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Biomedical subjects

Z Darzynkiewicz

Publications and source records attributed to Z Darzynkiewicz.

At least 235 records · Page 13Linked to original sources
PubMed1986

Flow cytometry of colorectal carcinoma with three-year follow-up.

Flow cytometry DNA measurements were carried out on colorectal carcinomas from a series of 33 patients who have since been followed for three to four years. The tumors could be subclassified by this technique into 15 that had near-diploid DNA and 18 that were aneuploid. Prior reports had suggested that the near-diploid tumors carry a better prognosis than aneuploid tumors, but in this study the distribution by Dukes' stage and survival were the same in both groups.

Adult↗
PubMed1986

p53 content in relation to cell growth and proliferation in murine L1210 leukemia and normal lymphocytes.

The protein p53 has been reported to be associated with cell transformation and/or proliferation. Using p53 monoclonal antibodies we estimated by flow cytometry the relative content of this protein in individual L1210 leukemic cells from exponentially growing and plateau-phase cultures and compared it with that in normal thymocytes of parental DBA/2 mice and in mitogen-stimulation and nonstimulated human lymphocytes. Simultaneous differential staining of p53 vs DNA and p53 vs RNA, followed by bivariate analysis, made it possible to estimate p53 with respect to cell position in the cell cycle and correlate it with RNA (predominantly rRNA) content. The data show that in exponentially growing L1210 cells p53 is being progressively accumulated during the G1, S and G2 phases and that the content of p53 and RNA are highly correlated. In plateau L1210 cultures most cells are arrested in G1, some cells, however, still continue to progress through S and G2. In these cultures the p53 content of all cells, regardless of the phase of the cell cycle, is diminished and the decrease in p53 is more pronounced than that of RNA or total protein content. The normal thymocytes as well as the stimulated lymphocytes show bimodal distribution with respect to p53 expression, compatible with the assumption that the cycling cells have increased expression of this protein related to the G0 cells. Some cycling cells, however, have minimal p53. The quantitative p53 immunofluorescence data were confirmed by the immunoprecipitation and gel electrophoresis. The results suggest that expression of p53 in leukemic and normal cells is more correlated with cell growth than with entrance to the cell cycle or progression through particular phases of the cycle.

Animals↗
PubMed1986

Relationship between the pharmacological activity of antitumor drugs Ametantrone and mitoxantrone (Novatrone) and their ability to condense nucleic acids.

Ametantrone (HAQ) and mitoxantrone (DHAQ) are structurally similar antitumor drugs of the anthracenedione class. The cytostatic, cytotoxic, and antitumor activities of these drugs are different, with DHAQ being 10-100 times more potent, per molar basis. Both drugs are strong intercalators and intercalative modes of binding are suspected as relevant to their pharmacological activity. No significant differences, however, that could explain the differences in pharmacological activity are observed in their intercalative properties with respect to base specificity and binding affinity. A correlation, however, is evident between their potency and ability to condense nucleic acids inasmuch as DHAQ condenses nucleic acids at concentrations that are lower by a factor of 5-40 than those of HAQ and these effects can be observed at their pharmacological concentrations. The condensation is base- and sugar-specific and the long purine sequences of single-stranded RNA are the most sensitive. Electron microscopy of L1210 cells exposed a short time (90 min) to 0.21-21 microM DHAQ reveals segregation of nucleoli; the segregated granular portion shows increased electron opacity. In some preparations patchy areas of nuclear chromatin characterized by increased electron opacity can be seen. The results are compatible with the possibility that pharmacological effects of these antitumor drugs could involve condensation of nucleic acids, primarily of RNA in nucleoli.

Animals↗
PubMed1986

Stathmokinetic analysis of human epidermal cells in vitro.

Proliferation kinetics of cultured human epidermal cells is characterized in quantitative terms. Three distinct subpopulations of keratinocytes, two of which are cycling, have been discriminated by two parameter DNA/RNA flow cytometry. Based on mathematical modelling, the cell cycle parameters of the cycling subpopulations have been assessed from stathmokinetic data collected at different time points after initiation of cultures (7-15 days). The first subpopulation is composed of low-RNA cells which resemble basal keratinocytes of epidermis and which show some characteristics of stem cells; these cells have a mean generation time of approximately 100 hr. The second subpopulation consists of high-RNA cells, resembling stratum spinosum cells of epidermis, which have an average generation time of approximately 40 hr. The third subpopulation consists of non-cycling cells with G0/G1 DNA content, with cytochemical features similar to those of cells in granular layer of epidermis. The results based on modelling can reproduce with acceptable accuracy the actual growth curve of the cultured cell population. Analysis of kinetics and differentiation of human keratinocytes is of interest in view of the recent application of cultured epidermal cell sheets for transplantation onto burn wounds. The results of this study also reveal the existence of regulatory mechanisms associated with proliferation and differentiation in the cultured epidermal cell population.

Acridine Orange↗
PubMed1986

Human keratinocyte culture. Identification and staging of epidermal cell subpopulations.

Stratification of human epidermal cells into multilayered sheets composed of basal and suprabasal layers (resembling the stratum germinativum and stratum spinosum of the epidermis) was studied in a dermal component-free culture system. Although no stratum corneum developed in vitro, this culture system provided a method to study early events in human keratinocyte differentiation. Multiparameter flow cytometric analysis of acridine orange-stained epidermal cells from these cultures revealed three distinct subpopulations differing in cell size, RNA content, and cell cycle kinetics. The first subpopulation was composed of small basal keratinocytes with low RNA content and a long generation time. The second subpopulation consisted of larger keratinocytes, having higher RNA content and a significantly shorter generation time. Finally, the third subpopulation contained the largest cells, which did not divide, and represent the more terminally differentiated keratinocytes. This in vitro approach provides discriminating cytochemical parameters by which the maturity of the epidermal cell sheets can be assessed prior to grafting onto human burn patients.

Burns↗
PubMed1986

Cytostatic and cytotoxic properties of pyronin Y: relation to mitochondrial localization of the dye and its interaction with RNA.

Pyronin Y (PY) is an intercalating cationic dye that shows specificity towards RNA. In viable cells this dye also accumulates in mitochondria. The cytostatic and cytotoxic effects of PY on L1210 and Chinese hamster ovary cells were studied in relation to its intracellular localization and compared with the affinity of PY to bind to double-stranded DNA and RNA and its propensity to condense single-stranded DNA and RNA. Antitumor properties of PY were tested on L1210 leukemia and Sarcoma 180 ascites in mice. At a concentration of 1.7 to 3.3 microM, PY was localized almost exclusively in mitochondria of cultured cells, similar to another mitochondrial probe, rhodamine 123. At that concentration PY was not toxic but suppressed cell growth, arresting cells in G1. At a concentration of 6.7 to 33.0 microM, PY was also localized in nucleoli and uniformly in cytoplasm, bound to the RNase-sensitive material therein. At that high concentration PY induced cell arrest in G2 and S and was cytotoxic. The dye exhibited a propensity to bind and condense (precipitate) single-stranded nucleic acids, and condensation could be measured by the appearance of light-scattering products. Among a variety of natural and synthetic nucleic acids the most sensitive were the RNA polymer, polyriboadenylate, and the copolymer, polyriboadenylate and polyriboguanylate, which underwent condensation at a PY concentration of 6.6 to 10.0 microM. Natural and synthetic DNA polymers were resistant to condensation. The data suggest that the cytostatic (G2 and S arrest) and cytotoxic (inability to exclude trypan blue, loss of clonogenicity) effects of PY seen at 6.7 to 33.0 microM concentration may be a consequence of the dye binding to RNA. PY may intercalate to double-stranded RNA and/or cause the specific condensation of single-stranded RNA; the polyadenylated sections of mRNA appear to be the most sensitive cellular targets to undergo condensation. PY showed antitumor properties extending survival of L1210 leukemic mice by 50% and slowing growth of Sarcoma 180 ascites tumor. The possibility that certain antitumor drugs, generally believed to act via intercalation to DNA, may exert chemotherapeutic effects via their interactions with RNA is discussed.

Animals↗
PubMed1985

Interactions of antitumor agents Ametantrone and Mitoxantrone (Novatrone) with double-stranded DNA.

Interactions of Mitoxantrone and Ametantrone with natural and synthetic nucleic acids in aqueous medium [0.15 NaCl, 5 mM 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (Hepes), pH 7.0, 25 degrees] have been studied using computer-aided spectrophotometric techniques. Absorption spectra of the drugs in monomeric and dimeric form and their complexes with DNAs at low drug/phosphate ratios (D/P) have been established. The latter were red-shifted and had lower amplitude as compared with the spectra of the free ligand's monomer; the change is consistent with the already well-established intercalative mode of drug-nucleic acid interaction. Drug-DNA equilibria have been studied using the McGhee-von Hippel model of noncooperative ligand-polymer interaction, with the correction for dimerization of drugs. Although Mitoxantrone is two orders of magnitude more potent an antitumor drug than Ametantrone, the intrinsic association constants (Ki) of both drugs were of similar magnitude. Also, no significant DNA-base specificity for either of the drugs (measured as Ki value for various homopolymers) was observed. Therefore, no correlation was apparent between the intercalative mode of binding to DNA, regardless of base composition, and the pharmacological activity of these drugs. At higher D/P ratios, a secondary mode of binding was detected by both spectroscopy and light-scattering measurement. Homopolymer-pairs and polymers containing only dI and dC were especially susceptible to this secondary type of binding. The possibility that this secondary type of binding may be responsible for the antitumor properties of the drugs is considered.

Anthraquinones↗
PubMed1985

Correlated measurements of DNA, RNA, and protein in individual cells by flow cytometry.

A cytochemical method was developed to differentially stain cellular DNA, RNA, and proteins with fluorochromes Hoechst 33342, pyronin Y, and fluorescein isothiocyanate, respectively. The fluorescence intensities, reflecting the DNA, RNA, and protein content of individual cells, were measured in a flow cytometer after sequential excitation by three lasers tuned to different excitation wavelengths. The method offers rapid analysis of changes in the cellular content of RNA and protein as well as in the RNA-protein, RNA-DNA, and protein-DNA ratios in relation to cell cycle position for large cell populations. An analysis of cycling cell populations (exponentially growing CHO cultures) and noncycling CHO cells arrested in the G1 phase by growth in isoleucine-free medium demonstrated the potential of the technique.

Animals↗
PubMed1985

Denaturation and condensation of DNA in situ induced by acridine orange in relation to chromatin changes during growth and differentiation of Friend erythroleukemia cells.

DNA in situ is progressively denatured when the cells or nuclei are treated with increasing concentration of acridine orange (AO). This transition can be monitored by flow cytometry as a decrease in green fluorescence. The complexes of denatured DNA and AO undergo immediate condensation and aggregation; this step is manifested by appearance of red luminescence and formation of precipitates that can be detected by electron microscopy. The precipitates form preferentially in heterochromatin as well as in ribosomes and polysomes. Their formation and further aggregation affects cellular light scatter properties in both the forward and right-angle direction. The AO-induced DNA denaturation and condensation was studied in nuclei of Friend erythroleukemia cells from exponentially growing, differentiated or quiescent cells. The DNA in nuclei of quiescent cells, from plateau-phase cultures, was the most sensitive to denaturation; it denatured (measured by changes in luminescence) at an AO concentration between 50 and 80 microM with the midpoint of the transition (Cd) at 70 microM. DNA in nuclei of differentiated cells (dimethyl-sulfoxide-induced erythroid differentiation) was more resistant (Cd = 77-83 microM), whereas DNA in exponentially growing cells was the most resistant (Cd = 86 microM). Extraction of proteins with 0.1 M HCl at 0 degree C abolished the differences between the cells and shifted the transition to a lower AO concentration (Cd = 46 microM). For comparison, the midpoint transitions representing condensation of free, nucleic acids measured as light scatter changes occurred at 13, 22, 31 and 53 microM of AO, for rRNA, tRNA, and denatured and native-calf thymus DNA, respectively. Denaturation and condensation of DNA, which can be induced by AO either in isolated nuclei or viable permeabilized or fixed cells provides a new approach to discriminate cell subpopulations with different chromatin structure by flow cytometry. The molecular mechanisms of this phenomenon are discussed.

Acridine Orange↗
PubMed1985

RNA content and chromatin structure of CHO cells arrested in metaphase by colcemid.

To evaluate the stability of cells arrested in metaphase, cell viability, RNA content, and chromatin structure (the latter probed by the DNA in situ sensitivity to acid-induced denaturation) were studied in uniform-age mitotic CHO cell populations maintained either at 37 degrees C (in the presence of Colcemid) or at 0-4 degrees C for up to 6 h. No significant changes in cell viability and RNA content were seen throughout the experiment for both groups of cells. The sensitivity of DNA in situ to denaturation was significantly increased during the initial 40 min of cell arrest in mitosis. However, no further chromatin changes for up to 6 h were evident regardless of whether cells were kept at 37 degrees C with Colcemid or at 0-4 degrees C in its absence. The data indicate that neither significant deterioration of metaphase cells nor progressive chromatin changes are expected during stathmokinesis experiments in vitro or during the metaphase cell arrest in cytogenetic studies lasting up to 6 h. Also, no RNA turnover can be detected in mitotic cells during this time interval.

Animals↗
PubMed1985

Monitoring intravesical bacillus Calmette-Guerin treatment of bladder carcinoma with flow cytometry.

Flow cytometry of bladder irrigation specimens was studied in 22 patients with low stage bladder carcinoma who were treated by transurethral resection of visible tumor followed in 3 to 5 weeks by a course of intravesical bacillus Calmette-Guerin. The most informative examinations were just before the first bacillus Calmette-Guerin treatment, 6 weeks after completing a 6-week course of treatment (3 months) and at 9 months. Of the patients 10 had recurrent tumors after therapy; recurrence was anticipated correctly by flow cytometry at the 12-week followup examination in 6 of the 10 patients and suspected in another. Of 12 patients who remained clinically free of disease for a minimum of 15 months after bacillus Calmette-Guerin therapy flow cytometry identified correctly 7 at 12 weeks, while 1 had a partial response and the remaining 4 reverted to a negative status at 9 months. Of interest, only 4 of the 22 patients were free of disease by flow cytometry at the start of bacillus Calmette-Guerin treatment despite attempted ablation of the tumor by transurethral resection, suggesting that intravesical administration of bacillus Calmette-Guerin destroys existing carcinoma in situ in some cases.

BCG Vaccine↗
PubMed1985

Normal and perturbed Chinese hamster ovary cells: correlation of DNA, RNA, and protein content by flow cytometry.

Quantitative, correlated determinations of DNA, RNA, and protein, as well as RNA to DNA and RNA to protein ratios, were performed on three-color stained cells using a multiwavelength-excitation flow cytometer. DNA-bound Hoechst 33342 (blue), protein-fluorescein isothiocyanate (green), and RNA-bound pyronin Y (red) fluorescence measurements were correlated as each stained cell intersected three spatially separated laser beams. The analytical scheme provided sensitive and accurate fluorescence determinations by minimizing the effects of overlap in the spectral characteristics of the three dyes. Computer analysis was used to generate two-parameter contour density profiles as well as to obtain numerical data for subpopulations delineated on the basis of cellular DNA content. Such determinations allowed for analysis of RNA to DNA and RNA to protein ratios for cells within particular regions of the cell cycle. The technique was used to study the interrelationship of DNA, RNA, and protein contents in exponentially growing Chinese hamster ovary cells as well as in cell populations progressing the cell cycle after release from arrest in G1 phase. The sensitivity of the method for early detection of conditions of unbalanced growth is demonstrated in the comparison of the differential effects of the cycle-perturbing agent, adriamycin, on cells treated either during exponential growth or while reversibly arrested in G1 phase.

Animals↗
PubMed1985

Binding of anti-Z-DNA antibodies in quiescent and activated lymphocytes: relationship to cell cycle progression and chromatin changes.

Although regions of DNA reacting with anti-Z-DNA antibodies have been identified in the polytene chromosomes of Drosophila spp. and the metaphase chromosomes from a number of different mammalian species, the biological role of this DNA is unknown. Flow cytometry was used in the present studies to quantitate the binding of anti-Z-DNA antibodies in quiescent and activated human peripheral blood lymphocytes; the antibody binding was then correlated with cell cycle phase. The data show that quiescent (G0 or G1Q) lymphocytes are heterogeneous with respect to their reaction with anti-Z-DNA antibodies. The transition from quiescence (G1Q) into the cell cycle (G1), which involves decondensation of chromatin, did not result in any significant change in binding of these antibodies. In contrast, progression of cells from G1 through S and G2 is correlated with a 27% decrease in anti-Z-DNA antibody reactivity relative to total DNA content. No significant change was observed during the transition from G2 to mitosis (M).

Antibodies↗
PubMed1985

Rhodamine 123 alters the mitochondrial ultrastructure of cultured L1210 cells.

Exposure of exponentially growing L1210 cells in vitro to 5-10 micrograms/ml of rhodamine 123 (R123) for 16-48 hr inhibits cell proliferation and induces cell arrest in the G1A phase of the cell cycle. The cells remain viable during the arrest and resume growth after removal of R123; extended exposure to R123 is cytotoxic. Exposure to R123 results in morphological alterations in mitochondria of all cells observed; specifically, mitochondria of R123-treated cells are characterized by a distention of the intracristal spaces and a significant increase in the number of matrix granules. Gross morphological changes of mitochondria include formation of extended organelles and the appearance of doughnut-shaped structures.

Animals↗
PubMed1985

Detection of 5-bromodeoxyuridine (BrdUrd) incorporation by monoclonal antibodies: role of the DNA denaturation step.

Immunochemical detection of cells that incorporate 5-bromodeoxyuridine (BrdUrd) requires prior denaturation of DNA in situ to make BrdUrd binding sites accessible to the antibodies. A technique is described in which the DNA denaturation step is facilitated by a) prior dissociation of histones from DNA and b) the use of low ionic strength buffer in which the cells are suspended during heating. Dissociation of histones is achieved by cell treatment with 0.08N HCl at 0 degree C, which a) increases accessibility of DNA to propidium iodide (and following the denaturation to the antibodies); b) lowers stability of DNA to thermal denaturation; c) decreases differences between various cell types due to variability in chromatin structure; and d) ensures more complete DNA denaturation. Cell heating (80-95 degrees C) at low ionic strength (1 mM Na+) eliminates the need for formamide and results in extensive and rapid DNA denaturation. The method was applied in Friend leukemia, L1210 and HL-60 cell lines, and to bone marrow, experimental animal tumor and primary human tumor cells.

Animals↗
PubMed1984

Analysis of a cell cycle model based on unequal division of metabolic constituents to daughter cells during cytokinesis.

We demonstrate that the unequal division of RNA during cytokinesis explains the dispersion of cell generation times in CHO cell cultures. Experimental cytometric results reported previously serve as a basis for a probabilistic model of cytokinesis. Unequal RNA division to daughter cells, together with two simple laws of RNA production, are used as a source of randomness within the cell cycle. The model reproduces the experimental growth of the CHO cell population, including the observed variability in RNA content. The model has stabilizing properties which explain why a cell population with increased RNA content characteristics, a few cell cycles, to the original pattern. Other cell cycle characteristics, like sister-to-sister and mother-to-daughter generation time correlations implied by the model, are close to their experimental analogs. The conceptual basis of the model is general enough to include unequal division of factors other than RNA (cell mass, cell proteins, etc.) as sources of generation time variability. It seems that the observed dispersion of cell generation times, explained previously in the terms of random transitions in some part of the cell cycle (the Smith & Martin A and B state hypothesis), can be reduced to the single random event of unequal division. This supplies a new convenient tool in the investigation of cell cycle kinetics.

Animals↗
PubMed1984

Accessibility of DNA in situ to various fluorochromes: relationship to chromatin changes during erythroid differentiation of Friend leukemia cells.

Friend leukemia cells from exponentially growing or differentiated (DMSO-induced) cultures were permeabilized and their DNA was stained with 4'6-diamidino-2-phenylindole (DAPI), Hoechst 33342, acridine orange, ethidium bromide, propidium iodide, quinacrine, 7-amino-actinomycin D, mithramycin, or chromomycin A3. Accessibility of DNA to each of the above fluorochromes was compared in differentiated and nondifferentiated cells before and after nuclear proteins, mostly histones, were extracted with 0.1N HCl. A decrease in the accessibility of DNA to several dyes, especially pronounced in the case of some intercalators, was observed in differentiated cells. After extraction of nuclear proteins with HCl there was an increase in DNA accessibility, of varying degree depending on the fluorochrome and the difference between differentiated and nondifferentiated cells was abolished for most of the intercalating dyes. The increase was the lowest for DAPI (45%), the highest for 7-amino-actinomycin D (13-fold), and in general was higher for the intercalating dyes that unwind DNA than for dyes binding externally to the double helix. The results are discussed in terms of the mode of interactions between DNA and the fluorochromes and factors associated with chromatin structure that may affect accessibility of DNA in situ in exponentially growing and differentiated cells.

Animals↗