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Cellular morphology changes, which appear related to dedifferentiation (despecialization), have been produced in vitro in the nucleated red blood cell of the frog. This has been achieved by controlled alterations in the electrochemical environment of these living cells, both by a selective modification of the ionic concentrations of an isotonic amphibian Ringer solution, and by the electromagnetic induction of pulsating current having specific waveform parameters. Laser flow microfluorometry shows that the modified Ringer solution is able, per se, to partially trigger the process in the same time interval that certain induced current waveforms can significantly affect the number of cells in the so-called dedifferentiated state. It has also been found that, for a given waveform, the repetition rate appears to have a significant effect on the rate of cell change. Preliminary automated image analysis of cell smears suggests that dedifferentiated and normal cells have the same integrated optical density but different nuclear areas. In conclusion, it appears that, after the initial electrochemical trigger, the early stage of the process, when the cells move from a state of specialized function to one of less specific activity, is the unfolding of their chromatin supercoil, not involving DNA synthesis. Then cytofluorometry allowed us to identify, for the first time, fundamental modifications which occur in the cell nucleus under electromagnetic exposure.

The conclusions that we would like to draw from this review are the following: (a) Chromatin structure and function are exceedingly sensitive to changes in the proliferative state of a cell. Differences can be detected between cells in mitosis, G1 and S, and even between G0 and G1 cells. (b)These differences are very unlikely to be artifactual, since similar changes can also be demonstrated in intact nuclei. (c) Some of these differences can be abolished by extraction of chromatins with low concentrations of salt. (d) Differences between chromatins of normal and neoplastic cells can also be detected, but they are largely related to differences in the extent of cell proliferation. (e) A number of laboratories have been very busy in trying to elucidate chromatin structure with different technologies. Sometimes a change in a macromolecule cause by a physiological stimulus can tell us as much about its structure as a thousand instruments. The changes occuring in chromatin of proliferating cells could perhaps be profitably used to know more about chromatin structure.

Recently the discrete model of the cell cycle, described as a series of metabolic progressions through four distinct phases, has been challenged by a few curves of percent labeled mitoses (PLM) and related autoradiographic data, which have been questionably interpreted in terms of a "continuous" cell cycle model. This conclusion is based in part on a few questionable observations which we summarize, along with our objections and explanations. We present a critical analysis of the autoradiographic data obtained with the PLM, in terms of the limitations and inconveniences of the technique itself. Some attention is paid to the usefulness of the "radioautographic transfer function," which seems an arbitrary mathematical expression of old autoradiographic observations. Finally, a review is presented of the old (by autoradiography) and new (by physical, chemical, and computer image analysis) evidences of the existence of dicrete phases in the cell cycle, which in turn support the idea of an all-or-none phenomenon for DNA synthesis, and which rule out the hypothesis of a continuous cell cycle model with a varying DNA synthesis rate function.

For a better understanding of data provided by DNA alkaline elution technique, a new analytical model has been developed which takes into consideration both the physicochemical properties of in situ DNA strand (length and flexibility/superpacking) and the geometric and hydrodynamic configuration of the elution apparatus (flow and filter conditions). Simulation by this model of experimental data previously obtained before and after carcinogens administration, has shown that for constant flow and filter conditions elution profiles are dependent, not only from DNA molecular weight, but also from a parameter critically related to modifications in chain flexibility/superpacking. This has been confirmed by several independent observations, including the time-dependent changes in non-denaturing lysing solution monitored by hydroxylapatite and alkaline elution techniques.

When resting confluent monolayers of WI-38 fibrolasts are trypsinized and replated at a lower density they are stimulated to proliferate again with an interval of 18 hours between replating and the onset of DNA synthesis. Trypsinization of resting cells causes a 40% loss of nuclear proteins as well as of cytoplasmic proteins. The amount of nuclear proteins remains low for the first six hours after the cells have been replated and then it increases rapidly, reaching the same level of non-trypsinized resting cells by ten hours after plating. The proteins that are lost from the nucleus immediately after trypsinization are chromatin-associated proteins and most of them are non-histone chromosomal proteins, although a modest loss of histones cannot be ruled out. The loss of non-histone chromosomal proteins from cells that have been trypsinized causes changes in the structure of chromatin that can be detected by circular dichroism and by viscosity measurements. These results show that cell trypsinization causes an extensive loss of proteins from chromatin and that the loss is restored only several hours after the cells have been replated at a lower density.

The possibility of controlling the activity of biological processes by means of an electromagnetic (em) field is well established in the literature, and already leads to clinical applications (1).

Differential scanning calorimetry of chromatin isolated from rat liver cells revealed three discrete thermal transitions whose temperatures and melting enthalpies depend on ionic strength in the range 0 to 600 millimolar NaCl. Intact nuclei showed a fourth thermal transition at a lower temperature and different melting enthalpies for the other three transitions still present at temperatures similar to those obtained in isolated chromatin. The data are discussed in terms of the tertiary, quaternary, and quinternary structures of chromatin DNA.

Smears of synchronized HeLa S3 cells at 1, 3, 5, 8 and 12 hours after mitosis were Feulgen-stained with 1N HCl at 60° C for 15, 60 and 120 minutes. For each cell population the integrated optical density, nuclear perimeter and area, measured by using the image analyzer Quantimet 720-D on line with a PDP11/40 computer, show a striking differential dependence upon hydrolysis time. IOD frequency distributions of log-phase HeLa cells are statistically significant at the three hydrolysis times; specifically, both the coefficient of variation of “G1” peak and the fraction of cells with “G1 DNA content” increase with hydrolysis time. Laser flow microfluorimetric studies were also conducted on the same log-phase HeLa cells, Feulgen-stained with acriflavine, respectively at 1N HCl at 60°C for 5, 10, 20, 60, and 120 minutes; 4N HCl at 25°C for 15, 60 and 120 minutes and 5N HCl at 37°C for 15, 60 and 120 minutes. The decompositon of the DNA histograms yields dramatic increases of “G1” fractions, accompanied by increases of the coefficients of variation of the “G1” peaks, whenever the modal fluorescence per cell decreases as a result of variation in HC1 normality, temperature or time of hydrolysis. This differential hydrolysis dependence of the Feulgen reaction is compatible with the difference in chromatin supercoiling for cells in different phases and subphases of the cycle. Optimal hydrolysis conditions are outlined, stressing the limitations of utilization of Feulgen reactions for a quantative assay of DNA content and for FMF cell cycle analysis.

The dramatic progress and findings made at the molecular and cellular level, discussed in the previous chapter, now permit a molecular description of the neoplastic process that involves lower- and higher-order nuclear structures, genes and cytoskeleton, and that confirms what was believed in the early days, namely that cancer is the final product of a multistep process (Farber and Cameron, 1980; Pitot, 1979; Pontecorvo, 1975; Heston, 1975). The questions that are still unanswered or inconclusively proven are: What are the steps in the cancer process? Which of them are limiting? Which of the steps are more susceptible to intervention, either through treatment following early detection (the object of Chapter 3) or through prevention (the object of this chapter).

A typical mammalian cell contains DNA strand a few meters long; this DNA, covered with acidic and basic proteins, is folded into the cell nucleus whose diameter is only a few microns. Likely DNA structure and cell function are intimately and causally related. The question then arises as to how DNA is geometrically organized from the angstrom up to the micron level within the nucleus, and how histone and non-histone proteins interact with DNA to modulate its structure and conversely cell function.

When quiescent WI-38 cells are stimulated to proliferate by nutritional changes, the chromatin of stimulated cells shows an increased ellipticity in circular dichroism spectra and an increased ability to bind ethidium bromide. The differences between the chromatins of quiescent and stimulated cells are abolished if both chromatins are treated with 0.25 M NaCl. However, when salt-treated chromatins are reconstituted by dialysis agains 10 mM Tris-HCl, the difference in circular dichroism spectra and ethidium bromide binding re-appear. Furthermore, addition of the 0.25 M NaCl extract from chromatin of stimulated cells to chromatin of quiescent cells causes an increased ellipticity in circular dichroism spectra similar to that of stimulated chromatin. Since only proteins (mostly nonhistones) are detectable in the 0.25 M NaCl extract, these results strongly suggest that the chromosomal proteins extractable with low concentrations of salt are responsible for the functional and structural changes occurring in chromatin of WI-38 cells stimulated to proliferate.

In the last 20 years there has been a worldwide explosion in legislation aimed toward establishing and expanding a coordinated and intensive approach to the cancer problem. In the United States, the National Cancer Act of 1971 was instrumental in dramatically increasing funds for the National Cancer Institute (up to $1 billion in 1980) and for the creation of regional comprehensive cancer centers (now about 20) that coordinate interdisciplinary efforts in research, teaching, and patient care. This increased not only the number of scientists focusing on cancer, but also the number of physician cancer specialists (i.e., oncologists) from less than 100 in the late 1960s to more than 2800 in 1980.

In all physico-chemical and functional characterization of isolated chromatin, there is of course a large degree of artifact introduced during the isolation process. In this respect abundant evidence exists in the literature that the structural and functional properties of native chromatin are modified by physical alterations introduced during chromatin preparation (1,2,3). In order to bypass such limitations, one approach has been to analyze the geometry and densitometric textures of the nuclear images produced by differential staining of chromatin-DNA in situ (such as by a Feulgen reaction). For a detailed account of the most recent findings during image analysis on the study of chromatin during cell cycle, visus transformation and cell proliferation, the readers should refer to recent publications (4–7). Here we intend only to outline a critical overview of the state of the art with advantages and limitations.

Due to their particulate nature, native biopolymers (such as membrane proteins or chromatin) tend to form an intensely light-scattering suspension. Scattered light is deviated from the beam axis at an angle great enough to miss the detector, resulting in (a) anomalously high optical density and (b) circular intensity differential scattering (CIDS), i.e., left and right circurlarly polarized light are scattered with different efficiences out of the photomultiplier (PM) window.

In vivo Proton NMR imaging is becoming an increasingly popular tool in diagnostic medicine for its non-invasive nature and for its high resolution (1,2). In most clinical applications the 3-D reconstructed human images are based on local measurements of T1 and T2 proton relaxation times. Increases in these relaxation times in tumors (3) have been attributed to a change in the water concentration at the cytoplasmic and/or extracellular level (4) or to a change in the physical state of water (3,5). Similarly, during the cell cycle the variations in T1, i.e., its shortening during the Gl-S transcription (6), have been correlated with cellular changes in water concentration (7). More importantly, it has been found (7) that prior to mitosis an increase in relaxation times of water protons preceeds by 4 hours the increase in water concentration, suggesting that a change in the physical state of water may preceed cell division.

Classical cytological analysis always enjoyed a very important advantage over biochemical approaches based on fractionation, purification, and subsequent biochemical study of some cell constituents. This advantage consists of the possibility of examining the properties of single cells instead of averages from an entire bulk of cells and correlating these properties with a “space” dimension. The main disadvantages, in respect to a biochemical approach, have always been the poor understanding of the kinetics (not to mention molecular mechanisms) of the interactions between dyes and cellular receptors which are usually controlled only qualitatively; as well as the classification of the observed cells, not on the basis of quantitative measurements, but according to the qualitative and subjective judgement of a human eye-brain system.

Differential scanning calorimetry (DSC) and gel electrophoresis have been carried out on intact nuclei and on the corresponding chromatin extracted, using the two most common procedures, from rat hepatocytes, as a function of shearing, ionic strength and DNA-ase digestion. These studies, along with those obtained on free DNA fragments of widely different length, suggest that the chromatin prepared by limited nuclease digestion lacks the topological constraints present in situ, which appear instead to be preserved even at low ionic strength (0.01 M) by lysis of the native nuclei in cold hypotonic buffer and by subsequent gentle resuspension (without shearing) of the viscous chromatin mass. While both unsheared chromatin preparations contain a similar repeating structure, only the chromatin prepared by limited nuclease digestion is lacking the higher molecular-weight bands present in native nuclei. Furthermore only “cold water” chromatin reveals significant cell-cycle-related thermodynamic alterations which exactly mimic those apparent in situ from the heat capacity profiles of corresponding nuclei. Results are discussed in terms of DNA packing/supercoil in vivo which could modulate during cell-cycle progression and which could be maintained by the topological constraints existing along native nucleofilaments.

A recent review (1,2) indicates that significant correlation exist between the structure of chromatin and the extent of cell proliferation (aging, serum stimulation, virus transformation, cell-cycle phases, etc.). Chromatin, of course, refers to the diffuse interphase form of chromosome isolated from eukaryotic cells and the question remains open as to whether observed chromatin changes are reflected in the intact cell.

SV-40 virus-transformed human diploid fibroblasts (2RA) were grown in a monolayer on plastic Petri dishes in an aqueous medium deuterated to different concentrations of deuterium oxide: 10, 20, 30, up to 60%. The cells must be acclimatized to concentrations higher than 20% D2O by stepping them from a lower initial concentration during their exponential growth. The increase of cell doubling time with increasing deuterium concentrations seems to correlate, at least at 20% D2O, with an initial period of suspended cell growth (lag-phase), and is qualitatively similar to that previously reported for Escherichia coli.

The alkaline elution technique, developed by Kohn and coworkers (Kohn and Ewig, 1973; Kohn et al., 1976; Kohn, 1979), provides a sensitive measurement of DNA single-strand size distribution in mammalian cells and represents an advanced alternative to the technique of alkaline sucrose gradient sedimentation. Alkaline elution of DNA from cell lysates allows size measurement of DNA single strands longer than the sizes that can be measured by sedimentation in alkaline sucrose gradients. The elution technique requires relatively simple and not expensive apparatuses, and is less time-consuming in comparison with the latter. Moreover, it gives more repeatable results and is more suitable to assess DNA damage from various tissues following in vivo exposure to DNA interacting agents. In the last few years, this technique has been widely applied by different groups of researchers to the study of various DNA lesions induced by several physical or chemical agents (ranging from carcinogens to antineoplastic agents), and a large series of results have been collected. The aims of this review are: to discuss the physical basis that governs DNA elution, to summarize the possible application of this method, to revise the use of alkaline elution and its reliability in the assessment of DNA fragmentation in various organs of mice and rats following in vivo exposure to chemical agents, and to evaluate critically its role as a screening procedure for carcinogenic compounds.

In a recent review1 we have presented evidence for the organization of DNA from its lowest (bp sequence) to its highest structure, as emerging from a wide variety of biophysical and biochemical measurements conducted on cells of diversified functional states. The changes occurring at the level of the nuclear structure were determinant in building a firm basis for the high order chromatin structure, from the nucleus to the genes, namely the “fibrosome” model which identified within the 300 A chromosome-sized fiber a fundamental repeating unit about 17 Kbp long, arranged drapery-like near the nuclear envelope or the nucleoli and capable of undergoing transitions between three discrete states of DNA superpackingl1. Without such a working model to be constantly challenged and updated, any exploration of the numerous modifications occurring during cell proliferation and transformation could be nothing more than a fishing expedition. Some of the changes induced are indeed horrifying in their complexity (cycles within cycles) and heterogeneity (membrane versus cytoskeleton, cytoplasmic proteins versus nuclear chromatin, acetylation versus phosphorylation of histones, HMG versus H1). An excellent account of most recent findings is given in multi-authored books2–4. Despite the jungle of “descriptive” cellular events (with recurrent logic where viral oncogenes are now substituting for serum stimulation) and the “zoo” of specific molecules (such as cyclic nucleotides, HMG, “growth factors”, H1, viral proteins, microtubules, glycoproteins), critical steps can be identified, and where feasible, will be summarized here within a coherent framework for gene expression compatible with the above model for chromatin and gene structure.

Chromatin was isolated from rat liver cells at 0, 3, 5, 11, 18 and 24 h following partial hepatectomy. Consistent with findings in cultured cells stimulated to proliferate, there was an increase in chromatin molar ellipticity measured at 276 nm, and a decrease in thermal stability 3 to 8 h after surgery. These events occured prior to the onset of DNA synthesis. These early changes be­ tween non-proliferating (G0) and proliferating (G1) cells, as well as later chromatin conformational changes observed at S and G2 phases, mimic changes in template activity. Results with sheared and unsheared chromatin (both with in vitro and in vivo systems) prove that structural and functional changes can be caused by even the slightest shearing during chromatin preparation, suggesting the loss of native chromatin organization. To eliminate this problem, experiments were also conducted using chromatin in situ. A flow cytometer (FCM) was used to study unfixed liver cell suspensions stained with ethidium bromide (EB). Fluorescence was mea­ sured in the green spectral range after addition of increasing amounts of EB. Experimental evidence is provided that the same alteration in chromatin conformation can be best detected using low molar ratios of EB per unit DNA due to greater fluorescence emission in G1 respect to G0 cells. These correlated studies demonstrate that the same changes controlling chromatin organization in situ are detected also in the tertiary-quaternary structure of “isolated” chromatin. These changes in chromatin conformation are macromolecular events related to cell proliferation both at the G0 -G1 and G1 -S transitions.

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.

Today in the biological sciences there is a growing interest and use in technologies capable of remote in vivo quantization of cellular constituents. The biophysical application of two such technologies, as scanning (1) and flow (2) cytometry, to elucidate the in vivo mechanisms of proliferation, differentiation and expression of metastatic potential in mammalian cell systems, has been recently introduced (for an updated review see also the chapters by Nicolini, Mendelsohn and Casperson in the previous book of this NATO-ASI series (3)). These technologies have made possible the detailed and quantitatively accurate characterization of “native interphase chromatin” (1,4,5) and membrane (2,6). This characterization has in turn led to a new model for normal and abnormal cell growth, that is related to and dependent upon the quinternary nuclear organization of the chromatin (1,4).

Early in this book distinguished scientists have summarized the experimental evidences on the orderly organization of the genetic material within mammalian cells from the primary structure (base sequences) to the secondary (the various forms of DNA double helix) structure. While the tertiary structure — wrapping of DNA around octamer histones to form the nucleosome (1) — is rather clear in details, large uncertainty still exists on the exact three-dimensional geometry of the higher order DNA foldings (from the supernucleosomes on). In this chapter we will attempt to summarize the present understanding of chromatin -DNA (2,3) higher order structure and dynamics, emphasizing recently introduced biophysical probes — the only ones capable to cope with the inherent difficulties of an “in situ” characterization.

The problem of determining the physical properties of giant DNA molecules in native conditions from intact mammalian nuclei (or cells) remains one of the most challenging and intriguing, especially considering their critical dependence on cell proliferation and transformation: DNA length (1), structure (2), and intrinsic viscosity (3) have been shown to be critically related to early events occurring during chemical carcinogenesis. Progressively lower amounts of “putative” single DNA strand breaks are detectable by alkaline sucrose gradients (1), alkaline elution (4), and viscometry (3), but numerous questions concerning the role of higher order structure (dealt with in a separate communication (5)) or the relative contribution of elastic and viscous components on the latter more sensitive viscoelastic measurements remain unanswered. Mammalian DNA molecules “in situ” have a pronounced tendency to associate to form gelled networks of nucleofilament fibers (2) with high elastic modulus and intrinsic viscosity which lend themselves to quantitative measurements (6,7).

Circular intensity differential scattering (CIDS) has been proven a powerful method in determining the higher-order structure of large biopolymers, such as chromatin. Theoretical predictions of the expected differential light scattering of circularly polarized light have previously been made for chromatin, either within the Born approximation, treating nucleosomes as noninteracting, oblate ellipsoids, or within a multiple dipole approximation, treating nucleosomes as interacting spheres. In order to conduct a meaningful interpretation of the CIDS signal in terms of given geometric parameters of the chiral structure, we have in this paper combined the two approaches considering the mutual interactions of ellipsoidal nucleosomes. In the process we have also found a confirmation for rthe validity of the Born approximation itself.

Changes in CD spectra of 5-deoxybromouridine (brUdRib)-substituted chromatin depend on the extent of thymidine replacement by brUdRib. With 20 and 14 per cent replacement, a blue shift and a marked increase in positive ellipticity are reported in the CD spectra of brUdRib-substituted chromatin, while with 4.9 per cent replacement little effect is noticed. BrUdRib-substitution does not affect the number of ethidium bromide primary binding sites of chromatin.

G0, G1, and mammalian cells and nuclei were shortly digested with either micrococcal nuclease or DNAse I, both before and after mild fixation, either before (G0) or after (G1) partial hepatectomy. Cells were Feulgen stained and examined by high resolution light microscopy. In metabolically active G1 nuclei, intranuclear DNA appears organized at least in two distinct domains, whereby, the highly dispersed one is large enough to be detected at the resolution of the light microscope and appears preferentially attacked by limited DNAse I digestion. The action of the enzyme is readily apparent only in the nuclei that are first digested and then fixed. Spectroscopic characterization of the same nuclei reveals that the fixation causes a sizeable removal of proteins, mostly in the soluble chromatin subfraction. Results are discussed in terms of two control levels for gene expression and for higher order DNA structure.

In this work, we report on the use of a computer-based mathematical model which allows us to quantitate the in vivo effects of various chemotherapeutic agents on both malignant and normal cells. The usefulness of the model is shown by obtaining the dose response of the small intestinal crypt cells to Ara-C. The use of such an approach in determining optimal chemotherapeutic protocols for cancer treatment is discussed.

In this paper we describe a computerized system for data recovery from differential scanning calorimetry of mammalian cells and their biopolymers. The 'in-house'-designed preamplifier, digital acquisition, control card and its real-time software provide us with a powerful workstation to acquire and analyze large quantities of data on-line and off-line. The final data are obtained after eliminating noise interference using both hardware and software filters. Fourier analysis is also performed for a more refined thermodynamic characterization. All software is written in Basic and Fortran 77 under the DOS 3.10 operating system on a personal computer.

A successful method has been developed for isolating viable single cell suspensions of intestinal crypt cells from the small bowel of the mouse. The lumen of the intestine was perfused with a 0.2% trypsin solution that dissociated the lining epithelial cells. Crypt cell isolation, which proves to be extremely critical, occurred under optimal mechanical and chemical configurations about 75 min after the beginning of the procedure.

The cellular and nuclear morphometry of synchronized CHO cells growing on glass in the presence or absence of 10 −3 M of cAMP has been determined. Measurements were made at various time intervals following selective mitotic detachment. The changes in nuclear morphometry during the cycle of cells growing on glass confirms previous findings on similar cells growing in suspension. When cells are grown on glass, the cell shape also changes. Specifically, when cells are treated with cAMP, the cytoplasmic cell shape and condensation is directly coupled with nuclear chromatin shape and condensation, but they are uncoupled for the transformed cells.

One-parameter (nuclear DNA) and two-parameter (nuclear DNA and protein or cellular light scatter) measurements of cervical smears were performed using an ICP 11 and a cytofluorograf 4800 respectively. A total of about 1000 cases was analyzed. For the estimation of nuclear DNA alone two fluorochromes were tested (ethidium bromide (EB) and mithramycin (MMC)) combined with three different methods of cell preparation. For the two-parameter measurements cells were double stained with EB and fluorescein isothiocyanate (FITC). Red fluorescence (EB) versus green fluorescence (FITC) or red fluorescence versus scatter were recorded. A computer analysis of the one-parameter histograms was performed using discriminant analysis and the results were compared with the cytodiagnosis of microscopic specimens stained with the Papanicolaou technique. The error rates of the flow cytometric (FCM) data were as follows: (a) standard EB staining, 11% false negative, 26% false positive, 6% unsatisfactory results; (b) pepsination of vital cells and EB staining, 12% false negative, 14% false positive and 4% unsatisfactory results; (c) MMC staining, 10% false negative, 65% false positive and 5% unsatisfactory results. Our two-parameter measurements prove that, as confirmed by cell sorting, red fluorescence versus scatter allows separation of at least three subpopulations in most analyzed samples: (a) anucleated cells; (b) leukocytes; and (c) intermediate and superficial cells.

Computer-enhanced analysis of electron micrographs of thin-sectioned rat liver nuclei, combined with three-dimensional reconstruction of the same Feulgen-stained nuclei, points to a unique clustering of chromatin DNA fibres near the nuclear border. Computer-enhanced image analysis has been applied to electron micrographs of the envelopes of the same rat liver nuclei prepared by freeze etching and a few essential geometrical parameters characterizing the pores and their distribution have been determined. During interphase, clusters of nuclear pores, closely paralleling the clustering of membrane-attached chromatin fibres, have been identified on the envelope, the number of these being similar to the number of homologus pairs of metaphase chromosomes. Furthermore, rapid changes induced in chromatin distribution appear to be associated with rapid changes in pore number, but not in the number of pore clusters.

Twelve methods for analysing FCM-histograms were compared using the same set of data. Some of the histograms that were analysed were simulated by computer and some were taken from experiments. Simulated data were generated assuming asynchronously growing cell populations and (i) measurement coefficients of variation (CV) from 2 to 16%; (ii) constant measurement CV or CV's increasing from G1 to G2 phase, and (iii) varying fractions of cells in each phase. Simulated data were also generated assuming synchronous cell populations in which a block in early S phase was applied and released. DNA histograms were measured for L-929 cells at various times after mitotic selection. Labelling indices were also measured for these cells at the same time. The fractions of cells in the G1, S, and (G2+ M) phases were calculated by each analytical method and compared with the actual fractions used for simulation, or in case of experimental data, with autoradiographic results. Generally, all methods yielded reasonably accurate fractions of cells in each phase with relative errors in the range of 10–20%. However, most methods tended to overestimate G1 fractions and underestimate S fractions. In addition, variations in the shape of the S phase distribution often caused considerable errors. Phase fractions were also calculated for histograms of kinetically perturbed populations, simulated as well as experimental The errors were only slightly larger than for histograms from asynchronously growing cell populations.

of Part A.- Section I: What is the Chromatin?.- Properties and Composition of Isolated Chromatin.- Expressed and Nonexpressed Portions of the Genome: Their Separation and Their Characterization.- Discussion.- Section II: Physical, Chemical and Biological Techniques for Studying Nucleosome, Chromatin, Chromosome and Nuclei.- Electron Microscopy: A Tool for Visualizing Chromatin.- Transcriptional Control of Native Chromatin.- Circular Dichroism of DNA, Protein and Chromatin.- Important Hydrodynamic and Spectroscopic Techniques in the Field of Chromatin Structure.- Preparation and Analysis of Core Particles and Nucleosomes: A Conveinient Method For Studying the Protein Composition of Nucleosomes Using Protamine-Release into Triton-Acid-Urea Gels.- The Interaction of Histones with DNA: Equilibrium Binding Studies.- Nucleosome Shape and Structure in Solution from Flow Birefringence.- Scattering and Diffraction by Neutrons and X-rays in the Study of Chromatin.- Nuclear Magnetic Resonance Studies of Nucleic Acids and Proteins.- Techniques for Cytochemical Studies of the Nucleus and its Substructures.- Chromatin Study in Situ: I. Image Analysis.- Chromatin Study in Situ: II. Static and Flow Microfluorimetry.- Chromatin Study in Situ: III. Differential Effects of Feulgen Hydrolysis.- Scanning and Flow Photometry of Chromosomes.- Discussion.- Section III: Various Levels of Chromatin Organization and Mechanisms for Transcriptional Control.- Histones Assembly and Their Structural Role for Nucleosome Core.- Nuclease Digestion and the Structure of Chromatin.- Reconstitution of Nucleosomes.- Conformation of Polynucleosomes in Low Ionic Strength Solution.- Chromatin Structure: Relation of Nucleosomes of DNA Sequences.- Histone Complexes, Nucleosomes, Chromatin and Cell-Cycle Dependent Modification of Histones.- Evidence for Superstructures of Wet Chromatin.- Chromatin Fractionation and the Properties of Transcriptionally Active Regions of Chromatin.- Chromatin Reconstitution and Non-Histone Proteins.- Discussion.- Section IV: Structure-Function of the Genetic Apparatus and Cell Cycle, Aging, Neoplastic Transformation, Differentiation, Chemical Carcinogenesis.- The Structure and Function of Chromatin in Lower Eukaryotes.- Chromatin Structure from Angstrom to Micorn Levels, and Its Relationship to Mammalian Cell Proliferation.- Chromatin Pattern in Situ: Dependence upon Cell Cycle, Preimplantation and Development, and Cellular Aging in Vitro.- Neoplastic Transformation: The Relevance of in Vitro Studies for the Understanding of Tumor Pathenogenesis and Neoplastic Growth.- Cell Differentiation and Malignancy in Leukemia.- Cellular Morphometry in Transformation, Differentiation and Aging.- Basic Mechanisms in Chemical Carcinogenesis.- Carcinogen Induced Alteration in Gene Packing and Its Possible Significance in Carcinogenesis.- Covalent Binding of a Carcinogen to DNA as a Probe of Chromatin Structure.- Carcinogenesis, DNA Repair and Chromatin.- Electromagnetic Induction of Electrochemical Information at Cell Surfaces: Application to Chromatin Structure Modification.- Discussion.- Section V: Review and Summary of the Genetic Apparatus.- Session I: Basic Components of the Genetic Apparatus.- Session II: The Second Level of Organization - Chromatin.- Session III: The Third Level of Organization.- Session IV: Generalized Biological Effects.

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.

NEWCHEM, an artificial intelligence system for the control of cancer cell growth, is described. This system takes into account the most recent advances in molecular and cellular biology and in cell-drug interaction, and aims to develop optimal strategies for the selective control of cancer cell through qualitative reasoning from first principles at cellular level.

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.