (HR) or non‑homologous end joining (NHEJ) is a reasonable strategy for sensitizing glioblastoma cells to these agents. The results of this work show that HR down‑regulation in glioblastoma cells impairs the repair of temozolomide (TMZ)‑induced DSB. HR down‑regulation greatly sensitizes cells to cell death following O6‑methylating (TMZ) or O6‑chlorethylating (nimustine) treatment, but not following ionizing radiation. The RNAi mediated inhibition in DSB repair and chemo‑sensitization was proportional to the knockdown of the HR protein RAD51. Chemo‑sensitization was demonstrated for several HR proteins, in glioma cell lines proficient and mutated in p53. Evidence is provided showing that O6MeG is the primary lesion responsible for the increased sensitivity of glioblastoma cells following TMZ treatment, and that inhibition of the resistance marker MGMT restores the chemo‑sensitization achieved by HR down‑regulation. Data are also provided to show that inhibition of DNA‑PK dependent NHEJ does not significantly sensitized glioblastoma cells to TMZ treatment. Finally, the data also show that PARP inhibition with olaparib additionally sensitized HR down‑regulated glioma cells to TMZ. Collectively, the data show that processing of O6MeG through two rounds of DNA replication is required for DSB formation, checkpoint activation and apoptosis induction, and that O6MeG‑triggered apoptosis is also executed in subsequent generations. Furthermore, the data provide proof of principle evidence that down‑regulation of HR is a reasonable strategy for sensitizing glioma cells to killing by O6‑alkylating chemotherapeutics.

Prognostic significance of apoptotic cell death in bladder cancer: a tissue microarray study on 179 urothelial carcinomas from cystectomy specimens

To assess the prognostic significance of apoptosis related markers in bladder cancer.

Cell death modulation by intravenous immunoglobulin

The induction of cell death in immune cells by naturally occurring antibodies specific for death receptors may present an important antiinflammatory mechanism of intravenous immunoglobulin (IVIG). Conversely, the protection of tissue cells from death receptor-mediated apoptosis by blocking antibodies is thought to contribute to the beneficial effects of IVIG in certain inflammatory disorders such as toxic epidermal necrolysis, also known as Lyell's syndrome. In this review, we focus on recent insights into the role of functional antibodies against Fas, sialic acid-binding immunoglobulin-like lectin (Siglec)-8, and Siglec-9 receptors in IVIG-mediated cell survival or death effects. In addition, we examine a variety of factors in inflammatory disease that may interplay with these cellular events and influence the therapeutic efficacy or potency of IVIG. These involve activation status of the target cell, cytokine microenvironment, pathogenesis and stage of disease, individual genetic determinants, species characteristics, and batch-to-batch variations of IVIG preparations.