In Vitro Assessment of the Role of p53 on Chemotherapy Treatments in Neuroblastoma Cell Lines.

Neuroblastoma is the most frequent malignant extracranial solid tumor of infancy. The overall objective of this work consists of determining the presence of alterations in the p53/MDM2/p14ARF signaling pathway in neuroblastoma cell lines and deciphering their possible relationship with resistance to known antineoplastic drugs and to differentiation agents. Firstly, we characterized 10 neuroblastoma cell lines for alterations at the p53/MDM2/p14ARF signaling pathway by analysis of TP53 point mutations, MYCN and MDM2 amplification, and p14ARF methylation, homozygous deletions, and expression.

Changes in gene expression profiling of apoptotic genes in neuroblastoma cell lines upon retinoic acid treatment.

To determine the effect of retinoic acid (RA) in neuroblastoma we treated RA sensitive neuroblastoma cell lines with 9-cis RA or ATRA for 9 days, or for 5 days followed by absence of RA for another 4 days. Both isomers induced apoptosis and reduced cell density as a result of cell differentiation and/or apoptosis. Flow cytometry revealed that 9-cis RA induced apoptosis more effectively than ATRA.

Promoter Methylation of RASSF1A Associates to Adult Secondary Glioblastomas and Pediatric Glioblastomas.

While allelic losses and mutations of tumor suppressor genes implicated in the etiology of astrocytoma have been widely assessed, the role of epigenetics is still a matter of study. We analyzed the frequency of promoter hypermethylation by methylation-specific PCR (MSP) in five tumor suppressor genes (PTEN, MGMT, RASSF1A, p14(ARF), and p16(INK4A)), in astrocytoma samples and cell lines. RASSF1A was the most frequently hypermethylated gene in all grades of astrocytoma samples, in cell lines, and in adult secondary GBM.

Inhibition of the sonic hedgehog pathway by cyplopamine reduces the CD133+/CD15+ cell compartment and the in vitro tumorigenic capability of neuroblastoma cells.

Sonic hedgehog (Hh) developmental pathway deregulation has been proven to play an essential role in several malignancies as neuroblastoma. We found that Hh signaling is active in neuroblastoma, as most pathway components, including GLI1, were expressed in cell lines and tumor samples. Furthermore, SHH ligand expression was found in cell lines and tumors, and GLI1 up-regulation was achieved in response to SHH treatment, suggesting an autocrine mechanism of aberrant activation.

Analysis of stemness gene expression and CD133 abnormal methylation in neuroblastoma cell lines.

Neuroblastoma is the most common extracranial solid tumor in children, accounting for up to 10% of all childhood malignancies. Cellular heterogeneity is a hallmark of this embryonal cancer, as distinct neural crest lineages can be found within the same tumor sample. The aim of our study was to investigate the presence of a subpopulation of immature cells with features of cancer-like stem cells in 10 neuroblastoma cell lines.

RASSF1A, BLU, NORE1A, PTEN and MGMT expression and promoter methylation in gliomas and glioma cell lines and evidence of deregulated expression of de novo DNMTs.

Methylation of CpG islands in gene promoters can lead to gene silencing. Together with deletion or mutation, it may cause a loss of function of tumor suppressor genes. RASSF1A (3p21.

Detection of methylation in promoter sequences by melting curve analysis-based semiquantitative real time PCR.

Background: We present two melting curve analysis (MCA)-based semiquantitative real time PCR techniques to detect the promoter methylation status of genes. The first, MCA-MSP, follows the same principle as standard MSP but it is performed in a real time thermalcycler with results being visualized in a melting curve. The second, MCA-Meth, uses a single pair of primers designed with no CpGs in its sequence.

Loss of heterozygosity and microsatellite instability on chromosome arm 10q in neuroblastoma.

Tumor suppressor genes can be inactivated by various mechanisms, including promoter hypermethylation and loss of heterozygosity. We screened the 10q locus for loss of heterozygosity and the promoter methylation status of PTEN, MGMT, MXI1, and FGFR2 in neuroblastic tumors and neuroblastoma cell lines. Expression of these genes in cell lines was analyzed with reverse transcriptase-polymerase chain reaction.

Frequent promoter hypermethylation of RASSF1A and CASP8 in neuroblastoma.

Background: Epigenetic alterations and loss of heterozygosity are mechanisms of tumor suppressor gene inactivation. A new carcinogenic pathway, targeting the RAS effectors has recently been documented. RASSF1A, on 3p21.

Homozygous deletion and expression of PTEN and DMBT1 in human primary neuroblastoma and cell lines.

Neuroblastoma is the most common pediatric solid tumor. Although many allelic imbalances have been described, a bona fide tumor suppressor gene for this disease has not been found yet. In our study, we analyzed 2 genes, PTEN and DMBT1, mapping 10q23.