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Non-invasiveness and relative safety of photothermal therapy, which enables local hyperthermia of target tissues using a near infrared (NIR) laser, has attracted increasing interest. Due to their biocompatibility, amenability of synthesis and functionalization, gold nanoparticles have been investigated as therapeutic photothermal agents. In this work, hollow gold nanoparticles (HGNP) were coated with poly-L-lysine through the use of COOH-Poly(ethylene glycol)-SH as a covalent linker. The functionalized HGNP, which peak their surface plasmon resonance at 800 nm, can bind thrombin. Thrombin-conjugated HGNP conduct in situ fibrin polymerization, facilitating the process of generating photothermal matrices. Interestingly, the metallic core of thrombin-loaded HGNP fragmentates at physiological temperature. During polymerization process, matrices prepared with thrombin-loaded HGNP were loaded with genetically-modified stem cells that harbour a heat-activated and ligand-dependent gene switch for regulating transgene expression. NIR laser irradiation of resulting cell constructs in the presence of ligand successfully triggered transgene expression in vitro and in vivo., This work was supported by grant PI15/01118 from Instituto de Salud Carlos III (ISCIII)-Fondos FEDER, Ministry of Economy and Competitiveness (MINECO), Spain, grants RTI2018- 095159-B-I00 and SAF2013-50364-EXP from MINECO, grant Roche- IdiPAZ from the intramural funding program of Foundation for Biomedical Research of La Paz University Hospital-IdiPAZ, grant ERC-2013-CoG-614715 (NANOHEDONISM) from ERC Consolidator Grant program and by HSF Pharmaceuticals S.A. C.E-D. was the re- cipient of predoctoral grant FI14/00447 from ISCIII-Fondos FEDER, MINECO. N.V. is supported by Program I2 from CAM

Comparative modeling of the DNA-binding domain of human HSF1 facilitated the prediction of possible binding pockets for small molecules and definition of corresponding pharmacophores. In silico screening of a large library of lead-like compounds identified a set of compounds that satisfied the pharmacophoric criteria, a selection of which compounds was purchased to populate a biased sublibrary. A discriminating cell-based screening assay identified compound 001, which was subjected to systematic analysis of structure–activity relationships, resulting in the development of compound 115 (IHSF115). IHSF115 bound to an isolated HSF1 DNA-binding domain fragment. The compound did not affect heat-induced oligomerization, nuclear localization and specific DNA binding but inhibited the transcriptional activity of human HSF1, interfering with the assembly of ATF1-containing transcription complexes. IHSF115 was employed to probe the human heat shock response at the transcriptome level. In contrast to earlier studies of differential regulation in HSF1-naïve and -depleted cells, our results suggest that a large majority of heat-induced genes is positively regulated by HSF1. That IHSF115 effectively countermanded repression in a significant fraction of heat-repressed genes suggests that repression of these genes is mediated by transcriptionally active HSF1. IHSF115 is cytotoxic for a variety of human cancer cell lines, multiple myeloma lines consistently exhibiting high sensitivity.

The aim of this work was the generation of a multifunctional nanopolymeric system that incorporates IR-780 dye, a near-infrared (NIR) imaging probe that exhibits photothermal and photodynamic properties; and a derivate of α-tocopheryl succinate (α-TOS), a mitochondria-targeted anticancer compound. IR-780 was conjugated to the hydrophilic segment of copolymer PEG-b-polyMTOS, based on poly(ethylene glycol) (PEG) and a methacrylic derivative of α-tocopheryl succinate (MTOS), to generate IR-NP, self-assembled nanoparticles (NPs) in aqueous media which exhibit a hydrophilic shell and a hydrophobic core. During assembly, the hydrophobic core of IR-NP could encapsulate additional IR-780 to generate derived subspecies carrying different amount of probe (IR-NP-eIR). Evaluation of photo-inducible properties of IR-NP and IR-NP-eIR were thoroughly assessed in vitro. Developed nanotheranostic particles showed distinct fluorescence and photothermal behavior after excitation by a laser light emitting at 808 nm. Treatment of MDA-MB-453 cells with IR-NP or IR-NP-eIR resulted in an efficient internalization of the IR-780 dye, while subsequent NIR-laser irradiation led to a severe decrease in cell viability. Photocytoxicity conducted by IR-NP, which could not be attributed to the generation of lethal hyperthermia, responded to an increase in the levels of intracellular reactive oxygen species (ROS). Therefore, the fluorescence imaging and inducible phototoxicity capabilities of NPs derived from IR-780-PEG-b-polyMTOS copolymer confer high value to these nanotheranostics tools in clinical cancer research. Statement of Significance Multifunctional polymeric nanoparticles (NPs) that combine imaging and therapeutic properties are highly valuable in cancer treatment. In this paper we describe the development of NPs that are fluorescent in the near-infrared (NIR). This is important for their visualization in living tissues that present low absorption and low autofluorescence in this wavelength region (between 700 and 1000 nm). Moreover, NPs present photothermal and photodynamic properties when NIR irradiated: the NPs produce an efficient increment of temperature and increase the intracellular reactive oxygen species (ROS) when laser irradiated at 808 nm. These tuneable photoinduced properties make the NPs highly cytotoxic after NIR irradiation and provide a new tool for highly precise cancer treatment.

One of the most important complications of total joint arthroplasty is failure associated with periprosthetic osteolysis, a process mainly initiated by the biological response to wear-derived products from the biomaterials in service. The inflammatory mediator interleukin-6 (IL-6) plays a key role in the establishment and progression of aseptic loosening. Metal particles specifically up-regulate IL-6 production in bone-forming cells and implant-bone interfacial tissues. The use of statins has been recently associated with a significantly reduced risk of revision in patients that undergo total hip arthroplasty. We hypothesized that simvastatin (Simv) could modulate the osteoblastic response to titanium particles (Ti) by attenuating the production of IL-6. Pre-treatment of human osteoblastic cells with Simv down-regulated Ti particle-induced IL-6 gene expression at mRNA and protein levels. The effect of Simv on Ti-induced IL-6 production in osteoblastic cells could not be explained by inhibition of the internalization of metal particles. The mechanism involved in this down-regulation is based in the inhibition of the HMG-CoA/GGPP/RhoA/ROCK pathway, independently of Simv effects in the cholesterol synthesis. The cytokine-lowering property of Simv has been observed in Saos-2 cells and human primary osteoblasts (hOBs) exposed to Ti particles, and was further enhanced when hOBs were co-cultured with macrophages.

Gold nanoparticles (Au NPs) are promising vectors for gene delivery applications. In order to gain insight on the influence of particle size on cell transfection, Au NPs were combined with poly(ethylenimine) (PEI) to prepare two sets of PEI-coated Au NPs having particle-size distributions centered at about 6 nm (

Biphasic calcium phosphates (BCPs) consist of a mixture of hydroxyapatite and β-tricalcium phosphate and are recommended as alternatives or additives to autogenous bone for orthopaedic and dental applications. There is clinical evidence showing particle release from bioceramics, which might impair the ability of human mesenchymal stem cells (hMSC) from bone marrow to proliferate or mature into a functional osteoblast phenotype. This study analyses the influence of BCP particles and their precursors, calcium-deficient apatite (CDA) particles, on in vitro hMSC behaviour. Both types of particles were efficiently internalized by hMSC. Cell viability, morphology and actin cytoskeleton reorganization were unaffected by exposure of hMSC to BCP or CDA particles. Direct exposure to BCP particles impaired hMSC osteogenic differentiation and bone matrix mineralization to a lesser extent than CDA, as assayed by evaluation of alkaline phosphatase activity, osteopontin secretion and mineralized nodule formation. The ability of bioceramic particles to affect osteogenic maturation through modification of soluble factors in media was assayed in an in vitro system that avoids direct cell–particle contact. Indirect exposure to CDA particles severely impaired hMSC osteogenic maturation owing to the uptake of Ca2+ from the culture media. Lower textural properties of BCP and the lack of calcium deficiency in its composition prevented Ca2+ uptake, allowing the development of a functional osteoblast phenotype.

It is feasible to restrict transgene expression to a tissue or region in need of therapy by using promoters that respond to focusable physical stimuli. The most extensively investigated promoters of this type are radiation-inducible promoters and heat shock protein gene promoters that can be activated by directed, transient heat. Temporal regulation of transgenes can be achieved by various two- or three-component gene switches that are triggered by an appropriate small molecule inducer. The most commonly considered gene switches that are reviewed herein are based on small molecule-responsive transactivators derived from bacterial tetracycline repressor, insect or mammalian steroid receptors, or mammalian FKBP12/FRAP. A new generation of gene switches combines a heat shock protein gene promoter and a small molecule-responsive gene switch and can provide for both spatial and temporal regulation of transgene activity.

7 páginas, 7 figuras -- PAGS nros. 24970-24976, Infection of HeLa cells with adenovirus-carrying HSF1+ cDNA, which encodes a mutated form of HSF1 with constitutive transactivation capacity, increased multidrug resistance 1 (MDR1) mRNA level and P-glycoprotein (P-gp) cell surface content and stimulated rhodamine 123 accumulation and vinblastine efflux activity. On the other hand, infection with adenovirus-carrying HSP70 andHSP27 cDNAs did not increase MDR1/P-gp expression. HSF1 regulates MDR1/P-gp expression at the transcriptional level, since HSF1+ bound the heat-shock consensus elements (HSEs) in the MDR1 gene promoter and also activated the expression of an MDR1 promoter-driven reporter plasmid (pMDR1(−1202)). In addition, heat-shock increased pMDR1(−1202) promoter activity but not the activity of a similar reporter plasmid with point mutations at specific HSEs, and the heat-induced increase was totally inhibited by co-transfection with an expression plasmid carrying HSF1−, a dominant negative mutant of HSF1. The stress inducers arsenite, butyrate, and etoposide also increased pMDR1(−1202) promoter activity, but the increase was not inhibited (in the case of butyrate) or was only partially inhibited (in the case of arsenite and etoposide) by HSF1−. These results demonstrate that HSF1 regulates MDR1 expression, and that the HSEs present in the −315 to −285 region mediate the heat-induced activation of the MDR1 promoter. However, other factors may also participate in MDR1 induction by stressing agents. The acquisition of the multidrug resistance (MDR)1 phenotype, defined as increased resistance against cytotoxic drugs with unrelated structures, represents one of the major obstacles for chemotherapy of tumors and other malignancies. One of the mechanisms that may account for MDR is the surface accumulation of the P-glycoprotein (P-gp, also called the multidrug transporter), which blocks the influx and/or increases the efflux of many hydrophobic agents, including some of the most commonly used anticancer drugs (for a review, see Ref. 1). In humans, P-gp is encoded by the MDR gene family, composed of two members, only one of which (MDR1) seems to be functionally linked to the development of the MDR phenotype (2). P-gp is expressed in some normal tissues, such as epithelial cells of kidney, liver, pancreas, and intestinal mucosa and capillaries of brain and testis. In these tissues, P-gp probably plays a physiological role, promoting the excretion of xenobiotics or preventing their absorption and also possibly acting as a chloride channel (reviewed in Refs. 1 and 3). As a consequence, cancers derived from these tissues may constitutively express P-gp and be intrinsically drug-resistant. However, in most cases P-gp-derived resistance is acquired during chemotherapy,e.g. in breast, bladder, lung, and ovary tumors, among others (for a review, see Ref. 3). Prolonged exposure to cytotoxic drugs may lead to the selection of cells with MDR1 gene amplification, at least under in vitro conditions (2, 4-6). However, even when amplification occurs, it does not always adequately explain the increased P-gp expression and drug resistance (6, 7). On the other hand, P-gp expression may also be de novo induced by short term exposures to cytotoxic agents such as UV light (8), actinomycin D (9), chemotherapeutic drugs (10), and inducers of the stress response (11, 12)., The heat-shock transcription factors (HSFs) were originally characterized as regulators of the expression of heat-shock protein (HSP) genes, through the binding to specific sequences (“heat-shock elements,” or HSEs) present in the promoter of these genes. The HSF family contains three members in humans, namely HSF1, HSF2, and HSF4 (13-15), among which HSF1 is specifically responsible for the stress-mediated HSP induction (16, 17). In unstressed cells, HSF1 is present in the cytoplasm as a monomer or forming heteromeric complexes. Upon treatment with stress inducers, HSF1 homotrimerizes, translocates to the nucleus, and binds the HSE (16, 17) to further acquire the transactivation capacity (18). Indirect proofs have suggested that HSFs could also participate in MDR1 gene expression. In fact, (a) HSEs have been identified in theMDR1 gene promoter (11, 12); (b) typical stress inducers such as heat-shock and arsenite, which induce HSPgene expression, also induce MDR1 gene expression in some cell types (11, 12); (c) some multidrug-resistant cell lines exhibit constitutively high HSF-DNA binding activity (19); and (d) quercetin, which inhibits HSF-HSE binding, also inhibits HSF-DNA binding and P-gp expression in multidrug-resistant cells (20). However, the problem is far from being clear, since (a) some work suggests that the activation of MDR1 expression by heat-shock and other stressing agents may be mediated by DNA sequences and transcription factors other than HSE and HSFs (21-23); (b) the possibility cannot be excluded that MDR1gene induction by stressing agents, instead of being a direct response, is mediated by increased HSPs expression; and (c) even if HSFs directly regulate MDR1 gene expression, it is not known which member of the family is actually implicated. In the present work, we analyzed the regulation of MDR1 gene expression by HSF1 using direct gene transfer procedures. This was made possible by the use of specific HSF1 mutants, namely a construction encoding an active mutant (HSF1+) (a deleted form of HSF1 with constitutive binding and transactivation capacities) and a construction encoding a dominant negative mutant (HSF1−) (a deleted form that constitutively binds the HSP gene promoters but is unable to transactivate, even under stressing conditions) (18), This work was supported by Dirección General de Enseñanza Superior e Investigación Cientı́fica (Spain) Grant PM97–0144 and Comunidad Autónoma de Madrid (Spain) Grant 08.1/0027/1997.The costs of publication of this article were defrayed in part by the payment of page charges

9 p.- 8 fig., Earlier studies have indicated that Jun/AP-1 activity is associated with, and probably required for apoptosis induction by DNA-damaging and stress-inducing agents in human myeloid cells. To investigate this possibility, we examined the capacity of continuous treatments with etoposide (10 μM) and camptothecin (0.4 μM), and pulse treatments with X-rays (20 Gy), heat (2 h at 42.5 °C) and cadmium chloride (2 h at 200 μM) followed by recovery, to provoke apoptosis and to simulate c-jun and c-fos expression and AP-1 binding in U-937 human promonocytic cells. All these treatments generated apoptosis with similar efficacy (50–60% apoptotic cells at 6 h of treatment or recovery). However, the capacity to increase c-jun and c-fos mRNA levels and to stimulate AP-1 binding was very different, ranging from more than a twelve-fold increase in the case of cadmium, to almost no increase in the case of heat-shock and etoposide. When the cells were pre-conditioned with a soft heat shock (1 h at 42 °C) the cadmium-provoked apoptosis was greatly inhibited, but the stimulation of AP-1 binding was not affected. The administration of cAMP-increasing agents also reduced the etoposide- and cadmium-provoked apoptosis. However, cAMP greatly stimulated c-jun and c-fos expression and AP-1 binding when applied together with etoposide (which itself was ineffective), and potentiated the cadmium-induced AP-1 binding. Conversely, retinoic acid abrogated the cadmium-provoked stimulation of AP-1 binding and transactivation capacity, and greatly inhibited the stimulation of binding caused by camptothecin and X-rays. However, retinoic acid did not inhibit the induction of apoptosis by these agents. These results indicate that Jun/AP-1 activity is not necessarily coupled with apoptosis, nor required for apoptosis induction by DNA-damaging and stress-inducing agents in human promonocytic cells, This work was partially supported by Grant PB97 ± 0144 from the DireccioÂn General de Enseñanza Superior e Investigación Científica, Spain, and Grant 08.1/0027/1997 from the Comunidad Autónoma de Madrid, Spain. A. Galán is the recipient of a predoctoral fellowship from the Ministerio de Educación y Cultura, Spain

A major challenge in regenerative medicine is to develop methods for delivering growth and differentiation factors in specific spatial and temporal patterns, thereby mimicking the natural processes of development and tissue repair. Heat shock (HS)-inducible gene expression systems can respond to spatial information provided by localized heating, but are by themselves incapable of sustained expression. Conversely, gene switches activated by small molecules provide tight temporal control and sustained expression, but lack mechanisms for spatial targeting. Here we combine the advantages of HS and ligand-activated systems by developing a novel rapamycin-regulated, HS-inducible gene switch that provides spatial and temporal control and sustained expression of transgenes such as firefly luciferase and vascular endothelial growth factor (VEGF). This gene circuit exhibits very low background in the uninduced state and can be repeatedly activated up to 1 month. Furthermore, dual regulation of VEGF induction in vivo is shown to stimulate localized vascularization, thereby providing a route for temporal and spatial control of angiogenesis.

We explore the synergistic effect of photothermal therapy and gene therapy, simultaneously triggered by silica-gold nanoshells (NS) or hollow gold nanoparticles (HGNPs) in human HeLa cells following near-infrared (NIR) light irradiation. Thermal transfer from NS was higher than that displayed by HGNPs, owing to a differential interaction of the nanomaterial with the biological environment. Under sublethal photothermal conditions, NS and HGNPs effectively modulated the expression levels of a DsRed-monomer reporter gene controlled by the highly heat-inducible human HSP70B promoter, as a function of nanomaterial concentration and length of laser exposure. Hyperthermia treatments at doses that do not promote cell death generated a lethal outcome in HeLa cells harboring the fusogenic GALV-FMG transgene under the control of the HSP70B promoter. Combination of lethal photothermia with the triggering of the cytotoxic transgene resulted in a dramatic increase of the cell-ablation area as a result of the synergistic activity established. From the Clinical Editor In this study photothermal therapy and gene therapy, simultaneously triggered by silica-gold nanoshells or hollow gold nanoparticles, was investigated in human HeLa cells following near-infrared (NIR) light irradiation. It is shown that the combination of lethal photothermia with the triggering of the cytotoxic transgene at sublethal levels results in a synergistic cytotoxic effect in vitro.

Wear particle-mediated osteolysis is one of the major problems affecting long-term survival of orthopaedic prostheses, frequently progressing to failure of fixation and revision surgery. Upon challenging with wear particles, macrophages and various other types of cells release soluble factors that stimulate the resorptive activity of osteoclasts and impair the function and activity of osteoblasts. Extracellular Hsp72 has been reported to activate macrophages and up-regulate pro-inflammatory cytokine production, although its role in osteolysis has not been established yet. The purpose of our study was to evaluate the involvement of this protein in the inflammatory response to wear particles that leads to periprosthetic osteolysis. To this end, we used interfacial tissues and blood samples from patients undergoing revision surgery due to aseptic loosening of cementless acetabular cups. Confocal microscopy indicated that Hsp72 co-localises with CD14(+) cells of interfacial tissues. Levels of Hsp72 in the culture media from periprosthetic membranes cultured ex vivo decreased along culture time and Hsp72 levels in sera from patients were lower and under the assay detection limit compared with those from age-matched control subjects. This suggests that interfacial tissues are not actively producing the protein but likely recruit it from peripheral circulation. Incubation of human macrophages with titanium (Ti) particles decreased the release of Hsp72 into culture media. Treatment with recombinant human Hsp72 enhanced considerably IL-6 levels in culture media which were not modified after macrophage co-stimulation with Ti particles, while pre-incubation with Hsp72 increased the Ti particle-induced TNF-α and IL-1β production. Altogether, these data indicate that extracellular Hsp72 amplifies the inflammatory response to wear debris by interacting with resident macrophages in periprosthetic tissues.

Osteosarcoma-derived cells have been routinely used for studying osteoblastic functions, but it remains unclear to what extent they mimic the behavior of primary osteoblasts in the study of cells and materials interactions. This study reports comparatively on the responses of three human osteosarcoma cell lines, MG-63, Saos-2 and U-2 OS, and human primary osteoblasts cultured on Ti6Al4V surfaces or exposed to Ti particles. Phenotypic characterization of the cell lines revealed that Saos-2 cells and primary osteoblasts displayed similar expression patterns of Cbfa1, SP7 and osteocalcin. Unlike primary cells, the cell lines expressed markers of undifferentiated cells, had high proliferative rates and poor fibronectin matrix assembly. None of the three cell lines faithfully reproduced the adhesive behavior of primary osteoblasts when cultured on Ti6Al4V surfaces or exposed to Ti particles. Differences in cell growth between the cell lines and primary osteoblasts cultured on Ti6Al4V surfaces were also observed. Ti particles inhibited the growth of Saos-2 cells and primary osteoblasts to a similar extent, while no such effect was observed in U-2 OS and MG-63 cells. Saos-2 cells reproduced the alkaline phosphatase (ALP) activity profile of primary osteoblasts cultured on metallic surfaces or exposed to particles. Altogether, these results show that none of the osteoblast-like cells studied perfectly mimic the behavior of human osteoblast cells (hOB) on Ti6Al4V surfaces or exposed to Ti particles. Saos-2 cells reproduce some of the hOB responses such as the profile of enzymatic ALP activity when cultured on the surfaces or treated with particles as well as cell growth inhibition when exposed to Ti particles. Although in vitro cytocompatibility studies involve the evaluation of multiple parameters, Saos-2 cells may be used as representative of human osteoblasts when these standard tests are evaluated.

Patterned surfaces direct cell spatial dynamics, yielding cells oriented along the surface geometry, in a process known as contact guidance. The Rho family of GTPases controls the assembly of focal adhesions and cytoskeleton dynamics, but its role in modulating bone-cell alignment on patterned surfaces remains unknown. This article describes the interactions of two human cell types involved in osseointegration, specifically mesenchymal stem cells and osteoblasts, with submicron- or nano-scale Ti6Al4V grooved surfaces generated by mechanical abrasion. The surface chemistry of the alloy was not affected by grinding, ensuring that the differences found in cellular responses were exclusively due to changes in topography. Patterned surfaces supported cell growth and stimulated mesenchymal stem cell viability. Anisotropic surfaces promoted cell orientation and elongation along the grates. Both cell types oriented on nanometric surfaces with grooves of 150 nm depth and 2 μm width. The number of aligned cells increased by approximately 30% on submicrometric grooves with sizes of about 1 μm depth and 10 μm width. Cells were treated with drugs that attenuate the activities of the GTPase RhoA and one of its downstream effectors, Rho-associated kinase (ROCK), and contact guidance of treated cells on the grooved surfaces was investigated. The data indicate that the RhoA/ROCK pathway is a key modulator of both mesenchymal stem cell and osteoblast orientation on nanometric surface features. RhoA and its effector participate in the alignment of mesenchymal stem cells on submicrometric grooves, but not of osteoblasts. These findings show that RhoA/ROCK signaling is involved in contact guidance of bone-related cells on metallic substrates, although to a varying extent depending on the specific cell type and the dimensions of the pattern. © 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Magnetic nanoparticles coated with materials having unique properties, such as ordered pore structures and large surface areas, hold great potential for multimodal therapies. This study reports on the biocompatibility of composites of maghemite nanoparticles embedded in an ordered mesoporous silica-matrix to form magnetic microspheres (MMS), and on their ability to conduct magnetic hyperthermia upon exposure to a low-frequency alternating magnetic field (AMF). MMS particles were efficiently internalized by human A549, Saos-2 and HepG2 cells, and were excluded from the nuclear compartment. MMS treatment did not interfere with morphological features or metabolic activities of the cells, indicating good biocompatibility of the material. MMS did not affect the endogenous heat-shock response of a HeLa-derived cell line that precisely reports the intensity of thermal stresses through changes in the activities of a stably integrated hsp70B promoter and a constitutive viral promoter. Maximum temperature in MMS suspensions increased to a range above 42°C as a function of the amounts of particles exposed to AMF. Cell culture experiments showed that, by adjusting the amount of MMS and the time of exposure to AMF, heat treatments of mild to very high intensities could be achieved. Cell viability dropped as a function of the intensity of the heat treatment achieved by MMS and AMF exposures. The possibility of fine-tuning the heating power output, together with efficient uptake by tumor cells in vitro, makes MMS a promising agent by which to provide hyperthermia treatments aimed toward remission of solid tumors.

Diamond-like carbon (DLC) coatings produced using the plasma-accelerating filtered pulsed arc discharge (FPAD) method display excellent adherence to the substrate and improve its corrosion resistance. This article reports the interactions of human osteoblastic cells with DLC and two DLC polymer hybrid (DLC-p-h) coatings deposited on smooth, matt and rough silicon wafers by the FPAD method. The DLC-p-h materials were DLC–polytetrafluoroethylene hybrid (DLC-PTFE-h) and DLC–polydimethylsiloxane hybrid (DLC-PDMS-h) coatings. The biocompatibility of the coatings was assayed by using mesenchymal stem cells, primary osteoblasts and Saos-2 cells. Human mesenchymal stem cells proliferated when cultured on DLC and DLC-PTFE-h, but their numbers diminished on DLC-PDMS-h. In all three cell types studied, phalloidin–TRITC staining disclosed cell-type organization typical of an actin cytoskeleton on DLC and DLC-PTFE-h, but minimal and disorganized stress fibers on cells cultured on DLC-PDMS-h. The microtubular cytoskeleton was similarly disorganized on DLC-PDMS-h. Cells on DLC-PDMS-h developed a peculiar form of membrane damage, with nuclear staining by propidium iodide associated with granular calcein staining of the cytoplasm. Active caspase-3 labeling was only seen in cells cultured on DLC-PDMS-h, indicating that these cells undergo apoptosis induced by defective cell adhesion. Results suggest that DLC-PDMS-h coatings might be useful in orthopedic applications where an implant or implant-facet should be protected against bone overgrowth while DLC and DLC-PTFE-h coatings might improve osseointegration.

Titanium (Ti) and its alloys are widely used in biomedical devices as bone tissue replacements due to their advantageous bulk mechanical properties and biocompatibility. It is known that particles released from Ti-based implants impair essential functions of osteoblasts, which for survival require attachment to specific extracellular matrix proteins at the bone surface. This study investigates whether Ti particles of micrometric sizes affect the osteoblast attachment machinery. Exposure of human osteoblastic Saos-2 cells to Ti particles impaired their adhesion strength, migration and proliferation. Attenuation of these functions was associated with reduced cell spreading, cell membrane disruptions and loss of cell shape. Cell exposure to Ti particles led to changes in cytoskeletal structures, including reduced ventral stress fibers combined with a disorderly arrangement of β-tubulin and acetylated α-tubulin fibers. Cytoskeleton disassembly was associated with a reduction in overall cell adhesion area, characterized by fewer centrally localized focal adhesions and shorter focal contacts at the periphery. Paxillin adaptor protein redistributed to peripheral corner regions, colocalizing with poorly organized actin fibers at attachment sites. Total focal adhesion kinase (FAK) protein amounts, as well as its degree of phosphorylation on the active form p-FAK (Tyr-397), decreased, which was accompanied by a lesser extent of co-localization with paxillin in focal contacts. On the other hand, p-FAK (Tyr-407), an inhibitory form of FAK, accumulated in the focal contacts of Ti-treated cells. Pyk2 phosphorylated on Tyr-402 colocalized with paxillin in focal contacts of untreated cells, while it was barely detected upon exposure to particles. In summary, changes in the phosphorylation states of both FAK and Pyk2 tyrosine kinases at focal contacts underlie impaired bone-forming cell attachment after exposure to Ti particles of micrometric sizes.

This article reports on the construction and analysis in vitro and in vivo of novel gene switches that can be used to achieve spatial as well as temporal control over the expression of a transgene of interest. The switches are expected to be functional in virtually any tissue and cell type. They consist of (a) a foreign or modified transactivator expressed under the dual control of a promoter or promoter cassette that is responsive to heat and the transactivator and (b) a promoter responsive to the transactivator for controlling the transgene of interest. A preferred gene switch of this type incorporated a mifepristone-dependent transactivator. This gene switch could be activated by a transient heat treatment in the presence of mifepristone. Activity increased with the intensity of the activating heat treatment and was found to persist for more than 6 days. The gene switch was essentially inactive prior to an activating heat treatment, in the absence or presence of mifepristone. Activated gene switch could be silenced by removal/withdrawal of mifepristone.

Cdc6 is a key regulator of the strict alternation of S and M phases during the mitotic cell cycle. In mammalian and plant cells that physiologically become polyploid, cdc6 is transcriptionally and post-translationally regulated. We have recently reported that Cdc6 levels are maintained in megakaryoblastic HEL cells, but severely downregulated by ectopic expression of transcriptional repressor Drosophila melanogaster escargot. Here, we show that cdc6 promoter activity is upregulated during megakaryocytic differentiation of HEL endoreplicating cells, and that Escargot interferes with such activation. Transactivation experiments showed that a 1.7 kb region located at 2800 upstream cdc6 transcription initiation site behaved as a potent enhancer in endoreplicating cells only. This activity was mainly dependent on a novel cis-regulatory element composed by an E2 box overlapping a GATA motif. Ectopic Escargot could bind this regulatory element in vitro and endogenous GATA-1 and E2A formed specific complexes in megakaryoblastic cells as well as in primary megakaryocytes. Chromatin Immunoprecipitation analysis revealed that both transcription factors were occupying the E2 box/GATA site in vivo. Altogether, these data suggest that cdc6 expression could be actively maintained during megakaryocytic differentiation through transcriptional mechanisms involving specific cis- and trans-regulatory elements. © Oxford University Press 2004; all rights reserved., This work was supported by grants PM98-0046 from the Spanish Ministry of Science and Technology, CAM 08.3/0037/2001 from the ‘Comunidad Autónoma de Madrid’ and FIS 01/0090-02 from ‘Fondo de Investigaciones Sanitarias’ to C.C. N.V. was partly supported by a Postdoctoral Fellowship from ‘Comunidad Autónoma de Madrid’ (Spain).

12 pages, 7 figures., Endomitosis is the process by which mammalian megakaryocytes become polyploid during terminal differentiation. As in other endoreplicating cells, cyclin-cdk complexes are distinctly regulated, probably to overcome the strict mechanisms that prevent rereplication in most somatic cells. We have asked whether key factors involved in the assembly and licensing of replication origins are equally regulated during endomitosis. Cdc6, cdt1, and geminin expression was analyzed during differentiation of two human megakaryoblastic cell lines, HEL and K562, which respectively do and do not establish endoreplication cycles. Geminin was downregulated, whereas cdt1 levels were maintained upon differentiation of both cell lines, independently of whether cells entered extra S-phases. In contrast, cdc6 was present and remained nuclear only in differentiated endoreplicating cells. Interestingly, cdc6 protein expression was reestablished in K562 cells that underwent endomitosis after transient or stable cyclin E overexpression. The high levels of cyclin E reached in these cells appeared to influence the stabilization of cdc6 protein rather than its RNA transcription rate. Finally, cdc6 overexpression drove HEL cells into endoreplication cycles in the absence of differentiation stimuli. Our results show that both cdt1 and cdc6 are differentially regulated during megakaryocytic differentiation and suggest an active role of cdc6 in endomitosis., This work was supported by a grant from the Ministry of Education to C.C. (PM98–0046). N.V. was supported by a Postdoctoral Fellowship from “Comunidad Autónoma de Madrid” (Spain).

Certain cell types escape the strict mechanisms imposed on the majority of somatic cells to ensure the faithful inheritance of parental DNA content. This is the case in many embryonic tissues and certain adult cells such as mammalian hepatocytes and megakaryocytes. Megakaryocytic endomitosis is characterized by repeated S phases followed by abortive mitoses, resulting in mononucleated polyploid cells. Several cell cycle regulators have been proposed to play an active role in megakaryocytic polyploidization; however, little is known about upstream factors that could control endomitosis. Here we show that ectopic expression of the transcriptional repressor escargot interferes with the establishment of megakaryocytic endomitosis. Phorbol ester-induced polyploidization was inhibited in stably transfected megakaryoblastic HEL cells constitutively expressing escargot. Analysis of the expression and activity of different cell cycle factors revealed that Escargot affects the G(1)/S transition by influencing Cdk2 activity and cyclin A transcription. Nuclear proteins that specifically bind the Escargot-binding element were detected in endomitotic and non-endomitotic megakaryoblastic cells, but down-regulation occurred only during differentiation of cells that become polyploid. As Escargot was originally implicated in ploidy maintenance of Drosophila embryonic and larval cells, our results suggest that polyploidization in megakaryocytes might respond to mechanisms conserved from early development to adult cells that need to escape normal control of the diploid state.

9 p.- 8 fig., Treatment of U-937 human promonocytic cells with the stress inducers cadmium chloride (2 h at 200 μM), heat (2 h at 42.5 °C) or X-rays (20 Gy), followed by recovery, caused death by apoptosis and stimulated caspase-3 activity. In addition, all stress agents caused intracellular oxidation, as measured by peroxide and/or anion superoxide accumulation. However, while pre-incubation with antioxidants (N-acetyl-L-cysteine or butylated hydroxyanisole) inhibited the induction of apoptosis by cadmium and X-rays, it did not affect the induction by heat-shock. Pre-incubation for 24 h with the GSH-depleting agent L-buthionine-[S,R]-sulfoximine (BSO) switched the mode of death from apoptosis to necrosis in cadmium-treated cells. By contrast, BSO only caused minor modifacions in the rate of apoptosis without affecting the mode of death in heat- and X-rays-treated cells. BSO potentiated peroxide accumulation in cells treated with both cadmium and X-rays. However, while the accumulation of peroxides was stable in the case of cadmium, it was transient in the case of X-rays. Moreover, the administration of antioxidants during the recovery period sufficed to prevent necrosis and restore apoptosis in BSO plus cadmium-treated cells. Cadmium and X-rays caused a decrease in intracellular ATP levels, but the decrease was similar in both apoptotic and necrotic cells. Taken together, these results demonstrate that (i) stress inducers cause intracellular oxidation, but oxidation is not a general requirement for apoptosis; and (ii) the duration of the oxidant state seems to be critical in determining the mode of death, This work was supported by Grant PB97 ± 0144 from the Dirección General de Enseñanza Superior e Investigación Científica (Spain) and Grant 08.1/0027/1997 from the Comunidad Autonóma de Madrid (Spain). A. Galán and A. Troyano are recipients of fellowships from the Ministerio de Educación y Cultura, Spain

9 páginas, 5 figuras -- PAGS nros. 38-46, Treatment for 2 h with 200 μM cadmium chloride, followed by recovery, caused apoptosis and induced heat-shock protein 70 (HSP70) expression in U-937 promonocytic cells. However, pre-incubation with the GSH depleting agent l-buthionine-[S,R]-sulfoximine (BSO, 1 mM for 24 h) caused necrosis instead of apoptosis and failed to induce HSP70 expression. This failure was a consequence of necrosis instead of GSH depletion, since BSO allowed or even potentiated HSP70 induction when used in combination with heat shock (2 h at 42.5°C) or with 50 μM cadmium, which caused apoptosis. The administration of N-acetyl-l-cysteine (NAC) at the beginning of recovery after BSO/200 μM cadmium treatment prevented the execution of necrosis and restored the execution of apoptosis, but did not restore HSP70 induction, indicating that the inhibition by BSO of HSP70 expression is an early regulated event. This contrasted with the capacity of NAC to prevent the alterations caused by BSO/200 μM cadmium in other proteins, namely the suppression of Bax expression and the increase in Bcl-2 and HSP-60 expression. Finally, it was observed that treatment with 200 μM cadmium rapidly increased the HSP70 mRNA level and stimulated heat-shock factor 1 (HSF1) trimerization and binding, and that these effects were prevented by pre-incubation with BSO. Taken together, these results indicate that the stress response is compatible with apoptosis but not with necrosis in cadmium-treated promonocytic cells. The suppression of the stress response is specifically due to the early inhibition of HSF1 activation, This work was supported by Grant PB97-0144 from the Dirección General de Enseñanza Superior e Investigación Cientı́fica, and 08.1/0027/1997 from the Comunidad Autónoma de Madrid, Spain. A.G. and A.T. are recipients of predoctoral fellowships from the Ministerio de Educación y Cultura, Spain

10.1074/jbc.275.15.11418 áginas, 9 figuras -- PAGS nros. 1418-11424, Pulse treatment of U-937 promonocytic cells with cadmium chloride (2 h at 200 μm) provoked apoptosis and induced a rapid phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) as well as a late phosphorylation of extracellular signal-regulated protein kinases (ERK1/2). However, although the p38MAPK-specific inhibitor SB203580 attenuated apoptosis, the process was not affected by the ERK-specific inhibitor PD98059. The attenuation of the cadmium-provoked apoptosis by SB203580 was a highly specific effect. In fact, the kinase inhibitor did not prevent the generation of apoptosis by heat shock and camptothecin, nor the generation of necrosis by cadmium treatment of glutathione-depleted cells, nor the cadmium-provoked activation of the stress response. The generation of apoptosis was preceded by intracellular H2O2 accumulation and was accompanied by the disruption of mitochondrial transmembrane potential, both of which were inhibited by SB203580. On the other hand, the antioxidant agent butylated hydroxyanisole-inhibited apoptosis but did not prevent p38MAPK phosphorylation. In a similar manner, p38MAPK phosphorylation was not affected by the caspase inhibitors Z-VAD and DEVD-CHO, which nevertheless prevented apoptosis. These results indicate that p38MAPK activation is an early and specific regulatory event for the cadmium-provoked apoptosis in promonocytic cells. Cadmium and other heavy metals are frequent environmental contaminants with well known mutagenic, carcinogenic, and teratogenic effects (1). Another property of heavy metals (and of other physical and chemical agents, such as heat and inhibitors of energy metabolism) is the capacity to induce the stress response, characterized by the synthesis and accumulation of heat-shock proteins (HSPs)1 (2). The stimulation of HSP gene expression by stress inducers in higher eukaryotes seems to be specifically regulated by heat-shock factor 1 (HSF1). Thus, under stressful conditions, HSF1 undergoes trimerization, translocation to the nucleus, binding to the heat-shock consensus elements, and finally hyperphosphorylation to fully acquire the transactivation capacity (3). In addition, stress inducers may provoke cell death, either apoptotic or necrotic, depending on the intensity of the treatments (4, 5). The morphological characteristics of apoptosis and necrosis are well known (6-8). Thus, during apoptosis the cells undergo nuclear and cytoplasmic shrinkage, the chromatin is partitioned into multiple fragments, and the cells are broken into multiple membrane-surrounded bodies (“apoptotic bodies”), but the cell membrane retains its integrity during the process. By contrast, necrosis is characterized by cell swelling and a rapid loss of membrane integrity. However, the regulation of apoptosis and necrosis and the factors that decide the selection of one or the other mode of death are still poorly known., One of the most relevant aspects in the regulation of both the stress response and apoptosis is the involvement of mitogen-activated protein kinases (MAPKs), a family of serine/threonine kinases that mediate intracellular signal transduction in response to different stimuli (9). The MAPK family members are themselves activated by reversible dual phosphorylation on a Thr-Xaa-Tyr conserved motif, by specific mitogen-activated protein kinases kinases. To date, three major MAPKs have been identified, namely the extracellular signal-regulated kinases (ERK1/2, p44/p42), the stress-activated protein kinases (c-Jun NH2-terminal kinases, stress-activated protein kinase 1), and the p38 mitogen-activated protein kinases (p38MAPK, stress-activated protein kinase 2). ERK1/2 are mainly (although not exclusively) activated by growth factors (10, 11) and are critically involved in the regulation of mitogenesis. On the other hand, c-Jun NH2-terminal kinases and p38MAPK are mainly activated by cytotoxic insults and are often associated with apoptosis (12-18). It was recently reported that cadmium chloride induced the stress response and caused a dose-dependent activation of ERK1/2 and p38MAPK (but not of c-Jun NH2-terminal kinases) in association with mitogenesis and apoptosis, respectively, in rat brain tumor cells (19). Whereas it was demonstrated that both kinases were involved in the regulation of the stress response, it is not clear whether they were also required to induce cell death. In the present work we investigated the capacity of cadmium chloride to induce p38MAPK and ERK1/2 activation and to cause cell death in U-937 human promonocytic cells. It was concluded that p38MAPK activation is an early and specific requirement for the cadmium-provoked apoptosis, which precedes and probably modulates the expression of other regulatory events, This work was supported by Grant PB97-0144 from the Dirección General de Enseñanza Superior e Investigación Cientı́fica, Grant 08.1/0027/1997 from the Comunidad Autónoma de Madrid, Spain (to P. A.), Grant RPG-97-092-01-CNE from the American Cancer Society, and Grant ROI-CA 74197-01 from the National Institutes of Health (to M. G. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The administration of sodium butyrate at 0.75 mM induced the functional differentiation of U-937 human promonocytic leukemia cells with negligible cell mortality. However, the drug rapidly caused cell death with characteristics of apoptosis when used at concentrations of 5 mM and above. In addition, butyrate stimulated the expression of the stress-responsive heat-shock protein 70 (HSP70) gene when applied at both differentiation-inducing and apoptosis-inducing concentrations. The induction of HSP70 by butyrate was inhibited by the simultaneous addition of cAMP-increasing agents (dibutyryl cAMP or the combination of forskolin plus theophylline). However, these agents did not prevent differentiation and only partially reduced apoptosis. Moreover, the DNA topoisomerase II inhibitor etoposide, which provoked U-937 cell differentiation and apoptosis with the same or greater efficiency than butyrate, failed to stimulate HSP70 expression. Finally, it was observed that cAMP-increasing agents also abrogated the induction of HSP70 and reduced the apoptosis caused by cadmium chloride, a typical inducer of the stress response. Taken together, these results indicate that HSP70 expression is not required for differentiation of promonocytic cells, as earlier proposed, and that butyrate probably triggers the stress response in these cells.

The treatment of U-937 human promonocytic cells with the differentiation inducer sodium butyrate (0.75 mM) transiently increased heat-shock protein 70 (HSP70) mRNA levels between 3 and 6 h, and heat-shock protein 27 (HSP27) mRNA levels between 12 and 24 h, as indicated by northern blot assays. Gel retardation assays indicated that butyrate also stimulated heat-shock factor (HSF) binding activity between 3 and 6 h, suggesting that the activation of HSP70 gene expression was mediated by the heat-shock factor DNA response element (HSE). In addition, the treatment provoked a biphasic alteration of the c-fos mRNA level, consisting of a slight increase between 0.5 and 3 h followed by a greater increase between 12 and 48 h, while it caused a single increase between 12 and 48 h in c-jun mRNA level. The possible involvement of the heat-shock protein genes in the butyrate-induced differentiation of U-937 cells is discussed.

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.