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The argyrophylic staining of the nucleolar organizer regions (AgNOR positive response) in interphase nuclei is often related directly to the cellular demand for ribosome biogenesis and is considered of relevance in studies of tumor pathology. Transformation of human breast epithelial MCF-10A cells by the c-Ha-ras oncogene results in altered growth, invasiveness and tumorigenicity in nude mice. Since ras transformation may be associated with a more intense nucleolar activity, we examined the influence of transfection by the Ha-ras oncogene on AgNOR staining response in MCF-10A cells. Following assessment of the AgNOR response with video image analysis, the AgNOR-positive areas and the AgNOR area/nuclear area ratio, but not the number of AgNOR aggregates or dots per nucleus, were found to be much higher after ras transformation. A role of the Ha-ras transformation on the nucleolar activity of the MCF-10A is thus suggested as assessed by the AgNOR staining. Based on data in the literature, it is also hypothesized that a decreased wild-type p53 level, possibly promoted by the ras transformation, may be associated with the increased AgNOR response.

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‐rasoncogene. 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 withMspI andHpaII restriction enzymes, which distinguish between methylated and non‐methylated DNA. The amount of methylated CpG sequences not digested byHpaII 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 byrastransformation in the mentioned NIH 3T3 cell lines.

OBJECTIVE: To assess continuous recording of the microscope status for quality assurance. STUDY DESIGN: Using a special microscope with coordinate registration and objective decoding, 8,653 cervical smears were read by five experienced cytotechnologists. Cytotechnologists could check their screening pattern on a TV monitor. Stage position and magnification were registered every 20 msec. RESULTS: The average screening time per case was 3.5 minutes followed by an approximately two-minute intercase interval. Daily workload profiles were generated to check compliance with workload regulations. Average screening time over a day and over a week was rather constant. CONCLUSION: Our experience demonstrates that continuous coordinate registration can be easily implemented as a method of quality assurance in routine cytology.

The discrimination of nuclear phenotypes of Feulgen-stained ras-transformed and nontransformed NIH 3T3 cells previously determined by image analysis using the scanning microspectrophotometry system, was investigated with the video system. A reasonable matching of the Feulgen-DNA values obtained with the two methods was demonstrated. Chromatin texture evaluated on the basis of the scatter diagram relating condensed chromatin relative areas vs. an average absorption ratio could also be assessed with the video system. The video image analysis system was effective in distinguishing nuclear phenotypes which differ in chromatin supraorganization in ras-transformed as well as in nontransformed NIH 3T3 cells, in distinguishing phenotypes not visually discernible from each other (transformed vs. nontransformed cells) and in discriminating among the cell lines used.

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.