Independent domains for recruitment of PRC1 and PRC2 by human XIST

XIST establishes inactivation across its chromosome of origin, even when expressed from autosomal transgenes. To identify the regions of human XIST essential for recruiting heterochromatic marks we generated a series of overlapping deletions in an autosomal inducible XIST transgene present in 8p of the HT1080 male fibrosarcoma cell line. We examined the ability of each construct to enrich its unified XIST territory with the histone marks established by PRC1 and PRC2 as well as the heterochromatin factors MacroH2A and SMCHD1. Chromatin enrichment of ubH2A by PRC1 required four distinct regions of XIST, and these were completely distinct from the two domains crucial for enrichment of H3K27me3 by PRC2. Both the domains required, as well as the impact of PRC1 and PRC2 inhibitors, suggest that PRC1 is required for SMCHD1 while PRC2 function is necessary for MacroH2A recruitment, although incomplete overlap of regions implicates roles for additional factors. This cooperativity between factors contributes to the requirement for multiple separate domains being required for each feature examined. The independence of the PRC1/PRC2 pathways was observed when XIST was expressed both autosomally or from the X chromosome suggesting that these observations are not purely a result of the context in which XIST operates. Although independent domains were required for the PRC1 and PRC2 pathways overall all regions tested were important for some aspect of XIST functionality, demonstrating both modularity and cooperativity across the XIST lncRNA.

The characterization of exosomes from fibrosarcoma cell and the useful usage of Dynamic Light Scattering (DLS) for their evaluation

Exosomes are a type of extracellular vesicles containing mRNA, miRNA, and proteins of origin cells, which can control the characteristics of other cells or surroundings. Despite increasing evidence on oncogenic properties of tumor-derived exosomes, fibrosarcoma-derived exosomes remain largely unrevealed. While the proper extraction and characterization of exosomes is critical in exosomes research, there are various limitations in techniques to measure the size and homogeneity of exosomes. Here, we analyzed exosomes from a fibrosarcoma cell line WEHI-164 compared with a breast cancer cell line MDA-MD-231 as a control. Results from dot blot and western blot analysis demonstrated that GM1 ganglioside, and TSG101, HSC70 and GAPDH proteins were contained in exosomes from the WEHI-164 fibrosarcoma cell line. The existence of tetraspanins such as CD81, CD63 and CD9 was confirmed in the exosomes by ExoView analysis. The results obtained from TEM showed their sphere-like shapes of around 50 to 70 nm in radius. Through DLS, we found out that the mean radius of the exosomes derived from WEHI-164 and MDA-MB-231 cell lines was 94.4 nm and 107.8 nm, respectively, with high homogeneity. When comparing the radius measured by TEM with the radius measured by DLS, it was revealed that the difference between the two methods was about 40 nm. This study has significance in characterizing the molecular properties of exosomes from a fibrosarcoma, which has not been researched much before, and in providing clear evidence that DLS can be used as an efficient, convenient and noninvasive technique to simply check the homogeneity and size of exosomes.

Combination Therapy of a Host Defense-Like Lytic Peptide and Continuous Low-Dose Doxorubicin Inhibits Tumor Growth in a Syngeneic Immunocompetent Murine Fibrosarcoma (BFS-1) Model

Background: The concept of a multimodality therapy in the treatment of soft tissue sarcomas (STS) has been discussed with controversy. Surgical resection with clear margins and radiation therapy remain gold standard in STS therapy. It is still questionable whether a systemic therapy with chemotherapeutics has a positive impact on the overall survival rate especially in early stages of disease, because the therapeutic effect in the treatment of STS is limited by its toxicities and its low responding rates. Treatment options are rare. As a result the search for combination therapies by using low dose approaches is of high importance. Recent studies showed the therapeutic efficiency of a designer host defense-like lytic D,L- amino acid peptide [D]-K 3 H 3 L 9 . Therefore we tested a combination of this peptide with Doxorubicin on two different sarcoma cell lines in vitro and also in a syngeneic immunocompetent murine fibrosarcoma mouse model. Methods: In vitro the human synovial sarcoma cell line SW 982 and the murine fibrosarcoma cell line BFS-1 were exposed to the oncolytic peptide [D]-K 3 H 3 L 9 , to the Anthracycline Doxorubicin and to both agents simultaneously. In vivo the murine fibrosarcoma cell line BFS-1 was injected subcutaneously into the syngeneic mice. When the tumors engrafted the oncolytic designer peptide [D]-K 3 H 3 L 9 , Doxorubicin or a combination of both was administered thrice a week for a three weeks’ follow-up. Results: The combination treatment approach using an oncolytic designer host defense peptide and Doxorubicin inhibited the in vitro sarcoma cell proliferation significantly. The single therapies, either with local intratumoral application of [D]-K 3 H 3 L 9 or with intraperitoneal application of Doxorubicin in the syngeneic mouse model, inhibited at least the tumor progression. The combination of both substances revealed a significant inhibition of tumor growth and weight. Conclusion: The in vivo low dose combination schedule inhibited the tumor growth significantly. Histological analyses of the tumor sections revealed an antiproliferative and antiangiogenic effect. So, these results demonstrate the effectiveness of combined low-dose application forms with designer host defense-like lytic peptides and chemotherapeutics.

Telomerase activity, mTert gene expression and the telomere length in mouse mesenchymal stem cells in the late period after γ- and γ,n-irradiation and in the tumors developed from these cells

In proliferating normal and tumor cells, the telomere length (TL) is maintained by high telomerase activity (TA). In the absence of TA the TL maintenance involves a mechanism of alternative lengthening of telomeres (ALT). The aim of this study was to investigate the level of TA, the mTert expression and TL in cultured normal and transformed by γ- and γ,n-irradiation mesenchymal stem cells (MSCs) from mouse bone marrow, in sarcomas that developed after the transplantation of these cells into syngeneic mice, and in fibrosarcoma cell lines obtained from these tumors to find out the role of AT or ALT in maintaining TL in these cells. During prolonged cultivation of normal and transformed under the influence of γ- (1 Gy and 6 Gy) and γ,n-irradiation (0.05 Gy, 0.5 Gy, and 2 Gy) MSCs from mouse bone marrow, a decrease in TA was detected in irradiated cells. Even deeper decrease in TA was found in sarcomas developed after administration of transformed MSCs to syngeneic mice and in fibrosarcoma cell lines isolated from these tumors in which TA was either absent or was found to be at a very low level. TL in three of the four lines obtained was halved compared to the initial MSCs. With absent or low TA and reduced TL, the cells of all the obtained fibrosarcoma lines successfully proliferated without signs of a change in survival. The mechanism of telomere maintainance in fibrosarcoma cell lines in the absence of TA needs further investigation and it can be assumed that it is associated with the use of the ALT. The detected decrease or absence of TA in transformed under the action of irradiation MSCs with the preservation or even an increase in the telomerase gene expression may be associated with the formation of inactive splicing variants, and requires further study. The obtained lines of transformed MSCs and fibrosarcomas with TA and without the activity of this enzyme can be a useful model for studying the efficacy of TA and ALT inhibitors in vitro and in vivo.

Arf6, JIP3, and dynein shape and mediate macropinocytosis

Macropinocytosis is an actin-driven form of clathrin-independent endocytosis that generates an enlarged structure, the macropinosome. Although many studies focus on signaling molecules and phosphoinositides involved in initiating macropinocytosis, the commitment to forming a macropinosome and the handling of that membrane have not been studied in detail. Here we show in HT1080 cells, a human fibrosarcoma cell line, a requirement for microtubules, dynein, the JIP3 microtubule motor scaffold protein, and Arf6, a JIP3 interacting protein, for the formation and inward movement of the macropinosome. While actin and myosin II also play critical roles in the formation of ruffling membrane, microtubules provide an important tract for initiation, sealing, and transport of the macropinosome through the actin- and myosin-rich lamellar region.