Z Darzynkiewicz

..Critically assessed is interpretation of the data as to whether the observed DDS events report induction of a particular type of DNA lesion...

..While HA has multitude of functions its anti-oxidant capabilities are often overlooked but may be of significance in preservation of integrity of stem and germ cells genome...

..We have recently reported that brief exposure of human pulmonary adenocarcinoma A549 cells or normal human bronchial epithelial cells (NHBE) to cigarette smoke (CS) induced phosphorylation of H2AX...

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..Several examples are presented that illustrate incorporation of EdU or EU in cells subjected to DNA damage detected as histone H2AX phosphorylation that have been analyzed by flow or laser scanning cytometry...

..e. exfoliated cells, dermal cell models, etc.) hold great promise for robust and exciting developments in MN assay automation as a high-content high-throughput analysis procedure...

..One of the dominating motors for this progress will be further development of cytometry instrumentation and the applicability of this instrumentation, methods, and probes...

..Described also are controls used to ensure accuracy of DNA ploidy determination, the principles in ploidy assessment and possible pitfalls in analysis...

..Antitumor strategies are being designed to treat cancers having particular defects in their DDR, concurrent with protecting normal cells...

..We presume that FQs mediate this effect via a combination of ROS-scavenging and mitochondrial suppression and therefore may protect against the onset or may slow the progression of numerous oxidative pathophysiological conditions...

..Concurrent staining of DNA with propidium or 4',6-diamidino-2-phenylindole (DAPI) and multiparameter analysis of cells by flow- or laser scanning cytometry enables one to correlate induction of apoptosis with the cell cycle phase...

..Concurrent measurement of cellular DNA content and phosphorylated proteins followed by multiparameter cytometric analysis allows one to correlate extent of their phosphorylation with cell cycle phase...

..As sensitive and specific indicators of the cell's proliferative potential, the cyclins, in particular D-type cyclins, are expected to be key prognostic markers in neoplasia...

..It is expected that flow cytometry will be the dominant methodology for necrobiology...

..By virtue of this attribute LSC appears to be the instrument of choice for analysis of apoptosis...

..g., for archival preservation or additional analysis. Potential future applications of LSC are discussed...

..The most common pitfalls and improper use of the methodology in analysis of cell death and in data interpretation are also discussed...

..The data suggest that the "unscheduled" expression of cyclin B1 might be a common defect in neoplasia...

..Because this LY-induced suppression was stronger in Jurkat cells (>70%) than in lymphocytes (20%), the proapoptotic effects of PKB/Akt down-regulation appear to be selective towards tumor cells...

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..FLICA can also be used to study intracellular localization of caspases, including their translocation...

..The capability of LSC to measure the protein translocation between nucleolus and nucleoplasm can be used to study the function and regulatory mechanisms of other proteins that reside in these compartments...

..In conclusion, a significant fraction of myocytes divides repeatedly from birth to senescence, counteracting the continuous death of cells in the aging mammalian rat heart...

..It also offers an assay to screen in vivo inhibitors of kinase(s) or phosphatase(s) involved in H3 phosphorylation or dephosphorylation, and it provides a valuable marker to identify mitotic cells by cytometry...

..The aim of this study was to seek further evidence in support of this postulate, namely to test whether the levels of CAA and CHP correlate with cells metabolic activity...

..The high level of H3 phosphorylation in monocytes may serve as a marker of these cells and is being explored as a possible diagnostic and prognostic tool in monocytic leukemias...

..Both agents (1) caused an apparent accumulation of cells in G(1) at the ED(50) concentration, (2) induced apoptosis at higher concentrations, and (3) decreased expression of the androgen receptor in LNCaP cells...

..The method can be extended for multiparameter analysis of cell populations stained with other fluorochromes of the same-wavelength emission but targeted (e.g., immunocytochemically) for different cell constituents...

..Therefore, the assay of CHP by multiparameter cytometry provides the means to measure effects of antioxidants and metabolic inhibitors on endogenous oxidative DNA damage in relation to cell cycle phase...

..Determining the mechanism by which PUFAs can inhibit melanoma growth will be an important first step in the rational use of PUFAs as antitumor agents...

..Our results suggest that different morphologic variants of apoptosis might be common in different tumor types and that cytometric quantification might provide biologically useful information not otherwise easy to obtain...

..The propensity of Onc to potentiate the activation-induced apoptosis suggests that this drug may have clinical utility as immunomodulating agent, e.g., to suppress transplant rejection or treat autoimmune diseases...

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..Mitosis in some cell types may proceed without ATM activation and H2AX phosphorylation...

..mitoxantrone, may contribute to formation of DSBs and to triggering of apoptosis...

..The above effects should be taken into account when interpreting data obtained with the use of cells synchronized by inhibitors of DNA replication...

..The quality of the staining provided by this methodology is comparable to conventional cell staining in dye solutions...

..The ability to automatically and rapidly discriminate mitotic cells combined with the possibility of their subsequent identification by image analysis makes LSC the instrument of choice for the FLM analysis...

..The data suggest that frequency of activated ATM and phosphorylated H2AX molecules, per apoptotic cell, is comparable...

..Such specialization may facilitate heterophagic degradation of nucleic acids during apoptosis...

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..Interestingly, HU inhibits the constitutive ("programmed") level of ATM phosphorylation in untreated cells. However, DNA fragmentation during apoptosis activates ATM and dramatically increases level of H2AX phosphorylation...

..The respective kinetics of p53-Ser15 phosphorylation versus ATM and Chk2 activation suggest that in response to DNA damage by TPT or MXT, Chk2 rather than ATM mediates p53 phosphorylation...

..g., maximal pixel) offers unique advantage in studies of the response of different cell constituents in relation to cell cycle position...

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..Due to the low toxicity of BrU and BrdU, this approach can be used in analysis of gene transcription or DNA replication in vivo...

..The present approach offers a unique capability to accurately estimate the kinetics of cell transition to apoptosis, revealing the unbiased cumulative apoptotic index over a long time span after induction of apoptosis...

..Cytometric analysis of CHP and CAA provides the means to measure the effectiveness of exogenous factors, which either through lowering aerobic metabolism or neutralizing radicals may protect DNA from such damage...

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..3 microM CEP for 24 h. The data strongly suggest that the scavenging properties of CEP provide a protection of DNA from the radicals generated endogenously during oxidative metabolism...

..Since no significant degradation of HA upon its exposure in tissue culture medium to H2O2 was detected the scavenging may occur intracellularly...

..The data indicate that supravital use of Ho 42, DRAQ5, and DCV induces various degrees of DDR, including activation of ATM, Chk2 and p53, which may have significant consequences on regulatory cell cycle pathways and apoptosis...

..These findings provide evidence for a previously unknown mechanism for the nuclear transport of RFC40 and also for a novel mechanism for cellular proliferation...

..Protocols are given for analysis of fixed cells, tissue samples, live cells (for subsequent sorting and culture), archival paraffin-embedded tissue, and apoptotic cells...

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..When DNA content measurements are coupled with immunostaining for cyclin expression, it is possible to reliably detect even more stages in progression through the cell cycle...

..Therefore, excess of chlorophyll in the diet, by sequestering aromatic mutagens (or antitumor drugs with a heterocyclic structure, if taken orally), may inhibit their accessibility to cells, thereby reducing their activity...

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..Because neither induction of H2AX phosphorylation nor apoptosis were observed at equimolar concentrations of ASA, the NO moiety attached to ASA appeared to mediate these effects...

..The observed H2AX phosphorylation in lymphocytes may reflect their DNA damage by the superoxide ions propagating from the neighboring granulocytes and/or monocytes...

..The present findings, however, demonstrate the feasibility of assessment of DDR during the treatment of leukemias with drugs targeting DNA...

..Detection of DNA damage in tumour cells during radio- or chemotherapy may provide an early marker predictive of response to treatment...

..In each case, these compounds have independent activities which may partly contribute to the biological activity of PC-SPES...

..The cytometric assessment of CS-induced DDR provides a means to estimate the genotoxicity of CS and to explore the mechanisms of the response as a function of cell cycle phase and cell type...

..Its cellular level appeared to be under control during the cell cycle progression of normal lymphocytes, but was found to be deregulated in HL-60 cells which may be associated with the tumor transformed cell phenotypes...

..The method offers a rapid and simple means of detecting genotoxic insult on cells...

..These post-mitotic cells were subsequently arrested in G(1) but maintained normal elongated morphology and were viable for at least 21 days. We conclude that in SMC PTX causes post-mitotic cell cycle arrest rather than cell death...

..Induction of gammaH2AX in G(1), G(2) and M is likely a response to the primary DSBs generated during UV exposure and/or DNA repair. It is unclear why the latter process was more pronounced in HeLa than in HL-60 cells...

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..When the primary drug-induced lesions do not involve ds DNA breaks, but ds DNA breaks are formed during DNA repair, as in the case of CP, analysis of H2AX phosphorylation may reflect extent of the repair process...

..The subsequent induction of apoptosis appears to be facilitated by functional p53. CPFX may therefore have a potential use as a chemotherapeutic agent in the treatment of lymphoblast-derived cancer...

..Our data suggest that the antileukemic activities of arsenic may be a result of mitotic arrest which culminates in apoptosis...

..The data also indicate that regardless whether treated with inhibitors of topo1 or topo2, at comparable levels of dsDNA breaks, the cells replicating DNA have a higher proclivity to undergo apoptosis compared to G1 or G2/M cells...

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..These analytical capabilities of LSC may be helpful not only to score MN but also to study mechanisms by which clastogenic agents induce MN...

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..The present assay to detect and measure DSBs induced by tobacco products complements other mutagenicity assays and may be applied to test potential carcinogens in other products...

..Given the multiplicity of commercially available reagents it is possible to label DNA strand breaks with a dye of any fluorescence color and excitation wavelength...

..These findings provide mechanistic explanation for LA activity and suggest that it may be considered as a chemopreventive agent and its anticancer properties should be further explored...

..Intensity of cellular green (FITC) and red (PI) fluorescence is measured by flow cytometry and bivariate analysis of the data is used to correlate the presence of DSBs with the cell cycle phase...

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..Particular attention is given to the use of multiparameter cytometry in analysis of the mechanism by which antitumor drugs affect cell cycle progression and induce cell cycle phase-specific apoptosis...

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..This unit provides full details on the correct use of acridine orange together with an excellent discussion of the pitfalls and problems...

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..Semin Hematol 38:179-193...

..A caution, therefore, should be exercised in interpreting data of the experiments in which PRT is used with the intention to specifically prevent activation of NF-kappaB...

..The present study demonstrates, however, that depending on prevailing conditions, COX-2-derived prostanoids may also inhibit VSMC proliferation...

..The phenomenon offers novel experimental model for induction of polyploidy and provides alternative to cytocholasin B to prevent cytokinesis in the mutagenicity cytokinesis-blocked micronucleus (CBMN) assay...

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..Each measured cell can be relocated for imaging or measurement after subsequent staining with other probes...

..Because many aspects of DNA content analysis are common to protocols in UNITS 7.3, 7.6, 7.16, 7.20, 7.23, & 7.25, certain parts of this unit provide information redundant with commentaries in these units...

..abstract_text> ..

..it is expected that such a combined analysis, especially involving estimates of the G1 and G2 cyclins, will be of high prognostic value, and will be used during treatment, to monitor tumor response. ..

..This in turn will help assess the cellular features predicting susceptibility to particular drugs and aid in new drug design...

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