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Analytical Cellular Pathology
Volume 20, Issue 4, Pages 163-171

Restriction Enzyme Analysis of DNA Methylation in “Condensed” Chromatin of Ha-Ras-Transformed NIH 3T3 Cells

Maria Luiza S. Mello,1,2 Ann F. Chambers,3 Benedicto C. Vidal,1,2 Wolfgang Planding,2 and Ulrich Schenck2

1Department of Cell Biology, Institute of Biology, UNICAMP, 13083‐970 Campinas, SP, Brazil
2Laboratory of Clinical Cytology, Institute of Pathology, Technical University of Munich, 81675 Munich, Germany
3Division of Experimental Oncology, Department of Oncology, University of Western Ontario and London Regional Cancer Centre, London, Ontario, Canada N6A 4L6

Received 1 February 2000; Accepted 1 October 2000

Copyright © 2000 Hindawi Publishing Corporation. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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.