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Biomedical subjects

F Traganos

Publications and source records attributed to F Traganos.

At least 73 records · Page 4Linked to original sources
PubMed1994

A selective procedure for DNA extraction from apoptotic cells applicable for gel electrophoresis and flow cytometry.

In cells undergoing apoptosis (programmed cell death), a fraction of nuclear DNA is fragmented to the size equivalent of DNA in mono- or oligonucleosomes. When such DNA is analyzed by agarose gel electrophoresis it generates the characteristic "ladder" pattern of discontinuous DNA fragments. Such a pattern of DNA degradation generally serves as a marker of the apoptotic mode of cell death. We developed a simple, rapid, and selective procedure for extraction of the degraded, low-molecular-weight DNA from apoptotic cells. The cells are prefixed in 70% ethanol, DNA is extracted with 0.2 M phosphate-citrate buffer at pH 7.8, and the extract is sequentially treated with RNase A and proteinase K and then subjected to electrophoresis. The ladder pattern was detected from DNA extracted from 1-2 x 10(6) HL-60 cells, of which as few as 8% were apoptotic, by flow cytometric criteria, as well as from blood and bone marrow samples from leukemic patients undergoing chemotherapy. The method is rapid and uses nontoxic reagents (no phenol, chloroform, etc.). This approach permits the analysis of DNA extracted from the very same cell population that is subjected to measurements by flow cytometry to estimate DNA ploidy, the cell cycle distribution of nonapoptotic cells, the percentage of apoptotic cells, or other parameters. Furthermore, the cells may be stored in 70% ethanol for at least several weeks before analysis without any significant DNA degradation. Treatment with ethanol also inactivates several pathogens, thereby increasing the safety of sample handling. The method is applicable to clinical samples, which can be fixed in ethanol and then stored and/or safety transported prior to analysis.

Apoptosis↗
PubMed1994

Flow cytometric detection of apoptosis: comparison of the assays of in situ DNA degradation and chromatin changes.

The aim of this study was to compare three methods of detection of apoptotic cells: (1) the method based on elution of low molecular weight DNA from the ethanol fixed cells followed by cell staining with DAPI (diamidino-2-phenylindole) or propidium iodide as the DNA fluorochromes, (2) the method of in situ labeling of DNA strand breaks with biotinylated dUTP, utilizing exogenous terminal deoxyribonucleotide transferase, and (3) the method of analysis of DNA denaturation in situ using acridine orange to differentially stain denatured and double-stranded DNA sections following cell exposure to 0.1 M HCl. Cells of the human promyelocytic HL-60 line, treated in vitro with the DNA topoisomerase I inhibitor camptothecin, which selectively triggers apoptosis of S-phase cells, were chosen as a model. The method based on analysis of changes in DNA denaturability was the most sensitive in terms of detection of the earliest changes in chromatin of cells undergoing apoptosis; the increased sensitivity of DNA to denaturation in S-phase cells was measured as early as 100 min after addition of camptothecin. DNA cleavage, assayed either by the univariate measurement of DNA content following extraction of low molecular weight DNA, or by labeling DNA strand breaks with biotinylated dUTP, was detected in S-phase cells after 120 min incubation with camptothecin. The percentage of apoptotic cells at the late stage of apoptosis, the kinetics of cell transition to apoptosis, and kinetics of the loss of S phase cells were all essentially similar when measured by any method.(ABSTRACT TRUNCATED AT 250 WORDS)

Apoptosis↗
PubMed1994

Effect of staurosporine on MOLT-4 cell progression through G2 and on cytokinesis.

Staurosporine (SSP) is an inhibitor of a variety of protein kinases with an especially high affinity towards protein kinase C. Whereas SSP has been shown to halt the cell cycle progression of various normal, nontransformed cell types in G1, most virus transformed or tumor cells are unaffected in G1 but arrest in G2 phase. SSP has also been observed to increase the appearance of cells with higher DNA content, suggestive of endoreduplication, in cultures of tumor cells. Using multivariate flow cytometry (DNA content vs. expression of cyclin B, nuclear p120 protein, or protein reactive with Ki-67 antibody) which makes it possible to discriminate cells with identical DNA content but at different phases of the cycle, we have studied the cell cycle progression of human lymphocytic leukemic MOLT-4 cells in the presence of 0.1 microM SSP. MOLT-4 cells did not arrest in G1 or G2 phase in the presence of the inhibitor. Rather, they failed to undergo cytokinesis, entering G1 phase at higher DNA ploidy (tetraploidy; G1T), and then progressed through ST (rereplication) into G2T and MT. The rates of entrance to G2 and G2T were essentially identical, indicating that the rates of cell progression through S and ST as well as through G2 and G2T, respectively, were similar. Cells entrance to mitosis and mitotic chromatin condensation were also similar at the diploid and tetraploid DNA content level and were unaffected by 0.1 microM SSP. No evidence of growth imbalance (altered protein or RNA to DNA ratio) was observed in the case of tetraploid cells. The data show that, in the case of MOLT-4 cells, all events associated with the chromosome or DNA cycle were unaffected by SSP; the only target of the inhibitor appears to be kinase(s) controlling cytokinesis.

Alkaloids↗
PubMed1994

Apoptotic cell death during treatment of leukemias.

The apoptosis-associated DNA strand breaks were detected in situ, in individual leukemic cells in peripheral blood and bone marrow of over 110 patients with different types of leukemia (ALL, AML, CML in blastic crisis, APL), prior to and during routine chemotherapy. The DNA strand breaks were labeled with digoxigenin- or biotin-conjugated dUTP in the reaction catalyzed by exogenous terminal deoxynucleotidyl transferase, and the cells, counterstained for DNA, were analyzed by bivariate flow cytometry. The proportion of cells with DNA strand breaks prior to therapy, most likely reflecting spontaneous apoptosis, varied from 0.1 to 16%, but in the large majority of cases was below 3%. Administration of drugs of different classes, which included DNA topoisomerase I (Topotecan) and II (mitoxantrone, VP-16) inhibitors, antimetabolite (ara-C) or microtubule poison (Taxol), all triggered the appearance of cells with extensive DNA breakage, typical of apoptosis, to up to 80%. The peak of the response, measured as maximal percent of cells with DNA strand breaks, which varied between individual patients by as much as factor 10, was generally seen between 8 to 24 h after the initial administration of DNA topoisomerase inhibitors, and somewhat later (48-72 h) during the response to Taxol or ara-C. Thus, the data show that the response to treatment with a variety of drugs, in terms of induction of apoptosis, can be conveniently measured by the present method. The prognostic value of the apoptotic index, before, as well as during treatment, is being estimated for each type of leukemia, in the ongoing prospective studies.

Antineoplastic Agents↗
PubMed1994

Altered susceptibility of differentiating HL-60 cells to apoptosis induced by antitumor drugs.

It has been reported that human promyelocytic leukemic HL-60 cells which undergo differentiation fail to respond by apoptosis when treated with antitumor drugs, predominantly DNA topoisomerase inhibitors. Because S phase cells are selectively sensitive to these drugs, and during differentiation there is a reduction in the proportion of cells in S phase, the reported decrease in the number of apoptotic cells could simply be a reflection of the paucity of sensitive cells in these cultures. Using cytometric methods which allow apoptosis to be related to cell cycle position, we have compared the apoptotic response of HL-60 cells growing exponentially and induced to myeloid differentiation by dimethyl sulfoxide (DMSO). The cells were treated with: (i) the DNA topoisomerase I inhibitor camptothecin (CAM), which selectively triggers apoptosis or S phase cells; (ii) the nucleoside antimetabolite 5-azacytidine (AZC) and hyperthermia, both of which preferentially affects G1 cells; and (iii) gamma radiation, which causes apoptosis predominantly of G2 + M cells. The cells exposed to 1.4% DMSO for 24 or 48 h were significantly more resistant to response by apoptosis, regardless of the nature of the agent and regardless of their position in the cell cycle. Thus, induction of differentiation lowers the cell's ability to respond to a variety of damaging agents by apoptosis and this effect is not correlated with cell cycle position. In addition, the difference in response was unrelated to expression of the apoptosis-modulating protein bcl-2, which appeared unchanged following 48 h exposure to DMSO. On the other hand, when the cells were pretreated with low concentrations of CAM or AZC, washed free of drug, and then treated with DMSO, the proportion of cells undergoing apoptosis was markedly increased, relative to drug-treated cells returned to DMSO-free medium. The present data may indicate that while the drug-induced damage screening mechanisms, which are linked to triggering apoptosis, may be more proficient in proliferating cells, the effectors of apoptosis are more expressed in cells undergoing differentiation. The data also suggest that the efficiency of chemotherapeutic agents or radiation may be reduced if a differentiating agent is used in combination therapy and is administered first. An enhancement of apoptosis, however, may be expected if the differentiating drug is administered in the reverse sequence.

Antineoplastic Agents↗
PubMed1993

Simultaneous analysis of cell cycle kinetics at two different DNA ploidy levels based on DNA content and cyclin B measurements.

The methods of cell cycle analysis that rely on DNA content measurements cannot discriminate between cells at different phases of the cycle if these cells have similar DNA content. This limitation can be circumvented by measurement of another cell cycle phase-specific cell constituent in addition to DNA content, followed by bivariate analysis of the correlated data. The aim of the present study was to explore the utility of a monoclonal antibody against the G2- and M phase-specific regulatory protein cyclin B for discrimination of cell populations with overlapping DNA content. This analysis, which was based on correlated DNA/cyclin B content measurements by flow cytometry, was applied to human lymphocytic leukemic MOLT-4 cells. The onset of cyclin B synthesis was observed in the last one third of S phase with its maximum accumulation in G2 and M phases; cells in G1 and early- and mid-S phases were negative. Cells arrested in metaphase by vinblastine expressed high levels of this protein, although not as high as in cells arrested in G2 by the DNA topoisomerase II inhibitor m-AMSA. Disruption of cytokinesis by the protein kinase inhibitor staurosporine led to DNA rereplication, cell progression through the chromatin cycle at higher DNA ploidy, and induction of polyploidy. It was possible, utilizing the cyclin B antibody, to discriminate between G2 + M cells with a 2C level of DNA and G1 cells with 4C DNA, as well as to distinguish doublets of G1 cells with a 2C DNA level. Thus, the rate of cell entrance to G1 at the 4C DNA level and the rates of progression through the cycle at both the 2C and 4C DNA levels could be simultaneously estimated. The data indicate that, in the presence of 0.1 microM staurosporine, cytokinesis of all MOLT-4 cells is impaired and the cells enter to and progress through the chromosome cycle at 4C DNA at the same rates as at 2C DNA. This approach can be helpful in the analysis of DNA ploidy and the cell cycle of human tumors when there is an overlap in DNA content values between normal stromal or infiltrating cells and aneuploid tumor cell population and may be the method of choice to investigate the activity of antitumor drugs which impair cytokinesis but do not interfere with progression of cells through the chromatin cycle.

Alkaloids↗
PubMed1993

Caffeine prevents apoptosis and cell cycle effects induced by camptothecin or topotecan in HL-60 cells.

Caffeine (3,7-dihydro-1,3,7,-trimethyl-1H-purine-6,6-dione; CAF) is known to potentiate the cytotoxic effects of DNA damaging agents such as ionizing radiation and alkylating agents. In contrast, however, the cytotoxic and cytostatic activity of aromatic, DNA-intercalating, DNA topoisomerase II inhibitors such as Adriamycin, ellipticine, or mitoxantrone are diminished in the presence of CAF. To resolve whether the protective effect of CAF is associated with a particular mechanism of drug interaction (e.g., intercalation into DNA, inhibition of DNA topoisomerase II), or the aromatic nature of the drug structure, per se, we have presently studied the effects of CAF on the cytostatic and cytotoxic action of camptothecin (CAM) and its less toxic but more water soluble derivative topotecan (TPT) on HL-60 human myelogenous leukemia cells: both drugs have aromatic structures but are nonintercalating inhibitors of DNA topoisomerase I. By using spectroscopy and titration microcalorimetry, we have also studied the direct interaction between CAF and TPT in solution. Low (20 nM) concentrations of CAM or TPT perturbed progression of HL-60 cells through S-phase, whereas higher concentrations (0.15 microM) of these drugs induced apoptosis; both effects were easily demonstrable after 4 h of treatment. When added simultaneously with CAM or TPT, CAF prevented both effects. The protective effect of CAF was concentration dependent and evident within the concentration range of 1-5 mM; nearly total protection was seen at a CAF concentration of 5 mM. The bathochromic and hypochromic shift in the absorption spectrum of the water soluble compound TPT upon addition of CAF indicated that CAF and TPT interact (stack) in a fashion similar to that previously observed for CAF and DNA intercalators. Microcalorimetric measurements of TPT titration with CAF indicate an exothermic reaction between these compounds (the enthalpy change was delta H degree = -4.2 kcal/mol), which is consistent with a stacking model of CAF-TPT interaction. Thus, the ability of CAF to protect HL-60 cells against the cell kinetic effects of CAM or TPT, as in the case of DNA intercalating topoisomerase II inhibitors, is most likely due to formation of complexes between CAF and these aromatic molecules, which result in reducing the effective concentration of the free form of these drugs available to the cells.

Antineoplastic Agents↗
PubMed1993

The cell cycle related differences in susceptibility of HL-60 cells to apoptosis induced by various antitumor agents.

The studies were aimed to detect the cell cycle-associated differences in the susceptibility of HL-60 cells to apoptosis induced by diverse agents. Exponentially growing HL-60 cells were treated with the DNA topoisomerase I inhibitor camptothecin; the DNA topoisomerase II inhibitors teniposide, m-AMSA, Mitoxantrone, or Fostriecin; the presumed tyrosine kinase inhibitor genistein; a serine/threonine kinase inhibitor H7; the protein synthesis inhibitor cycloheximide; the DNA replication inhibitor hydroxyurea; the nucleoside antimetabolites 1-beta-D-arabinofuranosylcytosine and 5-azacytidine; and the alkylating agent nitrogen mustard, cisplatin, hyperthermia, and gamma irradiation. Endonucleolysis, which accompanied apoptosis induced by these agents, was assessed by two different flow cytometric methods, one based on DNA content measurements following extraction of low molecular weight DNA, and another using exogenous terminal deoxynucleotidyl transferase to label in situ DNA strand breaks. Each method allowed for both identification of apoptotic cells and analysis of the cell cycle distribution of the unaffected cell population; the method using terminal transferase also allowed for identification of the cell cycle position of apoptotic cells. Confirmed by analysis of DNA degradation by gel electrophoresis and changes in cell morphology, apoptosis was observed as early as 3 h after administration of most drugs and for some drugs was cell cycle phase specific. Cells progressing through S phase were selectively susceptible when treated with camptothecin, teniposide, m-AMSA, Mitoxantrone, H7, hydroxyurea, and 1-beta-D-arabinofuranosylcytosine. Cells in G2-M preferentially underwent apoptosis in cultures treated with H7 or with gamma-irradiation. Cells in G1 phase were preferentially affected by 5-azacytidine, nitrogen mustard, and hyperthermia. No significant cell cycle specificity was observed in the case of Fostriecin, genistein, cycloheximide, or cisplatin. The cell cycle related difference in susceptibility to apoptosis may be a reflection of both the severity of the lesion induced by a given drug and the ability of the cells to repair that lesion; both can vary depending on the cell cycle phase.

Antineoplastic Agents↗
PubMed1993

Presence of DNA strand breaks and increased sensitivity of DNA in situ to denaturation in abnormal human sperm cells: analogy to apoptosis of somatic cells.

The presence of sperm cells characterized by increased sensitivity of DNA in situ to denaturation and by abnormal morphology (shape) in semen is associated with low fertility. Such cells often appear in increasing numbers following exposure to toxic or mutagenic agents. In the present study, the sensitivity of DNA in situ to acid-induced denaturation was assayed in sperm cells of 25 patients attending a human fertility center. The same samples were also subjected to analysis of DNA strand breaks. A new method for detecting DNA strand breaks in situ by labeling 3'-OH termini with biotinylated dUTP in a reaction employing exogenous terminal deoxynucleotidyl transferase has been applied. Both methods utilized flow cytometry. A good correlation was observed in all samples between the percentage of sperm cells with DNA strand breaks and the percentage of cells characterized by an increased sensitivity of DNA to denaturation (r = 0.87; P < = 0.05). It is likely, thus, that the sperm cells characterized by increased DNA sensitivity to denaturation have extensive DNA strand breakage. The above properties of abnormal sperm cells, which in all probability are dead in terms of their reproductive capacity, resemble properties of apoptotic somatic cells. Activation of the endogenous endonuclease which causes extensive DNA breakage, thus, is characteristic to both the programmed death of somatic cells (apoptosis) and functional elimination (of possibly defective) germ cells from the reproductive pool.

Apoptosis↗
PubMed1992

Effects of genistein on the growth and cell cycle progression of normal human lymphocytes and human leukemic MOLT-4 and HL-60 cells.

Genistein (GEN) is an isoflavone known to inhibit both tyrosine protein kinases and DNA topoisomerase II. The effects of GEN on cell proliferation and cell cycle kinetics of human myelogenous leukemia HL-60 and lymphocytic leukemia MOLT-4 cell cultures were studied, and the data were compared to results obtained with normal human lymphocytes stimulated to proliferate with phytohemagglutinin. GEN concentrations greater than 50 micrograms/ml (185 microM) were cytotoxic to HL-60 and MOLT-4 cells following exposure for 24 h; in HL-60 cell cultures, a population of cells with decreased DNA content and nuclear fragmentation characteristic of apoptosis was observed within 8 h. The 50% inhibition concentration after 24 h of exposure for HL-60 and MOLT-4 cells was 8.5 and 13.0 micrograms/ml, respectively. Normal proliferating lymphocytes survived a 24-h exposure of up to 200 micrograms/ml GEN. Short-term (4-8 h) exposures of MOLT-4 or HL-60 cells to 5-20 micrograms/ml GEN resulted in a suppression of cell progression through S or through both S and G2 phases, respectively, while equivalent treatment had no effect on proliferating lymphocytes. A stathmokinetic experiment using MOLT-4 cells revealed that as little as 5 micrograms/ml GEN suppressed cell exit from S to G2 phase by 40%, with a terminal point of action at or near the S-G2 border. Cell progression through the very early portion of G1 phase (G1A, characterized by postmitotic chromatin decondensation) was also suppressed by approximately 40%, whereas cell advancement through the remainder of the G1 phase was not markedly affected. Longer (24 h) exposure of proliferating lymphocytes to 20 micrograms/ml GEN led to an S-phase arrest, while similar treatment of leukemic cells caused cell arrest in G2 phase and an increase in the number of cells entering the cycle at higher DNA ploidy. The mitogen-induced transition of lymphocytes from G0 to G1 phase was extremely sensitive to inhibition by GEN; the 50% inhibition concentration was 1.6 micrograms/ml. The chemotherapeutic value of GEN may be due to the fact that, in terms of cytotoxicity, this agent is more active against proliferating leukemic cells than against normal proliferating lymphocytes. The sensitivity of the G0 to G1 transition in normal lymphocyte cultures and the suppressive effect of GEN on the G1A exit in MOLT-4 cells both suggest that protein kinases involved in chromatin decondensation may be a target of this drug. In light of the observation that lymphocyte stimulation is sensitive to the presence of GEN, the drug is expected to be a strong immunosuppressant.

Apoptosis↗
PubMed1992

Cytostatic and cytotoxic effects of fostriecin on human promyelocytic HL-60 and lymphocytic MOLT-4 leukemic cells.

Exposure of exponentially growing human promyelocytic of lymphocytic leukemic cells to the putative DNA topoisomerase II inhibitor fostriecin (FST), at a concentration of 1 microM, results in the suppression of their rate of progression through the S and G2 phases of the cell cycle. At concentrations between 5 microM and 0.5 mM, FST triggers endonucleolytic DNA degradation in human promyelocytic leukemia cells, resulting in apoptotic cell death; this effect is not selective for any particular phase of the cell cycle. Little or no apoptotic cell death is observed in lymphocytic leukemic cells at any FST concentration. Because FST, unlike other inhibitors of topoisomerase II, such as teniposide (TN) or amsacrine (m-AMSA), does not stabilize cleavable DNA-topoisomerase complexes, the observed differences between the effects of FST versus TN or m-AMSA on the cell cycle may provide clues regarding the role of such complexes in the kinetic effects of these inhibitors. The present results, therefore, are compared with our earlier data on the effects of TN and m-AMSA on the same cells. The only observed difference is the loss of cell cycle phase-specific triggering of DNA degradation by FST in human promyelocytic leukemia cells, compared to the S phase-specific effects of TN and m-AMSA. Therefore, stabilization of the DNA-topoisomerase cleavable complexes may be essential in the selectivity of cell kill during S phase. However, it appears that the presence of stabilized complexes is not essential to the suppression of cell progression through S or G2 or the induction of apoptotis or necrosis, in general, by topoisomerase II inhibitors.

Alkenes↗
PubMed1992

Different effects of staurosporine, an inhibitor of protein kinases, on the cell cycle and chromatin structure of normal and leukemic lymphocytes.

Staurosporine, a microbial alkaloid, is a strong inhibitor of protein kinases. The effects of staurosporine on the cell cycle progression and nuclear morphology of normal human lymphocytes stimulated to proliferate by phytohemagglutinin were studied and compared with the effects of this drug on human lymphocytic leukemic MOLT-4 cells. Exposure of normal lymphocytes to either 5-10 or 50-100 ng/ml of staurosporine resulted in the preferential accumulation of cells in G1 or G1 and G2 phases of the cell cycle, respectively. In contrast, regardless of the concentration (5-100 ng/ml), staurosporine arrested MOLT-4 cells initially in G2; these cells then initiated additional rounds of DNA replication, without division. Staurosporine (5-100 ng/ml) induced severe changes in the nuclear morphology of MOLT-4 cells, manifested as nuclear elongation, deep invaginations of the nuclear membrane, extensive fragmentation, and micronucleation. At concentrations of 5-10 ng/ml, staurosporine had no apparent effect on the nuclear morphology of normal lymphocytes and at 50-100 ng/ml it produced minor changes in the nuclear shapes of these cells. The data indicate that the kinase(s) involved in the regulation of cell exit from G1 and G2, respectively, in normal and leukemic lymphocytes may have different sensitivities to staurosporine, which suggests that the mechanisms controlling exit from G1 in these cells may be different. In MOLT-4 cells the staurosporine-sensitive kinase(s) appear to also be involved in phosphorylation of nuclear constituents essential for organization of gross chromatin structure. The different response of normal versus leukemic lymphocytes to staurosporine, if confirmed on clinical material, opens new strategies of tumor treatment.

Alkaloids↗
PubMed1992

DNA stainability in aneuploid breast tumors: comparison of four DNA fluorochromes differing in binding properties.

The aim of this study was to evaluate whether or not the differences in chromatin structure between diploid stromal cells or lymphocytes, which are often used as DNA ploidy standard, and aneuploid breast tumor cells can significantly affect the estimates of the DNA index of these tumors. To this end, the DNA content estimates of 34 aneuploid breast tumors, differing in size, degree of differentiation, and presence or absence of estrogen and progesterone receptors and metastases, were compared using four common DNA fluorochromes: DAPI, Hoechst 33342, propidium iodide, and acridine orange. These dyes differ in their mode of interaction with DNA (binding to minor groove or intercalation) and for each of them binding to DNA is restricted to a different degree by nuclear proteins. It was expected, therefore, that if differences in chromatin structure play a role in DNA content estimates, the DNA index of the measured tumors may vary depending on the dye. The cell nuclei were isolated from the tumors using a detergent-based procedure and stained with each of the dyes and the DNA index was estimated using peripheral blood lymphocytes as a DNA content standard. For each of the tumors, the DNA index estimates with all four dyes correlated very well. When the results obtained with individual dyes were compared in pairs, the correlation coefficients (r) of DNA indices were all above 0.96 (correlation at p less than 0.001). The best concordance was seen between specimens stained with Hoechst 33342 and DAPI (r = 0.99), and the least between those stained with Hoechst 33342 and propidium iodide (r = 0.96). The data indicate that DNA content analysis of unfixed nuclei, utilizing the above fluorochromes, is not significantly biased by differences in chromatin structure of the measured cells.

Acridine Orange↗
PubMed1992

Features of apoptotic cells measured by flow cytometry.

The present review describes several methods to characterize and differentiate between two different mechanisms of cell death, apoptosis and necrosis. Most of these methods were applied to studies of apoptosis triggered in the human leukemic HL-60 cell line by DNA topoisomerase I or II inhibitors, and in rat thymocytes by either topoisomerase inhibitors or prednisolone. In most cases, apoptosis was selective to cells in a particular phase of the cell cycle: only S-phase HL-60 cells and G0 thymocytes were mainly affected. Necrosis was induced by excessively high concentrations of these drugs. The following cell features were found useful to characterize the mode of cell death: a) Activation of an endonuclease in apoptocic cells resulted in extraction of the low molecular weight DNA following cell permeabilization, which, in turn, led to their decreased stainability with DNA-specific fluorochromes. Measurements of DNA content made it possible to identify apoptotic cells and to recognize the cell cycle phase specificity of the apoptotic process. b) Plasma membrane integrity, which is lost in necrotic but not apoptotic cells, was probed by the exclusion of propidium iodide (PI). The combination of PI followed by Hoechst 33342 proved to be an excellent probe to distinguish live, necrotic, early- and late-apoptotic cells. c) Mitochondrial transmembrane potential, assayed by retention of rhodamine 123 was preserved in apoptotic but not necrotic cells. d) The ATP-dependent lysosomal proton pump, tested by the supravital uptake of acridine orange (AO) was also preserved in apoptotic but not necrotic cells. e) Bivariate analysis of cells stained for DNA and protein revealed markedly diminished protein content in apoptotic cells, most likely due to activation of endogenous proteases. Necrotic cells, having leaky membranes, had minimal protein content. f) Staining of RNA allowed for the discrimination of G0 from G1 cells and thus made it possible to reveal that apoptosis was selective to G0 thymocytes. g) The decrease in forward light scatter, paralleled either by no change (HL-60 cells) or an increase (thymocytes) of right angle scatter, were early changes during apoptosis. h) The sensitivity of DNA in situ to denaturation, was increased in apoptotic and necrotic cells. This feature, probed by staining with AO at low pH, provided a sensitive and early assay to discriminate between live, apoptotic and necrotic cells, and to evaluate the cell cycle phase specificity of these processes. i) The in situ nick translation assay employing labeled triphosphonucleotides can be used to reveal DNA strand breaks, to detect the very early stages of apoptosis.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Changes in nuclear chromatin related to apoptosis or necrosis induced by the DNA topoisomerase II inhibitor fostriecin in MOLT-4 and HL-60 cells are revealed by altered DNA sensitivity to denaturation.

The antitumor drug fostriecin (phosphotrienin, FST) has been reported to exert its cytostatic and cytotoxic effects via inhibition of DNA topoisomerase II. The sensitivity of human lymphocytic leukemic MOLT-4 and promyelocytic HL-60 leukemic cells to a wide range of FST concentrations was studied by analyzing the cell cycle-specific effects and changes in nuclear chromatin induced by this inhibitor. The latter was evaluated by assaying the sensitivity of DNA in situ to acid-induced denaturation cytofluorimetrically, with the use of the metachromatic fluorochrome acridine orange (AO), which differentially stains double-stranded and denatured DNA. The cytostatic effects were observed soon after addition of FST (at concentrations of 1-30 microM for MOLT-4 cultures and 1-5 microM for HL-60 cultures) as a perturbation of cell progression through S and G2 phases of the cell cycle. Cell progression through the cycle was halted at greater than 30 microM FST in MOLT-4 cultures and at greater than 5 microM in HL-60 cultures; the effect was instantaneous and affected all phases of the cycle, so that no changes in the cell cycle distribution were apparent with increasing length of exposure to the drug. Instead, at these high FST concentrations, immediate cytotoxic effects became evident, manifesting either as cell apoptosis or necrosis. Apoptosis was observed only in the case of HL-60 cells, at FST concentrations of 5-100 microM, and was characterized by markedly increased sensitivity of DNA to denaturation combined with a decrease in overall DNA stainability, either with the DNA-specific dye DAPI or with AO, indicative of the activation of endogenous nucleases. Necrotic cell death was observed at FST concentrations of 1 mM and at greater than 30 microM for HL-60 and MOLT-4 cells, respectively: in both cases the overall DNA stainability, with either DAPI or AO, was unchanged compared to the control, but their DNA was very sensitive to denaturation. Interestingly, DNA in G2 and late S phase MOLT-4 cells, which were undergoing necrotic death, was much more sensitive to denaturation than was DNA in G1 cells of this lineage. The data indicate that chromatin changes induced by DNA topoisomerase II inhibitors in cells that undergo apoptotic or necrotic death can be conveniently monitored by the assay of DNA in situ sensitivity to denaturation.

Acridine Orange↗