Telomere Shortening Sensitizes Cancer Cells to Selected Cytotoxic Agents: In Vitro and In Vivo Studies and Putative Mechanisms

Background

Telomere/telomerase system has been recently recognized as an attractive target for anticancer therapy. Telomerase inhibition results in tumor regression and increased sensitivity to various cytotoxic drugs. However, it has not been fully established yet whether the mediator of these effects is telomerase inhibition per se or telomere shortening resulting from inhibition of telomerase activity. In addition, the characteristics and mechanisms of sensitization to cytotoxic drugs caused by telomerase inhibition has not been elucidated in a systematic manner.

Methodology/Principal Findings

In this study we characterized the relative importance of telomerase inhibition versus telomere shortening in cancer cells. Sensitization of cancer cells to cytotoxic drugs was achieved by telomere shortening in a length dependent manner and not by telomerase inhibition per se. In our system this sensitization was related to the mechanism of action of the cytotoxic drug. In addition, telomere shortening affected also other cancer cell functions such as migration. Telomere shortening induced DNA damage whose repair was impaired after administration of cisplatinum while doxorubicin or vincristine did not affect the DNA repair. These findings were verified also in in vivo mouse model. The putative explanation underlying the phenotype induced by telomere shortening may be related to changes in expression of various microRNAs triggered by telomere shortening.

Conclusions/Significance

To our best knowledge this is the first study characterizing the relative impact of telomerase inhibition and telomere shortening on several aspects of cancer cell phenotype, especially related to sensitivity to cytotoxic drugs and its putative mechanisms. The microRNA changes in cancer cells upon telomere shortening are novel information. These findings may facilitate the development of telomere based approaches in treatment of cancer.     

Use of circumflex scapular vessels as a recipient pedicle for autologous breast reconstruction: a report of 40 consecutive cases

The use of the circumflex scapular pedicle as a recipient vessel for breast reconstruction in a series of 40 consecutive cases in 37 patients is reported. There were 3 bilateral reconstructions and 34 unilateral reconstructions. Twenty-one cases were immediate reconstructions, and 19 cases were secondary reconstructions. The diameter of the artery varied from 1.5 mm to 3 mm and systematically matched with the diameter of the epigastric artery. The artery was a branch of the subscapular system in 82.5 percent of cases (33 of 40). In 17.5 percent of cases (7 of 40), the artery was a direct branch of the axillary artery. The length of available pedicle between the axillary vessel and the distal part where it can be divided (on its division between scapular and parascapular artery) was of 76 +/- 13 mm for the artery and 72 +/- 12 mm for the vein. The vein was unique in 77.5 percent of cases. The diameter was similar to the artery diameter when unique. There was a dual venous system in 21 of 40 cases (52.5 percent) but in 15 cases (37.5 percent), one of the two veins was dominant. In the seven cases for which the veins were dual and of equivalent diameter, the epigastric veins were also dual and allowed a second anastomosis. Clinically, the anastomosis was always possible on the artery. In one case of reconstruction after Halstedt mastectomy, no vein could be found, because all the veins had been ligated previously. One venous thrombosis (2.5 percent) and one arterial thrombosis were experienced. Both were treated by revised anastomoses and did not compromise late results. The circumflex scapular pedicle is a reliable and simple recipient site for breast reconstruction. It allows a unique site of dissection in immediate reconstruction and avoids division of the thoracodorsal pedicle. The technique is now used exclusively at this institution.     

The role of Bacillus thuringiensis Cry1C and Cry1E separate structural domains in the interaction with Spodoptera littoralis gut epithelial cells

The Bacillus thuringiensis delta-endotoxins Cry1C and Cry1E share toxicity against several important lepidopteran species. Their combined use to delay development of resistance in target insects depends on their differential interaction with the gut epithelial cells. The three structural domains and combinations of two consecutive domains of Cry1C and Cry1E were separately expressed in Escherichia coli, and their interactions with the brush border membrane vesicles (BBMV) of Cry1E-tolerant and -susceptible Spodoptera littoralis larvae were studied. About 80% reduction in binding of Cry1E and each of its separate domains to BBMV of Cry1E-tolerant larvae was observed, whereas Cry1C was toxic to all larvae and bound equally to BBMV derived from both Cry1E-tolerant and -susceptible larvae. These results suggest differential interactions of the two toxins with BBMV encompassing all three domains. Comparable binding assays performed with fluorescent Cry1C and Cry1C domain II showed that Cry1C has higher Bmax and lower Kd than Cry1C domain II and further supported the existence of toxin multisite interactions. Competitive binding assays were used to estimate the sequence of interaction events. Cry1C domain II could compete with domain III binding, whereas domain III did not interfere with domain II binding, indicating sequential interactions of domain III and then domain II with the same membrane site. No competition between domain II of Cry1C and Cry1E was observed, confirming the existence of different domain II binding sites for the two toxins. Taken together, all three domains specifically interact with the epithelial cell membrane. The folding of the three-domain toxin probably dictates the sequence of interaction events.     

Early molecular events in the assembly of the focal adhesion-stress fiber complex during fibroblast spreading

Cell adhesion to the extracellular matrix triggers the formation of integrin-mediated contact and reorganization of the actin cytoskeleton. Examination of nascent adhesions, formed during early stages of fibroblast spreading, reveals a variety of forms of actin-associated matrix adhesions. These include: (1). small ( approximately 1 microm), dot-like, integrin-, vinculin-, paxillin-, and phosphotyrosine-rich structures, with an F-actin core, broadly distributed over the ventral surfaces of the cells; (2). integrin-, vinculin-, and paxillin-containing "doublets" interconnected by short actin bundles; (3). arrays of actin-vinculin complexes. Such structures were formed by freshly plated cells, as well as by cells recovering from latrunculin treatment. Time-lapse video microscopy of such cells, expressing GFP-actin, indicated that long actin cables are formed by an end-to-end lining-up and apparent fusion of short actin bundles. All these structures were prominent during cell spreading, and persisted for up to 30-60 min after plating. Upon longer incubation, they were gradually replaced by stress fibers, associated with focal adhesions at the cell periphery. Direct examination of paxillin and actin reorganization in live cells revealed alignment of paxillin doublets, forming long and highly dynamic actin bundles, undergoing translocation, shortening, splitting, and convergence. The mechanisms underlying the assembly and reorganization of actin-associated focal adhesions and the involvement of mechanical forces in regulating their dynamic properties are discussed.     

Responses to drought of five Brachiaria species. II. Water relations and leaf gas exchange

Neotropical savannas are exposed to recurrent dry periods of varied duration, and forage grasses must be able to cope with such temporal stresses to maintain productive pastures. This study compared leaf water relations and net photosynthesis under drought of five perennial Brachiaria species: the tufted B. brizantha (CIAT 6780), the semi-stoloniferous B. decumbens (CIAT 606) and B. mutica, and the stoloniferous B. humidicola (CIAT 679) and B. dictyoneura (CIAT 6133). Plants of the five grasses were grown in large pots and subjected to drought by suspending watering until first wilting symptoms (14 days for B. brizantha, B. decumbens and B. mutica, and 29 days for B. humidicola and B. dictyoneura). Afterwards, they were re-watered and a second soil dry cycle was imposed. Time trends in leaf water potential (_l), relative water content (RWC), osmotic potential at full turgor (₀ ¹⁰⁰), stomatal conductance (Gs) and net photosynthesis (A) of stressed (DT) plants were compared to those of well-irrigated (CT) plants. Predawn _l in DT plants decreased to a minimum of –1.5 and –2.0 MPa in B. brizantha and B. mutica, compared to –2.5 to –3.0 MPa in B. decumbens, B. humidicola and B. dictyoneura. RWC decreased up to 50% in B. brizantha, compared to 75% in the other species. In B. humidicola, B. dictyoneura and in a lesser extent, B. decumbens, leaves of DT plants adjusted osmotically, by an apparent accumulation of nutrient solutes, at a rather constant ratio of turgid to dry weight of the tissue. Calculated osmotic adjustment ranged between 0.38 (B. decumbens) to 0.87 MPa (B. humidicola). This adjustment in ₀ ¹⁰⁰ was in some cases maintained 7 days after re-watering. In B. brizantha and B. mutica, Gs and A were significantly affected by drought, with maximum reduction percentages at the second drought period of 65 and 80%, respectively. The corresponding reduction in B. decumbens was 53 and 55%, respectively; whereas in B. humidicola and B. dictyoneura Gs and A were reduced less than 20%. In all species, re-watering allowed for the water relations (except ₀ ¹⁰⁰) and photosynthetic activity of leaves of DT plants to reach values comparable to those of CT plants. Results are discussed in term of root morphology and soil water extraction pattern, as well as leaf traits that may contribute to withstand drought under moderate soil water stress.     

Glyphosate inhibits the translocation of green fluorescent protein and sucrose from a transgenic tobacco host to<Emphasis Type="Italic"> Cuscuta campestris</Emphasis> Yunk.

The parasitic plant Cuscuta campestris is dependent on its host for water, assimilates and amino acids. It can be controlled by the herbicide glyphosate, which inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), resulting in shikimate accumulation. In this study, C. campestris was parasitic on transgenic tobacco plants expressing green fluorescent protein (GFP) in the phloem. Changes in [¹⁴C]sucrose and GFP accumulation in the parasite were used as indicators of the herbicides effect on translocation between the host and parasite. Host plants were treated with glyphosate 22 days after sowing. Shikimate accumulation in the parasite 1 day after glyphosate treatment (DAGT) confirmed EPSPS inhibition in C. campestris. No damage was visible in the host plants for the first 3 DAGT, while during that same time, a significant reduction in [¹⁴C]sucrose and GFP accumulation was observed in the parasite. Thus, we propose that the parallel reduction in GFP and sucrose accumulation in C. campestris is a result of a glyphosate effect on the parasites ability to withdraw assimilates from the host.Abbreviations CLSM Confocal laser-scanning microscope - DAGT Days after glyphosate treatment - DAS Days after sowing - EPSPS 5-Enolpyruvylshikimate-3-phosphate synthase - GFP Green fluorescent protein     

Transmembrane domain I of the gamma-aminobutyric acid transporter GAT-1 plays a crucial role in the transition between cation leak and transport modes

The sodium- and chloride-dependent gamma-aminobutyric acid (GABA) transporter is essential for synaptic transmission by this neurotransmitter. GAT-1 expressed in Xenopus laevis oocytes exhibits sodium-dependent GABA-induced inward currents reflecting electrogenic sodium-coupled transport. In lithium-containing medium, GAT-1 mediates GABA-independent currents, the relationship of which to the physiological transport process is poorly understood. In this study, mutants are described that appear to be locked in this cation leak mode. When Gly(63), located in the middle of the highly conserved transmembrane domain I, was mutated to serine or cysteine, sodium-dependent GABA currents were abolished. Strikingly, these mutants exhibited robust inward currents in lithium- as well as potassium-containing media. Membrane-impermeant sulfhydryl reagents inhibited these currents of the cysteine but not of the serine mutant, indicating that this position was accessible to the external aqueous medium. The cation leak currents mediated by wild-type GAT-1 were inhibited by low millimolar sodium concentrations in a noncompetitive manner. Mutations at other positions of transmembrane domain I increased or decreased the apparent sodium affinity, as monitored by the sodium-dependent steady-state GABA currents or transient currents. In parallel, the ability of sodium to inhibit the cation leak currents was increased or decreased, respectively. Thus, transmembrane domain I of GAT-1 contains determinants controlling both sodium-coupled GABA flux and the cation leak pathway as well as the interconversion of these distinct modes. Our observations suggest the possibility that the permeation pathway in both modes shares common structural elements.     

Neural networks applied to the prediction of fed-batch fermentation kinetics of Bacillus thuringiensis

This paper proposes using a new recurrent neural network model (RNNM) to predict and control fed batch fermentations of Bacillus thuringiensis. The control variables are the limiting substrate and the feeding conditions. The multi-input multi-output RNNM proposed has twelve inputs, seven outputs, nineteen neurons in the hidden layer, and global and local feedbacks. The weight update learning algorithm designed is a version of the well known backpropagation through time algorithm directed to the RNNM learning. The error approximation for the last epoch of learning is 2% and the total learning time is 51 epochs, where the size of an epoch is 162 iterations. The RNNM generalization was carried out reproducing a B. thuringiensis fermentation not included in the learning process. It attains an error approximation of 1.8%.     

Generation of ligand-receptor alliances by "SEA" module-mediated cleavage of membrane-associated mucin proteins

A mechanism is described whereby one and the same gene can encode both a receptor protein as well as its specific ligand. Generation of this receptor-ligand partnership is effected by proteolytic cleavage within a specific module located in a membrane resident protein. It is postulated here that the "SEA" module, found in a number of heavily O-linked glycosylated membrane-associated proteins, serves as a site for proteolytic cleavage. The subunits generated by proteolytic cleavage of the SEA module reassociate, and can subsequently elicit a signaling cascade. We hypothesize that all membrane resident proteins containing such a "SEA" module will undergo cleavage, thereby generating a receptor-ligand alliance. This requires that the protein subunits resulting from the proteolytic cleavage reassociate with each other in a highly specific fashion. The same SEA module that serves as the site for proteolytic cleavage, probably also contains the binding sites for reassociation of the resultant two subunits. More than one type of module can function as a site for proteolytic cleavage; this can occur not only in one-pass membrane proteins but also in 7-transmembrane proteins and other membrane-associated proteins. The proposal presented here is likely to have significant practical consequences. It could well lead to the rational design and identification of molecules that, by binding to one of the cleaved partners, will act either as agonists or antagonists, alter signal transduction and, hence, cellular behavior.     

Storage stability of Anagrapha falcifera nucleopolyhedrovirus in spray-dried formulations

A multiply embedded nucleopolyhedrovirus isolated from Anagrapha falcifera (Kirby) (AfMNPV) can lose insecticidal activity during months of dry storage in ambient room conditions. We tested the spray-dried AfMNPV formulations after storage for up to 1 year at room temperatures for insecticidal activity against neonate Trichoplusia ni (Hübner). Experimental formulations were made using combinations of corn flours, lignin, and sucrose, and were selected based on previous work which demonstrated that these formulations resisted solar degradation in field experiments. Twelve experimental formulations (organized in three groups of four formulations) compared the effect of (1) the ratio of formulation ingredients (lignin and corn flour) to virus concentration, (2) different sources of lignin, or (3) different corn flours and sugar. Based on a single-dose plant assay with these 12 formulations, none of the formulations lost significant activity due to the drying process, when compared with the unformulated wet AfMNPV. Samples of the 12 dried formulations were stored at room (22+/-3 degrees C) and refrigerated (4 degrees C) temperatures. Insecticidal activity (LC(50)) was determined with a dosage-response assay for neonates fed on treated cotton-leaf disks. After 6 (or 9) and 12 months storage, refrigerated samples maintained insecticidal activity better than corresponding samples stored at room temperatures with LC(50)s that averaged 2.0 x 10(6) polyhedral inclusion bodies per milliliter (pibs/ml) for refrigerated samples and 5.4 x 10(6) pibs/ml for samples stored at room temperatures. Compared with unformulated stock virus stored frozen, six formulations stored at room temperature and 10 formulations stored in the refrigerator did not lose significant insecticidal activity after 1 year based on overlapping 90% confidence intervals. Changing the ratio of virus to formulation ingredients did not provide a clear trend over the range of concentrations tested, and may be less important for shelf-life of virus activity compared with formulations made with different ingredients. Two of the four formulations made with different lignins were about 15 times less active after 1 year at room temperature compared with refrigerated samples, indicating that specific formulation ingredients can affect storage stability. Formulations that contained sugar generally maintained activity during storage better than formulations without sugar. Unformulated virus stock maintained insecticidal activity (ranged from 0.20 to 2.5 x 10(6) pibs/ml) better during storage than dried formulations with LC(50)s that ranged from 0.39 to 27 x 10(6) pibs/ml. Unformulated virus stock, which is essentially a suspension of virus occlusion bodies in homogenized insect cadavers, did not lose activity when stored at refrigerated or room temperature. We believe that stability of AfMNPV insecticidal activity during storage as dry formulations is related to the general composition of the formulation and that sugar may play a critical role in maintaining insecticidal activity.