1,25(OH)2Vitamin D3 induces elevated expression of the cell cycle inhibitor p18 in a squamous cell carcinoma cell line of the head and neck

BACKGROUND: 1Alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2) Vitamin D(3)] induces growth inhibition in squamous cell carcinoma (SCC) cell lines of the head and neck by arresting the cells in the G0/G1 phase of the cell cycle, probably due to an enhanced expression of p21, which could be demonstrated in other cell lines (JPPA, SCC9) before. In SCC25, a SCC cell line isolated from tongue, growth inhibition but no overexpression of p21 was detected. The retinoblastoma gene, as a direct target of G1 cyclin-CDK complexes, showed an obvious shift from the hyperphosphorylated to the hypophosphorylated form under 1,25(OH)(2)Vitamin D(3), which indicates that the growth inhibition takes place in the G0/G1 phase. To explore the possible pathway of growth inhibition in SCC25 we investigated other cell cycle inhibitors (p18, p19, p27). METHODS: Synchronized cells were treated with 1,25(OH)(2)Vitamin D(3) over 96 h. The cell cycle status and expression of cell cycle-regulating proteins was determined by fluorescence-activated cell sorting (FACS) and Western blotting. An overexpression of p18 in 1,25(OH)(2)Vitamin D(3) vs. ethanol-treated cells was determined until 30 h in SCC25. No influence was detectable on the expression of p27 and p19. CONCLUSION: One mechanism by which 1,25(OH)(2)Vitamin D(3) controls cell growth might be the upregulation of p21. As p21 was unsusceptible to 1,25(OH)(2)Vitamin D(3) in SCC25, other inhibiting proteins were necessary to be tested. The proven upregulation of p18 seems to be the responsible step for growth inhibition of 1,25(OH)(2)Vitamin D(3) in SCC25.     

Regulation of osteoblast differentiation by Pasteurella multocida toxin (PMT): a role for Rho GTPase in bone formation.

The role of the Rho-Rho kinase signaling pathway on osteoblast differentiation was investigated using primary mouse calvarial cells. The bacterial toxin PMT inhibited, whereas Rho-ROK inhibitors stimulated, osteoblast differentiation and bone nodule formation. These effects correlated with altered BMP-2 and -4 expression. These data show the importance of Rho-ROK signaling in osteoblast differentiation and bone formation. INTRODUCTION: The signal transduction pathways controlling osteoblast differentiation are not well understood. In this study, we used Pasteurella multocida toxin (PMT), a unique bacterial toxin that activates the small GTPase Rho, and specific Rho inhibitors to investigate the role of Rho in osteoblast differentiation and bone formation in vitro. MATERIALS AND METHODS: Primary mouse calvarial osteoblast cultures were used to investigate the effects of recombinant PMT and Rho-Rho kinase (ROK) inhibitors on osteoblast differentiation and bone nodule formation. Osteoblast gene expression was analyzed using Northern blot and RT-PCR, and actin rearrangements were visualized after phalloidin staining and confocal microscopy. RESULTS: PMT stimulated the proliferation of primary mouse calvarial cells and markedly inhibited the differentiation of osteoblast precursors to bone nodules with a concomitant inhibition of osteoblastic marker gene expression. There was no apparent causal relationship between the stimulation of proliferation and inhibition of differentiation. PMT caused cytoskeletal rearrangements because of activation of Rho, and the inhibition of bone nodules was completely reversed by the Rho inhibitor C3 transferase and partly reversed by inhibitors of the Rho effector, ROK. Interestingly, Rho and ROK inhibitors alone potently stimulated osteoblast differentiation, gene expression, and bone nodule formation. Finally, PMT inhibited, whereas ROK inhibitors stimulated, bone morphogenetic protein (BMP)-2 and -4 mRNA expression, providing a possible mechanism for their effects on bone nodule formation. CONCLUSIONS: These results show that PMT inhibits osteoblast differentiation through a mechanism involving the Rho-ROK pathway and that this pathway is an important negative regulator of osteoblast differentiation. Conversely, ROK inhibitors stimulate osteoblast differentiation and may be potentially useful as anabolic agents for bone.     

Quality of life on treatment with Metoprolol in Dilated Cardiomyopathy: Results from the MDC trial

Summary Quality of life in heart failure patients is receiving increased attention as a reflection of a treatment's potential secondary benefit of general well-being and daily functioning. The Metoprolol in Dilated Cardiomyopathy (MDC) trial was conducted as a large, multicenter trial to establish the effects of metoprolol on mortality and need for heart transplantation in patients with symptomatic idiopathic cardiomyopathy. It was found that metoprolol was well tolerated, improved symptoms and cardiac function, and prevented clinical deterioration in patients with symptomatic idiopathic dilated cardiomyopathy. Quality of life was evaluated as a secondary endpoint in 345 out of 383 randomized patients using a disease-specific questionnaire, the Quality of Life in Heart Failure Questionnaire, depicting physical activity, somatic symptoms, emotions, and life satisfaction. In a comparison of patients treated with metoprolol or placebo, patients treated with metoprolol noted a significantly more favorable response than those treated with placebo in terms of the overall treatment evaluation (p<0.05). Additionally, an analysis of the changes from baseline to 18 months, using 95% confidence intervals, revealed that patients treated with metoprolol showed a significant improvement from baseline to 18 months in life satisfaction, physical activity, and the total score, while patients treated with placebo did not change at all. The improvement in quality of life was supported by the correlations with improvement in traditional clinical parameters.     

Prediction of cancer mortality in the Nordic countries in 2005: effects of various interventions.

Rough estimates of the effect in 2005 of various preventive measures aimed at reducing cancer mortality in the Nordic countries were made using the American software CAN*TROL. The effect was measured as the percentage reduction in cancer mortality in 2005. The calculations were performed for changes in the smoking, dietary and sunbathing habits of the population (primary prevention), earlier diagnosis (secondary prevention) and improvements in survival resulting from better treatment (tertiary prevention). The calculations incorporate many assumptions, some of them more firmly based than others, such as uniformity of incidence trend in all the Nordic countries and also concerning the causality of various relations. For lack of evaluated Nordic data, we have used American figures concerning stage distributions and stage-specific relative survival rates. These assumptions should be borne in mind when drawing conclusions from the results obtained. The results show that there is a potential of up to several tens of percent for reducing total cancer mortality by the year 2005.     

Effects of nitric oxide synthase inhibition on blood flow and survival in experimental skin flaps.

The aim of the present study was to examine the effect of the nitric oxide synthase (NOS) inhibitor N(omega)-nitro-l-arginine methyl ester (l-NAME) on skin and flap blood flow, NOS activity and flap survival in an ischaemic dorsal flap model in the rat. Fifty-four rats were used in the study. l-NAME or the inactive enantiomere d-NAME was given intravenously either pre-, per- and postoperatively or only postoperatively. Controls received saline treatment. Blood pressure and skin and flap blood flow were monitored. NOS activity was measured in intact skin before and after l-NAME and d-NAME infusion and in flap tissue 48h postoperatively. Forty-eight hours postoperatively flap survival was determined in all rats. l-NAME treatment caused: (1) a marked attenuation of constitutive Ca(2+) dependent NOS activity in intact skin (p<0.001), (2) an increase in blood pressure (p<0.05), (3) a decrease in blood flow in intact skin and in skin flaps (p<0.05), and (4) a decrease in flap survival (p<0.05). In saline and d-NAME treated animals no change in blood pressure, blood flow or NOS activity in intact skin was noted. In conclusion this study shows that l-NAME attenuates constitutive Ca(2+) dependent NOS activity in intact skin, decreases skin and flap blood flow and decreases the survival of skin flaps. These results indicate that constitutive nitric oxide synthase is important for basal blood flow in skin and flap tissue and for the survival of skin flaps.     

Reduced genital sensitivity in female patients with multiple system atrophy of parkinsonian type.

According to the consensus statement on the diagnosis of multiple system atrophy (MSA), erectile dysfunction is required for male patients to fulfil the urinary incontinence criterion. However, there is no equivalent item for female patients. We questioned 19 female patients with MSA of the parkinsonian type (MSA-P), 28 female patients with Parkinson's disease (PD), and 27 healthy controls on their genital sensitivity. A total of 47% of the MSA patients but only 4% of the PD patients and 4% of the control group admitted to reduced genital sensitivity, a highly significant difference (P < 0.001). Moreover, the appearance of reduced genital sensitivity in female MSA patients showed a close temporal relation to the onset of the disease. If these preliminary results can be confirmed and further specified in a larger sample, a historical item of reduced genital sensitivity in female patients might become a diagnostic feature for MSA, comparable to erectile dysfunction in male patients.     

Checklist for the qualitative evaluation of clinical studies with particular focus on external validity and model validity

Background  

It is often stated that external validity is not sufficiently considered in the assessment of clinical studies. Although tools for its evaluation have been established, there is a lack of awareness of their significance and application. In this article, a comprehensive checklist is presented addressing these relevant criteria.     

An association between variants in the IGF2 gene and Beckwith-Wiedemann syndrome: interaction between genotype and epigenotype

Beckwith-Wiedemann syndrome (BWS) is a fetal overgrowth disorder involving the deregulation of a number of genes, including IGF2 and CDKN1C, in the imprinted gene cluster on chromosome 11p15.5. In sporadic BWS cases the majority of patients have epimutations in this region. Loss of imprinting of the IGF2 gene is frequently observed in BWS, as is reduced CDKN1C expression related to loss of maternal allele-specific methylation (LOM) of the differentially methylated region KvDMR1. The causes of epimutations are unknown, although recently an association with assisted reproductive technologies has been described. To date the only genetic mutations described in BWS are in the CDKN1C gene. In order to screen for other genetic predispositions to BWS, the conserved sequences between human and mouse differentially methylated regions (DMRs) of the IGF2 gene were analyzed for variants. Four single nucleotide polymorphisms (SNPs) were found in DMR0 (T123C, G358A, T382G and A402G) which occurred in three out of 16 possible haplotypes: TGTA, CATG and CAGA. DNA samples from a cohort of sporadic BWS patients and healthy controls were genotyped for the DMR0 SNPs. There was a significant increase in the frequency of the CAGA haplotype and a significant decrease in the frequency of the CATG haplotype in the patient cohort compared to controls. These associations were still significant in a BWS subgroup with KvDMR1 LOM, suggesting that the G allele at T382G SNP (CAGA haplotype) is associated with LOM at KvDMR1. This indicates either a genetic predisposition to LOM or interactions between genotype and epigenotype that impinge on the disease phenotype.