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Biomedical subjects

Z Darzynkiewicz

Publications and source records attributed to Z Darzynkiewicz.

At least 307 records · Page 17Linked to original sources
PubMed1980

Effects of ellipticine on cell survival and cell cycle progression in cultured mammalian cells.

The effects of ellipticine [5,11-dimethyl-6H-pyrido(4,3-b)carbazole; NSC 71795] on cell viability, growth, and colony formation were investigated in suspension (Friend leukemia and L1210) and adherent [Chinese hamster ovary (CHO)]tumor cell systems as well as in mitogen-stimulated human peripheral blood lymphocyte cultures. Cell cycle progression and the terminal point of action of the drug were monitored by flow cytometry. Ellipticine was cytostatic for all cell lines tested, blocking cells in G2 phase following 24 hr constant exposure at concentrations in the range of 1.0 microgram/ml. A 10 times higher drug concentration was required to block cells in G2 if the cells were exposed for only 30 min to the drug followed by 23.5 hr culture in drug-free medium. Formation of CHO cell colonies was inhibited by 50% following exposure to ellipticine for 2 hr at 6.0 microgram/ml or for 24 hr at 0.3 microgram/ml. Fifty % cell kill in asynchronously growing Friend leukemia and L1210 cells was obtained following exposure to ellipticine for 24 hr at 2.0 microgram/ml and 1.15 microgram/ml, respectively, whereas human peripheral blood lymphocytes required 66 hr exposure to 1.0 microgram/ml to kill 50% of the cells. Phytohemagglutinin-stimulated lymphocytes were remarkably resistant to the cytotoxic effect of ellipticine but did display a dose-dependent inhibition of stimulation and accumulation in G2 whether the drug was added prior to our during active cell proliferation. Ellipticine, at cytostatic concentrations, had a marked effect on cellular RNA content. Friend leukemia cells, blocked in G2 by the drug, doubled their RNA content compared to control cells. L1210 and CHO cells, but not lymphocytes, also increased in RNA content following ellipticine treatment. Drug concentrations which blocked cells in G2 also led in the case of Friend leukemia and L1210 but not CHO cells to an increase in the proportion of cells with greater than 4C amounts of DNA.

Alkaloids↗
PubMed1980

Increase in cellular RNA in cells infected with DNA oncogenic viruses.

The amount of RNA per cell has been measured by flow microfluorometry in cultured cells stimulated by serum or infected with DNA oncogenic viruses (SV40, polyoma virus, and adenovirus). While all four agents stimulate, although to a different extent, cellular DNA synthesis in quiescent cells, the accumulation of cellular RNA varies. Serum, SV40, and polyoma virus cause a marked increase in total cellular RNA that is already apparent in G1 cells before entry into S. On the other hand, adenovirus 2, while capable of stimulating cellular DNA synthesis, fails to detectably increase the amount of RNA per cell. These results suggest that adenovirus 2 may act on quiescent cells through mechanisms different from those of serum, SV40, or polyoma virus.

Acridines↗
PubMed1980

Action of dihydroxyanthraquinone on cell cycle progression and survival of a variety of cultured mammalian cells.

Dihydroxyanthraquinone, 1,4-dihydroxy-5,8-bis(( (2-[(2-hydroxyethyl)amino]ethyl)amino))-9,10-anthracenedione (NSC 279836), was observed to alter the cell cycle kinetics of a variety of mammalian cell lines as monitored by flow cytometry. Continuous exposure of Friend leukemia, L1210, and Chinese hamster cells to the drug in vitro at concentrations of 1.0 to 10 ng/ml resulted in the accumulation of cells in G2 by 24 hr in culture. When cells were exposed to dihydroxyanthraquinone for 30 min, washed free of drug, and cultured in fresh medium for 24 hr, a 10 times higher drug concentration was required to produce a G2 block identical to that observed during continuous exposure. Stimulation of human lymphocytes by phytohemagglutinin could be inhibited in a dose-dependent manner by brief pretreatment of cells with the drug. However, while previously stimulated but as yet noncycling lymphocytes were profoundly affected by much lower concentrations, proliferating lymphocytes were refractory to treatment with the drug up to a concentration of 1 microgram/ml. Exposure to the drug for 24 hr, at concentrations as low as 3.2 ng/ml, inhibited colony formation of exponentially growing Chinese hamster cells by 50%, whereas stationary culture required an 8-fold higher concentration to produce the same results. Drug concentrations in the range of 0.3 to 0.8 micrograms/ml over a period of 24 hr reduced the viability of Friend leukemia and L1210 cells by 50% as measured by trypan blue dye exclusion. In contrast, human lymphocyte viability was only mildly affected following 24 hr incubation with up to 5.0 micrograms dihydroxyanthraquinone per ml. There was a marked effect on cellular RNA content in two of the cell lines tested. Friend leukemia and L1210 cells blocked in G2 by the drug manifested a 140 and 70% increase in RNA content, respectively, when compared to control cells. In addition, though suboptimal concentrations of the drug resulted in a transient accumulation of cells in G2, optimal drug concentrations not only blocked cells in G2 but in the case of Friend leukemia and L1210 cells led to an increase in the proportion of cells with greater than 4C amounts of DNA. The results obtained with dihydroxyanthraquinone were compared to those obtained previously with a nonhydroxylated analog, anthracenedione (NCS 287513).

Animals↗
PubMed1980

Discrimination of human leukemia subtypes by flow cytometric analysis of cellular DNA and RNA.

A newly developed flow cytometry technique for simultaneous measurements of three features of individual cells--DNA, RNA, and nuclear diameter--using acridine orange as a fluorescent metachromatic dye, has been applied to cell-cycle analysis. DNA stemline determination, and to classification of 102 cases of human leukemias in adults. Acute lymphoblastic leukemia (L1-2) was characterized by moderately increased RNA of G0/1 cells as compared to normal lymphocytes; acute nonlymphoblastic leukemia (M 1-5) by very high RNA of G0/G1 cells. Both had either diploid or aneuploid DNA stemlines. Chronic lymphocytic leukemia showed diploid DNA, very low proliferation, and low RNA, similar to that found by use to be typical for normal B cells. In chronic myelogenous leukemia, two cell populations were distinguished, one with high RNA, the other with very low RNA and elongated nuclear diameter due to stripped, unfolded nuclei of polymorphonuclear leukocytes. The number of leukemic blast cells, identified by aneuploid DNA values, correlates well with conventional microscopy counts and could be followed during the course of treatment. Thus, acridine orange flow cytometry can be used to discriminate subtypes of human leukemias, to determine cell cycle stages, and to detect and monitor aneuploid leukemia stemlines.

Blood Cell Count↗
PubMed1980

Effects of 9,10-anthracenedione, 1,4-bis[[2-[(2-hydroxyethyl)amino]-ethyl]amino]-, diacetate on cell morphology and nucleic acids of friend leukemia cells.

Treatment of Friend leukemia cells for 18 hours with 9,10-anthracenedione, 1,4-bis[[(2-hydroxyethyl)amino]ethyl]amino]-, diacetate (ANT) at concentrations up to 1.0 microgram/ml induced significant changes in cell metabolism and structure. Alterations in cell nucleic acid content were detected in cells stained with acridine orange under conditions such that DNA and RNA contents could be measured simultaneously by flow cytometry. Cells treated for 18 hours with ANT at concentrations of 0.05-0.1 microgram/ml became partially blocked at the G2 phase. In addition, about 30% of the cells became polyploid and demonstrated diplochromosomes at the 8C level of mitosis. The nuclear chromatin of blocked cells had an altered structure as reflected by a change in sensitivity of DNA in situ to denaturation induced by low pH. All viable cells treated with ANT for 18 hours at concentrations of 0.4-1.0 microgram/ml were blocked in G2 phase. These cells had significantly more RNA than did untreated cells. Transmission electron microscopic observations of thin-sectioned cells suggested that this increased RNA content in ANT-treated cells was mostly due to an approximately 50% increased cell diameter and partly due to a disproportionate increase in nucleolar size. In addition, electron microscopy revealed that ANT caused increased chromatin condensation and granulation. The drug had no apparent effect on production of the endogenous Friend murine leukemia virus.

Animals↗
PubMed1980

Effects of prospidine on survival and growth of mammalian cells in culture.

The effects of prospidine, chemically known as 3, 12-diaza-6,9 diazoniadispiro[5.2.5.2]hexadecane,3,12-bis(3-chloro-2-hydroxypropyl)-dichloride (NSC-166100), on cell viability, growth, and colony formation were investigated in several mammalian cell lines. Cell cycle progression and the terminal point of action of the drug were monitored by flow cytometry. Prospidine was cytostatic for two suspension cultures (Friend leukemia and L1210 cells) at a concentration of 10 mg/ml during the first cell cycle after exposure to the drug. Cells were blocked in G2 at lower concentrations of prospidine (e.g., 1.0 mg/ml) but only after 12--24 hours of continuous exposure, i.e., during the second cell cycle in the presence of drug. Cells could be observed to accumulate in G2 by 24 hours even if prospidine (0.1 mg/ml) was removed after 12 hours. A short incubation with a liver cytosol fraction (30 min) or with cultured cells (12 hr) failed to enhance the potency of the drug. Formation of colonies of the adherent Chinese hamster ovary cell line was inhibited by 50% following 24-hour exposure to 1.1 mg prospidine/ml. Under culture conditions in which cells were blocked in G2, their RNA content increased only slightly, but the incorporation of [5-(3)H]uridine into RNA was suppressed by 15--20%. Incubation of cells with prospidine increased the stability of DNA in situ to acid-induced denaturation, which thus suggested that the drug may interact with cellular DNA.

Animals↗
PubMed1980

Ellipticine-induced changes in cell growth and nuclear morphology.

Twenty-four-hour exposure of Friend leukemia (FL) and L1210 cells to 1.0 micrograms ellipticine/ml resulted in a slowdown in cell proliferation, accumulation of cells in G2 phase, an increase in cellular RNA content, and an increase in the proportion of cells with greater than 4C (tetraploid) content of DNA in both cell lines. Removal of the drug and subsequent cell culturing in fresh medium for 24 hours led to a further increase in the proportion of cells having more than a 4C content of DNA, with no change in cell number, in FL cell cultures. Distinct micronucleation and an increase in cell size without any sign of cell division were seen in FL cells so treated. In contrast, L1210 cells after transfer to fresh medium reentered the normal cell cycle, continued to proliferate, and demonstrated only a slight increase in cells with greater than a 4C content of DNA within 24 hours of removal of the drug.

Alkaloids↗
PubMed1979

Subpopulations of human peripheral blood cells: analysis of granulocytic progenitor cells by flow cytometry and immunologic surface markers.

Normal human peripheral blood cells were separated into different populations based upon isopycnic sedimentation, E rosetting, and EAC rosetting. Each population was characterized according to morphology, surface markers, granulocytic colony formation in semi-solid media, and stainable RNA content by acridine orange (AO) flow cytometry. These techniques enrich for a population of cells that is characterized by a lymphoid morphology, a high granulocytic-macrophage progenitor cell cloning efficiency, a lack of surface markers, and a high stainable RNA content not found in the other two populations of peripheral blood lymphocytes (T cells and B cells). The stainable RNA content serves as a new metabolic marker for the population of cells in which the preponderance of granulocytic progenitor cells reside.

B-Lymphocytes↗
PubMed1979

Correlation between cell cycle duration and RNA content.

The metachromatic fluorochrome acridine orange was used to differentially stain DNA and RNA in Chinese hamster ovary (CHO) cells and in mitogen-stimulated human lymphocytes during their progression through the cell cycle. Green and red fluorescence of individual cells, representing cellular DNA and RNA, respectively, was measured by flow cytometry. CHO cells were synchronized by selective detachment at mitosis. Their rate of progression through G1 and subsequently through S phase correlated with the content of stainable RNA. The mean duration of the G1 phase was 5.2 hours for cells with high RNA content (highest 25 percentile population) and 8.1 hours for cells with low RNA (lowest 25 percentile). The duration of S phase was 5.9 and 7.5 hours for high- and low-RNA, 25 percentile subpopulations, respectively. Lymphocytes synchronized at the G1/S boundary by hydroxyurea or 5-fluorodeoxyuridine showed extremely high intercellular variation with respect to content of stainable RNA. After release from the block they traversed S phase at rates linearly proportional to the content of stainable RNA. The duration of S phase was five hours for cells with high RNA-, six to nine hours for cells with moderate RNA- and up to 27 hours for cells with minimal RNA-content. The data suggest that the rate of progression through the cell cycle of individual cells within a population may be correlated with the number of ribosomes per cell.

Animals↗
PubMed1979

Cell-cycle distribution of urothelial tumour cells as measured by flow cytometry.

The fraction of cells in S + G2 + mitosis from 54 urothelial tumours was calculated by flow cytometry after acridine orange (AO) staining of cells obtained by bladder irrigation or biopsy. Fluorescence signals emitted by the AO-stained DNA and RNA of each cell were separated optically and measured for 5,000 cells per specimen. The patients were classified by the histology of their tumours and clinical data into 5 diagnostic categories: NED (no evidence of disease, but history of bladder tumour), 3; papilloma, 8; non-invasive papillary carcinoma, 8; carcinoma in situ, 17 and invasive carcinoma, 18. The fraction of cells with DNA values in S + G2 + M of the cell cycle varied between 7 and 57% of the total, with a wide range within each diagnostic category, but no statistically significant differences between the groups. The proportion of cells in S + G2 + M from an individual tumour was not correlated with histologic grade or clinical behaviour. The possibility that some tumour cells with DNA values above G1 level are quiescent cells arrested at S or G2 is discussed.

Cell Count↗
PubMed1979

Relationship between RNA content and progression of lymphocytes through S phase of cell cycle.

A flow cytometric technique for simultaneous measurements of RNA and DNA in individual cells has been applied to correlate the content of cellular RNA with the rate of progression of cells through the S phase. Human peripheral lymphocytes stimulated with phytohemagglutinin were blocked at the G1/S phase boundary by hydroxyurea or 5-fluorodeoxy-uridine treatment. Cells in the G1 phase as well as cells blocked at the G1/S phase boundary showed high heterogeneity with respect to stainable RNA content. After release from the block, the cells traversed the S phase at rates proportional to the quantity of stainable RNA per cell. Cells with the highest RNA content completed DNA replication 5 hr after release from the block; the cells with minimal RNA traversed the S phase at one-fifth of this rate. The large intercellular variation in stainable RNA and length of the S phase may be due to functional heterogeneity in the lymphocyte population. Our results suggest a correlation between the number of ribosomes and the rate of DNA replication in lymphocytes.

Cell Cycle↗
PubMed1979

Human epidermal cell cultures: growth and differentiation in the absence of differentiation in the absence of dermal components or medium supplements.

Human epidermal cells grew and differentiated in vitro, provided that the pH of the culture medium was at 5.6-5.8, the seeding density was optimal (approximately 2.5 x 10(5) cells per cm2), and the incubation temperature was maintained at 35-37 degrees C. Under these conditions, epidermal cells from many different skin locations grew to confluency within 15-20 days and formed multi-layered sheets whose differentiated structure resembled that of the full depth of skin epidermis. Cell proliferation and differentiation did not require a feeder layer, a collagen substrate, a high concentration of fetal bovine serum, or added hormones. The sheets of differentiated epidermal cells could be dissociated from the plastic surfaces of the tissue culture flasks. The use of such cultured cells for wound dressing is proposed.

Cell Differentiation↗
PubMed1979

Identification of polymorphonuclear leukocytes in cytologic samples for flow cytometry.

Inflammatory cells are commonly present in cytologic specimens obtained for flow cytometry, and may interfere with the analysis of epithelial cells. We have found that detergent (Triton X-100) pretreatment in the two-step acridine orange staining procedure disrupts granulocyte cell membranes to yield bare nuclei; bladder epithelial and squamous cells on the other hand are quite resistant to the detergent treatment. Being deprived of their cytoplasmic RNA, the granulocytes lose red fluorescence. Moreover, the shearing forces in the cytometer extend the multisegmented granulocyte nuclei and align them in the direction of flow. Thus, they present as elongated objects in the measuring system, giving a large DNA fluorescence pulsewidth (nuclear size). These two phenomena make it possible to identify granulocytes in the recorded data, where they are discernible from the mononucleated leukocytes and from epithelial cells. By data selection the granulocytes can be excluded, rendering epithelial cell populations more amenable to analysis. This method may make it unnecessary to remove physically leukocytes from the specimen before flow cytometry; it may also provide a way to analyze the morphology of granulocyte nuclei and to assess methods to manipulate their membrane stability. Full protection from membrane disruption is accomplished by alcohol fixation, and partial protection by 20-30% serum.

Cytological Techniques↗
PubMed1979

Different sensitivity of chromatin to acid denaturation in quiescent and cycling cells as revealed by flow cytometry.

The properties of DNA in situ as reflected by its staining with acridine orange are different in quiescent nonstimulated lymphocytes as compared with interphase lymphocytes that have entered the cell cycle after stimulation by mitogens. The difference is seen after cell treatment with buffers at pH 1.5 (1.3-1.9 range) followed by staining with acridine orange at pH 2.6 (2.3-2.9). Under these conditions the red metachromatic fluorescence of the acridine orange-DNA complex is higher in quiescent cells than in the cycling lymphocytes while the orthochromatic green fluorescence is higher in the cycling, interphase cells. The results suggest that DNA in condensed chromatin of quiescent lymphocytes (as in metaphase chromosomes) is more sensitive to acid-denaturation than DNA in dispersed chromatin of the cycling interphase cells. The phenomenon is used for flow cytometric differentiation between G0 and G1 cells and between G2 and M cells. In contrast to normal lymphocytes the method applied to neoplastic cells indicates the presence of cell subpopulations with condensed chromatin but with DNA content characteristic not only of G1 but also of S and G2 cells. The possibility that these cells represent quiescent (resting) subpopulations, arrested in G1, S and/or G2, is discussed.

Cell Cycle↗
PubMed1979

Regional lymph node reactivity in explanted bladder cancer of mice as measured by flow cytometry.

The reactivity of lymphocytes in lymph nodes draining the site of a transplantable experimental bladder tumor (MBT2 in C3H/HeJ mice) has been measured in a multiparameter flow cytometry system. Acridine orange was used as a nucleic acid probe. This dye intercalates in helical DNA, emitting green (530 nm) fluorescence upon exposure to blue light; it stacks to single-stranded RNA, emitting red (640 nm) fluorescence. The relative magnitude of the increase of lymphocyte DNA and RNA has been evaluated simultaneously in tumor-draining nodes, in nondraining nodes of the same animal, and in untreated control animals. Stimulation of the regional node lymphocytes could be observed after 20 days but not after 10 days. It was uniformly high at 35 days. The transcriptive response (increased proportion of lymphocytes with high RNA) was more pronounced than the proliferative (increased proportion of lymphocytes with more than diploid DNA). The histological changes in the stimulated nodes resembled closely those described by others in human tumor-draining nodes. The described method has the advantage of being simple, rapid, and able to measure a representative part of the whole-cell population.

Animals↗
PubMed1979

Lymph node reactivity to experimental bladder tumor in preimmunized animals as measured by two parameter flow cytometry.

Lymph node lymphocyte reaction to an explanted, transplantable mouse bladder tumor (MBT-2) was investigated by flow cytometry in animals previously immunized with irradiated tumor cells. Nodal lymphocytes in representative samples from four different lymph node sites were differentially stained for DNA and RNA with the fluorescent dye acridine orange; cell proliferation and the increase in RNA content were measured. Immunization abrogated tumor growth; one immunization reduced tumor take to 25 per cent of the animals, and two immunizations to 14 per cent. Lymphocyte reactivity to the tumor was reflected both by an increase of DNA synthesizing cells and by diploid cells with high RNA. The latter response was more pronounced and thus the more sensitive parameter for measuring immunologic lymph node reactivity. The juxtatumoral node displayed the most pronounced reactivity, but all node sites showed some degree of reaction.

Animals↗