MODULATION OF RAS TRANSFORMATION AFFECTING CHROMATIN SUPRAORGANIZATION AS ASSESSED BY IMAGE-ANALYSIS

Changes in chromatin supraorganization defined in terms of patterns of chromatin texture were studied by video image analysis in Feulgen-stained revertants of LTR-ras-transformed NIH 3T3 cells and in cell lines obtained by transfection of these revertants with sense and antisense constructs of the lysyl oxidase gene (also named Lox or ''ras recision gene''). The objective was to determine whether changes in expression of the Lox gene, which have been assumed to modulate cell transformation by ras, could also affect the chromatin supraorganization changes known to be elicited in NM 3T3 cells by ras transformation. The image analysis results revealed that, although a nuclear phenotype visually similar to the most frequent one (III) in ras-transformed NM 3T3 cells also appeared in the revertant, it contained a remarkably less tight chromatin packing state. This situation was also found in the revertant transfected with the sense construct of the Lox gene, but in the revertant transfected with the Lox antisense constructs the chromatin texture of the III phenotype was equal to or close to that of the ras-transformed cells. With regard to the nuclear phenotype characterized by abundant loosely packed chromatin and less represented in the transformed cell lines (I'), changes in the various cell lines, although detectable, were not as drastic as those reported for the III phenotype, The enhancement in chromatin condensation of the type III nuclei, which affects euchromatin, is probably associated with a limited transcription of the genome. Although the image analysis results are mostly in agreement with previously published data on the molecular biology and tumorigenicity of the same cell lines, it appears that the phenomenon of chromatin condensation once established in NM 3T3 cells by LTR-ras transformation could not be totally reverted by simply affecting Lox expression. (C) 1995 Academic Press, Inc.