bluuebunny/crossref_metadata_2025 huggingface dataset:
Aminopeptidase N reduces basolateral Na+-K+-ATPase in proximal tubule cells
Authors: Kotlo Kumar , Shukla Sagar , Tawar Urmila , Skidgel Randal A. , Danziger Robert S.
Published: July 2007

Aminopeptidase N/CD13 (Anpep) is a membrane-bound protein that catalyzes the formation of natriuretic hexapeptide angiotensin IV (ANG IV) from ANG III. We previously reported that Anpep is more highly expressed in the kidneys of Dahl salt-resistant (SR/Jr) than salt-sensitive (SS/Jr) rats, Anpep maps to a quantitative trait locus for hypertension, and that the Dahl SR/Jr rat contains a functional polymorphism of the gene. This suggests that renal Anpep may be linked to salt sensitivity; however, its effect on renal Na handling has not been determined. Here, we examined regulation of basolateral Na+-K+-ATPase, a preeminent basolateral Na+transporter in proximal tubule cells, by Anpep in LLC-PK1 cells. Treatment of the cells with Anpep siRNA increased total cellular Na+-K+-ATPase activity and basolateral Na+-K+-ATPase abundance by approximately twofold. Conversely, Anpep overexpression reduced Na+-K+-ATPase activity and basolateral abundance by ∼50%. Similar effects were observed after treatment with ANG IV (10 nM, ×30 min and 12 h). ANG IV receptor (AGTRIV) knockdown via specific siRNA relieved the decreases in basolateral Na+-K+-ATPase levels and activity induced by Anpep overexpression. In sum, these results demonstrate that Anpep reduces basolateral Na+-K+-ATPase levels via ANG IV/AGTRIV signaling. This novel pathway may be important in renal adaptation to high salt.
Renal inflammation is modulated by potassium in chronic kidney disease: possible role of Smad7
Authors: Wang Wansheng , Soltero Liliana , Zhang Ping , Huang Xiao R. , Lan Hui Y. , Adrogue Horacio J.
Published: July 2007

High-potassium diets have been shown to be beneficial in cardiovascular disease partly because of a blood pressure-lowering effect. The effect of potassium on inflammation has not been studied. We investigated the influence of potassium supplementation on the degree of renal inflammation and the intracellular signaling mechanisms that could mediate inflammation in chronic kidney disease (CKD). CKD was created in male Sprague-Dawley rats by subtotal nephrectomy. Two groups of CKD rats were pair fed with diets containing 2.1% potassium (potassium-supplemented diet) or 0.4% potassium (basal diet). Body weight, blood pressure, and blood and urine electrolytes were measured biweekly. The animals were euthanized at week 8, and the remnant kidneys were analyzed by histology, immunohistochemistry, Western blotting, and real-time quantitative PCR. In the CKD pair-fed groups, blood potassium concentration did not differ significantly, but blood pressure was lower in the potassium-supplemented group. Compared with the basal diet, potassium supplementation decreased renal tubulointerstitial injury and suppressed renal inflammation as evidenced by decreased macrophage infiltration, lower expression of inflammatory cytokines, and decreased NF-κB activation. These renoprotective effects were associated with downregulation of renal transforming growth facto-β, upregulation of renal Smad7, and lower blood pressure. Our results show that potassium supplementation can reduce renal inflammation and hence, could modulate the progression of kidney injury in CKD.
Basic fibroblast growth factor modulates proliferation and collagen expression in urinary bladder smooth muscle cells
Authors: Imamura Masaaki , Kanematsu Akihiro , Yamamoto Shingo , Kimura Yu , Kanatani Isao , Ito Noriyuki , Tabata Yasuhiko , Ogawa Osamu
Published: July 2007

Bladder hypertrophy is a general consequence of bladder outlet obstruction (BOO) and a typical phenomenon observed in clinical urologic diseases such as benign prostatic hyperplasia and neurogenic bladder. It is characterized by smooth muscle hyperplasia, altered extracellular matrix composition, and increased contractile function. Various growth factors are likely involved in hypertrophic pathophysiology, but their functions remain unknown. In this report, the role of basic fibroblast growth factor (bFGF) was investigated using a rat bladder smooth muscle cell (BSMC) culture system and an original animal model, in which bFGF was released from a gelatin hydrogel directly onto rat bladders. bFGF treatment promoted BSMC proliferation both in vitro and in vivo. In vitro, bFGF downregulated the expression of type I collagen, but upregulated type III collagen. ERK1/2, but not p38MAPK, was activated by bFGF, whereas inhibition of ERK1/2 by PD98059 reversed bFGF-induced BSMC proliferation, type I collagen downregulation, and type III collagen upregulation. In the in vivo release model, bFGF upregulated type III collagen and increased the contractile force of treated bladders. In parallel with these findings, hypertrophied rat bladders created by urethral constriction showed increased urothelial bFGF expression, BSMC proliferation, and increased type III collagen expression compared with sham-operated rats. These data suggest that bFGF from the urothelium could act as a paracrine signal that stimulates the proliferation and matrix production of BSMC, thereby contributing to the hypertrophic remodeling of the smooth muscle layer.
Inflammatory cytokines disrupt LDL-receptor feedback regulation and cause statin resistance: a comparative study in human hepatic cells and mesangial cells
Authors: Chen Yaxi , Ruan Xiong Z. , Li Qiu , Huang Ailong , Moorhead John F. , Powis Stephen H. , Varghese Zac
Published: July 2007

LDL receptor (LDLr) is widely expressed in both liver and peripheral tissue. We aimed to clarify tissue-specific regulation of LDLr in hepatic cell line (HepG2) cells and human kidney mesangial cells (HMCs) under physiological and inflammatory conditions. We have demonstrated that the concentration of LDL required for 50% inhibition of LDLr mRNA expression (IC50) in HepG2 was 75 μg/ml, but only 30 μg/ml in HMCs. The concentration of mevastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, which achieved 200% upregulation of LDLr (UC200) in HepG2 cells, was 0.7 μM, which is much lower than 2.8 μM in HMCs. Inflammatory stress increased IC50to 80 and 75 μg/ml of LDL, UC200to 2.8 μM, and 4.2 μM of mevastatin in HepG2 and HMCs. There was obvious sterol-regulatory element binding protein cleavage-activating protein accumulation in the Golgi in HepG2 cells, but not in HMCs in the presence of high concentration of LDL. IL-1β further increased sterol-regulatory element binding protein cleavage-activating protein accumulation in HepG2 and HMCs in the presence of high concentration of LDL. These results indicate that LDLr in HepG2 cells have a relative resistant phenotype for downregulation, while LDLr in HMCs is very sensitive for downregulation. Inflammatory cytokine disrupts LDLr negative feedback regulation induced by intracellular cholesterol in both cell types, to a greater degree in HMCs, which could be one reason why HMCs are more prone to become foam cells under inflammatory stress. Inflammation also causes statin resistance; therefore, a high concentration of statin may be required to achieve the same biological effect.
Exercise decreases oxidative stress and inflammation and restores renal dopamine D1 receptor function in old rats
Authors: Asghar Mohammad , George Liza , Lokhandwala Mustafa F.
Published: July 2007

Recently, we reported that oxidative stress decreases D1 receptor numbers and G protein activation in renal proximal tubules (RPT), resulting in diminished natriuretic response to dopamine in old rats. We tested the hypothesis that exercise in old rats will decrease oxidative stress and restore natriuretic response to D1 receptor agonist, SKF 38393. Old (23 mo) rats were subjected to rest (sedentary) or to treadmill exercise followed by measurement of oxidative stress [malondialdehyde (MDA)], inflammation [C-reactive protein (CRP)], anti-inflammation (IL-10), antioxidant enzyme [superoxide dismutase (SOD)], and natriuretic response to SKF 38393. We found that MDA levels decreased and total SOD activity and Cu/ZnSOD protein increased in RPT of exercised rats. Exercise increased the nuclear levels of Nrf2 transcription factor (which binds to anti-oxidant response elements) in RPT. The levels of CRP decreased and IL-10 increased in RPT of exercised rats. Furthermore, exercise increased the basal bindings of [3H]SCH 23390 and [35S]GTPγS (indexes of D1 receptor number and G protein activation, respectively) together with D1 receptor and Gαq proteins in RPT membranes. Moreover, SKF 38393 increased sodium excretion in exercised rats. Also, exercise decreased the levels of proteins in the urine of old rats. These results demonstrate that exercise decreases oxidative stress, inflammation, and proteinuria and increases anti-oxidant defense and D1 receptor function in old rats. Therefore, exercise may prove beneficial in preventing age-associated increases in oxidative stress, inflammation, and preserving kidney function, in general, and renal D1 receptor responsiveness, in particular.
Serotonin Concentrations in the Lumbosacral Spinal Cord of the Adult Rat Following Microinjection or Dorsal Surface Application
Authors: Brumley Michele R. , Hentall Ian D. , Pinzon Alberto , Kadam Brijesh H. , Blythe Anthony , Sanchez Francisco J. , Taberner Annette M. , Noga Brian R.
Published: July 2007

Application of neuroactive substances, including monoamines, is common in studies examining the spinal mechanisms of sensation and behavior. However, affected regions and time courses of transmitter activity are uncertain. We measured the spatial and temporal distribution of serotonin [5-hydroxytryptamine (5-HT)] in the lumbosacral spinal cord of halothane-anesthetized adult rats, following its intraspinal microinjection or surface application. Carbon fiber microelectrodes (CFMEs) were positioned at various locations in the spinal cord and oxidation currents corresponding to extracellular 5-HT were measured by fast cyclic voltammetry. Intraspinal microinjection of 5-HT (100 μM, 1–3 μl) produced responses that were most pronounced at CFMEs positioned ≤800 μm from the drug micropipette: 5-HT concentration was significantly higher (1.43 vs. <0.28% of initial concentration) and response latency was shorter (67.1 vs. 598.2 s) compared with more distantly positioned CFMEs. Treatment with the selective 5-HT reuptake inhibitor clomipramine only slightly affected the spread of microinjected 5-HT. Surface application over several segments led to a transient rise in concentration that was usually apparent within 30 s and was dramatically attenuated with increasing depth: 0.25% of initial concentration (1 mM) within 400 μm of the dorsal surface and <0.001% between 1,170 and 2,000 μm. This initial response to superfusion was sometimes followed by a gradual increase to a new concentration plateau. In sum, compared with bath application, microinjection can deliver about tenfold higher transmitter concentrations, but to much more restricted areas of the spinal cord.
Generation of Slow Network Oscillations in the Developing Rat Hippocampus After Blockade of Glutamate Uptake
Authors: Cattani Adriano Augusto , Bonfardin Valérie Delphine , Represa Alfonso , Ben-Ari Yehezkel , Aniksztejn Laurent
Published: July 2007

Cell-surface glutamate transporters are essential for the proper function of early cortical networks because their dysfunction induces seizures in the newborn rat in vivo. We have now analyzed the consequences of their inhibition by dl-TBOA on the activity of the developing CA1 rat hippocampal network in vitro. dl-TBOA generated a pattern of recurrent depolarization with an onset and decay of several seconds' duration in interneurons and pyramidal cells. These slow network oscillations (SNOs) were mostly mediated by γ-aminobutyric acid (GABA) in pyramidal cells and by GABA and N-methyl-d-aspartate (NMDA) receptors in interneurons. However, in both cell types SNOs were blocked by NMDA receptor antagonists, suggesting that their generation requires a glutamatergic drive. Moreover, in interneurons, SNOs were still generated after the blockade of NMDA-mediated synaptic currents with MK-801, suggesting that SNOs are expressed by the activation of extrasynaptic NMDA receptors. Long-lasting bath application of glutamate or NMDA failed to induce SNOs, indicating that they are generated by periodic but not sustained activation of NMDA receptors. In addition, SNOs were observed in interneurons recorded in slices with or without the strata pyramidale and oriens, suggesting that the glutamatergic drive may originate from the radiatum and pyramidale strata. We propose that in the absence of an efficient transport of glutamate, the transmitter diffuses in the extracellular space to activate extrasynaptic NMDA receptors preferentially present on interneurons that in turn activate other interneurons and pyramidal cells. This periodic neuronal coactivation may contribute to the generation of seizures when glutamate transport dysfunction is present.
Serotonin Induces Memory-Like, Rapamycin-Sensitive Hyperexcitability in Sensory Axons ofAplysiaThat Contributes to Injury Responses
Authors: Weragoda Ramal M. S. , Walters Edgar T.
Published: July 2007

The induction of long-term facilitation (LTF) of synapses of Aplysia sensory neurons (SNs) by serotonin (5-HT) has provided an important mechanistic model of memory, but little is known about other long-term effects of 5-HT on sensory properties. Here we show that crushing peripheral nerves results in long-term hyperexcitability (LTH) of the axons of these nociceptive SNs that requires 5-HT activity in the injured nerve. Serotonin application to a nerve segment induces local axonal (but not somal) LTH that is inhibited by 5-HT–receptor antagonists. Blockade of crush-induced axonal LTH by an antagonist, methiothepin, provides evidence for mediation of this injury response by 5-HT. This is the first demonstration in any axon of neuromodulator-induced LTH, a phenomenon potentially important for long-lasting pain. Methiothepin does not reduce axonal LTH induced by local depolarization, so 5-HT is not required for all forms of axonal LTH. Serotonin-induced axonal LTH is expressed as reduced spike threshold and increased repetitive firing, whereas depolarization-induced LTH involves only reduced threshold. Like crush- and depolarization-induced LTH, 5-HT–induced LTH is blocked by inhibiting protein synthesis. Blockade by rapamycin, which also blocks synaptic LTF, is interesting because the eukaryotic protein kinase that is the target of rapamycin (TOR) has a conserved role in promoting growth by stimulating translation of proteins required for translation. Rapamycin sensitivity suggests that localized increases in translation of proteins that promote axonal conduction and excitability at sites of nerve injury may be regulated by the same signals that increase translation of proteins that promote neuronal growth.
Insulin-like Growth Factor Receptor as a Therapeutic Target in Head and Neck Cancer
Authors: Barnes Christopher J. , Ohshiro Kazufumi , Rayala Suresh K. , El-Naggar Adel K. , Kumar Rakesh
Published: July 2007

Purpose: Insulin-like growth factor type I receptor (IGF-IR) plays critical roles in epithelial cancer cell development, proliferation, motility, and survival, and new therapeutic agents targeting IGF-IR are in development. Another receptor tyrosine kinase, the epidermal growth factor receptor (EGFR), is an established therapeutic target in head and neck cancer and IGF-IR/EGFR heterodimerization has been reported in other epithelial cancers. The present study was undertaken to determine the effects of anti–IGF-IR therapeutic targeting on cell signaling and cancer cell phenotypes in squamous cell carcinomas of the head and neck (SCCHN).

Experimental Design: The therapeutic efficacy of the human anti–IGF-IR antibody IMC-A12 alone and in combination with the EGFR blocking antibody cetuximab (C225) was tested in SCCHN cell lines and in tumor xenografts.

Results: IGF-IR was overexpressed in human head and neck cancer cell lines and tumors. Pretreatment of serum-starved 183A or TU159 SCCHN cell lines with A12 (10 μg/mL) blocked IGF-stimulated activation of IGF-IR, insulin receptor substrate (IRS)-1 and IRS-2, mitogen-activated protein kinase, and phosphatidylinositol 3-kinase. A12 induced G0-G1 cell cycle arrest and blocked cell growth, motility, and anchorage-independent growth. Stimulation of head and neck cancer cells with either IGF or EGF resulted in IGF-IR and EGFR heterodimerization, but only IGF caused activating phosphorylation of both receptors. Combined treatment with A12 and the EGFR blocking antibody C225 was more effective at reducing cell proliferation and migration than either agent alone. Finally, TU159 tongue cancer cell xenografts grown in athymic nude mice were treated thrice weekly for 4 weeks with vehicle, A12 (40 mg/kg i.p.), C225 (40 mg/kg i.p.), or both agents (n = 8 mice per group; 2 tumors per mouse). Linear regression slope analysis showed significant differences in median tumor volume over time between all three treatment groups and the control group. Complete regression was seen in 31% (A12), 31% (C225), and 44% (A12 + C225) of tumors.

Conclusion: Here we found the overexpression of IGF-IR, the functional heterodimerization of IGF-IR and EGFR, and effective therapeutic targeting of these receptors in human head and neck cancer xenografts.
Tamoxifen Induces Heparanase Expression in Estrogen Receptor–Positive Breast Cancer
Authors: Cohen Irit , Maly Bella , Simon Itamar , Meirovitz Amichay , Pikarsky Eli , Zcharia Eyal , Peretz Tamar , Vlodavsky Israel , Elkin Michael
Published: July 2007

Purpose: Mammalian heparanase degrades heparan sulfate, the main polysaccharide of the basement membrane. Heparanase is an important determinant in cancer progression, acting via the breakdown of extracellular barriers for invasion, as well as release of heparan sulfate–bound angiogenic and growth-promoting factors. The present study was undertaken to elucidate molecular mechanisms responsible for heparanase overexpression in breast cancer.

Experimental Design: To characterize heparanase regulation by estrogen and tamoxifen and its clinical relevance for breast tumorigenesis, we applied immunohistochemical analysis of tissue microarray combined with chromatin immunoprecipitation assay, reverse transcription-PCR, and Western blot analysis.

Results: A highly significant correlation (P < 0.0001) between estrogen receptor (ER) positivity and heparanase overexpression was found in breast cancer. Binding of ER to heparanase promoter accompanied estrogen-induced increase in heparanase expression by breast carcinoma cells. Surprisingly, heparanase transcription was also stimulated by tamoxifen, conferring a proliferation advantage to breast carcinoma cells grown on a naturally produced extracellular matrix. Heparanase overexpression was invariably detected in ER-positive second primary breast tumors, developed in patients receiving tamoxifen for the initial breast carcinoma. The molecular mechanism of the estrogenlike effect of tamoxifen on heparanase expression involves recruitment of transcription coactivator AIB1 to the heparanase promoter.

Conclusions: Heparanase induction by ligand-bound ER represents an important pathway in breast tumorigenesis and may be responsible, at least in part, for the failure of tamoxifen therapy in some patients. Our study provides new insights on breast cancer progression and endocrine therapy resistance, offering future strategies for delaying or reversing this process.
RAD001 Inhibits Human Ovarian Cancer Cell Proliferation, Enhances Cisplatin-Induced Apoptosis, and Prolongs Survival in an Ovarian Cancer Model
Authors: Mabuchi Seiji , Altomare Deborah A. , Cheung Mitchell , Zhang Lili , Poulikakos Poulikos I. , Hensley Harvey H. , Schilder Russell J. , Ozols Robert F. , Testa Joseph R.
Published: July 2007

Purpose: mTOR (mammalian target of rapamycin) plays a central role in regulating cell growth and cell cycle progression and is regarded as a promising therapeutic target. We examined whether mTOR inhibition by RAD001 (everolimus) is therapeutically efficacious in the treatment of ovarian cancer as a single agent and in combination with cisplatin.

Experimental Design: Using four human ovarian cancer cell lines, we determined the effect of RAD001 by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Western blot, and apoptosis assays. We evaluated the association between phospho-AKT/mTOR activity and RAD001 sensitivity. We also determined the effect of RAD001 on tumor growth and malignancy using mice inoculated with human ovarian cancer cells.

Results: RAD001 markedly inhibited cell proliferation of human ovarian carcinoma cells with high AKT activity (OVCAR10 and SKOV-3), but the effect was minimal in cells with low AKT activity (OVCAR4 and OVCAR5). Sensitivity to RAD001 was independent of p53 expression. RAD001 inhibited the phosphorylation of downstream 4E-BP1 and p70S6 kinase and attenuated the expression of Myc. RAD001 also attenuated the expression of HIF-1α and vascular endothelial growth factor, important factors in angiogenesis and tumor invasiveness. RAD001 enhanced cisplatin-induced apoptosis in cells with high AKT/mTOR activity, with minimal effect in cells with low AKT-mTOR activity. Mouse xenografts of SKOV-3 cells revealed that RAD001 inhibits tumor growth, angiogenesis, and i.p. dissemination and ascites production and prolongs survival. Moreover, treatment with RAD001 significantly enhanced the therapeutic efficacy of cisplatin in vivo.

Conclusion: These results indicate that RAD001 could have therapeutic efficacy in human ovarian cancers with hyperactivated AKT/mTOR signaling.
Regulation of p53 Expression in Response to 5-Fluorouracil in Human Cancer RKO Cells
Authors: Ju Jingfang , Schmitz John C. , Song Bo , Kudo Kenji , Chu Edward
Published: July 2007

Purpose: The purpose of the study is to investigate the regulation of p53 expression in response to 5-fluorouracil (5-FU) in human colon cancer cells.

Experimental Design: Human colon cancer RKO cells were used as our model system. The levels of p53 expression and p53 protein stability in response to 5-FU and doxorubicin were investigated. In addition, the acetylation and phosphorylation status of p53 after 5-FU and doxorubicin treatment was analyzed by Western immunoblot analysis.

Results: Treatment of human colon cancer RKO cells with 10 μmol/L 5-FU resulted in significantly increased levels of p53 protein with maximal induction observed at 24 h. The level of acetylated p53 after 5-FU exposure remained unchanged, whereas the phosphorylated form of p53 was expressed only after 24 h drug treatment. Northern blot analysis revealed no change in p53 mRNA levels after 5-FU treatment. No differences were observed in the half-life of p53 protein in control and 5-FU–treated cells, suggesting that the increase in p53 was the direct result of newly synthesized protein. In contrast, the maximal induction of p53, in response to doxorubicin, occurred at an earlier time point (4 h) when compared with cells treated with 5-FU (24 h). No corresponding change in p53 mRNA was observed. Levels of both the acetylated and phosphorylated forms of p53 were markedly increased upon doxorubicin exposure when compared with treatment with 5-FU, resulting in a significantly prolonged half-life of p53 (120 versus 20 min).

Conclusion: These results, taken together, suggest that the regulatory mechanisms controlling p53 expression, in response to a cellular stress, are complex and are dependent upon the specific genotoxic agent. With regard to 5-FU, we show that translational regulation is an important process for controlling p53 expression. Studies are under way to define the specific mechanism(s) that control 5-FU–mediated translational regulation of p53.
Association of Cysteine-Rich Secretory Protein 3 and β-Microseminoprotein with Outcome after Radical Prostatectomy
Authors: Bjartell Anders S. , Al-Ahmadie Hikmat , Serio Angel M. , Eastham James A. , Eggener Scott E. , Fine Samson W. , Udby Lene , Gerald William L. , Vickers Andrew J. , Lilja Hans , Reuter Victor E. , Scardino Peter T.
Published: July 2007

Purpose: It has been suggested that cysteine-rich secretory protein 3 (CRISP-3) and β-microseminoprotein (MSP) are associated with outcome in prostate cancer. We investigated whether these markers are related to biochemical recurrence and whether addition of the markers improves prediction of recurring disease.

Experimental Design: Tissue microarrays of radical prostatectomy specimens were analyzed for CRISP-3 and MSP by immunohistochemistry. Associations between marker positivity and postprostatectomy biochemical recurrence [prostate-specific antigen (PSA) >0.2 ng/mL with a confirmatory level] were evaluated by univariate and multivariable Cox proportional hazards regression. Multivariable analyses controlled for preoperative PSA and pathologic stage and grade.

Results: Among 945 patients, 224 had recurrence. Median follow-up for survivors was 6.0 years. Patients positive for CRISP-3 had smaller recurrence-free probabilities, whereas MSP-positive patients had larger recurrence-free probabilities. On univariate analysis, the hazard ratio for patients positive versus negative for CRISP-3 was 1.53 (P = 0.010) and for MSP was 0.63 (P = 0.004). On multivariable analysis, both CRISP-3 (P = 0.007) and MSP (P = 0.002) were associated with recurrence. The hazard ratio among CRISP-3–positive/MSP-negative patients compared with CRISP-3–negative/MSP-positive patients was 2.38. Adding CRISP-3 to a base model that included PSA and pathologic stage and grade did not enhance the prediction of recurrence, but adding MSP increased the concordance index minimally from 0.778 to 0.781.

Conclusion: We report evidence that CRISP-3 and MSP are independent predictors of recurrence after radical prostatectomy for localized prostate cancer. However, addition of the markers does not importantly improve the performance of existing predictive models. Further research should aim to elucidate the functions of CRISP-3 and MSP in prostate cancer cells.
The Effect of Repeated Administration of Cyclophosphamide on Cytochrome P450 2B in Rats
Authors: Afsharian Parvaneh , Terelius Ylva , Hassan Zuzana , Nilsson Christina , Lundgren Stefan , Hassan Moustapha
Published: July 2007

Purpose: The prodrug cyclophosphamide (CPA) is activated by cytochrome P450 (CYP) enzymes. CPA is one of the corner stones in all cancer treatment. We have studied the effect of repeated doses of CPA given at different time intervals on the mRNA, protein levels, and enzyme activity of CYPs in rats.

Experimental Design: Two groups of animals (A-75 and A-150) were treated with four doses of CPA (75 and 150 mg/kg, respectively) at short time intervals (6 h). The third group of animals (B-150) was treated with 150 mg/kg at 24-h intervals. Three animals were killed 30 min after administration, and three animals immediately before the next dose.

Results: CYP2B1 and CYP2B2 mRNAs were significantly induced at 6 h after each dose in group A-75 (maximum of 2100-fold and 60-fold after the third dose, respectively), whereas the mRNA levels measured at 6 h postadministration in group A-150 were 1,490-fold and 36-fold after the second dose. In group B-150, no significant induction of mRNA levels was observed. CYP2B1 and CYP2B2 protein levels also increased with increased mRNAs. Plasma levels of 4-hydroxy-CPA measured at 30 min after dose correlated well with the increase in protein levels.

Conclusion: Up-regulation of CYP2B mRNA, with a concomitant increase in protein expression and activity, were observed after repeated administration of low doses of CPA compared with that found using higher doses, possibly due to toxicity counteracting induction. These results may help in designing more effective dosing schedules for CPA.
Detection of Circulating Tumor Cells in Early-Stage Breast Cancer Metastasis to Axillary Lymph Nodes
Authors: Nakagawa Taku , Martinez Steve R. , Goto Yasufumi , Koyanagi Kazuo , Kitago Minoru , Shingai Tatsushi , Elashoff David A. , Ye Xing , Singer Frederick R. , Giuliano Armando E. , Hoon Dave S.B.
Published: July 2007

Purpose: Clinical and pathologic prognostic factors do not always accurately predict disease outcome. Patients with early-stage breast cancer may harbor clinically significant but undetected systemic disease. We hypothesized that a multimarker quantitative real-time reverse transcription-PCR (qRT) assay could detect circulating tumor cells (CTC) in patients with early-stage breast cancer and correlate with sentinel lymph node (SLN) and non-SLN metastasis status.

Experimental Design: Blood samples from 90 women with the American Joint Committee on Cancer stages I to III breast cancer and 39 age-matched normal healthy volunteers were assessed by qRT for mRNA expression of three markers: stanniocalcin-1 (STC-1), N-acetylgalactosaminyltransferase (GalNacT), and melanoma antigen gene family-A3 (MAGE-A3). CTC biomarker detection was correlated with overall axillary LN (ALN), SLN, and non-SLN histopathology status.

Results: CTCs were detected in 39 of 90 (43%) patients, but not in normal volunteers. At least one CTC biomarker was detected in 10 of 35 (29%) stage I patients, 19 of 42 (45%) stage II patients, and 10 of 13 (77%) stage III patients. In multivariate analysis, only lymphovascular invasion and ≥2 CTC biomarkers detected significantly correlated with ALN metastasis [odds ratio (OR), 12.42; 95% confidence interval (95% CI), 3.52-43.77, P < 0.0001; and OR, 3.88; 95% CI, 1.69-8.89, P = 0.001, respectively]. The number of CTC biomarkers detected similarly correlated with SLN and non-SLN metastasis status (P = 0.0004). At least one CTC biomarker was detected in 10 of 11 (91%) patients with non-SLN metastases.

Conclusion: The detection of CTCs offers a novel means to assess the presence of systemic disease spreading relative to SLN and ALN histopathology status.
Overexpression of the Centrosomal Protein Aurora-A Kinase is Associated with Poor Prognosis in Epithelial Ovarian Cancer Patients
Authors: Landen Charles N. , Lin Yvonne G. , Immaneni Anand , Deavers Michael T. , Merritt William M. , Spannuth Whitney A. , Bodurka Diane C. , Gershenson David M. , Brinkley William R. , Sood Anil K.
Published: July 2007

Purpose: To assess the clinical significance of Aurora-A kinase, a centrosome-regulating serine-threonine kinase, in ovarian carcinoma.

Experimental Design: Aurora-A kinase expression was assessed by Western blot (cell lines) or immunohistochemistry (high-grade epithelial ovarian cancers), and clinical variables were collected by retrospective chart review. Centrosome amplification was assessed by immunofluorescence in cell lines, and by immunohistochemistry in patient samples.

Results: All ovarian cancer cell lines exhibited significant Aurora-A kinase protein overexpression, and all except A2780-par had centrosome amplification, a characteristic of mitotic dysregulation leading to genomic instability. Fifty-eight of 70 patient samples (82.8%) exhibited Aurora-A kinase overexpression compared with normal ovarian surface epithelium. High Aurora-A kinase expression was strongly associated with supernumerary centrosome count in tumor cells (P < 0.001). Tumors with the greatest Aurora-A overexpression (n = 24) had decreased patient survival (median survival, 1.44 versus 2.81 years; P = 0.01). High Aurora-A expression and suboptimal surgical cytoreduction remained predictors of poor survival (P < 0.05) by multivariate analysis.

Conclusions: Aurora-A kinase is overexpressed by a substantial proportion of ovarian cancers and is associated with centrosome amplification and poor survival. It may be a useful prognostic marker and target in ovarian cancer.
Polymorphic TP53BP1 and TP53 Gene Interactions Associated with Risk of Squamous Cell Carcinoma of the Head and Neck
Authors: Chen Kexin , Hu Zhibin , Wang Li-E , Zhang Wei , El-Naggar Adel K. , Sturgis Erich M. , Wei Qingyi
Published: July 2007

Purpose: Tumor protein 53-binding protein 1 (TP53BP1) and TP53 interact during TP53-mediated transcriptional activation and during checkpoint activation in response to DNA damage. Because suboptimal repair of tobacco-induced DNA damage is associated with risk of squamous cell carcinoma of the head and neck (SCCHN), we hypothesized that potentially functional polymorphisms in TP53BP1 and TP53 may contribute jointly to SCCHN risk.

Experimental Design: In a case-control study, DNA samples from age- and sex-matched SCCHN patients (n = 818) and cancer-free controls (n = 821) were genotyped for the presence of three variants of TP53BP1 (T-885G, Glu353Asp, and Gln1136Lys) and three variants of TP53 (Arg72Pro, PIN3, and MspI). Multivariate logistic regression was used to assess the adjusted odds ratios (OR) and 95% confidence intervals (95% CI).

Results: Although none of these six genetic variants alone was associated with SCCHN risk, the combined TP53BP1 genotypes were associated with a significant, dose response–dependent decrease in SCCHN risk among carriers of TP53Pro72Pro, TP53PIN3del/del, and TP53Msp1AA genotypes (trend test: P = 0.024, 0.016, and 0.016, respectively). Furthermore, TP53BP1 variant haplotype GGC carriers who were also TP53 variant homozygotes had a significantly lower risk of SCCHN than did TP53BP1 haplotype TCA carriers (adjusted OR, 0.48; 95% CI, 0.25-0.94 for TP53Pro72Pro; adjusted OR, 0.17; 95% CI, 0.04-0.69 for TP53PIN3del/de; and adjusted OR, 0.16; 95% CI, 0.04-0.65 for TP53Msp1AA). There was statistical evidence of interaction between TP53BP1 and TP53 diplotypes (P = 0.017).

Conclusion: Our data suggest that TP53BP1 variants may have protective effects on SCCHN risk but such effects were confined to TP53 variant allele/haplotype carriers.
Synergistic Interactions between Vorinostat and Sorafenib in Chronic Myelogenous Leukemia Cells Involve Mcl-1 and p21CIP1 Down-Regulation
Authors: Dasmahapatra Girija , Yerram Nitin , Dai Yun , Dent Paul , Grant Steven
Published: July 2007

Purpose: Interactions between the multikinase inhibitor sorafenib (Bay 43-9006) and the histone deacetylase inhibitor vorinostat were examined in chronic myelogenous leukemia (CML) cells sensitive and resistant to imatinib mesylate.

Experimental Design: K562, LAMA 84, and primary CML patient-derived CD34+ mononuclear cells were exposed to vorinostat followed by sorafenib, after which effects on cell viability and various survival signaling pathways were monitored by flow cytometry, clonogenic assays, and Western blotting. Real-time reverse transcription-PCR was used to monitor gene expression, and the functional contribution of p21CIP1 and Mcl-1 down-regulation were determined in cells transfected with corresponding constructs.

Results: Pretreatment (24 h) with vorinostat followed by sorafenib optimally induced mitochondrial injury and cell death in Bcr/Abl+ cells (e.g., K562 and LAMA 84). Similar results were obtained in imatinib mesylate–resistant cells expressing activated Lyn as well as in primary CD34+ bone marrow cells obtained from CML patients. This regimen also markedly inhibited CML cell colony formation. Combined but not individual treatment of CML cells with vorinostat and sorafenib triggered pronounced mitochondrial dysfunction (i.e., cytochrome c, Smac, and AIF release), caspase activation, poly(ADP-ribose) polymerase cleavage, and down-regulation of Mcl-1. Sorafenib also blocked vorinostat-mediated induction of p21CIP1. Down-regulation of Mcl-1 was caspase and transcription independent, whereas p21CIP1 down-regulation was partially caspase and transcription dependent. Enforced expression of p21CIP1 and particularly Mcl-1 significantly attenuated vorinostat/sorafenib-mediated lethality.

Conclusions: These findings suggest that combined treatment with vorinostat and sorafenib synergistically induces apoptosis in CML cells through a process that involves Mcl-1 down-regulation and inhibition of p21CIP1 induction.
A Lagrangian Objective Analysis Technique for Assimilating In Situ Observations with Multiple-Radar-Derived Airflow
Authors: Ziegler Conrad L. , Buban Michael S. , Rasmussen Erik N.
Published: July 2007

A new Lagrangian analysis technique is developed to assimilate in situ boundary layer measurements using multi-Doppler-derived wind fields, providing output fields of water vapor mixing ratio, potential temperature, and virtual potential temperature from which the lifting condensation level (LCL) and relative humidity (RH) fields are derived. The Lagrangian analysis employs a continuity principle to bidirectionally distribute observed values of conservative variables with the 3D, evolving boundary layer airflow, followed by temporal and spatial interpolation to an analysis grid. Cloud is inferred at any grid point whose height z > zLCL or equivalently where RH ≥ 100%. Lagrangian analysis of the cumulus field is placed in the context of gridded analyses of visible satellite imagery and photogrammetric cloud-base area analyses. Brief illustrative examples of boundary layer morphology derived with the Lagrangian analysis are presented based on data collected during the International H2O Project (IHOP): 1) a dryline on 22 May 2002; 2) a cold-frontal–dryline “triple point” intersection on 24 May 2002. The Lagrangian analysis preserves the sharp thermal gradients across the cold front and drylines and reveals the presence of undulations and plumes of water vapor mixing ratio and virtual potential temperature associated with deep penetrative updraft cells and convective roll circulations. Derived cloud fields are consistent with satellite-inferred cloud cover and cloud-base locations.
A Case Study of Subtropical Frontogenesis during a Blocking Event
Authors: Chen George Tai-Jen , Wang Chung-Chieh , Wang An-Hsiang
Published: July 2007

During 8–14 June 2000, a 500-hPa blocking event occurred over Mongolia and northern China (near 45°N, 108°E), which was the only case over this region in June since 1981. As the block developed, the initially weak low-level mei-yu front over southern China evolved into a system with strong baroclinity and subsequently moved south. The frontal passage over Taiwan caused temperatures to drop by 10°C, the largest in June over two decades. Using gridded analyses, manually analyzed weather maps, and satellite and surface data, the present study investigates the evolution of this mei-yu front under the influence of the block. The 925-hPa frontogenetical function is computed and effects of different processes are discussed. As the blocking event developed, concurrent ridge–trough amplification in the lower–midtroposphere produced a reversed thermal pattern. The lower-tropospheric high moved southward, and large-scale confluence and deformation were enhanced between the northerly flow and the prefrontal southwesterly flow. The location of the block, to the west-southwest of the Okhotsk Sea area, allowed it to affect the front over southern China and caused it to penetrate inside 20°N, unusual for the month of June. The distribution of the frontogenetical function indicated that the mei-yu frontogenesis and the maintenance of the front were attributed to both deformation and convergence. These two processes together counteracted the strong frontolysis along the frontal zone from diabatic effects, caused by evaporative cooling of frontal precipitation on the warm side and stronger sensible heat transfer (and daytime heating over less cloudy areas) on the cold side of the front. When deformation, convergence, and diabatic effects were all combined, the net total frontogenesis peaked slightly ahead of the frontal zone, thus contributing to the southward propagation of the front in addition to the advection by postfrontal cold air in the present case. When the front moved into the South China Sea, the cross-frontal thermal gradient diminished rapidly, mainly due to the frontolytic effect from sensible heat flux over warm waters.
Verification of an Ensemble Prediction System against Observations
Authors: Candille G. , Côté C. , Houtekamer P. L. , Pellerin G.
Published: July 2007

A verification system has been developed for the ensemble prediction system (EPS) at the Canadian Meteorological Centre (CMC). This provides objective criteria for comparing two EPSs, necessary when deciding whether or not to implement a new or revised EPS. The proposed verification methodology is based on the continuous ranked probability score (CRPS), which provides an evaluation of the global skill of an EPS. Its reliability/resolution partition, proposed by Hersbach, is used to measure the two main attributes of a probabilistic system. Also, the characteristics of the reliability are obtained from the two first moments of the reduced centered random variable (RCRV), which define the bias and the dispersion of an EPS. Resampling bootstrap techniques have been applied to these scores. Confidence intervals are thus defined, expressing the uncertainty due to the finiteness of the number of realizations used to compute the scores. All verifications are performed against observations to provide more independent validations and to avoid any local systematic bias of an analysis. A revised EPS, which has been tested at the CMC in a parallel run during the autumn of 2005, is described in this paper. This EPS has been compared with the previously operational one with the verification system presented above. To illustrate the verification methodology, results are shown for the temperature at 850 hPa. The confidence intervals are computed by taking into account the spatial correlation of the data and the temporal autocorrelation of the forecast error. The revised EPS performs significantly better for all the forecast ranges, except for the resolution component of the CRPS where the improvement is no longer significant from day 7. The significant improvement of the reliability is mainly due to a better dispersion of the ensemble. Finally, the verification system correctly indicates that variations are not significant when two theoretically similar EPSs are compared.
Estimation of Predictability with a Newly Derived Index to Quantify Similarity among Ensemble Members
Authors: Yamada Tomohito J. , Koster Randal D. , Kanae Shinjiro , Oki Taikan
Published: July 2007

This study reveals the mathematical structure of a statistical index, Ω, that quantifies similarity among ensemble members in a weather forecast. Previous approaches for quantifying predictability estimate separately the phase and shape characteristics of a forecast ensemble. The diagnostic Ω, on the other hand, characterizes the similarity (across ensemble members) of both aspects together with a simple expression. The diagnostic Ω is thus more mathematically versatile than previous indices.
Climatology of Cyclone Size Characteristics and Their Changes during the Cyclone Life Cycle
Authors: Rudeva Irina , Gulev Sergey K.
Published: July 2007

Climatology of the atmospheric cyclone sizes and their change over the cyclone life cycle is analyzed on the basis of tracking 57 yr of NCEP–NCAR reanalysis sea level pressure data over the Northern Hemisphere. To quantify the atmospheric cyclone sizes a coordinate transform was used, which allows for the collocation of the cyclone center with the virtual pole and for the establishment of a unique coordinate system for the further determination of cyclone geometry. This procedure was incorporated into a numerical cyclone tracking scheme and provided quantitative estimation of cyclone geometry at every stage of the cyclone development. Climatological features of the distribution of the cyclone size characteristics (effective radius, asymmetry) are considered for the cyclones with different central pressure, deepening rate, and lifetime. Mean effective cyclone radius may experience significant changes, ranging from 300–400 km over the continents to more than 900 km over the oceans. There is found to be a strong dependence of the cyclone effective radius on the cyclone lifetime and intensity, implying the largest cyclone sizes for the most intense and long-living transients. Analysis of size changes during the cyclone life cycle implies that the cyclone radius increases during the development stage from 50% to 150%. Size evolution during the cyclone life cycle implies a universal dependence of the normalized cyclone effective radius and the normalized cyclone age. The actual maximum cyclone radius can be determined from these two nondimensional parameters and cyclone central pressure. Further application of the analysis of cyclone size and shape are discussed.
The Prediction of Extratropical Storm Tracks by the ECMWF and NCEP Ensemble Prediction Systems
Authors: Froude Lizzie S. R. , Bengtsson Lennart , Hodges Kevin I.
Published: July 2007

The prediction of extratropical cyclones by the European Centre for Medium-Range Weather Forecasts (ECMWF) and the National Centers for Environmental Prediction (NCEP) ensemble prediction systems (EPSs) has been investigated using an objective feature tracking methodology to identify and track the cyclones along the forecast trajectories. Overall the results show that the ECMWF EPS has a slightly higher level of skill than the NCEP EPS in the Northern Hemisphere (NH). However in the Southern Hemisphere (SH), NCEP has higher predictive skill than ECMWF for the intensity of the cyclones. The results from both EPSs indicate a higher level of predictive skill for the position of extratropical cyclones than their intensity and show that there is a larger spread in intensity than position. Further analysis shows that the predicted propagation speed of cyclones is generally too slow for the ECMWF EPS and shows a slight bias for the intensity of the cyclones to be overpredicted. This is also true for the NCEP EPS in the SH. For the NCEP EPS in the NH the intensity of the cyclones is underpredicted. There is small bias in both the EPS for the cyclones to be displaced toward the poles. For each ensemble forecast of each cyclone, the predictive skill of the ensemble member that best predicts the cyclone’s position and intensity was computed. The results are very encouraging showing that the predictive skill of the best ensemble member is significantly higher than that of the control forecast in terms of both the position and intensity of the cyclones. The prediction of cyclones before they are identified as 850-hPa vorticity centers in the analysis cycle was also considered. It is shown that an indication of extratropical cyclones can be given by at least 1 ensemble member 7 days before they are identified in the analysis. Further analysis of the ECMWF EPS shows that the ensemble mean has a higher level of skill than the control forecast, particularly for the intensity of the cyclones, from day 3 of the forecast. There is a higher level of skill in the NH than the SH and the spread in the SH is correspondingly larger. The difference between the ensemble mean error and spread is very small for the position of the cyclones, but the spread of the ensemble is smaller than the ensemble mean error for the intensity of the cyclones in both hemispheres. Results also show that the ECMWF control forecast has ½ to 1 day more skill than the perturbed members, for both the position and intensity of the cyclones, throughout the forecast.