ABSTRACT
This report summarises selected presentations that focused on aspects of biomarker discovery and validation, imaging in preclinical and clinical development and biomarkers in clinical drug development, as discussed during the Third Annual Biomarkers Congress held in Manchester, UK on 14 - 15 May 2008. More than 140 delegates attended this event to discuss the application of biomarkers to all facets of the drug development process. The conference consisted of five intensive streams discussing current and future directions in: i) biomarker discovery and validation; ii) molecular diagnostics, data integration, data analysis, modelling and bioinformatics; iii) biomarkers in clinical drug development; iv) biomarker discovery and validation: therapeutic areas; and v) imaging in preclinical and clinical development and safety biomarkers. This review will focus on selected presentations from the first, third and fifth streams.
ABSTRACT
The p21Waf1/Cip1/Sdi1 cyclin-dependent kinase inhibitor is a key regulator of cell cycle progression and has also been observed to influence the expression of genes associated with several age-related disorders. Previous work has shown that expression of p21 in tumour cells mediates an antiapoptotic and mitogenic paracrine effect, which is in contrast to the arrested state of p21-expressing cells. Here, we have employed SELDI-MS technology to characterise, at a proteomic level, factors released from HT-1080 human fibrosarcoma cells displaying inducible p21 expression. Conditioned media from induced and noninduced cells were profiled on a range of diverse ProteinChip arrays and subjected to SELDI-MS analysis. Evaluation of proteins binding onto IMAC, Q10 or CM10 surfaces led to the discovery of a number of putative p21-regulated factors. We further validated three p21-regulated proteins observed at 10.2, 11.7 and 13.4 kDa. Using Q Ceramic HyperD fractionation columns, we were able to selectively enrich for each of these three proteins. Subsequent SDS-PAGE and MS analysis of tryptic digests identified the 13.4 kDa protein as cystatin C and the 10.2 kDa protein as pro-platelet basic protein (PPBP). Judging by the apparent MW and the pI of the 11.7 kDa protein, we reasoned that it may be beta-2-microglobulin, which was confirmed by subsequent identification. Increased levels of cystatin C and beta-2-microglobulin in conditioned media from p21-expressing cells was confirmed by antibody capture experiments using anticystatin C and anti-beta-2-microglobulin antibodies on preactivated PS-20 arrays. Western blot analysis demonstrated increased expression of intracellular and extracellular cystatin C and beta-2-microglobulin in p21-expressing cells, compared to noninduced controls. Increased levels of PPBP were validated in cell lysates from p21-expressing cells. The three secreted factors that we have identified in this study, have all been shown previously to have growth modulating effects and, as such, may contribute to the observed mitogenic and anti-apoptotic paracrine activity of p21-expressing [corrected] cells.
Subject(s)
Cyclin-Dependent Kinase Inhibitor p21/metabolism , Proteomics , Blotting, Western , Cell Line, Tumor , Electrophoresis, Polyacrylamide Gel , Humans , Mass SpectrometryABSTRACT
The incidence of melanoma is increasing rapidly, with advanced lesions generally failing to respond to conventional chemotherapy. Here, we utilized DNA microarray-based gene expression profiling techniques to identify molecular determinants of melanoma progression within a unique panel of isogenic human melanoma cell lines. When a poorly tumorigenic cell line, derived from an early melanoma, was compared with two increasingly aggressive derivative cell lines, the expression of 66 genes was significantly changed. A similar pattern of differential gene expression was found with an independently derived metastatic cell line. We further examined these melanoma progression-associated genes via use of a tailored TaqMan Low Density Array (LDA), representing the majority of genes within our cohort of interest. Considerable concordance was seen between the transcriptomic profiles determined by DNA microarray and TaqMan LDA approaches. A range of novel markers were identified that correlated here with melanoma progression. Most notable was TSPY, a Y chromosome-specific gene that displayed extensive down-regulation in expression between the parental and derivative cell lines. Examination of a putative CpG island within the TSPY gene demonstrated that this region was hypermethylated in the derivative cell lines, as well as metastatic melanomas from male patients. Moreover, treatment of the derivative cell lines with the DNA methyltransferase inhibitor, 2'-deoxy-5-azacytidine (DAC), restored expression of the TSPY gene to levels comparable with that found in the parental cells. Additional DNA microarray studies uncovered a subset of 13 genes from the above-mentioned 66 gene cohort that displayed re-activation of expression following DAC treatment, including TSPY, CYBA and MT2A. DAC suppressed tumor cell growth in vitro. Moreover, systemic treatment of mice with DAC attenuated growth of melanoma xenografts, with consequent re-expression of TSPY mRNA. Overall, our data support the hypothesis that multiple genes are targeted, either directly or indirectly, by DNA hypermethylation during melanoma progression.
Subject(s)
DNA Methylation , Gene Expression Regulation, Neoplastic , Melanoma, Experimental/genetics , Skin Neoplasms/genetics , Animals , Azacitidine/analogs & derivatives , Azacitidine/pharmacology , Azacitidine/therapeutic use , Biomarkers, Tumor , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , DNA Modification Methylases/antagonists & inhibitors , DNA Modification Methylases/genetics , DNA Modification Methylases/metabolism , Decitabine , Disease Progression , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Epigenesis, Genetic , Female , Gene Expression Profiling , Humans , Melanoma, Experimental/metabolism , Melanoma, Experimental/prevention & control , Mice , Mice, Nude , Oligonucleotide Array Sequence Analysis , RNA, Messenger/metabolism , RNA, Neoplasm/metabolism , Skin Neoplasms/prevention & control , Skin Neoplasms/secondary , Transplantation, Heterologous , Tumor Cells, CulturedABSTRACT
The fibulins are a family of secreted glycoproteins, which are characterised by repeated epidermal-growth-factor-like domains and a unique C-terminal structure. Six distinct fibulin genes, encoding at least nine protein products generated by alternative splicing, have been identified. Considerable evidence is available pointing towards a structural role for fibulins within the extracellular matrix. Fibulins have been shown to modulate cell morphology, growth, adhesion and motility. The dysregulation of certain fibulins occurs in a range of human disorders, including cancer. Indeed, both tumour suppressive and oncogenic activities have been proposed for members of the fibulin family. Herein, we discuss the possible roles of fibulins in cancer, in addition to their diagnostic and therapeutic potential.
