Mass spectrometry is being used to find disease-related patterns in mixtures of proteins derived from biological fluids. Questions have been raised about the reproducibility and reliability of peak quantifications using this technology. We collected nipple aspirate fluid from breast cancer patients and healthy women, pooled them into a quality control sample, and produced 24 replicate SELDI spectra. We developed a novel algorithm to process the spectra, denoising with the undecimated discrete wavelet transform (UDWT), and evaluated it for consistency and reproducibility. UDWT efficiently decomposes spectra into noise and signal. The noise is consistent and uncorrelated. Baseline correction produces isolated peak clusters separated by flat regions. Our method reproducibly detects more peaks than the method implemented in Ciphergen software. After normalization and log transformation, the mean coefficient of variation of peak heights is 10.6%. Our method to process spectra provides improvements over existing methods. Denoising using the UDWT appears to be an important step toward obtaining results that are more accurate. It improves the reproducibility of quantifications and supplies tools for investigation of the variations in the technology more carefully. Further study will be required, because we do not have a gold standard providing an objective assessment of which peaks are present in the samples.

Polypyrimidine tract-binding protein 1 (PTBP1) is a multi-functional RNA-binding protein that is aberrantly overexpressed in glioma. PTBP1 and its brain-specific homologue polypyrimidine tract-binding protein 2 (PTBP2) regulate neural precursor cell differentiation. However, the overlapping and non-overlapping target transcripts involved in this process are still unclear. To determine why PTBP1 and not PTBP2 would promote glial cell-derived tumours, both PTBP1 and PTBP2 were knocked down in the human glioma cell lines U251 and LN229 to determine the role of these proteins in cell proliferation, migration, and adhesion. Surprisingly, removal of both PTBP1 and PTBP2 slowed cell proliferation, with the double knockdown having no additive effects. Decreased expression of both proteins individually and in combination inhibited cell migration and increased adhesion of cells to fibronectin and vitronectin. A global survey of differential exon expression was performed following PTBP1 knockdown in U251 cells using the Affymetrix Exon Array to identify PTBP1-specific splicing targets that enhance gliomagenesis. In the PTBP1 knockdown, previously determined targets were unaltered in their splicing patterns. A single gene, RTN4 (Nogo) had significantly enhanced inclusion of exon 3 when PTBP1 was removed. Overexpression of the splice isoform containing exon 3 decreased cell proliferation to a similar degree as the removal of PTBP1. These results provide the first evidence that RNA-binding proteins affect the invasive and rapid growth characteristics of glioma cell lines. Its actions on proliferation appear to be mediated, in part, through alternative splicing of RTN4.

Background: Tumor-predominant splice isoforms were identified during comparative in silico sequence analysis of EST clones, suggesting that global aberrant alternative pre-mRNA splicing may be an epigenetic phenomenon in cancer. We used an exon expression array to perform an objective, genome-wide survey of glioma-specific splicing in 24 GBM and 12 nontumor brain samples. Validation studies were performed using RT-PCR on glioma cell lines, patient tumor and nontumor brain samples.

Normal human prostate (NHP) epithelial cells undergo senescence in vitro and in vivo, but the underlying molecular mechanisms remain obscure. Here we show that the senescence of primary NHP cells, which are immunophenotyped as intermediate basal-like cells expressing progenitor cell markers CD44, ␣2␤1, p63, hTERT, and CK5/CK18, involves loss of telomerase expression, up-regulation of p16, and activation of p53. Using genetically defined manipulations of these three signaling pathways, we show that p16 is the primary determinant of the NHP cell proliferative capacity and that hTERT is required for unlimited proliferative life span. Hence, suppression of p16 significantly extends NHP cell life span, but both p16 inhibition and hTERT are required to immortalize NHP cells. Importantly, immortalized NHP cells retain expression of most progenitor markers, demonstrate gene expression profiles characteristic of proliferating progenitor cells, and possess multilineage differentiation potential generating functional prostatic glands. Our studies shed important light on the molecular mechanisms regulating the proliferative life span of NHP progenitor cells.The prostatic glands contain neuroendocrine (NE) 7 cells and two epithelial cells: 1) luminal cells expressing cytokeratin 8 (CK8) and CK18, androgen receptor (AR), prostate-specific antigen (PSA), prostatic acid phosphatase, CD26, CD57, and 15-lipoxygenase 2 (15-LOX2) and 2) basal cells expressing CK5/CK14, CD44, CD104 (integrin ␤4), Bcl-2, p63, telomerase, and glutathione S-transferase-(1-3). It has been proposed that a common stem/progenitor cell may generate both basal and luminal cells (4). Alternatively, basal cells may function as progenitors to luminal cells (5, 6).The adult rodent prostate possesses regenerative stem cells (SCs) (6 -8). Whether adult human prostate contains definitive SCs is less certain, although there exists strong evidence that the basal cell layer harbors regenerative cells (1, 6, 9 -12), and several candidate populations of human prostate stem/progenitor cells, preferentially localized in the basal layer, have been reported. These include the cells that preferentially express CD44 (13), ␣2␤1 (i.e. ␣2␤1 hi ) (14), or CD133 (15, 16) and the side population (17), whose phenotype is mediated by multidrug resistance family proteins, such as MDR-1 and ABCG2 (18). Interestingly, the ABCG2 ϩ cells in the benign prostate constitute Ͻ1% of total basal cell population and share essentially the same transcriptome as the side population cells (19 * This work was supported, in whole or in part, by National Institutes of Health Grants R01-AG023374, R01-ES015888, and R21-ES015893-01A1. This work was also supported by American Cancer Society Grant RSG MGO-105961, Department of Defense Grants W81XWH-07-1-0616 and PC073751, the Prostate Cancer Foundation, the Elsa Pardee Foundation (to D. G. T.), and two Center Grants, CCSG-5 P30 CA166672 and ES07784. The costs of publication of this article were defrayed in part by the payment of page charges. This article m...