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Biomedical subjects

H Hoehn

Publications and source records attributed to H Hoehn.

At least 73 records · Page 4Linked to original sources
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

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

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

Clonal analysis of stable chromosome rearrangements in Bloom's syndrome fibroblasts.

In situ cytogenetic analysis was performed on colonies derived from single cells of cultured skin fibroblast-like strains from two patients with Bloom's syndrome (GM 1492 and GM 2520). Metaphases in all of the colonies displayed structural chromosome rearrangements. Among 212 metaphases from 24 colonies of GM 1492, only 16% were pseudodiploid, and there was a high incidence of de novo rearrangements within individual colonies. There were two "families" of 16 and five colonies, respectively, each containing identical or related aneusomies, and these could be arranged into pedigrees showing clonal evolution. The heterochromatic region of chromosome #1 and the telomeric regions of chromosome arms 2q, 3q, 4p, and 11q were most frequently involved in the rearrangements. In contrast, strain GM 2520 showed less intraclonal variation, was primarily pseudodiploid, and displayed only three clonal types, one of which had extensive subclonal variation (19 of 24 clones). A remarkable finding in GM 2520 was that, in some clones, extra copies of specific chromosome segments were present as translocations. These results caution against the use of strain GM 1492 as a prototype Bloom's syndrome strain for cell biological studies.

Bloom Syndrome↗
PubMed1983

Fragile X expression studied by clonal analysis and somatic cell hybridization.

The expression of the fragile site on the X chromosome was examined in euploid somatic cell hybrids to determine if a normal genome could complement the abnormal genome and suppress fragile X expression. Fibroblasts from the patient showed the fragile X in 8% to 12% of cells, and fibroblasts from the normal individual showed no fragile X expression. Six fibroblast clones from the patient showed a variable frequency of fragile X expression (from 6.6% to 12%), suggesting that the fragile X is not expressed in a clonal fashion. Three clones from the normal individual did not show the fragile X. Four hybrid clones showed the fragile X in 5.6%, 4.0%, 7.0%, and 4.0% of cells, respectively, indicating that fragile X expression is not completely suppressed by the normal genome.

Cells, Cultured↗
PubMed1982

Cultivated cells from mid-trimester amniotic fluids: IV. Cell type indentification via one and two-dimensional electrophoresis of clonal whole cell homogenates.

Clones of cultivated amniotic fluid cells that have distinct morphologic and growth characteristics (F, AF and E-type) were examined by one-dimensional SDS polyacrylamide gel electrophoresis (SDS-PAGE) and by two-dimensional electrophoresis employing isoelectric focusing and SDS-PAGE (IEF-PAGE). No qualitative differences in band pattern were observed in SDS-PAGE between the various amniotic fluid cell types, but consistent quantitative differences in the ratios of four bands of presumed filamentous proteins provided good distinction between amniotic fluid cells and postnatal skin fibroblast-like cells. By adding separation on the basis of electrical charge to that of molecular size (IEF-PAGE), we observed reproducible qualitative differences in the protein spot patterns between F and both AF and E-type amniotic fluid cells. At least eight discrete proteins appear not be synthesized by prenatal F-type cells in comparison with their isogenic AF and E counterparts under identical culture conditions. The two-dimensional electrophoretic patterns thus confirm that F and AF amniotic cells, in spite of their morphologic and growth kinetic similarities, are developmentally distinct cell types that retain their differentiated states in culture.

Amniotic Fluid↗
PubMed1982

Interphase flow-cytogenetics: correlation of DNA fluorescence to aneuploidy in human fibroblast cultures.

Improvements in the accuracy of DNA content determination by flow cytometry might allow the detection of aneuploidy in interphase cells to serve as an adjunct to conventional cytogenetics. We compared alternate methods of staining and fluorescence standardization in analyses of human diploid and constitutionally aneuploid cultured fibroblast-like cells. Optimum results were obtained using the dye DAPI (4,6-diamidino-2-phenylindole) and an avian red blood cell standard. The standard error of the estimate of DNA content by this technique was 0.7%-0.96%, approaching more closely than previously the range necessary for reliable discrimination of numerical chromosomal aberrations.

Aneuploidy↗
PubMed1981

Effects of radical-scavenging enzymes and reduced oxygen exposure on growth and chromosome abnormalities of Werner syndrome cultured skin fibroblasts.

Two sister strains of skin fibroblast-like (FL) cells from a patient with Werner's syndrome (adult progeria) were grown in regular tissue culture medium or medium supplemented with the radical-scavenging enzymes superoxide dismutase and catalase. Differences in proliferative potential were shown to be due to strain variation rather than to treatment with the enzymes. Two independent strains were also grown in ambient (18%) or reduced (1%) concentrations of oxygen. All cultures (untreated, treated with enzymes, treated with reduced oxygen exposure) displayed the limited in vitro growth potential and cytogenetic abnormality characteristic of Werner's syndrome FL cells; thus the underlying defect in Werner's syndrome does not appear to be related to defective protection against or repair of damage by free radicals.

Catalase↗
PubMed1981

Systematic growth studies, cocultivation, and cell hybridization studies of Werner syndrome cultured skin fibroblasts.

The growth of 20 independently derived skin fibroblastlike (FL) cell strains from three individuals with Werner syndrome (adult progeria) was compared with the growth of ten FL cell strains from normal individuals. Population growth rates and total replicative life spans of Werner syndrome strains averaged 53% and 27%, respectively, of the growth rates and life spans of non-Werner strains. In the first few passages, four Werner syndrome strains demonstrated population growth rates in the low normal range, but the longest-lived Werner syndrome strain had only 75% of the total replicative potential of the shortest-lived normal strain. Exponential growth rates, cloning efficiencies, and saturation densities of Werner strains were also reduced, whereas cell attachment was normal. Viable cells (identified by dye exclusion) were maintained in post-replicative Werner syndrome and control cultures for periods of at least 10 months; there was no evidence of accelerated post-replicative senescence of cell death of Werner syndrome FL cells. Cocultivation of Werner syndrome and normal strains did not influence population growth rates of either strain. Two proliferating hybrid clones were obtained from fusions of normal and Werner syndrome FL cell strains and these hybrids displayed the reduced growth potential typical of Werner syndrome FL cells. These studies confirm that low growth rate and sharply reduced replicative life span are characteristic of cultured skin FL cells from patients with Werner syndrome, and they suggest that these characteristics are not affected by complementation with non-Werner FL cells.

Cell Division↗
PubMed1981

Flow-cytogenetics. II. High-resolution ploidy measurements in human fibroblast cultures.

high-resolution flow-cytometric measurements of ethidium bromide/mithramycin-stained human fibroblast cultures reveal discrepancies between fluorescence pulse-height distributions and genome size as a function of cell strain, culture conditions, culture age, and proliferative stage. These discrepancies were quantitatively assessed by (1) evaluation of the variation of the widths of the 2c fluorescence pulse-height histogram peaks, (2) comparisons between sample to standard fluorescence ratios of 45,XO and 49,XXXXY cell strains, and (3) comparison of fluorescence intensities among cell populations of identical genome size but differing degrees of chromatin condensation (mitotic vs. nonmitotic cells of diploid and tetraploid cell strains). As in our previous studies with lymphocytes, our results suggest caution in equating fluorescence intensity with "DNA content" in flow measurements of nonhomogeneous cell populations. Conditions of cell culture and sample preparation must be standardized in order to compare "DNA content" differences by flow techniques. Remaining sources of variation presently limit detection to differences in DNA content of greater than 2%.

Cell Line↗