One, two, three—p53, p63, p73 and chemosensitivity

Molecular links between apoptosis, tumorigenesis and drug resistance provide starting points for new therapeutic approaches and for a targeted cancer therapy. The discovery of the p53-related genes p63 and p73 raised the possibility that they may be cancer-associated genes and as a consequence that p53 is not the only component in predicting prognosis and response to chemotherapy, but instead the status of a network that contains p53, p73 and p63. This review focuses on the status and interrelationship of the p53 family members in human cancer as critical elements for tumor progression and response to therapy. Literature up to December 2006 is reviewed. p63 and p73--as well as p53--each use multiple promoters and alternative splicing to generate an array of isoforms, including full-length isoforms with a transactivation (TA-) domain homologous to that of full-length p53, and amino-terminally truncated (DeltaN-) isoforms. Whereas the full-length TA isoforms of p63 and p73 can activate downstream target genes and induce apoptosis, the DeltaN isoforms which lack the transactivation domain can act as dominant inhibitors of the full-length forms of p53, p63 and p73, inhibiting transactivation of target genes and induction of apoptosis. Deregulated dominant negative p63 and p73 isoforms play an oncogenic role in human cancer and contribute to chemoresistance. Thus, therapeutic modulation of TAp63/DeltaNp63, TAp73/DeltaNp73 and mutant p53 levels might be used to target the large percentage of human tumors that harbor p53 mutations and/or overexpress DeltaNp63 or DeltaNp73. Interfering with the expression or function of DeltaNp63 and/or DeltaNp73 and/or mutant p53 in tumor cells may render such tumors more responsive to therapy and reduce their aggressiveness and metastatic capacity.     

Targeting CDK9 by wogonin and related natural flavones potentiates the anti‐cancer efficacy of the Bcl‐2 family inhibitor ABT‐263

Tumor initiation, progression and resistance to therapies are tightly associated with over‐expression of anti‐apoptotic proteins Bcl‐2, Bcl‐x_L, Bcl‐w and Mcl‐1. ABT‐263 (Navitoclax), an orally bio‐available small‐molecule mimetic of the Bcl‐2 homology domain 3, inhibits Bcl‐2, Bcl‐x_L, and Bcl‐w and has shown anti‐cancer effects mainly on lymphomas and lymphocytic leukemia. Despite promising results obtained from the clinical trials, the use of ABT‐263 in patients is dose‐limited due to causing thrombocytopenia via inhibition of Bcl‐x_L in platelets. ABT‐199 specifically inhibits Bcl‐2; however, its use is limited to tumors over‐expressing only Bcl‐2. Besides, many tumors resist treatment due to high levels of Mcl‐1 expression or develop resistance via up‐regulation of Mcl‐1 during long‐term exposure. These obstacles highlight the demand to improve the ABT‐263‐based therapy. In this study, we show that anti‐cancer flavones, e.g., wogonin, baicalein, apigenin, chrysin and luteolin enhance ABT‐263‐induced apoptosis in different cancer cell lines and in primary AML and ALL cells by down‐regulation of Mcl‐1 expression. Importantly, wogonin does not enhance the toxicity of ABT‐263 to proliferating normal T cells and thrombocytes. Wogonin also potentiates the lethality of ABT‐263 in cancer cells which have acquired resistance to ABT‐263. Furthermore, we show that combination of wogonin with ABT‐263 promotes in vivo tumor regression in a human T‐cell leukemia xenograft mouse model. Our study demonstrates that wogonin (and related flavones) reduce the effective dose of ABT‐263 thereby possibly decreasing the risk of adverse side effects.     

Rocaglamide and a XIAP inhibitor cooperatively sensitize TRAIL-mediated apoptosis in Hodgkin's lymphomas

Although most of the patients with Hodgkin's lymphoma (HL) can be cured by the current regimen of high-dose multiagent chemotherapy, the treatment causes high risks of later toxicities including secondary malignancies. Therefore, new rational strategies are needed for HL treatment. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent due to its tumor selectivity and its lack of toxicity for normal cells. Unfortunately, many cancers remain resistant to TRAIL including HL. HL is characterized by enhanced expression of cellular caspase-8 (FLICE)-inhibitory protein (c-FLIP) and X-linked inhibitor of apoptosis (XIAP), which block receptor-mediated apoptosis by inhibiting caspase-8 and caspase-3, respectively. We have recently discovered the herbal compound Rocaglamide, which breaks TRAIL-resistance in acute T cell leukemia through inhibition of c-FLIP expression. We have also shown that small molecule XIAP inhibitors can sensitize TRAIL-mediated apoptosis in several resistant tumors. However, whether targeting XIAP or c-FLIP is also a suitable strategy to prime HL cells for TRAIL-induced apoptosis has not yet been investigated. In our study, we show that Rocaglamide suppresses c-FLIP expression in HL cells in a dose- and time-dependent manner. However, downregulation of c-FLIP alone was not sufficient to sensitize TRAIL-induced apoptosis in HL cells. Similarly, treatment of HL cells with a small molecule XIAP inhibitor resulted in a moderate induction of apoptosis. However, inhibition of XIAP alone was also not sufficient to enhance TRAIL-induced cell death. Synergistic increase in TRAIL-mediated killing of HL cells was only obtained by combination of Rocaglamide and XIAP inhibitors. Our study demonstrates that targeting both c-FLIP and XIAP are necessary for an efficient treatment of HL.     

Clonal deletion of major histocompatibility complex class I-restricted CD4+CD8+ thymocytes in vitro is independent of the CD95 (APO-1/Fas) ligand

The CD95 (APO-1/Fas) ligand (CD95L) mediates apoptosis in sensitive target cells, Ca²⁺-independent cytotoxicity of cells from perforin knock-out mice, and peripheral deletion of activated T cells through engagement of its cognate receptor CD95. Double-positive thymocytes show a high constitutive expression of CD95. Therefore, we used a model system and investigated whether negative selection through apoptosis might involve CD95/CD95L. We analyzed whether CD95L may induce antigen-specific deletion of double-positive thymocytes from mice transgenic for a lymphocytic choriomeningitis virus (LCMV)/H2^b-specific T cell receptor (TCR). These cells are deleted in vitro upon addition of the LCMV-peptide 33–41 in a major histocompatibility complex-class I-restricted fashion. Deletion was not blocked by soluble mouse and human CD95-Fc receptor decoys. CD95-Fc receptor decoys, however, were effective in blocking apoptosis induced by mouse CD95L-transfected L929 cells in sensitive CD95⁺ target cells and in thymocytes. These results suggest that TCR-induced deletion of immature thymocytes in vitro is independent of CD95L. Thus, our data argue against a role of CD95L in negative selection of MHC-class I-restricted autoreactive thymocytes.     

Komplikationen winkelstabiler Plattenosteosynthesen am Humeruskopf

From January 2002 to April 2005, 200 proximal humeral head fractures in 196 patients with an average age of 67 years (±16) were treated with an angular-stable proximal humeral plate (PHILOS, Synthes). A total of 162 patients were followed-up at a mean interval of 13.8 months. The mean constant score was 66±19 points. With 81 registered complications, 40 revision operations were necessary. Most of these were due to technical errors such as implant-dislocation of the screws (22) or plates (6). Intraoperative axial varus deviations (7) or insufficient reduction of the tubercula (8) were also found. Secondary implant loosening in the humeral head or in the shaft was registered in seven times. Necroses of the humeral head (5), partial necroses (9) or sintering (4) appeared to be due to the fracture type and should be rated more as a consequence of a blood circulation disturbance rather than a technical failure. Despite the considerable rate of complications, the proximal locking humeral plate offers good potential for fixing dislocated fracture parts, thus permitting early functional treatment.     

Investigating Machine Learning Techniques for Predicting the Process Characteristics of Stencil Printing

Stencil printing is the most crucial process in reflow soldering for the mass assembly of electronic circuits. This paper investigates different machine learning-based methods to predict the essential process characteristics of stencil printing: the area, thickness, and volume of deposited solder paste. The training dataset was obtained experimentally by varying the printing speed (from 20 to 120 mm/s), the size (area ratio from 0.35 to 1.7) of stencil apertures, and the particle size (characterized by a log-normal distribution) in the solder paste. Various machine learning-based methods were assessed; ANFIS–adaptive neuro-fuzzy inference systems; ANN artificial neural networks (with different learning methods); boosted trees, regression trees, SVM–support vector machines. Each method was optimized and fine-tuned with hyperparameter optimization, and the overfitting phenomenon was also prevented with cross-validation. The regression tree was the best performing approach for modelling the stencil printing, while ANN with the Bayesian regularization learning method was only slightly worse. The presented methodology for fine-tuning, parameter optimization, and the comparison of different machine learning-based methods can easily be adapted to any application field in electronics manufacturing.     

Correlation of histological and ex-vivo confocal tumor thickness in malignant melanoma

The ex-vivo confocal laser scanning microscopy (ex-vivo CLSM) is a novel diagnostic method for fresh tissue examination, which has already shown promising results in the evaluation of healthy skin and different skin tumors. In malignant melanoma, the histological tumor thickness plays an essential role for further treatment strategies. The immediate perioperative measurement of tumor thickness by means of ex-vivo CLSM might accelerate the decision for further operating procedures in malignant melanoma. Ten histologically confirmed malignant melanomas from various donor sites were blindly examined by two investigators via ex-vivo CLSM and conventional light microscopy. The histopathological tumor thickness (HTT) and confocal tumor thickness (CTT) were measured independently and evaluated using correlation curves, Spearman’s correlation coefficient, and Bland-Altman plots. Bland-Altman plots for HTT and reflectance-mode CTT, as well as for fluorescence-mode CTT, showed high correlations. Spearman’s correlation coefficient of HTT and CTT was 1.00 in FM and RM. The mean difference of RM-CTT and FM-CTT versus HTT was 0.09?±?0.30 mm and 0.19?±?0.35 mm. In one case, the HTT was identical to the CTT in both modes. This pilot study shows high conformity of CTT and HTT measured in malignant melanoma underlining the potential of ex-vivo CLSM for perioperative decisions on safety margin excisions of malignant melanoma in the future.     

Hemagglutinin stalk antibodies elicited by the 2009 pandemic influenza virus as a mechanism for the extinction of seasonal H1N1 viruses

After the emergence of pandemic influenza viruses in 1957, 1968, and 2009, existing seasonal viruses were observed to be replaced in the human population by the novel pandemic strains. We have previously hypothesized that the replacement of seasonal strains was mediated, in part, by a population-scale boost in antibodies specific for conserved regions of the hemagglutinin stalk and the viral neuraminidase. Numerous recent studies have shown the role of stalk-specific antibodies in neutralization of influenza viruses; the finding that stalk antibodies can effectively neutralize virus alters the existing dogma that influenza virus neutralization is mediated solely by antibodies that react with the globular head of the viral hemagglutinin. The present study explores the possibility that stalk-specific antibodies were boosted by infection with the 2009 H1N1 pandemic virus and that those antibodies could have contributed to the disappearance of existing seasonal H1N1 influenza virus strains. To study stalk-specific antibodies, we have developed chimeric hemagglutinin constructs that enable the measurement of antibodies that bind the hemagglutinin protein and neutralize virus but do not have hemagglutination inhibition activity. Using these chimeric hemagglutinin reagents, we show that infection with the 2009 pandemic H1N1 virus elicited a boost in titer of virus-neutralizing antibodies directed against the hemagglutinin stalk. In addition, we describe assays that can be used to measure influenza virus-neutralizing antibodies that are not detected in the traditional hemagglutination inhibition assay.