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Biomedical subjects

Z Darzynkiewicz

Publications and source records attributed to Z Darzynkiewicz.

At least 271 records · Page 15Linked to original sources
PubMed1983

Denaturation of RNA and DNA in situ induced by acridine orange.

The products of interaction of acridine orange (AO) with single-stranded (ss) nucleic acids are precipitates which exhibit red luminescence. Titration of rRNA or thymus DNA with AO results in formation of such products suggesting that the dye, per se, denatures double-stranded (ds) sections of these biopolymers. This transition, measured as the increase of red luminescence, a concomitant decrease of green fluorescence, and followed by an increase of light scatter of the AO-nucleic acid complexes, is cooperative and at 0.15 N NaCl occurs at 4-20 and 10-50 microM range of AO concentration for rRNA and DNA, respectively. The changes in stainability of nucleic acids in situ, in permealized cells, occur at higher AO concentration. Thus, the transition of RNA in situ is biphasic and seen at 20-120 microM AO. In the presence of EDTA, however, the change is monophasic and shifted to the 10-30 microM range of AO concentration. The change in stainability of DNA also shows two phases: one at 30-60 microM and another at 70-120 microM of AO. Extraction of basic proteins with 0.08 N HCl shifts the transition of DNA to the 30-60 microM AO concentration and makes it monophasic. The observed differences in denaturability of RNA vs DNA explain the specificity of AO in differential staining of these bipolymers in histochemical reactions. In living cells the products of interaction of AO with nucleic acids are detected by electron microscopy. In the cytoplasm of interphase cells the formation of dense precipitates within ribosomes and polysomes, simultaneous with a specific retraction of ribosome-polysome complexes from the periphery of the cell to the nucleus is evident. The latter suggests higher order organization of these particles involving their association with each other or with the nucleus via polyanionic macromolecules which collapse upon binding with AO. The DNA in heterochromatin is more sensitive to AO-induced denaturation, as evidenced by the fact that the dense complexes are formed preferentially in the regions of condensed chromatin of the interphase nucleus, or in metaphase chromosomes.

Acridine Orange↗
PubMed1983

Secretion of albumin and alpha-foetoprotein by dimethylsulphoxide-stimulated hepatocellular carcinoma cells.

Exposure of BW77-1 and BW77-2 mouse hepatic tumour cells to the polar solvent dimethylsulphoxide (DMSO) altered extracellular accumulation of albumin and alpha-foetoprotein (AFP) and perturbed their cell cycle kinetics. The amount of albumin secreted into the culture growth medium was dependent on the concentration of DMSO used. Hepatic tumour cells cultured in 1 and 2% DMSO accumulated 50% and 111% more albumin, respectively, than non-DMSO-stimulated cells during the final 24 h of a 4-day exposure to the polar solvent. Commitment of mouse hepatoma cells to increased albumin secretion was temporally dependent, requiring a minimum of 48 h in the presence of DMSO. The AFP level in 1% DMSO-treated cultures was also significantly increased, compared with control cells. Unlike albumin secretion, however, exposure of hepatic tumour cells to 2% DMSO did not further increase (but slightly decreased) extracellular AFP accumulation. Treatment of BW77-1 cells with DMSO resulted in a gradual decline in the percentage of 2C DNA content cells (diploid G1 population) and in a corresponding increase in the proportion of cells with a 4C DNA content (generation of either a G2 or tetraploid G1 population). The extent of this shift directly reflected the concentration of polar solvent in the medium and paralleled the DMSO-induced stimulation in albumin secretion. DMSO-stimulated hepatic tumour cells, therefore, may prove useful in the elucidation of specific regulatory events underlying control of gene expression during the hepatocyte cell cycle.

Albumins↗
PubMed1982

Flow cytometry of low stage bladder tumors: correlation with cytologic and cystoscopic diagnosis.

Flow cytometry examinations (FCM) were carried out on 110 bladder irrigation specimens from 84 urologic outpatients who had had prior conservative treatment for low stage bladder tumors. The specimens were easily obtained, and adequately cellular in all cases. Of 60 examinations on patients with no cytoscopic abnormalities, FCM and conventional cytology were both negative in 30 and both positive in 12 instances; there were 18 with positive FCM and negative cytology. Of 15 instances with cystoscopically benign appearing papillary lesions, FCM and cytology were both negative in three and both positive in three cases; FCM was positive and cytology negative in nine. Of 35 instances with suspicious cystoscopic findings, FCM and cytology were both positive in 19 and both negative in four; FCM was positive and cytology negative in 12. There were no examples of positive conventional cytology and negative FCM. These findings indicate that, after conservative treatment of low stage tumors, FCM of bladder irrigation specimens may be a more sensitive measure of cytologic abnormalities than is conventional cytology. Specimen collection is feasible as part of the routine urologic examination in an outpatient clinic.

Carcinoma in Situ↗
PubMed1982

Luminescence of the solid complexes of acridine orange with RNA.

The products of interaction between acridine orange (AO) and natural RNA, or the synthetic RNA homopolymers are precipitates insoluble over a wide range of ionic strength. These complexes have a composition of 1 AO molecule per 1 phosphate. The reaction is highly cooperative and the complex exhibits metachromatic luminescence. Significant differences in the luminescence spectra, related to base composition of RNA, characterize the insoluble complexes. This observation suggests that dye-base interactions take place in the AO-RNA complexes. During titration of poly(rA) with AO and simultaneous measurement of the luminescence and light scatter a two-step formation of the particles could be detected. The cooperative binding of the ligand at D/P less than 0.6 coincides with formation of small particles (molecular aggregates or micelles) and is followed at D/P greater than 0.6 by a cooperative agglomeration; the product of this agglomeration is particles of the size 0.35 micrometer and larger. Evidence is presented that suggests that the long wave length luminescence (approximately 650 nm) is a consequence of the solute-solid state transition of the AO-RNA interaction product rather than the classic dye-dye interactions previously visualized in the stacking model. Since these novel observations cannot be fully explained by the previously postulated molecular mechanisms of AO binding, an alternative model is advanced. Its implications in quantitative cytochemistry of nucleic acid as applied to flow cytometry are discussed.

Acridine Orange↗
PubMed1982

The ratio of RNA to total nucleic acid content as a quantitative measure of unbalanced cell growth.

Use of the metachromatic dye, acridine orange, to stain cells in suspension for flow cytometry allows for the simultaneous measurement of DNA and RNA content in individual cells. The relative RNA content as a function of total cellular nucleic acid content [alpha r = RNA/(RNA + DNA)] is a constant value, characteristic for particular cell lines during their exponential growth under optimal conditions. This ratio can be estimated for the G1A, G1B, S, and G2 + M cell cycle compartments. Changes in growth rate or the addition of antitumor drugs induces characteristic changes in the ratio either evenly throughout or at a particular phase of the cell cycle. Under such conditions, measurement of cellular DNA and RNA content provides a sensitive assay of any deviation from balanced cell growth. Unbalanced growth caused by suboptimal culture conditions or as a result of incubation with various antitumor agents is illustrated. Examples of unbalanced growth which are not correlated with cell viability as measured by cell clonogenicity are discussed.

Acridine Orange↗
PubMed1982

Changes in DNA content of human blood mononuclear cells with senescence.

The mean DNA content per cell and heterogeneity of cell populations with respect to DNA content were measured in samples of peripheral blood mononuclear cells from donors of different ages. Nearly all of the cells in these populations are found in the G1Q phase of the cell cycle. While the mean DNA content was the same for all age groups, a significant increase in intercellular variability, reflected by an increase in coefficient of variation of the mean, was observed in the group of subjects between 67 and 83 years of age in comparison with the group of donors between 20 and 30 years of age. The difference was observed regardless of whether cells were stained with acridine orange or propidium iodide, and under conditions where the intercellular variability cannot be attributed to differences in chromatin structure or quantity of mitochondrial DNA. Increasing cell heterogeneity may reflect the age associated deterioration of the fidelity of DNA replication and/or repair and provide a quantitative marker of the accumulated defects in the DNA of a cell population. The observed changes may be responsible for the decline in immune function with age.

Adult↗
PubMed1982

Characterization of bladder papilloma by two-parameter DNA-RNA flow cytometry.

Two-parameter flow cytometry (FCM) studies of 0.9% NaCl solution bladder irrigation specimens were performed on 48 patients with histologically orderly or atypical papilloma of the urinary bladder in order to assess the value of RNA as a possible second parameter, along with DNA, in the detection of bladder tumors. DNA, RNA, and nuclear diameter measurements were obtained for each of 5000 cells/sample, and analyses were based on the distributions of those values. With the use of DNA content alone, 22 cases (46%) were classified positive by FCM. With RNA content as an additional parameter, 40 cases (83%) were positive. Two cases were suspicious, and 6 cases were normal by both parameters. Of 28 patients with papillomas showing histological atypia, 16 patients had positive DNA histograms, including 3 patients with aneuploid stemlines, but 24 of the 28 patients had positive RNA histograms. Of 20 patients with orderly papillomas, 6 patients had positive DNA histograms, including 3 patients with aneuploid DNA stem cell lines, but 16 of the 20 patients had positive RNA histograms. Thus, the probability of positive DNA histograms is higher in atypical papillomas (57%) than in orderly papillomas (30%), whereas elevated (positive) RNA is more characteristic of all papillomas without distinction between those that are histologically atypical (86% positive) or orderly (80% positive). For patients at risk of developing papillary bladder tumors, two-parameter DNA-RNA FCM appears to offer greater diagnostic sensitivity than does FCM based on DNA content alone.

Cell Nucleus↗
PubMed1982

Flow cytometry of lung carcinoma: a comparison of DNA stemline and cell cycle distribution with histology.

Flow cytometry studies of the DNA distribution of 33 lung tumors were carried out. All of the carcinomas (32 cases) had aneuploid DNA modal values, ranging from 2.15c to 5.05c; in the single case of carcinoid studied, the tumor cells were diploid. DNA ploidy levels tended to be higher for epidermoid than adenocarcinoma; they were the same in lymph node metastases as in the primary tumor. Cell cycle distributions calculated from the tumor cell DNA values showed considerable variation, ranging from 9% to 58% for the S phase and from less than 1% to 29% for the G2M phase. Whether these variations have clinical significance is not known at this time.

Adenocarcinoma↗
PubMed1982

Interaction of rhodamine 123 with living cells studied by flow cytometry.

The cationic fluorochrome rhodamine 123 (R123), reported to bind specifically to mitochondria of living cells, was presently investigated with respect to its uptake by a variety of cell types in various functional states and the subsequent effect of the dye on cell growth. The emission spectrum of R123 taken up by cells undergoes a 12-nm red shift, suggesting formation of a complex. Cells accumulate R123 rapidly; near maximum binding is reached after 5 to 10 min, regardless of the temperature (0-37 degrees) of incubation. There is a dose-dependent relationship between R123 concentration in the medium and the dye accumulation in the cell that covers the range of 0.1 to 10.0 and 0.1 to 5.0 microgram of R123 per ml under equilibrium and nonequilibrium conditions, respectively. Some leakage of the dye from cells occurs, following their transfer into dye-free medium. Despite the leakage, the intracellular dye can be detected after at least two cell divisions, thus indicating that: (a) the R123-labeled cells divide; (b) during division, labeled mitochondria are distributed into the daughter cells; and (c) R123 may be used as a cell tracer. Cell death often is accompanied by a transient increase in R123 fluorescence. Dead cells exhibit either uniform, strong fluorescence or show a patchy labeling pattern suggesting swollen mitochondria. With time (4 to 8 hr), dead cells lose ability to retain R123 and lyse. Uptake of R123 by living cells is increased during the transition from quiescence into the cycle, and a decrease is seen when Friend leukemia cells undergo erythroid differentiation; in all cases, changes in R123 uptake are correlated with changes in cellular RNA content. Simultaneous cell staining with R123 and ethidium or propidium provides a rapid assay of the viability of the cells and their metabolic state, i.e., as related to proliferation or motility. Pulse-labeling of cells with up to 10 microgram of R123 per ml has no significant effect on their immediate growth and cloning efficiency. In the continuous presence of R123, however, cells become specifically arrested in the G1A compartment, i.e., in early G1 phase. Detailed analysis of the cell cycle kinetics reveals that cell progression through all phases is slowed 4 hr after addition of R123. Cell exit from G1A, however, is affected as early as 2 hr following addition of R123, and with time the cells are unable to leave this compartment at all. Uncharged rhodamine dyes (rhodamine 110 and rhodamine B) do not accumulate in mitochondria and are without effect on the cell cycle. The cytostatic effect of R123 is discussed in light of the dye specificity for mitochondrial membranes and the disruption of cell energy metabolism, resulting in the inability of the cells to attain a critical content of essential components (i.e., ribosomal RNA), necessary for cell entrance into the prereplicative (G1B) compartment of G1 phase.

Animals↗
PubMed1982

Flow cytometry of breast carcinoma. III. Possible altered kinetics in axillary lymph node metastases.

The cell cycle distribution of human mammary carcinoma was determined by flow cytometry in 21 cases and compared with that of the same tumor concurrently metastatic in regional lymph nodes. The percentage of S-phase cells in the nodal metastasis as compared to the primary tumor was significantly lower in estrogen-binding-positive tumors than in estrogen-binding-negative tumors. Since we do not have evidence of clonal evolution in the metastases, it is possible that there is a local inhibitory effect of the lymph node on tumor cell proliferation in the case of some estrogen-binding-positive tumors.

Breast Neoplasms↗
PubMed1981

Flow cytometry of breast carcinoma: I. Relation of DNA ploidy level to histology and estrogen receptor.

Flow cytometry studies of the DNA distribution of tumor cells from 92 human breast cancers showed measurable aneuploidy (hyperploidy) in 83 cases (92%). The DNA ploidy values were unimodal in each case, but there was a bimodal distribution for the entire series. One group of tumors had a diploid or near diploid DNA distribution and a second group had ploidy levels from triploid to tetraploid or higher. The tumors with lower DNA ploidy (at or near diploid) tended to be histologically low grade, cytologically more orderly and estrogen-binding positive; those with higher DNA ploidy were more likely to be higher grade, more anaplastic, and estrogen-binding negative.

Breast Neoplasms↗
PubMed1981

Flow cytometry of breast carcinoma: II. Relation of tumor cell cycle distribution to histology and estrogen receptor.

Flow cytometry analysis of cell cycle distribution was carried out on acridine-orange-stained cell suspensions freshly prepared from carcinoma of the breast of 90 women, and compared with control, benign breast specimens from 10 women. There was a significantly greater proportion of cells in S (9.1% +/- 5.8) and G2 + M (8.0 +/- 6.0) phases of the cycle in breast cancer specimens than in the S (1.5% +/- 0.6) and G2 + M (2.5 +/- 0.9) phases of the cycle in benign, control specimens of breast tissue. Great variation was noted among the breast cancers, with medullary carcinomas having the highest percentage of cells in S + G2 + M, and papillary, tubular, colloid and well-differentiated duct carcinomas having the lowest. Tumors from premenopausal women classified as estrogen receptor negative had significantly more cells in S + G2 + M than did tumors from postmenopausal women that were estrogen-receptor positive.

Age Factors↗