bluuebunny/crossref_metadata_2025 huggingface dataset:
Nitric Oxide and Peroxynitrite in Health and Disease
Authors: Pacher Pál , Beckman Joseph S. , Liaudet Lucas
Published: January 2007

The discovery that mammalian cells have the ability to synthesize the free radical nitric oxide (NO) has stimulated an extraordinary impetus for scientific research in all the fields of biology and medicine. Since its early description as an endothelial-derived relaxing factor, NO has emerged as a fundamental signaling device regulating virtually every critical cellular function, as well as a potent mediator of cellular damage in a wide range of conditions. Recent evidence indicates that most of the cytotoxicity attributed to NO is rather due to peroxynitrite, produced from the diffusion-controlled reaction between NO and another free radical, the superoxide anion. Peroxynitrite interacts with lipids, DNA, and proteins via direct oxidative reactions or via indirect, radical-mediated mechanisms. These reactions trigger cellular responses ranging from subtle modulations of cell signaling to overwhelming oxidative injury, committing cells to necrosis or apoptosis. In vivo, peroxynitrite generation represents a crucial pathogenic mechanism in conditions such as stroke, myocardial infarction, chronic heart failure, diabetes, circulatory shock, chronic inflammatory diseases, cancer, and neurodegenerative disorders. Hence, novel pharmacological strategies aimed at removing peroxynitrite might represent powerful therapeutic tools in the future. Evidence supporting these novel roles of NO and peroxynitrite is presented in detail in this review.
Heat-inducible in vivo gene therapy of colon carcinoma by human mdr1 promoter–regulated tumor necrosis factor-α expression
Authors: Walther Wolfgang , Arlt Franziska , Fichtner Iduna , Aumann Jutta , Stein Ulrike , Schlag Peter M.
Published: January 2007

The promoter of the human multidrug resistance gene (mdr1) harbors defined heat-responsive elements, which could be exploited for construction of heat-inducible expression vectors. To analyze the hyperthermia inducibility of the mdr1 promoter in vitro and in vivo, we used the pcDNA3-mdrp-hTNF vector construct for heat-induced tumor necrosis factor α (TNF-α) expression in transfected HCT116 human colon carcinoma cells at mRNA level by quantitative real-time reverse transcription-PCR and at protein level by TNF-α ELISA. For the in vitro studies, the pcDNA3-mdrp-hTNF–transfected tumor cells were treated with hyperthermia at 43°C for 2 h. In the animal studies, stably transfected or in vivo jet-injected tumor-bearing Ncr:nu/nu mice were treated for 60 min at 42°C to induce TNF-α expression. Both the in vitro and in vivo experiments show that hyperthermia activates the mdr1 promoter in a temperature- and time-dependent manner, leading to an up to 4-fold increase in mdr1 promoter–driven TNF-α expression at mRNA and an up to 3-fold increase at protein level. The in vivo heat-induced TNF-α expression combined with Adriamycin (8 mg/kg) treatment leads to the inhibition of tumor growth in the animals. These experiments support the idea that heat-induced mdr1 promoter–driven expression of therapeutic genes is efficient and feasible for combined cancer gene therapy approaches. [Mol Cancer Ther 2007;6(1):236–43]
Disrupting insulin-like growth factor signaling as a potential cancer therapy
Authors: Sachdev Deepali , Yee Douglas
Published: January 2007

The type I insulin-like growth factor receptor (IGF-IR) plays multiple roles in several cancers and increased circulating levels of insulin-like growth factor-I (IGF-I) are associated with increased risk of breast, colon, and prostate cancers. Because IGF-II and insulin signal via the insulin receptor (IR) to stimulate the growth of cancer cells, inhibition of IR might be necessary to totally disrupt the action of IGFs and their receptors. This review describes the well-recognized roles of IGF-IR in driving the malignant phenotype, examines the evidence that perhaps IR should also be targeted to inhibit the effects of the IGF ligands and insulin in cancer, describes the strategies to disrupt IGF signaling in cancer, and highlights some key issues that need to be considered as clinical trials targeting IGF-IR proceed. [Mol Cancer Ther 2007;6(1):1–12]
Prodigiosin induces the proapoptotic gene NAG-1 via glycogen synthase kinase-3β activity in human breast cancer cells
Authors: Soto-Cerrato Vanessa , Viñals Francesc , Lambert James R. , Kelly Julie A. , Pérez-Tomás Ricardo
Published: January 2007

Prodigiosin (2-methyl-3-pentyl-6-methoxyprodigiosene) is a bacterial metabolite that has anticancer and antimetastatic properties. However, the molecular mechanisms responsible for these abilities are not fully understood. Gene expression profiling of the human breast cancer cell line MCF-7 treated with prodigiosin was analyzed by cDNA array technology. The majority of the significantly modified genes were related to apoptosis, cell cycle, cellular adhesion, or transcription regulation. The dramatic increase of the nonsteroidal anti-inflammatory drug-activated gene 1 (NAG-1) made this gene an interesting candidate regarding the possible mechanism by which prodigiosin induces cytotoxicity in MCF-7 cells. Our results show that prodigiosin triggers accumulation of the DNA-damage response tumor-suppressor protein p53 but that NAG-1 induction was independent of p53 accumulation. Moreover, prodigiosin caused AKT dephosphorylation and glycogen synthase kinase-3β (GSK-3β) activation, which correlated with NAG-1 expression. Prodigiosin-induced apoptosis was recovered by inhibiting GSK-3β, which might be due, at least in part, to the blockade of the GSK-3β–dependent up-regulation of death receptors 4 and 5 expression. These findings suggest that prodigiosin-mediated GSK-3β activation is a key event in regulating the molecular pathways that trigger the apoptosis induced by this anticancer agent. [Mol Cancer Ther 2007;6(1):362–9]
Cell cycle phenotype-based optimization of G2-abrogating peptides yields CBP501 with a unique mechanism of action at the G2 checkpoint
Authors: Sha Shi-Ken , Sato Takuji , Kobayashi Hidetaka , Ishigaki Machiyo , Yamamoto Sayaka , Sato Hitoshi , Takada Asako , Nakajyo Sigetosi , Mochizuki Yasuo , Friedman Jonathan M. , Cheng Fong-Chi , Okura Takashi , Kimura Ryohei , Kufe Donald W. , VonHoff Daniel D. , Kawabe Takumi
Published: January 2007

Cell cycle G2 checkpoint abrogation is an attractive strategy for sensitizing cancer cells to DNA-damaging anticancer agent without increasing adverse effects on normal cells. However, there is no single proven molecular target for this therapeutic approach. High-throughput screening for molecules inhibiting CHK1, a kinase that is essential for the G2 checkpoint, has not yet yielded therapeutic G2 checkpoint inhibitors, and the tumor suppressor phenotypes of ATM and CHK2 suggest they may not be ideal targets. Here, we optimized two G2 checkpoint-abrogating peptides, TAT-S216 and TAT-S216A, based on their ability to reduce G2 phase accumulation of DNA-damaged cells without affecting M phase accumulation of cells treated with a microtubule-disrupting compound. This approach yielded a peptide CBP501, which has a unique, focused activity against molecules that phosphorylate Ser216 of CDC25C, including MAPKAP-K2, C-Tak1, and CHK1. CBP501 is >100-fold more potent than TAT-S216A and retains its selectivity for cancer cells. CBP501 is unusually stable, enters cells rapidly, and increases the cytotoxicity of DNA-damaging anticancer drugs against cancer cells without increasing adverse effects. These findings highlight the potency of CBP501 as a G2-abrogating drug candidate. This report also shows the usefulness of the cell cycle phenotype-based protocol for identifying G2 checkpoint-abrogating compounds as well as the potential of peptide-based compounds as focused multitarget inhibitors. [Mol Cancer Ther 2007;6(1):147–53]
D-501036, a novel selenophene-based triheterocycle derivative, exhibits potentin vitroandin vivoantitumoral activity which involves DNA damage and ataxia telangiectasia–mutated nuclear protein kinase activation
Authors: Juang Shin-Hun , Lung Chia-Chi , Hsu Pi-Chen , Hsu Kuo-Shun , Li Yu-Chen , Hong Pao-Chiung , Shiah Her-Shyong , Kuo Ching-Chuan , Huang Ching-Wei , Wang Yu-Chin , Huang Leeyuan , Chen Tom S. , Chen Shyh-Fong , Fu Kuo-Chu , Hsu Cheng-Li , Lin Meng-Ju , Chang Ching-jer , Ashendel Curtis L. , Chan Thomas C.K. , Chou Kai-Ming , Chang Jang-Yang
Published: January 2007

D-501036 [2,5-bis(5-hydroxymethyl-2-selenienyl)-3-hydroxymethyl-N-methylpyrrole] is herein identified as a novel antineoplastic agent with a broad spectrum of antitumoral activity against several human cancer cells and an IC50 value in the nanomolar range. The IC50 values for D-501036 in the renal proximal tubule, normal bronchial epithelial, and fibroblast cells were >10 μmol/L. D-501036 exhibited no cross-resistance with vincristine- and paclitaxel-resistant cell lines, whereas a low level of resistance toward the etoposide-resistant KB variant was observed. Cell cycle analysis established that D-501036 treatment resulted in a dose-dependent accumulation in S phase with concomitant loss of both the G0-G1 and G2-M phase in both Hep 3B and A-498 cells. Pulsed-field gel electrophoresis showed D-501036–induced, concentration-dependent DNA breaks in both Hep 3B and A-498 cells. These breaks did not involve interference with either topoisomerase-I and topoisomerase-II function or DNA binding. Rapid reactive oxygen species production and formation of Se-DNA adducts were evident following exposure of cells to D-501036, indicating that D-501036–mediated DNA breaks were attributable to the induction of reactive oxygen species and DNA adduct formation. Moreover, D-501036–induced DNA damage activated ataxia telangiectasia–mutated nuclear protein kinase, leading to hyperphosphorylation of Chk1, Chk2, and p53, decreased expression of CDC25A, and up-regulation of p21WAF1 in both p53-proficient and p53-deficient cells. Collectively, the results indicate that D-501036–induced cell death was associated with DNA damage–mediated induction of ataxia telangiectasia–mutated activation, and p53-dependent and -independent apoptosis pathways. Notably, D-501036 shows potent activity against the growth of xenograft tumors of human renal carcinoma A-498 cells. Thus, D-501036 is a promising anticancer compound that has strong potential for the management of human cancers. [Mol Cancer Ther 2007;6(1):193–202]
Molecular correlates of gefitinib responsiveness in human bladder cancer cells
Authors: Shrader Marissa , Pino Maria Simona , Brown Gordon , Black Peter , Adam Liana , Bar-Eli Menahse , Dinney Colin P.N. , McConkey David J.
Published: January 2007

We characterized the effects of the small molecule epidermal growth factor receptor (EGFR) inhibitor gefitinib (ZD1839, Iressa) on cell proliferation in a panel of 17 human bladder cancer cell lines. Gefitinib inhibited DNA synthesis in a concentration-dependent fashion in 6 of 17 lines. Growth inhibition was associated with p27Kip1 accumulation and decreased cyclin-dependent kinase 2 activity. Gefitinib also inhibited baseline EGFR, AKT, and extracellular signal-regulated kinase (ERK) phosphorylation in the EGFR-dependent cells maintained in serum-free medium, whereas it had no effect on baseline EGFR or ERK phosphorylation in the EGFR-independent cells. Analyses of candidate markers of EGFR dependency revealed that the gefitinib-sensitive cells expressed higher surface EGFR levels than the gefitinib-resistant lines. Gefitinib-sensitive cells generally expressed higher levels of E-cadherin and lower levels of vimentin than the gefitinib-resistant cells, but these correlations were not perfect, suggesting that these markers of epithelial-mesenchymal transition cannot be used by themselves to prospectively predict EGFR-dependent growth. Together, our results show that bladder cancer cells are markedly heterogeneous with respect to their sensitivity to EGFR antagonists. Although surface EGFR levels and epithelial-mesenchymal transition status seem to roughly correlate with responsiveness, they cannot be used by themselves to identify bladder tumors that will be sensitive to EGFR-directed therapy. However, comparing levels of p27Kip1 or DNA synthesis before and after gefitinib exposure does identify the drug-sensitive cells. [Mol Cancer Ther 2007;6(1):277–85]
Effects of External Electrical Stimulation on Laser-Beam-Induced Experimental Thrombosis
Authors: Aguejouf O. , Doutremepuich F. , Doutremepuich C.
Published: January 2007

External electrical stimulation (EES) has demonstrated venous antithrombotic properties. The aim of this investigation was to study its antithrombotic properties using unfractionated heparin (UFH) as a reference in arterial and venous mesenteric microvessels. For this purpose a rat model of laser-beam-induced thrombosis was used. Four groups of rats were investigated (n = 7): control, UFH (5 mg /kg), UFH (2.5 mg/kg) and electrical stimulation [e-vascular®, E = 10, alternate polarity, in two protocols: (i) 30 min before and during thrombosis induction and (ii) only 30 min before thrombosis induction as preventive stimulation]. This effect was tested in arterial and venous microvessels in the four groups using the continuous protocol and only in arterial microvessels using the preventive protocol. Three parameters were studied: the number of laser beams needed to induce platelet thrombus formation, the number of emboli and the duration of embolization. Induced hemorrhagic time (IHT), platelet aggregation induced by ADP and coagulation tests (activated partial thromboplastin time, aPTT, prothrombin time, pTT, and fibrinogen level) were also determined. Continuous electrical stimulation decreased the number of emboli and the duration of embolization both in arterioles and in venules. It also significantly reduced the amplitude and velocity of the ex vivo platelet aggregation induced by ADP. Contrary to UFH, neither aPTT nor pTT were affected in platelet-poor plasma collected after the induction of thrombosis by laser beam, nor did it modify IHT. Preventive electrical stimulation produced similar results with fewer effects on induced venous thrombosis. These data suggest that EES has a potent antithrombotic effect on arterial and venous thrombosis without any hemorrhagic adverse effects, making it a very promising tool in the prevention and treatment of venous and arterial thromboembolic complications.
Mutations and Polymorphisms in Genes Affecting Haemostasis Components in Children with Thromboembolic Events
Authors: Komitopoulou A. , Platokouki H. , Kapsimali Z. , Moschovi M. , Kattamis A. , Pergantou H. , Aronis S.
Published: January 2007

The distribution of mutations/polymorphisms in genes affecting haemostasis [factor V Leiden (FVL), FV H1298R (FVR2), FII 20210A, b-Fib 455G→A, FXIII V34L, PAI-1 4G, HPA-1b] among 141 children with thrombosis at various sites and 103 controls was compared. Additionally, the carriage of these mutations/polymorphisms was associated with the levels of their corresponding proteins in thrombosed children. Thrombosis was more frequent in boys (p = 0.021). No studied mutation/polymorphism was found to be a risk factor for thrombosis, except for FVL (odds ratio 3.8, 95% CI 1.4–10.6). The risk of thrombosis for FVL carriers was twice as high in children with an idiopathic thrombosis (odds ratio 5.4) than in thrombosed children with an underlying disease or a triggering event (odds ratio 2.7). FVL carriers had an odds ratio of 5.9 (95% CI 1.8–19.6) when FVR2 was absent. In thrombosed children, the activated protein C resistance ratio was significantly lower in the presence of FVL (p < 0.001). Prothrombin and fibrinogen levels, although higher in FII 20210A and b-Fib 455G→A carriers, did not reach statistical significance.
An Experimentally Antithrombotic Strawberry Variety Is Also Effective in Humans
Authors: Naemura Aki , Ohira Hideo , Ikeda Masamichi , Koshikawa Kaneyuki , Ishii Hiromitsu , Yamamoto Junichiro
Published: January 2007

Prevention of arterial thrombotic diseases is of high priority in developed countries. As inappropriate diet is regarded as an important risk factor of thrombotic events, daily intake of an antithrombotic diet may offer a convenient and effective way of prevention. Earlier we used animal models of thrombosis to find fruits and vegetables with potential antithrombotic activity. Among various strawberry varieties tested, a particular variety (KYSt-4, Nohime) showed a significant antithrombotic effect. The aim of the present investigation was to extend this study to humans, by testing the experimentally active KYSt-4 and inactive KYSt-10 variety for effectiveness in humans after oral intake. Filtrates of strawberries were prepared and administered orally. Thrombotic status was tested by a novel global test (Gorog Thrombosis Test). The strawberry variety (KYSt-4; Nohime) which earlier inhibited experimental thrombosis showed antithrombotic effects in humans, while the experimentally inactive variety (KYSt-10) as well as the relevant control (water) were ineffective.
Cue Exposure Therapy for the Treatment of Opiate Addiction: Results of a Randomized Controlled Clinical Trial
Authors: Marissen Marlies A.E. , Franken Ingmar H.A. , Blanken Peter , van den Brink Wim , Hendriks Vincent M.
Published: January 2007

Background: Persistent cue reactivity to drug-related stimuli is a well-known phenomenon among abstinent drug users and has been found to be a predictor of relapse. Cue exposure therapy (CET) aims to reduce this cue reactivity by exposing abstinent drug users to conditioned drug-related stimuli while preventing their habitual response, i.e. drug use. Methods: 127 abstinent heroin-dependent Dutch inpatients were randomized to CET (n = 65; 55 completers) and placebo psychotherapy treatment (PPT) (n = 62; 59 completers). It was examined whether CET would lead to a decrease in drug-related cue reactivity (using mixed-design ANOVA) and subsequently to lower dropout and relapse rates (using logistic regression) compared to PPT. Results: Both groups responded with a similar decrease in self-reported cue reactivity (craving, mood). The CET group did show a significant decrease in physiological reactivity (skin conductance) compared to PPT. However, dropout and relapse rates were, contrary to our expectations, significantly higher in the CET group. Conclusions: This is the first randomized controlled trial showing that CET, compared to a non-specific psychotherapy, might increase dropout and relapse rates among abstinent heroin-dependent clients in a drug-free setting. Caution is warranted when applying CET in this specific context.
Pantoprazole 40 mg Is as Effective as Esomeprazole 40 mg to Relieve Symptoms of Gastroesophageal Reflux Disease after 4 Weeks of Treatment and Superior regarding the Prevention of Symptomatic Relapse
Authors: Glatzel Dirk , Abdel-Qader Muwafeg , Gatz Gudrun , Pfaffenberger Bernd
Published: January 2007

Background/Aim: Little is known about the symptom course during and after treatment of gastroesophageal reflux disease (GERD). Here we address this question in patients with erosive GERD treated with pantoprazole or esomeprazole 40 mg once daily using the validated reflux questionnaire ReQuestTM. Methods: Of 585 patients enrolled, 561 (intention-to-treat; ITT) patients with endoscopically confirmed GERD grades A–D (Los Angeles Classification) were randomized. To assess the GERD symptomatology, the patients completed the ReQuest daily, and analysis was done prior to (7 days), during (28 days), and after treatment (7 days). The mean scores (last 3 treatment days) of the subscale ReQuestTM-GI (gastrointestinal complaints) were compared between both groups. After the end of treatment, the number of symptom episodes and the rate of relapses were calculated. Results: Noninferiority of pantoprazole versus esomeprazole during treatment was shown (mean ReQuest-GI score). During the posttreatment period, the proportion of patients experiencing a symptomatic relapse (51 vs. 61%, p = 0.0216, ITT) and the number of symptom episodes (0.56 vs. 0.74, p = 0.0095, ITT) were significantly lower on pantoprazole than on esomeprazole. Conclusions: Pantoprazole 40 mg was at least as effective as esomeprazole 40 mg for relieving GERD symptoms. During the posttreatment phase, patients on pantoprazole had a significantly lower risk to relapse and experienced significantly fewer symptom episodes.
Determination and Prediction of P-Glycoprotein and Multidrug-Resistance-Related Protein Expression in Breast Cancer with Double-Phase Technetium-99m Sestamibi Scintimammography
Authors: Kim In-Ju , Bae Young-Tae , Kim Seong-Jang , Kim Yong-Ki , Kim Dong-Soo , Lee Jung Sub
Published: January 2007

Purpose: To determine and predict P-glycoprotein (Pgp) and multidrug-resistance-related protein (MRP) expression in untreated breast cancer patients by visual and quantitative indices of double-phase 99mTc MIBI scintimammography (DSMM). Patients and Methods: Eighty-two patients with untreated breast cancer received DSMM. Pgp and MRP expression was assessed by immunohistochemical (IHC) staining of surgical specimens. Visual and quantitative analyses were compared with the results of IHC to determine and predict Pgp and MRP. Results: The early and delayed tumor to normal tissue ratio (T/N) of the Pgp-negative and MRP-negative group had significantly higher values than those of the Pgp-positive and MRP-positive group. However, there were no statistically significant differences in washout rate (WR, in %) according to the expression of Pgp and MRP. The optimal T/N ratios were ≤2.23 for early and ≤1.75 for delayed image for Pgp expression and ≤2.45 for early and ≤1.96 for delayed image for MRP expression. The optimal visual grades of DSMM for the determination of Pgp and MRP expression were ≤3. With logistic regression analysis, potent predictors of DSMM were visual assessment for Pgp expression and early T/N for MRP expression. Conclusion: In conclusion, despite a moderate sensitivity and specificity, visual and quantitative indices of DSMM could be used to determine and predict Pgp and MRP expression in untreated breast cancer. However, these findings need confirmation in a larger patient cohort to enable a better validation of Pgp and MRP expression to determine optimal early and delayed T/Ns and to investigate predictors of Pgp and MRP expression in breast cancer.
Antitumor Effects of Two Less-Calcemic Vitamin D Analogs (Paricalcitol and QW-1624F2-2) in Squamous Cell Carcinoma Cells
Authors: Alagbala Adebusola A. , Johnson Candace S. , Trump Donald L. , Posner Gary H. , Foster Barbara A.
Published: January 2007

The active metabolite of vitamin D3 (1α,25-dihydroxyvitamin D3, calcitriol) has potent antitumor activities in vitro and in vivo in multiple cancers. Concerns about induction of hypercalcemia by calcitriol and the desire for more potent agents have prompted development of less-calcemic vitamin D analogs. These studies demonstrate that two vitamin D analogs, 19-nor-1α,25-dihydroxyvitamin D2 (paricalcitol) and 1α-hydroxymethyl-16-ene-24,24-difluoro-25-hydroxy-26,27-bis-homovitamin D3 (QW-1624F2-2, QW), have anticancer effects in the calcitriol-responsive squamous cell carcinoma (SCC) cell line. Paricalcitol (GI50 = 0.7 nM) and QW (GI50 = 0.001 nM) inhibited SCC cell growth; however, QW was more potent. Paricalcitol (10 nM) and QW (10 nM) induced G₀/G1 cell cycle arrest and inhibited DNA synthesis by ∼95%. The vitamin D analogs modulated cell cycle regulators, including decreasing mRNA and protein levels of p21Waf1/Cip1 (p21) and cyclin-dependent kinase 2 (cdk2), and increasing p27Kip1 (p27) protein expression. Vitamin D analogs induced apoptosis, caspase-3 cleavage and increased expression of pro-apoptotic MEKK-1. Phosphorylation of Akt, MEK and ERK1/2 that promote cell growth and survival were inhibited by vitamin D analogs. The anticancer effects of paricalcitol and QW are comparable to the effect of calcitriol. These less-calcemic vitamin D analogs are as effective as calcitriol in vitro and are promising for prevention and treatment of cancer and other diseases.
Overexpression of the Orotate Phosphoribosyl-Transferase Gene Enhances the Effect of 5-Fluorouracil on Gastric Cancer Cell Lines
Authors: Taomoto Jyunnya , Yoshida Kazuhiro , Wada Yoshiyuki , Tanabe Kazuaki , Konishi Kazuo , Tahara Hidetoshi , Fukushima Masakazu
Published: January 2007

Objective: Orotate phosphoribosyl-transferase (OPRT) is the initial enzyme of the 5-fluorouracil (5-FU) metabolic pathway, converting 5-FU into 5-fluorouridinemonophosphate, which is the most important mechanism of 5-FU activation. We therefore investigated whether overexpression of the OPRT gene enhances sensitivity to 5-FU. Methods: An expression vector of the OPRT gene (pTARGET-OPRT) was transfected into two gastric cancer cell lines, TMK-1 and MKN-45, with low baseline expression levels of OPRT. The sensitivity to and anti-tumor activity of 5-FU were then investigated in vitro and in vivo in these two transfected clones (TMK-OPRT and MKN-OPRT). Results: Although cell growth was unaltered compared to parent cells, overexpression of the OPRT gene was confirmed by Western blotting in both the TMK-OPRT and MKN-OPRT cells. OPRT enzyme activity increased 38-fold in TMK-OPRT cells and 8.0 fold in MKN-OPRT cells compared to their parent cells. Interestingly, although the sensitivity to Adriamycin, cis-platinum, mitomycin C and paclitaxel was unaltered in the transfected clones, the sensitivity to 5-FU was increased 14.2- and 6.0-fold in TMK-OPRT and MKN-OPRT cells, respectively, compared to their parent cells. Moreover, enhanced sensitivity was also confirmed in the in vivo study. Conclusion: The results indicate that overexpression of the OPRT gene plays an important role in the antiproliferative effect of 5-FU and might therefore be a predictive factor of response to 5-FU in gastric cancer patients.
Extracorporeal Photodynamic Image Detection of Mouse Osteosarcoma in Soft Tissues Utilizing Fluorovisualization Effect of Acridine Orange
Authors: Satonaka Haruhiko , Kusuzaki Katsuyuki , Matsubara Takao , Shintani Ken , Wakabayashi Toru , Matsumine Akihiko , Uchida Atsumasa
Published: January 2007

Various imaging methods have been employed for the extracorporeal detection of malignant tumors in the human body, such as scintigraphy and PET; however, none is sufficiently accurate and all are also very expensive. To resolve these issues, we attempted to develop a new imaging technique of photodynamic diagnosis (PDD) with acridine orange (AO). AO has the ability to rapidly and specifically accumulate in malignant tumors and emit brilliant green fluorescence after blue light excitation. In this study, we investigated the feasibility of PDD utilizing the fluorovisualization effect of AO, for the extracorporeal detection of mouse osteosarcoma inoculated into the soft tissues. At 2 h after intravenous administration of 0.1, 0.2, 0.5, 1.0, 2.0 and 5.0 mg/kg AO, the tumor and the surrounding normal tissues were illuminated by blue light. The visual fluorescence contrast and ratio (X) of the difference in fluorescence intensity between the tumor and the surrounding normal tissues were evaluated using a high-resolution digital camera equipped with an absorption filter. In addition, the fluorescence contrast was also detected sequentially at 0.5, 1, 2, 3, 6 and 12 h after intravenous administration of AO at 1.0 mg/kg. The results revealed that the optimal condition for clear detection of the tumor was evaluation 2 h after intravenous injection of AO at 0.1 mg/kg, because it provided the best visual contrast on the digital images, and the fluorescence intensity as well as the value of X were higher as compared to the values under other conditions of dose and timing. Based on the results of an acute toxicity study of AO, the estimated LD50 of this substance following intravenous administration was 27.30 mg/kg. In conclusion, we believe that PDD using AO administered intravenously may be feasible for the detection of human musculoskeletal sarcomas in the soft tissues at extremities, and this technique might be a less invasive, less expensive, quicker and more accurate imaging modality than other previously reported imaging methods for this purpose.
Cysteine-674 of the Sarco/Endoplasmic Reticulum Calcium ATPase Is Required for the Inhibition of Cell Migration by Nitric Oxide
Authors: Ying Jia , Tong XiaoYong , Pimentel David R. , Weisbrod Robert M. , Trucillo Mario P. , Adachi Takeshi , Cohen Richard A.
Published: January 2007

Objectives—

Nitric oxide inhibits smooth muscle cell migration after arterial injury, but the detailed mechanism is not fully understood. The sarco/endoplasmic reticulum calcium ATPase (SERCA) lowers cell Ca
2+
by increasing intracellular Ca
2+
uptake and inhibiting extracellular Ca
2+
influx. Our previous studies showed that NO causes cyclic GMP-independent arterial relaxation by increasing SERCA activity by inducing reversible
S
-glutathiolation at cysteine-674. Because Ca
2+
is an important second messenger for cell migration, we hypothesized that NO also inhibits cell migration through redox regulation of SERCA activity via cysteine-674.

Methods and Results—

To test our hypothesis, overexpression of either wild type (WT) or mutant SERCA in which cysteine-674 was mutated to serine was accomplished by stable transfection of HEK 293 or adenoviral expression in rat aortic smooth muscle cells (VSMCs). In the cell models expressing mutant SERCA, biotinylated-iodoacetamide (BIAM) and biotinylated-glutathione labeling of SERCA was decreased, and NO failed to increase SERCA activity or decrease Ca
2+
influx, thus validating that the expression of mutant SERCA prevents its redox-dependent activation. In the absence of NO, fetal bovine serum stimulated migration of both cell types expressing WT or C674S SERCA at similar rates. The NO donor S-nitrosopenicillamine inhibited migration of cells with WT SERCA, but had no effect on the migration of either HEK cells or VSMCs with C674S SERCA. The same result was obtained in VSMCs in which endogenous NO was produced by iNOS induced by interleukin (IL)-1β. Blocking cyclic GMP did not prevent the inhibition of migration by NO.

Conclusions—

In cells overexpressing SERCA, the cyclic GMP-independent, redox regulation of SERCA cysteine-674 is required for the inhibition of cell migration by both exogenous and endogenously generated NO.
Mineralocorticoid Receptor Activation Promotes Vascular Cell Calcification
Authors: Jaffe Iris Z. , Tintut Yin , Newfell Brenna G. , Demer Linda L. , Mendelsohn Michael E.
Published: January 2007

Objective—

Clinical studies demonstrate that mineralocorticoid receptor (MR) antagonism improves outcomes in cardiovascular patients and that vascular calcification correlates with adverse cardiac events. We have recently demonstrated that human vascular smooth muscle cells (VSMCs) express functional MRs that, in response to aldosterone, modulate expression of osteogenic genes including alkaline phosphatase (ALP) and bone morphogenetic protein-2 (BMP2). This study examines the effects of MR activation by aldosterone on the process of in vitro vascular calcification.

Methods and Results—

Using immunoblotting and adenoviral promoter-reporter assays, we demonstrated that calcifying vascular cells (CVCs), an in vitro model of vascular calcification, express MRs that mediate both aldosterone- and cortisol-stimulated gene transcription. In this model, aldosterone stimulated ALP activity, an early marker of osteoblastic differentiation, as well as mineralization. Aldosterone antagonism with spironolactone abolished both effects implicating CVC MRs in the mechanism of aldosterone-stimulated vascular calcification. Inhibition of BMP2 signaling by overexpression of dominant negative BMP2 receptor did not attenuate aldosterone-induced osteoblastic differentiation.

Conclusions—

Aldosterone activation of MR promotes osteoblastic differentiation and mineralization of VSMCs independent of BMP2 signaling. These data provide a mechanistic link between hormone-mediated VSMC MR activation and vascular calcification, two processes associated with increased risk of cardiovascular ischemic events in humans.
Age-Dependent Impairment of Endothelial Progenitor Cells Is Corrected by Growth Hormone Mediated Increase of Insulin-Like Growth Factor-1
Authors: Thum Thomas , Hoeber Sarah , Froese Sabrina , Klink Ivonne , Stichtenoth Dirk O. , Galuppo Paolo , Jakob Marten , Tsikas Dimitrios , Anker Stefan D. , Poole-Wilson Philip A. , Borlak Jürgen , Ertl Georg , Bauersachs Johann
Published: January 2007

Aging is associated with an increased risk for atherosclerosis. A possible cause is low numbers and dysfunction of endothelial progenitor cells (EPC) which insufficiently repair damaged vascular walls. We hypothesized that decreased levels of insulin-like growth factor-1 (IGF-1) during age contribute to dysfunctional EPC. We measured the effect of growth hormone (GH), which increases endogenous IGF-1 levels, on EPC in mice and human subjects. We compared EPC number and function in healthy middle-aged male volunteers (57.4±1.4 years) before and after a 10 day treatment with recombinant GH (0.4 mg/d) with that of younger and elderly male subjects (27.5±0.9 and 74.1±0.9 years). Middle-aged and elderly subjects had lower circulating CD133
+
/VEGFR-2
+
EPC with impaired function and increased senescence. GH treatment in middle-aged subjects elevated IGF-1 levels (126.0±7.2 ng/mL versus 241.1±13.8 ng/mL;
P
<0.0001), increased circulating EPC with improved colony forming and migratory capacity, enhanced incorporation into tube-like structures, and augmented endothelial nitric oxide synthase expression in EPC comparable to that of the younger group. EPC senescence was attenuated, whereas telomerase activity was increased after GH treatment. Treatment of aged mice with GH (7 days) or IGF-1 increased IGF-1 and EPC levels and improved EPC function, whereas a two day GH treatment did not alter IGF-1 or EPC levels. Ex vivo treatment of EPC from elderly individuals with IGF-1 improved function and attenuated cellular senescence. IGF-1 stimulated EPC differentiation, migratory capacity and the ability to incorporate into forming vascular networks in vitro via the IGF-1 receptor. IGF-1 increased telomerase activity, endothelial nitric oxide synthase expression, phosphorylation and activity in EPC in a phosphoinositide-3-kinase/Akt dependent manner. Small interference RNA-mediated knockdown of endothelial nitric oxide synthase in EPC abolished the IGF-1 effects. Growth hormone-mediated increase in IGF-1 reverses age-related EPC dysfunction and may be a novel therapeutic strategy against vascular disorders with impairment of EPC.
Ca
2+
/Calmodulin Kinase II-Dependent Phosphorylation of Ryanodine Receptors Suppresses Ca
2+
Sparks and Ca
2+
Waves in Cardiac Myocytes
Authors: Yang Dongmei , Zhu Wei-Zhong , Xiao Bailong , Brochet Didier X.P. , Chen S.R. Wayne , Lakatta Edward G. , Xiao Rui-Ping , Cheng Heping
Published: January 2007

The multifunctional Ca
2+
/calmodulin-dependent protein kinase II δ
C
(CaMKIIδ
C
) is found in the macromolecular complex of type 2 ryanodine receptor (RyR2) Ca
2+
release channels in the heart. However, the functional role of CaMKII-dependent phosphorylation of RyR2 is highly controversial. To address this issue, we expressed wild-type, constitutively active, or dominant-negative CaMKIIδ
C
via adenoviral gene transfer in cultured adult rat ventricular myocytes. CaMKII-mediated phosphorylation of RyR2 was reduced, enhanced, or unaltered by dominant-negative, constitutively active, or wild-type CaMKIIδ
C
expression, whereas phosphorylation of phospholamban at Thr17, an endogenous indicator of CaMKII activity, was at 73%, 161%, or 115% of the control group expressing β-galactosidase (β-gal), respectively. In parallel with the phospholamban phosphorylation, the decay kinetics of global Ca
2+
transients was slowed, accelerated, or unchanged, whereas spontaneous Ca
2+
spark activity was hyperactive, depressed, or unchanged in dominant-negative, constitutively active, or wild-type CaMKIIδ
C
groups, respectively. When challenged by high extracellular Ca
2+
, both wild-type and constitutively active CaMKIIδ
C
protected the cells from store overload-induced Ca
2+
release, manifested by a ≈60% suppression of Ca
2+
waves (at 2 to 20 mmol/L extracellular Ca
2+
) in spite of an elevated sarcoplasmic reticulum Ca
2+
content, whereas dominant-negative CaMKIIδ
C
promoted Ca
2+
wave production (at 20 mmol/L Ca
2+
) with significantly depleted sarcoplasmic reticulum Ca
2+
. Taken together, our data support the notion that CaMKIIδ
C
negatively regulates RyR2 activity and spontaneous sarcoplasmic reticulum Ca
2+
release, thereby affording a negative feedback that stabilizes local and global Ca
2+
-induced Ca
2+
release in the heart.
p38-MAPK Induced Dephosphorylation of α-Tropomyosin Is Associated With Depression of Myocardial Sarcomeric Tension and ATPase Activity
Authors: Vahebi Susan , Ota Asuka , Li Manxiang , Warren Chad M. , de Tombe Pieter P. , Wang Yibin , Solaro R. John
Published: January 2007

Our objective in work presented here was to understand the mechanisms by which activated p38α MAPK depresses myocardial contractility. To test the hypothesis that activation of p38 MAPK directly influences sarcomeric function, we used transgenic mouse models with hearts in which p38 MAPK was constitutively turned on by an upstream activator (MKK6bE). These hearts demonstrated a significant depression in ejection fraction after induction of the transgene. We also studied hearts of mice expressing a dominant negative p38α MAPK. Simultaneous determination of tension and ATPase activity of detergent-skinned fiber bundles from left ventricular papillary muscle demonstrated a significant inhibition of both maximum tension and ATPase activity in the transgenic-MKK6bE hearts. Fibers from hearts expressing dominant negative p38α MAPK demonstrated no significant change in tension or ATPase activity. There were no significant changes in phosphorylation level of troponin-T3 and troponin-T4, or myosin light chain 2. However, compared with controls, there was a significant depression in levels of phosphorylation of α-tropomyosin and troponin I in fiber bundles from transgenic-MKK6bE hearts, but not from dominant negative p38α MAPK hearts. Our experiments also showed that p38α MAPK colocalizes with α-actinin at the Z-disc and complexes with protein phosphatases (PP2α, PP2β). These data are the first to indicate that chronic activation of p38α MAPK directly depresses sarcomeric function in association with decreased phosphorylation of α-tropomyosin.
Contrasting Gene Expression Profiles in Two Canine Models of Atrial Fibrillation
Authors: Cardin Sophie , Libby Eric , Pelletier Patricia , Le Bouter Sabrina , Shiroshita-Takeshita Akiko , Le Meur Nolwenn , Léger Jean , Demolombe Sophie , Ponton André , Glass Leon , Nattel Stanley
Published: January 2007

Gene-expression changes in atrial fibrillation patients reflect both underlying heart-disease substrates and changes because of atrial fibrillation-induced atrial-tachycardia remodeling. These are difficult to separate in clinical investigations. This study assessed time-dependent mRNA expression-changes in canine models of atrial-tachycardia remodeling and congestive heart failure. Five experimental groups (5 dogs/group) were submitted to atrial (ATP, 400 bpm ×24 hours, 1 or 6 weeks) or ventricular (VTP, 240 bpm ×24 hours or 2 weeks) tachypacing. The expression of ≈21,700 transcripts was analyzed by microarray in isolated left-atrial cardiomyocytes and (for 18 genes) by real-time RT-PCR. Protein-expression changes were assessed by Western blot. In VTP, a large number of significant mRNA-expression changes occurred after both 24 hours (2209) and 2 weeks (2720). In ATP, fewer changes occurred at 24 hours (242) and fewer still (87) at 1 week, with no statistically-significant alterations at 6 weeks. Expression changes in VTP varied over time in complex ways. Extracellular matrix-related transcripts were strongly upregulated by VTP consistent with its pathophysiology, with 8 collagen-genes upregulated >10-fold, fibrillin-1 8-fold and MMP2 4.5-fold at 2 weeks (time of fibrosis) but unchanged at 24 hours. Other extracellular matrix genes (eg, fibronectin, lysine oxidase-like 2) increased at both time-points (≈10, ≈5-fold respectively). In ATP, mRNA-changes almost exclusively represented downregulation and were quantitatively smaller. This study shows that VTP-induced congestive heart failure and ATP produce qualitatively different temporally-evolving patterns of gene-expression change, and that specific transcriptomal responses associated with atrial fibrillation versus underlying heart disease substrates must be considered in assessing gene-expression changes in man.
Pharmacologically Preconditioned Skeletal Myoblasts Are Resistant to Oxidative Stress and Promote Angiomyogenesis via Release of Paracrine Factors in the Infarcted Heart
Authors: Niagara Muhammad Idris , Haider Husnain Kh. , Jiang Shujia , Ashraf Muhammad
Published: January 2007

Strategies to enhance skeletal myoblast (SkM) survival after transplantation in the ischemic heart have achieved little success. We posit that preconditioned (PC) SkMs show improved survival and promote repair of the infarcted myocardium via paracrine signaling after transplantation. SkMs from male Fischer-344 rats (rSkMs) were PC for 30 minutes with 200 μmol/L diazoxide. Treatment of PC rSkMs with 100 μmol/L H
2
O
2
for 2 hours resulted in significantly reduced cell injury, as shown by lactate dehydrogenase–release assay, and prevented apoptosis, as demonstrated by cytochrome
c
translocation, TUNEL, annexin V staining, and preservation of mitochondrial membrane potential. PC rSkMs expressed elevated phospho-Akt (1.85-fold), basic fibroblast growth factor (1.44-fold), hepatocyte growth factor (2.26-fold), and cyclooxygenase-2 (1.33-fold) as compared with non-PC rSkMs. For in vivo studies, female Fischer-344 rats after permanent coronary artery ligation were grouped (n=12/group) to receive 80 μL of basal medium without rSkMs (group 1) or containing 1.5×10
6
non-PC (group 2) or PC (group 3) rSkMs. Real-time PCR for
sry
gene 4 days after transplantation (n=4/group) showed 1.93-fold higher survival of rSkMs in group 3 as compared with group 2. Four weeks later, echocardiography revealed improved indices of left ventricular function, including ejection fraction and fractional shortening in group 3 (
P
<0.02) as compared with groups 1 and 2. Blood vessel count per surface area (at ×400 magnification) was highest in scar and periscar areas in group 3 as compared with the other groups (
P
<0.05). We conclude that activation of signaling pathways of preconditioning in SkMs promoted their survival by release of paracrine factors to promote angiomyogenesis in the infarcted heart. Transplantation of PC SkMs for heart cell therapy is an innovative approach in the clinical perspective.
Mechanisms of Abnormal Calcium Homeostasis in Mutations Responsible for Catecholaminergic Polymorphic Ventricular Tachycardia
Authors: Iyer Vivek , Hajjar Roger J. , Armoundas Antonis A.
Published: January 2007

Catecholaminergic polymorphic ventricular tachycardia is a heritable arrhythmia unmasked by exertion or stress and is characterized by triggered activity and sudden cardiac death. In this study, we simulated mutations in 2 genes linked to catecholaminergic polymorphic ventricular tachycardia, the first located in calsequestrin (CSQN2) and the second in the ryanodine receptor (RyR2). The aim of the study was to investigate the mechanistic basis for spontaneous Ca
2+
release events that lead to delayed afterdepolarizations in affected patients. Sarcoplasmic reticulum (SR) luminal Ca
2+
sensing was incorporated into a model of the human ventricular myocyte, and CSQN2 mutations were modeled by simulating disrupted RyR2 luminal Ca
2+
sensing. In voltage-clamp mode, the mutant CSQN2 model recapitulated the smaller calcium transients, smaller time to peak calcium transient, and accelerated recovery from inactivation seen in experiments. In current clamp mode, in the presence of β stimulation, we observed delayed afterdepolarizations, suggesting that accelerated recovery of RyR2 induced by impaired luminal Ca
2+
sensing underlies the triggered activity observed in mutant CSQN2-expressing myocytes. In current-clamp mode, in a model of mutant RyR2 that is characterized by reduced FKBP12.6 binding to the RyR2 on β stimulation, the impaired coupled gating characteristic of these mutations was modeled by reducing cooperativity of RyR2 activation. In current-clamp mode, the mutant RyR2 model exhibited increased diastolic RyR2 open probability that resulted in formation of delayed afterdepolarizations. In conclusion, these minimal order models of mutant CSQN2 and RyR2 provide plausible mechanisms by which defects in RyR2 gating may lead to the cellular triggers for arrhythmia, with implications for the development of targeted therapy.
Dynamics of Model Error: The Role of the Boundary Conditions
Authors: Nicolis C.
Published: January 2007

The different modes of the early stages of the response of a forecasting model to a small error in the boundary conditions are analyzed. A general formulation of the problem based on the use of Green’s functions is developed and implemented on systems in which the operators acting on the spatial coordinates of the fields involved are diffusion-like and advection-like. It is shown that the generic behavior displays a nonanalytic structure not reducible to a power of time for short times.
Angular Momentum Conservation and Gravity Wave Drag Parameterization: Implications for Climate Models
Authors: Shaw Tiffany A. , Shepherd Theodore G.
Published: January 2007

The robustness of the parameterized gravity wave response to an imposed radiative perturbation in the middle atmosphere is examined. When momentum is conserved and for reasonable gravity wave drag parameters, the response to a polar cooling induces polar downwelling above the region of the imposed cooling, with consequent adiabatic warming. This response is robust to changes in the gravity wave source spectrum, background flow, gravity wave breaking criterion, and model lid height. When momentum is not conserved, either in the formulation or in the implementation of the gravity wave drag parameterization, the response becomes sensitive to the above-mentioned factors—in particular to the model lid height. The spurious response resulting from nonconservation is found to be nonnegligible in terms of the total gravity wave drag–induced downwelling.