Nitric oxide cooperates with glucocorticoids in thymic epithelial cell-mediated apoptosis of double positive thymocytes.

T cell development in the thymus is controlled by thymic epithelial cells (TE). While it is accepted that TE interact with maturing T cells, the mechanisms by which they trigger 'death by neglect' of double-positive (DP) thymocytes are poorly understood. We and others have demonstrated a role for TE-derived glucocorticoids (GCs) in this process. We have studied TE-induced apoptosis using an in vitro system based on co-culturing a thymic epithelial cell line (TEC) with DP thymic lymphoma cells or thymocytes (DP thymic cells). Here, we demonstrate that nitric oxide (NO*) is also involved in this death process. The inducible nitric oxide synthase (iNOS) inhibitors N(G)-methyl-L-arginine and 1,4-PBIT attenuated TEC-induced apoptosis of DP thymic cells. Co-cultivation of TEC with DP thymic cells increased the expression of iNOS in TEC. A concomitant increase in NO* was detected by staining with DAF-FM diacetate. Moreover, the iNOS-regulating cytokines IL-1alpha, IL-1beta and IFNgamma were up-regulated upon interaction of TEC with DP thymic cells. Neutralizing IL-1R or IFNgamma reduced TEC-induced apoptosis of DP thymic cells. Cardinally, NO* synergizes with GCs in eliciting apoptosis of DP thymic cells. Our data indicate that a cross-talk between DP thymic cells and TEC is required for proper induction of iNOS-up-regulating cytokines with a subsequent increase in iNOS expression and NO* production in TEC. NO*, in turn, cooperates with GCs in promoting death by neglect. We suggest that NO* together with GCs fine-tune the T cell selection process.

Mechanisms regulating the susceptibility of hematopoietic malignancies to glucocorticoid-induced apoptosis.

Glucocorticoids (GCs) are commonly used in the treatment of hematopoietic malignancies owing to their ability to induce apoptosis of these cancerous cells. Whereas some types of lymphoma and leukemia respond well to this drug, others are resistant. Also, GC-resistance gradually develops upon repeated treatments ultimately leading to refractory relapsed disease. Understanding the mechanisms regulating GC-induced apoptosis is therefore uttermost important for designing novel treatment strategies that overcome GC-resistance. This review discusses updated data describing the complex regulation of the cell's susceptibility to apoptosis triggered by GCs. We address both the genomic and nongenomic effects involved in promoting the apoptotic signals as well as the resistance mechanisms opposing these signals. Eventually we address potential strategies of clinical relevance that sensitize GC-resistant lymphoma and leukemia cells to this drug. The major target is the nongenomic signal transduction machinery where the interplay between protein kinases determines the cell fate. Shifting the balance of the kinome towards a state where Glycogen synthase kinase 3alpha (GSK3alpha) is kept active, favors an apoptotic response. Accumulating data show that it is possible to therapeutically modulate GC-resistance in patients, thereby improving the response to GC therapy.

4D imaging of protein aggregation in live cells.

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances. The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state, target them for degradation by the ubiquitin-proteasome system, or direct them to protective aggregation inclusions. In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment. Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure. We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein (Ubc9(ts)) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9(ts) is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 ° C, or when the cell faces misfolding stress, Ubc9(ts) misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.

A role for bcl-2 in notch1-dependent transcription in thymic lymphoma cells.

Notch1 is a transcription factor important for T-cell development. Notch1 is active in double negative (DN) thymocytes, while being depressed in double positive (DP) thymocytes. Synchronously, the expression of Bcl-2 becomes downregulated during the transition from DN to DP thymocytes. We previously observed that overexpression of an intracellular active Notch1 (ICN) in Bcl-2-positive 2B4 T cells leads to the transcription of Notch1-regulated genes. However, these genes were not induced in Bcl-2-negative DP PD1.6 thymic lymphoma cells overexpressing ICN. Here we show that, when Bcl-2 is simultaneously introduced into these cells, Notch-regulated genes are transcribed. Only in the presence of both Bcl-2 and ICN, PD1.6 thymic lymphoma cells become resistant to glucocorticoid (GC)-induced apoptosis. Our data suggest that Bcl-2 plays a role in modulating Notch1 function in T cells.

Confinement to organelle-associated inclusion structures mediates asymmetric inheritance of aggregated protein in budding yeast.

The division of the S. cerevisiae budding yeast, which produces one mother cell and one daughter cell, is asymmetric with respect to aging. Remarkably, the asymmetry of yeast aging coincides with asymmetric inheritance of damaged and aggregated proteins by the mother cell. Here, we show that misfolded proteins are retained in the mother cell by being sequestered in juxtanuclear quality control compartment (JUNQ) and insoluble protein deposit (IPOD) inclusions, which are attached to organelles. Upon exposure to stress, misfolded proteins accumulate in stress foci that must be disaggregated by Hsp104 in order to be degraded or processed to JUNQ and IPOD. Cells that fail to deliver aggregates to an inclusion pass on aggregates to subsequent generations.

Glycogen synthase kinase-3 plays a central role in mediating glucocorticoid-induced apoptosis.

It is still unclear how glucocorticoids (GCs) induce apoptosis of thymocytes and T lymphoma cells. Emergence of GC-resistant lymphoma cells is a major obstacle in GC therapy, emphasizing the need for novel strategies that maintain the sensitivity of lymphoma cells to the proapoptotic effects of GC. We have undertaken a kinome study to elucidate the signal transduction pathways involved in mediating GC-induced apoptosis. Our study shows that glycogen synthase kinase (GSK3) plays a central role in promoting GC-induced apoptosis. In the absence of a ligand, GSK3alpha, but not GSK3beta, is sequestered to the glucocorticoid receptor (GR). Exposure to GCs leads to dissociation of GSK3alpha from GR and subsequent interaction of GSK3alpha and GSK3beta with the proapoptotic Bim protein, an essential mediator of GC-induced apoptosis. Chemical inhibition of GSK3 by SB216763, BIO-Acetoxime, or LiCl and GSK3 inhibition using a dominant-negative mutant of GSK3 impede this cell death process, indicating that GSK3 is involved in transmitting the apoptotic signal. GC resistance in lymphoma cells can be relieved by inhibiting the phosphatidylinositol-3 kinase-Akt survival pathway, which inactivates GSK3. Notch1, a transcription factor frequently activated in T acute lymphoblastic leukemia cells, confers GC resistance through activation of Akt. Altogether, this study illuminates the link connecting upstream GR signals to the downstream mediators of GC-induced apoptosis. Our data suggest that targeting protein kinases involved in GSK3 inactivation should improve the outcome of GC therapy.

Protein kinase networks regulating glucocorticoid-induced apoptosis of hematopoietic cancer cells: fundamental aspects and practical considerations.

Glucocorticoids (GCs) are integral components in the treatment protocols of acute lymphoblastic leukemia, multiple myeloma, and non-Hodgkin lymphoma owing to their ability to induce apoptosis of these malignant cells. Resistance to GC therapy is associated with poor prognosis. Although they have been used in clinics for decades, the signal transduction pathways involved in GC-induced apoptosis have only partly been resolved. Accumulating evidence shows that this cell death process is mediated by a communication between nuclear GR affecting gene transcription of pro-apoptotic genes such as Bim, mitochondrial GR affecting the physiology of the mitochondria, and the protein kinase glycogen synthase kinase-3 (GSK3), which interacts with Bim following exposure to GCs. Prevention of Bim up-regulation, mitochondrial GR translocation, and/or GSK3 activation are common causes leading to GC therapy failure. Various protein kinases positively regulating the pro-survival Src-PI3K-Akt-mTOR and Raf-Ras-MEK-ERK signal cascades have been shown to be activated in malignant leukemic cells and antagonize GC-induced apoptosis by inhibiting GSK3 activation and Bim expression. Targeting these protein kinases has proven effective in sensitizing GR-positive malignant lymphoid cells to GC-induced apoptosis. Thus, intervening with the pro-survival kinase network in GC-resistant cells should be a good means of improving GC therapy of hematopoietic malignancies.