Significance of NOTCH1 Expression in the Progression of Human Lung and Colorectal Cancers.

Lung and colorectal cancers are the most common types of cancer characterized by a poor prognosis and a high mortality rate. Mutations in the genes encoding components of the main intra- and extracellular signaling pathways, in particular the NOTCH1 gene (Notch1, a member of the Notch family of receptors), play one of the key roles in progression of these malignancies. Notch signaling is involved in maintaining homeostasis of the intestinal epithelium and structural and functional lung elements. Therefore, it is not surprising that the constitutive activity and hyperactivity of Notch signaling due to somatic mutations in genes coding for the products directly involved into its activation, could lead to the progression of these cancer types. The aim of our study was to investigate how the NOTCH1 downregulation via RNA interference (RNAi) affects the phenotype, characteristics, and Notch-dependent signaling of human A549 lung and HCT116 colorectal carcinoma cells. Several small harpin RNAs (shRNAs) were selected using the bioinformatic analysis and tested for their ability to suppress the NOTCH1 expression. The most efficient one was used to produce the A549 and HCT116 cells with NOTCH1 knockdown. The obtained cell lines demonstrated decreased proliferation rates, reduced colony-forming capacity under adhesive conditions, and decreased migration activity in a Boyden chamber. The NOTCH1 knockdown also significantly decreased expression of some Notch signaling target genes potentially involved in the acquisition and maintenance of more invasive and malignant cell phenotype. In vivo experiments in immunodeficient athymic female Balb/c nu/nu mice confirmed the results obtained in vitro: the NOTCH1 inhibition decreased the growth rates of the subcutaneous xenografts formed by A549 and HCT116 tumor cells. Therefore, downregulation of the gene encoding the Notch1 receptor potentially reduces malignant characteristics of human lung and colorectal carcinoma cells.

Correlation of the regenerative potential of dermal fibroblasts in 2D culture with the biological properties of fibroblast-derived tissue spheroids.

In situ 3D bioprinting is a new emerging therapeutic modality for treating human skin diseases. The tissue spheroids have been previously suggested as a powerful tool in rapidly expanding bioprinting technology. It has been demonstrated that the regenerative potential of human dermal fibroblasts could be quantitatively evaluated in 2D cell culture and confirmed after implantation in vivo. However, the development of unbiassed quantitative criteria of the regenerative potential of 3D tissue spheroids in vitro before their in situ bioprinting remains to be investigated. Here it has been demonstrated for the first time that specific correlations exist between the regenerative potential of human dermal fibroblasts cultured in vitro as 2D cell monolayer with biological properties of 3D tissue spheroids fabricated from these fibroblasts. In vitro assessment of biological properties included diameter, spreading and fusion kinetics, and biomechanical properties of 3D tissue spheroids. This comprehensive characterization could be used to predict tissue spheroids' regenerative potential in vivo.

Myogenic potential of human alveolar mucosa derived cells.

Difficulties related to the obtainment of stem/progenitor cells from skeletal muscle tissue make the search for new sources of myogenic cells highly relevant. Alveolar mucosa might be considered as a perspective candidate due to availability and high proliferative capacity of its cells. Human alveolar mucosa cells (AMC) were obtained from gingival biopsy samples collected from 10 healthy donors and cultured up to 10 passages. AMC matched the generally accepted multipotent mesenchymal stromal cells criteria and possess population doubling time, caryotype and immunophenotype stability during long-term cultivation. The single myogenic induction of primary cell cultures resulted in differentiation of AMC into multinucleated myotubes. The myogenic differentiation was associated with expression of skeletal muscle markers: skeletal myosin, skeletal actin, myogenin and MyoD1. Efficiency of myogenic differentiation in AMC cultures was similar to that in skeletal muscle cells. Furthermore, some of differentiated myotubes exhibited contractions in vitro. Our data confirms the sufficiently high myogenic potential and proliferative capacity of AMC and their ability to maintain in vitro proliferation-competent myogenic precursor cells regardless of the passage number.

Bioceramics composed of octacalcium phosphate demonstrate enhanced biological behavior.

Bioceramics are used to treat bone defects but in general do not induce formation of new bone, which is essential for regeneration process. Many aspects related to bioceramics synthesis, properties and biological response that are still unknown and, there is a great need for further development. In the most recent research efforts were aimed on creation of materials from biological precursors of apatite formation in humans. One possible precursor is octacalcium phosphate (OCP), which is believed to not only exhibit osteoconductivity but possess osteoinductive quality, the ability to induce bone formation. Here we propose a relatively simple route for OCP ceramics preparation with a specifically designed microstructure. Comprehensive study for OCP ceramics including biodegradation, osteogenic properties in ortopic and heterotopic models and limited clinical trials were performed that demonstrated enhanced biological behavior. Our results provide a possible new concept for the clinical applications of OCP ceramics.

Octacalcium phosphate ceramics combined with gingiva-derived stromal cells for engineered functional bone grafts.

Biocompatible ceramic fillers are capable of sustaining bone formation in the proper environment. The major drawback of these scaffolding materials is the absence of osteoinductivity. To overcome this limitation, bioengineered scaffolds combine osteoconductive components (biomaterials) with osteogenic features such as cells and growth factors. The bone marrow mesenchymal stromal cells (BMMSCs) and the ß-tricalcium phosphate (ß-TCP) are well-known and characterized in this regard. The present study was conducted to compare the properties of novel octacalcium phosphate ceramic (OCP) granules with ß-TCP (Cerasorb(®)), gingiva-derived mesenchymal stromal cells (GMSCs) properties with the BMMSCs and osteogenic and angiogenic properties of a bioengineered composite based on OCP granules and the GMSCs. This study demonstrates that GMSCs and BMMSСs have a similar osteogenic capacity. The usage of OCP ceramic granules in combination with BMMSCs/GMSCs significantly affects the osteo- and angiogenesis in bone grafts of ectopic models.

Repression of sestrin family genes contributes to oncogenic Ras-induced reactive oxygen species up-regulation and genetic instability.

Oncogenic mutations within RAS genes and inactivation of p53 are the most common events in cancer. Earlier, we reported that activated Ras contributes to chromosome instability, especially in p53-deficient cells. Here we show that an increase in intracellular reactive oxygen species (ROS) and oxidative DNA damage represents a major mechanism of Ras-induced mutagenesis. Introduction of oncogenic H- or N-Ras caused elevated intracellular ROS, accumulation of 8-oxo-2'-deoxyguanosine, and increased number of chromosome breaks in mitotic cells, which were prevented by antioxidant N-acetyl-L-cysteine. By using Ras mutants that selectively activate either of the three major targets of Ras (Raf, RalGDS, and phosphatidylinositol-3-kinase) as well as dominant-negative Rac1 and RalA mutants and inhibitors of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases kinase-1 and p38 MAPKs, we have shown that several Ras effectors independently mediate ROS up-regulation. Introduction of oncogenic RAS resulted in repression of transcription from sestrin family genes SESN1 and SESN3, which encode antioxidant modulators of peroxiredoxins. Inhibition of mRNAs from these genes in control cells by RNA interference substantially increased ROS levels and mutagenesis. Ectopic expression of SESN1 and SESN3 from lentiviral constructs interfered with Ras-induced ROS increase, suggesting their important contribution to the effect. The stability of Ras-induced increase in ROS was dependent on a p53 function: in the p53-positive cells displaying activation of p53 in response to Ras, only transient (4-7 days) elevation of ROS was observed, whereas in the p53-deficient cells the up-regulation was permanent. The reversion to normal ROS levels in the Ras-expressing p53-positive cells correlated with up-regulation of p53-responsive genes, including reactivation of SESN1 gene. Thus, changes in expression of sestrins can represent an important determinant of genetic instability in neoplastic cells showing simultaneous dysfunctions of Ras and p53.

Transformation by RAS oncogene decreases the width of substrate-spread fibroblasts but not their length.

We compared morphometric parameters of the contours of cells in four pairs of non-transformed mouse and rat lines and of the same lines transformed by oncogenes of the RAS family. As expected, the mean areas of all RAS-transformed lines were much smaller than those of their non-transformed counterparts. At the same time the average length of cell projection did not regularly decrease after transformation. These results show that transformation induced by expression of RAS oncogene selectively affect only one component of spreading, namely transversal spreading and not longitudinal spreading; these changes result in an increase of antero-posterior polarity of transformed fibroblasts.

ROS up-regulation mediates Ras-induced changes of cell morphology and motility.

Expression of activated Ras causes an increase in intracellular content of reactive oxygen species (ROS). To determine the role of ROS up-regulation in mediation of Ras-induced morphological transformation and increased cell motility, we studied the effects of hydrogen peroxide and antioxidant NAC on morphology of REF52 rat fibroblasts and their ability to migrate into the wound in vitro. Treatment with low dosages of hydrogen peroxide leading to 1.5- to 2-fold increase in intracellular ROS levels induced changes of cell shape, actin cytoskeleton organization, cell adhesions and migration resembling those in Ras-transformed cells. On the other hand, treatment with NAC attenuating ROS up-regulation in cells with conditional or constitutive expression of activated Ras led to partial reversion of morphological transformation and decreased cell motility. The effect of ROS on cell morphology and motility probably results from modulation of activity of Rac1, Rho, and cofilin proteins playing a key role in regulation of actin dynamics. The obtained data are consistent with the idea that ROS up-regulation mediates two key events in Ras-induced morphological transformation and cell motility: it is responsible for Rac1 activation and is necessary (though insufficient) for realization of Ras-induced cofilin dephosphorylation.

Cell type-specific effects of asbestos on intracellular ROS levels, DNA oxidation and G1 cell cycle checkpoint.

Exposure to asbestos fibers increases the risk of development of mesotheliomas and lung carcinomas, but not fibrosarcomas. We present data suggesting that resistance of fibroblasts to asbestos-induced carcinogenesis is likely to be connected with their lower ability to generate reactive oxygen species (ROS) in response to asbestos exposure and stricter control of proliferation of cells bearing asbestos/ROS-induced injuries. In fact, chrysotile (Mg6Si4O10(OH)8) asbestos exposure (5-10 microg/cm2) increased intracellular ROS and 8-oxo-guanine contents in rat pleural mesothelial cells, but not in lung fibroblasts. Simultaneously, moderate dosages of chrysotile and other agents increasing ROS levels (hydrogen peroxide, H2O2 and ethyl-methanesulfonate, EMS) inhibited cell cycle progression, in particular G1-to-S transition, in fibroblasts, but not in mesothelial cells. The arrested fibroblasts underwent cell death, while the majority of chrysotile-treated mesothelial cells survived. The differences in cell cycle response to asbestos/ROS-induced injuries correlated with distinct activity of p53-p21Cip1/Waf1 pathway in the two cell types. Chrysotile, H2O2 and EMS caused p53 upregulation in both cell types, but mesothelial cells, unlike fibroblasts, showed no accumulation of p21Cip1/Waf1. Of note, treatment with doxorubicin caused similar p53-dependent p21Cip1/Waf1 upregulation and cell cycle arrest in both cell types. This suggests differential response of fibroblasts and mesothelial cells specifically to asbestos/ROS exposure rather than to all DNA-damaging insults.