Restriction enzyme analysis of DNA methylation in "condensed" chromatin of Ha-ras-transformed NIH 3T3 cells.

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.

Image analysis of Feulgen-stained transformed NIH 3T3 cells differing in p21 expression and ras-induced metastatic ability.

Nuclear phenotypes of clonal and polyclonal T24 H-ras-transformed NIH 3T3 cells differing in p21 expression and experimental metastatic ability were studied in Feulgen-stained preparations by image analysis. The objective was to determine if these cells varied in their degree of chromatin condensation, as previously reported with cell transformation, or in any other chromatin texture property highlighted by image analysis parameters. The majority of nuclei in all of these ras-transformed cells exhibited increased levels of chromatin condensation, independent of ras levels or metastatic properties. This chromatin texture characteristic was assumed to be related to the transformed phenotype. No significant changes in chromatin supraorganization that could be correlated directly with ras levels or metastatic ability were found, with the exception of an increased frequency of a relatively rare phenotype in highly metastatic cells. This phenotype was characterized by an extreme contrast in packing state between condensed and noncondensed chromatin. It is suggested that ras transformation results in alterations in chromatin structure but that there is not a simple relationship between ras p21 expression levels or ras-induced metastatic ability and the degree of these changes.