Br-cAMP induction of apoptosis in synchronized CHO cells.

The proliferation of suspension cultures of malignant CHO cells was inhibited by 0.5 mM Br-cAMP treatment and restored by its removal. This treatment also inhibited histone H1 phosphorylation completely, reduced histones H2A and H4 phosphorylations, induced DNA degradation, and produced cells containing micronuclei. Agarose gel electrophoresis of the degraded DNA fragments produced a "ladder" pattern confirming these cells were undergoing apoptosis. Cell cycle synchrony experiments demonstrated culture growth inhibition was the result of two different cell cycle-specific processes: [1] arrested cell cycle traverse at a restriction point in mid-G1, and [2] rapid apoptosis following cell division. Br-cAMP did not stop cells in late-G1, S, G2, or M from traversing the cell cycle and dividing, but rather, induced apoptosis following mitosis. The restriction point of Br-cAMP arrest was located in the middle of a wider band of G1 arrest induced by isoleucine deprivation. The cells synchronized in G1 before the restriction point were held in G1-arrest by Br-cAMP and spared apoptotic death. These studies support the further study of cAMP derivatives as agents to induce tumor regression by apoptosis and reverse transformation.

Morphometric comparisons of rat alveolar macrophages, pulmonary interstitial macrophages, and blood monocytes.

Pulmonary interstitial macrophages (IM) account for a substantial fraction of the total pulmonary macrophage (PM) population in the mammalian lung, with the remaining balance of extravascular mononuclear phagocytes being mainly alveolar macrophages (AM). Unlike the AM that can be harvested readily by bronchoalveolar lavage, the lung's IM subpopulation of PM has been characterized less well, primarily because of its relative inaccessibility. Recently we developed a method to isolate viable IM from rat lungs using an Fc gamma receptor affinity technique in conjunction with multiparameter flow cytometry. Using this approach, we undertook the present investigation to characterize quantitatively the structural features of the IM and to compare the morphologic attributes of this subpopulation of PM to those of flow cytometrically sorted AM and blood monocytes (BM). Measured or calculated parameters for each population included mean cellular equivalent circular diameter, cell area and volume, and nuclear, mitochondrial, cytoplasmic, and lysosomal volume densities in each cell type. Lysosomal volume densities were subcategorized further into primary lysosomes, small secondary lysosomes, large secondary lysosomes, lipid droplets, and vacuoles. Additionally, the shape, form, and surface irregularity of the cells and various subcellular components were determined. Comparisons of the size and other structural features of the AM, IM, and BM have indicated that (1) the morphologic phenotypes of these three populations of mononuclear phagocytes distinctly differ from one another, (2) the IM and BM are morphologically and morphometrically more akin to one another than they are to AM, and (3) the IM are more similar to the AM than are the BM. These findings suggest that the IM may represent a transitional stage of maturation between BM and AM. Our findings, however, do not rule out the possibility that at least some of the lung's IM are a discrete, BM-independent population of macrophages.

Airway intra-luminal macrophages: evidence of origin and comparisons to alveolar macrophages.

Airway intra-luminal macrophages (AI-LM) are a little-studied subpopulation of pulmonary macrophages that are located on the surfaces of the conducting airways of the lower respiratory tract. In this study, we: (1) developed a flow cytometric approach by which AI-LM can be viably isolated in high purity from cell suspensions obtained by airway washings; (2) comparatively examined various functional, biochemical, biophysical, and morphologic features of the rat's AI-LM and alveolar macrophage (AM) phenotypes, and (3) investigated the origin of the AI-LM in the rat. Airway cells were harvested from the tracheas of adult Fischer-344 rats, and AM were obtained from the lungs by conventional bronchopulmonary lavage or via prosthetic airway circuits that supplanted the removed tracheas. Flow cytometric analyses of lavaged airway cells revealed that the AI-LM fell within the range of the electro-optical phenotype of AM, and subsequent cell-sorting experiments demonstrated that virtually all viable AI-LM could be sorted from contaminating airway epithelial cells in greater than 95% purity based on their electro-optical characteristics, e.g., electronic volume, axial light loss, 90 degrees light scatter, and blue and green autofluorescence signals. In Fc gamma receptor-mediated phagocytic studies, approximately 90% of AM engulfed opsonized erythrocytes (EIgG) whereas only 60% of the AI-LM were able to do so. Comparisons of the numbers of EIgG in phagocytic AM and in phagocytic AI-LM indicated the AI-LM were less phagocytic. Densitometric analyses of sorted AI-LM and of sorted AM stained for acid phosphatase, nonspecific esterase, and beta-glucuronidase indicated that the activities of these enzymes were generally less in the AI-LM than in the AM. Morphometric comparisons of sorted AM and of sorted AI-LM showed that the AI-LM were generally larger than the AM and that the surfaces and nuclei of the AI-LM were more regular than those of the AM. The AI-LM were found to strongly label with the monoclonal antibody ED1, which recognizes an antigen on the surfaces of rat AM, but the AI-LM did not label with the monoclonal antibody ED2, which recognizes an antigen on the surfaces of rat peribronchial and pulmonary perivascular macrophages. Over the course of alveolar phase clearance of a lung burden of polystyrene microspheres, the frequency distributions of the particles in AI-LM and in AM were found to be virtually identical.(ABSTRACT TRUNCATED AT 400 WORDS)

Migratory behaviors of alveolar macrophages during the alveolar clearance of light to heavy burdens of particles.

We investigated the unstimulated and stimulated migratory activities of lavaged alveolar macrophages (AMs) in vitro over the course of alveolar clearance of three different mass lung burdens of microspheres. Our intent was to uncover potentially important relationships between the migratory behaviors of the AM and the retention kinetics of particles. Groups of adult, male Fischer-344 rats were intratracheally instilled with approximately 86 micrograms (low burden, LB), approximately 1 mg (medium burden, MB), or approximately 3.7 mg (high burden, HB) of polystyrene microspheres (2.13 microns diameter), or with carrier vehicle (phosphate buffered saline, PBS) alone. The lung retention kinetics of the particles were determined over an approximately 170 day period. On days 14, approximately 57, and approximately 85, lavaged AMs were assessed for their abilities to migrate through 5-microns pore membranes in response to inactivated rat serum (unstimulated condition) and yeast-activated rat serum (stimulated condition). The retention characteristics of the three burdens could be satisfactorily described by two-component, negative exponential equations. The kinetics of retention of the LB and MB were similar, although some evidence indicated the MB slightly retarded the lung clearance process. Deposition of the HB resulted in more marked prolongations of both the early, more rapid, and the slower, longer term components of alveolar clearance. The unstimulated migration indices of AMs from the particle-instilled lungs were generally not significantly different from those of AMs from PBS-instilled lungs except for a significant increase in the migration indices of LB AMs at the last assay time. The stimulated migration indices of MB and HB AMs were significantly decreased on assay days 14 and approximately 57. On day approximately 85, however, the migration indices of LB, MB, and HB AMs were all increased above the PBS AMs. Comparisons of the frequency distributions of particles in the unstimulated and stimulated AM that migrated to those in corresponding parent AM populations consistently indicated a preferential migration of particle-free AMs and of AMs with lesser loads of microspheres. The overall results of this study suggest that the unstimulated and stimulated migratory activities of particle-laden AMs are depressed in vitro. Our results also suggest that the migratory activities of generally particle-free AMs may be enhanced over a prolonged period of time following the deposition of particles in the lung.

Radiobiology of ultrasoft X rays. III. Normal human fibroblasts and the significance of terminal track structure in cell inactivation.

Ultrasoft characteristic X rays from carbon (0.28 keV) are severely attenuated as they pass through biological material, causing a nonuniform distribution of dose to cell nuclei. Complications of studying ultrasoft X rays can be minimized in this context by using cells with very thin cytoplasm and nuclei (e.g., less than the attenuation length of the X rays), and which exhibit a more nearly exponential dose response to cell killing, such as normal human fibroblasts compared with V79 cells. Using this cell system, we report the relative biological effectiveness (RBE) of A1-K and C-K X rays to be near unity. Previous studies of cell inactivation by characteristic carbon X rays gave RBEs of 3 to 4, supporting the idea that localized energy depositions from secondary electrons and primary track ends represent the principal mode of biological action for other low-LET radiations. In part, the reported high RBEs result from the use of mean dose to describe energy deposited within the cell nuclei by these poorly penetrating radiations. Implicit in the use of mean dose is that cellular damage varies linearly with dose within a critical target(s), an assumption that is of questionable validity for cells that exhibit pronounced curvilinear dose responses. The simplest interpretation of the present findings is that most energy depositions caused by track-end effects are not necessarily more damaging than the sparsely ionizing component.

Quantitative cytochemistry of 3 beta-hydroxysteroid dehydrogenase activity in avian granulosa cells during follicular maturation.

Previous studies have shown that biosynthesis of progesterone, the major steroid product of hen granulosa cells, increases during follicular maturation. However, the contribution of individual granulosa cells to the total progesterone production of each follicle is not known. The objective of the present study was to determine the presence and relative activity of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) in individual granulosa cells isolated from each of the five largest yolk-filled preovulatory follicles of laying hens. 3 beta-HSD cytochemistry in the presence or absence of pregnenolone substrate was performed on digitonin-permeabilized granulosa cells in suspension. The stained cells were fixed in a 70% ethanol solution until 1) the percentage of cells from each follicle that stained dark blue-indicating the presence of 3 beta-HSD activity-was determined by counting under light microscopy, and 2) the intensity of staining-indicating the relative amount of enzyme activity-was quantified using video image analysis. There were three findings. First, 100% of granulosa cells from each of the five largest preovulatory follicles stained positively for the presence of 3 beta-HSD activity. Second, the amount of 3 beta-HSD activity was normally distributed among granulosa cells in the population from each follicle. Third, as follicles matured from the fifth largest to the largest follicle, 3 beta-HSD activity increased steadily in individual cells, as indicated by increased staining intensities. The results indicate uniformity in the steroidogenic capacity of cells in the granulosa layer of hen preovulatory follicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Radiobiology of ultrasoft X rays. II. Cultured C3H mouse cells (10T1/2).

In the first paper of this series (Radiat. Res. 110, 396-412 (1987], using V79 cells, we reported that the relative biological effectiveness (RBE) of ultrasoft X rays was found to increase with decreasing energy, and the oxygen enhancement ratio (OER) was found to decrease with decreasing energy. In this report, we present RBE and OER results for 10T1/2 cells that are known to grow uniformly flat and are considerably thinner than V79 cells. Thus the variation in dose across the cell nucleus is considerably reduced. The OER results agree well with our earlier V79 results. However, the RBE values for 10T1/2 cells compared to V79 cells are systematically less for all soft X rays and especially for 0.28 keV carbon-K (1.3 compared to 3.4 for V79 cells). Some plausible explanations are presented to reconcile the apparent discrepancy between V79 and 10T1/2 results.

Transmission electron microscopy of small numbers of sorted cells.

A new method that allows the transmission electron microscopic examination of as few as 1 x 10(4) cells obtained by flow cytometric sorting is described. The approach involves "sandwiching" fixed cells in an agarose case by a microcentrifugation system consisting of small-diameter cell-centrifugation tubes and subsequent processing of the cells by conventional techniques. The advantages offered by this method are discussed.

Radiobiology of ultrasoft X rays. I. Cultured hamster cells (V79).

Ultrasoft X rays (approximately less than keV) provide a useful probe for the study of the physical parameters associated with the induction of biological lesions because the spatial scale of their energy depositions is of nanometer dimensions, comparable to that of critical structures within the cell. We report on cell-killing experiments using cultured hamster cells (V79) exposed to carbon K (0.28 keV), aluminum K (1.5 keV), copper K (8.0 keV), and 250 kVp X rays, under oxic and hypoxic conditions, and as a function of cell-cycle phase. Our principal results are: RBE increases with decreasing X-ray energy; OER decreases with decreasing X-ray energy; and cell-cycle response is similar for all X-ray energies. Our RBE results confirm earlier observations using ultrasoft X rays on mammalian cells. The shapes of fitted curves through the data for each energy are statistically indistinguishable from one another, implying that the enhanced effectiveness is purely dose modifying. The results reported herein generally support the view that single-track effects of radiation are predominantly due to very local energy depositions on the nanometer scale, which are principally responsible for observed radiobiological effects.

Preparation of viable single cell suspensions of tracheal epithelial cells.

This paper reports a procedure used for isolating the entire epithelial lining of the rat trachea. Isolated trachea was initially filled with 0.2% hyaluronidase and incubated at 37 degrees C for 30 min. Tracheas were flushed with medium and then reinflated with 0.5 microgram/ml cytochalasin B and re-incubated for 60 min. The tracheal lumens were again flushed and reinstilled with 24 iu/ml pronase and incubated for a further 30 min. The tracheas were flushed again and the cells removed enumerated and viability assessed by trypan blue dye exclusion. Cell yields (X 10(6)) from 30 consecutive Fischer 344 rats were 5.06 +/- 0.16 (s.e.m.) and the mean percentage of viable cells was 83.13 +/- 1.10 (s.e.m.). This cell yield was close to the estimated tracheal cell population (5.3 X 10(6)). The suspensions were predominantly single cells which apparently retained a normal ultrastructural appearance.

Separation of underivatized gangliosides by ion exchange high performance liquid chromatography.

A procedure is described which separates neutral glycolipids from gangliosides, and which separates the gangliosides into classes, based on their number of sialic acid residues. In addition to separation into mono-, di- and trisialoganglioside classes, there is purification of individual disialoganglioside species. The procedure uses a commercially available--NH2 high performance liquid chromatography (HPLC) column, which normally is used as an adsorption column. In this method the column is modified by protonation, to pH 5.4, so that it exhibits ion exchange as well as adsorption properties. Sensitive, nondestructive detection of eluent peaks is accomplished by monitoring continuously at 210 nm, a wavelength near which underivatized glycosphingolipids have absorbance maxima and which also will clearly detect most possible contaminants. An entire run, including re-equilibration of the column, takes two hours.

Preparation of histone variants and high-mobility group proteins by reversed-phase high-performance liquid chromatography.

Methods have been developed for the preparation of histone variants and high-mobility group (HMG) proteins by high-performance liquid chromatography (HPLC). The individual HPLC fractions were recovered as a dry powder in 95% yield by direct lyophilization from the column effluent. Perchloric acid-soluble H1 variants and HMG proteins from Chinese hamster cells (line CHO) were separated on a mu Bondapak CN column using a 0-50% linear acetonitrile gradient in water containing 0.2% trifluoroacetic acid (TFA). The proteins were eluted in the following order: HMG-E/G (an HMG-14/17 class proteins from CHO cells), HlO, Hl, HMG-2, and HMG-l. HMG-E/G, Hl, and an unidentified protein were recovered electrophoretically pure. HlO contained contaminants which could be removed by subsequent chromatography on a mu Bondapak C18 Radial-Pak column, but HMG-l and HMG-2 could not be completely resolved. Nucleosomal core histones were fractionated on a mu Bondapak C18 Radial-Pak column using a 30-55% linear acetonitrile gradient containing 0.2-0.3% TFA. They were eluted in the following order: H2B, (LHP)H2A, (MHP)H2A, H4, LHP(H3), and (MHP)H3, (where LHP and MHP refer to less-hydrophobic and more-hydrophobic variants). If the gradient containing 0.3% TFA was interrupted with an isocratic elution at 43% acetonitrile, the H2B, (LHP)H2A, (MHP)H2A, and H4 proteins were completely resolved, thus providing a good preparative method for these proteins. The H2A class of Drosophila histones was also fractionated on a mu Bondapak C18 Radial-Pak column using a 30-35% linear acetonitrile gradient containing 0.2% TFA. Drosophila melanogaster H2A, obtained as a single fraction by chromatography on Biol-Gel P-100, was eluted from the C18 column as three proteins. The order of elution was identified by electrophoresis to be: H2Aox (an oxidized form of H2A), D2 (a Drosophila-specific subtype), and H2A.