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PubMed2003

Ectopic chromosome around centrosome in metaphase cells as a marker of high-risk human papillomavirus-associated cervical intraepithelial neoplasias.

This report describes the appearance of ectopic chromosome around centrosome (ECAC) in metaphase cell nuclei of high-risk HPV-associated cervical neoplasms. ECAC are clearly visible on HE-stained sections as a tiny (approx. 0.7 micro), round, dark structure or an aggregate of filamentous chromosome, often symmetrical at bilateral centrosomes. They appear in CINs from an early stage (CIN I), with the highest incidence in HPV16-associated CIN II-III (75%), and are less common in HPV-related invasive carcinomas (21%) and in lesions associated with high-risk HPV types other than 16. Rates for ECAC-positive nuclei in metaphase preparations (ECAC rate) for each lesion ranged 3.6-30%, the average being 14.5%. ECAC appeared very rarely in CINs associated with intermediate-risk HPVs and was never encountered in HPV6/11-induced condylomas or HPV-unrelated neoplasms in other organs. ECAC may be morphologic evidence of HPV-induced chromosomal instability working as a background mechanism in cervical carcinogenesis and also a useful marker for the histopathologic differentiation of high-risk CINs.

Aneuploidy↗
PubMed2003

Centrosomal abnormality is common in and a potential biomarker for bladder cancer.

Centrosomal abnormalities have been implicated in chromosomal segregation aberrations that result from the formation of multipolar mitotic spindles and lead to aneuploidy. Aneuploidy is a characteristic of neoplasia and underlies the development and progression of bladder cancer. Therefore, centrosomal abnormality may play a key role in urothelial tumor transformation. The purpose of our investigation was to determine whether centrosomal abnormalities are present in malignant urothelial cells, define the relationship between centrosomal abnormalities and aneuploidy and determine whether the presence of centrosomal abnormalities might be a potential diagnostic marker for bladder cancer. Bladder wash specimens obtained from patients with and without a history of urothelial carcinoma were analyzed for centrosomal abnormalities using an immunoassay with a gamma-tubulin antibody. FISH with centromeric probes for chromosomes 4 and 9 and DNA ploidy image analysis were performed to detect aneuploidy. Defective centrosomes were found in 40 of 45 bladder wash specimens from patients with bladder cancer but in none of the 10 samples from patients without it. A large percentage (69%) of grade 1 tumors were positive for centrosomal abnormalities, and these abnormalities were increasing in numbers and size in grade 2 (93%) and grade 3 (100%) specimens. Centrosomal abnormalities and numerical chromosomal aberrations frequently appeared concomitantly in the same malignant cells. All of the specimens showing aneuploidy also exhibited centrosomal abnormalities: centrosomal defects and aneuploidy occurred together in 80% of malignant bladder tumors, with an especially high percentage in higher-grade tumors. The overall positivity of centrosomal abnormalities was higher than that of aneuploidy (88% vs. 80%), especially in grade 1 tumors (69% vs. 46%), whereas aneuploidy was strongly associated with grade 2 and grade 3 tumors. Centrosomal abnormalities are common in bladder cancer, even in low-grade tumors, and strongly associated with cancer grade and aneuploidy, especially in high-grade neoplasms. Centrosomal abnormalities appear to be intrinsic to aneuploidy and tumorigenesis and may be potential markers for early detection of bladder cancer.

Aneuploidy↗
PubMed1994

Nuclear DNA content, proliferating-cell nuclear antigen (PCNA) and p53 immunostaining in predicting progression of laryngeal cancer in situ lesions.

Squamous epithelial cancer in situ (CIS) of the upper aerodigastric tract is a histopathologically well-defined condition. However, in clinical practice, morphological grading of dysplasia is difficult and shows large variability. The biology of CIS remains enigmatic, and there is yet no reliable way to predict whether a CIS lesion will progress to invasive cancer, remain stable or regress. In the search for markers able to foretell clinical outcome, we performed image DNA cytometry (ICM) and immunohistochemical staining for PCNA as well as p53 in 38 laryngeal CIS lesions, of which 9 progressed to invasive cancer. The majority of the CIS lesions displayed high-grade DNA aberration, a high PCNA-positive rate, and every third lesion was p53-positive by immunostaining. The lesions which progressed to invasive cancer showed a clear tendency towards more pronounced DNA aberration, a higher percentage of intense PCNA staining and more frequent p53 positivity. By combining the results from the analyses of DNA, PCNA and p53 in a prognostic index for each individual case, we correctly classified 82% of the lesions as progressors or non-progressors.

Adult↗
PubMed1995

Immunofluorescence and confocal laser scanning microscopy studies of osteoblast growth and phenotypic expression in three-dimensional degradable synthetic matrices.

In the development of three-dimensional cell-polymer synthetic matrices for tissue regeneration, visualization of cells growing in these porous structures can be difficult. The focus of this study was the development and use of a novel method that would allow for visualization of osteoblasts inside opaque matrices. The morphologic responses and phenotypic characterization of osteoblasts as they attach, spread, and migrate through a porous three-dimensional biodegradable polymer-ceramic matrix in vitro were studied using immunofluorescence and confocal laser scanning microscopy (CLSM). CLSM offers several advantages over the most commonly used imaging methods [traditional light microscopy and scanning electron microscopy (SEM)]. CLSM filters out-of-focus background and provides more structural details of cells. In addition, CLSM does not require extensive sample preparation as does SEM. When used in conjunction with fluorescence-labeled antibodies to identify cells and their products, it can characterize morphology of growing cells and successfully determine phenotypic function. Using monoclonal antibody to osteocalcin, a bone cell-specific protein, cells throughout the matrix were found to have preserved osteoblast-like phenotype with growth. The morphology of cells throughout the matrix was found to be similar to osteoblast cells grown on tissue culture polystyrene and consisted of spread polygonal forms. Using the technique of CLSM with immunofluorescent antibodies, we have demonstrated for the first time that these three-dimensional degradable polymer matrices can support osteoblast growth and phenotypic expression throughout its structure.

Alkaline Phosphatase↗
PubMed1992

Fluorescence cytometry of microtubules and nuclear DNA during cell-cycle and reverse-transformation.

Synchronized CHO-K1 cells and their dibutyryl c-AMP treated counterparts have been characterized by means of static and flow fluorescence cytometry at the level of nuclear DNA and cytoplasmic microtubules. In order to confirm earlier findings on synchronized population, Carnoy fixed and hydrolyzed, several new findings are here reported at the level of single intact cell. The fluorescence intensity of DAPI-stained glutaraldehyde fixed 2C cells correlates well with the average absorbance of the corresponding Feulgen-stained cells, thereby appearing also to be a measure of chromatin condensation during the G1 phase. In the early part of G1, the drastic alteration in anti-beta tubulin immunostaining is shown to parallel microtubule depolymerization induced by calcium or colcemide. The known 1-2 h lengthening of the G1 period after reverse-transformation appears to correlate with a similar delay in the abrupt chromatin decondensation. The above results are discussed in terms of the role of microtubules and nuclear morphometry (and their coupling) in the control of cell cycle progression of transformed vs. fibroblast-like cells.

Animals↗
PubMed1993

Premalignant lesions of the upper aerodigestive tract: biomarkers of genetic alterations, proliferation, and differentiation.

The normal distribution of cell division in squamous mucosa is in the basal or adjacent suprabasal cell layers. Migration of cells toward the epithelial surface results in cell differentiation, most often expressed by high molecular weight keratin intermediate filaments and components of the cornified envelope, including "involucrin." These latter expressions of terminal differentiation are common in keratinizing dysplasia and invasive squamous cell carcinomas. However, they are less common in the non-keratinizing dysplasias, which fail to express evidence of epithelial maturation. Cell proliferation occurs in or near the basal layer in normal or reactive/reversible hyperplasias. In dysplasia (both keratinizing and non-keratinizing), cell proliferation is observed at all levels of the epithelium. Concomitant with these abnormalities in proliferation and differentiation are nuclear changes characterized by large hyperchromatic nuclei. The enlarged nuclei reflect increased DNA content, as documented by flow cytometry and image analysis techniques. DNA aneuploidy represents a spectrum of genomic alterations reflecting steps toward the progression to invasive carcinoma, which for the most part, have not yet been identified.

Animals↗
PubMed2001

Regulatory volume decrease (RVD) by isolated and in situ bovine articular chondrocytes.

Articular chondrocytes in vivo are exposed to a changing osmotic environment under both physiological (static load) and pathological (osteoarthritis) conditions. Such changes to matrix hydration could alter cell volume in situ and influence matrix metabolism. However the ability of chondrocytes to regulate their volume in the face of osmotic perturbations have not been studied in detail. We have investigated the regulatory volume decrease (RVD) capacity of bovine articular chondrocytes within, and isolated from the matrix, before and following acute hypotonic challenge. Cell volumes were determined by visualising fluorescently-labelled chondrocytes using confocal laser scanning microscopy (CLSM) at 21 degrees C. Chondrocytes in situ were grouped into superficial (SZ), mid (MZ), and deep zones (DZ). When exposed to 180mOsm or 250mOsm hypotonic challenge, cells in situ swelled rapidly (within approximately 90 sec). Chondrocytes then exhibited rapid RVD (t(1/2) approximately 8 min), with cells from all zones returning to approximately 3% of their initial volume after 20 min. There was no significant difference in the rates of RVD between chondrocytes in the three zones. Similarly, no difference in the rate of RVD was observed for an osmotic shock from 280 to 250 or 180mOsm. Chondrocytes isolated from the matrix into medium of 380mOsm and then exposed to 280mOsm showed an identical RVD response to that of in situ cells. The RVD response of in situ cells was inhibited by REV 5901. The results suggested that the signalling pathways involved in RVD remained intact after chondrocyte isolation from cartilage and thus it was likely that there was no role for cell-matrix interactions in mediating RVD.

Animals↗
PubMed2005

Regional estimates of hair cells and supporting cells in the human crista ampullaris.

Regional estimates of type I and type II vestibular hair cells (HC) and supporting cell (SC) numbers were obtained from the horizontal crista ampullaris by using design-based stereology in human. Cristae were microdissected from temporal bones obtained post-mortem (N=16, age range 26-98 years). Three groups were made according to age: group 1, n=5, ages between 26 and 67 years, average age 51 years; group 2, n=4, average age 84 years; and group 3, n=7, average age 94 years. For group 1, the average total HC number was 8,005+/-214, corresponding to 4,119+/-107 type I HC, 3,886+/-117 type II HC, and 10,274+/-224 SC. The type I:type II HC ratio was 1.06+/-0.01, and HC density was 0.80 cells/100 microm2. For group 2, the average total HC number was 7,074+/-489, corresponding to 3,733+/-212 type I HC, 3,341+/-314 type II HC, and 9,321+/-858 SC. The type I:II HC ratio was 1.12+/-0.06, and HC density was 0.75 cells/100 microm2. For group 3, the average HC number was 6,009+/-327, corresponding to 3,380+/-223 type I HC, 2,628+/-235 type II HC, and 10,185+/-182 SC. The type I:II HC ratio was 1.34+/-0.10, and HC density was 0.63 cells/100 microm2. A significant decline in type I, type II, and total HC number and density was found in groups 2 and 3, with individuals exceeding the average human life span.

Adult↗
PubMed2005

Three-dimensional topography of the motor endplates of the rat gastrocnemius muscle.

Spatial distribution of motor endplates affects the shape of the electrical activity recorded from muscle. In order to provide information for realistic models of action potential propagation within muscles, we assembled three-dimensional maps of the motor endplates of the rat medial gastrocnemius (MGM) and lateral gastrocnemius (MGL) muscles. The maps were assembled from histological cross sections stained for acetylcholinesterase activity. Within MGL, the motor endplates formed three columns along its longitudinal axis. Within MGM, the motor endplates were arranged in a leaf-like body that shifted obliquely from proximal to distal. As inferred from the proximo-distal distribution of the cross-sectional projection area, the majority of the motor endplates were concentrated in the middle of MGL and in the distal third of MGM. Regions of maximal motor endplate concentration are considered most suitable for injections of neuroactive substances, such as neuronal tracers. The assembled maps of the gastrocnemius muscles can be used as guides for such injections within the motor endplate zones.

Acetylcholine↗
PubMed1995

Identification of a low-risk group of stage I breast cancer patients by cytometrically assessed DNA and nuclear texture parameters.

Image cytometrical measurements were performed on Feulgen-stained cells from 329 stage I breast cancers (pT1pN0,M0,R0). For each patient, several DNA (ploidy, S-phase fraction, exceeding rates, 2c deviation index, ploidy balance, entropy, and histogram typing), morphometric (area and radius of nuclei), and textural parameters (mainly co-occurrence and run-length) were calculated. The prognostic value of these parameters was investigated by multivariate Cox regression analysis, considering a distant recurrence-free survival of 8 years as the prognostic criterion. In the multivariate analysis, one DNA parameter (histogram type) and two textural parameters (co-occurrence and variation of the average heterochromatin area) were proven to have independent prognostic value. Using a linear combination of these variables, a prognostic factor was calculated for each individual patient. Patients were stratified using this factor into several groups according to their risk for distant recurrence. Thus, a low-risk group of stage I patients was identified, remaining distant recurrence-free for 8 years. In addition, a group of patients with a worse prognosis and an 8-year recurrence rate of about 26 per cent was identified, compared with the average distant recurrence rate of all stage I patients of 13 per cent. A combination of DNA and textural parameters can provide powerful prognostic information in stage I breast carcinomas and may allow a better selection of patients for different therapy protocols.

Adult↗
PubMedDate not supplied

Lengths measurements in microvascular corrosion castings: two-dimensional versus three-dimensional morphometry.

In the present study we compared measurements of vessel lengths from (a) single-digital scanning electron microscope (SEM) images of microvascular corrosion casts (VCCs) of gill filters of tadpoles of Xenopus laevis Daudin by two-dimensional (2-D) morphometry (Optimas 6.5, Optimas Corp., Bothell, Wash., USA; planar measurements) and (b) digital stereopairs by three-dimensional (3-D) morphometry (3D-Morphometry, Minnich and Muska OEG, Salzburg). Depending on the spatial orientation of the vessels measured, we found a maximum difference of 58.84% (100 [3-D]-41.16 [2-D]) in vessel lengths by 3-D morphometry versus 2-D morphometry, which, in multiple (segmental) lengths measurements or when determining space angles, might be even higher. Based on results we consider 3-D morphometry of VCCs to be the method of choice for lengths measurements.

Animals↗
PubMed1999

Path reconstruction as a tool for actin filament speed determination in the in vitro motility assay.

The in vitro motility assay is used to measure speed of actin filaments moving over a glass surface coated with heavy meromyosin. In this paper a new method, the path reconstruction method, is presented to evaluate observed speeds. The method is compared with the commonly used centroid method, in which the centroids of the filaments are followed from frame to frame. Instead, in the path reconstruction method speed is evaluated from determination of perimeters of the filaments in each frame and by reconstruction of the traversed paths of the filaments over a number of frames. Biases in the determination of speed occurring in the centroid method due to curvature of paths and to video noise and Brownian motion are eliminated in the path reconstruction method, allowing measurement over a range of frame rates from 5 to 25 per second. The path reconstruction method leads to a clear separation of motile and nonmotile filaments provided that filaments are analyzed over at least 10 successive frames and allows easier separation of uniform and nonuniform sliding behavior.

Actins↗
PubMed1999

Apoptosis during iron chelator-induced differentiation in F9 embryonal carcinoma cells.

We have previously demonstrated that three potent iron chelators, hinokitiol, dithizone and deferoxamine, induce differentiation of F9 embryonal carcinoma cells, as do other well-known morphogens such as retinoic acid (RA) and sodium butyrate (NaB). In this study, we compared the patterns of cell proliferation, cell death and cell cycle arrest during the process of differentiation induced by these five agents. When F9 cells were cultured with the agents at their individual differentiation-inducing concentrations, cell proliferation was rapidly inhibited by treatment with the iron chelators and NaB. In contrast, RA did not influence the rate of increase of cell number at the concentration of 1 microm. The three chelators also caused a marked reduction in cell viability, and the treated cells exhibited internucleosomal DNA fragmentation, whereas cells treated with NaB showed no apoptotic characteristics. RA induced apoptosis weakly at 1 microm and strongly at higher concentrations. In addition, all the iron chelators hindered cell cycle progression, resulting in an arrest at the G1-S interface or S phase. The phenomena observed in chelator-treated cells were considerably different from those in RA- or NaB-treated cells. It is concluded that the three iron chelators cause both severe apoptotic cell death and cell cycle arrest of proliferating F9 cells via cellular iron deprivation, and that this apoptotic change may be independent of the process of differentiation.

Animals↗
PubMed1998

Cellular and cytoskeleton morphology and strength of adhesion of cells on self-assembled monolayers of organosilanes.

The objective of this study was to explore the potential use of self-assembled monolayers (SAMs) of alkylamine and arylalkyamine as well-defined, homogeneous, tailored in vitro model surfaces for exploring the effect of hydrodynamic flow on morphology and strength of adhesion of human umbilical vein endothelial cells. The cell surface area, shape, f-actin distribution, and adhesion strength of human umbilical vein endothelial cells cultured on self-assembled monolayers of organosilanes were found to be dependent on the chemical composition of the organosilane film and the magnitude of wall shear stress. The direct effects of the differences in chemistry between the two silanes, in modulating cellular response, are probably only secondary to the modulation of cellular functions mediated by differential protein adsorption and conformation on the two silanes. For short seeding times (30 min), prior to application of flow, both substrate chemistry and shear stress modulated the cellular morphology and cytoskeletal organization. For longer seeding times (24 h), prior to application of flow, the chemistry of the underlying surface was the dominant variable in modulating cellular morphology, while the hydrodynamic shear stress modulated the cytoskeleton organization. Cells on N-(2-aminoethyl)-3-aminopropyl trimethoxysilane (EDA) were pleomorphic, while cells on ((((aminoethyl)amino)methyl)phenylethyl)trimethoxysilane (PEDA) expressed a rounded morphology. Application of an incrementally loaded flow regime (0.07-1.25 ml/s) resulted in a time- and shear stress-dependent (10-180 dyn/cm2) detachment of cells, with the cells on EDA depicting higher resistance to wall shear stress. Cellular morphology correlated with the strength of adhesion; cells with rounded morphology on a hydrophobic silane, PEDA, were less tightly bound to the silane, while spread cells on a hydrophilic silane, EDA, were more tightly bound. The higher surface free energy of EDA is speculated to influence the increased cell spreading and strength of adhesion observed in these studies. The presence of the phenyl group in PEDA reduces the surface free energy and may account for the reduced spreading and lower strength of adhesion. The use of well-defined systems, such as monolayer organosilanes, with tunable surface physicochemical properties may serve to deconstruct the complex interaction of cells with extracellular matrix components: surface charge, surface hydrophobicity, and other short- and long-range forces can be individually controlled and correlated with cellular functions. The organosilane monolayers could serve as the building blocks for sequential addition of proteins or cell adhesive/cell repulsive cues to stepwise engineering and construction of more complex systems resembling ECM matrices.

Biocompatible Materials↗
PubMed1999

The Schrödinger's cat quandary in cell biology: integration of live cell functional assays with measurements of fixed cells in analysis of apoptosis.

The existing cytometric methodologies do not allow one to directly correlate, within the same cells, functional cell attributes that are revealed supravitally with features that require cell fixation to be detected or measured. Taking advantage of the "file merge" feature of the laser-scanning cytometer, we have been able to correlate the supravital changes that occur during apoptosis, namely the drop in mitochondrial transmembrane potential (Delta Psim) and generation of the reactive oxygen intermediates (ROIs), with features revealed by analysis of fixed cells: the cell cycle position and DNA fragmentation. The cell cycle position was established based on the cell's stainability with propidium iodide while DNA fragmentation was assessed by in situ DNA strand break labeling using exogenous terminal deoxynucleotidyltransferase. During apoptosis of HL-60 cells induced by the DNA topoisomerase I inhibitor camptothecin (CPT), the dissipation of Delta Psim occurred preferentially in S-phase cells and preceded the appearance of DNA strand breaks. Essentially all cells with DNA strand breaks had dissipated Delta Psim. Compared to the decrease of Delta Psim, the CPT-induced rise in ROIs during apoptosis was less restricted to S-phase cells. Furthermore, no elevation of ROIs was detected in a significant proportion of cells with DNA strand breaks. The data suggest that DNA fragmentation may occur in some cells prior to the increase in ROIs and thus, unlike the dissipation of Delta Psim, the oxidative stress may not be a prerequisite for activation of an endonuclease. Alternatively, the oxidative stress may be a transient event, occupying a narrow "time window" during the apoptotic process. The approach opens a possibility to study direct relationships, within the same cells, between cellular changes (e.g., occurring during apoptosis, mitogenesis, differentiation, etc.) detected by functional assays of live cells and changes that cannot be analyzed supravitally.

Apoptosis↗
PubMed2000

A detailed analysis of cyclin A accumulation at the G(1)/S border in normal and transformed cells.

The temporal relationship between cyclin A accumulation and the onset of DNA replication was analyzed in detail. Five untransformed and nine transformed asynchronously growing cell cultures were investigated using a triple immunofluorescence staining protocol combined with computerized evaluation of staining intensities in individual cells. The simultaneous staining of BrdU, cyclin A, and cyclin E made it possible to determine the cell cycle position of each cell investigated. Cells at the G(1)/S border were identified on the basis of cyclin E content and were further analyzed with respect to cyclin A and BrdU content. A method was developed to calculate objective thresholds defining the highest staining intensity found in the negative cells in the population. Using the thresholds we could distinguish cells with minute amounts of cyclin A and BrdU from truly negative cells. We show that the onset of cyclin A accumulation and the start of DNA replication occurs at the same time, or deviating by a few minutes at the most. We also show that cyclin A accumulates continuously during S. This study clearly demonstrates that nuclear cyclin A can be used as a reliable marker for the S and G(2) phases in both normal and transformed interphase cells.

Antibodies↗
PubMed2001

Integrin alpha3beta1 engagement disrupts intercellular adhesion.

During tissue morphogenesis and tumor invasion, epithelial cells must undergo intercellular rearrangement in which cells are repositioned with respect to one another and the surrounding mesenchymal extracellular matrix. Using three-dimensional aggregates of squamous epithelial cells, we show that such intercellular rearrangements can be triggered by activation of beta1 integrins after their ligation with extracellular matrices. On nonadherent substrates, multicellular aggregates (MCAs) formed rapidly via E-cadherin junctional complexes and over time became compacted spheroids exhibiting a more epithelial phenotype. After MCAs were replated on culture substrates, the spheroids collapsed to yield tightly arranged cell monolayers. Cell-cell contact induced rapid elevation in E-cadherin levels, which was due to an increase in the metabolic stability of junctional receptors. During MCA remodeling of cell-cell adhesions, and monolayer formation, their E-cadherin levels fell rapidly. Similar behavior was obtained regardless of which ECM ligand-collagen type I, fibronectin, or laminin 1-MCAs were seeded on. In contrast, when seeded onto a matrix elaborated by squamous epithelial cells, cells in the MCA attached, spread, lost cell-cell junctions, and dispersed. Analysis identified laminin 5 as the active ECM ligand in this matrix, and MCA dispersion required functional beta1 integrin and specifically alpha3beta1. Furthermore, substrate-immobilized anti-integrin antibody effectively reproduced the epithelial-mesenchymal-like transition induced by the laminin 5 matrix. During the early stages of aggregate rearrangement and collapse, cells on laminin 5 substrates, but not those on collagen I substrates, exhibited intense cortical arrays of F-actin, microspikes, and fascin accumulation at their peripheral surfaces. These results suggest that engagement of specific integrin-ligand pairs regulates cadherin junctional adhesions during events common to epithelial morphogenesis and tumor invasion.

Antibodies, Monoclonal↗
PubMed2002

New vascular tissue rapidly replaces neural parenchyma and vessels destroyed by a contusion injury to the rat spinal cord.

Blood vessels identified by laminin staining were studied in uninjured spinal cord and at 2, 4, 7, and 14 days following a moderate contusion (weight drop) injury. At 2 days after injury most blood vessels had been destroyed in the lesion epicenter; neurons and astrocytes were also absent, and few ED1+ cells were seen infiltrating the lesion center. By 4 days, laminin associated with vessel staining was increased and ED1+ cells appeared to be more numerous in the lesion. By 7 days after injury, the new vessels formed a continuous cordon oriented longitudinally through the lesion center. ED1+ cells were abundant at this time point and were found in the same area as the newly formed vessels. Astrocyte migration from the margins of the lesion into the new cordon was apparent. By 14 days, a decrease in the number of vessels in the lesion center was observed; in contrast, astrocytes were more prominent in those areas. In addition to providing a blood supply to the lesion site, protecting the demise of the newly formed vascular bridge might provide an early scaffold to hasten axonal regeneration across the injury site.

Animals↗