Potential of a cyclone prototype spacer to improve in vitro dry powder delivery

Copyright The Author(s) 2013. This article is published with open access at Springerlink.com. This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are creditedPurpose: Low inspiratory force in patients with lung disease is associated with poor deagglomeration and high throat deposition when using dry powder inhalers (DPIs). The potential of two reverse flow cyclone prototypes as spacers for commercial carrierbased DPIs was investigated. Methods: Cyclohaler®, Accuhaler® and Easyhaler® were tested with and without the spacers between 30-60 Lmin-1. Deposition of particles in the next generation impactor and within the devices was determined by high performance liquid chromatography. Results: Reduced induction port deposition of the emitted particles from the cyclones was observed due to the high retention of the drug within the spacers (e.g. salbutamol sulphate (SS): 67.89 ± 6.51 % at 30 Lmin-1 in Cheng 1). Fine particle fractions of aerosol as emitted from the cyclones were substantially higher than the DPIs alone. Moreover, the aerodynamic diameters of particles emitted from the cyclones were halved compared to the DPIs alone (e.g. SS from the Cyclohaler® at 4 kPa: 1.08 ± 0.05 μm vs. 3.00 ± 0.12 μm, with and without Cheng 2, respectively) and unaltered with increased flow rates. Conclusion: This work has shown the potential of employing a cyclone spacer for commercial carrier-based DPIs to improve inhaled drug delivery.Peer reviewe

Telomerase activity in melanoma and non-melanoma skin cancer

Telomeres are specialized structures consisting of repeat arrays of TTAGGGn located at the ends of chromosomes. They are essential for chromosome stability and, in the majority of normal somatic cells, telomeres shorten with each cell division. Most immortalized cell lines and tumours reactivate telomerase to stabilize the shortening chromosomes. Telomerase activation is regarded as a central step in carcinogenesis and, here, we demonstrate telomerase activation in premalignant skin lesions and also in all forms of skin cancer. Telomerase activation in normal skin was a rare event, and among 16 samples of normal skin (one with a history of chronic sun exposure) 12.5% (2 out of 16) exhibited telomerase activity. One out of 16 (6.25%) benign proliferative lesions, including viral and seborrhoeic wart samples, had telomerase activity. In premalignant actinic keratoses and Bowen's disease, 42% (11 out of 26) of samples exhibited telomerase activity. In the basal cell carcinoma and cutaneous malignant melanoma (CMM) lesions, telomerase was activated in 77% (10 out of 13) and 69% (22 out of 32) respectively. However, only 25% (3 out of 12) of squamous cell carcinomas (SCC) had telomerase activity. With the exception of one SCC sample, telomerase activity in a positive control cell line derived from a fibrosarcoma (HT1080) was not inhibited when mixed with the telomerase-negative SCC or CMM extracts, indicating that, overall, Taq polymerase and telomerase inhibitors were not responsible for the negative results. Mean telomere hybridizing restriction fragment (TRF) analysis was performed in a number of telomerase-positive and -negative samples and, although a broad range of TRF sizes ranging from 3.6 to 17 kb was observed, a relationship between telomerase status and TRF size was not found

Short Telomeres Initiate Telomere Recombination in Primary and Tumor Cells

Human tumors that lack telomerase maintain telomeres by alternative lengthening mechanisms. Tumors can also form in telomerase-deficient mice; however, the genetic mechanism responsible for tumor growth without telomerase is unknown. In yeast, several different recombination pathways maintain telomeres in the absence of telomerase—some result in telomere maintenance with minimal effects on telomere length. To examine non-telomerase mechanisms for telomere maintenance in mammalian cells, we used primary cells and lymphomas from telomerase-deficient mice (mTR−/− and Eμmyc+mTR−/−) and CAST/EiJ mouse embryonic fibroblast cells. These cells were analyzed using pq-ratio analysis, telomere length distribution outliers, CO-FISH, Q-FISH, and multicolor FISH to detect subtelomeric recombination. Telomere length was maintained during long-term growth in vivo and in vitro. Long telomeres, characteristic of human ALT cells, were not observed in either late passage or mTR−/− tumor cells; instead, we observed only minimal changes in telomere length. Telomere length variation and subtelomeric recombination were frequent in cells with short telomeres, indicating that length maintenance is due to telomeric recombination. We also detected telomere length changes in primary mTR−/− cells that had short telomeres. Using mouse mTR+/− and human hTERT+/− primary cells with short telomeres, we found frequent length changes indicative of recombination. We conclude that telomere maintenance by non-telomerase mechanisms, including recombination, occurs in primary cells and is initiated by short telomeres, even in the presence of telomerase. Most intriguing, our data indicate that some non-telomerase telomere maintenance mechanisms occur without a significant increase in telomere length

Pharmacokinetic properties and antitumor efficacy of the 5-fluorouracil loaded PEG-hydrogel

<p>Abstract</p> <p>Background</p> <p>We have studied the <it>in vitro </it>and <it>in vivo </it>utility of polyethylene glycol (PEG)-hydrogels for the development of an anticancer drug 5-fluorouracil (5-FU) delivery system.</p> <p>Methods</p> <p>A 5-FU-loaded PEG-hydrogel was implanted subcutaneously to evaluate the drug retention time and the anticancer effect. For the pharmacokinetic study, two groups of male rats were administered either an aqueous solution of 5-FU (control group)/or a 5-FU-loaded PEG-hydrogel (treated group) at a dose of 100 mg/kg. For the pharmacodynamic study, a human non-small-cell lung adenocarcinoma (NSCLC) cell line, A549 was inoculated to male nude mice with a cell density of 3 × 10⁶. Once tumors start growing, the mice were injected with 5-FU/or 5-FU-loaded PEG-hydrogel once a week for 4 weeks. The growth of the tumors was monitored by measuring the tumor volume and calculating the tumor inhibition rate (IR) over the duration of the study.</p> <p>Results</p> <p>In the pharmacokinetic study, the 5-FU-loaded PEG-hydrogel gave a mean residence time (MRT) of 8.0 h and the elimination half-life of 0.9 h; these values were 14- and 6-fold, respectively, longer than those for the free solution of 5-FU (p < 0.05). In the pharmacodynamic study, A549 tumor growth was significantly inhibited in the 5-FU-loaded PEG-hydrogel group in comparison to the untreated group beginning on Day 14 (p < 0.05-0.01). Moreover, the 5-FU-loaded PEG-hydrogel group had a significantly enhanced tumor IR (p < 0.05) compared to the free 5-FU drug treatment group.</p> <p>Conclusion</p> <p>We suggest that 5-FU-loaded PEG-hydrogels could provide a useful tool for the development of an anticancer drug delivery system.</p

Phosphorothioate oligonucleotides, suramin and heparin inhibit DNA-dependent protein kinase activity

Phosphorothioate oligonucleotides and suramin bind to heparin binding proteins including DNA polymerases, and inhibit their functions. In the present study, we report inhibition of DNA-dependent protein kinase activity by phosphorothioate oligonucleotides, suramin and heparin. Inhibitory effect of phosphorothioate oligonucleotides on DNA-dependent protein kinase activity was increased with length and reached a plateau at 36-mer. The base composition of phosphorothioate oligonucleotides did not affect the inhibitory effect. The inhibitory effect by phosphorothioate oligodeoxycytidine 36-mer can be about 200-fold greater than that by the phosphodiester oligodeoxycytidine 36-mer. The inhibitory effect was also observed with purified DNA-dependent protein kinase, which suggests direct interaction between DNA-dependent protein kinase and phosphorothioate oligonucleotides. DNA-dependent protein kinase will have different binding positions for double-stranded DNA and phosphorothioate oligodeoxycytidine 36-mer because they were not competitive in DNA-dependent protein kinase activation. Suramin and heparin inhibited DNA-dependent protein kinase activity with IC50 of 1.7 μM and 0.27 μg ml−1 respectively. DNA-dependent protein kinase activities and DNA double-stranded breaks repair in cultured cells were significantly suppressed by the treatment with suramin in vivo. Our present observations suggest that suramin may possibly result in sensitisation of cells to ionising radiation by inactivation of DNA-dependent protein kinase and the impairment of double-stranded breaks repair

A Novel Checkpoint and RPA Inhibitory Pathway Regulated by Rif1

Cells accumulate single-stranded DNA (ssDNA) when telomere capping, DNA replication, or DNA repair is impeded. This accumulation leads to cell cycle arrest through activating the DNA–damage checkpoints involved in cancer protection. Hence, ssDNA accumulation could be an anti-cancer mechanism. However, ssDNA has to accumulate above a certain threshold to activate checkpoints. What determines this checkpoint-activation threshold is an important, yet unanswered question. Here we identify Rif1 (Rap1-Interacting Factor 1) as a threshold-setter. Following telomere uncapping, we show that budding yeast Rif1 has unprecedented effects for a protein, inhibiting the recruitment of checkpoint proteins and RPA (Replication Protein A) to damaged chromosome regions, without significantly affecting the accumulation of ssDNA at those regions. Using chromatin immuno-precipitation, we provide evidence that Rif1 acts as a molecular “band-aid” for ssDNA lesions, associating with DNA damage independently of Rap1. In consequence, small or incipient lesions are protected from RPA and checkpoint proteins. When longer stretches of ssDNA are generated, they extend beyond the junction-proximal Rif1-protected regions. In consequence, the damage is detected and checkpoint signals are fired, resulting in cell cycle arrest. However, increased Rif1 expression raises the checkpoint-activation threshold to the point it simulates a checkpoint knockout and can also terminate a checkpoint arrest, despite persistent telomere deficiency. Our work has important implications for understanding the checkpoint and RPA–dependent DNA–damage responses in eukaryotic cells

Extensive telomere erosion is consistent with localised clonal expansions in Barrett’s metaplasia

Barrett’s oesophagus is a premalignant metaplastic condition that predisposes patients to the development of oesophageal adenocarcinoma. However, only a minor fraction of Barrett’s oesophagus patients progress to adenocarcinoma and it is thus essential to determine bio-molecular markers that can predict the progression of this condition. Telomere dysfunction is considered to drive clonal evolution in several tumour types and telomere length analysis provides clinically relevant prognostic and predictive information. The aim of this work was to use high-resolution telomere analysis to examine telomere dynamics in Barrett’s oesophagus. Telomere length analysis of XpYp, 17p, 11q and 9p, chromosome arms that contain key cancer related genes that are known to be subjected to copy number changes in Barrett’s metaplasia, revealed similar profiles at each chromosome end, indicating that no one specific telomere is likely to suffer preferential telomere erosion. Analysis of patient matched tissues (233 samples from 32 patients) sampled from normal squamous oesophagus, Z-line, and 2 cm intervals within Barrett’s metaplasia, plus oesophago-gastric junction, gastric body and antrum, revealed extensive telomere erosion in Barrett’s metaplasia to within the length ranges at which telomere fusion is detected in other tumour types. Telomere erosion was not uniform, with distinct zones displaying more extensive erosion and more homogenous telomere length profiles. These data are consistent with an extensive proliferative history of cells within Barrett’s metaplasia and are indicative of localised clonal growth. The extent of telomere erosion highlights the potential of telomere dysfunction to drive genome instability and clonal evolution in Barrett’s metaplasia

Amplification of telomerase (hTERT) gene is a poor prognostic marker in non-small-cell lung cancer

Telomerase reactivation is a hallmark of human carcinogenesis. Increased telomerase activity may result from gene amplification and/or overexpression. This study evaluates the prognostic value of hTERT gene amplification and mRNA overexpression in 144 resectable non-small-cell lung cancer (NSCLC) specimens. The hTERT gene copy number was assessed by quantitative polymerase chain reaction (qPCR) on laser-capture microdissected tumour cells of 81 tumours, and by fluorescence in situ hybridisation (FISH) on a subset of 59 tumours. hTERT mRNA level was determined by reverse transcription (RT)–qPCR in 130 tumours. In total, 57% of (46 out of 81) primary NSCLC specimens demonstrated hTERT amplification, which was significantly more common (P<0.001) in adenocarcinoma (30 out of 40) than in squamous cell carcinoma (13 out of 37). The hTERT mRNA overexpression was noted in 74% (94 out of 130) of tumours; it was more frequent in squamous cell than in adenocarcinoma (87 vs 68%, P=0.03). Overexpression was significantly associated with amplification (P=0.03), especially in adenocarcinoma. The hTERT gene amplification was prognostic for shorter recurrence-free survival (hazard ratio=2.16, P=0.03). These data indicate that gene amplification is an important mechanism for hTERT overexpression in lung adenocarcinoma and is an independent poor prognostic marker for disease-free survival in NSCLC

Cell Proliferation in the Presence of Telomerase

BACKGROUND: Telomerase, which is active early in development and later in stem and germline cells, is also active in the majority of human cancers. One of the known functions of telomerase is to extend the ends of linear chromosomes, countering their gradual shortening at each cell division due to the end replication problem and postreplication processing. Telomerase concentration levels vary between different cell types as well as between different tumors. In addition variable telomerase concentrations will exist in different cells in the same tumor when telomerase inhibitors are used, because of limitations of drug delivery in tissue. Telomerase extends short telomeres more frequently than long telomeres and the relation between the extension frequency and the telomere length is nonlinear. METHODOLOGY/PRINCIPAL FINDINGS: Here, the biological data of the nonlinear telomerase-telomere dynamics is incorporated in a mathematical theory to relate the proliferative potential of a cell to the telomerase concentration in that cell. The main result of the paper is that the proliferative capacity of a cell grows exponentially with the telomerase concentration. CONCLUSIONS/SIGNIFICANCE: The theory presented here suggests that long term telomerase inhibition in every cancer progenitor or cancer stem cell is needed for successful telomere targeted cancer treatment. This theory also can be used to plan and assess the results of clinical trials targeting telomerase