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RNA relocation and the incidence of nucleolus‐like bodies accumulated during mitosis were studied cytochemically in benzo[a]pyrene (BP)‐transformed human breast epithelial MCF‐10F cells after microcell‐mediated transfer of normal chromosomes 11 and 17. The changes resulting from the transfer of these two chromosomes in tumorigenic MCF‐10F cells (BP1‐E cell line) were examined, since alterations in these chromosomes are involved in the expression of the transformed and tumorigenic phenotypes in the MCF‐10F cell series. In addition, the frequency of nucleolus‐like bodies decreases drastically with transformation and tumorigenicity in MCF‐10F cells, thus being conceivable that it would be affected in presence of normal chromosomes 11 or 17. The pattern of RNA relocation associated with the mitotic spindle did not vary in the cell lines analyzed. The introduction of chromosome 17 in BP1‐E cells either decreased or did not affect the frequency of persistent nucleolus‐like bodies. In contrast, in cells which received a normal chromosome 11, the frequency of nucleolus‐like bodies was closer to that of non‐transformed MCF‐10F cells. These results suggest that a normal chromosome 11 but not chromosome 17 contributes to the maintenance of an RNA surplus which accumulates in nucleolus‐like bodies during cell division of the human breast epithelial cells, at least in vitro. Some loci which were retained in the BP1‐E cells which received a normal chromosome 11 are probably involved with the control of RNA transcript production.

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.

When transfected to benzo[a]pyrene (BP)‐transformed MCF‐10F human breast epithelial cells (BP1 cell line) the c‐Ha‐ras oncogene has proven to enhance the neoplastic changes initiated by exposure to BP, giving rise to an aggressive tumorigenic cell line, BP1‐Tras. We have previously demonstrated by image analysis that BP affects the DNA content and the chromatin supraorganization of MCF‐10F cells. Here Feulgen‐stained BP1‐Tras cells were studied by image analysis in order to evaluate possible additional changes in DNA content and chromatin texture induced by insertion of the ras oncogene. A high variability in DNA content also including polyploidy or near‐polyploidy, and an increase in the packing states of the chromatin which became still condensed in BP1 cells were found in BP1‐Tras cells. The results differed from those reported for the BP1‐E1 cell line which is also an aggressive tumorigenic cell line, but was attained through progressive passages of BP‐transformed cells. It was demonstrated that different patterns of changes in DNA content and chromatin organization may be involved in equally aggressive tumorigenic BP‐transformed cell lines originated from the same cell line by different mechanisms.

Changes in nuclear and nucleolar morphometric parameters were investigated by image analysis procedures in human breast MCF-10F epithelial cells expressing different stages of the tumourigenic progression after benzo[a]pyrene (BP) transformation (BP1, BP1-E, and BP1-E1 cell lines), and additionally transfected with the c-Ha-ras oncogene (BP1-Tras cell line). Nuclear pleomorphism was evident in all the transformed cells. The analysis of different morphometric parameters did not show a clear relationship between specific nuclear and nucleolar changes and the expression of the different stages of the tumourigenesis, with the exception of the nucleolar size, which could be associated to the expression of the tumourigenic phenotype, and a nucleolar area/nuclear area ratio, which discriminated the immortalized, the transformed, and the tumourigenic phenotypes from one another. The nuclear morphometric data established for the BP-transformed cells and for the cells additionally transfected with the c-Ha-ras oncogene were suggestive of complex and distinct morphofunctional mechanisms involving the in vitro transformation of the MCF-10F cells. The nuclear changes found in the BP1-Tras cell line were assumed to be related to the additional effects and/or enhanced genomic instability induced by transfection with the ras oncogene.