Biomedical subjects

Z Darzynkiewicz

Publications and source records attributed to Z Darzynkiewicz.

At least 253 records · Page 14Linked to original sources

PubMed1984

Compound 48/80 impairs cytokinesis in murine leukemic cells.

Compound 48/80 (poly-p-methoxyphenethylmethylamine), an agent commonly used to trigger degranulation of mast cells, at concentrations of 5-20 micrograms/ml suppresses the proliferation of L1210 and Friend leukemic cells in vitro, inducing the formation of giant cells, which are polykaryons. Both the proportion of polykaryons in cultures and their size (which reflects the number of nuclei per polykaryon) increase during growth in the presence of 48/80 up to 48 hr; thereafter, the cells lose viability. A predominant number of nuclei in these polykaryons contain a 4C, or higher DNA content. The data indicate that compound 48/80 impairs the cleavage (cytokinesis) and perhaps mitotic processes. Mechanisms by which compound 48/80 induces the described effects are unknown but may be related to the polycationic nature of the polymer and its interaction with the cell membrane. Certain attributes of compound 48/80 suggest that this or similar polymers may have value as research tools for the study of regulatory mechanisms involved in cell division.

Animals↗

PubMed1984

Condensation of nucleic acids by intercalating aromatic cations.

Certain intercalating aromatic cations, such as the fluorochrome acridine orange or the antitumor drug Mitoxantrone, induce condensation of nucleic acids in solutions. The appearance of the condensed form during titration of nucleic acids with these intercalating ligands can be quantitatively monitored by light scatter measurements. The resulting highly reproducible light scatter transition curves are typical of the cooperative processes, and the transitions occur at different critical concentrations of the ligands depending upon both the ligand itself and the primary structure (base and sugar composition) and the secondary structure (single- or double-stranded) of the nucleic acids. The mechanism of condensation of nucleic acids by intercalating cationic ligands is discussed in light of the model of interactions occurring between certain intercalators and single-stranded nucleic acids and compared with the condensation induced by polyvalent "simple" cations such as Co3+ or spermine4+. The described phenomenon can have an application in analytical and preparative biochemistry for characterization of the primary and secondary structure of nucleic acids and for separation of the compounds. The possibility that the condensation plays a role in mutagenic and pharmacological effects of aromatic cations is considered.

Acridine Orange↗

PubMed1984

Denaturation of nucleic acids induced by intercalating agents. Biochemical and biophysical properties of acridine orange-DNA complexes.

At high binding densities acridine orange (AO) forms complexes with ds DNA which are insoluble in aqueous media. These complexes are characterized by high red- and minimal green-luminescence, 1:1 (dye/P) stoichiometry and resemble complexes of AO with ss nucleic acids. Formation of these complexes can be conveniently monitored by light scatter measurements. Light scattering properties of these complexes are believed to result from the condensation of nucleic acids induced by the cationic, intercalating ligands. The spectral and thermodynamic data provide evidence that AO (and other intercalating agents) induces denaturation of ds nucleic acids; the driving force of the denaturation is high affinity and cooperativity of binding of these ligands to ss nucleic acids. The denaturing effects of AO, adriamycin and ellipticine were confirmed by biochemical studies on accessibility of DNA bases (in complexes with these ligands) to the external probes. The denaturing properties of AO vary depending on the primary structure (sugar- and base-composition) of nucleic acids.

Acridine Orange↗

PubMed1984

Effects of [3H]UdR on the cell-cycle progression of L1210 cells.

Tritium-labelled uridine [( 3H]UdR) perturbs progression of L1210 cells through the mitotic cycle. The main effect manifests as a slowdown or arrest of a portion of cells in G2 and is already observed 2 hr after addition of 0.5-5.0 microCi/ml of [3H]UdR into cultures. At 2.5-5.0 microCi/ml of [3H]UdR a slowdown of cell progression through S is also apparent. Additionally, there is an increase in the number of cells with DNA values higher than 4C in cultures growing in the presence of [3H]UdR for 8-24 hr. A pulse of [3H]UdR of 2 hr duration labels predominantly (95%) cellular RNA. The first cell-cycle effects (G2 slowdown) are observed when the amount of the incorporated [3H]UdR is such that, on average there are fewer than thirty-six [3H] decays per cell which corresponds to approximately 12-19 rads of radiation. The S-phase slowdown is seen at a dose of incorporated [3H]UdR twice as high as that inducing G2 effects. The specific localization of [3H]UdR in nucleoli, peripheral nucleoplasm and in cytoplasm, as well as differences in the kinetics of the incorporation in relation to phases of the cell cycle are discussed in the light of the differences between the effects of [3H]UdR and [3H]thymidine. Mathematical modelling of the cell-cycle effects of [3H]UdR is provided.

Animals↗

PubMed1984

Effects of a prospective antitumor agent, 1,4-bis(2'-chloroethyl)-1,4-diazabicyclo-[2.2.1] heptane diperchlorate, on cultured mammalian cells.

The effects of 1,4-bis(2'-chloroethyl)-1,4-diazabicyclo-[2.2.1] heptane diperchlorate (CBH; NSC 57198) on cell viability, growth, progression through the cell cycle, survival, and differentiation were investigated in suspension cultures of murine lymphocytic leukemia (L1210) and erythroleukemic (FL) cells and normal human lymphocytes stimulated with phytohemagglutinin (PHA) and in adherent cultures of Chinese hamster ovary (CHO) cells. CBH was equally cytotoxic toward stationary and exponentially growing CHO cells. Cell viability was diminished by 50% following 24 hr exposure to approximately 50 micrograms CBH per ml. Treatment of quiescent human lymphocytes for 24 hr with up to 100 micrograms CBH per ml did not appreciably diminish cell viability though the subsequent stimulation of such lymphocytes with PHA was inhibited in a dose dependent fashion. L1210, FL cells, and PHA stimulated human lymphocytes were equally sensitive to CBH, 50% inhibition of growth was obtained following 24 hr treatment with 25 micrograms CBH per ml. Incubation for up to 48 hr with CBH did not result in differentiation of FL cells to mature hemoglobin containing cells. Constant exposure of L1210 cells and PHA-stimulated human lymphocytes to 10-50 micrograms CBH per ml resulted in accumulation of cells in G2 + M phase; higher drug concentrations resulted in cell arrest in mid to late S phase and G2 phase. A short 1-hr pulse of the drug resulted in a transient accumulation of L1210 cells in S and G2 phases. However, cells recovered from a short pulse of drug and by 48 hr, both cell proliferation and the cell cycle distribution appeared normal. A detailed analysis of cell cycle progression of L1210 cells in the presence of the drug indicated that the duration of G2 phase was extended at low concentrations (10 micrograms/ml) while the transit of cells through S was retarded with subsequent accumulation in late S and G2 phase at higher (50 micrograms/ml) concentrations. Concomitant with cell arrest in S and G2 phase an increase in cellular RNA content indicating unbalanced growth was observed. This state of unbalanced growth was reversible in cultures exposed to a 1-hr pulse of up to 100 micrograms CBH per ml; cellular RNA content returned to control values by 48 hr. No effect on nuclear chromatin as assayed by acid denaturation was observed. Though the exact mechanism of drug action is not known, the data are not incompatible with the drug acting as an alkylating agent.

Animals↗

PubMed1984

Immunological studies of aging. IX. Impaired proliferation of T lymphocytes detected in elderly humans by flow cytometry.

Lymphocytes from humans over the age of 65 incorporate approximately 50% less tritiated thymidine than do lymphocytes from young donors when cultured with phytohemagglutinin. Because lymphocytes from elderly humans are more sensitive to cell cycle arrest induced by tritiated thymidine, it was impossible to determine to what extent impaired thymidine incorporation reflected a defect in proliferation or the increased sensitivity to the radioactive isotope. Flow cytometry was used to measure the proliferative response of T cells from young and old donors in culture with PHA. It was found that 25 percent fewer lymphocytes from old as compared to young humans enter the G1 or complete the S phase of the cell cycle. However, the rate of progression through the cell cycle by activated cells from young and old humans is comparable. Thus, flow cytometry suggested that the difference in thymidine incorporation by lymphocytes from old and young donors is attributable equally to a proliferative defect and to cell cycle arrest induced by tritiated thymidine. This conclusion was supported by the fact that the relative impairment of thymidine incorporation by lymphocytes from old donors was only one-half as great when a 20-min instead of a 24-hr pulse of tritiated thymidine was used.

Adult↗

PubMed1984

Multiparameter flow cytometric analysis of hepatic nuclear RNA and DNA of normal and hepatotoxin-treated mice.

Combined immunohistologic and flow cytometric methods were used to monitor the regenerative response of the mouse liver following intraperitoneal administration of the hepatonecrotic agent carbon tetrachloride (CCl4). A slight increase in the percentage of liver nuclei engaged in DNA synthesis (S-phase population) was evident 1 day after CCl4. The percentage of S-phase nuclei increased thereafter, lasting until day 6 after CCl4 with a peak at day 2 after CCl4. The method of sampling used (every 24 hours) places the period of maximal DNA synthetic activity between 48 hours and 72 hours after inoculation of the hepatonecrotic agent; at 48 hours the number of S-phase nuclei was approximately five times higher than the nonregenerating liver. Modified multiparametric flow cytofluorimetric analysis of hepatic nuclei indicated that early repopulation of the liver in response to CCl4-induced necrosis involved the selective proliferation of a specific subpopulation of liver cells which contained diploid nuclei of high RNA content. These nuclei were also observed in control animals, suggesting the existence of a subcompartment of 2C DNA content G1 hepatocytes potentially programmed for replacement proliferation.

Animals↗

PubMed1984

RNA and DNA content of isolated nuclei from bladder irrigation specimens as measured by flow cytometry.

Cell nuclei were isolated from bladder irrigation specimens from urologic patients having neoplastic disease and distinct aneuploid tumor populations with ploidy levels greater than 2. 0c . The isolated nuclei were subsequently stained with acridine orange, and their fluorescence was measured by flow cytometry. The RNA-DNA frequency distributions of nuclei were compared to those of whole, intact cells from the identical specimens. A comparison of RNA content revealed distinct subpopulations of aneuploid G1 tumor cells with different RNA content. The low and high RNA subpopulations were present in both whole cells and intact nuclei. The overall profile regarding ploidy levels of tumors as well as cell cycle distributions were comparable in both whole cells and nuclei. The resolution of DNA content, however, was significantly better in nuclei preparations. This fact made it easier to distinguish aneuploid cells with near-diploid values of DNA content.

Aneuploidy↗

PubMed1984

Expression of Ca antigen in relation to cell cycle in cultured human tumor cells.

In order to explain the heterogeneity of Ca antigen (Ca) expression observed previously on human malignant cells, a relationship of antigen content with events in the cell cycle was studied by multiparameter flow cytometry on four continuous human cell lines, HeLaS3, C4l, HT29, and T24, grown exponentially in culture. In the four cell lines studied, there was an increase in Ca expression during the G1 and G2 + M phases whereas, during the S phase, the Ca expression was relatively constant. Although the level of Ca varied among the four cell lines, the mean amount of Ca expressed during the G2 + M phase was from 58 to 94% higher than the mean for the G1 phase. The G1 and G2 + M cell populations also displayed considerably greater variability of Ca content when compared with cells in the S phase. The level of Ca among cell lines appeared to have an inverse relationship to DNA content expressed as DNA index. Ca expression appeared to be related to RNA content and hence, presumably, ribosomal content, across the cell cycle. This study suggests that some of the heterogeneity of Ca expression in human cancer cells may be related to cell cycle events.

Antigens, Neoplasm↗

PubMed1984

Effects of retinoic acid versus dimethyl sulfoxide on Friend erythroleukemia cell growth. I. Cell proliferation, RNA content, and protein content.

The effect of all-trans-retinoic acid (RA), an oxidative product of vitamin A, on cell growth, cell cycle kinetics, RNA content, and protein content of exponentially growing Friend erythroleukemia (FL) cells was determined and compared with the results obtained with dimethyl sulfoxide [(DMSO) CAS: 67-68-5; methyl sulfoxide], an inducer of differentiation, and alpha-difluoromethylornithine (DFMO), a potent inhibitor of ornithine decarboxylase (EC 4.1.1.17) activity. Growth inhibition of FL cells was observed only during continuous treatment with RA. While RA did not prevent growth to a high cell density, if cultures were maintained in exponential growth, cell number was reduced by 37, 67.4, and 72.2% after 6-day exposure to 10(-7), 10(-6), and 10(-5) M RA, respectively. In comparison, 280 mM (2%) DMSO and 5 mM DFMO inhibited growth over the same time course by 92 and 97.3%, respectively. DMSO resulted in an early, transient (18-24 hr) accumulation of cells in G1 phase followed by a later (5 day), irreversible accumulation of G1 cells. RA required several cell generations (48-72 hr) before a dose-dependent G1 accumulation was observed. Two populations of RA-treated FL cells could be identified: one with an intermediate RNA content (T-cells) similar to near-plateau-phase control cultures and the other with low RNA content (Q-cells) similar to that observed for DMSO-differentiated (D) cells. The kinetics of the decrease in RNA content of Q-cells paralleled those of D-cells in DMSO-treated cultures; the proportion of Q- versus T-cells in RA-treated cultures was dependent on both concentration and length of exposure. DFMO treatment did not give rise to low-RNA-containing Q-cells. Protein content of RA-treated cells was also diminished and approached that observed for hemoglobin-containing D-cells. FL cells were recoverable from long-term (greater than 5 days) treatment with RA, though 2-3 days were required for reestablishment of exponentially growing cultures; apparently only moderate RNA-containing T-cells repopulated the culture. Neither RA nor DFMO treatment gave rise to benzedine-positive, hemoglobin-containing cells as compared to DMSO that induced differentiation in these cultures.

Animals↗

PubMed1984

Effects of retinoic acid versus dimethyl sulfoxide on Friend erythroleukemia cell growth. II. Induction of quiescent, nonproliferating cells.

Treatment of Friend erythroleukemia (FL) cells in vitro with 10(-7) to 10(-5) all-trans-retinoic acid (RA) leads to a concentration-dependent accumulation of a subpopulation of quiescent cells. This subpopulation, termed "Q-cells," contained markedly reduced RNA and protein levels and had a cell cycle distribution with a predominance of cells in G1 phase, which was nearly identical to that found in fully differentiated dimethyl sulfoxide (CAS:67-68-5; methyl sulfoxide)-induced FL cultures. The G1 cells in this RA-induced subpopulation (G1Q cells), though viable, did not enter S-phase, whereas the small percentage of Q-cells with S and G2 DNA content progressed very slowly through the cycle. While the Q-cell population did not contain the differentiation-associated chromatin protein H1 degrees, the cells did manifest a more condensed nuclear chromatin, altered sensitivity to acid denaturation, and reduced accessibility of the DNA in chromatin to acridine orange. The extent of chromatin condensation and the number of free ribosomes versus polysomes in RA-treated FL cells were intermediate between those in untreated and fully differentiated cells, whereas viral budding and the number of nucleoli remained unchanged from those seen in the untreated cell state. The non-Q-cell population in RA-treated cultures, termed "T" (transitional) cells, had an intermediate RNA and protein content and a cell cycle distribution similar to those of control cultures nearing the plateau phase of growth. In the absence of any late markers of differentiation, the Q-cell population was tentatively identified as a unique, quiescent cell population not previously described in the FL cell system.

Animals↗

PubMed1984

Cell cycle-specific effects of tumor necrosis factor.

The immediate effect of tumor necrosis factor (TNF) added to cultures of L-cells is cytostasis, manifested as cell arrest in G2. This effect prevails during the initial 4 hr when the number of G2 cells increases markedly in the absence of any significant cell death in the culture. Shortly thereafter, the cytolytic effect becomes apparent; extensive cell lysis can be detected after 7 hr of exposure to TNF. After 24 hr nearly all cells are lysed. Results of experiments in which the effects of TNF on the kinetics of cell progression through various phases of the cell cycle were studied indicate that in the presence of TNF cells do progress through G2, although with considerable delay, and reach mitosis. Most cells die (undergo lysis) specifically at late stages of mitosis (telophase) or soon after cytokinesis. Sensitivity of cells to the lytic effect of TNF is increased by cell arrest in mitosis with Colcemid or vinblastine. TNF exerts little effect on rates of cell progression through G1 or S phases of the cell cycle, although few cells die during S phase. Nonlysed cells from cultures treated with TNF do not show any changes in nuclear chromatin, suggesting that neither DNA nor nuclear proteins are the primary targets of the TNF. The present data implicate metabolic changes which occur during mitosis (cytokinesis), perhaps associated with synthesis or assembly of cell membrane components, as being responsible for increased cell sensitivity towards the cytolytic effect of TNF. The mechanism of the early cytostatic effect of the factor is unknown.

Animals↗

PubMed1983

Interactions of acridine orange with nucleic acids. Properties of complexes of acridine orange with single stranded ribonucleic acid.

Interactions between acridine orange (AO) and nucleic acids (calf thymus DNA, and homoribo- and homodeoxyribo-polynucleotides) were studied in solutions containing ethanol as a cosolvent. Light absorption, scattering and luminescence were measured as a function of AO concentration at different dye/phosphate (D/P) ratios, and the data were analyzed using the McGhee-von Hippel probabilistic model of the polymer-ligand interactions. The absorption spectra of AO complexes with four homoribopolymers are presented. The intrinsic association constants and cooperativity coefficients of the formation of the complexes were calculated. The effects of ethanol (up to 35%, v/v) on these interactions were concentration dependent and may be extrapolated to zero concentration of this cosolvent. The possibility of destabilization of the double helix of nucleic acids by AO at high D/P ratios is discussed in light of the available thermodynamic data.

Acridine Orange↗

PubMed1983

Increased accessibility of bases in DNA upon binding of acridine orange.

Acridine orange (AO) forms 1:1 complexes with dsDNA which are insoluble in aqueous media, exhibit red luminescence, have minimal green luminescence and resemble complexes of AO with ss nucleic acids. During formation and/or dissociation of these complexes, accessibility of DNA bases to two conformational probes, formaldehyde and diethyl pyrocarbonate is increased, suggesting that the base pairing is destroyed and DNA at least partially denatured. Adriamycin and Ellipticine, but not Ethidium Bromide exert similar destabilizing effects. The results confirm our earlier predictions based on thermodynamic calculations that the double helix undergoes destabilization upon binding an intercalator characterized by high cooperativity in interaction with ss nucleic acids. Thus, the highly cooperative ligand binding to ss sections during the "breathing" of the polymer may progressively destabilize the adjacent ds structure.

Acridine Orange↗

PubMed1983

Increased sensitivity of lymphocytes from people over 65 to cell cycle arrest and chromosomal damage.

Flow cytometry revealed that, in the presence of tritiated thymidine, a greater percentage of phytohemagglutinin-stimulated lymphocytes from old human donors were arrested in the G2 or M phase than were cells from young donors. Furthermore, lymphocytes from old donors showed significantly more chromosomal damage than did lymphocytes from young donors. Lymphocyte cultures from old or young donors not exposed to tritiated thymidine had the same percentage of cycling lymphocytes in G2 or M, although the number of lymphocytes stimulated by phytohemagglutinin to enter the cell cycle was significantly lower in cultures from old donors. Thus, the impaired incorporation of tritiated thymidine by phytohemagglutinin-exposed lymphocytes from old humans reflects both an impaired proliferative response to phytohemagglutinin and an increased sensitivity to the radiobiological effects of tritiated thymidine.

Adult↗

PubMed1983

RNA and DNA content of isolated cell nuclei measured by multiparameter flow cytometry.

Nuclei were isolated from various cell types including Chinese hamster ovary (CHO) and L1210 leukemia cell lines, primary cultures of fibroblasts, nonstimulated and stimulated human lymphocytes and mouse liver cells, by using different isolation techniques. The isolated nuclei were subsequently stained with acridine orange (AO) and their fluorescence was measured by flow cytometry. Various procedures designed to stain DNA versus RNA differentially with AO were tested, and the staining of isolated nuclei was compared with that of whole cells. Control incubations with RNase and DNase were performed to estimate in whole cells and in nuclei the contribution of DNA and RNA to the fluorescence intensity at the respective wavelength bands of maximum emission for DNA (F530) and RNA (F greater than 600). Depending on the cell type, 10-20% of total cell RNase-sensitive F greater than 600 is localized in the nuclei. The RNase-resistant portion of F greater than 600 of isolated nuclei represents the stainability of DNA. Suppression of cell proliferation in subconfluent cultures results in a decrease in both whole cell and in nuclear RNA content. Nonstimulated lymphocyte nuclei have considerably lower RNA content than nuclei from lymphocytes stimulated by pokeweed mitogen. Two subpopulations of nuclei having the same (2C) DNA content but differing in RNA content, are present in mouse liver; the cells entering S phase originate from the high RNA population.

Animals↗

PubMed1983

Distinction between 5-bromodeoxyuridine labeled and unlabeled mitotic cells by flow cytometry.

Exponentially growing Friend leukemia cells are exposed to 5-bromodeoxyuridine (BrdUrd) for the time period equivalent to one generation. After fixation and incubation with RNase, DNA in situ is partially denatured by acid and the cells are stained with acridine orange (AO). Staining with AO under these conditions reveals the extent of denatured versus double stranded DNA and enables one to distinguish mitotic from interphase cells. It is observed that all BrdUrd treated cells, regardless of cell cycle phase, have decreased both green and red fluorescence. This finding suggests that BrdUrd interacts with AO not only in the complexes of the dye with double stranded DNA, but also with the single stranded biopolymer. The BrdUrd-attributed suppression of cell fluorescence is large enough to separate totally BrdUrd labeled from unlabeled mitotic populations. The combination of these two flow cytometric techniques, one which allows discrimination between interphase and metaphase cells and the other which allows further subdivision of metaphase cells into those which have incorporated BrdUrd and those which have not, provides an alternative to the autoradiographic procedure necessary for obtaining the fraction of labeled mitoses.

Acridine Orange↗

PubMed1983

Do all daughter cells enter the "indeterminate" ("A") state of the cell cycle? Analysis of stathmokinetic experiments on L1210 cells.

The results of ten different stathmokinetic experiments on L1210 cells are analyzed to determine whether all the postmitotic cells enter the exponential AG1 compartment of the cell cycle characterized by exponentially distributed transit times. The analysis is based on a mathematical model coherent with the generalized A-B-transition hypothesis. An original fitting procedure is introduced to estimate the fraction of cells entering AG1 as well as other parameters of the cell cycle. Results of the analysis suggest that either all or nearly all postmitotic cells enter G1A. The results are discussed with respect to the validity of the A-B-transition hypothesis of the "probabilistic" model of the cell cycle. Some systematically occurring discrepancies that do not conform with generally accepted cell cycle models are apparent from the analysis of this data.

Animals↗