ABSTRACT
In this paper, three different clustering algorithms were applied to assemble infrared (IR) spectral maps from IR microspectra of tissues. Using spectra from a colorectal adenocarcinoma section, we show how IR images can be assembled by agglomerative hierarchical (AH) clustering (Ward's technique), fuzzy C-means (FCM) clustering, and k-means (KM) clustering. We discuss practical problems of IR imaging on tissues such as the influence of spectral quality and data pretreatment on image quality. Furthermore, the applicability of cluster algorithms to the spatially resolved microspectroscopic data and the degree of correlation between distinct cluster images and histopathology are compared. The use of any of the clustering algorithms dramatically increased the information content of the IR images, as compared to univariate methods of IR imaging (functional group mapping). Among the cluster imaging methods, AH clustering (Ward's algorithm) proved to be the best method in terms of tissue structure differentiation.
Subject(s)
Adenocarcinoma/chemistry , Adenocarcinoma/pathology , Colorectal Neoplasms/chemistry , Colorectal Neoplasms/pathology , Spectroscopy, Fourier Transform Infrared/methods , Algorithms , Cluster Analysis , Humans , Image Enhancement , Staining and LabelingABSTRACT
Profilin 1 (PFN1) is a regulator of the microfilament system and is involved in various signaling pathways. It interacts with many cytoplasmic and nuclear ligands. The importance of PFN1 for human tissue differentiation has been demonstrated by the findings that human cancer cells, expressing conspicuously low PFN1 levels, adopt a nontumorigenic phenotype upon raising their PFN1 level. In the present study, we characterize the ligand binding site crucial for profilin's tumor suppressor activity. Starting with CAL51, a human breast cancer cell line highly tumorigenic in nude mice, we established stable clones that express PFN1 mutants differentially defective in ligand binding. Clones expressing PFN1 mutants with reduced binding to either poly-proline-stretch ligands or phosphatidyl-inositol-4,5-bisphosphate, but with a functional actin binding site, were normal in growth, adhesion, and anchorage dependence, with only a weak tendency to elicit tumors in nude mice, similar to controls expressing wild-type PFN1. In contrast, clones expressing a mutant with severely reduced capacity to bind actin still behaved like the parental CAL51 and were highly tumorigenic. We conclude that the actin binding site on profilin is instrumental for normal differentiation of human epithelia and the tumor suppressor function of PFN1.
Subject(s)
Actins/chemistry , Contractile Proteins/physiology , Genes, Tumor Suppressor , Microfilament Proteins/physiology , Neoplasms/metabolism , Actin Cytoskeleton/metabolism , Actins/metabolism , Animals , Binding Sites , Cell Adhesion , Cell Division , Cell Line, Tumor , Cell Movement , Collagen/pharmacology , Cytoplasm/metabolism , Drug Combinations , Epithelium/metabolism , Female , Humans , Immunoblotting , Laminin/pharmacology , Ligands , Mice , Mice, Nude , Mutation , Neoplasm Transplantation , Phenotype , Phosphatidylinositol 4,5-Diphosphate/chemistry , Point Mutation , Profilins , Proteoglycans/pharmacology , Recombinant Proteins/chemistry , Signal Transduction , Time Factors , TransfectionABSTRACT
Aberrant glycosylation of membrane components due to specific alterations of glycosyltransferase activity is a common feature of carcinoma cells and is usually associated with invasion and metastasis. In a prospective study, the enzyme activity of the sialyltransferases ST6GAL-I and ST3GAL-III was studied in gastric cancer and normal mucosa in 55 patients by a radiometric assay. Cellular localization of sialyltransferase ST6GAL-I mRNA expression was studied by in situ hybridization. Sialyltransferase ST6GAL-I mRNA expression was mainly localized to epithelial cells. ST6GAL-I enzyme activity was enhanced within the tumor tissue. Significant correlations were found between the presence of signet ring cells and enhanced ST6GAL-I activity in the tumor tissue (p = 0.047) or in the mucosa (p = 0.024), and between signet ring cells and ST3GAL-III activity in the mucosa (p < 0.001). Multivariate Cox analysis demonstrated that only lymph node metastases (p = 0.044) had a significant influence on tumor-related survival. ST3GAL-III and ST6GAL-I activity showed no independent prognostic relevance in multivariate analysis, but high levels of ST3GAL-III and ST6GAL-I in the tumor tissue correlated with secondary local tumor recurrence (p = 0.005; p = 0.012). Interestingly, also the nonmalignant and uninvolved mucosa of tumor patients was altered on the molecular level and in some cases showed enhanced sialyltransferase levels indicative of the alteration of glycosylation very early during tumorigenesis.
