The esophageal epithelium is subject to damage from bile acid reflux that promotes normal tissue injury resulting in the development of Barrett's epithelium. There is a selection pressure for mutating p53 in this preneoplastic epithelium, thus identifying a physiologically relevant model for discovering novel regulators of the p53 pathway. Proteomic technologies were used to identify such p53 regulatory factors by identifying proteins that were overexpressed in Barrett's epithelium. A very abundant polypeptide selectively expressed in Barrett's epithelium was identified as anterior gradient-2. Immunochemical methods confirmed that anterior gradient-2 is universally up-regulated in Barrett's epithelium, relative to normal squamous tissue derived from the same patient. Cancer development is a multistep process involving sequential mutation in oncogenes and tumor suppressor genes that give selective advantage to the evolving cancer cells. The major clinical models that are giving novel molecular mechanistic insight into the multistep evolution of human neoplasia include colorectal and esophageal cancer. Colorectal cancer progression is perhaps the most well-characterized model whereby cancer development evolves through histopathological stages termed dysplasia, adenoma, and carcinoma, which can eventually metastasize. Classic molecular studies in colorectal cancer have indicated that RAS and APC mutations occur earlier in this progression sequence, while p53 mutations occur relatively later (1). Additional modifying factors include genome instability, (2), DNA methylation (3), and associated epigenetic changes in the expression of regulatory genes that can have profound effects on cancer incidence (4).Esophageal adenocarcinoma cancer progression also proceeds through a set of morphological intermediates termed metaplasia and dysplasia, which are collectively called Barrett's esophagus or Barrett's epithelium (5, 6). Barrett's epithelium is thought to develop as an adaptive response following exposure to gastric and duodenal contents refluxed into the esophagus. Barrett's epithelium is a premalignant lesion, and although the progression from Barrett's cell types to adenocarcinoma is not inevitable, the risk is estimated at an increase of 30-to 125-fold as compared with the normal population (7-9) The incidence of Barrett's epithelium has dramatically increased over the last decade; however, more alarming is the parallel increase in adenocarcinoma of the esophagus over a similar period of time (10). Barrett's epithelium being hyperproliferative is believed to be a fertile field for malignant transformation, and such early premalignant lesions produce biological and genetic heterogeneity as seen in previous studies by p53 mutations, aneuploidy, and abnormal methylation resulting in stepwise changes in differentiation, proliferation, and apoptosis (11). However, the environmental, genetic, and metabolic factors that regulate stress responses in normal and Barrett's epithelium, as well as the associated transformation o...

Using comparative proteomic analysis we have identified over-expression of mortalin in colorectal adenocarcinomas. Mortalin, also known as mitochondrial heat-shock protein 70 (mhsp 70), is involved in cell cycle regulation with important roles in cellular senescence and immortalization pathways. It is known to bind to and inactivate wild-type tumour suppressor protein p53 and influences the Ras-Raf-MAPK pathway. By immunostaining a colorectal cancer tissue microarray linked to a patient database, we further found that mortalin over-expression correlates with poor patient survival and, in multivariate analysis, is independent of standard prognostic variables (p = 0.04). Our findings demonstrate that mortalin over-expression may predict outcome in colorectal cancer and suggest that this protein is involved in colorectal neoplasia. Our experimental approach emphasises the analytical power of combining proteomics with tissue microarray analysis in the context of a well-defined tumour database.

Heterogeneous ribonucleoprotein K (hnRNP K) is a member of the hnRNP family which has several different cellular roles including transcription, mRNA shuttling, RNA editing and translation. Several reports implicate hnRNP K having a role in tumorigenesis, for instance hnRNP K increases transcription of the oncogene c-myc and hnRNP K expression is regulated by the p53/MDM 2 pathway. In this study comparing normal colon to colorectal cancer by proteomics, hnRNP K was identified as being overexpressed in this type of cancer. Immunohistochemistry with a monoclonal antibody to hnRNP K (which we developed) on colorectal cancer tissue microarray, confirmed that hnRNP K was overexpressed in colorectal cancer (Po0.001) and also showed that hnRNP K had an aberrant subcellular localisation in cancer cells. In normal colon hnRNP K was exclusively nuclear whereas in colorectal cancer the protein localised both in the cytoplasm and the nucleus. There were significant increases in both nuclear (P ¼ 0.007) and cytoplasmic (P ¼ 0.001) expression of hnRNP K in Dukes C tumours compared with early stage tumours. In Dukes C patient's good survival was associated with increased hnRNP K nuclear expression (P ¼ 0.0093). To elaborate on the recent observation that hnRNP K is regulated by p53, the expression profiles of these two proteins were also analysed. There was no correlation between hnRNP K and p53 expression, however, patients who presented tumours that were positive for hnRNP K and p53 had a poorer survival outcome (P ¼ 0.045).

The global analysis of cellular proteins has recently been termed proteomics and is a key area of research that is developing in the post-genome era. Proteomics uses a combination of sophisticated techniques including two-dimensional (2D) gel electrophoresis, image analysis, mass spectrometry, amino acid sequencing, and bio-informatics to resolve comprehensively, to quantify, and to characterize proteins. The application of proteomics provides major opportunities to elucidate disease mechanisms and to identify new diagnostic markers and therapeutic targets. This review aims to explain briefly the background to proteomics and then to outline proteomic techniques. Applications to the study of human disease conditions ranging from cancer to infectious diseases are reviewed. Finally, possible future advances are briefly considered, especially those which may lead to faster sample throughput and increased sensitivity for the detection of individual proteins.

Exposure of Saccharomyces cerevisiae to 0.9 mM sorbic acid at pH 4.5 resulted in the upregulation of 10 proteins; Hsp42, Atp2, Hsp26, Ssa1 or Ssa2, Ssb1 or Ssb2, Ssc1, Ssa4, Ach1, Zwf1 and Tdh1; and the downregulation of three proteins; Ade16, Adh3 and Eno2. In parallel, of 6144 ORFs, 94 (1.53%) showed greater than a 1.4-fold increase in transcript level after exposure to sorbic acid and five of these were increased greater than two-fold; MFA1, AGA2, HSP26, SIP18 and YDR533C. Similarly, of 6144 ORFs, 72 (1.17%) showed greater than a 1.4-fold decrease in transcript level and only one of these, PCK1, was decreased greater than two-fold. Functional categories of genes that were induced by sorbic acid stress included cell stress (particularly oxidative stress), transposon function, mating response and energy generation. We found that proteomic analysis yielded distinct information from transcript analysis. Only the upregulation of Hsp26 was detected by both methods. Subsequently, we demonstrated that a deletion mutant of Hsp26 was sensitive to sorbic acid. Thus, the induction of Hsp26, which occurs during adaptation to sorbic acid, confers resistance to the inhibitory effects of this compound.

The chaperonins are key molecular complexes, which are essential in the folding of proteins to produce stable and functionally competent protein conformations. One member of the chaperonin group of proteins is TCP1 (chaperonin containing t-complex polypeptide 1, or CCT), but little is known about this protein in tumours. In this study, we used comparative proteomic analysis to show that t-complex protein subunits TCP1 beta and TCP1 epsilon are over-expressed in colorectal adenocarcinomas. Monoclonal antibodies to these proteins were developed and the expression and cellular localization of these two proteins in colorectal cancer were analysed by immunohistochemistry on a colorectal cancer tissue microarray. In colorectal cancer, TCP1 beta cellular localization was exclusively cytoplasmic, whereas TCP1 epsilon staining was seen in both the nucleus and the cytoplasm. Both cytoplasmic TCP1 beta and cytoplasmic TCP1 epsilon were significantly over-expressed (p < 0.001 for each protein) in primary colorectal cancer and also showed increased expression with advancing Dukes' stage (p = 0.018 for TCP1 beta and p = 0.045 for TCP1 epsilon). A trend was also identified between over-expression of cytoplasmic TCP1 beta and reduced patient survival (p = 0.05). These results show that both TCP1 beta and TCP1 epsilon are over-expressed in colorectal cancer and indicate a role for TCP1 beta and TCP1 epsilon in colorectal cancer progression.

Aims-Laser capture microdissection is a recent development that enables the isolation of specific cell types for subsequent molecular analysis. This study describes a method for obtaining proteome information from laser capture microdissected tissue using colon cancer as a model. Methods-Laser capture microdissection was performed on toluidine blue stained frozen sections of colon cancer. Tumour cells were selectively microdissected. Conditions were established for solubilising proteins from laser microdissected samples and these proteins were separated by two dimensional gel electrophoresis. Individual protein spots were cut from the gel, characterised by mass spectrometry, and identified by database searching. These results were compared with protein expression patterns and mass spectroscopic data obtained from bulk tumour samples run in parallel. Results-Proteins could be recovered from laser capture microdissected tissue in a form suitable for two dimensional gel electrophoresis. The solubilised proteins retained their expected electrophoretic mobility in two dimensional gels as compared with bulk samples, and mass spectrometric analysis was also unaVected. Conclusion-A method for performing two dimensional gel electrophoresis and mass spectrometry using laser capture microdissected tissue has been developed. (J Clin Pathol: Mol Pathol 2001;54:253-258)

In this study, we identified lysine residues in the fibrinogen A␣ chain that serve as substrates during transglutaminase (TG)-mediated cross-linking of plasminogen activator inhibitor 2 (PAI-2). Comparisons were made with ␣ 2 -antiplasmin (␣ 2 -AP), which is known to cross-link to lysine 303 of the A␣ chain. A 30-residue peptide containing Lys-303 specifically competed with fibrinogen for cross-linking to ␣ 2 -AP but not for crosslinking to PAI-2. Further evidence that PAI-2 did not cross-link via Lys-303 was the cross-linking of PAI-2 to I-9 and des-␣C fibrinogens, which lack 100 and 390 amino acids from the C terminus of the A␣ chain, respectively. PAI-2 or ␣ 2 -AP was cross-linked to fibrinogen and digested with trypsin or endopeptidase Glu-C, and the resulting peptides analyzed by mass spectrometry. Peptides detected were consistent with tissue TG (tTG)-mediated cross-linking of PAI-2 to lysines 148, 176, 183, 457 and factor XIIIa-mediated cross-linking of PAI-2 to lysines 148, 230, and 413 in the A␣ chain. ␣ 2 -AP was crosslinked only to lysine 303. Cross-linking of PAI-2 to fibrinogen did not compete with ␣ 2 -AP, and the two proteins utilized different lysines in the A␣ chain. Therefore, PAI-2 and ␣ 2 -AP can cross-link simultaneously to the ␣ polymers of a fibrin clot and promote resistance to lysis.