Ruxolitinib with resminostat exert synergistic antitumor effects in Cutaneous T-cell Lymphoma.

BACKGROUND: The combination of JAK/STAT and HDAC inhibitors exerted beneficial effects in haematological malignancies, presenting promising therapeutic CTCL targets. We aim to investigate the efficacy of JAK1/2i ruxolitinib in combination with HDACi resminostat in CTCL in vitro. MATERIAL & METHODS: Non-toxic concentrations of ruxolitinib and/or resminostat were administered to MyLa (MF) and SeAx (SS) cells for 24h. Cytotoxicity, cell proliferation and apoptosis were estimated through MTT, BrdU/7AAD and Annexin V/PI assay. Multi-pathway analysis was performed to investigate the effect of JAK1/2i and/or HDACi on JAK/STAT, Akt/mTOR and MAPK signalling pathways. RESULTS: Both drugs and their combination were cytotoxic in MyLa (p<0.05) and in SeAx cell line (p<0.001), inhibited proliferation of MyLa (p<0.001) and SeAx (p<0.001) at 24h, compared to untreated cells. Moreover, combined drug treatment induced apoptosis after 24h (p<0.001) in MyLa, and SeAx (p<0.001). The combination of drugs had a strong synergistic effect with a CI<1. Importantly, the drugs' combination inhibited phosphorylation of STAT3 (p<0.001), Akt (p<0.05), ERK1/2 (p<0.001) and JNK (p<0.001) in MyLa, while it reduced activation of Akt (p<0.05) and JNK (p<0.001) in SeAx. CONCLUSION: The JAKi/HDACi combination exhibited substantial anti-tumor effects in CTCL cell lines, and may represent a promising novel therapeutic modality for CTCL patients.

Synergistic inhibitory effects of low-dose decitabine in combination with bortezomib in the AML cell line Kasumi-1.

In the present study, the ability of the proteasome inhibitor bortezomib (BZ), an oxidative stress-inducing agent, to sensitize acute myeloid leukemia (AML) cells to decitabine (Dacogen®, DAC; a DNA methyltransferase inhibitor), in terms of cell viability and differentiation, was investigated. Kasumi-1 AML (M2) cells were treated with low-dose DAC (10, 50, 100, 200 or 400 nΜ), with or without BZ (10 nM). Apoptosis and the cell cycle were evaluated after 24 h of treatment through fluorescence-assisted cell sorting (FACS) with Annexin V/propidium iodide and DAPI staining, respectively. The expression levels of CD193, CD11b, CD13, CD14, CD15, CD16 and CD117 surface differentiation markers were evaluated by FACS after 6 days of treatment. The results indicated significant alterations in cell death and cell cycle phases in Kasumi-1 cells following DAC and BZ combination treatment compared to untreated cells and cells with single treatments. Low-dose DAC/BZ combinations significantly enhanced apoptosis and decreased the population of live Kasumi-1 cells, with 100 and 200 nM of DAC and 10 nM BZ appearing to have the most potent synergistic effect according to a combination index. Furthermore, cell cycle profiling revealed that DAC/BZ treatment synergistically led to G0/G1- and G2/M-phase arrest. By contrast, DAC appeared to promote monocytic and granulocytic differentiation of Kasumi-1 cells more effectively alone than in combination with BZ. BZ acted synergistically with low-dose DAC in vitro, leading to enhanced apoptosis and G0/G1- and G2/M-phase arrest in AML cells, hence prohibiting either DNA synthesis or mitosis. Although further in vivo investigation is necessary, these results provide a strong rationale for the implementation of a combination treatment with DAC and bortezomib in AML therapy, followed by DAC alone, which may achieve better clinical responses and possibly partially overcome the frequently encountered DAC resistance of patients with AML.

Upregulated hypoxia inducible factor 1α signaling pathway in high risk myelodysplastic syndrome and acute myeloid leukemia patients is associated with better response to 5-azacytidine-data from the Hellenic myelodysplastic syndrome study group.

5-azacytidine (5-AZA) is considered the standard of care for patients with high-risk myelodysplastic syndromes (MDS) and patients with acute myeloid leukemia (AML) not candidate for intensive chemotherapy. However, even after an initial favorable response, almost all patients relapse, with the exact mechanisms underlying primary or secondary 5-AZA resistance remaining largely unknown. Several reports have previously demonstrated the significance of hypoxia in the regulation of both physiological and malignant hematopoiesis. In MDS, high hypoxia inducible factor 1α (Hif-1α) expression has been correlated with poor overall survival and disease progression, while its involvement in the disease's pathogenesis was recently reported. We herein investigated the possible association of the Hif-1α signaling pathway with response to 5-AZA therapy in MDS/AML patients. Our data demonstrated that 5-AZA-responders present with higher Hif-1α mRNA and protein expression compared to 5-AZA-non-responders/stable disease patients, before the initiation of therapy, while, interestingly, no significant differences in Hif-1α mRNA expression at the 6-month follow-up were observed. Moreover, we found that 5-AZA-responders exhibited elevated mRNA levels of the Hif-1α downstream targets lactate dehydrogenase a (LDHa) and BCL2 interacting protein 3 like (BNIP3L), a further indication of an overactivated Hif-1a signaling pathway in these patients. Kaplan-Meier survival analysis revealed a significant correlation between high Hif-1α mRNA expression and better survival rates, while logistic regression analysis showed that Hif-1α mRNA expression is an independent predictor of response to 5-AZA therapy. From the clinical point of view, apart from proposing Hif-1α mRNA expression as a significant predictive factor for response to 5-AZA, our data offer new perspectives on MDS combinational therapies, suggesting a potential synergistic activity of 5-AZA and Hif-1α inducers, such as propyl hydroxylases inhibitors (PHDi).

Proteomic mapping of Drosophila transgenic elav.L-GAL4/+ brain as a tool to illuminate neuropathology mechanisms.

Drosophila brain has emerged as a powerful model system for the investigation of genes being related to neurological pathologies. To map the proteomic landscape of fly brain, in a high-resolution scale, we herein employed a nano liquid chromatography-tandem mass spectrometry technology, and high-content catalogues of 7,663 unique peptides and 2,335 single proteins were generated. Protein-data processing, through UniProt, DAVID, KEGG and PANTHER bioinformatics subroutines, led to fly brain-protein classification, according to sub-cellular topology, molecular function, implication in signaling and contribution to neuronal diseases. Given the importance of Ubiquitin Proteasome System (UPS) in neuropathologies and by using the almost completely reassembled UPS, we genetically targeted genes encoding components of the ubiquitination-dependent protein-degradation machinery. This analysis showed that driving RNAi toward proteasome components and regulators, using the GAL4-elav.L driver, resulted in changes to longevity and climbing-activity patterns during aging. Our proteomic map is expected to advance the existing knowledge regarding brain biology in animal species of major translational-research value and economical interest.

Revisiting Histone Deacetylases in Human Tumorigenesis: The Paradigm of Urothelial Bladder Cancer.

Urinary bladder cancer is a common malignancy, being characterized by substantial patient mortality and management cost. Its high somatic-mutation frequency and molecular heterogeneity usually renders tumors refractory to the applied regimens. Hitherto, methotrexate-vinblastine-adriamycin-cisplatin and gemcitabine-cisplatin represent the backbone of systemic chemotherapy. However, despite the initial chemosensitivity, the majority of treated patients will eventually develop chemoresistance, which severely reduces their survival expectancy. Since chromatin regulation genes are more frequently mutated in muscle-invasive bladder cancer, as compared to other epithelial tumors, targeted therapies against chromatin aberrations in chemoresistant clones may prove beneficial for the disease. "Acetyl-chromatin" homeostasis is regulated by the opposing functions of histone acetyltransferases (HATs) and histone deacetylases (HDACs). The HDAC/SIRT (super-)family contains 18 members, which are divided in five classes, with each family member being differentially expressed in normal urinary bladder tissues. Since a strong association between irregular HDAC expression/activity and tumorigenesis has been previously demonstrated, we herein attempt to review the accumulated published evidences that implicate HDACs/SIRTs as critical regulators in urothelial bladder cancer. Moreover, the most extensively investigated HDAC inhibitors (HDACis) are also analyzed, and the respective clinical trials are also described. Interestingly, it seems that HDACis should be preferably used in drug-combination therapeutic schemes, including radiation.

The indispensable contribution of s38 protein to ovarian-eggshell morphogenesis in Drosophila melanogaster.

Drosophila chorion represents a remarkable model system for the in vivo study of complex extracellular-matrix architectures. For its organization and structure, s38 protein is considered as a component of major importance, since it is synthesized and secreted during early choriogenesis. However, there is no evidence that proves its essential, or redundant, role in chorion biogenesis. Hence, we show that targeted downregulation of s38 protein, specifically in the ovarian follicle-cell compartment, via employment of an RNAi-mediated strategy, causes generation of diverse dysmorphic phenotypes, regarding eggshell's regionally and radially specialized structures. Downregulation of s38 protein severely impairs fly's fertility and is unable to be compensated by the s36 homologous family member, thus unveiling s38 protein's essential contribution to chorion's assembly and function. Altogether, s38 acts as a key skeletal protein being critically implicated in the patterning establishment of a highly structured tripartite endochorion. Furthermore, it seems that s38 loss may sensitize choriogenesis to stochastic variation in its coordination and timing.

Quantitative and qualitative analysis of regulatory T cells in B cell chronic lymphocytic leukemia.

Accumulated data indicate a significant role of T cell dysfunction in the pathogenesis of chronic lymphocytic leukemia. In CLL, regulatory T cells are significantly higher and show lower apoptotic levels compared to healthy donors. We demonstrate that CLL derived CD4+CD25-CD127- and CD4+CD25lowCD127- subpopulations share a common immunophenotypic profile with conventional Tregs and are associated with advanced stage disease. We further provide evidence that the increased number of Tregs contributes indirectly to the proliferation of the CLL clone, by suppressing the proliferation of Teffs which in turn suppress CLL cells. These data are further supported by our observations that CLL derived Tregs appear rather incapable of inducing apoptosis of both normal B cells and CLL cells, in contrast to normal Tregs, suggesting an immunoediting effect of CLL cells on Tregs which negatively affects the functionality of the latter and contributes to the failure of Tregs in CLL to efficiently eliminate the abnormal clone.

Data of sperm-entry inability in Drosophila melanogaster ovarian follicles that are depleted of s36 chorionic protein.

This paper presents data associated with the research article entitled "Targeted downregulation of s36 protein unearths its cardinal role in chorion biogenesis and architecture during Drosophila melanogaster oogenesis" [1]. Drosophila chorion is produced by epithelial follicle cells and one of its functional serving role is egg fertilization through the micropyle, a specialized narrow channel at the anterior tip of the egg [2]. Sperm entry during fertilization is necessary for the egg to complete meiosis [3]. D. melanogaster flies being characterized by severe downregulation of the s36 chorionic protein, specifically in the follicle-cell compartment of their ovary, appear with impaired fly fertility (Velentzas et al., 2016) [1]. In an effort to further investigate whether the observed infertility in the s36-targeted flies derives from a fertilization failure, such as the inability of sperm to pass through egg׳s micropyle, we mated females carrying s36-depleted ovaries with males expressing the GFP protein either in their sperm tails, or in both their sperm tails and sperm heads.

Quantitative Measurement of L1 Human Papillomavirus Type 16 Methylation for the Prediction of Preinvasive and Invasive Cervical Disease.

Background: Methylation of the human papillomavirus (HPV) DNA has been proposed as a novel biomarker. Here, we correlated the mean methylation level of 12 CpG sites within the L1 gene, to the histological grade of cervical precancer and cancer. We assessed whether HPV L1 gene methylation can predict the presence of high-grade disease at histology in women testing positive for HPV16 genotype. Methods: Pyrosequencing was used for DNA methylation quantification and 145 women were recruited. Results: We found that the L1 HPV16 mean methylation (±SD) significantly increased with disease severity (cervical intraepithelial neoplasia [CIN] 3, 17.9% [±7.2] vs CIN2, 11.6% [±6.5], P < .001 or vs CIN1, 9.0% [±3.5], P < .001). Mean methylation was a good predictor of CIN3+ cases; the area under the curve was higher for sites 5611 in the prediction of CIN2+ and higher for position 7145 for CIN3+. The evaluation of different methylation thresholds for the prediction of CIN3+ showed that the optimal balance of sensitivity and specificity (75.7% and 77.5%, respectively) and positive and negative predictive values (74.7% and 78.5%, respectively) was achieved for a methylation of 14.0% with overall accuracy of 76.7%. Conclusions: Elevated methylation level is associated with increased disease severity and has good ability to discriminate HPV16-positive women that have high-grade disease or worse.

Apoptosis Induction and Gene Expression Profile Alterations of Cutaneous T-Cell Lymphoma Cells following Their Exposure to Bortezomib and Methotrexate.

Mycosis fungoides (MF) and its leukemic variant Sézary syndrome (SS) comprise the majority of CTCL, a heterogenous group of non-Hodgkins lymphomas involving the skin. The CTCL's resistance to chemotherapy and the lack of full understanding of their pathogenesis request further investigation. With the view of a more targeted therapy, we evaluated in vitro the effectiveness of bortezomib and methotrexate, as well as their combination in CTCL cell lines, regarding apoptosis induction. Our data are of clinical value and indicate that the bortezomib/methotrexate combinational therapy has an inferior impact on the apoptosis of CTCL compared to monotherapy, with bortezomib presenting as the most efficient treatment option for SS and methotrexate for MF. Using PCR arrays technology, we also investigated the alterations in the expression profile of genes related to DNA repair pathways in CTCL cell lines after treatment with bortezomib or methotrexate. We found that both agents, but mostly bortezomib, significantly deregulate a large number of genes in SS and MF cell lines, suggesting another pathway through which these agents could induce apoptosis in CTCL. Finally, we show that SS and MF respond differently to treatment, verifying their distinct nature and further emphasizing the need for discrete treatment approaches.

Targeted Downregulation of s36 Protein Unearths its Cardinal Role in Chorion Biogenesis and Architecture during Drosophila melanogaster Oogenesis.

Drosophila chorion represents a model biological system for the in vivo study of gene activity, epithelial development, extracellular-matrix assembly and morphogenetic-patterning control. It is produced during the late stages of oogenesis by epithelial follicle cells and develops into a highly organized multi-layered structure that exhibits regional specialization and radial complexity. Among the six major proteins involved in chorion's formation, the s36 and s38 ones are synthesized first and regulated in a cell type-specific and developmental stage-dependent manner. In our study, an RNAi-mediated silencing of s36 chorionic-gene expression specifically in the follicle-cell compartment of Drosophila ovary unearths the essential, and far from redundant, role of s36 protein in patterning establishment of chorion's regional specialization and radial complexity. Without perturbing the developmental courses of follicle- and nurse-cell clusters, the absence of s36 not only promotes chorion's fragility but also induces severe structural irregularities on chorion's surface and entirely impairs fly's fertility. Moreover, we herein unveil a novel function of s36 chorionic protein in the regulation of number and morphogenetic integrity of dorsal appendages in follicles sporadically undergoing aged fly-dependent stress.

Global Proteomic Profiling of Drosophila Ovary: A High-resolution, Unbiased, Accurate and Multifaceted Analysis.

BACKGROUND: Drosophila melanogaster ovary serves as an attractive model system for the investigation of the cell cycle, death, signaling, migration, differentiation, development and stemness. By employing the 3750/+ heterozygote fly strain that carries specific functions in the follicle cell compartment, and a reliable control in GAL4/UAS-based transgenic technology, we herein characterized the protein-expression profiling of D. melanogaster ovary by applying high-resolution proteomic tools and bioinformatics programs. MATERIALS AND METHODS: Whole-cell total protein extracts derived from 3750/+ fly ovaries were prepared under highly denaturing conditions and after tryptic digestion, their cognate peptides were processed to liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis in a high-resolution LTQ Orbitrap Elite instrument. Obtained protein data were analyzed through use of UniProt, DAVID, KEGG and PANTHER bioinformatics platforms. RESULTS: The 7,583 unique peptides identified show that fly ovary contains at least 2,103 single proteins, which are distributed to all egg chamber compartments, in cytoplasm, membrane and nucleus, compartmentalized into major cellular organelles, and categorized into critical macromolecular assemblies. Among the recognized specific functions, nucleic acid binding, hydrolase, oxidoreductase, transporter and vesicle-mediated trafficking activities were the most prevalent. Determinants implicated in cellular metabolism and gene expression are represented by ~41% and ~17% of the ovarian proteome, respectively. Surprisingly, several proteins were found engaged in aging, immune response and neurogenesis. All major signaling pathways were detected, while apoptotic and non-apoptotic cell death programs were also identified. Remarkably, proteins involved in tumor formation, neurodegenerative and inflammatory diseases were also recognized. The successful remodeling of the proteasome and nearly complete molecular reconstruction of the citrate cycle and fatty acid degradation pathways demonstrate the efficacy, accuracy and fidelity of our combined proteomics/bioinformatics approach. CONCLUSION: Global proteomic characterization of D. melanogaster ovary allows the discovery of novel regulators and pathways, and provides a systemic view of networks that govern ovarian pathophysiology and embryonic development in fly species as well in humans.

Proteasome, but not autophagy, disruption results in severe eye and wing dysmorphia: a subunit- and regulator-dependent process in Drosophila.

Proteasome-dependent and autophagy-mediated degradation of eukaryotic cellular proteins represent the two major proteostatic mechanisms that are critically implicated in a number of signaling pathways and cellular processes. Deregulation of functions engaged in protein elimination frequently leads to development of morbid states and diseases. In this context, and through the utilization of GAL4/UAS genetic tool, we herein examined the in vivo contribution of proteasome and autophagy systems in Drosophila eye and wing morphogenesis. By exploiting the ability of GAL4-ninaE. GMR and P{GawB}Bx(MS1096) genetic drivers to be strongly and preferentially expressed in the eye and wing discs, respectively, we proved that proteasomal integrity and ubiquitination proficiency essentially control fly's eye and wing development. Indeed, subunit- and regulator-specific patterns of severe organ dysmorphia were obtained after the RNAi-induced downregulation of critical proteasome components (Rpn1, Rpn2, α5, ß5 and ß6) or distinct protein-ubiquitin conjugators (UbcD6, but not UbcD1 and UbcD4). Proteasome deficient eyes presented with either rough phenotypes or strongly dysmorphic shapes, while transgenic mutant wings were severely folded and carried blistered structures together with loss of vein differentiation. Moreover, transgenic fly eyes overexpressing the UBP2-yeast deubiquitinase enzyme were characterized by an eyeless-like phenotype. Therefore, the proteasome/ubiquitin proteolytic activities are undoubtedly required for the normal course of eye and wing development. In contrast, the RNAi-mediated downregulation of critical Atg (1, 4, 7, 9 and 18) autophagic proteins revealed their non-essential, or redundant, functional roles in Drosophila eye and wing formation under physiological growth conditions, since their reduced expression levels could only marginally disturb wing's, but not eye's, morphogenetic organization and architecture. However, Atg9 proved indispensable for the maintenance of structural integrity of adult wings in aged flies. In toto, our findings clearly demonstrate the gene-specific fundamental contribution of proteasome, but not autophagy, in invertebrate eye and wing organ development.

Detrimental effects of proteasome inhibition activity in Drosophila melanogaster: implication of ER stress, autophagy, and apoptosis.

In eukaryotes, the ubiquitin-proteasome machinery regulates a number of fundamental cellular processes through accurate and tightly controlled protein degradation pathways. We have, herein, examined the effects of proteasome functional disruption in Dmp53 (+/+) (wild-type) and Dmp53 (-/-) Drosophila melanogaster fly strains through utilization of Bortezomib, a proteasome-specific inhibitor. We report that proteasome inhibition drastically shortens fly life-span and impairs climbing performance, while it also causes larval lethality and activates developmentally irregular cell death programs during oogenesis. Interestingly, Dmp53 gene seems to play a role in fly longevity and climbing ability. Moreover, Bortezomib proved to induce endoplasmic reticulum (ER) stress that was able to result in the engagement of unfolded protein response (UPR) signaling pathway, as respectively indicated by fly Xbp1 activation and Ref(2)P-containing protein aggregate formation. Larva salivary gland and adult brain both underwent strong ER stress in response to Bortezomib, thus underscoring the detrimental role of proteasome inhibition in larval development and brain function. We also propose that the observed upregulation of autophagy operates as a protective mechanism to "counterbalance" Bortezomib-induced systemic toxicity, which is tightly associated, besides ER stress, with activation of apoptosis, mainly mediated by functional Drice caspase and deregulated dAkt kinase. The reduced life-span of exposed to Bortezomib flies overexpressing Atg1_RNAi or Atg18_RNAi supports the protective nature of autophagy against proteasome inhibition-induced stress. Our data reveal the in vivo significance of proteasome functional integrity as a major defensive system against cellular toxicity likely occurring during critical biological processes and morphogenetic courses.

Dasatinib inhibits proliferation and induces apoptosis in the KASUMI-1 cell line bearing the t(8;21)(q22;q22) and the N822K c-kit mutation.

Activating mutations of the c-kit gene are frequently found in CBF (core binding factor) leukemias. We evaluated the effect of tyrosine kinase inhibitor dasatinib in leukemic cell lines bearing or not c-kit mutations. Our data demonstrate that in the AML Kasumi-1 cell line, bearing the N822K c-kit mutation, dasatinib is a potent suppressor of c-kit and Src kinase activity and inhibits the phosphorylation of their downstream target AKT, possibly through the Src-mediated VEGF/VEGFR receptor type 2 pathway. Dasatinib also effectively blocks proliferation and induces apoptosis through caspase-3 activation in Kasumi-1 cells. These data further encourage the integration of dasatinib in the treatment of CBF AML with c-kit mutations in the context of clinical trials, which are eagerly anticipated.

Expression analysis of mir-17-5p, mir-20a and let-7a microRNAs and their target proteins in CD34+ bone marrow cells of patients with myelodysplastic syndromes.

Mir-17-5p and mir-20a, members of the mir-17-92 family, down-regulate E2F1, which is over-expressed in myelodysplastic syndromes (MDS). Moreover, let-7a down-regulates KRAS, which is aberrantly expressed in MDS. We evaluated the expression of the aforementioned microRNAs in CD34+ cells of 43 MDS patients using real-time PCR and their target proteins (E2F1, MYC, BCL2, CCND1, and KRAS) by Western blot. Mir-17-5p and mir-20a were under expressed in high risk MDS patients, compared to low risk MDS patients. Similarly, let-7a was under expressed in patients with intermediate or high-risk karyotype. Interestingly, there was an inverse correlation between microRNA and the expression levels of their targets. Importantly, mir-17-5p and mir-20a constitute favorable prognostic factors in MDS, since their expression was associated with increased overall survival of MDS patients.

Programmed cell death of the ovarian nurse cells during oogenesis of the ladybird beetle Adalia bipunctata (Coleoptera: Coccinellidae).

Programmed cell death (PCD) is an evolutionary conserved and genetically regulated form of cell death, in which the cell plays an active role in its own demise. It is widely recognized that PCD can be morphologically classified into three major types: type I, known as apoptosis, type II, called autophagy, and type III, specified as cytoplasmic cell death. So far, PCD has been morphologically analyzed in certain model insect species of the meroistic polytrophic ovary-type, but has never been examined before in insects carrying meroistic telotrophic ovaries. In the present study, we attempted to thoroughly describe the three different types (I, II and III) of PCD occurring during oogenesis in the meroistic telotrophic ovary of the Coleoptera species Adalia bipunctata, at different developmental ages of the adult female insects. We reveal that in the ladybird beetle A. bipunctata, the ovarian tropharia undergo age-dependent forms of apoptotic, autophagic and cytoplasmic (paraptotic-like) cell death, which seem to operate in a rather synergistic fashion, in accordance with previous observations in Diptera and Lepidoptera species. Furthermore, we herein demonstrate the occurrence of morphogenetically abnormal ovarioles in A. bipunctata female insects. These atretic ovarioles collapse and die through a PCD-mediated process that is characterized by the combined activation of all three types of PCD. Conclusively, the distinct cell death programs (I, II and III) specifically engaged during oogenesis of A. bipunctata provide strong evidence for the structural and functional conserved nature of PCD during insect evolution among meroistic telotrophic and meroistic polytrophic ovary-type insects.

Proteasome inhibition induces developmentally deregulated programs of apoptotic and autophagic cell death during Drosophila melanogaster oogenesis.

Ubiquitin/proteasome-mediated degradation of eukaryotic proteins is critically implicated in a number of signalling pathways and cellular processes. To specifically impair proteasome activities, in vitro developing Drosophila melanogaster egg chambers were exposed to the MG132 or epoxomicin proteasome inhibitors, while a GAL4/UAS binary genetic system was employed to generate double transgenic flies overexpressing ß2 and ß6 conditional mutant proteasome subunits in a cell type-specific manner. MG132 and epoxomicin administration resulted in severe deregulation of in vitro developing egg chambers, which was tightly associated with precocious induction of nurse cell-specific apoptotic and autophagic death programmes, featured by actin cytoskeleton disorganization, nuclear chromatin condensation, DRICE caspase activation and autophagosome accumulation. In vivo targeted overexpression of ß2 and ß6 conditional mutants, specifically in the nurse cell compartment, led to a notable up-regulation of sporadic apoptosis potency during early and mid-oogenesis 'checkpoints', thus reasonably justifying the observed reduction in eclosion efficiency. Furthermore, in response to the intracellular abundance of ß2 and ß6 conditional mutant forms, specifically in numerous tissues of third instar larval stage, the developmental course was arrested, and lethal phenotypes were obtained at this particular embryonic period, with the double transgenic heterozygote embryos being unable to further proceed to complete maturation to adult flies. Our data demonstrate that physiological proteasome function is required to ensure normal oogenesis and embryogenesis in D. melanogaster, since targeted and cell type-dependent proteasome inactivation initiates developmentally deregulated apoptotic and autophagic mechanisms.