Barnase as a New Therapeutic Agent Triggering Apoptosis in Human Cancer Cells

RNases are currently studied as non-mutagenic alternatives to the harmful DNA-damaging anticancer drugs commonly used in clinical practice. Many mammalian RNases are not potent toxins due to the strong inhibition by ribonuclease inhibitor (RI) presented in the cytoplasm of mammalian cells.In search of new effective anticancer RNases we studied the effects of barnase, a ribonuclease from Bacillus amyloliquefaciens, on human cancer cells. We found that barnase is resistant to RI. In MTT cell viability assay, barnase was cytotoxic to human carcinoma cell lines with half-inhibitory concentrations (IC(50)) ranging from 0.2 to 13 microM and to leukemia cell lines with IC(50) values ranging from 2.4 to 82 microM. Also, we characterized the cytotoxic effects of barnase-based immunoRNase scFv 4D5-dibarnase, which consists of two barnase molecules serially fused to the single-chain variable fragment (scFv) of humanized antibody 4D5 that recognizes the extracellular domain of cancer marker HER2. The scFv 4D5-dibarnase specifically bound to HER2-positive cells and was internalized via receptor-mediated endocytosis. The intracellular localization of internalized scFv 4D5-dibarnase was determined by electronic microscopy. The cytotoxic effect of scFv 4D5-dibarnase on HER2-positive human ovarian carcinoma SKOV-3 cells (IC(50) = 1.8 nM) was three orders of magnitude greater than that of barnase alone. Both barnase and scFv 4D5-dibarnase induced apoptosis in SKOV-3 cells accompanied by internucleosomal chromatin fragmentation, membrane blebbing, the appearance of phosphatidylserine on the outer leaflet of the plasma membrane, and the activation of caspase-3.These results demonstrate that barnase is a potent toxic agent for targeting to cancer cells

Bone collagen and muscle δ¹³C in relation to the timing of the migration of Garden Warblers Sylvia borin during return migration from Africa

In migratory birds, seasonal factors interacting at different stages of the annual cycle can influence individual life histories. These have been well documented for Nearctic-Neotropical birds. Yet, seasonal interactions between the wintering, migration, and breeding periods have not been demonstrated for Palearctic-African passerine migrants. We tested whether variation in long- and shortterm dietary choice of the Garden Warbler Sylvia borin can influence events during the subsequent spring migration from the African winter quarters to the Palearctic breeding grounds. Using bone collagen and muscle carbon stable isotope (d13C) analysis, we assessed the relationships between dietary history, body condition, and migration timing in the Garden Warbler during a return migration from Africa. We predicted that Garden Warblers arriving early will have significantly different muscle and collagen d13C relative to those arriving later. Whereas muscle d13C (referring to events in the immediate past) was not related to body condition, we found a strong association between body condition and collagen d13C signatures (representing the integration of long-term events). Collagen and muscle d13C indicate that birds passing through later originated from moister or cooler geographic areas. The observed variation in isotope signatures might relate to differences in habitat and geographic/population origin, or in dietary intake

Early Stages of we/we wal/wal Mouse Hair Morphogenesis: Light and Fluorescent Microscopy of the Whole-Mount Epidermis

In adult skin, hair follicles cyclically self-renew in a manner that recapitulates embryonic hair follicle morphogenesis. The most common pathology of hair in adults is alopecia, which is hair loss to different extent. There are a number of murine models of alopecia including spontaneous mutations. In the present study, we worked with double homozygous we/we wal/wal mice which demonstrate symptoms closely resembling human alopecia. Using whole-mount preparations of epidermis of E18.5 embryos we show that hair follicle defects can be revealed as early as during embryonic morphogenesis in these mutants. The number of hair follicles was reduced almost 1.5-fold in mutant skin. The shape of the early stage small follicles was altered in mutant animals as compared to control ones. Additionally, follicles of mutant embryos were wider at the point of conjunction with interfollicular epidermis. We believe that the mutant mice studied represent a fascinating model to address the problem of hair loss. We demonstrated alterations in the morphogenesis of embryonic hair follicle in we/we wal/wal double homozygous mice developing alopecia postnatally. We suppose that incorrect morphogenesis of hair follicles during embryogenesis is closely related to alopecia in the adult life. Unveiling the mechanisms involved in altered embryogenesis may elucidate the pathogenesis of alopecia

Subtype of Neuroblastoma Cells with High KIT Expression Are Dependent on KIT and Its Knockdown Induces Compensatory Activation of Pro-Survival Signaling

Neuroblastoma (NB) is a pediatric cancer with high clinical and molecular heterogeneity, and patients with high-risk tumors have limited treatment options. Receptor tyrosine kinase KIT has been identified as a potential marker of high-risk NB and a promising target for NB treatment. We investigated 19,145 tumor RNA expression and molecular pathway activation profiles for 20 cancer types and detected relatively high levels of KIT expression in NB. Increased KIT expression was associated with activation of cell survival pathways, downregulated apoptosis induction, and cell cycle checkpoint control pathways. KIT knockdown with shRNA encoded by lentiviral vectors in SH-SY5Y cells led to reduced cell proliferation and apoptosis induction up to 50%. Our data suggest that apoptosis induction was caused by mitotic catastrophe, and there was a 2-fold decrease in percentage of G2-M cell cycle phase after KIT knockdown. We found that KIT knockdown in NB cells leads to strong upregulation of other pro-survival growth factor signaling cascades such as EPO, NGF, IL-6, and IGF-1 pathways. NGF, IGF-1 and EPO were able to increase cell proliferation in KIT-depleted cells in an ERK1/2-dependent manner. Overall, we show that KIT is a promising therapeutic target in NB, although such therapy efficiency could be impeded by growth factor signaling activation

Intracellular Localization of Blattella germanica Densovirus (BgDV1) Capsid Proteins

Densovirus genome replication and capsid assembly take place in the nucleus of the infected cells. However, the mechanisms underlying such processes as the delivery of virus proteins to the nucleus and the export of progeny virus from the nucleus remain elusive. It is evident that nuclear transport signals should be involved in these processes. We performed an in silico search for the putative nuclear localization signal (NLS) and nuclear export signal (NES) motifs in the capsid proteins of the Blattella germanica Densovirus 1 (BgDV1) densovirus. A high probability NLS motif was found in the common C-terminal of capsid proteins together with a NES motif in the unique N-terminal of VP2. We also performed a global search for the nuclear traffic signals in the densoviruses belonging to five Densovirinae genera, which revealed high diversity in the patterns of NLSs and NESs. Using a heterologous system, the HeLa mammalian cell line expressing GFP-fused BgDV1 capsid proteins, we demonstrated that both signals are functionally active. We suggest that the NLS shared by all three BgDV1 capsid proteins drives the trafficking of the newly-synthesized proteins into the nucleus, while the NES may play a role in the export of the newly-assembled BgDV1 particles into the cytoplasm through nuclear pore complexes

Selective Inhibition of HDAC Class I Sensitizes Leukemia and Neuroblastoma Cells to Anticancer Drugs

The acquired resistance of neuroblastoma (NB) and leukemia cells to anticancer therapy remains the major challenge in the treatment of patients with these diseases. Although targeted therapy, such as receptor tyrosine kinase (RTK) inhibitors, has been introduced into clinical practice, its efficacy is limited to patients harboring mutant kinases. Through the analysis of transcriptomic data of 701 leukemia and NB patient samples and cell lines, we revealed that the expression of RTK, such as KIT, FLT3, AXL, FGFR3, and NTRK1, is linked with HDAC class I. Although HDAC inhibitors have antitumor activity, they also have high whole-body toxicity. We developed a novel belinostat derivative named hydrazostat, which targets HDAC class I with limited off-target effects. We compared the toxicity of these drugs within the panel of leukemia and NB cell lines. Next, we revealed that HDAC inhibition with hydrazostat reactivates NTRK1, FGFR3, ROR2, KIT, and FLT3 expression. Based on this finding, we tested the efficacy of hydrazostat in combination with RTK inhibitor imatinib. Additionally, we show the ability of hydrazostat to enhance venetoclax-induced apoptosis. Thus, we reveal the connection between HDACs and RTK and describe a useful strategy to overcome the complications of single-agent therapies

Flight muscle shape reliably predicts flight muscle mass of migratory songbirds : a new tool for field ornithologists

International audienceThe pectoral muscle is the biggest organ within a passerine bird. It provides flight locomotion and is known to act as a protein source during periods with increased protein demands or decreased protein availability. The mass of the flight muscle is dynamic and changes during juvenile growth, reproduction, seasonal acclimatization, fasting and migration. Thus, a tool that accurately and non-invasively quantifies this phenotypic flexibility in flight muscle mass is of interest to ornithologists. We provide a calibration and validation of a "muscle meter" device designed to accurately measure the shape of the flight muscle. For two species of different size, the European starling () and the garden warbler (), we compared the accuracy and precision of different linear regression models for predicting flight muscle mass. The multifactorial linear regression model with the most support for both species included "muscle meter score" (mm), tarsus length and body mass (), although a simpler model with mm and had as much support for predicting flight muscle mass of European starlings. A validation exercise revealed that flight muscle mass of these two species could be estimated with a relative error of about 3%. The muscle meter is a simple device, easy and quick to handle, that can reliably and non-invasively estimate flight muscle mass of captive and wild birds when used in conjunction with standard measurements of tarsus length and