[Show abstract][Hide abstract] ABSTRACT: Aims. Normal human pregnancy is a complex process of many immunoregulatory mechanisms which protect fetus from the activation of the maternal immune system. The aim of the study was to investigate the apoptosis of lymphocytes in peripheral blood of normal pregnant patients and healthy nonpregnant women. Methods. Sixty pregnant women and 17 nonpregnant women were included in the study. Lymphocytes were isolated and labeled with anti-CD3, anti-CD4, and anti-CD8 monoclonal antibodies. Apoptosis was detected by CMXRos staining and analyzed using the flow cytometric method.
Results. We found significantly higher apoptosis of total lymphocytes in peripheral blood of pregnant patients when compared to healthy nonpregnant women. The percentage of apoptotic T CD3+CD8+ cells in the first trimester was significantly higher when compared to the third trimester of normal pregnancy. The ratio of T CD3+CD4+ : T CD3+CD8+ apoptotic lymphocytes was significantly lower in the first trimester when compared to other trimesters of pregnancy and to both of the phases of the menstrual cycle. Conclusions. The higher apoptosis of T CD3+CD8+ lymphocytes and the lower ratio of T CD3+CD4+ : T CD3+CD8+ apoptotic cells in the first trimester of normal pregnancy may suggest a higher susceptibility of T CD3+CD8+ cells for apoptosis as a protective mechanism at the early stage of pregnancy.
Full-text · Article · Jul 2014 · Research Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: Benzalkonium chloride (BAK) is a cationic detergent with a very slow turnover. Because of its strong antibacterial activities, BAK is widely used especially in dentistry and ophthalmology. It is the most commonly used preservative in topical ophthalmic medications. Due to chronicity and widespread use of such treatments, BAK's side effects are of great importance. BAK toxicity for adherent cells, probably related to its pro-oxidative activities, is time- and dose-dependent. Although lymphocytes often infiltrate superficial eye tissues, the BAK influence on them is yet to be established. The aim of this study was to check BAK cytotoxicity on T lymphocytic Jurkat line cells and to verify the suggestion that BAK can induce G2M cell blocks. A dose- and time-dependent cytotoxic effect of BAK on lymphoid cells in relatively low concentrations was shown in this study. In lower concentrations, it shows a moderate apoptotic and minimal antiproliferative effect on Jurkat cells, while in higher concentrations it shows a rapid necrotic effect. No G2M cell blocks were observed. Our findings could suggest lymphoid dysfunction during intensive, prolonged topical BAK treatment, even at dosages relatively non-toxic to epithelial eye cells.
[Show abstract][Hide abstract] ABSTRACT: Potex constitutes a potato fiber preparation widely used as an ingredient to meat and bakery products which thermal treatment results in creation of new compounds. Melanoidins are high molecular weight brown end products of Maillard reaction, and few data presenting tumor cell growth inhibiting activity of melanoidins have been reported. Thus, in present study we utilized water extract of Potex roasted (180 °C for 2 h), whose chemical characterization revealed the presence of melanoidin complexes. Heated Potex extract inhibited C6 glioma cell proliferation in a dose-dependent manner measured by MTT method. High molecular weight components present in initial extract were responsible for stronger antiproliferative effect compared with low molecular weight fraction. Impaired MAPK (mitogen-activated protein kinase) and Akt signaling was found in cells treated with the extract. Moreover, flow cytometry analyses revealed the extract to induce G1/S arrest in glioma cells. Simultaneously, Western blot analysis showed elevated levels of p21 protein with concomitant decrease of cyclin D1. In conclusion, observed antiproliferative activity of melanoidins present in heated Potex was linked to disregulated MAPK and Akt signaling pathways, as well as to cell cycle cessation. These results suggest potential application of Potex preparation as a functional food ingredient and chemopreventive agent.
No preview · Article · Feb 2011 · Journal of Agricultural and Food Chemistry
[Show abstract][Hide abstract] ABSTRACT: Activation of caspases is a hallmark of apoptosis. Several methods, therefore, were developed to identify and count the frequency of apoptotic cells based on the detection of caspases activation. The method described in this chapter is based on the use of fluorochrome-labeled inhibitors of caspases (FLICA) applicable to fluorescence microscopy, and flow- and image-cytometry. Cell-permeant FLICA reagents tagged with carboxyfluorescein or sulforhodamine when applied to live cells in vitro or in vivo, exclusively label cells that are undergoing apoptosis. The FLICA labeling methodology is simple, rapid, robust, and can be combined with other markers of cell death for multiplexed analysis. Examples are presented on FLICA use in combination with a vital stain (propidium iodide), detection of the loss of mitochondrial electrochemical potential, and exposure of phosphatidylserine on the outer surface of plasma cell membrane using Annexin V fluorochrome conjugates. Following cell fixation and stoichiometric staining of cellular DNA, FLICA binding can be correlated with DNA ploidy, cell cycle phase, DNA fragmentation, and other apoptotic events whose detection requires cell permeabilization. The "time window" for the detection of apoptosis with FLICA is wider compared to that with the Annexin V binding, making FLICA a preferable marker for the detection of early phase apoptosis and more accurate for quantification of apoptotic cells.
Full-text · Article · Jan 2011 · Methods in molecular biology (Clifton, N.J.)
[Show abstract][Hide abstract] ABSTRACT: The long-term-treatment of glaucoma with topical medications is associated with side effects involving cornea damage. We examined the effect of glaucoma topical medications (bimatoprost, travoprost, latanoprost, timolol, betaxolol, dorzolamide, brinzolamide, brimonidine) on growth of cells of three human epithelial corneal lines.
The cells were cultured in 8-chamber slides, treated with different concentrations of the medications, and fixed at 24, 48, and 72 h. Cell number on slides to estimate viability and growth curves, frequency of apoptosis (FLICA and caspase-3 activation probes), and proliferation (BrdU incorporation assay) were measured by laser scanning cytometry (LSC).
Depending on concentration all examined medications induced cell necrosis or apoptosis and suppressed proliferation. Significant variability in proliferation and apoptosis was observed within the same cultures depending on local cell density, with cells in high density areas being more resistant. The data indicate that commonly used topical medications exert cytostatic and cytotoxic effects in cultures of corneal cells and suggest that caution should be exercised in their use, particularly, when the corneal diseases are accompanied by cell proliferation and regeneration, in long-term-treatment.
The present approach of using LSC makes it possible to assess and compare cytostatic and cytotoxic effects of different topical medications on the respective target cells.
Full-text · Article · Nov 2009 · Cytometry Part B Clinical Cytometry
[Show abstract][Hide abstract] ABSTRACT: B-cell chronic lymphocytic leukaemia (B-CLL) has been described as the progressive accumulation of mature-appearing B cells in peripheral blood and bone marrow, resulting from failed apoptosis rather than from alterations in cell cycle regulation. Recent investigations suggest that high WBC and lymphocyte counts result not only from defects in apoptosis, but also from cell proliferation. In this study, we aimed to examine the process of apoptosis in B-CLL patients before and during anti-cancer therapy, to answer the question of whether this parameter would presage the response to treatment and the clinical course of the disease. We found that ex vivo spontaneous apoptosis was higher in advanced-stage (III-IV acc. Rai) than in early-stage (I-II acc. Rai) patients. In I-II Rai stage patients the percentage of ex vivo apoptotic cells after chemotherapy was higher than that of apoptotic cells prior to treatment, whereas in III-IV Rai stage patients the percentage of ex vivo apoptotic cells after chemotherapy was lower than that of apoptotic cells before the anti-cancer therapy. The results of our study, in the context of the cited literature, suggest a relationship between higher ex vivo spontaneous apoptosis before treatment in advanced-stage patients with a higher proliferation of leukaemic cells and poor outcome.
No preview · Article · Jul 2008 · Oncology Reports
[Show abstract][Hide abstract] ABSTRACT: Unsatisfactory effects of treatment of laryngeal carcinoma patients stimulate the clinicians as well as researchers to develop new more effective treatment models and to find new reliable prognostic factors. The aim of the present study was the evaluation of the use of primary cell cultures of the laryngeal carcinoma and laser scanning cytometry (LSC) in the assessment of tumor reactivity to cisplatinum. Nineteen primary cultures of laryngeal carcinoma cells established from fragments of laryngeal carcinoma infiltrations were cultured with or without cisplatin, stained with monoclonal antibodies against P53 and BCL-2 proteins and analyzed by LSC. Cisplatin added to the culture medium leads to the significant increase of P53 expression and decrease of BCL-2 expression. Moreover, changes of P53 and BCL-2 expressions were significantly correlated. Our findings of apoptosis regulatory mechanisms could be useful in patient qualification for the chemotherapeutic follow-up treatment.
Full-text · Article · Jun 2008 · Folia Histochemica et Cytobiologica
[Show abstract][Hide abstract] ABSTRACT: Propionibacterium acnes (P. acnes) has been implicated in the pathogenesis of acne vulgaris which is the most common cutaneous disorder. It has a proinflammatory activity and takes part in immune reactions modulating the Th1/Th2 cellular response. The exposure of dendritic cells (DCs) to whole bacteria, their components, cytokines or other inflammatory stimuli and infectious agents induces differentiation from immature DCs into antigen-presenting mature DCs. The aim of the study was to evaluate the capability of P. acnes to induce the maturation of DCs. We stimulated monocyte derived dendritic cells (Mo-DCs) from acne patients with various concetrations of heat-killed P. acnes (10(6)-10(8) bacteria/ml) cultured from acne lesions. The results showed an increase in CD80+/CD86+/DR+ and CD83+/CD1a+/DR+ cells percentage depending on the concetration of P. acnes. The expression of CD83 and CD80 (shown as the mean fluorescence intensity - MFI) increased with higher concetrations of P. acnes. There were also significant correlations between MFI of CD83, CD80, CD86 and concetration of P. acnes. The study showed that P. acnes in the concetration of 10(8) bacteria/ml is most effective in the induction of Mo-DCs maturation. Futher studies concerning the influence on the function of T cells are needed.
Full-text · Article · Feb 2008 · Folia Histochemica et Cytobiologica
[Show abstract][Hide abstract] ABSTRACT: Common methods applicable to flow cytometry make it possible to: (1) identify and quantify dead or dying cells, (2) reveal a mode of cell death (apoptosis or necrosis), and (3) study mechanisms involved in cell death. Gross changes in cell morphology and chromatin condensation, which occur during apoptosis, can be detected by analysis with laser light beam scattering. Early events of apoptosis, dissipation of the mitochondrial transmembrane potential and caspase activation, can be detected using either fluorochrome reporter groups or appropriate antibodies. Exposure of phosphatidylserine on the exterior surface of the plasma membrane can be detected by the binding of fluoresceinated annexin V. Another apoptotic event, DNA fragmentation based on DNA content of cells with fractional ("sub-G1") or DNA strand-break labeling, TUNEL; or In Situ End Labeling, ISEL;. Still another hallmark of apoptosis is the activation of tissue transglutaminase (TGase), the enzyme that crosslinks protein and thereby makes them less immunogenic. The major advantage of flow cytometry in these applications is that it provides the possibility of multiparametric measurements of cell attributes.
No preview · Article · Jan 2008 · Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.]
[Show abstract][Hide abstract] ABSTRACT: Onconase (Onc), is a novel amphibian cytotoxic ribonuclease with antitumor activity, and is currently in a confirmatory phase III clinical trial for the treatment of malignant mesothelioma. It was recently reported that Rana pipiens oocytes contain still another ribonuclease, named Amphinase (Amph). Amph shows 38-40% amino acid sequence identity with onconase, presents as four variants varying between themselves from 87-99% in amino acid sequence identity and has a molecular mass approximately 13,000. In the present study we describe the effects of Amph on growth of several tumor cell lines. All four variants demonstrated cytostatic and cytotoxic activity against human promyelocytic HL-60-, Jurkat T-cell- and U-937 monocytic leukemia cells. The pattern of Amph activity to certain extent resembled that of Onc. Thus, cell proliferation was suppressed at 0.5-10.0 mug/ml (40-80 nM) Amph concentration with distinct accumulation of cells in G(1) phase of the cell cycle. In addition, the cells were undergoing apoptosis, which manifested by DNA fragmentation (presence of "sub-G1" cells, TUNEL-positivity), caspases and serine proteases activation as well as activation of transglutaminase. The cytostatic and cytotoxic effects of Amph required its ribonuclease activity: the enzymatically inactive Amph-2 having histidine at the active site alkylated was ineffective. The effectiveness and cell cycle specificity was generally similar for all four Amph variants and at the equimolar concentrations was somewhat more pronounced than that of Onc. The observed cytostatic and cytotoxic activity of Amph against tumor cell lines suggests that similar to Onc this cytotoxic ribonuclease may have antitumor activity and find an application in clinical oncology.
[Show abstract][Hide abstract] ABSTRACT: Onconase (ONC), an antitumor ribonuclease from oocytes of a frog Rana pipiens, capable of inducing apoptosis in many cell lines is synergistic with several other anticancer drugs. Since cytotoxic effects of numerous drugs are modulated by reactive oxygen intermediates (ROI), we have studied effects of ONC on the intracellular level of oxidants in several normal cell types as well as tumor cell lines. It is demonstrated for the first time that ONC substantially decreases the content of ROI in all cell lines studied. This effect depends on the ribonucleolytic activity of the enzyme and is due to both, decreased rate of ROI generation and accelerated rate of their degradation. Onconase decreases the mitochondrial transmembrane potential and consequently, generation of ATP. Simultaneously the enzyme decreases the expression of an antiapoptotic protein Bcl-2, and upregulates the proapoptotic Bax protein. These finding are consistent with the enzyme propensity to induce apoptosis. The observed antioxidant activity of ONC may be an important element of its cytotoxicity towards cancer cells. The enzyme seems to exert its biological activities by interfering with the redox system of cellular regulation.
No preview · Article · Oct 2007 · International Journal of Oncology
[Show abstract][Hide abstract] ABSTRACT: The cytometric methods for cell cycle analysis can be grouped into three categories. The first comprises methods that rely on a single time point measurement of the cell population. The second category is the methods that combine time-lapse measurements of populations of cells synchronized in the cycle or whose progression through the cycle was perturbed. Methods of the third category rely on analysis of DNA replication concurrent with measurement of DNA content. The time-lapse measurements of the cohort of 5'-bromo-2'-deoxyuridine (BrdUrd)-labeled cells allows one to estimate their rate of progression through different points of the cell cycle. Apoptotic cells often have fractional DNA content due to the fact that the fragmented (low MW) DNA undergoes extraction during the staining procedure. Some cells may also lose DNA (chromatin) by shedding apoptotic bodies. Only a fraction of DNA thus remains within apoptotic cells. The expression of proliferation-associated proteins often varies during the cell cycle, as well as is different in cycling and quiescent cells.
[Show abstract][Hide abstract] ABSTRACT: The laser scanning cytometer (LSC) is the microscope-based cytofluorometer that offers a plethora of analytical capabilities. Multilaser-excited fluorescence emitted from individual cells is measured at several wavelength ranges, rapidly (up to 5000 cells/min), with high sensitivity and accuracy. The following applications of LSC are reviewed: (1) identification of cells that differ in degree of chromatin condensation (e.g., mitotic or apoptotic cells or lymphocytes vs granulocytes vs monocytes); (2) detection of translocation between cytoplasm vs nucleus or nucleoplasm vs nucleolus of regulatory molecules such as NF-kappaB, p53, or Bax; (3) semiautomatic scoring of micronuclei in mutagenicity assays; (4) analysis of fluorescence in situ hybridization; (5) enumeration and morphometry of nucleoli; (6) analysis of phenotype of progeny of individual cells in clonogenicity assay; (7) cell immunophenotyping; (8) visual examination, imaging, or sequential analysis of the cells measured earlier upon their relocation, using different probes; (9) in situ enzyme kinetics and other time-resolved processes; (10) analysis of tissue section architecture; (11) application for hypocellular samples (needle aspirate, spinal fluid, etc.); (12) other clinical applications. Advantages and limitations of LSC are discussed and compared with flow cytometry.
Full-text · Article · Feb 2006 · Methods in Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: Trans arachidonic acid isomers (trans-AA) constitute a new group of trans fatty acids (trans-FA) generated in vivo via endogenous cis-trans isomerization stimulated by the NO2 radical. Because both NO2 and trans-FA have been implicated as causative factors in cancer, we studied the effect of the trans-AA isomers on proliferation and viability of human promyelocytic (HL-60) cells. The four trans arachidonic (trans-AA) acid isomers synthesized by us have been presently tested with respect to their competence to affect the proliferation and viability of human promyeolocytic HL-60 cells in culture. The data demonstrate that one of the isomers, 5,6-trans-AA, showed distinct activity by targeting cell progression through the cell cycle and inducing apoptosis. The effects were time- and concentration-dependent: the cytostatic effect of 5E-AA was observed at 10 microM following 72 h of treatment. This effect was manifested as a perturbation of cell progression through G1 phase, indicating the 'on' activation of the G1 checkpoint as evidenced by the flow- and laser scanning-cytometry techniques. Apoptotic cells were identified by comparison of their morphology, DNA fragmentation, caspase activation and collapse of mitochondrial potential with control cells. These observations suggested that 5E-AA induced a mitochondrial pathway of apoptosis. There was no evidence of cell-cycle phase specificity in induction of apoptosis by 5E-AA, as the cells showing highly fragmented DNA or caspase-3 activation were distributed in all phases of the cycle. The data suggest that 5E-AA may have at least two targets: one that is cell-cycle specific and associated with the observed arrest in the G1 phase and another, unrelated to the cell cycle, which is responsible for triggering apoptosis indiscriminately, regardless of cycle phase I.
No preview · Article · Dec 2005 · International Journal of Oncology
[Show abstract][Hide abstract] ABSTRACT: In this study, we show that protein phosphatase-1 (PP1) inhibitor-3 (Inh3) is localized to the nucleoli and centrosomes in interphase HEK 293 cells. Inh3 exhibited a specific co-localization to the nucleoli with PP1gamma1, and to the centrosomes with PP1alpha. These findings indicate that Inh3 may act as a modulator of PP1 functions in the processes of cytokinesis, as well as of nucleolar events. The specificity of the interaction of Inh3 with the PP1 isoforms was also demonstrated in vitro, where Inh3 co-immunoprecipitated with PP1alpha and PP1gamma1, but not with PP1beta. The nuclear localization signal of Inh3 was identified as a N-terminal basic cluster (33RKRK36), while nucleolar localization was shown to be dependent on a C-terminal basic cluster (94HRKGRRR100). The importance of the individual basic residues was quantitatively assessed by site-directed mutagenesis and a novel use of laser scanning cytometry.
No preview · Article · Dec 2005 · Archives of Biochemistry and Biophysics
[Show abstract][Hide abstract] ABSTRACT: The regulatory subunit (RIalpha) of cAMP-dependent Protein Kinase A (PKA) is overexpressed in a variety of tumors and carcinomas such as renal cell carcinomas, pituitary tumors of the rat, malignant osteoblasts, colon carcinomas, serous ovarian tumors and primary human breast carcinomas. However, the direct relation between overexpression of RIalpha and malignancy is still unclear. We have recently identified a novel interaction between RIalpha and RFC40, the second subunit of Replication Factor C (RFC), and have demonstrated that this interaction may be associated with cell survival. Coincidentally, RFC40 is overexpressed in gestational trophoblastic diseases such as choriocarcinomas. This study was undertaken to investigate a possible functional role for both these proteins together, in DNA replication and cellular proliferation. In the course of this study, a nonconventional nuclear localization signal was identified for RIalpha. Nuclear transport of RFC40 was found to be dependent on RIalpha, and this transport appeared to be a crucial step for cell cycle progression from G1 to S phase. Impairment in the nuclear transport of RFC40 by RIalpha arrested cells in G1 phase. These findings provide evidence for a previously unknown mechanism for the nuclear transport of RFC40 and also for a novel mechanism for cellular proliferation.
Full-text · Article · May 2005 · Cancer biology & therapy
[Show abstract][Hide abstract] ABSTRACT: Paclitaxel (PTX), a microtubule-active drug, causes mitotic arrest leading to apoptosis in certain tumor cell lines. Here we investigated the effects of PTX on human arterial smooth muscle cell (SMC) cells. In SMC, PTX caused both (a) primary arrest in G(1) and (b) post-mitotic arrest in G(1). Post-mitotic cells were multinucleated (MN) with either 2C (near-diploid) or 4C (tetraploid) DNA content. At PTX concentrations above 12 ng/ml, MN cells had 4C DNA content consistent with the lack of cytokinesis during abortive mitosis. Treatment with 6-12 ng/ml PTX yielded MN cells with 2C DNA content. Finally, 1-6 ng/ml of PTX, the lowest concentrations that affected cell proliferation, caused G(1) arrest without multinucleation. It is important that PTX did not cause apoptosis in SMC. The absence of apoptosis could be explained by mitotic exit and G(1) arrest as well as by low constitutive levels of caspase expression and by p53 and p21 induction. Thus, following transient mitotic arrest, SMC exit mitosis to form MN 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.
[Show abstract][Hide abstract] ABSTRACT: Described are four widely used procedures to analyze the cell cycle by flow cytometry. The first two are based on univariate analysis of cellular DNA content following cell staining with either propidium iodide (PI) or 4',6'-diamidino-2-phenylindole (DAPI) and deconvolution of the cellular DNA content frequency histograms. This approach reveals distribution of cells in three major phases of the cycle (G1 vs S vs G2/M) and makes it possible to detect apoptotic cells with fractional DNA content. The third approach is based on the bivariate analysis of DNA content and proliferation-associated proteins. The expression of cyclin D, cyclin E, cyclin A, or cyclin B1 vs DNA content is presented as an example. This approach allows one to distinguish, for example, G0 from G1 cells, identify mitotic cells, or relate expression of other intracellular proteins to the cell cycle position. The fourth procedure relies on the detection of 5'-bromo-2'-deoxyuridine (BrdU) incorporation to label the DNA-replicating cells.
Full-text · Article · Feb 2004 · Methods in Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: Common assays of drug-induced cytotoxicity on adherent cells rely on cell trypsinization followed by count of live and dead cells. To estimate the cell cycle effects, cellular DNA content is analyzed by flow cytometry. This procedure is laborious and time consuming. The alternative viability assays, e.g., based on reduction of 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide, although rapid and convenient, do not provide information about individual cells or cell cycle effects and may be biased by growth imbalance.
The bladder carcinoma T-24 cells were seeded onto eight-chamber microscope slide-based tissue culture vessels. The novel antitumor drug, the bis-intercalating anthracycline WP631, was administered at various concentrations to different chamber cultures on the same slide; the control cultures were left untreated. After 24, 48, and 72 h, the cultures were fixed, and cellular DNA was stained with 4,6-diamidino-2-phenyl indole (DAPI). The slides were scanned by laser scanning cytometry (LSC) to obtain the number of attached cells per culture chamber and reveal their cell cycle distribution.
The cell growth and viability plots in the absence and presence of WP621 were constructed from the frequency of the attached cells per chamber. A 50% reduction in cell number was observed at the 75 nM concentration of WP321. Mitotic and postmitotic cells were identified based on high intensity of maximal pixel of DAPI fluorescence. An increase in proportion of cells in G2 was seen at 75-300 nM of WP631. Relatively few (<12%) apoptotic cells, identified by the presence of DNA strand breaks, remained attached in the WP631-treated cultures.
Because late apoptotic cells detach during culturing, the cells that remain attached in the multi-chamber cultures represent predominantly live cells; the deficit in their number compared with the untreated cultures, recorded by LSC during scanning, provides information about the degree of cytostatic and cytotoxic effects of the studied drug. The possibility to demonstrate the cell cycle distribution, including a distinction between G2 and M cells, provides an additional advantage of this assay. Other parameters that may be associated with the cell cycle perturbation or with induction of apoptosis also can be measured in the same cultures by using the multiparameter capabilities of LSC. Each measured cell can be relocated for imaging or measurement after subsequent staining with other probes.