Calpain 5 (CAPN5) is a non-classical member of the calpain family. It lacks the EF-hand motif characteristic of the classical calpains, calpain 1 and 2, but retains catalytic and Ca2+ binding non EF domains. Tra-3, an ortholog of CAPN5, is involved in necrotic cell death in C.elegans; although specific role of CAPN5 has not been investigated in the mammalian CNS. I compared relative mRNA levels of calpains in rat CNS, which revealed that CAPN5 is the second most highly expressed calpain. We examined relative levels of CAPN5 from late embryonic day 18 to postnatal day 90 and found lower mRNA but higher protein levels during CNS development. Using X –gal staining in Capn5 +/- mice, immunostaining of rat brain sections and SH-SY5Y cells, and subcellular fractionation of rat brain cortex, we found that CAPN5 is a non-cytoplasmic calpain localized in the nucleus and enriched in synaptic mitochondria. Proteinase K treatment of mitochondria and mitoplasts from B35 rat neuroblastoma cells and rat synaptic mitochondria revealed CAPN5 was localized on the inner mitochondrial membrane and released from mitochondria on membrane permeabilization with alamethicin. We used immunolabelling, confocal imaging, nuclear subfractionation and transient transfections to evaluate the subnuclear localization of CAPN5. CAPN5 was detected in punctate domains and associated with promyelocytic leukemia (PML) protein, a tumor suppressor protein. We further demonstrated that CAPN5 carries a nonconventional bipartite nuclear localization signal. Together, these findings demonstrate that CAPN5 is a non-cytosolic calpain, abundant in the CNS and localized to the mitochondria inner membrane and nuclear PML bodies.

Cell fate decisions incorporate distinct and overlapping mechanisms. The activity of caspase 3 was initially understood to be a cell death restricted event, however numerous studies have implicated this enzyme in the regulation of both differentiation and proliferation. How the activity of caspase 3 promotes a non-death cell fate remains unclear. Here we examine the role caspase 3 activity plays during skeletal muscle differentiation; in particular we explore the hypothesis that the mechanism of inducing DNA strand breaks during cell death is also a key feature of differentiation, albeit with a distinctly different outcome. We delineate the transient formation of Caspase 3/Caspase activated DNase (CAD) dependent DNA strand breaks during differentiation. The formation of these breaks is essential for differentiation and the regulation of specific genes. In particular expression of the cell cycle inhibitor p21 is related to the formation of a DNA strand break within the gene’s promoter element. Further, we explored the genome wide association of CAD using Chromatin Immunoprecipitation coupled to high through put sequencing (ChIP-seq). This approach identified a potential role for Caspase3/CAD in regulating the expression of Pax7. Here, a CAD directed DNA strand break in the Pax7 gene is correlated with decreased Pax7 expression, an outcome that has been shown to be critical for progress of the myogenic differentiation program. The regulation of Pax7 expression through a CAD induced DNA strand break raises an intriguing connection between this regulation and oncogenic transformation observed in alveolar rhabdomyosarcoma. The putative site of CAD induced DNA strand breaks that promote decreased Pax7 expression during differentiation corresponds to site of chromosomal translocations responsible for Pax7 fusion events in alveolar rhabdomyosarcoma.

Fibroblast growth factor 2 (FGF2) is a multifunctional protein translated as CUG-initiated, high molecular weight (hi FGF2) or AUG-initiated, low molecular weight (lo FGF2) isoforms with potentially distinct functions. Previous work showed that overexpression of hi- but not lo FGF2 elicited chromatin compaction resulting in cell death, by an intracrine route. A series of studies were undertaken aimed at extending our understanding of the intracrine action of Hi FGF2. Major findings are as follows: a. Hi FGF2 overexpression induces apoptotic cell death, as indicated by increased TUNEL staining, and mitochondrial participation (cytochrome c release to cytosol, rescue of the hi FGF2 phenotype by the anti-apoptotic protein Bcl-2. b. Increased expression of pro-survival signals/proteins that are known to upregulate Bcl-2, such as nuclear Akt; the PIM-1 kinase; and the heat shock protein hsp70, also rescued the hi FGF2-induced phenotype. c. The hi-FGF2 effect was associated with sustained, intracrine, activation of ERK, and was blocked by ERK inhibitors. d. FGF2 isoform specific affinity chromatography followed by mass spectroscopy identified several proteins as potentially interacting with hi FGF2; of these, the p68 RNA helicase and the hsp70 were further confirmed as interacting partners, by co-immunoprecipitation. e. Increased nuclear co-localization, and possibly interaction, between hi FGF2 and overexpressed hsp70 correlated with rescue from hi FGF2 induced cell death. f. Factors associated with cardiac pathology (isoproterenol, angiotensin II, endothelin I) also upregulated endogenous hi FGF2 in cardiac cells in culture. Adriamycin-induced cardiotoxicity in the rat, known to be linked to increased incidence of apoptosis, was also associated with increased endogenous hi FGF2. g. Hi FGF2 is expressed in the human heart (atria) and localizes in both cytosol and nuclei, suggesting a participation in human heart physiology and pathophysiology. Work presented here is consistent with the notion that endogenous hi FGF2 up-regulation may play a role in promoting cell death during prolonged tissue stress and dysfunction. It follows that processes related to hi FGF2 upregulation, hi FGF2-nuclear protein interactions and mechanisms of hi FGF2 induced cell death, represent potential therapeutic targets for modulating cell death.

Tikslas. Ištirti įvairaus oligomerizacijos laipsnio Aβ1-40 ir Aβ1-42 peptidų agregatų poveikį smegenėlių ląstelėms ir išaiškinti toksinio veikimo mechanizmus. Uždaviniai: 1.Įvertinti Aβ1-40 ir Aβ1-42 monomerų, oligomerų ir fibrilių poveikį neuronų-glijos ląstelių kultūros gyvybingumui. 2.Nustatyti ryšį tarp Aβ1-42 oligomerų toksiškumo ir dydžio. 3.Ištirti Aβ1-42 oligomerų poveikį neuronų ir mikroglijos ląstelių plazminės membranos įtampos pokyčiams ir įvertinti, kokią reikšmę Aβ1-42 oligomerų sukeltoje neuronų žūtyje turi išorinio kalcio koncentracija, NMDA receptorių ir endocitozės aktyvumas. 4.Nustatyti Aβ1-42 oligomerų poveikį smegenėlių kultūrai, papildytai J774 makrofagais 5.Nustatyti Aβ1-42 oligomerų poveikį mitochondrijų oksidacinio fosforilinimo sistemai. Mokslinis naujumas Šiame darbe nustatytas ryšys tarp Aβ1-42 oligomerų dydžio ir toksiškumo neuronams. Pirmą kartą parodyta, kad maži (toksiški) Aβ1-42 oligomerai (1–2 nm), koncentracijose, kurios gali susidaryti smegenyse patologinėmis sąlygomis, yra toksiški ląstelių kultūroje esantiems neuronams, tačiau nekeičia kitų, ląstelių kultūroje esančių, ląstelių gyvybingumo (mikroglijos ir astrocitų). Pirmieji išsiaiškinome, kad maži ir dideli Aβ1-42 oligomerai gali sukelti neuronų žūtį skirtingais mechanizmais: maži tiesiogiai veikdami neuronus, o dideli veikdami per glijos ląsteles. Mūsų tyrimai atskleidė iki šiol neaprašytą reiškinį, kad maži Aβ1-42 oligomerai gali sąlygoti ne tik neuroninių ląstelių žūtį, bet ir jų... [toliau žr. visą tekstą] / Objective and tasks. The aim of this study is to investigate the effects of Aβ1-40 and Aβ1-42 peptide aggregates at various degrees of oligomerization on cultivated neurons of brain cells and their toxic mechanisms. 1.To investigate the effect of various Aβ1–40 and Aβ1-42 aggregates on the viability of neuronal-glial cell cultures. 2.To determine the relationship between the toxicity of Aβ1-42 oli-gomers and their size. 3.To analyze the effect of Aβ1-42 oligomers on membrane potential of neuronal and glial cells. To assess the effect of extracellular Ca2+ concentration, NMDA receptors and activity of endocytosis on neu¬ronal death caused by Aβ1-42 oligomers. 4.To determine the effect of Aβ1-42 oligomers (4–6 nm in size) on neurons in CGC cultures with added macrophages J774 cells. 5.To determine the effect of Aβ1-42oligomers on respiration of isolated brain mitochondria. Scientific novelty This study determined the relationship between size of amyloid and its toxicity to neurons. For the first time it was shown that small Aβ1-42 oligomers of the size 1–3 nm were toxic to neurons in cell cultures at the concentrations which occur in brain under pathological conditions. However, they did not affect the viability of other cells (microglia and astrocytes) present in the cell cultures. For the first time it has been shown that small and large Aβ1-42 oligo-mers can cause neuronal death by different mechanisms: small oligomers by affecting neurons directly, and large oligomers... [to full text]

Our lab recently demonstrated that retargeting an alkylating agent, chlorambucil (Cbl), to the mitochondrion is a viable strategy to restore drug activity and overcome drug resistance in cancer. The mechanism of action of the mitochondria-targeted chlorambucil (mt-Cbl) was studied using a cervical carcinoma model. It was discovered that mt-Cbl bound to mitochondrial DNA and various mitochondrial proteins. A ρ° model revealed that the toxicity of mt-Cbl is largely dependent on its protein targets. Damage induced by mt-Cbl was found to result in the activation of several modes of caspase-independent cell death including necrosis. In contrast, Cbl was found to only activate caspase-dependent cell death that is highly sensitive to caspase inhibition. These results illustrate that the ability of mt-Cbl to activate various orthogonal cell death pathways is what allows mt-Cbl to bypass several drug resistance mechanisms, thus making mitochondrial retargeting a lucrative strategy for future anticancer drug development.

Our lab recently demonstrated that retargeting an alkylating agent, chlorambucil (Cbl), to the mitochondrion is a viable strategy to restore drug activity and overcome drug resistance in cancer. The mechanism of action of the mitochondria-targeted chlorambucil (mt-Cbl) was studied using a cervical carcinoma model. It was discovered that mt-Cbl bound to mitochondrial DNA and various mitochondrial proteins. A ρ° model revealed that the toxicity of mt-Cbl is largely dependent on its protein targets. Damage induced by mt-Cbl was found to result in the activation of several modes of caspase-independent cell death including necrosis. In contrast, Cbl was found to only activate caspase-dependent cell death that is highly sensitive to caspase inhibition. These results illustrate that the ability of mt-Cbl to activate various orthogonal cell death pathways is what allows mt-Cbl to bypass several drug resistance mechanisms, thus making mitochondrial retargeting a lucrative strategy for future anticancer drug development.

This thesis summarises research performed with the intent of exploring the many facets of cell death. In the first part of the thesis, the fate of the formin-homology domain containing protein FHOD1 during apoptosis is examined (research performed in the laboratory of Dr. Sophie Roy) and evidence for the cleavage of FHOD1 by caspase-3 at the SVPD616 site is demonstrated. Moreover, the C-terminal FHOD1 cleavage product is shown to translocate to the nucleolus where it inactivates RNA polymerase I transcription. / In the second part of the thesis, the role of the RNA-binding protein HuR in cancer cell migration and invasion, as well as in multidrug resistance is determined using RNA interference to knockdown the expression of HuR in HeLa and KB-V1 cells respectively (research performed in the laboratory of Dr. Imed Gallouzi). In this part of the thesis, HuR is shown to promote cancer cell migration and invasion by stabilizing the beta-actin mRNA in a U-rich-dependent manner. Moreover, evidence is shown for the potential involvement of HuR in the phenomenon of multidrug resistance possibly through the post-transcriptional regulation of the multidrug resistance 1 gene.

Ultrasound has been identified as a possible non-invasive drug delivery device that could avoid many of the problems found in traditional therapeutics. Studies have shown that ultrasound can deliver molecules into cells; however, the applicability of ultrasound has been limited due to uncontrollable cellular viability losses after sonication. In this study, we sought to quantify the heterogeneous bioeffects of ultrasound in order to gain more insight into how ultrasound affects cells. We were also concerned with identifying the causes of and preventing programmed cell death caused by ultrasound exposure. In order to accomplish these objectives, we used flow cytometry to group cells into quantifiable characteristic populations. This allowed us to identify the relative importance of different forms of rapid cell death. We found that up to 65% of cells (at the highest ultrasound pressure studied) can lose viability rapidly and, for the first time, quantified them among three distinct populations: (1) cells that retain normal size but lose plasma membrane integrity; (2) intact nuclei surrounded by plasma membrane remnants; (3) debris resulting from cellular lysis. Our analysis was supported by mechanical sorting of these populations and subsequent imaging using confocal microscopy. We then monitored the viable populations for 6 h after ultrasound exposure. Results indicated that up to 15% of viable cells (at the highest ultrasound pressure studied) underwent apoptosis, which we showed was associated with an influx of intracellular Ca2+; therefore, we developed a method of chelating intracellular Ca2+ after sonication in an effort to maintain viability of those cells. Using this technique, we showed for the first time that cells could be saved, and we were able to prevent apoptosis by 50%, thereby increasing the overall viability of cells exposed to ultrasound. We conclude that ultrasound is a useful method to deliver molecules into cells and that appropriate selection of sonication conditions can minimize cell death by rapid and apoptotic mechanisms.

The precise structure of the adult Drosophila eye results from a coordinated process of cell sorting, differentiation and selective cell death in the retinal epithelium. Mutations in the gene echinus cause supernumerary pigment cells due to insufficient cell death. This study reports the identification of echinus and the characterization of its role in Drosophila retinal development. Using a combination of deletion mapping, gene expression analysis and genomic sequencing, echinus was cloned and several alleles were sequenced. echinus encodes a ~180kDa protein containing an ubiquitin hydrolase domain at its N-terminus and a polyglutamine tract at its C-terminus. echinus is expressed in the retina during pupal development and mutants of echinus have decreased levels of apoptosis during several stages of retinal development. Defects in the cell sorting process that precedes cell death are also observed in echinus loss-of-function mutants and echinus overexpression can cause defects in ommatidial rotation and the morphology of cone cells. echinus is a positive regulator of DE-cadherin and Enabled accumulation in adherens junctions of retinal epithelial cells. Genetic interactions were observed between echinus and the genes wingless, enabled and expanded. An immunofluorescence assay in Drosophila S2 cell cultured demonstrated that Echinus localizes to intracellular vesicles that do not appear to be endocytic in nature, and the C-terminal region of Echinus was shown to be necessary for this association. A protein interaction screen using an immunoprecipitation and mass spectrometry approach identified interactions between Echinus and the vesicle coat protein Clathrin, the scaffolding protein RACK1 and the casein kinase I epsilon (Dco). Co-immunoprecipitation additionally identified an interaction between Echinus and Enabled. This work has revealed echinus to be an important regulator of cell sorting and adherens junction formation in the developing retina and has identified multiple interactions between echinus and enabled, a regulator of the actin cytoskeleton.

The p53 protein is a primary mediator of apoptosis and growth arrest after exposure to DNA-damaging agents. Previous work has categorised a wild type p53 gene in the majority of childhood acute lymphoblastic leukaemia (ALL) cases, in which instance the p53 protein functions as a modulator of chemotherapy-induced cell death. In contrast, certain p53-induced proteins, such as p21WAF1, can act in an anti-apoptotic manner, and bestow resistance to chemotherapy. Previous studies of the p53 pathway in ALL have utilised cell lines and primary material. In this study a model of ALL was utilised that had previously been developed from a heterogeneous panel of patient biopsies established as xenografts in immune-deficient mice, and are adaptable for short term in vitro culture. A wild-type p53 protein response to etoposide and nutlin-3 exposure was a feature of the whole ALL xenograft panel, irrespective of clinical characteristics and disease biology. While a range of p53 target genes were induced in B-cell precursor (BCP)-ALL and T-ALL xenografts after etoposide exposure, there was negligible induction of p21WAF1 in T- ALL samples. Further work with the histone deacetylase inhibitor vorinostat facilitated p53-independent induction of p21WAF1 in BCP-ALL samples, yet failed to induce p21WAF1 in T- ALL. An association was observed between reduced p21WAF1 expression in the T-ALL samples and decreased histone H3 acetylation in the p21WAF1 promoter together with increased cytosine methylation in the first exon/intron of the p21WAF1 gene. These results suggest that p21WAF1 in T-ALL cells is subject to epigenetic modifications that cause transcriptional silencing. Defective induction of p21WAF1 in T-ALL xenografts was associated with increased sensitivity to the death-inducing effects of drugs, phosphatidylserine (PS) externalisation and caspase-3/-7 activity after drug exposure, indicating that p21WAF1 may exert an anti-apoptotic activity. As proof of principle, p21WAF1 was silenced in Nalm-6 cells by micro-RNA transduction and these cells exhibited increased sensitivity and rapid PS externalisation after drug exposure. A combination of a p21WAF1 inhibitory agent and vorinostat gave some pharmacological evidence to suggest that p21WAF1 inhibition could enhance drug efficacy. Overall, these investigations provide insight into the epigenetic regulation of p21WAF1 and demonstrate an anti-apoptotic role for p21WAF1 in childhood ALL cells.