ABSTRACT
The usefulness of computer analysis of two-dimensional electrophoresis gels has been investigated on the example of human keratinocytes transformation. For this purpose, the protein expression of various keratinocytes strains from normal to tumor cells has been analysed by two-dimensional electrophoresis. The resulting gels have been submitted to computer analysis, including various data analysis techniques allowing to select spots on the gels or to classify the gels themselves. The latter techniques appeared very useful, since they demonstrated that the major transition in words of variation of the protein expression lies at the normal cell/transformed cell transition rather than at the transformed cell/tumorigenic cell transition.
Subject(s)
Electrophoresis, Gel, Two-Dimensional , Image Processing, Computer-Assisted , Keratinocytes/metabolism , Neoplasm Proteins/biosynthesis , Neoplasms/metabolism , Protein Biosynthesis , Animals , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/pathology , Cell Line , Cell Line, Transformed , Cell Transformation, Neoplastic , Cell Transformation, Viral , Female , Gene Expression , Humans , Mice , Mice, Nude , Neoplasm Proteins/isolation & purification , Neoplasms/pathology , Proteins/isolation & purification , Simian virus 40 , Tumor Cells, Cultured , Uterine Neoplasms/metabolism , Uterine Neoplasms/pathology , Vulvar Neoplasms/metabolism , Vulvar Neoplasms/pathologyABSTRACT
Xeroderma pigmentosum (XP) and trichothiodystrophy (TTD) are two recessively transmitted human diseases characterized by DNA repair deficiency. While XP is associated with a very high incidence of cancer on skin exposed to sunlight, TTD is not a cancer-prone disease. Therefore, unrepaired UV-induced DNA lesions do not appear to be enough to give rise to tumors. In order to understand the differences between these two syndromes, we measured catalase activity in cellular extracts, UV irradiated or not, and quantified H2O2 production following in vitro UV irradiation. We confirmed on 21 different XP diploid fibroblast lines that catalase activity was decreased on average by a factor of five as compared to controls, while XP heterozygote lines exhibited intermediary responses. All seven TTD lines we tested were deficient in UV-induced lesion repair and exhibited a high level of catalase activity. However, molecular analysis of catalase transcription showed no difference between normal, XP and TTD cell lines. This was confirmed by Western blots where the amount of catalase subunits was identical in all cell lines studied. Finally, UV irradiation induces five and three times more H2O2 production in XP lines compared with TTD or controls respectively. These striking differences between TTD and XP indicate that UV light, directly or indirectly, together with defective oxidative metabolism may increase the initiation and/or the progression steps in the XP environment compared to TTD. This may partly explain the different tumoral phenotype observed between the two diseases.
Subject(s)
Acatalasia , DNA Repair , Hair Diseases/enzymology , Nail Diseases/enzymology , Xeroderma Pigmentosum/enzymology , Adolescent , Adult , Catalase/radiation effects , Cells, Cultured , Child , Child, Preschool , DNA Repair/radiation effects , Female , Hair Diseases/genetics , Heterozygote , Humans , Infant , Male , Nail Diseases/genetics , Oxygen Consumption/radiation effects , Xeroderma Pigmentosum/geneticsABSTRACT
Total nuclear proteins extracted from cell lines representing various stages of differentiation of mouse B lymphocytes were studied by computer analysis of two-dimensional gels. Of the 1438 spots present on the gels, 55 varied significantly in intensity during differentiation. The variations occurred most often in steps correlating with those classically defined for B-cell differentiation. Seventeen spots were not detectable in at least one of the stages (qualitative variations) and could represent switching on or off of genes coding for nuclear proteins. Detailed analysis of the 55 variable spots showed that they fall into small sets characterized by similar expression profiles, which argues for a combinatorial, multistep control mechanism of gene expression. In addition, analysis of the expression of all the nuclear proteins resolved on the gels clearly differentiated B-lineage cells from myeloid cells and suggested that the most important transition in B-cell differentiation occurs between the resting B cell and plasmocyte stages.
Subject(s)
B-Lymphocytes/cytology , Cell Differentiation , Nuclear Proteins/biosynthesis , Animals , Cell Line , Cell Transformation, Neoplastic , Electrophoresis, Gel, Two-Dimensional , Methionine/metabolism , Nuclear Proteins/isolation & purificationABSTRACT
Nuclear proteins of normal and heat-shocked Drosophila cells were analysed by two-dimensional electrophoresis. The computerized processing of the gels allowed us to detect 6 proteins strongly induced by the heat treatment, but which were different from the usually described heat-shock proteins. The possible role of these proteins in genetic regulation is discussed, as is the value of this type of approach for the study of other genetic regulation phenomena.
