Clusterin up-regulation following sub-lethal oxidative stress and lipid peroxidation in human neuroblastoma cells.

Clusterin/apolipoprotein J is a multifunctional protein up-regulated during various pathophysiological states. Since oxidative stress plays an important role in brain aging, and in many neurodegenerative disorders, to further understand the mechanistic underpinnings of clusterin expression, in this study, we examined clusterin expression in human neuroblastoma cells under conditions of increased production of reactive oxygen species and lipid peroxidation. Specifically, we analyzed clusterin mRNA and protein levels in human neuroblastoma IMR-32 and SH-SY5Y cells following exposure to sub-lethal amounts of iron-ascorbate to induce an increase in reactive oxygen species generation and lipid peroxidation. Under such conditions, we observed a time-dependent up-regulation of clusterin protein and mRNA levels, detected by immunoblot analysis and RT-PCR, respectively. Given the known roles of clusterin, the results of the present study support the notion that an increase in clusterin expression may be a physiological defence mounted to reduce cell damage and maintain cell viability during periods of increased oxidative stress.

p63 expression in salivary gland tumors: role of DeltaNp73L in neoplastic transformation.

To illustrate the role of p63 and its truncated variants in salivary gland tumors, 23 consecutive tumors and 6 normal salivary glands were studied immunohistochemically with anti-p63 antibody and by reverse transcriptase (RT) and nested polymerase chain reaction (PCR) to detect p63 isoform expression. Normal salivary glands: p63 antibody-stained basal and myoepithelial cells; by RT and nested PCR, the 2 main isoforms were present, whereas DeltaNp73L was absent. Tumors: p63 antibody was positive in the following: Warthin tumor (WT) (3/3), oncocytoma (OC) (1/1), pleomorphic adenoma (PA) (7/7), polymorphous-low-grade adenocarcinoma (PLGA) (3/3), adenoid-cystic carcinoma (ADCC)(3/4), epithelial-myoepithelial-cell carcinoma (EMC) (1/1), and myoepithelial-cell carcinoma (MCC) (1/1). By RT and nested PCR all tumors expressed p63 irrespective of their morphologic differentiation. The DeltaNp73L isoform was present in tumoral tissue but absent in normal salivary gland. These data suggest that p63, particularly its splice variant DeltaNp73L, is involved in the neoplastic transformation of salivary glands.

Acquisition of i(8q) as an early event in malignant triton tumors.

Malignant triton tumors (MTT) are rare soft-tissue tumors characterized by a mixture of cells with nerve sheath and skeletal muscle differentiation. MTT is a histological variant of malignant peripheral nerve sheath tumors (MPNST). No characteristic cytogenetic anomaly has been detected in MPNST or MTT. In this paper, we report on the cytogenetic findings of an MTT from a 20-year old male with neurofibromatosis (NF1). The tumoral karyotype showed the modal number to be near-diploid and an abnormal karyotype with a Robertsonian translocation and 4 markers: 49,XY,der(14;15)(q10;q10),+4mar. Spectral karyotyping revealed the karyotype: 49,XY, der(14;15)(q10;q10),+i(8)(q10)x4. Fluorescence in situ hybridization analysis of the tissue confirmed the presence of the additional i(8)(q10) in all tumoral cells. The sequence analysis of p53 revealed a polymorphism in exon 9, codon 329. The two alleles, TTC and TCC, codify for phenylalanine and serine, respectively. Our results indicate that all neoplastic cells have the same cytogenetic pattern, suggesting that both cell lines, which show nerve sheath and skeletal muscle differentiation, are derived from a unique stem cell. The acquired Robertsonian chromosomal recombinants might represent an event in the tumorigenesis of MTT, and the present data suggest that genes located on 8q can be involved in the development of MTT.

Pattern of p63 expression in squamous cell carcinoma of the oral cavity.

P63 is a recently discovered gene harbouring different isoforms by alternate splicing. The two main isoforms, TAp63 and Delta Np63, have opposite functions, being responsible for cell-cycle arrest and cell proliferation, respectively. In addition, new isoforms have been described with the same sequence as TAp63 and Delta Np63, but lacking exon 4 (Delta 4Tap63 and Delta Np73L). P63 as detected using immunohistochemistry is present in squamous cell carcinomas. To better define the role of p63 in squamous cell carcinomas of the oral cavity (OSCC), 39 patients were investigated using immunohistochemical analysis with a monoclonal antibody recognising all p63 isoforms and an anti-Ki67 antibody. Reverse-transcription polymerase chain reaction (PCR) and nested PCR were also performed using isoform-specific primers to evaluate the p63 mRNA expression pattern. Using immunohistochemistry, p63 was always present in OSCC, and its distribution was similar to that of Ki67. The percentage of positive cells increased from normal to neoplastic mucosa, but there was no relationship between the number of p63 positive cells and prognosis. P63 mRNA was found in all patients. The truncated isoforms Delta 4TAp63 and Delta Np73L were more frequently expressed in patients presenting with metastases. Delta Np73L was found in 66.6% of tumours with lymph-node metastases, but in only 33.3% of those devoid of lymph-node metastases at presentation. An impaired expression of the p63 isoforms might favour cell proliferation and indirectly enhance the metastasising capacity of OSCC.

Modeling T-cell activation using gene expression profiling and state-space models.

MOTIVATION: We have used state-space models to reverse engineer transcriptional networks from highly replicated gene expression profiling time series data obtained from a well-established model of T-cell activation. State space models are a class of dynamic Bayesian networks that assume that the observed measurements depend on some hidden state variables that evolve according to Markovian dynamics. These hidden variables can capture effects that cannot be measured in a gene expression profiling experiment, e.g. genes that have not been included in the microarray, levels of regulatory proteins, the effects of messenger RNA and protein degradation, etc. RESULTS: Bootstrap confidence intervals are developed for parameters representing 'gene-gene' interactions over time. Our models represent the dynamics of T-cell activation and provide a methodology for the development of rational and experimentally testable hypotheses. AVAILABILITY: Supplementary data and Matlab computer source code will be made available on the web at the URL given below. SUPPLEMENTARY INFORMATION: http://public.kgi.edu/~wild/LDS/index.htm