Cell arrest and cell death in mammalian preimplantation development

The causes, modes, biological role and prospective significance of cell death in preimplantation development in humans and other mammals are still poorly understood. Early bovine embryos represent a very attractive experimental model for the investigation of this fundamental and important issue. To obtain reference data on the temporal and spatial occurrence of cell death in early bovine embryogenesis, three-dimensionally preserved embryos of different ages and stages of development up to hatched blastocysts were examined in toto by confocal laser scanning microscopy. In parallel, transcript abundance profiles for selected apoptosis-related genes were analyzed by real-time reverse transcriptase-polymerase chain reaction. Our study documents that in vitro as well as in vivo, the first four cleavage cycles are prone to a high failure rate including different types of permanent cell cycle arrest and subsequent non-apoptotic blastomere death. In vitro produced and in vivo derived blastocysts showed a significant incidence of cell death in the inner cell mass (ICM), but only in part with morphological features of apoptosis. Importantly, transcripts for CASP3, CASP9, CASP8 and FAS/FASLG were not detectable or found at very low abundances. In vitro and in vivo, errors and failures of the first and the next three cleavage divisions frequently cause immediate embryo death or lead to aberrant subsequent development, and are the main source of developmental heterogeneity. A substantial occurrence of cell death in the ICM even in fast developing blastocysts strongly suggests a regular developmentally controlled elimination of cells, while the nature and mechanisms of ICM cell death are unclear. Morphological findings as well as transcript levels measured for important apoptosis-related genes are in conflict with the view that classical caspase-mediated apoptosis is the major cause of cell death in early bovine development

Visual function improvement using photocromic and selective blue-violet light filtering spectacle lenses in patients affected by retinal diseases

Background To evaluate functional visual parameters using photocromic and selective blue-violet light filtering spectacle lenses in patients affected by central or peripheral scotoma due to retinal diseases. Sixty patients were enrolled in this study: 30 patients affected by central scotoma, group 1, and 30 affected by peripheral scotoma, group 2. Black on White Best Corrected Visual Acuity (BW-BCVA), White on Black Best Corrected Visual Acuity (WB-BCVA), Mars Contrast Sensitivity (CS) and a Glare Test (GT) were performed to all patients. Test results with blue-violet filter, a short-pass yellow filter and with no filters were compared. Results All scores from test results increased significantly with blue-violet filters for all patients. The mean BW-BCVA increased from 0.30 ± 0.20 to 0.36 ± 0.21 decimals in group 1 and from 0.44 ± 0.22 to 0.51 ± 0.23 decimals in group 2 (Mean ± SD, p < 0.0001 in both cases). The mean WB-BCVA increased from 0.31 ± 0.19 to 0.38 ± 0.23 decimals in group 1 and from 0.46 ± 0.20 to 0.56 ± 0.22 decimals in group 2 (Mean ± SD, p < 0.0001 in both cases). The letter count for the CS test increased from 26.7 ± 7.9 to 30.06 ± 7.8 in group 1 (Mean ± SD, p = 0.0005) and from 31.5 ± 7.6 to 33.72 ± 7.3 in group 2 (Mean ± SD, p = 0.031). GT was significantly reduced: the letter count increased from 20.93 ± 5.42 to 22.82 ± 4.93 in group 1 (Mean ± SD, p < 0.0001) and from 24.15 ± 5.5 to 25.97 ± 4.7 in group 2 (Mean ± SD, p < 0.0001). Higher scores were recorded with the Blue filter compared to Yellow filter in all tests (p < 0.05). No significant differences in any test results could be detected between the Yellow filter and the No filter condition. Conclusions The use of a combination of photocromic lens with a selective blue-violet light filter showed functional benefit in all evaluated patients

Alpha-2-Macroglobulin Is Acutely Sensitive to Freezing and Lyophilization: Implications for Structural and Functional Studies.

Alpha-2-macroglobulin is an abundant secreted protein that is of particular interest because of its diverse ligand binding profile and multifunctional nature, which includes roles as a protease inhibitor and as a molecular chaperone. The activities of alpha-2-macroglobulin are typically dependent on whether its conformation is native or transformed (i.e. adopts a more compact conformation after interactions with proteases or small nucleophiles), and are also influenced by dissociation of the native alpha-2-macroglobulin tetramer into stable dimers. Alpha-2-macroglobulin is predominately present as the native tetramer in vivo; once purified from human blood plasma, however, alpha-2-macroglobulin can undergo a number of conformational changes during storage, including transformation, aggregation or dissociation. We demonstrate that, particularly in the presence of sodium chloride or amine containing compounds, freezing and/or lyophilization of alpha-2-macroglobulin induces conformational changes with functional consequences. These conformational changes in alpha-2-macroglobulin are not always detected by standard native polyacrylamide gel electrophoresis, but can be measured using bisANS fluorescence assays. Increased surface hydrophobicity of alpha-2-macroglobulin, as assessed by bisANS fluorescence measurements, is accompanied by (i) reduced trypsin binding activity, (ii) increased chaperone activity, and (iii) increased binding to the surfaces of SH-SY5Y neurons, in part, via lipoprotein receptors. We show that sucrose (but not glycine) effectively protects native alpha-2-macroglobulin from denaturation during freezing and/or lyophilization, thereby providing a reproducible method for the handling and long-term storage of this protein.Early Career Fellowship from the National Health and Medical Research Council GNT1012521(A.R.W.); Wellcome Trust Programme Grant (J.R.K., C.M.D.) 094425/Z/10/Z; Samsung GRO Grant (M.R.W.)This is the final version of the article. It first appeared from PLoS via http://dx.doi.org/10.1371/journal.pone.013003

Phellinus linteus sensitises apoptosis induced by doxorubicin in prostate cancer

It has been demonstrated that the Phellinus linteus (PL) mushroom, which mainly consists of polysaccharides, possesses antitumour activity. The mechanisms of PL against malignant growth remain unknown. The anticancer drug doxorubicin (Dox) has been shown to induce apoptosis via initiating a caspase cascade. In this investigation, we tested the effect of PL on Dox-induced apoptosis in prostate cancer LNCaP cells. We showed that PL or Dox, at relatively low doses, does not induce apoptosis in the cells. However, combination treatment with low doses of PL and Dox results in a synergistic effect on the induction of apoptosis. In this apoptotic process, caspases 8, 3 and BID are cleaved, and the addition of caspase inhibitor z-VADfmk completely blocks apoptosis. In addition, JNK is activated in response to PL or the combination treatment in LNCaP cells. The suppression of JNK partially inhibits the induction of apoptosis elicited by the co-treatment. These findings indicate that PL has a synergistic effect with Dox to activate caspases in prostate cancer LNCaP cells. Our study also suggests that PL has therapeutic potential to augment the magnitude of apoptosis induced by antiprostate cancer drugs

Survivin expression in ovarian cancer and its correlation with clinico-pathological, surgical and apoptosis-related parameters

We investigated the association of survivin expression with prognosis and other apoptosis-related biological factors in 110 primary ovarian cancer patients admitted to the Division of Gynecologic Oncology, Catholic University of Rome. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded sections by using polyclonal antibody ab469 for survivin, and mouse monoclonal antibodies (clone 124 and DO-7), for bcl-2 and p53, respectively. Cytoplasmic survivin immunoreaction was observed in 84.5% cases, while nuclear survivin immunostaining was observed in 29.1% cases. We failed to find any relationship between cytoplasmic survivin positivity rate and any of the parameters examined. Serous tumours showed a lower percentage of nuclear survivin positivity with respect to other histotypes (20.5 vs 48.6%, respectively; P-value=0.004). The percentage of nuclear survivin positivity was higher in cases subjected to primary tumour cytoreduction (43.5%), with respect to patients subjected to exploratory laparotomy (20%) (P=0.024). Bcl-2 and p53 were, respectively, expressed in 27.3 and 60.0% of the cases and their expression was not correlated with survivin status. During the follow-up period, progression and death of disease were observed in 68 (61.8%) and 53 (48.2%) cases, respectively. There was no difference in time to progression and overall survival according to survivin status in ovarian cancer patients. In conclusion, in our experience, the immunohistochemical assessment of survivin status does not seem to be helpful in the prognostic characterisation of ovarian cancer. A more in depth investigation of the complex physiology of divergent survivin variants is needed in order to clarify the biological and the clinical role of differentially located survivin isoforms

Histone deacetylase inhibitors strongly sensitise neuroblastoma cells to TRAIL-induced apoptosis by a caspases-dependent increase of the pro- to anti-apoptotic proteins ratio

BACKGROUND: Neuroblastoma (NB) is the second most common solid childhood tumour, an aggressive disease for which new therapeutic strategies are strongly needed. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in most tumour cells, but not in normal tissues and therefore represents a valuable candidate in apoptosis-inducing therapies. Caspase-8 is silenced in a subset of highly malignant NB cells, which results in full TRAIL resistance. In addition, despite constitutive caspase-8 expression, or its possible restoration by different strategies, NB cells remain weakly sensitive to TRAIL indicating a need to develop strategies to sensitise NB cells to TRAIL. Histone deacetylase inhibitors (HDACIs) are a new class of anti-cancer agent inducing apoptosis or cell cycle arrest in tumour cells with very low toxicity toward normal cells. Although HDACIs were recently shown to increase death induced by TRAIL in weakly TRAIL-sensitive tumour cells, the precise involved sensitisation mechanisms have not been fully identified. METHODS: NB cell lines were treated with various doses of HDACIs and TRAIL, then cytotoxicity was analysed by MTS/PMS proliferation assays, apoptosis was measured by the Propidium staining method, caspases activity by colorimetric protease assays, and (in)activation of apoptotic proteins by immunoblotting. RESULTS: Sub-toxic doses of HDACIs strongly sensitised caspase-8 positive NB cell lines to TRAIL induced apoptosis in a caspases dependent manner. Combined treatments increased the activation of caspases and Bid, and the inactivation of the anti-apoptotic proteins XIAP, Bcl-x, RIP, and survivin, thereby increasing the pro- to anti-apoptotic protein ratio. It also enhanced the activation of the mitochondrial pathway. Interestingly, the kinetics of caspases activation and inactivation of anti-apoptotic proteins is accelerated by combined treatment with TRAIL and HDACIs compared to TRAIL alone. In contrast, cell surface expression of TRAIL-receptors or TRAIL is not affected by sub-toxic doses of HDACIs. CONCLUSION: HDACIs were shown to activate the mitochondrial pathway and to sensitise NB cells to TRAIL by enhancing the amplitude of the apoptotic cascade and by restoring an apoptosis-prone ratio of pro- to anti-apoptotic proteins. Combining HDACIs and TRAIL could therefore represent a weakly toxic and promising strategy to target TRAIL-resistant tumours such as neuroblastomas

Conditional deletion of epithelial IKKβ impairs alveolar formation through apoptosis and decreased VEGF expression during early mouse lung morphogenesis

<p>Abstract</p> <p>Background</p> <p>Alveolar septation marks the beginning of the transition from the saccular to alveolar stage of lung development. Inflammation can disrupt this process and permanently impair alveolar formation resulting in alveolar hypoplasia as seen in bronchopulmonary dysplasia in preterm newborns. NF-κB is a transcription factor central to multiple inflammatory and developmental pathways including dorsal-ventral patterning in fruit flies; limb, mammary and submandibular gland development in mice; and branching morphogenesis in chick lungs. We have previously shown that epithelial overexpression of NF-κB accelerates lung maturity using transgenic mice. The purpose of this study was to test our hypothesis that targeted deletion of NF-κB signaling in lung epithelium would impair alveolar formation.</p> <p>Methods</p> <p>We generated double transgenic mice with lung epithelium-specific deletion of IKKβ, a known activating kinase upstream of NF-κB, using a cre-<it>loxP </it>transgenic recombination strategy. Lungs of resulting progeny were analyzed at embryonic and early postnatal stages to determine specific effects on lung histology, and mRNA and protein expression of relevant lung morphoreulatory genes. Lastly, results measuring expression of the angiogenic factor, VEGF, were confirmed <it>in vitro </it>using a siRNA-knockdown strategy in cultured mouse lung epithelial cells.</p> <p>Results</p> <p>Our results showed that IKKβ deletion in the lung epithelium transiently decreased alveolar type I and type II cells and myofibroblasts and delayed alveolar formation. These effects were mediated through increased alveolar type II cell apoptosis and decreased epithelial VEGF expression.</p> <p>Conclusions</p> <p>These results suggest that epithelial NF-κB plays a critical role in early alveolar development possibly through regulation of VEGF.</p

The clinical significance of splice variants and subcellular localisation of survivin in non-small cell lung cancers

Survivin is a member of the inhibitor of apoptosis protein family. Survivin has splice variants with different biological functions associated with tumorigenesis. We investigated 134 non-small cell lung cancers (NSCLCs) to study the clinical significance of wild-type survivin, survivin-2B, and survivin-deltaEx3. Real-time PCR analyses were performed for their gene expressions. The subcellular localisation of survivin proteins was evaluated by immunohistochemistry. The Ki-67 proliferation index and the apoptotic index were also evaluated. The survivin-deltaEx3 gene expression was significantly higher in stage II–III than in stage I (P=0.0174), and significantly correlated with the nuclear pan-survivin expression (P<0.0001). The Ki-67 index was significantly higher in wild-type survivin-positive tumours (P<0.0001), survivin-deltaEx3-positive tumours (P<0.0001), and tumours with positive expression of the nuclear pan-survivin (P=0.0047). In contrast, the apoptotic index was significantly lower only in wild-type survivin-positive tumours (P<0.0001). Thus, the wild-type survivin gene expression was associated with apoptotic inhibition and tumour proliferation. Furthermore, the survivin-deltaEx3 gene expression was strongly associated with tumour proliferation, especially in advanced stage NSCLCs. In contrast, the survivin-2B gene expression did not correlate with tumour proliferation or tumour apoptosis