Effects of gender on locomotor sensitivity to amphetamine, body weight, and fat mass in regulator of G protein signaling 9 (RGS9) knockout mice.

Regulator of G-protein signaling (RGS) protein 9-2 is enriched in the striatum where it modulates dopamine and opioid receptor-mediated signaling. RGS9 knockout (KO) mice show increased psychostimulant-induced behavioral sensitization, as well as exhibit higher body weights and greater fat accumulation compared to wild-type (WT) littermates. In the present study, we found gender influences on each of these phenotypic characteristics. Female RGS9 KO mice exhibited greater locomotor sensitization to amphetamine (1.0mg/kg) treatment as compared to male RGS9 KO mice. Male RGS9 KO mice showed increased body weights as compared to male WT littermates, while no such differences were detected in female mice. Quantitative magnetic resonance showed that male RGS9 KO mice accumulated greater fat mass vs. WT littermates at 5months of age. Such observations could not be explained by increased caloric consumption since male and female RGS9 KO mice demonstrated equivalent daily food intake as compared to their respective WT littermates. Although indirect calorimetry methods found decreased oxygen consumption and carbon dioxide production during the 12-hour dark phase in male RGS9 KO vs. WT mice which are indicative of less energy expenditure, male RGS9 KO mice exhibited lower levels of locomotor activity during this period. Genotype had no effect on metabolic activities when KO and WT groups were compared under fasting vs. feeding treatments. In summary, these results highlight the importance of factoring gender into the experimental design since many studies conducted in RGS9 KO mice utilize locomotor activity as a measured outcome.

A Tet-on system for DRD1-expressing cells.

Cells expressing the dopamine D1 receptor (DRD1) have significant functional roles in diverse physiological processes including locomotion and drug addiction. The present work presents a novel in vivo DRD1-Bacterial Artificial Chromosome (BAC) Tet-on system allowing for the inducible activation of tet-operated transgenes specifically within DRD1-expressing cells of transgenic mice. It is shown that the DRD1-rtTA BAC-driven expression of a tet-operated reporter is under tight regulation by doxycycline and is restricted to DRD1-expressing brain regions. The model will be a useful research tool in studies of movement and reward and associated pathologies such as Parkinson's disease and addiction.

Analysis and correction of crosstalk effects in pathway analysis.

Identifying the pathways that are significantly impacted in a given condition is a crucial step in understanding the underlying biological phenomena. All approaches currently available for this purpose calculate a P-value that aims to quantify the significance of the involvement of each pathway in the given phenotype. These P-values were previously thought to be independent. Here we show that this is not the case, and that many pathways can considerably affect each other's P-values through a "crosstalk" phenomenon. Although it is intuitive that various pathways could influence each other, the presence and extent of this phenomenon have not been rigorously studied and, most importantly, there is no currently available technique able to quantify the amount of such crosstalk. Here, we show that all three major categories of pathway analysis methods (enrichment analysis, functional class scoring, and topology-based methods) are severely influenced by crosstalk phenomena. Using real pathways and data, we show that in some cases pathways with significant P-values are not biologically meaningful, and that some biologically meaningful pathways with nonsignificant P-values become statistically significant when the crosstalk effects of other pathways are removed. We describe a technique able to detect, quantify, and correct crosstalk effects, as well as identify independent functional modules. We assessed this novel approach on data from four experiments involving three phenotypes and two species. This method is expected to allow a better understanding of individual experiment results, as well as a more refined definition of the existing signaling pathways for specific phenotypes.

Comparison of toxicity associated with early morning versus late afternoon radiotherapy in patients with head-and-neck cancer: a prospective randomized trial of the National Cancer Institute of Canada Clinical Trials Group (HN3).

PURPOSE: Based on our demonstration of a circadian rhythm in the human oral mucosa cell cycle, with most cells in the G(1) phase in the morning and M phase at night, we hypothesized that morning radiotherapy (RT) would lead to less oral mucositis than afternoon RT. METHODS AND MATERIALS: A total of 216 patients were randomized to morning (8-10 AM) vs. afternoon (4-6 PM) RT and stratified by radiation dose, smoking status, and center. Patients receiving primary or postoperative RT alone were eligible. Oral mucositis was scored using the Radiation Therapy Oncology Group (RTOG) criteria and a validated scoring system. RESULTS: Of 205 evaluable patients, 52.9% vs. 62.4% developed RTOG Grade 3 or greater mucositis after morning vs. afternoon RT, respectively (p = 0.17). Morning RT was also associated with significantly less weight loss after 5 months (p = 0.024). In a subgroup of 111 patients treated to a dose of 66-70 Gy in 33-35 fractions, exploratory analyses revealed a significant reduction in Grade 3 or greater mucositis with morning RT (44.6% vs. 67.3%, p = 0.022) and a longer interval to the development of Grade 3 or greater mucositis (median, >7.9 vs. 5.6 weeks, p = 0.033). In 53 patients, who smoked during therapy, a significant reduction was found in Grade 3 or greater mucositis with morning RT (42.9% vs. 76%, p = 0.025). CONCLUSION: In this proof of principle study, morning RT was associated with significantly less weight loss after 5 months and an apparent reduction in oral mucositis in a subset of patients receiving >/=66 Gy and in patients who smoked during therapy.

Signal transducer and activator of transcription-3 is required in hypothalamic agouti-related protein/neuropeptide Y neurons for normal energy homeostasis.

Signal transducer and activator of transcription (Stat)-3 signals mediate many of the metabolic effects of the fat cell-derived hormone, leptin. In mice, brain-specific depletion of either the long form of the leptin receptor (Lepr) or Stat3 results in comparable obese phenotypes as does replacement of Lepr with an altered leptin receptor locus that codes for a Lepr unable to interact with Stat3. Among the multiple brain regions containing leptin-sensitive Stat3 sites, cells expressing feeding-related neuropeptides in the arcuate nucleus of the hypothalamus have received much of the focus. To determine the contribution to energy homeostasis of Stat3 expressed in agouti-related protein (Agrp)/neuropeptide Y (Npy) arcuate neurons, Stat3 was deleted specifically from these cells, and several metabolic indices were measured. It was found that deletion of Stat3 from Agrp/Npy neurons resulted in modest weight gain that was accounted for by increased adiposity. Agrp/Stat3-deficient mice also showed hyperleptinemia, and high-fat diet-induced hyperinsulinemia. Stat3 deletion in Agrp/Npy neurons also resulted in altered hypothalamic gene expression indicated by increased Npy mRNA and decreased induction of suppressor of cytokine signaling-3 in response to leptin. Agrp mRNA levels in the fed or fasted state were unaffected. Behaviorally, mice without Stat3 in Agrp/Npy neurons were mildly hyperphagic and hyporesponsive to leptin. We conclude that Stat3 in Agrp/Npy neurons is required for normal energy homeostasis, but Stat3 signaling in other brain areas also contributes to the regulation of energy homeostasis.

Collective and individual functions of leptin receptor modulated neurons controlling metabolism and ingestion.

Two known types of leptin-responsive neurons reside within the arcuate nucleus: the agouti gene-related peptide (AgRP)/neuropeptide Y (NPY) neuron and the proopiomelanocortin (POMC) neuron. By deleting the leptin receptor gene (Lepr) specifically in AgRP/NPY and/or POMC neurons of mice, we examined the several and combined contributions of these neurons to leptin action. Body weight and adiposity were increased by Lepr deletion from AgRP and POMC neurons individually, and simultaneous deletion in both neurons (A+P LEPR-KO mice) further increased these measures. Young (periweaning) A+P LEPR-KO mice exhibit hyperphagia and decreased energy expenditure, with increased weight gain, oxidative sparing of triglycerides, and increased fat accumulation. Interestingly, however, many of these abnormalities were attenuated in adult animals, and high doses of leptin partially suppress food intake in the A+P LEPR-KO mice. Although mildly hyperinsulinemic, the A+P LEPR-KO mice displayed normal glucose tolerance and fertility. Thus, AgRP/NPY and POMC neurons each play mandatory roles in aspects of leptin-regulated energy homeostasis, high leptin levels in adult mice mitigate the importance of leptin-responsiveness in these neurons for components of energy balance, suggesting the presence of other leptin-regulated pathways that partially compensate for the lack of leptin action on the POMC and AgRP/NPY neurons.

Signal transducer and activator of transcription (stat) binding sites but not stat3 are required for fasting-induced transcription of agouti-related protein messenger ribonucleic acid.

Energy homeostasis depends on the regulation of hypothalamic neurons by leptin, an adipocyte hormone whose circulating levels communicate body energy stores. Leptin activates the transcription factor signal transducer and activator of transcription 3 (Stat3) in hypothalamic neurons, including neuronal subtypes producing Agouti-related protein (Agrp), a neuropeptide that stimulates feeding. Previous studies have suggested a model in which high levels of Agrp transcription during fasting represent a default state that is actively repressed by phospho-Stat3 induced by leptin signaling in the fed state. We identify putative Stat3 binding elements in the Agrp promoter that have been highly conserved during vertebrate evolution. Using a reporter assay in transgenic mice that faithfully recapitulates normal regulation of Agrp, we show that these sites are required, but in a way opposite to that predicted by the existing model: mutation of the sites leads to a default state characterized by a low level of Agrp transcription and insensitivity to fasting. We also find that removing activatable Stat3 from Agrp neurons has no detectable effect on steady-state levels of Agrp mRNA in the fed or fasted state. These results suggest a new model for transcriptional regulation of orexigenic neuropeptides in which the default level of expression is low in the fed state, and transcriptional activation in response to fasting is mediated by factors other than Stat3.

Compromised local control due to treatment interruptions and late treatment breaks in early glottic cancer: Population-based outcomes study supporting need for intensified treatment schedules.

PURPOSE: This population-based study describes the treatment of early glottic cancer in Ontario, Canada and assesses whether treatment variations were associated with treatment effectiveness. METHODS AND MATERIALS: We studied 491 T1N0 and 213 T2N0 patients. Data abstracted from charts included age, sex, stage, treatment details, disease control, and survival. RESULTS: The total dose ranged from 50 to 70 Gy, and the daily dose ranged from 1.9 to 2.8 Gy. In 90%, treatment duration was between 25 and 50 days. Field sizes, field reductions, beam arrangement, and beam energy varied. Late treatment breaks occurred in 13.6% of T1N0 and 27.1% of T2N0 cases. Local control was comparable to other reports for T1N0 (82% at 5 years), but was only 63.2% in T2N0. Variables associated with local failure in T1N0 were age less than 49 years (relative risk [RR], 3.21; 95% confidence interval [CI], 1.49-6.90) and >3 treatment interruption days (RR, 2.43; 95% CI, 1.00-5.91). In T2N0, these were field reduction (RR, 2.33; 95% CI, 1.23-4.42) and late treatment breaks (RR, 2.19; 95% CI, 1.09-4.41). CONCLUSION: Some aspects of treatment for early glottic cancer were associated with worse local control. Problems with protracted treatment are of particular concern, underscoring the need for randomized studies to intensify radiotherapy.

Obesity-associated mutations in the human melanocortin-4 receptor gene.

Mutations in the human melanocortin-4 receptor (MC4R) gene have been associated with severe obesity. Many of the mutations result in partial or complete loss-of-function based on the nature of the mutation or the function of mutated receptors when tested in heterologous expression systems. This review discusses the role of MC4R in the central regulation of body weight, the pathogenic mechanisms of the mutations, and the validity of MC4R as an anti-obesity drug target.

Microarray analysis of fluoro-gold labeled rat dopamine neurons harvested by laser capture microdissection.

The cellular heterogeneity of brain tissue presents a challenge to gene expression profiling of specific neuronal cell types. The present study employed a fluorescent neural tracer to specifically label midbrain dopamine neurons and non-dopamine cortical neurons. The labeled cells were then used to visually guide harvesting of the cells by laser capture microdissection (LCM). RNA extracted from the two populations of harvested cells was then amplified, labeled and co-hybridized to high density cDNA microarrays for two-color differential expression profiling. Many of the genes most highly enriched in the dopamine neurons were found to be genes previously known to define the dopamine neuronal phenotype. However, results from the microarray were only partially validated by quantitative RT-PCR analysis. The results indicate that LCM harvesting of specific neuronal phenotypes can be effectively guided in a complex cellular environment by specific pre-labeling of the target cell populations and underlie the importance of independent validation of microarray results.

Cell surface expression of the melanocortin-4 receptor is dependent on a C-terminal di-isoleucine sequence at codons 316/317.

Loss-of-function mutations in the human melanocortin-4 receptor (MC4R) are associated with obesity. Previous work has implicated a C-terminal di-isoleucine motif at residues 316/317 in MC4R cell surface targeting. It was therefore of interest to examine function and cell surface expression of an MC4R mutation found in an obese proband in which one of these isoleucines was substituted by threonine (I317T). Single mutant (I316T or I317T) and double mutant (I316T,I317T) forms of MC4R were constructed by oligonucleotide-directed mutagenesis and tested for function and cell surface expression in transfected cells. Function was assessed using assays for agonist, [Nle(4)-d-Phe(7)]alpha-melanocyte-stimulating hormone (NDP-alpha-MSH) or forskolin-stimulated cAMP accumulation. Cell surface expression was determined by whole-cell binding of [(125)I]NDP-alpha-MSH, fluorescence immunocytochemistry and fluorescence-activated cell sorting. Maximal cAMP generation of the single mutants was reduced by 40% of wild-type receptor; the double mutant further reduced function to 40% of control, effects that were mirrored by decreases in cell-surface expression. Quantitative RT-PCR showed that, relative to wild-type receptor, transcript levels for the mutated receptors were not reduced. The results further implicate the C-terminal di-isoleucines in cell surface expression of MC4R and suggest that mutations of residues 316 or 317 would predict MC4R hypofunction.

Management and outcome differences in supraglottic cancer between Ontario, Canada, and the Surveillance, Epidemiology, and End Results areas of the United States.

PURPOSE: We compared the management and outcome of supraglottic cancer in Ontario, Canada, with that in the Surveillance, Epidemiology, and End Results (SEER) Program areas in the United States. METHODS: Electronic, clinical, and hospital data were linked to cancer registry data and supplemented by chart review where necessary. Stage-stratified analyses compared initial treatment and survival in the SEER areas (n = 1,643) with a random sample from Ontario (n = 265). We also compared laryngectomy rates at 3 years in those patients 65 years and older at diagnosis. RESULTS: Radical surgery was more commonly used in SEER, with absolute differences increasing with increasing stage: I/II, 17%; III, 36%; and IV, 45%. The 5-year survival rates were 74% in Ontario and 56% in SEER for stage I/II disease (P =.01), 55.7% in Ontario and 46.8% in SEER for stage III disease (P =.40), and 28.5% in Ontario and 29.1% in SEER for stage IV disease (P =.28). Cancer-specific survival results mirrored the overall survival results with the exception of stage IV disease, for which 34.6% of Ontario patients survived their cancer compared with 38.1% in SEER (P =.10). This stage IV difference was more pronounced when we further controlled for possible cause of death errors by restricting the comparison to patients with a single primary cancer (P =.01). Three-year actuarial laryngectomy rates differed. In stage I/II, these rates were 3% in Ontario compared with 35% in SEER (P < 10(-3)). In stage III disease, the rates were 30% and 54%, respectively (P =.03), and in stage IV disease they were 33% and 64% (P =.002). CONCLUSION: There are large differences in the management of supraglottic cancer between the SEER areas of the United States and Ontario. Long-term larynx retention was higher in Ontario, where radiotherapy is widely regarded as the treatment of choice and surgery is reserved for salvage. In stages I to III, survival was similar in the two regions despite the differences in treatment policy. In stage IV, there may be a small survival advantage in the U.S. SEER areas related to the higher use of primary surgery.

Does Catecholamine Secretion from Head and Neck Paragangliomas Respond to Radiotherapy? Case Report and Literature Review.

We report a 38-year-old woman with a malignant catecholamine-secreting vagal paraganglioma. In the preceding year she had intermittent severe frontoparietal headaches. While she was receiving radiotherapy (35 Gy in 15 fractions) she developed palpitations, which steadily worsened over the following three years. A repeat CT scan showed no change in the size of the tumor. Urinary catecholamines were elevated and a MIBG scan showed increased uptake in the region of the vagal paraganglioma. She underwent near total resection of her tumor via a Fisch type C approach. Pathological examination showed a paraganglioma with metastasis involving a cervical lymph node. The palpitations and headaches resolved completely after surgery. This report and other published case reports suggest that catecholamine secretion from head and neck paragangliomas does not appear to respond to radiotherapy. Patients with cardiovascular symptoms from catecholamine-secreting paragangliomas are best managed surgically.

Association between smoking during radiotherapy and prognosis in head and neck cancer: a follow-up study.

BACKGROUND: The study objective was to confirm a previous finding that patients with stage III/IV squamous head and neck cancer (SHNC) who smoke during radiotherapy (RT) experience reduced survival. METHODS: An observational cohort study. Patients' smoking status was assessed weekly by questionnaire plus blood cotinine. Patients were assessed every 3 to 4 months for survival. Logistic regression and Cox proportional hazards analyses were used to detect the independent contribution of smoking on survival. RESULTS: Of 148 patients, 113 smoked during RT. Blood cotinine and smoking questionnaire responses were highly correlated (Spearman R = .69; p < .0005). Abstainers and very light smokers experienced better survival than light, moderate, and heavy smokers (median, 42 vs 29 months; p = .07). Tumor and nodal status and years smoked were the most important prognostic factors. Smoking during RT was not an independent predictor of survival, but baseline smoking status was (p = .016). CONCLUSION: Smoking status should be documented in all future trials of RT in SHNC to allow for pooled analyses with sufficient power to address this question.

A comparison of published head and neck stage groupings in laryngeal cancer using data from two countries.

The combination of T, N, and M classifications into stage groupings is meant to facilitate a number of activities including: the estimation of prognosis and the comparison of therapeutic interventions among similar groups of cases. We tested the UICC/AJCC fifth edition stage grouping and six other TNM-based groupings proposed for head and neck cancer for their ability to meet these expectations in laryngeal cancer using data from Ontario, Canada, and the area of Southern Norway surrounding Oslo. We defined four criteria to assess each grouping scheme: (1) the subgroups defined by T, N, and M comprising a given group within a grouping scheme have similar survival rates (hazard consistency); (2) the survival rates differ among the groups (hazard discrimination); (3) the prediction of cure is high (outcome prediction); and (4) the distribution of patients among the groups is balanced. We previously identified or derived a measure for each criterion, and the findings were summarized using a scoring system. The range of scores was from 0 (best) to 7 (worst). The data sets were population-based, with 861 cases from Ontario and 642 cases from Southern Norway. Clinical stage assignment was used and the outcome of interest was cause-specific survival. Summary scores across the seven schemes had similar ranges: 0.9 to 5.1 in Ontario and 1.8 to 5.7 in Southern Norway, but the ranking varied. Summing the scores across the two datasets, the TANIS-7 scheme (Head & Neck 1993;15:497-503) ranked first, and was ranked high in both datasets (first and second, respectively). The UICC/AJCC scheme ranked sixth out of seven schemes, and its ranking was fifth and seventh, respectively. UICC/AJCC stage groupings were defined without empirical investigation. When tested, this scheme did not perform best. Our results suggest that the usefulness of the TNM system could be enhanced by optimizing the design of stage groupings through empirical investigation.