published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

The resistance of cancer cells to traditional chemotherapeutic agents is a major obstacle in the successful treatment of cancer. Cancer cells manipulate a variety of signaling pathways to enhance resistance to anticancer agents; such mechanisms include disrupting the DNA damage response and hyperactivating survival signaling pathways. In an attempt to better understand the molecular mechanisms that underlie resistance to chemotherapeutic agents, we investigated multiple processes regulated by the Rad9-Rad1-Hus1 (9-1-1) complex and Bif-1. The 9-1-1 complex plays an integral role in the response to DNA damage and regulates many downstream signaling pathways. Overexpression of members of this complex has been described in several types of cancer and was shown to correlate with tumorigenicity. In this study, we demonstrate that disruption of the 9-1-1 complex, through loss of Hus1, sensitizes cells to DNA damaging agents by upregulating BH3-only protein expression. Moreover, loss of Hus1 results in release of Rad9 into the cytosol, which enhances the interaction of Rad9 with Bcl-2 to potentiate the apoptotic response. We also provide evidence that disruption of the 9-1-1 complex sensitizes cells to caspase-independent cell death in response to DNA damage. Furthermore, we found that loss of Hus1 enhances DNA damage-induced autophagy. As autophagy has been implicated in caspase-independent cell death, these data suggest that the enhanced autophagy observed in Hus1-knockout cells may act as an alternate cell death mechanism. However, inhibition of autophagy, through knockdown of Atg7 or Bif-1, did not suppress, but rather promoted DNA damage-induced cell death in Hus1-deficient cells, suggesting that in apoptosis-competent cells autophagy may be induced as a cytoprotective mechanism. The aberrant activation of survival signals, such as enhanced EGFR signaling, is another mechanism that provides cancer cells with resistance to DNA damage. We found that knockdown of Bif-1 accelerated the co-localization of EGF with late endosomes/lysosomes thereby promoting EGFR degradation. Our results suggest that Bif-1 may enhance survival not only by inducing autophagy, but also by regulating EGFR degradation. Taken together, the results from our studies indicate that the 9-1-1 complex and Bif-1 may be potential targets for cancer therapy as they both regulate sensitivity to DNA damage.

μ -Calpain is localized to the mitochondrial intermembrane space. Apoptosisinducing factor (AIF), which executes caspase-independent cell death, is also localized to the mitochondrial intermembrane space. Following processing at the N-terminus, AIF becomes truncated (tAIF) and is released from mitochondria. The protease responsible for AIF processing has not been established. The same submitochondrial localization of mitochondrial μ-calpain and AIF gives support to the hypothesis that mitochondrial μ-calpain may be responsible for processing AIF. Atractyloside-induced tAIF release in rat liver mitochondria was inhibited by cysteine protease inhibitor MDL28170, but not by calpain inhibitors PD150606 or calpastatin. Moreover, μ-calpain immunoreactivity was difficult to detect in rat liver mitochondria. In a mitochondrial fraction from SH-SY5Y cells, incubation with 5 mM Ca2+ resulted in the activation of mitochondrial μ-calpain but not in AIF truncation. Finally, in hippocampal neurons calpain activation did not induce AIF processing or nuclear translocation and AIF translocation to nucleus was calpain independent. The localization of μ-calpain to the mitochondrial intermembrane space is suggestive of its possible involvement in AIF processing, but direct experimental evidence supporting such a role has been elusive. We observed that mitochondrial μ-calpain required high Ca2+ for activation. We examined the hypothesis that the endogenous calpain inhibitor, calpastatin, may be present in the neuronal mitochondria. Calpastatin was detected in the mitochondriaenriched fraction obtained from rat cerebral cortex and SH-SY5Y cells. The mitochondrial calpastatin was resistant to proteinase K digestion, indicating localization internal to the outer mitochondrial membrane. Submitochondrial fractionation revealed that the calpastatin was localized to the mitochondrial intermembrane space and mitoplasts (inner mitochondrial membrane and matrix) but not to the mitochondrial outer membrane fraction. Mitochondrial calpastatin was not detected when mitoplasts were incubated with proteinase K, suggesting that calpastatin is not present in the matrix. The N-terminus of XL domain of calpastatin, when fused to GFP and transfected to SH-SY5Y cells showed mitochondrial localization and thus confirmed the presence of a mitochondrial targeting sequence in calpastatin. Together, these results demonstrate the presence of calpastatin in the neuronal mitochondrial intermembrane space, the same submitochondrial compartment as mitochondrial μ-calpain. This finding explains the high Ca2+ requirements for mitochondrial μ-calpain activation.

Η δεσμίνη, η μυοειδική πρωτεΐνη των ενδιαμέσων ινιδίων, δημιουργεί ένα εκτενές δίκτυο που συνδέει τη συσταλτή συσκευή των μυοκυττάρων με την κυτταροπλασματική μεμβράνη -στο επίπεδο των εμβόλιμων δίσκων για τα καρδιομυοκύτταρα- τον πυρήνα και διάφορα μεμβρανώδη κυτταρικά οργανίδια. Σκοπός αυτής της εργασίας ήταν η διερεύνηση του μηχανισμού καρδιοπροστασίας που παρέχει ο κυτταροσκελετός της δεσμίνης in vivo, καθώς και η γενικότερη συμμετοχή της στα μονοπάτια της απόπτωσης. Η αποδιοργάνωση του κυτταροσκελετού είναι μια βασική διαδικασία για την εξέλιξη του μονοπατιού της απόπτωσης. Τόσο τα μικροϊνίδια της ακτίνης, όσο και τα ενδιάμεσα ινίδια -συμπεριλαμβανομένων και των πυρηνικών λαμινών- αποτελούν υπόστρωμα πέψης για διάφορα μέλη της οικογένειας των κασπασών. Η δεσμίνη είναι γνωστό ότι πέπτεται in vitro από ανασυνδυασμένη κασπάση-6, στο 263 ασπαρτικό οξύ. Το αμινοτελικό προϊόν αυτής της πέψης δεν είναι ικανό να συμμετέχει στη συγκρότηση ινιδίων, αλλά αντίθετα παρεμποδίζει αυτή τη διαδικασία σχηματίζοντας συσσωματώματα. Ως in vivo μοντέλο επαγωγής της απόπτωσης χρησιμοποιήθηκαν διαγονιδιακά ποντίκια που υπερεκφράζουν, ειδικά στην καρδιά, τον Παράγοντα Νέκρωσης Όγκων (Tumor Necrosis Factor, TNF-α). O TNF-α έχει βρεθεί ότι επάγει διαφορετικά μονοπάτια κυτταρικού θανάτου ενεργοποιώντας, μεταξύ άλλων, μέλη της οικογένειας των πρωτεολυτικών ενζύμων, των κασπασών. Το μυοκάρδιο των ποντικιών που υπερεκφράζει TNF-α (ΜΗCsTNF) χαρακτηρίζεται αρχικά από ομόκεντρη υπερτροφία των καρδιομυοκυττάρων που ακολουθείται από διάταση τόσο των κόλπων, όσο και των κοιλιών. Τα ποντίκια αυτά πεθαίνουν από καρδιακή ανεπάρκεια σε ποσοστό 25% πριν τη συμπλήρωση του έκτου μήνα ζωής. Στο μυοκάρδιο των ΜΗCsTNF ποντικιών ελέγχθηκε αρχικά ο υποκυτταρικός εντοπισμός της δεσμίνης. Η δεσμίνη στην καρδιά των ΜΗCsTNF ποντικιών έχει χάσει τον εντοπισμό της στους εμβόλιμους δίσκους, αλλά διατηρεί, εν μέρει, την παρουσία της στους Ζ-δίσκους. Προκειμένου να διαπιστωθεί εάν εκτός από τη δεσμίνη, έχει αλλοιωθεί η παρουσία και άλλων πρωτεϊνών των εμβόλιμων δίσκων, μελετήθηκε στο μυοκάρδιο αυτών των ποντικιών, η υποκυτταρική κατανομή της δεσμοπλακίνης, της β-κατενίνης και της κοννεξίνης 43, που είναι χαρακτηριστικές πρωτεΐνες των δεσμοσωμάτων, των συνδέσμων πρόσδεσης και των χασματοσυνδέσμων, αντίστοιχα. Οι πρωτεΐνες αυτές αρχικά φαίνεται να αγκυροβολούνται στους εμβόλιμους δίσκους, παροδικά όμως απομακρύνονται και κατευθύνονται προς την πλευρική επιφάνεια των καρδιομυοκυττάρων. Μελέτη με ηλεκτρονικό μικροσκόπιο αποκάλυψε ότι εκτός από την κατανομή συγκεκριμένων πρωτεϊνών, είναι διαταραγμένη και η γενική αρχιτεκτονική των εμβόλιμων δίσκων. Επιπρόσθετα, πιστοποιήθηκε ότι η δεσμίνη πέπτεται in vivo και συσσωρεύεται σε συσσωματώματα. Η παρουσία συσσωματωμάτων δεσμίνης έχει συσχετιστεί με αρκετές κληρονομικές μυοπάθειες στον άνθρωπο, οι οποίες χαρακτηρίζονται ως Μυοπάθειες Σχετιζόμενες με τη Δεσμίνη (Desmin Related Myopathies, DRM). Μεταλλάξεις, αλλά και τροποποιήσεις στη δεσμίνη ή σε άλλες πρωτεΐνες με τις οποίες αλληλεπιδρά προκειμένου να δημιουργήσει το συνεχόμενο κυτταροσκελετικό δίκτυο, έχουν ως συνέπεια τη συγκέντρωση συσσωματωμάτων στο κυτταρόπλασμα και έχουν ενοχοποιηθεί για τη διατατική μυοκαρδιοπάθεια και την εξέλιξη της καρδιακής ανεπάρκειας. Στο μυοκάρδιο των ΜΗCsTNF ποντικιών πιστοποιήθηκε η παρουσία δύο διαφορετικών ειδών συσσωματωμάτων της δεσμίνης. Τα πρώτα εμφανίζουν διάχυτο κυτταροπλασματικό πρότυπο και η δεσμίνη σε αυτά συνεντοπίζεται με την HSP25, ενώ τα δεύτερα τείνουν να περιοριστούν κοντά στον πυρήνα και εδώ η δεσμίνη συνεντοπίζεται με την ουμπικουϊτίνη. Μελέτη της υπερδομής των συσσωματωμάτων έδειξε ότι ποικίλουν σε μέγεθος και σύσταση, καθώς περιέχουν κυστίδια, μιτοχόνδρια, μεμβρανώδες υλικό και υλικό υψηλής πυκνότητας, που φαίνεται ότι παρεμποδίζουν, εκτός από τη λειτουργία του πρωτεασώματος, την συσταλτική ικανότητα των μυοϊνιδίων και συμβάλλουν στην εξέλιξη της καρδιακής ανεπάρκειας. Προκειμένου να μελετηθεί η σπουδαιότητα της δεσμίνης στην παθολογία του μυοκαρδίου που υπερεκφράζει TNF-α, ΜΗCsTNF ποντίκια διασταυρώθηκαν με ποντίκια που δεν εκφράζουν δεσμίνη (des-/-). Τα des-/- ποντίκια χαρακτηρίζονται από μια παροδική υπερτροφία των καρδιομυοκυττάρων, η οποία ακολουθείται από ανάπτυξη διατατικής μυοκαρδιοπάθειας με εξέχοντα χαρακτηριστικά τις ανωμαλίες στα μιτοχόνδρια, το μαζικό κυτταρικό θάνατο και τις εκτενείς εναποθέσεις ασβεστίου στην εξωτερική επιφάνεια της καρδιάς και κολλαγόνου στο εσωτερικό της. Τα ΜΗCsTNF des-/- ποντίκια παρουσίασαν δραματική μείωση και στις δύο μορφές συσσωματωμάτων, ενώ η δεσμοπλακίνη και η β-κατενίνη -πρωτεΐνες που ανήκουν σε μια ενιαία και διαπλεκόμενη μορφή κυτταρικών συνδέσεων, την “area composita”- διατήρησαν σημαντικά την παρουσία τους στους εμβόλιμους δίσκους. Οι παρατηρήσεις αυτές υπογραμμίζουν το βαθμό στον οποίο η δεσμίνη συμμετέχει στην εξέλιξη του ΜΗCsTNF φαινοτύπου. Παραδόξως, στο μυοκάρδιο των ΜΗCsTNF des-/- ποντικιών δεν παρατηρούνται τα μορφολογικά χαρακτηριστικά που προκαλεί η έλλειψη της δεσμίνης, ενώ διατηρούνται αυτά της υπερέκφρασης του TNF-α, υποδηλώνοντας ενδεχόμενη προστατευτική δράση του TNF-α για τα des-/- καρδιομυοκύτταρα. Η διερεύνηση του μηχανισμού της πέψης της δεσμίνης από την κασπάση-6, αλλά και της απομάκρυνσής της από τους εμβόλιμους δίσκους, με τις συνακόλουθες συνέπειες, αποτέλεσε τον επόμενο στόχο αυτής της εργασίας. Για το σκοπό αυτό, δημιουργήθηκαν διαγονιδιακά ποντίκια που εκφράζουν τη μεταλλαγμένη δεσμίνη D263E, η οποία στο 263 αμινοξύ φέρει αντί για ασπαρτικό, γλουταμινικό οξύ. Η D263E δεσμίνη δεν πέπτεται από τις κασπάσες στο ΜΗCsTNF μυοκάρδιο, διατηρεί σε μεγάλο βαθμό την παρουσία της στους εμβόλιμους δίσκους και φαίνεται ότι συγκρατεί και τις υπόλοιπες πρωτεΐνες της “area composita” στη δομή αυτή. Επιπλέον, τα συσσωματώματα της ουμπικουϊτίνης μειώνονται σημαντικά και χάνεται ο συνεντοπισμός με τη δεσμίνη σε όσα από αυτά σχηματίζονται. Τέλος, η παρουσία της D263E δεσμίνης μείωσε τον αριθμό των αποπτωτικών καρδιομυοκυττάρων στο ΜΗCsTNF μυοκάρδιο και βελτίωσε σημαντικά την καρδιακή λειτουργία. Συνοψίζοντας, τα αποτελέσματα αυτής της εργασίας υπογραμμίζουν ότι η δεσμίνη είναι ένα μόριο με σημαντικό ρόλο στην εξέλιξη της μυοκαρδιοπάθειας και της καρδιακής ανεπάρκειας, στο μοντέλο που προκαλείται από την υπερέκφραση του TNF-α. Η συμμετοχή της στο μονοπάτι της απόπτωσης ως υπόστρωμα πέψης για τις κασπάσες και οι επακόλουθες συνέπειες, αποτελούν βάση μελέτης του ρόλου των ενδιαμέσων ινιδίων και για άλλες εκφυλιστικές νόσους. / Desmin, the muscle specific intermediate filament protein, forms a three-dimensional scaffold which links the contractile apparatus to the plasma membrane intercalated disks (IDs), the nucleus and also other membranous cellular organelles. The main goal of this study was to assess the mechanism of cardioprotection by the desmin cytoskeleton in vivo and its participation in the apoptotic pathway. Disruption of the cytoskeleton is a key event in apoptotic cell death pathways. Both actin microfilaments and intermediate filaments -including the nuclear lamins- are substrates for cleavage by different members of the caspase family. Desmin is known to be specifically cleaved in vitro, by caspase-6, at the 263 aspartic acid residue. The amino-terminal product of desmin cleavage is completely unable to assemble into filaments and forms intracellular aggregates. The in vivo model used for the induction of apoptosis was transgenic mice that overexpress specifically in the heart the Tumor Necrosis Factor-α (ΤΝF-α). ΤΝF-α has been described to induce several cell death pathways that converge on activation of different members of the caspase proteolytic enzyme family. The myocardium of mice that overexpress ΤΝF-α (ΜΗCsTNF) is characterized by concentric hypertrophy of cardiomyocytes followed by chamber dilation. 25% of ΜΗCsTNF mice die by heart failure before the age of sixth month. We examined the subcellular localization of desmin in the myocardium of ΜΗCsTNF mice. Desmin is absent from the IDs of ΜΗCsTNF myocardium whereas it preserves, in part, its presence at Z-disks. In order to delineate whether the relocalization of desmin influences the position of other ID proteins, we examined the subcellular localization of desmoplakin, β-catenin and connexin 43, which are characteristic proteins of desmosomes, adherent junctions and gap junctions respectively. At early stages, these proteins localize almost normally in MHCsTNF cardiomyocytes, however, in the 3-month old MHCsTNF mice, all studied proteins were primarily localized at the lateral, non-junctional, side of the cardiomyocytes. Ultrastructural study of the MHCsTNF myocardium revealed that the overall ID architecture is altered. Additionally, we show that desmin is cleaved in vivo and accumulates into cytoplasmic aggregates. The presence of desmin positive aggregates has been correlated with several human inherited myopathies called Desmin Related Myopathies (DRM). Mutations within its gene or in the genes of other desmin associated cytoskeletal proteins, result in accumulation of aggregates in the cytoplasm and have been linked to Dilated Cardiomyopathy (DCM) and heart failure. In the MHCsTNF myocardium we observe two distinct kinds of aggregates that are positive for desmin. The first ones are diffused in the cytoplasm and desmin colocalizes with HSP25, while in the second category the aggregates tend to be restricted near the nucleus and desmin colocalizes with ubiquitin. Ultrastructural study of the aggregates reveals that they are variable in size and composition, consisting of vesicles, mitochondria, membranous whorls and, occasionally, clumps of electron dense material, suggesting that, in addition to other functions such as proteasome activity, they may interfere with the sarcomere contraction thereby leading to impairment of cardiac function. In order to study the importance of desmin in the development of the ΜΗCsTNF pathology, we crossed ΜΗCsTNF mice with desmin null mice (des-/-). In desmin-deficient mice transient cardiomyocyte hypertrophy is followed by development of a DCM that is characterized by mitochondrial functional, ultrastructural and other defects, extensive cell death, fibrotic lesions and calcium deposition at the external surface of the heart. ΜΗCsTNF des-/- mice showed significant decrease in both kind of aggregates, whereas desmoplakin and β-catenin -proteins that belong to the uniform cardiomyocyte junctional structure “area composita”- preserve at high degree their presence at IDs. These observations underline the level at which desmin participates in the progress of ΜΗCsTNF phenotype. Surprisingly, the myocardium of ΜΗCsTNF des-/- mice eliminated the morphological defects caused by the absence of desmin while retained those caused by TNF-α overexpression, suggesting a possible counteracting action of TNF-α in the des-/- cardiomyocytes. To investigate the mechanism and the importance of desmin cleavage by caspase-6 and its withdrawal from IDs with the concomitant consequences, we generated transgenic mice with cardiac-restricted expression of a desmin mutant (D263E), harboring a substitution of the 263 aspartic acid (D) with a glutamic acid (E), which renders desmin resistant to caspase mediated cleavage. We showed that D263E desmin is indeed resistant to caspase cleavage in the ΜΗCsTNF myocardium, largely retains its proper ID localization and allows the same for the other proteins of the area composita. Additionally, the ubiquitin positive aggregates are diminished and the few generated do not colocalize with desmin. Importantly, D263E desmin expression attenuated cardiomyocyte apoptosis, prevented left ventricular wall thinning and improved function of MHCsTNF hearts. To summarize, the data presented in this study underline that desmin is a target and mediator of ΤΝF-α induced cardiomyopathy and heart failure. Its contribution in the pathway of apoptosis as a substrate for caspase cleavage and the subsequent participation in the formation of aggregates and the alteration of the ID architecture is the baseline for the study of the role of Intermediate Filaments in degenerative diseases.

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.

CD20 est une phosphoprotéine transmembranaire exprimée spécifiquement à la surface des lymphocytes B. Malgré les nombreuses études qui ont montré son implication dans le flux calcique, son rôle physiologique est assez mal connu. Cependant, des études récentes ont démontré que CD20 peut jouer un rôle important dans la mort cellulaire. D’ailleurs, le rituximab, un anticorps monoclonal chimérique dirigé contre CD20 humain, a montré son efficacité dans le traitement de nombreuses maladies auto-immunes. Cet anticorps est capable d’induire une profonde déplétion des lymphocytes B, qui va également interférer avec la coopération T et la sécrétion de cytokines. En plus, l’engagement du CD20 à la surface des cellules induit la mort cellulaire, alors que la partie cytoplasmique de cette molécule ne possède pas un motif de mort. Donc, il est possible que cette réponse soit médiée par des molécules qui semblent être associées au CD20 comme CD40. En effet, CD40, une glycoprotéine transmembranaire de type I, est un composant majeur du système immunitaire, dont l’engagement pourrait moduler la fonction cellulaire et même conduire à la mort rapide des cellules B. Le travail présenté dans ce mémoire porte sur l’étude de la mort cellulaire induite par un anti-CD20, le rituximab, ainsi que l’étude du rôle de l’association CD20/CD40 dans la mort cellulaire médiée par cet anticorps. Nos résultats montrent que la mort cellulaire induite par le rituximab varie en fonction du type cellulaire et du niveau d’expression du CD20, et que la présence du CD40 à la surface des cellules augmente l’activité de la mort cellulaire induite par le rituximab. En plus, CD20 et CD40 sont associés à la surface cellulaire, et la partie cytoplasmique n’est pas impliquée dans cette association mais semble être importante dans la mort cellulaire induite via CD20. / CD20 is a transmembrane phosphoprotein specifically expressed at the surface of B cells. Despite the many studies that have shown its involvement in calcium flux, its physiological role is poorly understood. However, recent studies have shown that CD20 can play an important role in cell death. Moreover, rituximab, a chimeric monoclonal antibody directed against human CD20, has shown efficacy in the treatment of many autoimmune diseases. Indeed, this antibody is able to induce a profound depletion of B cells and interfere in T cooperation and secretion of cytokines. In addition, the engagement of CD20 at the cell surface induces cell death, while the cytoplasmic portion of this molecule has no death domain. So, it is possible that this response is mediated by molecules that could be associated with CD20, like CD40. In fact, CD40, a type I transmembrane glycoprotein, is a major component of the immune system. The engagement of CD40 can modulate cellular function and may even lead to rapid B cell death. The work presented in this thesis will focus on the study of cell death induced by an anti-CD20, rituximab, and on the role of the association of CD40 with CD20 at the cell surface in susceptibility to cell death mediated by this antibody. Our results show that cell death induced by rituximab depends on cell type and on the levels of expression of CD20, and that the presence of CD40 at the cell surface increases cell death induced by rituximab. In addition, CD20 and CD40 are associated at the cell surface, and the cytoplasmic portion is not involved in this association but seems to be important in induction of cell death by CD20.

No description available.

No description available.

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.

HSP27, the Drosophila homolog of mammalian HspB1, is a nuclear sHsp that is both stress induced and developmentally regulated with a conserved cyto-protective function. It is multiply phosphorylated in vivo through an unconfirmed mechanism at unidentified residues. The effect of phosphorylation on its localization, oligomerization, and function is also not well understood. Here we report a genetic investigation into the role of Hsp27 in Drosophila development, and a preliminary investigation into the effect of phosphorylation on HSP27 localization and function in Drosophila S2 cells. Through a proteomic screen, a pro-apoptotic role for Hsp27 in embryonic developmentally regulated programmed cell death was suggested and supported by RNAi experiments, but not replicated using Hsp27null mutant stocks. These stocks were complicated by the intriguing appearance of multiple background mutations. Specific developmental defects in transgenic lines overexpressing phospho-mutant isoforms were then investigated. These too were subject to multiple independent incidences of background genetic mutation, which we believe may be related to Hsp27 mis-expression. We also studied the endogenous expression and localization pattern of HSP27 in stressed and unstressed Drosophila S2 cells. We found evidence that wild-type protein localization is influenced by stress. Finally, we took a first step toward understanding how phosphorylation might regulate HSP27 localization by examining the effect of targeted mutations of serine residues (S58, S71, and S75) on the localization pattern of exogenous HSP27. By characterizing the expression of endogenous and overexpressed HSP27 in Drosophila cells, we provide a foundation for future investigation into the regulated localization and function of HSP27 that can be extended to address the regulatory mechanisms that govern the protective capacities and oligomeric properties of phosphorylated HSP27 in Drosophila.