RESUMO
Increased amounts of chromatin condensation (i.e., localized areas of high DNA density, or chromatin higher order packing state) have been described in NIH 3T3 cells transformed with the Ha-ras oncogene. The structural basis for this oncogene-mediated alteration in nuclear organization is unknown. Since DNA methylation is likely to be involved in regulating the nucleosomal level of DNA packaging, we studied the role of DNA methylation in higher-order chromatin organization induced by Ha-ras. CpG-methylated DNA content was estimated in "condensed" chromatin of Ha-ras-transformed NIH 3T3 cell lines which differ in ras expression and ras-induced metastatic ability but present approximately the same values of "condensed" chromatin areas. The question posed was that if DNA methylation were involved with the chromatin higher-order organization induced by Ha-ras in these cell lines, the methylated DNA density in the "condensed" chromatin would also be the same. The DNA evaluation was performed by video image analysis in Feulgen-stained cells previously subjected to treatment with Msp I and Hpa II restriction enzymes, which distinguish between methylated and non-methylated DNA. The amount of methylated CpG sequences not digested by Hpa II in "condensed" chromatin regions was found to vary in the studied ras-transformed cell lines. DNA CpG methylation status is thus suggested not to be involved with the higher order chromatin condensation induced by ras transformation in the mentioned NIH 3T3 cell lines.
Assuntos
Cromatina/química , Metilação de DNA , DNA/metabolismo , Genes ras , Corantes de Rosanilina , Células 3T3 , Animais , Linhagem Celular Transformada , Cromatina/genética , Corantes , DNA-Citosina Metilases , Desoxirribonuclease HpaII , Processamento de Imagem Assistida por Computador , Camundongos , Microscopia de VídeoRESUMO
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 NIH 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 NIH 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 NIH 3T3 cells by LTR-ras transformation could not be totally reverted by simply affecting Lox expression.
Assuntos
Transformação Celular Neoplásica , Cromatina/fisiologia , Genes ras , Corantes de Rosanilina , Células 3T3 , Animais , Linhagem Celular , Núcleo Celular/ultraestrutura , Cromatina/ultraestrutura , Corantes , Humanos , Interferon Tipo I/farmacologia , Camundongos , Fenótipo , Proteína-Lisina 6-Oxidase/biossíntese , Proteína-Lisina 6-Oxidase/genética , Sequências Repetitivas de Ácido Nucleico , TransfecçãoRESUMO
This study is aimed at determining the relationship between polyploidy and the amount of nucleolar organiser region (AgNOR) positive dots or aggregates using high resolution image analysis. Liver imprints from mice in which hepatocyte polyploidy is very well documented and related to variation in nuclear area were used as a model for this investigation. A technical variant of the AgNOR method using a Triton X-100 treatment was developed for removal of some proteins from the cytoplasm, which produced a clearer and cleaner background. Feulgen-stained preparations were used to detect the association of various ploidy degrees with their respective values of nuclear area and to subsequently furnish a basis for the association of the nuclear areas in AgNOR-stained cell preparations with different ploidy levels. A high correlation between nuclear and AgNOR-stained areas was revealed and was demonstrated to be much higher than the correlation between nuclear area and the number of AgNOR positive dots or aggregates. The use of two different thresholds for segmentation of the grey levels of the AgNOR-stained material demonstrated the importance of the appropriate decision to obtain the best results to be associated with polyploidy in terms of the real biological event involved, the nuclear area being in this case correlated to Feulgen-DNA values.