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Biomedical subjects

M Kubbies

Publications and source records attributed to M Kubbies.

51 records · Page 3Linked to original sources
PubMed1988

The phytohemagglutinin response of human peripheral blood lymphocytes as a function of donor age: a re-examination using BrdU-Hoechst flow cytometry.

Mononuclear cells were isolated from peripheral blood by a standard Ficoll-Hypaque technique from 127 healthy donors, ranging in age from newborns to 86 years of age. As a measure of their in vitro growth response, the fraction of non-cycling cells was determined at 48 and 72 h after phytohemagglutinin (PHA) exposure by means of BrdU-Hoechst flow cytometry. This technique provides an optimal assay system for the non-cycling cell fraction, since all cycling cells will have incorporated BrdU thereby quenching the fluorescence of the Hoechst 33258 fluorochrome. Lymphocytes from prepubertal donors showed significantly decreased non-cycling cell fractions, as did lymphocytes from an additional group of 14 adults with hypogonadism due to the 45, XO condition (Turner-Syndrome). Much to our surprise, we found no definitive correlation between donor age and the non-cycling fraction of cells from the adult lymphocyte donors. Nor did we find any age-related increase in the variance of the non-cycling cell fraction. These observations suggest that the previously reported age-related decline in the PHA response of human PBL may reflect an increasing delay, rather than an overall diminution, of the PHA response as a function of donor age.

Adolescent↗
PubMed1987

Interphase cell flow cytometry as a means of monitoring genomic size in normal and neoplastoid cell cultures.

The DNA specific fluorescence of mass cultures and clones derived from human skin and bladder tumor tissue was assayed by flow cytometry. In order to detect and quantitate small fluorescence intensity changes, cytogenetically defined triploid or diploid human fibroblast strains were cocultivated, harvested, and stained with the cell strain of unknown karyotype. The triploid standard (derived from human abortus tissue) proved chromosomally unstable at high passage level. Fifteen male, female, and 45,X strains displayed target-to-standard cell fluorescence ratios commensurate with their respective chromosome constitutions. Interstrain variation was highest among the 45,X strains, although mosaicism could not be detected by conventional cytogenetics. Interclonal fluorescence variation was two- to ten-fold higher among the tumor-derived clones tested. Chromosome counts and subcloning experiments indicate that this increased fluorescence variation is due to genome size variation. The clonal evolution of genome size differences was observed in subclones of chromosomally divergent parental clones. These observations suggest that well controlled flow cytometry can adequately resolve subtle degrees of genome size variation in cultivated human cells. The technique is especially suited for monitoring genome size changes in cultivated tumor cells.

Cell Separation↗
PubMed1987

Confirmation of Fanconi's anemia and detection of a chromosomal aberration (1Q12-32 triplication) via BrdU/Hoechst flow cytometry.

Peripheral blood lymphocyte cultures of a patient with clinical evidence of Fanconi's anemia (FA) were investigated by means of BrdU/Hoechst flow cytometry. This technique can be used to differentiate cycling from noncycling cells after PHA stimulation; it also permits the quantitative assessment of cell cycle progression and cell-cycle arrest. A characteristic cell kinetic pattern of combined G2 phase prolongation and G2 phase arrest was observed in the present patient, confirming the clinical diagnosis of FA. An additional finding was the presence of a small subpopulation of peripheral blood cells, refractory to PHA stimulation, and displaying a DNA content 5% greater than that of the diploid cells. BrdU/Hoechst flow cytometry was crucial to the unequivocal demonstration of this subpopulation, which would not have been detected without enrichment by sequential depletion of PHA-responsive cells from the G0/G1 compartment during a 96-h stimulation experiment. The parallel finding of a presumptive triplication affecting the q12-32 segment of chromosome 1 in rare spontaneous divisions of a 40-h peripheral blood culture is consistent with the flow-cytometric detection of elevated DNA content if, as is common in FA, the abnormal cell lineage represents a preleukemic clone of monocytic origin.

Adolescent↗
PubMed1987

Differences in growth factor sensitivity between primary and transformed murine cell cultures revealed by BrdU/Hoechst flow cytometry.

Spontaneous cell transformation is a common feature of all murine cell cultures grown in vitro for extended periods of time. During early passages, these cultures show either progressively reduced growth or complete cessation of growth; after such a 'crisis' they display increasing growth rates and unlimited lifespan. Here we use a novel bromodeoxyuridine/Hoechst flow-cytometric technique to examine the growth response of diploid and transformed cells of the murine species Micromys minutus under a variety of growth conditions. After spontaneous transformation, growth factor exposure results in increased G0/G1 cell recruitment and higher growth rates than shown by the nontransformed diploid cell fraction. Despite clonal heterogeneity, this difference is seen at all fetal calf serum (FCS) concentrations, although it is most pronounced with low serum. Epidermal growth factor and insulin are shown to act synergistically and promote growth equal to exposures of transformed cultures to 10% FCS. The observed differences in growth factor response between diploid and aneuploid cells could explain the reported lack of a classical growth crisis in growth factor-supplemented media during the spontaneous in vitro transformation of primary cell cultures.

Aneuploidy↗
PubMed1986

Flow cytometry reveals a high degree of genomic size variation and mixoploidy in various strains of the acellular slime mold Physarum polycephalum.

High-resolution flow cytometry, using avian erythrocytes as an internal standard, was employed to study constitutive genome size variation of G2-phase nuclei of Physarum polycephalum strains during the macroplasmodial stage of their life cycle. Our results document a previously unknown extent of genome size variation and mixoploidy in this organism. The unimodal diploid strain Tu 291 displayed the largest genome of the strains tested; in contrast, the Colonia strain displayed only half of the Tu 291 G2-phase fluorescence, confirming its haploid nature. An additional strain, derived from a recent cross between Lu897 and Lu898 amoebae, must have arisen by selfing (propagation of only one of the parental genomes to the macroplasmodial stage), since its nuclei display close to the haploid G2-phase DNA content. The observation of a small fraction of corresponding diploid nuclei within the haploid population of this strain, while maintained as microplasmodia, supports the notion that meiosis in haploid strains may require the presence of diploid nuclei. Two of the descendants of the prototype haploid Colonia strain, which were kept for extended periods of time in submerse culture, proved to be near diploid and mixoploid. Polyploidization and subsequent loss of DNA thus seems to contribute to the extremes of genome size variation in Physarum. In addition to unimodal fluorescence distributions, a number of diploid strains displayed bi- and even trimodal distributions within harvests of a single G2-phase macroplasmodium. Analysis of these mixoploid strains by means of gaussian curve-fitting suggests that the smaller genome size differences in Physarum may arise in step-wise diminution of DNA in approximate units of 3-5% of the original Tu 291 genome.

DNA, Fungal↗
PubMed1986

The fate of the primary diploid population during spontaneous transformation of growth factor-supplemented murine cell cultures.

Primary cultures derived from lung and renal tissue of the newborn harvest mouse (Micromys minutus) were serially passaged in media supplemented with epidermal growth factor, hydrocortisone, transferrin, insulin, and triiodothyronine. Although these growth factor supplements eliminated the growth crisis commonly encountered during the initial stages of murine primary cultures, the original diploid cell fraction clearly underwent such a "crisis"; the truly diploid cells invariably disappeared as these cultures reached 20 to 40 population doublings. They were replaced, either gradually or precipitously, by various heteroploid cell fractions. In three of four independent cultures, these "established" cells were hypotetraploid and appeared to be derived from a small number of progenitors already present during the very early (precrisis) culture stages. In contrast to rather frequent DNA changes displayed by clones and subclones derived from the various heteroploid cell lineages, the predominant components of the established mass cultures displayed a highly constant DNA fluorescence pattern. Our results suggest that primary murine cell cultures develop heteroploid cell lineages even if the initial growth crisis is mitigated by growth factor supplements. These heteroploid cells appear to respond more efficiently to stimulation by various growth factors than the primary diploid cell population.

Animals↗
PubMed1985

BrdU-Hoechst flow cytometry reveals regulation of human lymphocyte growth by donor-age-related growth fraction and transition rate.

An improved BrdU-Hoechst flow assay was applied to cell kinetic studies of human lymphocyte cultures during a 24-96 hr interval after PHA stimulation. The assay shows that the duration of the initial lag phase and the proportions of noncycling cells increase as a function of donor age, whereas the rates of transition from each cell cycle compartment to the next decrease. Cell cycle arrest occurs in the first S and G2 phase after stimulation of lymphocytes from a 75-year-old donor but not from younger donors. The data are consistent with several models of cell cycle kinetics, so long as these models are modified to include a fraction of noncycling cells in each cell cycle compartment.

Aging↗
PubMed1985

Flow cytometric correlation between BrdU/Hoechst quench effect and base pair composition in mammalian cell nuclei.

72 h after growth stimulation in BrdU supplemented media Hoechst fluorescence histograms of mammalian euploid cell cultures display up to five distinct peaks which correspond to a consecutive series of cell maturity classes. This pattern is due to differential quenching of the Hoechst fluorescence by BrdU-substituted chromatin. There are constitutive differences in the efficacy of fluorescence quenching among species: Human and bovine cells display more quenching at unifilary as apposed to bifilary substitution. The reverse holds for the AT-rich genome of the murine species Micromys minutus. The theoretical residual fluorescence computed for an infinite number of replications in the presence of BrdU amounts to 14% in case of Micromys cells, but to 19% for human and bovine cells. Flow cytometric estimates of the AT-content, employing base pair specific dyes, yielded 62.0% for Micromys, 60.6% for human and 53.2% for bovine cells. While these figures are in accordance with published data, they do not sufficiently explain the concordant behavior of human and bovine cells, and the discordant behavior of Micromys cells, as regards the quenching of Hoechst fluorescence. Additional factors such as differences in DNA-sequence organisation (eg. base pair specific interspersion pattern, lateral asymmetry) must be invoked.

Animals↗
PubMed1985

Cell cycle kinetics by BrdU-Hoechst flow cytometry: an alternative to the differential metaphase labelling technique.

Human lymphocyte cell cycle kinetics was studied in parallel in whole blood and in isolated lymphocyte cultures by differential metaphase labelling and by flow cytometry, employing the principle of quenching of Hoechst fluorescence by BrdU substituted DNA. The BrdU-Hoechst flow technique yields information on the kinetics of cell recruitment and cell cycle progression superior to the differential metaphase staining, since it provides data from interphase cells, including cycle compartment durations, non-cycling cell fractions and transition probabilities. The Smith and Martin model, modified to include a fraction of non-cycling cells, yields excellent correspondence to the experimental data. We show that lymphocytes isolated from Ficoll gradients respond to PHA stimulation with a 4-6 hr delay compared to whole blood cultures or to cultures with autologous serum supplementation. A detailed study of the effects of such culture supplements on lag phase duration, cell cycle compartment length, non-cycling cell fractions and transition probabilities illustrates the application and reproducibility of the flow assay. The potential of the method is further documented with two examples showing the dependence of lymphocyte proliferation on donor age and donor genotype.

Adult↗
PubMed1985

Endogenous blockage and delay of the chromosome cycle despite normal recruitment and growth phase explain poor proliferation and frequent edomitosis in Fanconi anemia cells.

BrdU-Hoechst flow cytometry was employed to study the proliferation kinetics of blood lymphocytes from patients with Fanconi anemia (FA). Compared to controls, untreated FA lymphocytes show normal response to PHA stimulation, normal G0/G1 exit rates, and normal first S-phase durations. The G2 phase of the first cell cycle, however, is severely prolonged, and 24% of the recruited population become arrested during the first chromosome cycle (S, G2/M phases). The delay suffered during G2 appears to be compensated in part by a subsequent G1 phase duration that is unusually short for postnatal human cells (3.7 +/- 0.5 hrs). In analogy to what has been observed in other cell systems after experimental delays of the chromosome cycle, we therefore postulate that at least some FA cells enter their second growth phase without prior completion of the delayed chromosome cycle. Renewed replication would ensue in such cells without prior passing through mitosis and cytokinesis, leading to endoreduplication, which is a frequent finding in the FA syndrome.

Adolescent↗
PubMed1984

Flow cytometric analysis of small DNA content differences in heterogeneous cell populations: human amniotic fluid cells.

Accurate flow cytometric determination of nuclear DNA fluorescence has been attempted with human amniocentesis materials employing a detergent nuclear isolation protocol and 4,6-diamidino-2-phenylindole (DAPI) staining. Assay of native fluids yielded inadequate fluorescence histograms, whereas analysis of cultured amniocytes yielded unimodal histograms in 80 of 83 samples. Three fluids displayed unexpected bimodality, which may have resulted from the interaction between detergent and chromatin in rare cell types. Using a standardized protocol for preparation, staining, and assay, including brief periods of cocultivation of a human triploid reference standard, the percent experimental error in the flow cytometric DNA content estimate was as low as 0.2% in repeated assays of a single sample, and 0.4% in assays of a given genotype at different occasions. In two series of blindly assayed specimens, the percent standard error of the DNA content estimate ranged from 0.6 to 1.0%.

Amniotic Fluid↗
PubMed1983

Flow cytometric analysis of factors which influence the BrdUrd-Hoechst quenching effect in cultivated human fibroblasts and lymphocytes.

Human fibroblasts and lymphocytes were grown in bromodeoxyuridine (BrdUrd) containing medium, harvested after 27-71 hours and stained with the fluorochrome Hoechst 33258. Parameters that influence the BrdUrd quenching effect of Hoechst fluorescence at a given pH and ionic strength of the staining solution were determined. The parameters were determined in order to obtain high resolution histograms with differentiation of each cell cycle compartment for noncycling cells and for cycling cells in the first and second cycle. The BrdUrd concentration was found to be the most significant determinant, but cell density, cell type, dye concentration, type of Hoechst stain and incubation temperature during staining also contribute to the modulation of fluorescence of BrdUrd substituted nuclei. Optimization of these factors is required in order to achieve complete and well resolved cell cycle histograms by the BrdUrd-Hoechst flow cytometric technique.

Benzimidazoles↗
PubMed1983

Decreased oxygen supply enhances growth in culture of human mid-trimester amniotic fluid cells.

Human mid-trimester amniotic fluid cells were cultivated under conditions of decreased oxygen supply. Compared to control cultures the low-oxygen group showed improved growth which was quantitated by three independent assays (1) direct cell counts, (2) bromodeoxyuridine (BrdU)-Hoechst flow-cytometry, and (3) cloning efficiency. The growth promoting effects of lowered oxygen hold for all major morphologic categories of amniotic fluid cells.

Amniotic Fluid↗
PubMed1983

Mitotic cell cycle control in Physarum. Unprecedented insights via flow-cytometry.

High resolution flow-cytometric studies of isolated macroplasmodial nuclei of the myxomycete. Physarum polycephalum provide definite evidence for the persistence of natural synchrony at mitosis throughout the entire cell cycle, i.e. completely synchronous DNA replication and traverse of G2. Even if nuclei within a given macroplasmodium belong to two distinct genome size classes (mixoploidy), they cycle and traverse mitosis in strict synchrony. This cannot be explained by current models of regulation of division based solely upon nuclear size and/or nuclear/cytoplasm ratios. Constitutional DNA content variation was apparent among all tested strains, and loss of late-replicating, presumably AT-rich DNA accounts for this variation. A constant duration of the S phase is maintained, irrespective of DNA content, via differential slowdown of replication rates during the 2nd and 3rd hours of replication. A frequently described extension of nuclear replication into G2 could not be substantiated. Interference with DNA and protein synthesis provides the first evidence for a brief "G1 phase" equivalent of 3-4 min duration in asynchronous microplasmodial cultures, and temporally assigns a protein synthesis-dependent "transition point" for completion of mitosis and initiation of DNA synthesis at 5 min prior to actual division nuclei which have passed this point at the time of addition of cycloheximide replicate 5% of their DNA before they become arrested. These findings provide strong experimental support for the transition point concept of cell cycle control, and additionally are commensurate with some form of the replicon-set hypothesis in Physarum.

Cell Nucleus↗