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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.

The most recent findings on the molecular and cellular characterization of normal and abnormal cell proliferation are summarized. They include molecular spectroscopy, nucleic acid conformation, protein modifications, premature chromosome condensation, nucleoli changes, nuclear and cell morphometry, image analysis, flow microfluorimetry, and time-lapse cinematography. Biophysical and biochemical evidence in favor or against two cycles of chromatin condensation, followed by two abrupt random decondensations, per cell cycle are presented. Other biphasic changes at the molecular and cellular levels that favor the existence of two random transitions, or restriction points, per cell cycle are discussed. A comprehensive unitary model of the cell cycle is then outlined; this model is able to explain most findings on continuously dividing cells and on quiescent cells induced to proliferate. Within this analytical framework the physical-chemical and biological properties are given, in either normal or tumor cells, for the various types of "noncycling" cells that are here viewed as necessary steps in mammalian cell growth rather than separates states. The implications of the coupling of higher-order chromatin structure with cell geometry and growth, high in fibroblast-like cells but low in transformed cells, are also discussed. Molecular mechanisms likely responsible for the chromatin conformational changes occurring at the G0 leads to G1, G1 leads to S, G2 leads to M transitions are finally discussed in terms of polyelectrolyte theory.

Nuclear DNA-space images from Feulgen-stained HeLa cells synchronized at 1, 3, 5, 8, 12, 15, and 18 h following mitosis are digitized and their densitometric-geometric patterns are analyzed by means of a Quantimet 720-D image analyzer on line with a PDP11/40 computer. Frequency distributions of picture point optical densities for the phases and subphases as seen in nuclear images show that DNA packing changes are evident by means of ordinary optical microscopy. Radii of gyration of the images, and optical density profiles and distributions for several squashes of similar cells reveal that in particular instances chromatin DNA is distributed mostly towards the periphery, and usually with high circular isotropy. Cross power spectra of individual scan lines suggest that existence of higher order "quinternary" periodic structure for chromatin that modulates during the cell cycle. Three-dimensional reconstruction 2- micrometer sections of intact, Feulgen-stained mammalian tumor tissue show stainable material only toward the nuclear perimeter and not in the center (compatible with the evidence that initial thymidine incorporation in HeLa cells is generally at the nuclear border). Densitometric properties of reconstructed interphase chromatin-DNA bodies are highly coupled with similar properties of the whole nucleus, showing that a more condensed nucleus is always accompanied by a more condensed interphase chromatin DNA. The effect of micrococcal nuclease digestion on the digitized nuclear images is also presented. All the above data are then discussed in terms of a quinternary chromatin-DNA structure and its modulation during the cell cycle.

A theoretical approach to modeling Circular Intensity Differential Scattering (CIDS) of native chromatin as multiple scattering of dipoles is discussed without the Born approximation. The model can explain the experimental data in the literature. It is shown that CIDS contains more structural information than does total light scattering and to a good approximation is independent of the length of the scattering molecules. Finally, CIDS in conjunction with traditional light scattering measurements should aid in discriminating between various alternative models of higher order chromatin structure now being proposed. Generalization of this theoretical study to other complex biomolecular structures, is also briefly discussed.

In previous papers the existence of two cycles of chromosome condensation-decondensation per cell cycle was suggested based on experiments involving nuclear morphometry measurements of Feulgen-stained nuclei. This conclusion can be criticized since its assumption of a relationship between nuclear morphology and chromatin structure is derived from indirect evidence. In this paper, we report simultaneous measurements of nuclear area and nuclear fluorescence intensity on individual cells stained with the intercalating dye, acridine orange (AO). Using cells in various stages of G1 and synchronized by two different methods, our results demonstrate a linear correlation between nuclear area and fluorescence intensity. They also indicate two cycles of chromatin condensation-decondensation during the G1 period, as assayed by the number of chromatin primary, intercalating AO binding sites. Finally, they show that the first of these cycles involves a transition in early G1 from a very small condensed nucleus (immediately after telephase) to a relatively large, dispersed nucleus that occurs abruptly.

Using HeLa S-3 cells synchronized by selective detachment, in this paper we report a parallel study of nuclear morphology and autoradiography grain patterns between middle G1 and middle S phases. Our results show two distinct [3H]-thymidine labeling patterns. The first "peripheral" labeling pattern has a characteristic nuclear size distribution, in contrast to the heterogeneous and varying size distributions of Feulgen-stained nuclei, and apparently is characteristic of very early S phase. The sizes of the second labeling pattern--homogeneous or inhomogeneous grain distribution throughout the nucleus--are equal or larger than the first and vary with S phase progression. Together, the corresponding nuclear sizes of the labeled nuclei represent the larger extreme of nuclear areas, and the labeling index closely parallels the fraction of nuclei with areas larger than the minimum size of the labeled nuclei. These results suggest a characteristic nuclear size (reflecting unique intranuclear DNA distribution) as a necessary, if not sufficient, requirement for S phase initiation. Parallel experimentation with rat liver cells-synchronized in vivo by partial hepatectomy and analyzed by thin section autoradiography--confirms the existence of a peripheral labeling pattern in both the very early part and the very late part of S phase, which reconciles our data with previous results and points to the fact that both initiation and termination sites for DNA replication are near the nuclear periphery.