Search

not Available.

The administration of 150 nM etoposide, an inhibitor of DNA topoisomerase II activity, decreased the proliferation and induced the differentiation of U937 human promonocytic cells, as determined by nitroblue tetrazolium reduction, surface accumulation of CD11b/CD18 and CD11c/CD18 integrins, and c-fms protooncogene expression. The expression of these differentiation markers started to be detected at 24 h of treatment. Etoposide caused little cell damage, as determined by trypan blue exclusion and by apoptotic-like DNA degradation, which was slightly initiated at 48 h. The treatment induced a transient increase in c-fos, c-jun, and jun B mRNA levels, with maximum values at 12 h, a transient increase in collagenase mRNA level, with maximum value at 48 h, and a progressive increase in vimentin and lamin A and C mRNAs. These changes were qualitatively similar to those produced by 12-O-tetradecanoylphorbol-13-acetate. Etoposide also caused a transient increase of total AP-1 binding activity, with maximum value at 12 h of treatment, as determined by gel retardation assays. The drug produced an early transient activation (3-6 h) of membrane-bound protein kinase C, followed by the later activation (48 h) of both the membrane and cytosolic enzyme. The protein kinase C inhibitors, sphinganine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), attenuated the induction of differentiation markers by etoposide. These results suggest that protein kinase C and AP-1-dependent gene expression could be involved in myeloid cell differentiation by DNA topoisomerase II inhibitors.

Pulse treatments of U-937 human promonocytic leukemia cells with the DNA topoisomerase-II inhibitors 4′-(9-acridynilamino)methanesulfon-m-anisidide (amsacrine, mAMSA) or etoposide (VP-16) caused growth inhibition, G2-arrest, increase in cell size and expression of differentiation markers. All these effects were greatly reduced by the presence of 5–10 mM caffeine. In addition, caffeine partially prevented the increase in the number of topoisomerase-DNA cleavable complexes caused by the topoisomerase inhibitors, as determined by SDS/CIK precipitation assays; it caused chromatin condensation, as determined by flow cytometry assays, and interacted with mAMSA in solution, as suggested by spectrophotometric assays. Pulse treatment with caffeine greatly inhibited RNA synthesis but not DNA or protein synthesis, as indicated by labelled precursor incorporation assays. The transcription inhibitor 5,6-dichloro-1-β-D-ribofuranosylbenzymidazole reduced the mAMSA- and VP-16-produced growth inhibition in a similar manner. It is concluded that RNA synthesis inhibition is one of the possible mechanisms by which caffeine protects cells from the action of topoisomerase-II inhibitors. © 1994 Wiley-Liss, Inc.

It ought to be possible to recruit normal cellular defenses to mitigate ischemia/reperfusion damage and to reduce toxicity of chemotherapeutic drugs. Stress-preconditioned cells acquire a tolerant state characterized by increased resistance to such insults. This state is widely believed to be mediated, partially, by heat shock proteins (Hsps). Indirect evidence suggests that stress-induced Hsp expression is controlled by heat shock transcription factor 1 (Hsf1), which factor may therefore represent a preferred target for therapeutic modulation of tolerance. In support, positively acting (Hsf1(+)) and negatively acting (Hsf1(-)) mutants of Hsf1 were identified. Inhibition of endogenous Hsf1 activity by Hsf1(-) prevents stress-induced Hsp synthesis and development of tolerance. Hsf1(+) drastically enhances expression of major Hsps in the absence of stress and induces tolerance against heat, simulated ischemia and toxicity by cyclophosphamide. Where compared, tolerance induced was slightly better than that produced by heat preconditioning. Thus, development of the tolerant state is dependent on increased levels of the cohort of Hsps induced by stress preconditioning, and Hsf1 can induce accumulation of a typical set of Hsps, which proteins are alone capable of providing tolerance at a similar level as heat preconditioning. These findings make Hsf1 a preferred target for pharmacological intervention to deliberately induce tolerance.

Treatment of U-937 promonocytic cells with the DNA topoisomerase II inhibitor etoposide rapidly caused death by apoptosis, as determined by changes in chromatin structure, production of DNA breaks, nucleosome-sized DNA degradation, decrease in mitochondrial membrane potential and phosphatidyl serine translocation in the plasma membrane, and at the same time induced intracellular acidification. Both the execution of the apoptotic process and the intracellular acidification were reduced by the addition of forskolin plus theophylline or other cAMP increasing agents. These agents also attenuated the induction of apoptosis by camptothecin, heat-shock, cadmium chloride and X-radiation. Although etoposide slightly increased the production of reactive oxygen intermediates, this increase was not prevented by forskolin plus theophylline, and the addition of antioxidant agents failed to inhibit apoptosis. Etoposide caused a great increase in NF-(kappa)B binding activity, which was not prevented by forskolin plus theophylline, while AP-1 binding was little affected by the topoisomerase inhibitor. The treatments did not significantly alter the levels of Bcl-2 and Bax. By contrast, the expression of c-myc, which was very high in untreated U-937 cells and only partially inhibited by etoposide, was rapidly and almost totally abolished by the cAMP increasing agents. Finally, it was observed that etoposide caused a transient dephosphorylation of retinoblastoma (Rb), which was associated with cleavage of poly(ADP-ribose) polymerase (PARP). Both Rb dephosphorylation and PARP cleavage were inhibited by forskolin plus theophylline. The inhibition of Rb (type I) phosphatase and ICE/CED-3-like protease activities, and the abrogation of c-myc expression, are mechanisms which could explain the anti-apoptotic action of cAMP increasing agents in myeloid cells.

Heat-shock for 2 hours at 42 degrees C, or the administration for 3 hours of 100 or 150 microM cadmium chloride, inhibited the subsequent proliferation activity, induced the expression of functional differentiation markers, and caused an increase in the amount of the stress-responsive HSP70 protein in U-937 human promonocytic cells. In addition, both heat and cadmium produced an increase in the amount of the intermediate filament protein vimentin, as determined by immunoblot and immunofluorescence assays. By contrast, the amounts of actin and beta-tubulin were not significantly altered. The amount of vimentin mRNA was also increased during recovery from stress, indicating that vimentin expression was not exclusively regulated at the protein level. Although cadmium caused an early, transient stimulation of c-jun and c-fos expression and AP-1 binding activity, heat-shock failed to alter both protooncogene expression and transcription factor binding, indicating that the stress-induced vimentin increase was not the result of AP-1-mediated transcriptional activation. Finally, it was observed that the rate of decay of vimentin mRNA upon actinomycin D administration was decreased in heat- and cadmium-pretreated cells in comparison to untreated cells. These results indicate that stress treatments cause an increase in vimentin levels in promonocytic cells, which may be explained at least in part by transcript stabilization.

Fabricating polymeric scaffolds using cost-effective manufacturing processes is still challenging. Gas foaming techniques using supercritical carbon dioxide (scCO2) have attracted attention for producing synthetic polymer matrices; however, the high-pressure requirements are often a technological barrier for its widespread use. Compressed 1,1,1,2-tetrafluoroethane, known as Freon R134a, offers advantages over CO2 in manufacturing processes in terms of lower pressure and temperature conditions and the use of low-cost equipment. Here, we report for the first time the use of Freon R134a for generating porous polymer matrices, specifically polylactide (PLA). PLA scaffolds processed with Freon R134a exhibited larger pore sizes, and total porosity, and appropriate mechanical properties compared with those achieved by scCO2 processing. PLGA scaffolds processed with Freon R134a were highly porous and showed a relatively fragile structure. Human mesenchymal stem cells (MSCs) attached to PLA scaffolds processed with Freon R134a, and their metabolic activity increased during culturing. In addition, MSCs displayed spread morphology on the PLA scaffolds processed with Freon R134a, with a well-organized actin cytoskeleton and a dense matrix of fibronectin fibrils. Functionalization of Freon R134a-processed PLA scaffolds with protein nanoparticles, used as bioactive factors, enhanced the scaffolds' cytocompatibility. These findings indicate that gas foaming using compressed Freon R134a could represent a cost-effective and environmentally friendly fabrication technology to produce polymeric scaffolds for tissue engineering approaches., This work was supported by the Spanish Ministry of Economy and Competitiveness (MOTHER MAT2016-80826-R and Mol4Bio PID2019-105622RBI00); Spanish Ministry of Science, Innovation and Universities (RTI2018-095159-B-100); Instituto de Salud Carlos III (ISCIII)- European Regional Development Fund (ERDF); MINECO-AES (PI15/00752, PI15/01118 and PI18/00643); the Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN); CSIC through grant 2019AEP133; Comunidad Autónoma de Madrid (grant S2013/MIT-2862); Generalitat de Catalunya (grants 2017-SGR-918, 2017-SGR-229 and CERCA Programme); the Fundació Marató de TV3 (Nr. 201812); the COST Action CA15126 Between Atom and Cell; and the European Social Fund and EU to J.V., A.V. and I.R. (H2020-INFRAIA-2014-2015; NFFA-654360)., With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000917-S).