HTERT gene expression levels and telomerase activity in drug resistant MCF-7 cells

Cancer cells and some highly proliferative normal cells can stabilize telomere lengths by telomerase, which adds hexameric repeats to the ends of linear chromosomes. In this study, the activity of telomerase reverse transcriptase (hTERT) and its gene expression levels were investigated in paclitaxel, docetaxel, vincristine and doxorubicin resistant human MCF-7 breast adenocarcinoma cells. Materials and Methods: Resistant cell lines were developed by stepwise selection of cells (MCF-7/S) in increasing doses of paclitaxel (MCF-7/Pac), docetaxel (MCF-7/Doc), vincristine (MCF-7/Vinc) and doxorubicin (MCF-7/Dox). Antiproliferative effects of anticancer drugs were evaluated by XTT assay and IC50 values for different drugs were determined from cell proliferation curves. Expression levels of hTERT gene in sensitive and resistant cells were analyzed by RT-PCR. TRAP-Silver Staining assay was used to evaluate telomerase activities in these cells. Results: When drug resistant and sensitive MCF-7 cells were compared no significant differences were observed in hTERT expression levels and telomerase enzyme activities. Conclusion: This report demonstrates that drug resistance developed against paclitaxel, docetaxel, vincristine and doxorubicin in MCF-7 cells is independent of the expression of hTERT gene and telomerase activity.В опухолевых клетках, а также некоторых нормальных клетках с высоким пролиферативным потенциалом длина теломеров может стабилизироваться за счет фермента теломеразы, добавляющего гексамерные повторы к концам линейных хромосом. Цель: проанализировать активность обратной транскриптазы теломеразного комплекса и экспрессию гена теломеразы в клетках MCF-7 аденокарциномы молочной железы человека, устойчивых к паклитакселу, доцетакселу, винкристину и доксорубицину. Материалы и методы: сублинии клеток MCF-7, обладающих лекарственной резистентностью, были полу- MCF-7, обладающих лекарственной резистентностью, были полу -7, обладающих лекарственной резистентностью, были получены путем селекции исходных клеток при культивировании их в присутствии возрастающих доз паклитаксела (MCF-7/Pac), доцетаксела (MCF-7/Doc), винкристина (MCF-7/Vinc) и доксорубицина (MCF-7/Dox). Антипролиферативный эффект противоопухолевых препаратов определяли в ХТТ-тесте. Величины IC50 для различных препаратов определяли по кривым пролиферации клеток. Уровень экспрессии гена hTERT в чувствительных и резистентных клетках анализировали методом ОТ-ПЦР. Теломеразную активность определяли с помощью набора TRAPeze. Результаты: проанализированные резистентные линии клеток MCF-7 не отличались от исходной линии ни по уровню экспрессии гена hTERT, ни по уровню теломеразной активности. Выводы: развитие лекарственной резистентности к паклитакселу, доцетакселу, винкристину и доксорубицину в клетках MCF-7 не связано с изменениями экпрессии гена hTERT или уровня теломеразной активности в них

Pore timing:the evolutionary origins of the nucleus and nuclear pore complex

The name “eukaryote” is derived from Greek, meaning “true kernel”, and describes the domain of organisms whose cells have a nucleus. The nucleus is thus the defining feature of eukaryotes and distinguishes them from prokaryotes (Archaea and Bacteria), whose cells lack nuclei. Despite this, we discuss the intriguing possibility that organisms on the path from the first eukaryotic common ancestor to the last common ancestor of all eukaryotes did not possess a nucleus at all—at least not in a form we would recognize today—and that the nucleus in fact arrived relatively late in the evolution of eukaryotes. The clues to this alternative evolutionary path lie, most of all, in recent discoveries concerning the structure of the nuclear pore complex. We discuss the evidence for such a possibility and how this impacts our views of eukaryote origins and how eukaryotes have diversified subsequent to their last common ancestor

Sumoylation of the GTPase Ran by the RanBP2 SUMO E3 ligase complex

The SUMO E3 ligase complex RanBP2/RanGAP1*SUMO1/Ubc9 localizes at cytoplasmic nuclear pore complex (NPC) filaments and is a docking site in nucleocytoplasmic transport. RanBP2 has four Ran binding domains (RBDs), two of which flank RanBP2's E3 ligase region. We thus wondered whether the small GTPase Ran is a target for RanBP2-dependent sumoylation. Indeed, Ran is sumoylated both by a reconstituted and the endogenous RanBP2 complex in semi-permeabilized cells. Generic inhibition of SUMO isopeptidases or depletion of the SUMO isopeptidase SENP1 enhances sumoylation of Ran in semi-permeabilized cells. As Ran is typically associated with transport receptors, we tested the influence of Crm1, Imp beta, Transportin, and NTF2 on Ran sumoylation. Surprisingly, all inhibited Ran sumoylation. Mapping Ran sumoylation sites revealed that transport receptors may simply block access of the E2-conjugating enzyme Ubc9, however the acceptor lysines are perfectly accessible in Ran/NTF2 complexes. Isothermal titration calorimetry revealed that NTF2 prevents sumoylation by reducing RanGDP's affinity to RanBP2's RBDs to undetectable levels. Taken together, our findings indicate that RanGDP and not RanGTP is the physiological target for the RanBP2 SUMO E3 ligase complex. Recognition requires interaction of Ran with RanBP2's RBDs, which is prevented by the transport factor NTF2

Prostate stromal cells produce CXCL-1, CXCL-2, CXCL-3 and IL-8 in response to epithelia-secreted IL-1.

Contains fulltext : 81824schalken.pdf (publisher's version ) (Closed access)It is well accepted that tumor microenvironment is essential for tumor cells survival, cancer progression and metastasis. However, the mechanisms by which tumor cells interact with their surrounding at early stages of cancer development are largely unidentified. The aim of this study was to identify specific molecules involved in stromal-epithelial interactions that might contribute to early stages of prostate tumor formation. Here, we show that conditioned medium (CM) from immortalized non-transformed prostate epithelial cells stimulated immortalized prostate stromal cells to express cancer-related molecules. CM obtained from epithelial cells triggered stromal cells to express and secrete CXCL-1, CXCL-2, CXCL-3 and interleukin (IL)-8 chemokines. This effect was predominantly mediated by the cytokines of the IL-1 family secreted by the epithelial cells. Thus, prostate epithelial cells induced the secretion of proinflammatory and cancer-promoting chemokines by prostate stromal cells. Such interactions might contribute to prostatic inflammation and progression at early stages of prostate cancer formation

Mutant p53 gain of function underlies high expression levels of colorectal cancer stem cells markers

Emerging notion in carcinogenesis ascribes tumor initiation and aggressiveness to cancer stem cells (CSCs). Specifically, colorectal cancer (CRC) development was shown to be compatible with CSCs hypothesis. Mutations in p53 are highly frequent in CRC, and are known to facilitate tumor development and aggressiveness. Yet, the link between mutant p53 and colorectal CSCs is not well-established. In the present study, we set to examine whether oncogenic mutant p53 proteins may augment colorectal CSCs phenotype. By genetic manipulation of mutant p53 in several cellular systems, we demonstrated that mutant p53 enhances colorectal tumorigenesis. Moreover, mutant p53-expressing cell lines harbor larger sub-populations of cells highly expressing the known colorectal CSCs markers: CD44, Lgr5, and ALDH. This elevated expression is mediated by mutant p53 binding to CD44, Lgr5, and ALDH1A1 promoter sequences. Furthermore, ALDH1 was found to be involved in mutant p53-dependent chemotherapy resistance. Finally, analysis of ALDH1 and CD44 in human CRC biopsies indicated a positive correlation between their expression and the presence of oncogenic p53 missense mutations. These findings suggest novel insights pertaining the mechanism by which mutant p53 enhances CRC development, which involves the expansion of CSCs sub-populations within CRC tumors, and underscore the importance of targeting these sub-populations for CRC therapy. © 2018 The Author(s)

Modeling and X-ray Analysis of Defect Nanoclusters Formation in B4C under Ion Irradiation

In the presented work, B4C was irradiated with xenon swift heavy ions at the energy of 167 MeV. The irradiation of the substrate was done at room temperature to a fluence of 3.83 × 1014 ion/cm2. The samples were then analyzed with the X-ray diffraction technique to study the structural modification, as it can probe the region of penetration of xenon atoms due to the low atomic number of the two elements involved in the material under study. The nano-cluster formation under ion irradiation was observed. Positron lifetime (PLT) calculations of the secondary point defects forming nanoclusters and introduced into the B4C substrate by hydrogen and helium implantation were also carried out with the Multigrid instead of the K-spAce (MIKA) simulation package. The X-ray diffraction results confirmed that the sample was B4C and it had a rhombohedral crystal structure. The X-ray diffraction indicated an increase in the lattice parameter due to the Swift heavy ion (SHI) irradiation. In B12-CCC, the difference between τ with the saturation of H or He in the defect is nearly 20 ps. Under the same conditions with B11C-CBC, there is approximately twice the value for the same deviation