Asymmetric cancer cell division regulated by AKT.

Human tumors often contain slowly proliferating cancer cells that resist treatment, but we do not know precisely how these cells arise. We show that rapidly proliferating cancer cells can divide asymmetrically to produce slowly proliferating "G0-like" progeny that are enriched following chemotherapy in breast cancer patients. Asymmetric cancer cell division results from asymmetric suppression of AKT/PKB kinase signaling in one daughter cell during telophase of mitosis. Moreover, inhibition of AKT signaling with small-molecule drugs can induce asymmetric cancer cell division and the production of slow proliferators. Cancer cells therefore appear to continuously flux between symmetric and asymmetric division depending on the precise state of their AKT signaling network. This model may have significant implications for understanding how tumors grow, evade treatment, and recur.

Expressed isolated integrin beta1 subunit cytodomain induces endothelial cell death secondary to detachment.

Expression of isolated beta integrin cytoplasmic domains in cultured endothelial cells was reported to induce cell detachment and death. To test whether cell death was the cause or the consequence of cell detachment, we expressed isolated integrin beta1 cytoplasmic and transmembrane domains (CH1) in cultured human umbilical vein endothelial cells (HUVEC), and monitored detachment, viability, caspase activation and signaling. CH1 expression induced dose-dependent cell detachment. At 24 h over 90% of CH1-expressing HUVEC were detached but largely viable (>85%). No evidence of pro-caspase-8,-3, and PARP cleavage or suppression of phosphorylation of ERK, PKB and Ikappa-B was observed. The caspase inhibitor z-VAD did not prevent cell detachment. At 48 h, however, CH1-expressing cells were over 50% dead. As a comparison trypsin-mediated detachment resulted in a time-dependent cell death, paralleled by caspase-3 activation and suppression of ERK, PKB and Ikappa-B phosphoyrylation at 24 h or later after detachment. HUVEC stimulation with agents that strengthen integrin-mediated adhesion (i.e. PMA, the Src inhibitor PP2 and COMP-Ang1) did not prevent CH1-induced detachment. Expression of CH1 in rat carotid artery endothelial cells in vivo caused endothelial cell detachment and increased nuclear DNA fragmentation among detached cells. A construct lacking the integrin cytoplasmic domain (CH2) had no effect on adhesion and cell viability in vitro and in vivo. These results demonstrate that isolated beta1 cytoplasmic domain expression induces caspase-independent detachment of viable endothelial cells and that death is secondary to detachment (i.e. anoikis). They also reveal an essential role for integrins in the adhesion and survival of quiescent endothelial cells in vivo.

Insulin-secreting beta-cell dysfunction induced by human lipoproteins.

Diabetes is associated with significant changes in plasma concentrations of lipoproteins. We tested the hypothesis that lipoproteins modulate the function and survival of insulin-secreting cells. We first detected the presence of several receptors that participate in the binding and processing of plasma lipoproteins and confirmed the internalization of fluorescent low density lipoprotein (LDL) and high density lipoprotein (HDL) particles in insulin-secreting beta-cells. Purified human very low density lipoprotein (VLDL) and LDL particles reduced insulin mRNA levels and beta-cell proliferation and induced a dose-dependent increase in the rate of apoptosis. In mice lacking the LDL receptor, islets showed a dramatic decrease in LDL uptake and were partially resistant to apoptosis caused by LDL. VLDL-induced apoptosis of beta-cells involved caspase-3 cleavage and reduction in the levels of the c-Jun N-terminal kinase-interacting protein-1. In contrast, the proapoptotic signaling of lipoproteins was antagonized by HDL particles or by a small peptide inhibitor of c-Jun N-terminal kinase. The protective effects of HDL were mediated, in part, by inhibition of caspase-3 cleavage and activation of Akt/protein kinase B. In conclusion, human lipoproteins are critical regulators of beta-cell survival and may therefore contribute to the beta-cell dysfunction observed during the development of type 2 diabetes.

c-Myc controls the development of CD8alphaalpha TCRalphabeta intestinal intraepithelial lymphocytes from thymic precursors by regulating IL-15-dependent survival.

The murine gut epithelium contains a large population of thymus-derived intraepithelial lymphocytes (IELs), including both conventional CD4(+) and CD8alphabeta(+) T cells (expressing T-cell receptor alphabeta [TCRalphabeta]) and unconventional CD8alphaalpha(+) T cells (expressing either TCRalphabeta or TCRgammadelta). Whereas conventional IELs are widely accepted to arise from recirculation of activated CD4(+) and CD8alphabeta(+) T cells from the secondary lymphoid organs to the gut, the origin and developmental pathway of unconventional CD8alphaalpha IELs remain controversial. We show here that CD4-Cre-mediated inactivation of c-Myc, a broadly expressed transcription factor with a wide range of biologic activities, selectively impairs the development of CD8alphaalpha TCRalphabeta IELs. In the absence of c-Myc, CD4(-) CD8(-) TCRalphabeta(+) thymic precursors of CD8alphaalpha TCRalphabeta IELs are present but fail to develop on adoptive transfer in immunoincompetent hosts. Residual c-Myc-deficient CD8alphaalpha TCRalphabeta IEL display reduced proliferation and increased apoptosis, which correlate with significantly decreased expression of interleukin-15 receptor subunits and lower levels of the antiapoptotic protein Bcl-2. Transgenic overexpression of human BCL-2 resulted in a pronounced rescue of CD8alphaalpha TCRalphabeta IEL in c-Myc-deficient mice. Taken together, our data support a model in which c-Myc controls the development of CD8alphaalpha TCRalphabeta IELs from thymic precursors by regulating interleukin-15 receptor expression and consequently Bcl-2-dependent survival.

Deleterious Actions Of Vegf On The Blood Retinal Barrier And Photoreceptor Survival In The Light-damage Model

Purpose: The retinal balance between pro- and anti-angiogenic factors is critical for angiogenesis control, but is also involved in cell survival. We previously reported upregulation of VEGF and photoreceptor (PR) cell death in the Light-damage (LD) model. Preliminary results showed that anti-VEGF can rescue PR from cell death. Thus, we investigated the role of VEGF on the retina and we herein described the effect of anti-VEGF antibody delivered by lentiviral gene transfer in this model.Methods: To characterize the action of VEGF during the LD, we exposed Balb/c mice subretinally injected with LV-anti-VEGF, or not, to 5'000 lux for 1h. We next evaluated the retinal function, PR survival and protein expression (VEGF, VEGFR1/2, Src, PEDF, p38MAPK, Akt, Peripherin, SWL-opsin) after LD. We analyzed Blood retinal barrier (BRB) integrity on flat-mounted RPE and cryosections stained with β-catenin, ZO-1, N-cadherin and albumin.Results: Results indicate that the VEGF pathway is modulated after LD. LD leads to extravascular albumin leakage and BRB breakdown: β-catenin, ZO-1 and N-cadherin translocate to the cytoplasm of RPE cells showing loss of cell cohesion. This phenomenon is in adequacy with the VEGF time-course expression. Assessment of the retinal function reveals that PR rescue correlates with the level of LV-anti-VEGF expression. Rhodopsin content was higher in the LV-anti-VEGF group than in controls and measures of the ONL thickness indicate that LV-anti-VEGF preserves by 82% the outer nuclear layer from degeneration. Outer segments (OS) appeared well organized with an appropriate length in the LV-anti-VEGF group compared to controls, and the expression of SWL-opsin is maintained in the OS without being mislocalized as in the LV-GFP group. Finally, LV-anti-VEGF treatment prevents BRB breakdown and maintained RPE cell integrity.Conclusions: This study involves VEGF in LD and highlights the prime importance of the BRB integrity for PR survival. Taken together, these results show that anti-VEGF is neuroprotective in this model and maintains functional PR layer in LD-treated mice.

Fluorodeoxyuridine mediated cell cycle synchronization in S-phase increases the Auger radiation cell killing with 125I-iododeoxyuridine.

Aim: 125I-iododeoxyuridine is a potential Auger radiation therapy agent. Its incorporation in DNA of proliferating cells is enhanced by fluorodeoxyuridine. Here, we evaluated therapeutic activities of 125I-iododeoxyuridine in an optimized fluorodeoxyuridine pre-treatment inducing S-phase synchronization. Methods: After S-phase synchronization by fluorodeoxyuridine, cells were treated with 125I-iododeoxyuridine. Apoptosis analysis and S-phase synchronization were studied by flow cytometry. Cell survival was determined by colony-forming assay. Based on measured growth parameters, the number of decays per cell that induced killing was extrapolated. Results: Treatment experiments showed that 72 to 91% of synchronized cells were killed after 0.8 and 8 kBq/ml 125I-iododeoxyuridine incubation, respectively. In controls, only 8 to 38% of cells were killed by corresponding 125I-iododeoxyuridine activities alone and even increasing the activity to 80 kBq/ml gave only 42 % killing. Duplicated treatment cycles or repeated fluorodeoxyuridine pre-treatment allowed enhancing cell killing to >95 % at 8 kBq/ml 125I-iododeoxyuridine. About 50 and 160 decays per S-phase cells in controls and S-phase synchronization, respectively, were responsible for the observed cell killing at 0.8 kBq/ml radio-iododeoxyuridine. Conclusion: These data show the successful application of fluorodeoxyuridine that provided increased 125I-iododeoxyuridine Auger radiation cell killing efficacy through S-phase synchronization and high DNA incorporation of radio-iododeoxyuridine.

APRIL secreted by neutrophils binds to heparan sulfate proteoglycans to create plasma cell niches in human mucosa.

The bone marrow constitutes a favorable environment for long-lived antibody-secreting plasma cells, providing blood-circulating antibody. Plasma cells are also present in mucosa-associated lymphoid tissue (MALT) to mediate local frontline immunity, but how plasma cell survival there is regulated is not known. Here we report that a proliferation-inducing ligand (APRIL) promoted survival of human upper and lower MALT plasma cells by upregulating expression of the antiapoptotic proteins bcl-2, bcl-xL, and mcl-1. The in situ localization of APRIL was consistent with such a prosurvival role in MALT. In upper MALT, tonsillar epithelium produced APRIL. Upon infection, APRIL production increased considerably when APRIL-secreting neutrophils recruited from the blood infiltrated the crypt epithelium. Heparan sulfate proteoglycans (HSPGs) retained secreted APRIL in the subepithelium of the infected zone to create APRIL-rich niches, wherein IgG-producing plasma cells accumulated. In lower MALT, neutrophils were the unique source of APRIL, giving rise to similar niches for IgA-producing plasmocytes in villi of lamina propria. Furthermore, we found that mucosal humoral immunity in APRIL-deficient mice is less persistent than in WT mice. Hence, production of APRIL by inflammation-recruited neutrophils may create plasma cell niches in MALT to sustain a local antibody production.

The role of ubiquitin-proteasome system in glioma survival and growth.

High-grade gliomas represent a group of aggressive brain tumors with poor prognosis due to an inherent capacity of persistent cell growth and survival. The ubiquitin-proteasome system (UPS) is an intracellular machinery responsible for protein turnover. Emerging evidence implicates various proteins targeted for degradation by the UPS in key survival and proliferation signaling pathways of these tumors. In this review, we discuss the involvement of UPS in the regulation of several mediators and effectors of these pathways in malignant gliomas.

Peroxisome proliferator-activated receptor gamma is required in mature white and brown adipocytes for their survival in the mouse.

The peroxisome proliferator-activated receptor gamma (PPARgamma) mediates the activity of the insulin-sensitizing thiazolidinediones and plays an important role in adipocyte differentiation and fat accretion. The analysis of PPARgamma functions in mature adipocytes is precluded by lethality of PPARgamma(-/-) fetuses and tetraploid-rescued pups. Therefore we have selectively ablated PPARgamma in adipocytes of adult mice by using the tamoxifen-dependent Cre-ER(T2) recombination system. We show that mature PPARgamma-null white and brown adipocytes die within a few days and are replaced by newly formed PPARgamma-positive adipocytes, demonstrating that PPARgamma is essential for the in vivo survival of mature adipocytes, in addition to its well established requirement for their differentiation. Our data suggest that potent PPARgamma antagonists could be used to acutely reduce obesity.

Cell viability and noninvasive in vivo MRI tracking of 3D cell encapsulating self-assembled microcontainers.

Several molecular therapies require the implantation of cells that secrete biotherapeutic molecules and imaging the location and microenvironment of the cellular implant to ascertain its function. We demonstrate noninvasive in vivo magnetic resonance imaging (MRI) of self-assembled microcontainers that are capable of cell encapsulation. Negative contrast was obtained to discern the microcontainer with MRI; positive contrast was obtained in the complete absence of background signal. MRI on a clinical scanner highlights the translational nature of this research. The microcontainers were loaded with cells that were dispersed in an extracellular matrix, and implanted both subcutaneously and in human tumor xenografts in SCID mice. MRI was performed on the implants, and microcontainers retrieved postimplantation showed cell viability both within and proximal to the implant. The microcontainers are characterized by their small size, three dimensionality, controlled porosity, ease of parallel fabrication, chemical and mechanical stability, and noninvasive traceability in vivo.

Role of MicroRNAs in Islet Beta-Cell Compensation and Failure during Diabetes.

Pancreatic beta-cell function and mass are markedly adaptive to compensate for the changes in insulin requirement observed during several situations such as pregnancy, obesity, glucocorticoids excess, or administration. This requires a beta-cell compensation which is achieved through a gain of beta-cell mass and function. Elucidating the physiological mechanisms that promote functional beta-cell mass expansion and that protect cells against death, is a key therapeutic target for diabetes. In this respect, several recent studies have emphasized the instrumental role of microRNAs in the control of beta-cell function. MicroRNAs are negative regulators of gene expression, and are pivotal for the control of beta-cell proliferation, function, and survival. On the one hand, changes in specific microRNA levels have been associated with beta-cell compensation and are triggered by hormones or bioactive peptides that promote beta-cell survival and function. Conversely, modifications in the expression of other specific microRNAs contribute to beta-cell dysfunction and death elicited by diabetogenic factors including, cytokines, chronic hyperlipidemia, hyperglycemia, and oxidized LDL. This review underlines the importance of targeting the microRNA network for future innovative therapies aiming at preventing the beta-cell decline in diabetes.

Intravitreous injection of PLGA microspheres encapsulating GDNF promotes the survival of photoreceptors in the rd1/rd1 mouse.

PURPOSE: To evaluate the potential delay of the retinal degeneration in rd1/rd1 mice using recombinant human glial cell line-derived neurotrophic factor (rhGDNF) encapsulated in poly(D,L-lactide-co-glycolide) (PLGA) microspheres. METHODS: rhGDNF-loaded PLGA microspheres were prepared using a water in oil in water (w/o/w) emulsion solvent extraction-evaporation process. In vitro, the rhGDNF release profile was assessed using radiolabeled factor. In vivo, rhGDNF microspheres, blank microspheres, or microspheres loaded with inactivated rhGDNF were injected into the vitreous of rd1/rd1 mice at postnatal day 11 (PN11). The extent of retinal degeneration was examined at PN28 using rhodopsin immunohistochemistry on whole flat-mount retinas, outer nuclear layer (ONL) cell counting on histology sections, and electroretinogram tracings. Immunohistochemical reactions for glial fibrillary acidic protein (GFAP), F4/80, and rhodopsin were performed on cryosections. RESULTS: Significant delay of rod photoreceptors degeneration was observed in mice receiving the rhGDNF-loaded microspheres compared to either untreated mice or to mice receiving blank or inactivated rhGDNF microspheres. The degeneration delay in the eyes receiving the rhGDNF microspheres was illustrated by the increased rhodopsin positive signals, the preservation of significantly higher number of cell nuclei within the ONL, and significant b-wave increase. A reduction of the subretinal glial proliferation was also observed in these treated eyes. No significant intraocular inflammatory reaction was observed after the intravitreous injection of the various microspheres. CONCLUSIONS: A single intravitreous injection of rhGDNF-loaded microspheres slows the retinal degeneration processes in rd1/rd1 mice. The use of injectable, biodegradable polymeric systems in the vitreous enables the efficient delivery of therapeutic proteins for the treatment of retinal diseases.

Covalent cell surface functionalization of human fetal osteoblasts for tissue engineering.

The chemical functionalization of cell-surface proteins of human primary fetal bone cells with hydrophilic bioorthogonal intermediates was investigated. Toward this goal, chemical pathways were developed for click reaction-mediated coupling of alkyne derivatives with cellular azido-expressing proteins. The incorporation via a tetraethylene glycol linker of a dipeptide and a reporter biotin allowed the proof of concept for the introduction of cell-specific peptide ligands and to follow the reaction in living cells. Tuning the conditions of the click reaction resulted in chemical functionalization of living human fetal osteoblasts with excellent cell survival.

Ectopic lymphoid-organ development occurs through interleukin 7-mediated enhanced survival of lymphoid-tissue-inducer cells.

Development of Peyer's patches and lymph nodes requires the interaction between CD4+ CD3- IL-7Ralpha+ lymphoid-tissue inducer (LTi) and VCAM-1+ organizer cells. Here we showed that by promoting their survival, enhanced expression of interleukin-7 (IL-7) in transgenic mice resulted in accumulation of LTi cells. With increased IL-7 availability, de novo formation of VCAM-1+ Peyer's patch anlagen occurred along the entire fetal gut resulting in a 5-fold increase in Peyer's patch numbers. IL-7 overexpression also led to formation of multiple organized ectopic lymph nodes and cecal patches. After immunization, ectopic lymph nodes developed normal T cell-dependent B cell responses and germinal centers. Mice overexpressing IL-7 but lacking either RORgamma, a factor required for LTi cell generation, or lymphotoxin alpha1beta2 had neither Peyer's patches nor ectopic lymph nodes. Therefore, by controlling LTi cell numbers, IL-7 can regulate the formation of both normal and ectopic lymphoid organs.

Cell-permeable peptides induce dose- and length-dependent cytotoxic effects.

We have explored the threshold of tolerance of three unrelated cell types to treatments with potential cytoprotective peptides bound to Tat(48-57) and Antp(43-58) cell-permeable peptide carriers. Both Tat(48-57) and Antp(43-58) are well known for their good efficacy at crossing membranes of different cell types, their overall low toxicity, and their absence of leakage once internalised. Here, we show that concentrations of up to 100 microM of Tat(48-57) were essentially harmless in all cells tested, whereas Antp(43-58) was significantly more toxic. Moreover, all peptides bound to Tat(48-57) and Antp(43-58) triggered significant and length-dependent cytotoxicity when used at concentrations above 10 microM in all but one cell types (208F rat fibroblasts), irrespective of the sequence of the cargo. Absence of cytotoxicity in 208F fibroblasts correlated with poor intracellular peptide uptake, as monitored by confocal laser scanning fluorescence microscopy. Our data further suggest that the onset of cytotoxicity correlates with the activation of two intracellular stress signalling pathways, namely those involving JNK, and to a lesser extent p38 mitogen-activated protein kinases. These responses are of particular concern for cells that are especially sensitive to the activation of stress kinases. Collectively, these results indicate that in order to avoid unwanted and unspecific cytotoxicity, effector molecules bound to Tat(48-57) should be designed with the shortest possible sequence and the highest possible affinity for their binding partners or targets, so that concentrations below 10 microM can be successfully applied to cells without harm. Considering that cytotoxicity associated to Tat(48-57)- and Antp(43-58) bound peptide conjugates was not restricted to a particular type of cells, our data provide a general framework for the design of cell-penetrating peptides that may apply to broader uses of intracellular peptide and drug delivery.