S-phase progression in mammalian cells: modelling the influence of nuclear organization

The control of DNA replication is of fundamental importance as cell proliferation demands that identical copies of the genetic material are passed to the two daughter cells that form during mitosis. These genetic copies are generated in the preceding S phase, where the entire DNA complement of the mother cell must be copied exactly once. As part of this process, it is known that different regions of mammalian genomes are replicated at specific times of a temporally defined replication programme. The key feature of this programme is that active genes in euchromatin are replicated before inactive ones in heterochromatin. This separation of S phase into periods where different classes of chromatin are duplicated is important in maintaining changes in gene expression that define individual cell types. Recent attempts to understand the structure of the S-phase timing programme have focused on the use of genome-wide strategies that inevitably use DNA isolated from large cell populations for analysis. However, this approach provides a composite view of events that occur within a population without knowledge of the cell-to-cell variability across the population. In this review, we attempt to combine information generated using genome-wide and single cell strategies in order to develop a coherent molecular understanding of S-phase progression. During this integration, we have explored how available information can be introduced into a modelling environment that best describes S-phase progression in mammalian cells.     

Utility and limitations of flow cytometry DNA content analysis in neoplastic cells]

A review of the literature concerning the analysis of nuclear DNA content by flow cytometry is made and it is compared with own experience. The advantages and the limits of this technique are examined. The practical problems in the interpretation of the histograms and the value of the measurements are discussed. The importance of this analysis in diagnosis and in staging of many tumors and the clinical involvements are emphasized. In many tumors DNA ploidy represents a new independent prognostic variable that is useful to separate the cases with a potential worse behaviour in an early stage, when other classic parameter are not available. It is also stressed the importance of flow cytometric DNA analysis of tumor cells. This is made on the same tissues that pathologists use for histopathological diagnosis and the results are important in many cases for a diagnostic accuracy.     

Surface morphology of normal and neoplastic rat cells.

Nontumorigenic rat cells and their tumorigenic counterparts were studied with scanning electron microscopy under controlled conditions in vitro and with transmission electron microscopy after replantation in vivo to discern if external morphology reflected the cell's neoplastic state or the etiology of transformation. Interphase cells in six of seven nontumorigenic lines were flat and monolayered under confluent conditions and exhibited smooth, nonactive cell surfaces. A nontumorigenic cell line morphologically transformed with human adenovirus-2 consisted of spherical cells with blebbed surfaces. Cells from six tumorigenic lines transformed with avian sarcoma virus had highly active surfaces with many surface projections. Cells from two chemical carcinogen-transformed rat embryo lines were flat with no surface projections in subconfluent culture and rounded with only a few microvilli at high densities, but cells from a sarcoma chemically induced in an adult rat were villous. When villous cells were syngeneically replanted in vivo, they lost most microvilli. The external morphology of cells was influenced by a number of factors simultaneously, with no universal pattern associated with tumorigenic capacity or transforming agent.     

Light scattering from normal and dysplastic cervical cells at different epithelial depths: finite-difference time-domain modeling with a perfectly matched layer boundary condition

The finite-difference time-domain (FDTD) method provides a flexible approach to studying the scattering that arises from arbitrarily inhomogeneous structures. We implemented a three-dimensional FDTD program code to model light scattering from biological cells. The perfectly matched layer (PML) boundary condition has been used to terminate the FDTD computational grid. We investigated differences in angle-dependent scattering properties of normal and dysplastic cervical cells. Specifically, the scattering patterns and phase functions have been computed for normal and dysplastic cervical cells at three different epithelial depths, namely, basal/parabasal, intermediate, and superficial. Construction of cervical cells within the FDTD computational grid is based on morphological and chromatin texture features obtained from quantitative histopathology. The results show that angle-dependent scattering characteristics are different not only for normal and dysplastic cells but also for cells at different epithelial depths. The calculated scattering cross-sections are significantly greater for dysplastic cells. The scattering cross-sections of cells at different depths indicate that scattering decreases in going from the superficial layer to the intermediate layer, but then increases in the basal/parabasal layer. This trend for epithelial cell scattering has also been observed in confocal images of ex vivo cervical tissue.     

Functional morphology of breast APUD cells in dysplasia and neoplastic processes

Histochemical and immunohistochemical methods were used to examine 29 malignant tumors (18 lobular and 11 invasive carcinomas) and 34 fibroadenomas of the mammary gland (MG). APUD cells containing serotonin, melatonin, and beta-endorphine were shown to be present in the duct epithelium of the normal MG and its pericanalicular fibroadenoma. APUD cells were detected in 21 of the 29 malignant tumours of MG. Hormonal differences of APUD cells were found in poorly and well differentiated carcinomas: the former contained serotonin, melatonin, and beta-endorphine (inhibitors of proliferation), the latter--insulin and adrenocorticotropic hormone (stimulators of cell division). Such differences in the endocrine function of MG malignant tumors are likely to be significant in the clinical course and determination of prognosis for carcinomas of various differentiation.     

Light scattering from cells: the contribution of the nucleus and the effects of proliferative status

As part of our ongoing efforts to understand the fundamental nature of light scattering from cells and tissues, we present data on elastic light scattering from isolated mammalian tumor cells and nuclei. The contribution of scattering from internal structures and in particular from the nuclei was compared to scattering from whole cells. Roughly 55% of the elastic light scattering at high-angles (> 40 degrees) comes from intracellular structures. An upper limit of 40% on the fractional contribution of nuclei to scattering from cells in tissue was determined. Using cell suspensions isolated from monolayer cultures at different stages of growth, we have also found that scattering at angles greater than about 110 degrees was correlated with the DNA content of the cells. Based on model calculations and the relative size difference of nuclei from cells in different stages of growth, we argue that this difference in scattering results from changes in the internal structures of the nucleus. This interpretation is consistent with our estimate of 0.2 micron as the mean size of the scattering centers in cells. Additionally, we find that while scattering from the nucleus accounts for a majority of internal scattering, a significant portion must result from scattering off of cytoplasmic structures such as mitochondria.     

DNA and chromatin structure: Influence of incubation on the chromatin condensation and nuclear stability of human spermatozoa by flow cytometry

Flow cytometry analysis was used for the acurate and objectiveevaluation of sperm chromatin condensation and chromatin stabilityof sperm nuclei. It was also possible to determine the influenceof incubation on sperm chromatin. Different types of spermatozoawere studied: unprocessed spermatozoa at 1 and 45 min afterejaculation, after swim-up (migrated), spermatozoa incubatedfor 6 h in non-capacitating conditions (aged), or in B2 medium(capacitated) or B2 medium followed 1 h later with A23187 (reacted).All types of spermatozoa were analysed before and after treatmentwith various decondensation agents: sodium dodecyl sulphate(SDS), SDS plus EDTA and SDS plus disulphide-reducing agent[dithiotreitol (DTT)). Sperm nuclei were enzymatically isolatedand stained with propidium iodide. Three flow cytometric parameterswere then measured: forward light scatter (cellular size), sidelight scatter (cellular complexity) and fluorescence (uptakeof propidium iodide). Fluorescence was the most suitable parameterto study the degree of condensation and resistance to decondensationof DNA in the spermatozoa. Unprocessed spermatozoa 1 min afterejaculation underwent decondensation by all assessed treatments(anionic detergent, chelating or disulphidereducing agents).Unprocessed spermatozoa 45 min after ejaculation and migratedspermatozoa did not undergo decondensation with SDS treatment,but decondensation occurred after treatment with SDS + EDTAor SDS + DTT. Spermatozoa incubated for 6 h under both non-capacitating(aged spermatozoa) and capacitating conditions (capacitatedspermatozoa) and reacted spermatozoa were decondensed only aftertreatment with SDS + DTT. In conclusion, the post-ejaculationand incubation time have to be taken into account when clinicalinterpretation of the effect of different treatments on spermchromatin condensation is made.     

Transplacental induction of mouse lung tumors: stage of fetal organogenesis in relation to frequency, morphology, size, and neoplastic progression of N-nitrosoethylurea-induced tumors

Pregnant C3H/HeNCr MTV- mice were given a single intraperitoneal injection of 0.5 mmol N-nitrosoethylurea/kg on days 14, 16, or 18 of gestation. Six of the male offspring were sacrificed for study at the ages of 2, 4, 8, 16, 32, and 52 weeks. Grossly visible lung tumors were counted and all lungs were sectioned completely, saving every tenth section for histologic evaluation. All N-nitrosoethylurea-induced mouse lung tumors have previously been shown to originate from alveolar type II cells. Lung tumors were diagnosed as solid, papillary, or mixed solid/papillary types, and at the largest area of each tumor, the perimeter was measured and compared with the number of sections per tumor. The fraction of tumors detected grossly depended on size and, on average, only 51% of neoplasms present were detected macroscopically. A significant correlation was seen between the mean number of histological sections and perimeter length per tumor, in particular for small and medium sized papillary neoplasms. The growth of solid tumors was limited to a maximum size, after which they progressed towards papillary types. The numbers of transplacentally induced mouse lung tumors were distributed in direct proportion to the weight of the individual lung lobes, unrelated to day of treatment of type or tumor. Tumor biology depended on the day of treatment reflecting numbers of degree of differentiation of fetal alveolar type II cells, i.e., the target cell: most tumors developed in offspring treated on day 16, tumor size was greater and progression from solid to papillary neoplasms faster at earlier treatments, increase in tumor multiplicity postnatally was only seen in mice treated late in gestation, and mice treated on day 14 or day 16 showed a consistent ratio of solid to papillary tumors.     

The cytological features and DNA content of cervical adenocarcinoma

The relationship among cytological features, DNA content, and degree of histological differentiation of cervical adenocarcinoma was investigated in an attempt to discover a more accurate means of screening for this cancer. In highly differentiated adenocarcinoma (so-called adenoma malignum), the nuclei were only somewhat more irregular in size and shape than those of normal columnar epithelial cells. The cells were arranged in slightly multilayered clusters. The cells of well-differentiated adenocarcinoma were usually columnar in shape, and they exfoliated side by side in clusters. In moderately differentiated adenocarcinoma, solitary cells with markedly atypical nuclei were combined with multilayered cell clusters. The cells from poorly differentiated adenocarcinoma were roundish, occurred as solitary cells or irregularly overlapping cell clusters, and showed markedly atypical nuclei. As the degree of histological differentiation decreased, as determined by measurement of the DNA content of the cells, the DNA distribution covered a wider range in terms of ploidy, and the number of cells exceeding tetraploid DNA content increased.     

DNA content and nuclear size of megakaryocytes in thrombocythaemia

Total nuclear DNA content and nuclear size of megakaryocytes were studied in biopsies of the iliac bone marrow of individuals with normal or increased platelet counts. The DNA content was determined using Feulgen cytophotometry of bone marrow smears and the nuclear area by morphometric analysis of megakaryocytes of bone marrow sections. The mean DNA content and the mean nuclear area were both significantly larger in megakaryocytes of patients with thrombocytosis as a result of myeloproliferative disease than in patients with secondary thrombocytosis as well as in two control groups of individuals with normal platelets counts, one comprising healthy volunteers, the other with various non-haematological disorders. There was a statistically significant correlation between the DNA content and nuclear area of the megakaryocytes (r = 0.92) in the entire group of bone marrows studied.     

Sperm nuclear DNA damage and altered chromatin structure: effect on fertilization and embryo development

In the first part of this report we investigate whether chromatin anomalies in human spermatozoa can influence fertilization after intracytoplasmic sperm injection (ICSI). We have examined the sperm chromatin packaging quality using the chromomycin A3 (CMA3) fluorochrome and the presence of DNA damage in spermatozoa using in-situ nick translation. When comparing the spermatozoa of patients undergoing in-vitro fertilization (IVF) and ICSI distinct differences are evident in that ICSI males have a higher CMA3 fluorescence, indicating spermatozoa with loosely packed chromatin, and more spermatozoa containing endogenous DNA nicks. When examining the unfertilized oocytes of ICSI patients we found that men who had a high percentage of anomalies in their chromatin, i.e. > 30% CMA3 fluorescence and > 10% nicks, had more than double the number of unfertilized oocytes containing spermatozoa that had remained condensed. The observation that failed fertilized oocytes, injected with spermatozoa from patients with a higher percentage of sperm nuclear anomalies, contain more condensed spermatozoa indicates that a selection process against these spermatozoa may be in place at the time of fertilization. In the second part of the study we show that spare ICSI embryos have significantly lower rates of development to the blastocyst stage compared with those developed after routine IVF. These results show that a greater understanding of the molecular basis of male infertility is therefore needed to broaden our knowledge on the effect that abnormal spermatozoa have on fertilization and embryo development.     

The influence of senescence on craniofacial and cervical morphology in humans

This study discusses the morphologic evolution of the cranio-facial and cervical bone structures throughout life. A cephalometric study was made on lateral radiographs. The population studied included 84 males and 102 females. Ages ranged from 21 to 101. The cranial structures, superior facial structure, mandible and cervical vertebral column were successively examined. The anteroposterior diameter of the calvarium does not seem to undergo any modification during life. On the other hand, a highly significant increase of the thickness of this structure can be noted. The upper facial structure presents some modification, namely a significant increase of its posterior height. The palatine processus seems to change direction and pivot downwards and forwards. The maxillary sinus does not undergo any changes. The mandible, which is stable in its major axes, shows more malleable sectors which are more especially situated at the level of its body. The study of the cervical vertebral column reveals a loss of overall height, and an increase in the lordosis. The most numerous and most evident morphologic modifications were observed around the age of fifty in both males and females. The fact that these transformations are always commoned and greater in the latter reveals the plausible influence of the menopause. It appears that bone structures of membranous origin are the site of significant modifications compared with structures of endochondral origin, which benefit from a greater stability.     

Thymidine phosphorylase expression in progression of cervical cancer: correlation with microvessel count, proliferating cell nuclear antigen, and apoptosis

AIMS: To determine how epithelial and stromal thymidine phosphorylase expression affects angiogenesis, rapid tumour growth, and decreased apoptotic activity in cervical cancer at varying stages of progression. METHODS: Epithelial and stromal thymidine phosphorylase expression, the microvessel count (reflected by factor VIII related antigen), and proliferating cell nuclear antigen (PCNA) were assessed immunohistochemically in 25 specimens of normal cervical epithelium, 35 of carcinoma in situ (CIS), 34 of microinvasive carcinoma, and 34 of invasive cervical squamous cell carcinoma. Apoptosis was evaluated by the terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labelling (TUNEL) method. The relation of epithelial and stromal thymidine phosphorylase expression to microvessel count, PCNA index, and apoptotic index was examined. RESULTS: Epithelial and stromal thymidine phosphorylase expression progressively increased along a continuum from normal epithelium to invasive squamous cell carcinoma. Epithelial and stromal thymidine phosphorylase expression showed a significant positive correlation with microvessel counts. Within each histological stage, CIS cases with high stromal thymidine phosphorylase expression, invasive squamous cell carcinoma cases with high epithelial thymidine phosphorylase expression, and microinvasive carcinoma cases with high thymidine phosphorylase expression in both epithelium and stroma had a significantly higher microvessel count. High epithelial thymidine phosphorylase expression was associated with a significantly higher PCNA index in CIS and microinvasive carcinoma, but not in invasive squamous cell carcinoma. No significant correlation was seen between apoptotic index and either epithelial or stromal thymidine phosphorylase expression or microvessel count. CONCLUSIONS: Epithelial and stromal thymidine phosphorylase expression may combine to promote angiogenesis during progression of cervical cancer, and epithelial thymidine phosphorylase expression may stimulate tumour cell proliferation in the early stages.     

An attempt to influence DNA content in postmitotic Purkinje cells of the cerebellum

Feulgen-DNA content has been measured cytophotometrically in granule and Purkinje cells of the rat and mouse cerebellum. The study has confirmed that under normal conditions a very small part of the P. cell population in rats (less than 3%) possesses a Feulgen-DNA surplus ranging from 2C to 4C. In mice, the hyperdiploid (H2C) P. cells are even more rare. The occurrence of H2C-P. cells in rats and probably also in mice has not been substantially changed in animals exposed to factors interfering with chromatin structure and/or its template activity; the H2C-P. cells seem to be slightly more frequent after injecting mice with corticoids (Urbason) or in animals suffering from ectromelia or hereditary Purkinje cell degeneration. The incidence of H2C-P. cells has neither been substantially affected by experimental conditions which are known to lead to functional and/or metabolic stimulation of the cerebellum. Functional changes in the number of H2C-P. cell nuclei may, however, be short-term or transient in character and therefore might have excaped detection in our models. The findings rule out an impact of some reasons suspected for the artefactual origin of Feulgen H2C DNA values as e.g. compactness of the chromatin.     

Morphometric analysis of nuclear variations in normal and neoplastic mammary ductal cells

Morphometry is a method to detect changes in a variety of tissues through quantitative elements. The purpose of this study was to examine several nuclear morphologic characteristics in normal and neoplastic mammary ductal cells using a multivariable method and expression of estrogen receptors by immunohistochemical techniques. A total of 1879 nuclei were examined by a computerized program, following the detection of estrogen receptors. Nuclear area, perimeter, diameter, maximal and minimal radio were obtained in 439 normal ductal nuclei. The mean nuclear area was 14.45 with a range between 10.88 and 17.90. Variables showed adequate statistical correlation (r > 0.5). A total of 1440 neoplastic nuclei were classified as grades I, II and III, and a statistical significative difference was found between these three groups. We conclude that the nuclear area is a reliable variable for statistical correlation being the ductal nuclei anisotropic objects.     

Merkel cells, normal and neoplastic: an update

Merkel cells (MC) occur in the basal epidermal layer, hair follicles, and oral mucosa, as complexes with sensory axons. The axons transduce slowly adapting type I mechanoreception, and MC modulate their sensitivity. MC also determine and maintain the 3-dimensional epidermal structure. They have neuroendocrine granules, rigid spinous processes, and desmosomal junctions with each other and with keratinocytes. Rare MC are dermaWl. Current evidence supports a basal cell origin. Merkel cell carcinomas (MCC) occur mostly in sun-exposed skin in old age. Trabecular, intermediate, or small cell in pattern, MCC have neuroendocrine granules, intercellular junctions, rigid spinous processes, and a paranuclear collection of intermediate filaments staining for cytokeratin 20. Most MCC behave indolently, but those with the small cell pattern, and some with the intermediate pattern, are aggressive and rapidly fatal.     

Variability of cardiomyocyte DNA content, ploidy level and nuclear number in mammalian hearts

DNA content, ploidy level, cell size and nuclear number were investigated in 54 mammalian hearts from nine species. DNA content was determined biochemically and ploidy level of cells was studied by the means of Feulgen cytophotometry. Nuclear number was calculated by a new method, while cell size was determined by using ocular micrometry. In most mammals diploid cell nuclei predominate. Higher ploidy levels were found in the human and the pig hearts. The total amount of DNA correlated with the myocardial weight. Eight million heart muscle cell nuclei were found in mice (myocardial weight 160 mg), and 2600 million heart muscle cell nuclei in the human heart (myocardial weight 210 g), but in the hearts of horses up to 35 000 million heart muscle cell nuclei (myocardial weight 3.4 kg) were found. The number of heart muscle and connective tissue cell nuclei was correlated with myocardial weight. The ratio of connective tissue cell nuclei to heart muscle cell nuclei was between 2:1 and 3:1. In cardiac growth this ratio shifted towards connective tissue cell nuclei. Increased heart weight corresponds to an increase in cell size. Diameter between 11 m and 18 m may be an optimum for heart muscle cells of mammals.