Quantification of epithelial cells in coculture with fibroblasts by fluorescence image analysis

BACKGROUND: To demonstrate that senescent fibroblasts stimulate the proliferation and neoplastic transformation of premalignant epithelial cells (Krtolica et al.: Proc Natl Acad Sci USA 98:12072-12077, 2001), we developed methods to quantify the proliferation of epithelial cells cocultured with fibroblasts. METHODS: We stained epithelial-fibroblast cocultures with the fluorescent DNA-intercalating dye 4,6-diamidino-2-phenylindole (DAPI), or expressed green fluorescent protein (GFP) in the epithelial cells, and then cultured them with fibroblasts. The cocultures were photographed under an inverted microscope with appropriate filters, and the fluorescent images were captured with a digital camera. We modified an image analysis program to selectively recognize the smaller, more intensely fluorescent epithelial cell nuclei in DAPI-stained cultures and used the program to quantify areas with DAPI fluorescence generated by epithelial nuclei or GFP fluorescence generated by epithelial cells in each field. RESULTS: Analysis of the image areas with DAPI and GFP fluorescences produced nearly identical quantification of epithelial cells in coculture with fibroblasts. We confirmed these results by manual counting. In addition, GFP labeling permitted kinetic studies of the same coculture over multiple time points. CONCLUSIONS: The image analysis-based quantification method we describe here is an easy and reliable way to monitor cells in coculture and should be useful for a variety of cell biological studies.     

Morphometry of stallion spermatozoa by computer-assisted image analysis

Metric measurements of stallion spermatozoal heads were determined for live, unfixed spermatozoa and for Feulgen-stained spermatozoa by videomicroscopy and computerized image analysis. Two ejaculates were collected from each of five stallions of normal fertility. Air-dried semen smears were Feulgen-stained, and live, unfixed spermatozoa were examined as wet-mount preparations. For Feulgen-stained spermatozoa, videoimages (x3850) were captured, and sperm heads were detected via image segmentation and particle analysis. For live, unfixed spermatozoa, phase contrast videoimages (x3850) were measured to determine width and length of the sperm head. For Feulgen-stained spermatozoa, there were significant effects (P < 0.001) of stallion and ejaculate on measured parameters of area, circumference, and the length and width of the sperm head. For live, unfixed spermatozoa, there were significant effects of stallion on length and width and of ejaculate on length of the sperm heads. There was a very poor correlation between length and width of sperm heads between Feulgen-stained and live, unfixed spermatozoa. Two indices of sperm shape (oval factor and aspect ratio) were also determined. Both aspect ratio and oval factor were significantly affected by stallion (P < 0.001); however, oval factor was not affected by ejaculate and therefore may represent a less variable determination of sperm head shape across stallions. Overall, length and width of stallion sperm heads were larger (P < 0.01) for live, unfixed spermatozoa than for Feulgen-stained spermatozoa (length: 6.3 +/- 0.4 vs 5.08 +/- 0.44; width: 3.08 +/- 0.34 vs 2.71 +/- 0.28 mum, respectively). Computerized image analysis may be useful as a means to objectively measure sperm head dimensions in the stallion and could be useful in future studies to determine associations with stallion fertility.     

Morphometric analysis of LPS-stimulated human monocytes by computer-assisted image analysis.

This report describes the results of applying the computer-assisted image analysis system for the measurement of some cytological parameters of LPS-stimulated and nonstimulated human monocytes. The experiments were carried out by means of the digital cell image analysis of haematoxilyn stained monocytes. Five different parameters describing the morphology of monocytes and their nuclei were selected to quantitate the differences between control and activated cells area, perimeter, elongation, dispersion, and extension of images of cell projections. The results suggest that all of the analysed parameters can be used to discriminate stimulated from nonstimulated monocytes which permits detailed monitoring of the changes in cell morphology during monocyte activation.     

Quantitation of oncogene products by computer-assisted image analysis and flow cytometry

The use of antibodies permits the study of oncogene product expression in cells and tissues. However, quantitation of the levels of expression in immunohistochemical preparations is beset by difficulties, and the available scoring system provide semiquantitative data at best. Here we describe the use of computer-assisted image analysis for determination of oncoprotein levels in a model system and compare the results with those generated by flow cytometric analysis. The oncogene products measured are located in the nucleus (c-myc p62 and c-fos p55), the inner surface of the membrane (c-ras p21), and both sides of the membrane (c-erbB-2 p185). In each instance, both analytic modalities yielded concordant results. Our data indicate that computer-assisted image analysis is a useful tool for quantitating cell components in immunohistochemical preparations.     

Quantification of autolysis in Penicillium chrysogenum by semiautomated image analysis

An image analysis method is described for the characterization of empty (autolyzed and inactive) regions within the mycelia of filamentous fungi. It extends a previous method that characterized only regions filled with cytoplasm or vacuoles (i.e., the active biomass). The method is semiautomatic, requiring some manual editing before automated measurements. When the method was used for samples from a batch fermentation of an industrial strain of Penicillium chrysogenum, the empty regions were observed to constitute up to 15% (by projected area) of the biomass during the growth phase. After nutrient exhaustion, however, the proportion of empty regions rose rapidly, eventually representing more than 50% of the biomass by the end of fermentation. The increase in the percentage of empty regions coincided with a decrease in biomass (as measured by dry cell weight) and a fall in penicillin titre. Further morphological analysis revealed that fragmentation of mycelia, particularly clumps, coincided with increases in the levels of empty regions. This new image analysis method gave additional information on hyphal differentiation and a measure of autolysis. It was also a useful indicator of the processes leading to autolysis.     

An improved assay to quantitate the invasiveness of cells in modified Boyden chambers

The most commonly used means of assessing the invasiveness of cultured cells is the Boyden chamber assay, which requires that cells lyse Matrigel™, followed by migration through pores in a filter in response to a chemotactic gradient. This report describes a simple method, which greatly increases the speed and accuracy by which Boyden chamber assays can be analyzed, and permits the concurrent analysis of distinct cell subpopulations within specimens containing multiple-cell types.     

H1 variant synthesis in proliferating and quiescent human cells

The synthesis of histone H1 isoprotein species in human cells of several different types and in several different physiological states was studied. Up to five H1 and two H1 degrees isoprotein species could be resolved by two-dimensional electrophoresis. All five H1 isoprotein species were synthesized in exponentially growing cultures of IMR-90 human fibroblasts; in quiescent IMR-90 cells the synthesis of three H1 isoprotein species was greatly decreased while the synthesis of two others was much less affected. When DNA synthesis in exponentially growing cultures of IMR-90 was inhibited, the pattern of H1 isoprotein synthesis became similar to that found in quiescent cultures. Other human cells, isolated from blood, yielded similar results. These results suggest that the pattern of H1 synthesis is the same for cells in non-S phases of the cell cycle and in quiescent cells. Thus for histone H1 in human cells the relationship of the variant synthesis pattern to the growth state and DNA replication is similar to that of the core histone H3 but not that of H2A.     

Application of computer-assisted image analysis for studying viral structures

The application of CAIE has been shown to be useful for analyzing the structures of RNA viruses. Critical assessment of this method is essential for the selection of the micrographs of viruses. In our experience this procedure was helpful for resolving some questions concerning virus morphology.     

Immunofluorescence localization of Z-DNA in chromosomes: quantitation by scanning microphotometry and computer-assisted image analysis

Anti-Z-DNA polyclonal and monoclonal immunoglobulins raised against left-handed polynucleotides show various degrees of specificity for base sequence and substitution. Class 1 IgGs recognize all Z-DNA with equal affinity; class 2 IgGs show a preference for d(G-C)n sequences and class 3 IgGs for d(G-C)n sequences with substitutions at the C5 position of the pyrimidine. These antibodies served as probes for the localization of Z-DNA in polytene and metaphase chromosomes and in interphase chromatin by indirect immunofluorescence. A quantitative assessment of the binding of anti-Z-DNA IgGs to polytene chromosomes of Chironomus and Drosophila was made by scanning microphotometry and by computer-assisted image analysis of double immunofluorescence and DNA-specific dye fluorescence images. The three classes of antibodies bind to most of the bands in acid fixed polytene chromosomes of C. thummi; however, preferential binding of one class of antibody over another can be observed in certain regions. These differences can be quantitated by arithmetic division or subtraction of the normalized digital images. If a class 2 antibody is first bound at saturating concentrations the binding of class 1 antibody is reduced throughout most bands by 40-50%. However, the telomeres of the three large chromosomes bind greater than 10 times as much class 1 antibody as class 2 antibody, indicating that the Z-DNA tracts in these regions are comprised largely of alternating sequences containing the A X T basepair, e.g., A-C. High-resolution image analysis of class 1 and class 2 immunofluorescence patterns and the total DNA distribution from polytene chromosomes of D. melanogaster show that the two antibody distributions are very similar in a large majority of the bands, but they often deviate from the mean DNA distribution profile. Z-DNA sequences of both G-C and A-C type are detectable at all levels of ploidy from 2n to 2(13)n and in species as diverse as insects and man. We conclude that the vast majority of polytene chromosome bands (genes) contain one or a few DNA sequences with potential for undergoing the B----Z transition and contain both alternating purine-pyrimidine G-C and A-C tracts or mixed sequences. Highly heterochromatic bands and telomeres have more Z potential sequences than do other bands.     

Pattern of collagen synthesis and chemotactic response of fibroblasts derived from mucopolysaccharidosis patients

For comparative studies on the migratory potential we screened fibroblast strains derived from mucopolysaccharidosis (MPS) patients regarding their differential response to chemotactic stimuli and analysed their production of extracellular matrix components. Indirect immunofluorescence staining of MPS-fibroblasts showed the same distribution of type I and type III collagen and of fibronectin as in controls. Biochemical quantification of type I and type III collagen demonstrated an unaltered ratio of these collagen types, although the total amount of newly synthesized collagens was slightly reduced in fibroblasts from MPS patients. Whereas the synthesis of major extracellular matrix components was close to normal, the response of the MPS cells to chemotactic stimuli was greatly affected. Chemotactic migration was improved when fibroblasts were pretreated with medium conditioned by normal fibroblasts, although they never reached normal levels.     

Quantification of exocytosis kinetics by DIC image analysis of cortical lawns

Cortical lawns prepared from sea urchin eggs have offered a robust in vitro system for study of regulated exocytosis and membrane fusion events since their introduction by Vacquier almost 40 years ago (Vacquier in Dev Biol 43:62–74, 1975). Lawns have been imaged by phase contrast, darkfield, differential interference contrast, and electron microscopy. Quantification of exocytosis kinetics has been achieved primarily by light scattering assays. We present simple differential interference contrast image analysis procedures for quantifying the kinetics and extent of exocytosis in cortical lawns using an open vessel that allows rapid solvent equilibration and modification. These preparations maintain the architecture of the original cortices, allow for cytological and immunocytochemical analyses, and permit quantification of variation within and between lawns. When combined, these methods can shed light on factors controlling the rate of secretion in a spatially relevant cellular context. We additionally provide a subroutine for IGOR Pro® that converts raw data from line scans of cortical lawns into kinetic profiles of exocytosis. Rapid image acquisition reveals spatial variations in time of initiation of individual granule fusion events with the plasma membrane not previously reported.     

Induction of proliferating cell nuclear antigen (PCNA) complex formation in quiescent fibroblasts from a xeroderma pigmentosum patient.

Accumulated evidence indicates that proliferating cell nuclear antigen (PCNA) is an auxiliary protein of DNA polymerase delta and forms tight association with DNA replication sites during DNA replication or DNA repair synthesis. In this study, such PCNA complex formation was investigated by the indirect immunofluorescence method, using both normal human fibroblasts and those derived from a xeroderma pigmentosum group A (XP-A) patient. XP-A fibroblasts in both proliferating and quiescent states did not show any differences from normal fibroblasts in the properties of PCNA-staining in the untreated conditions. The PCNA complex formation was induced in quiescent normal fibroblasts by both ultraviolet light (UV)- and X-irradiation, whereas in XP-A fibroblasts it was induced by X-irradiation, but not by UV-irradiation. However, PCNA complex was induced in quiescent XP-A fibroblasts by UV-irradiation when the cells had previously incorporated 5-bromodeoxyuridine (BrdU). These observations indicate a close correlation of PCNA complex formation and unscheduled DNA synthesis (UDS). Thus, it was concluded that PCNA complex formation was commonly induced in at least three conditions to produce UDS in spite of different types of DNA damages and DNA repair mechanisms.     

Quantification of angiogenesis by a computerized image analysis system in renal cell carcinoma

OBJECTIVE: To ascertain whether tumor angiogenesis quantitated by a computerized image analysis system correlates with clinical outcome in renal cell carcinoma. STUDY DESIGN: Microvessels were immunohistochemically labeled with antibodies to CD34 in sections from 62 cases of renal cell carcinoma. Computerized image analysis was used to evaluate the mean microvessel count (MMC) and mean percentage microvessel area (MPMA). RESULTS: MMC ranged from 19.3 to 315.0, while MPMA was 0.6-17.9%. There was a highly significant correlation between MMC and MPMA (r = .867, P < .01). Although MMC and MPMA decreased with increasing nuclear grade and TNM stage, this difference failed to achieve statistical significance. No statistically significant differences in survival were found for MMC or MPMA. CONCLUSION: Our results indicate that computerized image analysis can evaluate accurately tumor angiogenesis, but tumor angiogenesis in renal cell carcinoma does not provide significant prognostic information in renal cell carcinoma.     

High efficiency protein transduction of quiescent and proliferating primary hematopoietic cells

Primary hematopoietic cells are relatively refractory to DNA transfection methodologies. This is particularly so when they are quiescent or terminally differentiated and no longer able to divide. However, whole proteins can be introduced into such cells by protein transduction. We have modified the protein transduction domain (PTD) from the HIV-TAT protein used by other investigators. Using green fluorescent protein (GFP) as a reporter, we show that this new sequence allows more efficient transduction of recombinant fusion protein into a variety of hematopoietic cells tested compared with the native HIV TAT domain. This is true for peripheral blood CD34+ cells, dendritic cells, granulocytes, monocytes and lymphocytes all of which are quiescent or terminally differentiated. Furthermore, we were able to transduce myeloblasts from patients with acute myeloid leukemia (AML). In all cell types tested transduction efficiency was almost 100%. Transduction is maximal 15-30 s after addition of PTD or TAT-GFP fusion proteins as tested on quiescent T lymphocytes. This method will allow us to study of the effects of a variety of gene products in cell types that were previously resistant to gene transfection studies.     

Phosphorylation of nuclear and DNA-binding proteins in proliferating and quiescent mammalian cells.

The dependence of cell proliferation on nuclear protein phosphorylation was studied with exponential-phase and stationary-phase cultures of Chinese-hamster ovary cells. Nuclear proteins were fractionated, according to their DNA-binding affinities, by using sequential extractions of isolated nuclei with increasing concentrations of NaCl. When viable whole cells were labelled with H332PO4, phosphorylation of nuclear proteins was found to be lower in quiescent cells than in proliferating cells. Phosphorylation of nuclear proteins soluble in 0.30M-NaCl (less than 50% of these proteins bind to DNA) was greater than for those proteins soluble in higher salt concentrations (80-100% of these proteins bind to DNA). Cyclic AMP enhanced the phosphorylation of nuclear proteins soluble in 0.3 m-NaCl by 40-50%, and this stimulation was independent of cell growth. Cyclic AMP also increased the phosphorylation of nuclear proteins soluble in 0.6M-NaCl and 2.0M-NaCl by 40-50% in exponential-phase cultures, but not in stationary-phase cultures. Several examples of specific phosphorylation in response to cyclic AMP were observed, including a 35000-mol.wt. protein in the 0.30 M-NaCl-soluble fraction and several proteins larger than 100000 molecular weight within this fraction. A major peptide of molecular weight approx. 31000 extracted with 0.6M-NaCl was also phosphorylated. Its phosphorylation was independent of cyclic AMP in exponential-phase cultures, and it was not phosphorylated in plateau-phase cells. These changes in cell-growth-dependent phosphorylation occurred in the absence of any apparent qualitative changes in the nuclear protein molecular-weight distributions. These data demonstrate that (1) phosphorylation of nuclear proteins is dependent on the culture's proliferative status, (2) both cyclic AMP-dependent and cyclic AMP-independent specific phosphorylation occurs, and (3) the cyclic AMP-dependent growth-independent phosphorylation that occurs does not appear to be a modification of DNA-binding proteins, whereas the cyclic AMP-dependent growth-dependent phosphorylation does involve modification of DNA binding proteins.