Aberrant epigenetic silencing of neuronatin is a frequent event in human osteosarcoma.

The paternally imprinted neuronatin (NNAT) gene has been identified as a target of aberrant epigenetic silencing in diverse cancers, but no association with pediatric bone cancers has been reported to date. In screening childhood cancers, we identified aberrant CpG island hypermethylation in a majority of osteosarcoma (OS) samples and in 5 of 6 human OS cell lines studied but not in normal bone-derived tissue samples. CpG island hypermethylation was associated with transcriptional silencing in human OS cells, and silencing was reversible upon treatment with 5-aza-2'-deoxycytidine. Expression of NNAT was detectable in osteoblasts and chondrocytes of human bone, supporting a potential role in bone homeostasis. Enforced expression of NNAT in human OS cells lacking endogenous expression resulted in significant reduction in colony formation and in vitro migration compared to nonexpressor control cells. We next analyzed the effect of NNAT expression on intracellular calcium homeostasis and found that was associated with an attenuated decay of calcium levels to baseline following ATP-induced release of calcium from endoplasmic reticulum (ER) stores. Furthermore, NNAT expression was associated with increased cytotoxicity in OS cells from thapsigargin, an inhibitor of calcium reuptake into ER and an inducer of the ER stress response. These results suggest a possible tumor suppressor role for NNAT in human osteosarcoma. Additional study is needed ascertain sensitization to ER stress-associated apoptosis as a mechanism of NNAT-dependent cytotoxicity. In that case, epigenetic modification therapy to effect NNAT transcriptional derepression may represent a therapeutic strategy potentially of benefit to a majority of osteosarcoma patients.

WT1 regulates cyclin A1 expression in K562 cells.

The restricted expression of Wilms tumor 1 (WT1) and cyclin A1 (CCNA1) in normal tissues, as opposed to their abnormal expression in leukemia demonstrates the applicability of WT1 and CCNA1 as cancer antigens for immunotherapy, and as markers for prognosis and relapse. In this study, the WT1 and CCNA1 mRNA levels were found to be elevated in bone marrow samples from pediatric acute promyelocytic leukemia (APL or AML­M3) patients, and to be quite varied in pediatric acute lymphocytic leukemia (ALL) patients, compared to non­leukemic bone marrow controls. Consistent with the observed upregulation of both WT1 and CCNA1 in APL, WT1 overexpression elevated the CCNA1 mRNA levels in K562 leukemia cells. Treatment with curcumin decreased the WT1 levels in K562 cells, and also decreased CCNA1 protein expression. The examination of the CCNA1 promoter identified potential canonical WT1 binding sites within the 3­kb region upstream of the transcription start site. Chromatin immunoprecipitation and luciferase reporter assays confirmed WT1 binding and the activation of the CCNA1 promoter. Furthermore, the GC­rich core CCNA1 promoter region provided additional non­canonical WT1 activation sites, as revealed by promoter assays. The importance of the GC­rich core region of the CCNA1 promoter was confirmed by treating the K562 cells with mithramycin A, which blocks the binding of zinc finger transcription factors to GC­rich sequences. Mithramycin A subsequently suppressed both CCNA1 promoter activity and protein expression in the K562 cells. Taken together, the data from the WT1 overexpression, and curcumin and mithramycin A treatment experiments, as well as those from chromatin binding assays, along with inferences from patient RNA analyses, establish a plausible link between WT1 and CCNA1, and support the functional significance of an elevated WT1 expression in leukemia, which may also affect CCNA1 expression.

An unusual form of superficially disseminated glioma in children: report of 3 cases.

Three children, aged 4, 5, and 9 years, had an insidious onset of ataxia. Magnetic resonance imaging (MRI) showed hydrocephalus and countless foci of high T2 signal coating the cerebellum, basilar cisterns, brainstem, and fourth ventricle. Similar lesions were present in the spinal cord. Symptoms were relatively mild given the massive tumor burden. Biopsies were composed of superficially infiltrating cells with oligodendroglioma-like features (perinuclear halos and cytologic monotony) and microcysts. Classical cytogenetic analysis of 2 cases showed normal karyotypes. Chromosome fluorescence in situ hybridization revealed 1p36 deletion with intact 19q in 2 cases and no abnormality in one. A similar combination of clinical, MRI, and histopathologic findings has been reported previously in 10 other cases. The pathologic findings suggest a glioma with diffuse or multifocal superficial origin and do not correspond to a described entity in the current World Health Organization (WHO) classification of brain tumors.

Hypermethylation of the imprinted NNAT locus occurs frequently in pediatric acute leukemia.

Recent studies have demonstrated imprinting of the human neuronatin (NNAT) gene. NNAT maps to 20q11.2-q12, a region exhibiting loss of heterozygosity in acute myeloid leukemia and myelodysplastic/myeloproliferative disease. To investigate possible epigenetic dysregulation of genes in this region relevant to leukemogenesis, we analyzed methylation of the NNAT gene in normal tissues and in leukemias. We found a differential methylation pattern, typical of imprinted genes, at sites in the CpG island containing NNAT exon 1 in normal pituitary, peripheral blood cells and bone marrow-derived CD34-positive hematopoietic progenitor cells. Substantial or complete loss of the unmethylated NNAT allele was observed in leukemia cell lines and in 20 of 29 (69%) acute myeloid or lymphoid leukemia samples. While most highly expressed in brain, NNAT mRNA was also detected in normal hematopoietic progenitor cells and in leukemia cells exhibiting the normal methylation pattern, although not in hypermethylated leukemia cells. Demethylation by treatment of hypermethylated leukemia cells with 5-aza-2'-deoxycytidine resulted in reactivation of NNAT expression, concomitant with a reversion to the normal methylation pattern. The data demonstrate that hypermethylation of the NNAT locus is a frequent event in both myeloid and lymphoid acute leukemias of childhood. Aberrant hypermethylation of the NNAT locus suggests that the dysregulation of genes at 20q11.2-q12 in leukemia may be the result of epigenetic as well as genetic events.