Vitamin D and its analog EB1089 induce p27 accumulation and diminish association of p27 with Skp2 independent of PTEN in pituitary corticotroph cells

Disruption of the gene for the cyclin dependent kinase inhibitor (CDKI) p27/kip1 results in pituitary corticotroph hyperplasia while diminished expression of this protein has been described in aggressive human pituitary tumors. We have previously shown that 1,25-vitamin D3 (VD) hypophosphorylates p27 and interferes with the degradation of this CDKI in thyroid carcinoma cells. In this study we investigated whether VD/EB1089 can induce p27 accumulation and cause growth arrest of pituitary corticotroph cells. VD and EB1089 exhibited a significant reduction in AtT20 corticotroph but not PRL235 lactotroph cell growth. These changes were accompanied by selective accumulation of p27 in AtT20 but not in PRL235 cells. As p27 levels are highly dependent on protein degradation, we examined the effect of VD/EB1089 on p27 association with factors that target this CDKI to the proteasome. VD/EB1089 significantly restricted the association of p27 with Skp2 as well as with cyclin dependent kinase 2 (CDK2). As the tumor suppressor and phosphatase PTEN has been implicated in p27 regulation, we tested whether the effects of VD/EB1089 on p27 accumulation in corticotrophs could be mediated through this pathway. VD/EB1089 did not appreciably alter PTEN expression. Moreover, transfection of PTEN did not influence the effect of VD on p27 accumulation in corticotrophs. We conclude that VD/EB1089 can selectively arrest pituitary corticotroph growth and induce p27 accumulation.This effect is mediated at least partially through diminished p27 association with Skp2 and with CDK2. In contrast to other cell systems, PTEN does not participate in the regulation of corticotroph p27 and is not involved in mediating the effect of VD on p27 in these cells. Our findings highlight p27 and VD analogs as targets for manipulation and drug development respectively in the treatment of inoperable corticotroph adenomas.     

Hypothalamic paraventricular nucleus inhibition decreases renal sympathetic nerve activity in hypertensive and normotensive rats

Activity of the hypothalamic paraventricular nucleus (PVN) is essential for the maintenance of vasomotor sympathetic nerve discharge (SND) and blood pressure even in the anesthetized rat. Inactivation of the paraventricular nucleus results in a large depressor and sympathoinhibitory response. The current study was designed to examine the regulation of renal sympathetic nerve activity by the paraventricular nucleus in both hypertensive and normotensive rats. Experiments were performed in anesthetized, artificially ventilated spontaneously hypertensive (SH) and Wistar-Kyoto (WKY) rats. Renal sympathetic nerve activity, blood pressure and heart rate were recorded. Bilateral microinjections of the GABA(A) receptor agonist, muscimol (1 nmol in 100 nl), were made into the paraventricular nucleus. Decreases in blood pressure (SHR: from 111+/-3 to 54+/-4 mm Hg; WKY: 84+/-2 to 48+/-3 mm Hg), heart rate (SHR: 336+/-8 to 289+/-12 bpm; WKY 309+/-7 to 258+/-13 bpm) and renal sympathetic nerve activity (to 46+/-11% and 33+/-7% of control in the WKY and SHR, respectively) were observed. The renal nerve response to inactivation of the paraventricular nucleus was not different between the strains, indicating that modulation of renal sympathetic nerve activity by the paraventricular nucleus is similar in these rat strains. This is different from the previously reported effect of paraventricular nucleus inhibition on lumbar sympathetic nerve activity [Hypertension 39 (2002) 275]. Overall, we demonstrate that the paraventricular nucleus plays a critical role in the regulation of renal SND even under basal conditions in anesthetized animals.     

Profiles and time course of acute radiation toxicity symptoms during conformal radiotherapy for cancer of the prostate

Symptoms of gastrointestinal toxicity are dose-limiting for pelvic radiotherapy (RT). Existing toxicity registrations (RTOG/EORTC) are helpful in defining maximal tolerated doses, but tend to underestimate the total toxicity burden by excluding several minor complaints. We have applied a more detailed and quantitative recording of symptoms and related these scores to RT-induced endoscopic and histopathologic changes. Prevalence and severity of specific toxicity symptoms were recorded before, during (weeks 2 and 6) and 2 and 8 weeks after RT in 96 patients undergoing external beam RT for localized prostate cancer. RTOG/EORTC acute toxicity and ad hoc total toxicity scores (TTS) were recorded. TTS scores were calculated by adding scores based on visual analog scale (VAS) grading of individual symptoms. Fifty of the patients also underwent sequential proctoscopy with mucosal biopsy. Individual symptoms increased, but differed in prevalence and intensity during and after RT. TTS increased during the entire treatment course in spite of normalizing histopathologic and endoscopic changes from week 2 onwards. Twenty-seven patients had no RTOG/EORTC toxicity, four had grade 3 and none had grade 4 toxicity. All patients with grade 0 had increased TTS. Thus, TTS appeared more sensitive than RTOG/EORTC scoring. The study demonstrates that multiple toxicity symptoms contribute to total toxicity in response to pelvic RT. TTS is a feasible and sensitive method for detecting and quantifying acute toxicity and unveils morbidity which remains hidden with the RTOG/EORTC score system. The development and timing of symptoms may give clues to pathogenesis, treatment, and prophylaxis.     

Smoking and the risk of gastric cancer in the European Prospective Investigation Into Cancer and Nutrition (EPIC)

Smoking has recently been recognised as causally associated with the development of gastric cancer (GC). However, evidence on the effect by sex, duration and intensity of smoking, anatomic subsite and cessation of smoking is limited. Our objective was to assess the relation between tobacco use and GC incidence in the European Prospective Investigation into Cancer and Nutrition (EPIC). We studied data from 521,468 individuals recruited from 10 European countries taking part in the EPIC study. Participants completed lifestyle questionnaires that included questions on lifetime consumption of tobacco and diet in 1991-1998. Participants were followed until September 2002, and during that period 305 cases of stomach cancer were identified. After exclusions, 274 were eligible for the analysis, using the Cox proportional hazard model. After adjustment for educational level, consumption of fresh fruit, vegetables and preserved meat, alcohol intake and body mass index (BMI), there was a significant association between cigarette smoking and gastric cancer risk: the hazard ratio (HR) for ever smokers was 1.45 (95% confidence interval [CI] = 1.08-1.94). The HR of current cigarette smoking was 1.73 (95% CI = 1.06-2.83) in males and 1.87 (95% CI = 1.12-3.12) in females. Hazard ratios increased with intensity and duration of cigarette smoked. A significant decrease of risk was observed after 10 years of quitting smoking. A preliminary analysis of 121 cases with identified anatomic site showed that current cigarette smokers had a higher HR of GC in the cardia (HR = 4.10) than in the distal part of the stomach (HR = 1.94). In this cohort, 17.6 % (95% CI = 10.5-29.5 %) of GC cases may be attributable to smoking. Findings from this large study support the causal relation between smoking and gastric cancer in this European population. Stomach cancer should be added to the burden of diseases caused by smoking.     

Early onset of rejection in concordant hamster xeno hearts display signs of necrosis, but not apoptosis, correlating to the phosphocreatine concentration

BACKGROUND: The importance of apoptosis contra necrosis for ischemia/reperfusion (RP) and acute rejection in concordant rodent xenotransplantation is largely unknown. We explored this question by comparing rodent allo and concordant xenotransplants with different morphological methods to detect apoptosis and biochemical data on the levels of high-energy phosphates obtained with in vitro 31Phosphorous Magnetic Resonance Spectroscopy (31P MRS). More specifically, we applied a hitherto unused method in transplantation research, apoptosis specific biotin labeled oligonucleotides designed with a 10 base pair stem region and a 20 nucleotides large loop that form a hairpin like shape. The results obtained with this method were compared to results obtained with the more widely used in situ 3'-end labeling of DNA (TUNEL) assay and extraction and gel electrophoresis of labeled DNA (DNA laddering). METHODS: Cervical heart transplantations were performed between inbred Lewis (L) (RT1l) to L, L to DA (RT1a) rats, hamster (H) to H and H to L (X) (n=5 for all groups except for X, n=9). All hearts were subjected to 30 min of cold ischemia (+4 degrees C) and 6 h of RP before explantation. In vitro 31P MRS was used to determine the phosphocreatine (PCr), beta-adenosine triphosphate (beta-ATP) concentrations and the PCr/beta-ATP ratio of the transplants. We correlated the biochemical data to haematoxylin and eosin (H & E) stained tissue slides scored for rejection, infiltration of antibodies and complement depositions, DNA extraction and gel electrophoresis of labeled DNA (DNA laddering), in situ 3'-end labeling of DNA (TUNEL) and the apoptosis specific hairpin probe assays scoring. RESULTS: The rejection score of the xeno grafts differed significantly compared to their syngeneic hamster to hamster controls (2.40 +/- 0.25 vs. 1.20 +/- 0.20; P=0.005) and they had a significantly higher TUNEL score, 228 +/- 15 vs. 2.44 +/- 0.32 (P=0.009), that correlated to changes in PCr concentration (P<0.001) and to the PCr/beta-ATP ratio (P=0.01). The uptake was mainly (90-95%) located to 1-2 microm large extra cellular 'granule'. A picture resembling early necrosis was seen on the H & E stainings and reflected in the Billingham rejection score above. CONCLUSIONS: After 6 h of RP the onset of acute rejection in the concordant hamster xeno hearts displayed features of early, possibly mitochondrial, necrosis, but not apoptosis, which correlated to changes in the PCr concentration and the PCr/beta-ATP ratio. The mechanism for the early rejection observed is unclear and might be caused by other factors in the sera apart from cellular components, antibodies and complement factors. Identification of the underlying mechanisms could enable us to design rational therapies that prevent activation of the recipient's innate immune response.     

Vasoactive intestinal peptide and nitric oxide promote survival of adult rat myenteric neurons in culture

Several motility disorders originate in the enteric nervous system (ENS). Our knowledge of factors governing survival of the ENS is poor. Changes in the expression of vasoactive intestinal peptide (VIP) and nitric oxide synthase (NOS) in enteric neurons occur after neuronal injury and in intestinal adaptation. The aim of this study was to evaluate whether VIP and nitric oxide (NO) influence survival of cultured, dissociated myenteric neurons. Neuronal survival was evaluated after 0, 4, and 8 days in culture. Influence of VIP and NO on neuronal survival was examined after culturing in the presence of VIP, NO donor, VIP antiserum, or NOS inhibitor. A marked loss of neurons was noted during culturing. VIP and NO significantly promoted neuronal survival. Corroborating this was the finding of an enhanced neuronal cell loss when cultures were grown in the presence of VIP antiserum or NOS inhibitor.