Epigenetic silencing of miRNA-9 is associated with HES1 oncogenic activity and poor prognosis of medulloblastoma.

BACKGROUND: microRNA-9 is a key regulator of neuronal development aberrantly expressed in brain malignancies, including medulloblastoma. The mechanisms by which microRNA-9 contributes to medulloblastoma pathogenesis remain unclear, and factors that regulate this process have not been delineated. METHODS: Expression and methylation status of microRNA-9 in medulloblastoma cell lines and primary samples were analysed. The association of microRNA-9 expression with medulloblastoma patients' clinical outcome was assessed, and the impact of microRNA-9 restoration was functionally validated in medulloblastoma cells. RESULTS: microRNA-9 expression is repressed in a large subset of MB samples compared with normal fetal cerebellum. Low microRNA-9 expression correlates significantly with the diagnosis of unfavourable histopathological variants and with poor clinical outcome. microRNA-9 silencing occurs via cancer-specific CpG island hypermethylation. HES1 was identified as a direct target of microRNA-9 in medulloblastoma, and restoration of microRNA-9 was shown to trigger cell cycle arrest, to inhibit clonal growth and to promote medulloblastoma cell differentiation. CONCLUSIONS: microRNA-9 is a methylation-silenced tumour suppressor that could be a potential candidate predictive marker for poor prognosis of medulloblastoma. Loss of microRNA-9 may confer a proliferative advantage to tumour cells, and it could possibly contribute to disease pathogenesis. Thus, re-expression of microRNA-9 may constitute a novel epigenetic regulation strategy against medulloblastoma.

Bone morphogenetic protein-7 is a MYC target with prosurvival functions in childhood medulloblastoma.

Medulloblastoma (MB) is the most common malignant brain tumor in children. It is known that overexpression and/or amplification of the MYC oncogene is associated with poor clinical outcome, but the molecular mechanisms and the MYC downstream effectors in MB remain still elusive. Besides contributing to elucidate how progression of MB takes place, most importantly, the identification of novel MYC-target genes will suggest novel candidates for targeted therapy in MB. A group of 209 MYC-responsive genes was obtained from a complementary DNA microarray analysis of a MB-derived cell line, following MYC overexpression and silencing. Among the MYC-responsive genes, we identified the members of the bone morphogenetic protein (BMP) signaling pathway, which have a crucial role during the development of the cerebellum. In particular, the gene BMP7 was identified as a direct target of MYC. A positive correlation between MYC and BMP7 expression was documented by analyzing two distinct sets of primary MB samples. Functional studies in vitro using a small-molecule inhibitor of the BMP/SMAD signaling pathway reproduced the effect of the small interfering RNA-mediated silencing of BMP7. Both approaches led to a block of proliferation in a panel of MB cells and to inhibition of SMAD phosphorylation. Altogether, our findings indicate that high MYC levels drive BMP7 overexpression, promoting cell survival in MB cells. This observation suggests the potential relevance of targeting the BMP/SMAD pathway as a novel therapeutic approach for the treatment of childhood MB.

Quassinoids: From traditional drugs to new cancer therapeutics.

Quassinoids are a group of compounds extracted from plants of the Simaroubaceae family, which have been used for many years in folk medicine. These molecules gained notoriety after the initial discovery of the anti-leukemic activity of one member, bruceantin, in 1975. Currently over 150 quassinoids have been isolated and classified based on their chemical structures and biological properties investigated in vitro and in vivo. Many molecules display a wide range of inhibitory effects, including anti-inflammatory, anti-viral, anti-malarial and anti-proliferative effects on various tumor cell types. Although often the exact mechanism of action of the single agents remains unclear, some agents have been shown to affect protein synthesis in general, or specifically HIF-1α and MYC, membrane polarization and the apoptotic machinery. Considering that future research into chemical modifications is likely to generate more active and less toxic derivatives of natural quassinoids, this family represents a powerful source of promising small molecules targeting key prosurvival signaling pathways relevant for diverse pathologies. Here, we review available knowledge of functionality and possible applications of quassinoids and quassinoid derivatives, spanning traditional use to the potential impact on modern medicine as cancer therapeutics.

Telomere maintenance as therapeutic target in embryonal tumours.

Embryonal tumours most commonly occur in the first few years of life and account for approximately 30% of childhood malignancies. Knowledge of these tumours' genetics has already impacted on their clinical management and further knowledge of their cellular immortalization will hopefully result in novel therapies. The ends of human chromosomes are capped and protected by telomeres; cellular replication, however, causes their loss. A critical length of telomere repeats is required to ensure proper telomere function and avoid the activation of DNA damage pathways that result in senescence and cell death. To proliferate beyond the senescence checkpoint, cells must restore their telomere length. Hence stabilization of telomere is an important step in cell immortalization and carcinogenesis. Telomere maintenance is evident in virtually all types of malignant cells, including embryonal tumours, where either a telomerase-dependent or alternative lengthening of telomeres (ALT) mechanism is employed in order to ensure their limitless replicative potential. For this reason effective strategies targeting telomere maintenance in cancer cells require a combination of telomerase and ALT inhibitors. In this review, we are giving an overview about telomere maintenance in childhood tumours and discussing its potential as a new therapeutic target.

Which clinical and biological tumor markers proved predictive in the prospective multicenter trial HIT'91--implications for investigating childhood medulloblastoma.

BACKGROUND: Most recent studies analyzing candidate biological prognostic factors in childhood medulloblastoma (MB) are limited by small patient numbers due to dependence on fresh-frozen tumor material. By contrast, large archives of formalin-fixed, paraffin-embedded MB samples exist from homogeneously treated patients. PATIENTS AND METHODS: We have optimized RNA and DNA isolation from formalin-fixed paraffin-embedded MB samples. We then analyzed archived tumor samples from well-documented patients treated within the prospective randomized multicenter trial HIT'91 for DNA amplification of c-myc and N-myc, and mRNA expression of c-myc and trkC. RESULTS: TrkC and c-myc mRNA expression were identified as independent prognostic factors by multivariate analysis. Three risk groups were identified: 1) Favorable risk group: All 8 patients (2 metastatic) with elevated trkC and reduced c-myc mRNA expression (compared to levels of human cerebellum) remained relapse-free (7-year EFS 100%). 2) Poor risk group: 10 of 15 patients with metastatic disease and high c-myc and low trkC mRNA expression relapsed (7-year EFS 33%). 3) Intermediate risk group: The 7-year EFS of the remaining 78 patients was 65%. CONCLUSIONS: While the collection of fresh-frozen tumor samples is remaining a major challenge in large clinical trials, routinely processed paraffin-embedded tissue samples can be used to quantitate biological prognostic factors on the DNA and RNA level. Upon prospective validation of cut-off levels, this may lead to better risk-based stratification systems for children with medulloblastoma.

Interferon-gamma mediated up-regulation of caspase-8 sensitizes medulloblastoma cells to radio- and chemotherapy.

Loss of caspase-8 expression - which has been demonstrated in a subset of Medulloblastoma (MB) - might block important apoptotic signalling pathways and therefore contribute to treatment resistance. In this study, IFN-gamma mediated up-regulation of caspase-8 in human MB cells was found to result in chemosensitization to cisplatin, doxorubicin and etoposide, and sensitisation to radiation. These effects were more prominent in D425 and D341 MB cells (low basal caspase-8 expression) when compared to DAOY MB cells (high basal caspase-8 expression). IFN-gamma mediated chemosensitization and radiosensitization effects were reduced by treatment with the caspase-8 specific inhibitor z-IETD-fmk. Treatment of IFN-gamma resulted in activation of STAT1 in DAOY MB cells and to a lesser extent in D425, but not in D341, indicating that IFN-gamma acts in MB cells through STAT1-dependent and -independent signalling pathways. Taken together, our results demonstrate that IFN-gamma mediated restoration of caspase-8 in MB cells might enhance apoptotic pathways relevant to the response to chemo- and radiotherapy.

Antisense treatment of IGF-IR induces apoptosis and enhances chemosensitivity in central nervous system atypical teratoid/rhabdoid tumours cells.

Central nervous system (CNS) atypical teratoid/rhabdoid tumours (AT/RT) are among the paediatric malignant tumours with the worst prognosis and fatal outcome. Insulin-like growth factor I receptor (IGF-IR) protects cancer cells from apoptosis induced by a variety of anticancer drugs and radiation. In the present study, IGF-IR was expressed in 8/8 primary AT/RT as detected by immunohistochemistry. Moreover, we found IGF-I and IGF-II mRNA in BT-16 CNS AT/RT cells and IGF-II mRNA in BT-12 CNS AT/RT cells, and autophosphorylated IGF-IR in both cell lines, indicating the potential presence of an autocrine/paracrine IGF-I/II/IGF-IR loop in CNS AT/RT. IGF-IR antisense oligonucleotide treatment of human CNS AT/RT cells resulted in significant down-regulation of IGF-IR mRNA and protein expression, induction of apoptosis, and chemosensitisation to doxorubicin and cisplatin. These studies provide evidence for the influence of IGF-IR on cellular responses to chemotherapy and raise the possibility that curability of selected CNS AT/RT may be improved by pharmaceutical strategies directed towards the IGF-IR.

Telomerase inhibition, telomere shortening, cell growth suppression and induction of apoptosis by telomestatin in childhood neuroblastoma cells.

Neuroblastoma is a tumour derived from primitive cells of the sympathetic nervous system and is the most common extracranial solid tumour in childhood. Unfavourable tumours are characterised not only by structural changes, including 1p deletion and amplification of the MYCN proto-oncogene, but also by high telomerase activity. Telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to inhibit telomerase activity. In this study, we examined telomestatin, a G-quadruplex interactive agent, for its ability to inhibit telomere maintenance of neuroblastoma cells. Telomere length was determined by the terminal restriction fragment method, telomerase activity was measured by a quantitative telomeric repeat amplification protocol, and the expression of human telomerase by quantitative real-time polymerase chain reaction (RT-PCR). Short-term treatment with telomestatin resulted in dose-dependent cytotoxicity and induction of apoptosis. Long-term treatment with telomestatin at non-cytotoxic, but still telomerase activity-inhibiting, concentrations resulted in telomere shortening, growth arrest and induction of apoptosis. These results suggest that the effect of telomestatin is dose-dependent and at least 2-fold. Prolonged low-dose treatment with telomestatin limits the cellular lifespan of NB cells through disruption of telomere maintenance.

Famotidine for infant gastro-oesophageal reflux: a multi-centre, randomized, placebo-controlled, withdrawal trial.

BACKGROUND: Gastro-oesophageal reflux afflicts up to 7% of all infants. Histamine-2 receptor antagonists are the most commonly prescribed medications for this disorder, but few controlled studies support this practice. AIM: To evaluate the safety and efficacy of famotidine for infant gastro-oesophageal reflux disease. METHODS: Thirty-five infants, 1.3-10.5 months of age, entered an 8-week, multi-centre, randomized, placebo-controlled, two-phase trial: first 4 weeks, observer-blind comparison of famotidine 0.5 mg/kg and famotidine 1.0 mg/kg; second 4 weeks, double-blind withdrawal comparison (safety and efficacy) of each dose with placebo. RESULTS: No serious adverse events were reported. Eleven patients had 16 non-serious, possibly drug-related adverse experiences: 6 patients with agitation or irritability (manifested as head-rubbing in two), 3 patients with somnolence, 2 patients with anorexia, 2 with headache, 1 patient with vomiting, 1 patient with hiccups, and 1 patient with candidiasis. Of the 35 infants, 27 completed Part I. There were significant score improvements for famotidine 0.5 mg/kg in regurgitation frequency (P = 0.04), and for famotidine 1.0 mg/kg in crying time (P = 0.027) and regurgitation frequency (P = 0.004) and volume (P = 0.01). Eight infants completed Part II on double-blind treatment, which was insufficient for meaningful comparisons. CONCLUSIONS: Histamine-2 receptor antagonists may cause agitation and headache in infants. A possibly efficacious famotidine dose for infants is 0.5 mg/kg (frequency adjusted for age). As 1.0 mg/kg may be more efficacious in some, the dosage may require individualization based on response. Further sizeable placebo-controlled evaluations of histamine-2 receptor antagonists in infants with gastro-oesophageal reflux disease are warranted.

The regulatory subunit of a cGMP-regulated protein kinase A of Trypanosoma brucei.

This study reports the identification and characterization of the regulatory subunit, TbRSU, of protein kinase A of the parasitic protozoon Trypanosoma brucei. TbRSU is coded for by a single copy gene. The protein contains an unusually long N-terminal domain, the pseudosubstrate site involved in binding and inactivation of the catalytic subunit, and two C-terminally located, closely spaced cyclic nucleotide binding domains. Immunoprecipitation of TbRSU coprecipitates a protein kinase activity with the characteristics of protein kinase A: it phosphorylates a protein kinase specific substrate, and it is strongly inhibited by a synthetic protein kinase inhibitor peptide. Unexpectedly, this kinase activity could not be stimulated by cAMP, but by cGMP only. Binding studies with recombinant cyclic nucleotide binding domains of TbRSU confirmed that both domains bind cGMP with Kd values in the lower micromolar range, and that up to a 100-fold excess of cAMP does not compete with cGMP binding.

cAMP signalling in Trypanosoma brucei.

Cyclic AMP was the first second messenger to be identified. After five decades of research, much is currently known about its biological functions and clinical implications. Several components of the cAMP signalling pathways, such as the G-protein coupled receptors and the phosphodiesterases, have become sensitive and specific drug targets for a host of clinical applications. Surprisingly, very little effort has been invested so far into the study of cAMP signalling in parasites, and its significance in host/parasite interaction. Our laboratory has embarked on a study of cAMP signalling in Trypanosoma brucei. A newly identified adenylyl cyclase, GRESAG4.4B, a member of a small family of closely related genes, is being used as a model molecule for investigating the mechanisms which control cyclase activity in the T. brucei cell. On the other hand, a number of genes for different families of cAMP-specific phosphodiesterases have been identified and characterised. One enzyme, TbPDE1, is coded for by a single-copy gene. Knock-outs of this gene display an almost normal phenotype in culture, indicating that TbPDE1 is not an essential enzyme under culture conditions. A second phosphodiesterase which is being studied in detail, TbPDE2A, is clearly different from TbPDE1, and it is coded for by a member of a small gene family containing about six similar, but non-identical genes. TbPDE2A, as TbPDE1, is specific for cAMP. In its N-terminal, it contains a GAF domain which may represent an allosteric cGMP-binding site. The other members of the TbPDE2 family all exhibit strongly conserved catalytic domains, but vary widely in their N-terminal regulatory domains. With regard to downstream signalling by the cAMP generated through the interplay of adenylyl cyclases and phosphodiesterases, we have recently identified a single-copy gene (TbRSU1) which codes for a putative regulatory subunit of the cAMP-regulated protein kinase A. This protein exhibits considerable similarity with its mammalian counterparts. Immunoprecipitation co-precipitates a protein kinase activity with the characteristics of protein kinase A.

Nucleoside diphosphate kinase of Trypanosoma brucei.

Nucleoside diphosphate kinase (NDPK) is a highly conserved, multifunctional enzyme. Its originally described function is the phosphorylation of nucleoside diphosphates to the corresponding triphosphates, using ATP as the phosphate donor and a high-energy phosphorylated histidine residue as the reaction intermediate. More recently, a host of additional functions of NDPK have been discovered. Some of these correlate with the capacity of NDPK to transphosphorylate other proteins, in a manner reminiscent of bacterial two-component systems. Other functions may be mediated by direct DNA-binding of NDPK. This study describes the identification of NDPK from the parasitic protozoon Trypanosoma brucei. The genome of this major disease agent contains a single gene for NDPK. The predicted amino acid sequence of the trypanosomal enzyme is highly conserved with respect to all other species. The protein is constitutively expressed and is present in procyclic and in bloodstream forms. Immunofluorescence and immuno-electron microscopy demonstrate that trypanosomal NDPK (TbNDPK) is predominantly localized in the cell nucleus. Histidine phosphorylation of TbNDPK is essentially resistant to the experimental compound LY266500, a potent inhibitor of histidine phosphorylation of trypanosomal succinyl coenzyme A synthase.

The spectrum of pediatric eosinophilic esophagitis beyond infancy: a clinical series of 30 children.

OBJECTIVES: Eosinophilic esophagitis, previously confused with esophageal inflammation due to gastroesophageal reflux, has recently begun to be distinguished from it. We undertook this analysis of our large series of children with the condition to clarify its spectrum: its presenting symptoms; its relation to allergy, respiratory disease, and reflux; its endoscopic and histological findings; and its diagnosis and therapy. METHODS: We analyzed the details of our clinical series of 30 children with eosinophilic esophagitis, defining it as > or =5 eosinophils per high power field in the distal esophageal epithelium. Retrospective chart review was supplemented by prospective, blinded, duplicate quantitative evaluation of histology specimens, and by telephone contact with some families to clarify subsequent course. Presentation and analysis of the series as a whole is preceded by a case illustrating a typical presentation with dysphagia and recurrent esophageal food impactions. RESULTS: Presenting symptoms encompass vomiting, pain, and dysphagia (some with impactions or strictures). Allergy, particularly food allergy, is an associated finding in most patients, and many have concomitant asthma or other chronic respiratory disease. A subtle granularity with furrows or rings is newly identified as the endoscopic herald of histological eosinophilic esophagitis. Histological characteristics include peripapillary or juxtaluminal eosinophil clustering in certain cases. Association with eosinophilic gastroenteritis occurs, but is not common. Differentiation from gastroesophageal reflux disease is approached by analyzing eosinophil density and response to therapeutic trials. Therapy encompasses dietary elimination and anti-inflammatory pharmacotherapy. CONCLUSION: Awareness of the spectrum of eosinophilic esophagitis should promote optimal diagnosis and treatment of this elusive entity, both in children and in adults.

Isolated lower esophageal sphincter relaxation as "wave-suppressed" secondary peristalsis.

Esophageal venting following air insufflation may occur by secondary peristalsis or by isolated transient lower esophageal sphincter relaxation (TLESR). To identify factors determining venting by these two mechanisms, we analyzed the responses to esophageal air insufflation in 4 infants and in 2 adults. We used a nine-lumen dual-Dent-sleeve manometric catheter with an air insufflation esophageal side hole, identifying swallowing by pharyngeal manometry or submental electromyography. The time from the venting lower esophageal sphincter relaxation (whether part of a secondary peristalsis or an isolated TLESR) to the next swallow (whether spontaneous, in the infants, or on command, in the adults) was characterized as > or = 15 sec or < 15 sec. Of the 25 evaluable trials, the subsequent swallow was > or = 15 sec after the venting response in 9 instances and < 15 sec afterward in 16 instances. Eight of the 9 trials with delayed swallows (> or = 15 sec) were vented by secondary peristalsis, whereas 11 of the 16 with early swallows (< 15 sec) were vented by TLESR (X2 p < 0.01). TLESRs may be induced by esophageal stimuli, in which case they may represent "wave-suppressed" secondary peristaltic complexes.

Reflux symptoms in 100 normal infants: diagnostic validity of the infant gastroesophageal reflux questionnaire.

To identify the prevalence of reflux symptoms in normal infants, to characterize the diagnostic validity of a previously described 138-item Infant Gastroesophageal Reflux Questionnaire (I-GERQ) for separating normal infants from those with gastroesophageal reflux disease (GERD), and to identify potentially provocative caretaking practices, we administered the questionnaire to 100 infants attending a well-baby clinic (normals) and to 35 infants referred to the Gastroenterology Division for evaluation for GERI) and testing positive on esophageal pH probe or biopsy (GERD infants). Differences were analyzed by Chi-square, and odds ratios were defined. The diagnostic validity of a 25-point I-GERQ GERD score based on 11 items on the questionnaire was evaluated by calculating its sensitivity, specificity, and positive and negative predictive values. We found that normal infants had a high prevalence of reflux symptoms, such as daily regurgitation (40%), respiratory symptoms, crying more than an hour a day (17%), arching (10%), or daily hiccups (36%) but that many symptoms were significantly more prevalent in the GERD than in the normal infants (Chi-square P < .05), and odds ratios were above 3 for nearly 20 items. The positive and negative predictive values for the 25-point I-GERQ score were 1.00 and .94-.98, respectively. Environmental smoke exposure did not quite reach significance as a provocative factor for GERD. Although normal infants have a high prevalence of symptoms suggesting GERD, a simple questionnaire-based score is a valid diagnostic test with high positive and negative predictive values.

Quantitative computer-assisted image analysis of suction biopsy in pediatric gastroesophageal reflux.

Gastroesophageal reflux is a disorder with well-characterized histopathological features in the adult, but the incidence and pathogenesis of epithelial injury in children are poorly understood. Esophageal suction biopsies from 80 infants exhibiting symptoms of reflux were studied by computer-assisted image analysis and the results compared to routine histological scoring. The histological features evaluated were the number of intraepithelial lymphocytes and eosinophils, papillary height, interpapillary basal cell height, and a novel measure "integrated basal cell height." We quantitatively evaluated these histological criteria by computer-assisted image analysis and compared these results to four subjective grades of esophagitis: low, mild, moderate and high. In this report we now describe this quantitative histopathologic method for the evaluation of pediatric esophageal biopsies. Utilizing this method we demonstrate that both inter- and intra-observer variability were sufficiently low to stratify the mucosal changes reliably into at least four categories. The reliability of this objective analytic technique will permit studies into the disease progression and regression in pediatric reflux esophagitis.

Reliability and validity of an infant gastroesophageal reflux questionnaire.

To improve history-taking of infants with suspected gastroesophageal reflux, we developed an Infant Gastroesophageal Reflux Questionnaire consisting of 161 items covering demographics, symptoms (regurgitation, weight deficit, respiratory difficulties, fussiness, apnea, and pain or bleeding of esophagitis), and possible causes (feeding volume and frequency, allergy, infection, colic, central nervous system abnormalities, positioning, and smoke exposure). The questionnaire was completed by primary caretakers of 69 infants aged 1 to 58 weeks suspected of having reflux. Median time to complete the questionnaire was 20 minutes. The median internal consistency of 29 pairs of redundant questions was 0.94. Median test-retest consistency of 110 items for nine respondents was 0.88. Median interobserver consistency, evaluated for 129 items in 35 questionnaires also filled out by secondary caretakers, was 0.85. The median accuracy of four externally validated items was 1.00. This questionnaire can aid pediatricians in making decisions regarding diagnoses and treatment in this common but complex disorder.

Thickened feedings as a cause of increased coughing when used as therapy for gastroesophageal reflux in infants.

To determine whether thickening of infant formula feedings with rice cereal increases coughing, we studied 25 infants from birth to 6 months of age, referred for evaluation of gastroesophageal reflux. Coughing was blindly quantified after each of a pair of isocaloric meals (one thickened and one unthickened). Coughing was more frequent after thickened feedings than after unthickened feedings.

Viable cryopreserved aortic homograft for aortic valve endocarditis and annular abscesses.

Six consecutive patients with active aortic valve endocarditis, including 2 with extensive subannular aortic root abscess, were successfully treated with viable cryopreserved homograft aortic valve replacement. Two patients required extensive aortic root reconstruction with an appropriately trimmed aortic homograft to cover large abscess cavities. All patients showed resolution of infection with no perioperative mortality or clinically significant morbidity. Three patients had a minor degree of aortic insufficiency on postoperative echo-Doppler study. On follow-up at 6 to 48 months, all patients were in New York Heart Association functional class I. The resistance of the unstented homograft to infection makes it an attractive choice for patients requiring aortic valve replacement for active endocarditis. The results of surgical intervention in patients with extensive aortic root involvement may be further improved by the flexibility afforded by the homograft to be "custom-fit" to the abnormal aortic root and the ability to achieve secure abnormal aortic root and the ability to achieve secure valve fixation without use of prosthetic material.