bluuebunny/crossref_metadata_2025 huggingface dataset:
fMLP induces Hsp27 expression, attenuates NF-κB activation, and confers intestinal epithelial cell protection
Authors: Carlson Ryan M. , Vavricka Stephan R. , Eloranta Jyrki J. , Musch Mark W. , Arvans Donna L. , Kles Keri A. , Walsh-Reitz Margaret M. , Kullak-Ublick Gerd A. , Chang Eugene B.
Published: December 2006

Sustained expression of cytoprotective intestinal epithelial heat shock proteins (Hsps), particularly Hsp27, depends on stimuli derived from bacterial flora. In this study, we examined the role of the bacterial chemotactic peptide fMLP in stimulating colonic epithelial Hsp expression at concentrations encountered in a physiological milieu. Treatment of the polarized human intestinal epithelial cell line Caco2bbe with physiological concentrations of fMLP (10–100 nM) induced expression of Hsp27, but not Hsp72, in a time- and concentration-dependent manner. Induction of Hsp27 by fMLP was specific since the fMLP analogs MRP and MLP were not effective. Hsp27 induction by fMLP was blocked by the fMLP-receptor antagonist BOC-FLFLF and was blocked when the dipeptide transporter PepT1, an entry pathway for fMLP, was silenced. fMLP activated both the p38 and ERK1/2 MAP kinase pathways in Caco2bbe cells, but not the SAPK/JNK pathway. The p38 inhibitor SB203580, but not the MEK-1 inhibitor PD98059, blocked Hsp27 induction by fMLP. fMLP treatment inhibited actin depolymerization and decreased transepithelial resistance caused by the oxidant monochloramine, and this inhibition was reversed by silencing Hsp27 expression. fMLP pretreatment also inhibited activation of proinflammatory transcription factor NF-κB by TNF-α in Caco2bbe cells, reducing induction of NF-κB target genes by TNF-α both in human intestinal biopsies and Caco2bbe cells. In conclusion, fMLP may contribute to the maintenance of intestinal homeostasis by mediating physiological expression of Hsp27, enhancing cellular protection, and negatively regulating the inflammatory response.
KATP channel knockout worsens myocardial calcium stress load in vivo and impairs recovery in stunned heart
Authors: Gumina Richard J. , O'Cochlain D. Fearghas , Kurtz Christopher E. , Bast Peter , Pucar Darko , Mishra Prasanna , Miki Takashi , Seino Susumu , Macura Slobodan , Terzic Andre
Published: December 2006

Gene knockout of the KCNJ11-encoded Kir6.2 ATP-sensitive K+ (KATP) channel implicates this stress-response element in the safeguard of cardiac homeostasis under imposed demand. KATP channels are abundant in ventricular sarcolemma, where subunit expression appears to vary between the sexes. A limitation, however, in establishing the full significance of KATP channels in the intact organism has been the inability to monitor in vivo the contribution of the channel to intracellular calcium handling and the superimposed effect of sex that ultimately defines heart function. Here, in vivo manganese-enhanced cardiac magnetic resonance imaging revealed, under dobutamine stress, a significantly greater accumulation of calcium in both male and female KATP channel knockout (Kir6.2-KO) mice compared with sex- and age-matched wild-type (WT) counterparts, with greatest calcium load in Kir6.2-KO females. This translated, poststress, into a sustained contracture manifested by reduced end-diastolic volumes in KATP channel-deficient mice. In response to ischemia-induced stunning, male and female Kir6.2-KO hearts demonstrated accelerated time to contracture and increased peak contracture compared with WT. The outcome on reperfusion, in both male and female Kir6.2-KO hearts, was a transient reduction in systolic performance, measured as rate-pressure product compared with WT, with protracted increase in left ventricular end-diastolic pressure, exaggerated in female knockout hearts, despite comparable leakage of creatine kinase across groups. Kir6.2-KO hearts were rescued from diastolic dysfunction by agents that target alternative pathways of calcium handling. Thus KATP channel deficit confers a greater susceptibility to calcium overload in vivo, accentuated in female hearts, impairing contractile recovery under various conditions of high metabolic demand.
Rosiglitazone attenuates hypoxia-induced pulmonary arterial remodeling
Authors: Crossno Joseph T. , Garat Chrystelle V. , Reusch Jane E. B. , Morris Kenneth G. , Dempsey Edward C. , McMurtry Ivan F. , Stenmark Kurt R. , Klemm Dwight J.
Published: December 2006

Thiazolidinediones (TZDs) are insulin-sensitizing agents that also decrease systemic blood pressure, attenuate the formation of atherosclerotic lesions, and block remodeling of injured arterial walls. Recently, TZDs were shown to prevent pulmonary arterial (PA) remodeling in rats treated with monocrotaline. Presently we report studies testing the ability of the TZD rosiglitazone (ROSI) to attenuate pathological arterial remodeling in the lung and prevent the development of pulmonary hypertension (PH) in rats subjected to chronic hypoxia. PA remodeling was reduced in ROSI-treated animals exposed to hypoxia compared with animals exposed to hypoxia alone. ROSI treatment blocked muscularization of distal pulmonary arterioles and reversed remodeling and neomuscularization in lungs of animals previously exposed to chronic hypoxia. Decreased PA remodeling in ROSI-treated animals was associated with decreased smooth muscle cell proliferation, decreased collagen and elastin deposition, and increased matrix metalloproteinase-2 activity in the PA wall. Cells expressing the c-Kit cell surface marker were observed in the PA adventitia of untreated animals exposed to hypoxia but not in ROSI-treated hypoxic rats. Right ventricular hypertrophy and cardiomyocyte hypertrophy were also blunted in ROSI-treated hypoxic animals. Interestingly, mean PA pressures were elevated equally in the untreated and ROSI-treated groups, indicating that ROSI had no effect on the development of PH. However, mean PA pressure was normalized acutely in both groups of hypoxia-exposed animals by Fasudil, an agent that inhibits RhoA/Rho kinase-mediated vasoconstriction. We conclude that ROSI can attenuate and reverse PA remodeling and neomuscularization associated with hypoxic PH. However, this agent fails to block the development of PH, apparently because of its inability to repress sustained Rho kinase-mediated arterial vasoconstriction.
Model-projected mechanistic bases for sex differences in growth hormone regulation in humans
Authors: Farhy Leon S. , Bowers Cyril Y. , Veldhuis Johannes D.
Published: December 2006

Models of physiological systems facilitate rational experimental design, inference, and prediction. A recent construct of regulated growth hormone (GH) secretion interlinks the actions of GH-releasing hormone (GHRH), somatostatin (SRIF), and GH secretagogues (GHS) with GH feedback in the rat (Farhy LS, Veldhuis JD. Am J Physiol Regul Integr Comp Physiol 288: R1649–R1663, 2005). In contrast, no comparable formalism exists to explicate GH dynamics in any other species. The present analyses explore whether a unifying model structure can represent species- and sex-defined distinctions in the human and rodent. The consensus principle that GHRH and GHS synergize in vivo but not in vitro was explicable by assuming that GHS 1) evokes GHRH release from the brain, 2) opposes inhibition by SRIF both in the hypothalamus and on the pituitary gland, and 3) stimulates pituitary GH release directly and additively with GHRH. The gender-selective principle that GH pulses are larger and more irregular in women than men was conferrable by way of 4) higher GHRH potency and 5) greater GHS efficacy. The overall construct predicts GHRH/GHS synergy in the human only in the presence of SRIF when the brain-pituitary nexus is intact, larger and more irregular GH pulses in women, and observed gender differences in feedback by GH and the single and paired actions of GHRH, GHS, and SRIF. The proposed model platform should enhance the framing and interpretation of novel clinical hypotheses and create a basis for interspecies generalization of GH-axis regulation.
Gastrocnemius muscle fascicle behavior during stair negotiation in humans
Authors: Spanjaard M. , Reeves N. D. , van Dieën J. H. , Baltzopoulos V. , Maganaris C. N.
Published: December 2006

The aim of the present study was to establish the behavior of human medial gastrocnemius (GM) muscle fascicles during stair negotiation. Ten healthy male subjects performed normal stair ascent and descent at their own comfortable speed on a standard-dimension four-step staircase with embedded force platforms in each step. Kinematic, kinetic, and electromyographic data of the lower limbs were collected. Real-time ultrasound scanning was used to determine GM muscle fascicle length changes. Musculotendon complex (MTC) length changes were estimated from ankle and knee joint kinematics. The GM muscle was mainly active during the push-off phase in stair ascent, and the muscle fascicles contracted nearly isometrically. The GM muscle was mainly active during the touch-down phase of stair descent where the MTC was lengthened; however, the GM muscle fascicles shortened by ∼7 mm. These findings show that the behavior and function of GM muscle fascicles in stair negotiation is different from that expected on the basis of length changes of the MTC as derived from joint kinematics.
Contraction-relaxation coupling mechanism characterization in the thermodynamic phase plane: normal vs. impaired left ventricular ejection fraction
Authors: Wu Yue , Yu Yingbo , Kovács Sándor J.
Published: December 2006

Using simultaneous pressure-volume measurements obtained during cardiac catheterization, we employ the thermodynamic phase-plane (TPP) method to characterize global contraction-relaxation coupling (CRC) between normal and impaired left ventricular (LV) ejection fraction (LVEF) groups. The cardiac cycle inscribes a closed loop in the TPP defined by the coordinates “potential” power [V(dP/d t), ergs/s] and “kinetic” power [P(dV/d t), ergs/s]. The TPP-derived indexes κ and ρ define the chamber's contractile and CRC attributes, respectively. Data from 33 subjects dichotomized as normal control ( n = 22, >50% LVEF) and impaired LVEF ( n = 11, <50% LVEF) were analyzed. The results were as follows: κ = 3.0 ± 1.1 and ρ = −0.38 ± 0.21 for controls and κ = 5.4 ± 1.6 and ρ = −1.14 ± 0.47 for the impaired LVEF group; κ and ρ are significantly higher for impaired LVEF than for control ( P < 0.001 for both). As κ increased, ρ decreased ( r = −0.69) for all subjects. Hence, ventricles with impaired LVEF are thermodynamically less efficient because they require more potential power per unit of delivered kinetic power than controls. We conclude that TPP-derived indexes of CRC facilitate assessment of chamber efficiency in thermodynamic terms and elucidate the dominant differentiating features in terms of CRC indexes.
Doping dose of salbutamol and exercise: deleterious effect on cancellous and cortical bones in adult rats
Authors: Bonnet N. , Benhamou C. L. , Beaupied H. , Laroche N. , Vico L. , Dolleans E. , Courteix D.
Published: December 2006

Animal studies suggest that bone remodeling is under β-adrenergic control via the sympathetic nervous system. To our knowledge, the impact of β-agonist substances, at doping doses, has not been studied in adult rats. The purpose of this study was to examine the effects of salbutamol injections with or without treadmill exercise on trabecular and cortical bone in adult rats. Adult (36 wk of age) female Wistar rats ( n = 56) were treated with salbutamol (3 mg·kg−1·day−1 sc, 5 days/wk) or vehicle (sham) with or without subsequent treadmill exercise (13 m/min, 60 min/day, 5 days/wk) for 10 wk. Tibial and femoral bone mineral density was analyzed by dual-energy X-ray absorptiometry. Metaphysic trabecular bone structure was analyzed by micro-CT at the time of the animals' death. Bone cell activities were assessed histomorphometrically. After 10 wk, the increase in bone mineral density was less in salbutamol-treated than in sham rats (+3.3% vs. +12.4%, P < 0.05), and trabecular parameters were altered and bone resorption was increased in salbutamol-treated rats compared with controls. The negative effect on bone architecture in salbutamol-treated rats persisted, even with treadmill exercise. These results confirm the deleterious effect of β2-agonists on bone mass during chronic treatment and describe its effects on bone mechanical properties in adult rats. Bone loss occurred independently of a salbutamol-induced anabolic effect on muscle mass and was equally severe in sedentary and exercising rats, despite a beneficial effect of exercise on the extrinsic and intrinsic energy to ultimate strain. These bone effects may have important consequences in athletes who use salbutamol as a doping substance.
Increased insulin-stimulated Akt pSer473and cytosolic SHP2 protein abundance in human skeletal muscle following acute exercise and short-term training
Authors: Wadley Glenn D. , Konstantopoulos Nicky , Macaulay Lance , Howlett Kirsten F. , Garnham Andrew , Hargreaves Mark , Cameron-Smith David
Published: December 2006

The purpose of the present study was to determine in human skeletal muscle whether a single exercise bout and 7 days of consecutive endurance (cycling) training 1) increased insulin-stimulated Akt pSer473and 2) altered the abundance of the protein tyrosine phosphatases (PTPases), PTP1B and SHP2. In healthy, untrained men ( n = 8; 24 ± 1 yr), glucose infusion rate during a hyperinsulinemic euglycemic clamp, when compared with untrained values, was not improved 24 h following a single 60-min bout of endurance cycling but was significantly increased (∼30%; P < 0.05) 24 h following completion of 7 days of exercise training. Insulin-stimulated Akt pSer473was ∼50% higher ( P < 0.05) 24 h following the acute bout of exercise, with this effect remaining after 7 days of training ( P < 0.05). Insulin-stimulated insulin receptor and insulin receptor substrate-1 tyrosine phosphorylation were not altered 24 h after acute exercise and short-term training. Insulin did not acutely regulate the localization of the PTPases, PTP1B or SHP2, although cytosolic protein abundance of SHP2 was increased ( P < 0.05; main effect) 24 h following acute exercise and short-term training. In conclusion, insulin-sensitive Akt pSer473and cytosolic SHP2 protein abundance are higher after acute exercise and short-term training, and this effect appears largely due to the residual effects of the last bout of prior exercise. The significance of exercise-induced alterations in cytosolic SHP2 and insulin-stimulated Akt pSer473on the improvement in insulin sensitivity requires further elucidation.
Cancer Progression in the Transgenic Adenocarcinoma of Mouse Prostate Mouse Is Related to Energy Balance, Body Mass, and Body Composition, but not Food Intake
Authors: Huffman Derek M. , Johnson Maria S. , Watts Amanda , Elgavish Ada , Eltoum Isam A. , Nagy Tim R.
Published: December 2006

Calorie restriction can inhibit or delay carcinogenesis, reportedly due to a reduction in calorie intake rather than by concurrent changes in body mass and/or composition. Our objective was to test the hypothesis that body mass and/or composition have an important effect, independent of energy intake, on the benefits or hazards associated with calorie restriction or overeating, respectively. In the first experiment, transgenic mice that spontaneously develop prostate cancer [transgenic adenocarcinoma of mouse prostate (TRAMP)] were housed at 27°C or 22°C and pair fed the same diet for 21 weeks (95% of ad libitum intake at 27°C). In the second experiment, TRAMP mice were housed at 27°C or 22°C and fed the same diet ad libitum for 21 weeks. Despite a similar calorie intake, pair-fed mice at 27°C (PF27) were heavier (28.3 ± 3.3 versus 17.6 ± 1.6 g at 21 weeks; P < 0.001; mean ± SD) and had greater fat (6.4 ± 2.1 versus 1.9 ± 0.3 g; P < 0.001) and lean mass (P < 0.001) than pair-fed mice at 22°C. Furthermore, PF27 mice had greater levels of serum leptin (P < 0.001), lower levels of adiponectin (P < 0.05), and a greater frequency of prostatic adenocarcinoma (P < 0.05). In contrast, ad libitum–fed mice housed at 22°C consumed ∼30% more calories than ad libitum–fed mice at 27°C, but there was no difference between groups in body composition or cancer progression. These results imply that the ability of calorie restriction to inhibit or delay cancer incidence and progression is mediated in part by changes in energy balance, body mass, and/or body composition rather than calorie intake per se, suggesting that excess calorie retention, rather than consumption, confers cancer risk. [Cancer Res 2007;67(1):417–24]
Par-4-Dependent Apoptosis by the Dietary Compound Withaferin A in Prostate Cancer Cells
Authors: Srinivasan Sowmyalakshmi , Ranga Rama S. , Burikhanov Ravshan , Han Seong-Su , Chendil Damodaran
Published: December 2006

Deletion or mutation of the androgen receptor (AR) renders prostate tumors refractory to apoptosis by androgen ablation, the mainstay of prostate cancer therapy. To identify novel therapeutics that can induce apoptosis regardless of the AR status of prostate cancer cells, we screened dietary herbal compounds using a reporter assay for the prostate apoptosis response-4 (Par-4) gene, which induces p53- and PTEN-independent and cancer-selective apoptosis. One of the compounds, withaferin A (WA), a major constituent of the dietary compound Withania somnifera, induced Par-4-dependent apoptosis in androgen-refractory prostate cancer cells and regression of PC-3 xenografts in nude mice. Interestingly, restoration of wild-type AR in PC-3 (AR negative) cells abrogated both Par-4 induction and apoptosis by WA. Individually, WA and anti-androgens induced neither Par-4 nor apoptosis in androgen-responsive prostate cancer cells, yet in combination, WA and anti-androgen synergistically induced Par-4 and apoptosis in androgen-responsive prostate cancer cells. Thus, when judiciously combined with anti-androgens, WA inhibits survival of both androgen-responsive and androgen-refractory prostate cancer cells by a Par-4-dependent mechanism. As Par-4 up-regulation induces apoptosis in most tumor cells, our findings can be extended to high-throughput screens to identify synergistic combinations for both therapy-sensitive and therapy-resistant cancers. [Cancer Res 2007;67(1):246–53]
Molecular Mechanisms of Epidermal Growth Factor Receptor (EGFR) Activation and Response to Gefitinib and Other EGFR-Targeting Drugs
Authors: Ono Mayumi , Kuwano Michihiko
Published: December 2006

The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, including EGFR, HER2/erbB2, and HER3/erbB3, is an attractive target for antitumor strategies. Aberrant EGFR signaling is correlated with progression of various malignancies, and somatic tyrosine kinase domain mutations in the EGFR gene have been discovered in patients with non–small cell lung cancer responding to EGFR-targeting small molecular agents, such as gefitinib and erlotinib. EGFR overexpression is thought to be the principal mechanism of activation in various malignant tumors. Moreover, an increased EGFR copy number is associated with improved survival in non–small cell lung cancer patients, suggesting that increased expression of mutant and/or wild-type EGFR molecules could be molecular determinants of responses to gefitinib. However, as EGFR mutations and/or gene gains are not observed in all patients who respond partially to treatment, alternative mechanisms might confer sensitivity to EGFR-targeting agents. Preclinical studies showed that sensitivity to EGFR tyrosine kinase inhibitors depends on how closely cell survival and growth signalings are coupled with EGFR, and also with HER2 and HER3, in each cancer. This review also describes a possible association between EGFR phosphorylation and drug sensitivity in cancer cells, as well as discussing the antiangiogenic effect of gefitinib in association with EGFR activation and phosphatidylinositol 3-kinase/Akt activation in vascular endothelial cells.
Implications of Methylation Patterns of Cancer Genes in Salivary Gland Tumors
Authors: Williams Michelle D. , Chakravarti Nitin , Kies Merrill S. , Maruya Shin-Ichiro , Myers Jeffrey N. , Haviland Joie C. , Weber Randal S. , Lotan Reuben , El-Naggar Adel K.
Published: December 2006

Purpose: We investigated the methylation status and protein expression of four tumor suppressor genes to determine their role in salivary gland tumorigenesis.

Experimental Design: We performed methylation-specific PCR and protein analyses of 29 normal salivary glands, 23 benign, and 79 malignant salivary gland neoplasms to determine the pattern and potential diagnostic and/or biological role of the RASSF1, RARβ2, DAPK, and MGMT tumor suppressor gene methylation in these tumors.

Results: No methylation was detected in the normal tissues. Methylation occurred in 9 of 23 (39.1%) benign tumors; 3 (25.0%) pleomorphic adenomas and 6 (66.7%) Warthin's tumors at the MGMT, DAPK, or RASSF1 genes. Methylation occurred in 33 of 79 (41.8%) malignant tumors; 8 (30.8%) adenoid cystic carcinomas, 6 (33.3%) mucoepidermoid carcinomas, 6 (42.9%) acinic cell carcinomas, and 13 (62.0%) salivary duct carcinomas. RASSF1 and RARβ2 represented 75.8% of methylation events occurring most frequently in salivary duct and acinic cell carcinomas. Overall, we found no significant correlation between protein expression and methylation status of individual genes, but observed low or absent protein expression in several methylated tumors. Significant correlations were found between methylation and aggressive malignant phenotypes (P = 0.0004) and age (P = 0.05).

Conclusions: (a) Benign and malignant salivary tumors differed in the frequency and pattern of gene methylation; (b) high-grade carcinomas were significantly methylated compared with low-grade phenotypes; (c) RASSF1 and RARβ2 were highly methylated in malignant tumors and can be targeted for therapy; and (d) methylation pattern may serve as a diagnostic and biological marker in assessing these tumors.
Ki-67 Is an Independent Predictor of Bladder Cancer Outcome in Patients Treated with Radical Cystectomy for Organ-Confined Disease
Authors: Margulis Vitaly , Shariat Shahrokh F. , Ashfaq Raheela , Sagalowsky Arthur I. , Lotan Yair
Published: December 2006

Purpose: To determine the association of the cell proliferative marker Ki-67 with pathologic features and disease prognosis in patients with transitional cell carcinoma (TCC) of the urinary bladder.

Methods: Immunohistochemical staining for Ki-67 was done on serial cuts from tissue microarrays containing cystectomy specimens from 9 patients without bladder cancer and 226 consecutive patients with bladder TCC. We also assessed malignant lymph nodes from 50 of the 226 cystectomy patients.

Results: Ki-67 expression was increased in 42.5% cystectomy specimens and in 54% metastatic lymph nodes. In contrast, it was absent in all nine benign cystectomy specimens. Ki-67 overexpression was associated with advanced pathologic stage, higher grade, lymphovascular invasion, and metastases to lymph nodes (P = 0.001, 0.040, 0.031, and 0.036, respectively). In multivariate analyses, pathologic stage and lymph node metastases were independent predictors of disease recurrence and bladder cancer-specific mortality. In the subgroup of patients with organ-confined disease (<pT3 N0; n = 91), excluding patients who received neoadjuvant or adjuvant chemotherapy, Ki-67 status was an independent predictor of both disease recurrence (risk ratio, 7.591; P = 0.001) and bladder cancer-specific mortality (risk ratio, 4.045; P = 0.041).

Conclusions: Ki-67 overexpression is associated with features of aggressive bladder TCC and adds independent prognostic information to standard pathologic features for prediction of clinical outcome after radical cystectomy.
Enhancement of Immunologic Tumor Regression by Intratumoral Administration of Dendritic Cells in Combination with Cryoablative Tumor Pretreatment and Bacillus Calmette-Guerin Cell Wall Skeleton Stimulation
Authors: Udagawa Masaru , Kudo-Saito Chie , Hasegawa Go , Yano Kazuhito , Yamamoto Aiko , Yaguchi Masae , Toda Masahiro , Azuma Ichiro , Iwai Takehisa , Kawakami Yutaka
Published: December 2006

Purpose: We developed an effective immunotherapy, which could induce antitumor immune responses against shared and unique tumor antigens expressed in autologous tumors.

Experimental Design: Intratumoral administration of dendritic cells is one of the individualized immunotherapies; however, the antitumor activity is relatively weak. In this study, we attempted to enhance the antitumor efficacy of the i.t. dendritic cell administration by combining dendritic cells stimulated with Bacillus Calmette-Guerin cell wall skeleton (BCG-CWS) additionally with cryoablative pretreatment of tumors and analyzed the therapeutic mechanisms.

Results: These two modifications (cryoablation of tumors and BCG-CWS stimulation of dendritic cells) significantly increases the antitumor effect on both the treated tumor and the untreated tumor, which was distant at the opposite side, in a bilateral s.c. murine CT26 colon cancer model. Further analysis of the augmented antitumor effects revealed that the cryoablative pretreatment enhances the uptake of tumor antigens by the introduced dendritic cells, resulting in the induction of tumor-specific CD8+ T cells responsible for the in vivo tumor regression of both treated and remote untreated tumors. This novel combination i.t. dendritic cell immunotherapy was effective against well-established large tumors. The antitumor efficacy was further enhanced by depletion of CD4+CD25+FoxP3+ regulatory T cells.

Conclusions: This novel dendritic cell immunotherapy with i.t. administration of BCG-CWS–treated dendritic cells following tumor cryoablation could be used for the therapy of cancer patients with multiple metastases.
Active Antiviral T-Lymphocyte Response Can Be Redirected against Tumor Cells by Antitumor Antibody × MHC/Viral Peptide Conjugates
Authors: Cesson Valérie , Stirnemann Kathrin , Robert Bruno , Luescher Immanuel , Filleron Thomas , Corradin Giampietro , Mach Jean-Pierre , Donda Alena
Published: December 2006

Purpose: To redirect an ongoing antiviral T-cell response against tumor cells in vivo, we evaluated conjugates consisting of antitumor antibody fragments coupled to class I MHC molecules loaded with immunodominant viral peptides.

Experimental Design: First, lymphochoriomeningitis virus (LCMV)–infected C57BL/6 mice were s.c. grafted on the right flank with carcinoembryonic antigen (CEA)–transfected MC38 colon carcinoma cells precoated with anti-CEA × H-2Db/GP33 LCMV peptide conjugate and on the left flank with the same cells precoated with control anti-CEA F(ab′)2 fragments. Second, influenza virus–infected mice were injected i.v., to induce lung metastases, with HER2-transfected B16F10 cells, coated with either anti-HER2 × H-2Db/NP366 influenza peptide conjugates, or anti-HER2 F(ab′)2 fragments alone, or intact anti-HER2 monoclonal antibody. Third, systemic injections of anti-CEA × H-2Db conjugates with covalently cross-linked GP33 peptides were tested for the growth inhibition of MC38-CEA+ cells, s.c. grafted in LCMV-infected mice.

Results: In the LCMV-infected mice, five of the six grafts with conjugate-precoated MC38-CEA+ cells did not develop into tumors, whereas all grafts with F(ab′)2-precoated MC38-CEA+ cells did so (P = 0.0022). In influenza virus–infected mice, the group injected with cells precoated with specific conjugate had seven times less lung metastases than control groups (P = 0.0022 and P = 0.013). Most importantly, systemic injection in LCMV-infected mice of anti-CEA × H-2Db/cross-linked GP33 conjugates completely abolished tumor growth in four of five mice, whereas the same tumor grew in all five control mice (P = 0.016).

Conclusion: The results show that a physiologic T-cell antiviral response in immunocompetent mice can be redirected against tumor cells by the use of antitumor antibody × MHC/viral peptide conjugates.
Immunologic and Clinical Effects of Injecting Mature Peptide-Loaded Dendritic Cells by Intralymphatic and Intranodal Routes in Metastatic Melanoma Patients
Authors: Lesimple Thierry , Neidhard Eve-Marie , Vignard Virginie , Lefeuvre Claudia , Adamski Henri , Labarrière Nathalie , Carsin André , Monnier Delphine , Collet Brigitte , Clapisson Gilles , Birebent Brigitte , Philip Irène , Toujas Louis , Chokri Mohamed , Quillien Véronique
Published: December 2006

Purpose: A phase I/II trial was conducted to evaluate clinical and immunologic responses after intralymphatic and intranodal injections of mature dendritic cells.

Experimental Design: Fourteen patients with a metastatic melanoma received matured dendritic cells, loaded with Melan-A/MART-1 and/or NA17-A peptides and keyhole limpet hemocyanin. The cells were matured overnight with Ribomunyl, a toll-like receptor ligand, and IFN-γ, which ensured the production of high levels of interleukin-12p70. Dendritic cells were injected at monthly intervals, first into an afferent lymphatic and then twice intranodally. Immunologic responses were monitored by tetramer staining of circulating CD8+ lymphocytes and delayed-type hypersensitivity tests.

Results: Dendritic cell vaccination induced delayed-type hypersensitivity reactivity toward NA17-A-pulsed, keyhole limpet hemocyanin–pulsed, and Melan-A-pulsed dendritic cells in 6 of 10, 4 of 11, and 3 of 9 patients, respectively. Four of the 12 patients analyzed by tetramer staining showed a significantly increased frequency of Melan-A-specific T cells, including one patient vaccinated only with NA17-A-pulsed dendritic cells. Furthermore, 2 of the 12 analyzed patients had a significant increase of NA17-A-specific T cells, including one immunized after an optional additional treatment course. No objective clinical response was observed. Two patients were stabilized at 4 and 10 months and three patients are still alive at 30, 39, and 48 months.

Conclusions: Injections into the lymphatic system of mature peptide-loaded dendritic cells with potential TH1 polarization capacities did not result in marked clinical results, despite immunologic responses in some patients. This highlights the need to improve our understanding of dendritic cell physiology.
Inhibitory Effects of Castration in an Orthotopic Model of Androgen-Independent Prostate Cancer Can Be Mimicked and Enhanced by Angiogenesis Inhibition
Authors: Hammarsten Peter , Halin Sofia , Wikstöm Pernilla , Henriksson Roger , Rudolfsson Stina Häggström , Bergh Anders
Published: December 2006

Purpose: Today, the most important treatment of advanced prostate cancer is castration; unfortunately, however, the long-term effect of this therapy is insufficient. Recent studies suggest that castration-induced prostate involution could be caused by primary effects in the prostate vasculature; therefore, we examined if antivascular treatments could mimic the effects of castration.

Experimental Design: Androgen-independent AT-1 prostate cancer cells were grown inside the ventral prostate in adult rats. Tumor-bearing animals were treated with an inhibitor of vascular endothelial growth factor receptor 2 and epidermal growth factor receptor signaling, N-(4-bromo-2-fluorophenyl)-6-methoxy-7-[(1-methylpiperidin-4-yl)methoxy]quinazolin-4-amine (ZD6474, AstraZeneca, Södertälje, Sweden), and short-term effects (after 3 days) were compared with those induced by castration.

Results: Castration caused decreased vascular density in the normal tissue surrounding the tumor and consequently increased tumor hypoxia and apoptosis, and moderately decreased tumor growth. ZD6474 treatment resulted in decreased tumor vascular density accompanied by increased tumor hypoxia, apoptosis, and decreased tumor growth, suggesting that castration and antiangiogenic therapy work through similar mechanisms. Interestingly, castration or ZD6474 alone worked by reducing vascular density in the surrounding normal tissue and ZD6474 also in the tumor. Combined treatment with castration + ZD6474 was more effective than castration and ZD6474 alone in inducing tumor hypoxia, apoptosis, necrosis, and decreasing tumor vascular density.

Conclusion: These findings show that a drug that targets the vasculature in the tumor and in the surrounding ventral prostate lobe could mimic and even enhance the effects of castration. Our present findings thus suggest that castration + ZD6474 could be a particularly effective way to treat prostate tumors.
Targeting von Hippel-Lindau Pathway in Renal Cell Carcinoma
Authors: Patel Premal H. , Chadalavada Rajendrakumar S.V. , Chaganti R.S.K. , Motzer Robert J.
Published: December 2006

Inheritance of a defective copy of the von Hippel-Lindau (VHL) gene leads to the most common cause of inherited renal cell carcinoma (RCC). In addition, most patients with sporadic RCC have aberrant VHL. In the absence of VHL, hypoxia-inducible factor α accumulates, leading to production of several growth factors, including vascular endothelial growth factor and platelet-derived growth factor. We review here the biology of RCC and how a combination of proximal and distal block of VHL/hypoxia-inducible factor α pathway by novel targeted agents, including sunitinib, sorafenib, bevacizumab, everolimus, and temsirolimus, has led to significant improvements in progression-free survival.
Amplification of KIT, PDGFRA, VEGFR2, and EGFR in Gliomas
Authors: Puputti Marjut , Tynninen Olli , Sihto Harri , Blom Tea , Mäenpää Hanna , Isola Jorma , Paetau Anders , Joensuu Heikki , Nupponen Nina N.
Published: December 2006

Receptor tyrosine kinase aberrations are implicated in the genesis of gliomas. We investigated expression and amplification of KIT, PDGFRA, VEGFR2, and EGFR in 87 gliomas consisting of astrocytomas, anaplastic astrocytomas, oligodendrogliomas, or oligoastrocytomas in tumor samples collected at the time of the diagnosis and in samples of the same tumors at tumor recurrence. Gene amplifications were investigated using either chromogenic in situ hybridization or fluorescence in situ hybridization, and protein expression using immunohistochemistry. In samples collected at glioma diagnosis, KIT and PDGFRA amplifications were more frequent in anaplastic astrocytomas than in astrocytomas, oligodendrogliomas, and oligoastrocytomas [28% versus 5% (P = 0.012) and 33% versus 2% (P = 0.0008), respectively]. VEGFR2 amplifications occurred in 6% to 17% of the gliomas at diagnosis, and EGFR amplifications in 0% to 12%. Amplified KIT was more frequently present in recurrent gliomas than in newly diagnosed gliomas (P = 0.0066). KIT amplification was associated with KIT protein expression and with presence of PDGFRA and EGFR amplifications both at the time of the first glioma diagnosis and at tumor recurrence, and with VEGFR2 amplification at tumor recurrence. Three (4%) primary gliomas and 10 (14%) recurrent gliomas that were evaluable for coamplification of KIT, PDGFRA, and VEGFR2 showed amplification of at least two of these genes; the amplicon contained amplified KIT in all 13 cases. In conclusion, besides glioblastoma, amplified KIT, PDGFRA, and VEGFR may also occur in lower-grade gliomas and in their recurrent tumors. It is currently not known whether specific tyrosine kinase inhibitors are effective in the treatment of such gliomas. (Mol Cancer Res 2006;4(12):927–34)
Bub1 Up-Regulation and Hyperphosphorylation Promote Malignant Transformation in SV40 Tag–Induced Transgenic Mouse Models
Authors: Guo Conghui , Wu Guojun , Chin Joseph L. , Bauman Glenn , Moussa Madeleine , Wang Feng , Greenberg Norman M. , Taylor Stephen S. , Xuan Jim W.
Published: December 2006

Rodents do not naturally develop prostate cancer. Currently, most widely used genetically engineered mouse prostate cancer models use SV40 T/tag oncogene. To understand the mechanism underlying prostate cancer development in transgenic and knock-in SV40 Tag mouse models, we did cDNA microarray analyses, comparing gene expression profiles of prostate cancer tissues from early-, late-, and advance-stage androgen-independent prostate cancers. Of the 67 genes that were up-regulated by ≥10-fold, 40 are known to be required for chromosome stability. In particular, the spindle checkpoint component Bub1 was persistently up-regulated from early to advanced androgen-independent prostate cancer lesions. Significantly, Bub1, which is required for accurate chromosome segregation during mitosis, has recently been reported to bind SV40 Tag. Consistent with a spindle checkpoint defect, flow cytometry experiments indicate that advanced androgen-independent prostate cancer tumors exhibit aneuploidy, along with up-regulation of levels of both Bub1 mRNA and Bub1 protein or hyperphosphorylation. Importantly, up-regulation and hyperphosphorylation of Bub1 were also observed in established human prostate cancer cell lines and in clinical studies. Furthermore, analysis of human prostate cancer lines showed impaired spindle checkpoint function and endoreduplication following exposure to spindle toxins. Small interfering RNA–mediated repression of Bub1 in the human prostate cancer line PC-3 restrained cell proliferation, an effect mimicked by inhibition of mitogen-activated protein kinase, an upstream activator of Bub1. Thus, by perturbing Bub1 function, our observations suggest a new mechanism whereby the SV40 Tag oncoprotein promotes chromosomal instability and aneuploidy in transgenic mouse prostate cancer models. Whereas the exact details of this mechanism remain unclear, our novel findings raise the possibility of exploiting Bub1 as a new therapeutic target in the treatment of prostate cancer, the most common cancer in adult men in North America. (Mol Cancer Res 2006;4(12):957–69)
Fbw7 Isoform Interaction Contributes to Cyclin E Proteolysis
Authors: Zhang Wei , Koepp Deanna M.
Published: December 2006

The ubiquitin proteasome system plays important roles in regulating cell growth and proliferation. Many proteins that function in ubiquitin-mediated destruction have been linked to tumorigenesis. The putative tumor-suppressor protein Fbw7 (hAgo/hCdc4) is a specificity factor for the Skp1-Cul1-F-box protein ubiquitin ligase complex and targets a number of proto-oncogene products for ubiquitin-mediated destruction, including the cell cycle regulator cyclin E. In mammals, there are three splice variants of Fbw7 that use distinct first exons, resulting in proteins that have unique NH2 termini but are otherwise identical. Here, we show that the Fbw7 splice variants interact with each other through an NH2-terminal region common to all of the Fbw7 isoforms. Other F-box proteins have been shown to regulate substrate binding or turnover by forming homodimeric or heterodimeric complexes, which are dependent on a sequence motif called the D domain. Fbw7 and its orthologues exhibit significant sequence similarity to such F-box proteins, including the D domain. Fbw7 mutants that lack the region encompassing the D domain fail to bind other Fbw7 isoforms, despite being properly localized and binding both cyclin E and Skp1. Finally, we show the functional significance of this region as mutants lacking the NH2-terminal region involved in Fbw7 binding exhibit reduced rates of cyclin E protein turnover, indicating that Fbw7 isoform interaction is important for the efficiency of cyclin E turnover. Overall, this study contributes to the current understanding of the regulation of the Fbw7 tumor-suppressor protein. (Mol Cancer Res 2006;4(12):935–43)
Crack Cocaine: A Five-Year Follow-Up Study of Treated Patients
Authors: Ribeiro M. , Dunn J. , Sesso R. , Lima M.S. , Laranjeira R.
Published: December 2006

Objectives: To follow-up a group of 131 crack cocaine users and examine drug use, treatment experience, employment status, involvement in crime and mortality at 2 and 5 years. Methods: Consecutive crack-dependent patients who were admitted to a detoxification unit in São Paulo between 1992 and 1994 were re-interviewed on two occasions: 1995–1996 and 1998–1999. Results: 5 years after treatment information was obtained on 124 (95%) of the original cohort. 39.7% (n = 52) of the patients reported having been abstinent from cocaine for at least the last year, and 21.4% (n = 28) had used the drug. Of those subjects not using cocaine at 2 years, 19 (62%) were still abstinent at 5 years. Twenty-three (17.6%) patients had died by the 5-year follow-up with homicide, due to firearms or other weapons, being the commonest cause (n = 13). The annual adjusted mortality rate for the sample was 24.92 deaths/1,000 individuals, the excess mortality rate was of 21.64 deaths/1,000 individuals, and the standardized mortality ratio was 7.60. A history of injecting drug use, unemployment at the time of the index admission and administrative discharge at the index admission were factors that contributed to the risk of dying over the next 5 years. Conclusions: There was a progressive movement towards abstinence over the follow-up period, and there was evidence that once abstinence had been achieved it was maintained. On the other hand, the mortality rate was extremely high and was higher among those who were still using crack at 2 years.
Alcohol Screening in General Practices Using the AUDIT: How Many Response Categories Are Necessary?
Authors: Bischof G. , Grothues J. , Reinhardt S. , John U. , Meyer C. , Ulbricht S. , Rumpf H.-J.
Published: December 2006

Aims: The Alcohol Use Disorders Identification Test (AUDIT) is a common screening instrument. This study analyses if response categories of the AUDIT might be dichotomized without affecting the psychometric properties of the questionnaire. Methods: Participants between 18 and 65 years were recruited from general practices in two northern German cities. In total, 10,803 screenings were conducted (refusal rate: 5.9%). For those who were screened positive, the Munich-Composite International Diagnostic Interview (M-CIDI) was used for identification of 12-month Alcohol Use Disorders and at-risk consumption (exceeding 20/30 g per day). Abstinent subjects and screening positives without diagnostic interview were excluded from the analysis, leaving a sample of 7,112 subjects. ROC-Curves were calculated separately for each item in order to identify an optimal cut-off value. Finally, a version of the AUDIT based on dichotomized items was compared to the original version and its short-form, the AUDIT version based on three questions dealing with consumption AUDIT-C. Results: As an optimal cut-off value for items on consumption, drinking at least once a week, having more than 1–2 drinks per occasion, and drinking 6 or more drinks in one sitting at least once a month were identified. For all questions on alcohol-related problems or dependence symptoms, having ‘ever occurred’ differed best between subjects with and without Alcohol Use Disorders or at-risk consumption. Sensitivity and specificity of the dichotomized version of the AUDIT did not differ from the original version, and both full versions performed superior compared to the AUDIT-C. Conclusion: Data indicate that the AUDIT response categories may be dichotomized without affecting its validity.
The Relationship of Stress, Coping, Effect Expectancies and Craving
Authors: Grüsser Sabine Miriam , Mörsen Chantal Patricia , Wölfling Klaus , Flor Herta
Published: December 2006

Based on theoretical models of craving and addiction, this study investigated the association between stress-related variables and negatively and positively reinforcing dimensions of craving (relief and reward craving) in 150 opiate addicts, 150 alcohol addicts and 150 non-addicted controls. Stress-distress was the most powerful predictor of both dimensions of craving, followed by a lack of positive coping strategies whereas expectancies of substance effects seemed to be less important. Positive coping strategies were related to reduced craving only when they were accompanied by low stress-distress. In non-addicted subjects, only positive coping strategies were negatively related to craving. This study confirms the important role of stress-distress for the occurrence of craving in addicts.
Genomic Analysis of the Phosphotransferase System in Clostridium botulinum
Authors: Mitchell Wilfrid J. , Tewatia Parul , Meaden Philip G.
Published: December 2006

Clostridium botulinum is capable of fermenting carbohydrates, but there have been no detailed studies of the uptake of sugars and related substrates. In bacteria, a common and often predominant system of carbohydrate uptake is the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS). This multi-protein complex catalyses a group translocation involving both uptake and phosphorylation of carbohydrates, and is also known to play an important role in environmental sensing and metabolic regulation. The genome of C. botulinum encodes 15 PTSs which have a similar domain structure to the PTS in other bacteria. Based on phylogenetic relationships and analysis of gene clusters, the C. botulinum PTS appears to be involved in the uptake of hexoses, hexose derivatives and disaccharides. C. botulinum also contains the components of PTS-associated regulatory mechanisms which have been characterised in other bacteria. It therefore seems likely that the PTS plays a significant, and previously unrecognised, role in the physiology of this bacterium.
Conserved cis-Acting Elements Upstream of Genes Composing the Chitinolytic System of Streptomycetes Are DasR-Responsive Elements
Authors: Colson Séverine , Stephan Joachim , Hertrich Tina , Saito Akihiro , van Wezel Gilles P. , Titgemeyer Fritz , Rigali Sébastien
Published: December 2006

For soil-dwelling bacteria that usually live in a carbon-rich and nitrogen-poor environment, the ability to utilize chitin – the second most abundant polysaccharide on earth – is a decisive evolving advantage as it is a source for both elements. Streptomycetes are high-GC Gram-positive soil bacteria that are equipped with a broad arsenal of chitinase-degrading genes. These genes are induced when the streptomycetes sense the presence of chitooligosaccharides. Their expression is repressed as soon as more readily assimilated carbon sources become available. This includes for example glucose or N-acetylglucosamine, the monomer subunit of chitin. Historically, the first cis-acting elements involved in carbon regulation in streptomycetes were found more than a decade ago upstream of chitinase genes, but the transcriptional regulator had so far remained undiscovered. In this work, we show that these cis-acting elements consist of inverted repeats with multiple occurrences and are bound by the HutC/GntR type regulator DasR. We have therefore designated these sites as DasR-responsive elements (dre). DasR, which is also the repressor of the genes for the N-acetylglucosamine-specific phosphotransferase transport system, should therefore play a critical role in sensing the balance between the monomeric and polymeric forms of N-acetylglucosamine.
CcpA Mutants with Differential Activities in Bacillus subtilis
Authors: Sprehe Mareen , Seidel Gerald , Diel Marco , Hillen Wolfgang
Published: December 2006

CcpA is the master regulator for carbon catabolite regulation in Bacillus subtilis and regulates more than 300 genes by repression or activation. To revealthe effects of different functional domains of CcpA on various regulatory modes, we compared the activities of CcpA point mutants in activation (alsS, ackA) and repression (xynP, gntR). CcpA variants mutated at residues in the HPrSerP-binding region without allosteric functions are inactive. On the other hand, CcpA variants mutated at residues that change their conformation upon HPrSerP or CrhP binding regulate only ackA. Another set of mutants with alterations in the corepressor-binding region show glucose-independent regulation of xynP. The data presented here demonstrate the involvement of HPrSerP and/or CrhP in activation of ackA and alsS by CcpA. Furthermore, these data also indicatethat activation and repression mediated by CcpA may utilize different conformational changes of the protein.