A longevity study with enhancer substances (selegiline, BPAP) detected an unknown tumor-manifestation-suppressing regulation in rat brain.

AIMS: First proof to show that (-)-deprenyl/selegiline (DEP), the first selective inhibitor of MAO-B, later identified as the first ß-phenylethylamine (PEA)-derived synthetic catecholaminergic activity enhancer (CAE) substance and (2R)-1-(1-benzofuran-2-yl)-N-propylpentane-2-amine (BPAP), the tryptamine-derived presently known most potent, selective, synthetic enhancer substance, are specific markers of unknown enhancer-sensitive brain regulations. MAIN METHODS: Longevity study disclosing the operation of tumor-manifestation-suppressing (TMS) regulation in rat brain. Immonohistochemical identification of a fibromyxosarcoma in rats. Experiments with human medulloblastoma cell lines. Analysis of the mechanism of action of enhancer substances. KEY FINDINGS: Whereas 20/40 saline-treated rats manifested a fibromyxosarcoma, in groups of rats treated with 0.001mg/kg DEP: 15/40 rats; with 0.1mg/kg DEP: 11/40 rats (P<0.01); with 0.0001mg/kg BPAP: 8/40 rats (P<0.001); with 0.05mg/kg BPAP: 7/40 rats (P<0.01) manifested the tumor. Experiments with human medulloblastoma cell lines, HTB-186 (Daoy); UW-228-2, showed that BPAP was devoid of direct cytotoxic effect on tumor cells, and did not alter the direct cytotoxic effectiveness of temozolomide, cisplatin, etoposide, or vincristine. Interaction with distinct sites on vesicular monoamine-transporter-2 (VMAT2) is the main mechanism of action of the enhancer substances which clarifies the highly characteristic bi-modal, bell-shaped concentration-effect curves of DEP and BPAP. SIGNIFICANCE: Considering of the safeness of the enhancer substances and the finding that DEP and BPAP, specific markers of unknown enhancer sensitive brain regulations, detected the operation of an enhancer-sensitive TMS-regulation in rat brain, it seems reasonable to test in humans low dose DEP or BPAP treatment against the spreading of a malignant tumor.

The state of research into children with cancer across Europe: new policies for a new decade.

Overcoming childhood cancers is critically dependent on the state of research. Understanding how, with whom and what the research community is doing with childhood cancers is essential for ensuring the evidence-based policies at national and European level to support children, their families and researchers. As part of the European Union funded EUROCANCERCOMS project to study and integrate cancer communications across Europe, we have carried out new research into the state of research in childhood cancers. We are very grateful for all the support we have received from colleagues in the European paediatric oncology community, and in particular from Edel Fitzgerald and Samira Essiaf from the SIOP Europe office. This report and the evidence-based policies that arise from it come at a important junction for Europe and its Member States. They provide a timely reminder that research into childhood cancers is critical and needs sustainable long-term support.

Duplication of 7q34 is specific to juvenile pilocytic astrocytomas and a hallmark of cerebellar and optic pathway tumours.

BACKGROUND: Juvenile pilocytic astrocytomas (JPA), a subgroup of low-grade astrocytomas (LGA), are common, heterogeneous and poorly understood subset of brain tumours in children. Chromosomal 7q34 duplication leading to fusion genes formed between KIAA1549 and BRAF and subsequent constitutive activation of BRAF was recently identified in a proportion of LGA, and may be involved in their pathogenesis. Our aim was to investigate additional chromosomal unbalances in LGA and whether incidence of 7q34 duplication is associated with tumour type or location. METHODS AND RESULTS: Using Illumina-Human-Hap300-Duo and 610-Quad high-resolution-SNP-based arrays and quantitative PCR on genes of interest, we investigated 84 paediatric LGA. We demonstrate that 7q34 duplication is specific to sporadic JPA (35 of 53 - 66%) and does not occur in other LGA subtypes (0 of 27) or NF1-associated-JPA (0 of 4). We also establish that it is site specific as it occurs in the majority of cerebellar JPA (24 of 30 - 80%) followed by brainstem, hypothalamic/optic pathway JPA (10 of 16 - 62.5%) and is rare in hemispheric JPA (1 of 7 - 14%). The MAP-kinase pathway, assessed through ERK phosphorylation, was active in all tumours regardless of 7q34 duplication. Gain of function studies performed on hTERT-immortalised astrocytes show that overexpression of wild-type BRAF does not increase cell proliferation or baseline MAPK signalling even if it sensitises cells to EGFR stimulation. CONCLUSIONS AND INTERPRETATION: Our results suggest that variants of JPA might arise from a unique site-restricted progenitor cell where 7q34 duplication, a hallmark of this tumour-type in association to MAPK-kinase pathway activation, potentially plays a site-specific role in their pathogenesis. Importantly, gain of function abnormalities in components of MAP-Kinase signalling are potentially present in all JPA making this tumour amenable to therapeutic targeting of this pathway.

National registry of patients with juvenile idiopathic inflammatory myopathies in Hungary--clinical characteristics and disease course of 44 patients with juvenile dermatomyositis.

Idiopathic inflammatory myopathies (IIMs) are systemic autoimmune diseases characterized by chronic muscle inflammation resulting in progressive weakness and frequent involvement of internal organs, mainly the pulmonary, gastrointestinal and cardiac systems which considerably contribute to the morbidity and mortality of the IIMs. Aim of this study was to present clinical characteristics, disease course, frequency of relapses and survival in patients with juvenile dermatomyositis (DM). A national registry of patients with juvenile IIMs was elaborated by the authors in Hungary. We have summarized data of the register according to signs and symptoms, disease course, frequency of relapses and survival of patients with juvenile IIM. Analysis was performed using data of 44 patients with juvenile DM diagnosed between 1976 and 2004 according to Bohan and Peter's criteria. Survival probability was calculated by Kaplan-Meier method. Data of patients with juvenile DM were compared with data of 66 patients with adult DM. The most frequent cutaneous features were facial erythema and heliotrope rash. Extramuscular and extraskeletal manifestations of the disease were more frequent in adult patients. The most common extramuscular feature was arthralgia in both groups of patients with juvenile or adult DM. Cardiac manifestation of the disease was not observed in juvenile patients. Respiratory muscle involvement and interstitial lung disease (ILD) were more frequent among adult DM patients than cardiac manifestation of the myositis. In view of the disease course, the authors found that frequency of polycyclic and monophasic subtypes of the disease were mainly similar. The hazard of relapse was found higher during the first year after the remission. None of the juvenile patients died. Among adult patients four disease-specific deaths occurred. There was no correlation between relapse free survival and initial therapeutic regimen. Many of our patients had polycyclic or chronic disease. As relapses can occur after a prolonged disease-free interval, patients should be followed up for at least 2 years. Although we found favourable survival probability, further investigations are needed to assess functional outcome.

Disease course, frequency of relapses and survival of 73 patients with juvenile or adult dermatomyositis.

OBJECTIVE: Our aim is to present the disease course, frequency of relapses and survival of juvenile and adult dermatomyositis (JDM/DM) patients. METHODS: Analysis was performed using data on 73 patients. The median follow-up for 38 JDM patients was 32 months and 78 months for 35 adult DM patients. RESULTS: 23/38 JDM patients (60%) had monophasic, 12/38 (31.6%) had polycyclic and 3/38 (7.9%) had chronic disease. Among children treated only with glucocorticoids, 12/20 (60%) had monophasic and 8/20 (40%) had polycyclic disease. 10/17 (58.8%) children, who required second-line immunosuppressive agents, had monophasic and 4/17 (23.5%) had polycyclic disease. 18/35 DM (51.4%) patients had monophasic, 13/35 (37.1%) had polycyclic, 1/35 (2.9%) had chronic disease and 3/35 (8.6%) had fulminant myositis. Among DM patients requiring only glucocorticoids, 12/20 (60%) were monophasic and 8/20 (40%) were polycyclic. In patients requiring second-line immunosuppressive agents, 6/15 patients (40%) had monophasic and 5/15 (33.3%) had polycyclic disease. Among patients with polycyclic disease, the risk of relapse was higher during first year than later in the disease course. None of the JDM patients have died, while 4 disease-specific deaths occurred in adult patients. There was no significant difference between the survival of JDM and DM patients. DISCUSSION: There was no correlation between relapse-free survival and the initial therapeutic regimen. Many of our patients had polycyclic or chronic disease. As relapses can occur after a prolonged disease-free interval, patients should be followed for at least 2 years. Although we found a favourable survival rate, further investigations are needed to assess functional outcome.

Limited value of serum steroid measurements in identification of mild form of 21-hydroxylase deficiency.

BACKGROUND: ACTH stimulation test is widely used as a basic diagnostic method for non-classical congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD). However, the interpretation of this test has not been definitely established. To determine the cut-off values of basal and post-ACTH serum 17-OHP concentrations, data of patients with suspected 21-OHD has been analysed. PATIENTS AND METHODS: Two hundred and eighty-seven patients with postnatal/peripubertal virilization were investigated. Serum steroid concentrations were measured by RIA, urinary steroid profile was determined by capillary gas chromatography and mutation analysis of CYP21 gene was performed by allele specific PCR. 21-OHD was diagnosed by elevated serum 17-OHP concentrations, high level of the urinary 17-OHP metabolites and/or homozygosity for CYP21 mutations. RESULTS: Twenty-one patients of the total of 287 subjects (7.3 %) were identified as having 21-OHD. The numbers of 21-OHD patients compared to total numbers of patients with different ranges of serum 17-OHP were as follows: basal values below 3.5 ng/ml (mean + 1 SD) 0/225; between 3.5 - 6.6 ng/ml 3/41; above 6.6 ng/ml (mean + 2 SD) 18/21. Post-ACTH values below 6.4 ng/ml (mean + 1 SD) 0/226, between 6.4 - 10.3 ng/ml 0/35, above 10.3 ng/ml (mean + 2 SD) 21/26. CONCLUSION: There are patients with inappropriate peripubertal virilization who have slightly elevated 17-OHP concentrations. In this subgroup of patients more sensitive and specific methods are needed to establish the diagnosis of 21-OHD. Therefore we suggest performing an ACTH stimulation test in patients with a morning 17-OHP level above 3.5 ng/ml. Furthermore, urinary steroid profile and/or CYP21 gene analysis are needed in patients with a stimulated 17-OHP value between 10 and 30 ng/ml. These tests will distinguish between patients with non-classical 21-OHD and patients with other disorders.

PCR based diagnosis of 21-hydroxylase gene defects in Slovak patients with congenital adrenal hyperplasia.

OBJECTIVE: To analyse 21-hydroxylase gene for 8 most common mutations in patients with salt-wasting type of congenital adrenal hyperplasia. METHODS: Allele specific PCR performed on 8 salt-wasting CAH patients and their 23 healthy relatives. RESULTS: Two patients were homozygous for 8 bp deletion in exon 3, while 6 patients were homozygous for intron 2 splice mutation. Mutant allele for splice mutation was found also in both parents of patients with this type of mutation. CONCLUSIONS: These preliminary results show that only two mutations, 8 bp deletion in exon 3 and splice mutation in intron 2, were present in this group of Slovak patients with salt-wasting type of congenital adrenal hyperplasia.

Doxorubicin selectively inhibits brain versus atrial natriuretic peptide gene expression in cultured neonatal rat myocytes.

Doxorubicin is an antineoplastic agent with significant cardiotoxicity. We examined the effects of this agent on the expression of the natriuretic peptide (NP) genes in cultured neonatal rat atrial myocytes. Doxorubicin suppressed NP secretion, steady-state NP mRNA levels, and NP gene promoter activity. In each instance, brain NP (BNP) proved to be more sensitive than atrial NP (ANP) to the inhibitory effects of the drug. ICRF-187 and probucol reversed the inhibition by doxorubicin of ANP mRNA accumulation and ANP gene promoter activity while exerting no effect on BNP mRNA levels or promoter activity. This represents the first identification of the NP genes as targets of doxorubicin toxicity in the myocardial cell. This inhibition operates predominantly at a transcriptional locus and has more potent effects on BNP versus ANP secretion/gene expression. Measurement of BNP secretion/gene expression may provide a sensitive marker of early doxorubicin cardiotoxicity.

Screening for mutations of 21-hydroxylase gene in Hungarian patients with congenital adrenal hyperplasia.

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders, causing impaired secretion of cortisol and aldosterone from the adrenal cortex, with subsequent overproduction of adrenal androgens. The most common enzyme defect causing CAH is steroid 21-hydroxylase deficiency. To determine the mutational spectrum in the Hungarian CAH population, the CYP21 active gene was analyzed using PCR. A total of 297 Hungarian patients with 21-hydroxylase deficiency are registered in the 2nd Department of Pediatrics, Budapest, Hungary, and their clinical status was evaluated. Blood samples for CYP21 genotype determination could be obtained from 167 patients (representing 306 unrelated chromosomes and 56.2% of the total group of patients). Eight of the most common mutations were screened [In2 (intron 2 splice mutation), I172N, Del (Del: apparents large gene conversion), Q318X, R356W, 1761Tins, ClusterE6, V281L] using allele-specific amplification. The most frequent mutation in the Hungarian CAH population was found to be In2. Our results have shown a good genotype/phenotype correlation in case of most mutations; the In2 mutation is associated mostly with the severe form of the disease, whereas I172N was expressed in a wide spectrum of phenotypes. 1999)

Mechanical strain increases expression of the brain natriuretic peptide gene in rat cardiac myocytes.

Using a device that applies cyclical strain (1 Hz) to ventricular cardiocytes cultured on collagen-coated silicone elastomer surfaces, we have demonstrated strain-dependent increases in brain natriuretic peptide (BNP) secretion, BNP mRNA levels, and expression of a transiently transfected -1595 human BNP-luciferase reporter. When actinomycin D (10 microM) was introduced concomitantly with the strain stimulus, the strain-induced increase in BNP mRNA was eliminated, and the decay of transcripts was identical in the control and strained cells, indicating the lack of independent effects on transcript stability. Strain-dependent -1595 human BNP-luciferase activity was completely inhibited by chelerythrine, 2-aminopurine, genistein, and W-7 and only partially or not at all by KN-62, wortmannin, and H-89. The effects of these individual agents paralleled their effects on mitogen-activated protein kinase (MAPK) activity, but not c-Jun N-terminal kinase (JNK) activity, in the cells. Overexpression of wild-type MAPK and, to a lesser extent, JNK increased strain-dependent BNP promoter activity, whereas dominant-negative mutants of MAPK kinase, JNK kinase, or Ras completely blocked strain-dependent reporter activity. These findings provide the first demonstration that mechanical strain can increase myocardial gene expression through a transcriptional mechanism and suggest important roles for MAPK and JNK in mediating this effect.

An E-box motif conveys inhibitory activity on the atrial natriuretic peptide gene.

Atrial natriuretic peptide (ANP) is a potent diuretic, natriuretic, and vasorelaxant hormone that is expressed early in ventricular hypertrophy. Expression of human ANP is controlled by a series of regulatory elements located in the 5' flanking sequence of its gene. We generated a series of 5' deletion mutations extending from -2600 to -1150 relative to the transcription start site and linked them to a chloramphenicol acetyltransferase reporter gene. Using transient transfection analysis, we have identified a negative regulatory element between -1206 and -1152 relative to the start site. Each of a series of 5' deletion mutants, when introduced into fibroblast cultures, expressed the reporter function at a level that was significantly less (< 20%) than that seen with the -1152 reporter construct, whereas comparably transfected atrial cardiocytes demonstrated no change in reporter activity, implying that the repressor function is specific to cell type. The critical region (from -1206 to -1152) associates with a soluble protein present in cardiac fibroblast extracts in a sequence-specific fashion. Deoxyribonuclease I footprint analysis demonstrated the presence of several protected regions, including one that overlies an E-box motif (CAACTG), an element that in other systems has been implicated in promoting differentiation in the myocyte lineage. Site-directed mutagenesis of the E-box motif suppressed both the protein-binding and inhibitory activities of the 54-bp fragment. In summary, we have found a region in the 5' flanking sequence of the human ANP gene that represses transcriptional activity in nonmyocardial cells. This element may play an important role in the restriction of ANP gene expression to cardiac myocytes.

1,25(OH)2 vitamin D3, and retinoic acid antagonize endothelin-stimulated hypertrophy of neonatal rat cardiac myocytes.

1,25(OH)2 Vitamin D3 (VD3) and retinoic acid (RA) function as ligands for nuclear receptors which regulate transcription. Though the cardiovascular system is not thought to represent a classical target for these ligands, it is clear that both cardiac myocytes and vascular smooth muscle cells respond to these agents with changes in growth characteristics and gene expression. In this study we demonstrate that each of these ligands suppresses many of the phenotypic correlates of endothelin-induced hypertrophy in a cultured neonatal rat cardiac ventriculocyte model. Each of these agents reduced endothelin-stimulated ANP secretion in a dose-dependent fashion and the two in combination proved to be more effective than either agent used alone (VD3: 49%; RA:52%; VD3 + RA:80% inhibition). RA, at concentrations known to activate the retinoid X receptor, and, to a lesser extent, VD3 effected a reduction in atrial natriuretic peptide, brain natriuretic peptide, and alpha-skeletal actin mRNA levels. Similar inhibition (VD3:30%; RA:33%; VD3 + RA:59% inhibition) was demonstrated when cells transfected with reporter constructs harboring the relevant promoter sequences were treated with VD3 and/or RA for 48 h. These effects were not accompanied by alterations in endothelin-induced c-fos, c-jun, or c-myc gene expression, suggesting either that the inhibitory locus responsible for the reduction in the mRNA levels lies distal to the activation of the immediate early gene response or that the two are not mechanistically coupled. Both VD3 and RA also reduced [3H]leucine incorporation (VD3:30%; RA:33%; VD3 + RA:45% inhibition) in endothelin-stimulated ventriculocytes and, once again, the combination of the two was more effective than either agent used in isolation. Finally, 1,25(OH)2 vitamin D3 abrogated the increase in cell size seen after endothelin treatment. These findings suggest that the liganded vitamin D and retinoid receptors are capable of modulating the hypertrophic process in vitro and that agents acting through these or similar signaling pathways may be of value in probing the molecular mechanisms underlying hypertrophy.

Tissue-specific expression of the human brain natriuretic peptide gene in cardiac myocytes.

Brain natriuretic peptide (BNP) is a cardiac hormone constitutively expressed in the adult heart. To identify the cis-acting elements involved in regulation of the human BNP gene, we subcloned the full-length promoter (-1818 to +100) and deletions thereof upstream from a luciferase reporter gene and transiently transfected them into primary cultures of neonatal rat atrial and ventricular myocytes and myocardial fibroblasts. Luciferase activity of the full-length construct was higher in ventricular (39064 +/- 8488 relative light units, N=11) and atrial (11225 +/- 1907, N=17) myocytes than myocardial fibroblasts (329 +/- 113, n=5). Maximal promoter activity in ventricular and atrial myocytes was maintained by sequences positioned between -1818 and -1283 relative to the transcription start site. Deletion to -1175 resulted in a decrease, whereas further deletion to -500 effected an increase in reporter activity in both cell types. In ventricular and atrial myocytes, deletion from -500 to -40 reduced luciferase activity 20-fold and 2-fold, respectively, whereas in myocardial fibroblasts, deletion to -40 upregulated the BNP promoter 2-fold. Of note, deleting 16 bp between -127 and -111 reduced luciferase activity 7-fold and 4-fold in ventricular and atrial myocytes, respectively, but had essentially no effect on luciferase activity in fibroblasts. Placement of sequences lying between -127 and -40 upstream from a heterologous thymidine kinase promoter resulted in reporter expression that was 7.4-fold greater than the vector alone in ventricular myocytes, approximately 2-fold greater in atrial myocytes, and equivalent to the vector alone in fibroblasts. For study of activity of the human BNP promoter in adult myocytes, either 408 or 97 bp of 5' flanking sequence coupled to the luciferase reporter gene was injected into the apex of adult male Sprague-Dawley rat hearts. After 7 days, luciferase activity in the injected myocardium was 9.8-fold higher for the longer construct (70683 +/- 14744 versus 7223 +/- 3920, n=4, P < .01), consistent with our in vitro data. These data indicate that (1) the full-length human BNP promoter is more active in ventricular versus atrial myocytes and essentially inactive in fibroblasts, (2) the distal BNP promoter contains both positive and negative regulatory elements, (3) a region of the proximal BNP promoter located between -127 and -40 confers tissue specificity, and (4) the BNP promoter is active after injection into the adult rat heart.

1,25(OH)2D3 suppresses expression and secretion of atrial natriuretic peptide from cardiac myocytes.

We have examined the effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on the expression of the rat atrial natriuretic peptide (ANP) gene and the secretion of the encoded protein product in neonatal rat cardiac myocyte cultures. 1,25(OH)2D3 effected a dose- and time-dependent inhibition of agonist-stimulated ANP secretion, which was accompanied by a reduction in the levels of the ANP mRNA transcript. The latter effect appeared to derive, at least in part, from suppression of ANP gene transcription. Of interest, both the reduction in mRNA levels and the inhibition of transcriptional activity were amplified by simultaneous treatment with retinoic acid, suggesting that heterodimerization of liganded 1,25(OH)2D3 receptor and retinoic acid receptor (likely retinoid X receptor) may underlie the inhibitory mechanism in the cardiac myocyte. Neither the secretory effect nor the effect on transcription proved to be calcium dependent. 22-Oxacalcitriol, a nonhypercalcemic analogue of 1,25(OH)2D3, was equally effective in suppressing ANP mRNA levels and transcription of the gene. These findings add to a growing body of data that imply an important role for 1,25(OH)2D3 in the regulation of cardiovascular function.