Morphometric analysis of B2cAMP induced reverse transformation in synchronized CHO cells

Abstract

Synchronized tranformed and reverse-transformed (by 10(-3) M B2cAMP) CHO-K1 cells, growing adherent to plastic, are characterized by means of geometric and densitometric parameters at the level of both the entire cell and of the nuclei at various time intervals after selective miotic detachment. Transformed and reverse-transformed cells triple-stained with Feulgen, Napthol Yellow S, and periodic acid-Schiff appeared very similar in terms of integrated optical density (IOD), related to either polysaccharides, protein, or DNA amount. On the other hand, a shift from a polygonal to a spindle-shaped morphology is a accompanied by a significant decrease in both form factor and average optical density (AOD) of intact cell and nuclei, which are the most conspicuous measured changes caused by B2cAMP, in addition to a lengthening of the cell cycle duration. In both control and treated cells, important and parallel cell-cycle-dependent modulations of geometric and densitometric parameters are also observed, for both the cytoplasmic (i.e., cell morphometry) and DNA space (i e., nuclear morphometry). Specifically, the modulation in nulear morphometry during G1, S, G2, and M phases confirms previous findings on synchronized HeLa cells. The optical density threshold-dependence of geometric parameters shows that, while becoming fusiform, the cytoplasm of reverse-transformed cells had a particularly low optical density precisely in the polar area. Utilization of such an approach in the development of an objective morphological classification of all cell lines grown as monolayers "in vitro" is also discussed.