Biomedical subjects

Piotr Pozarowski

Publications and source records attributed to Piotr Pozarowski.

10 recordsLinked to original sources

PubMed2006

Laser scanning cytometry: principles and applications.

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.

Cell Nucleus↗

PubMed2005

Protein phosphatase-1 inhibitor-3 is co-localized to the nucleoli and centrosomes with PP1gamma1 and PP1alpha, respectively.

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.

Binding Sites↗

PubMed2005

RIalpha influences cellular proliferation in cancer cells by transporting RFC40 into the nucleus.

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.

Active Transport, Cell Nucleus↗

PubMed2005

5E, 8Z, 11Z, 14Z-eicosatetraenoic acid, a novel trans isomer of arachidonic acid, causes G1 phase arrest and induces apoptosis of HL-60 cells.

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.

Apoptosis↗

PubMed2004

Paclitaxel induces primary and postmitotic G1 arrest in human arterial smooth muscle cells.

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.

Aorta↗

PubMed2004

Simple, semiautomatic assay of cytostatic and cytotoxic effects of antitumor drugs by laser scanning cytometry: effects of the bis-intercalator WP631 on growth and cell cycle of T-24 cells.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Antineoplastic Agents↗

PubMed2004

Analysis of cell cycle by flow cytometry.

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.

Apoptosis↗

PubMed2003

NF-kappaB inhibitor sesquiterpene parthenolide induces concurrently atypical apoptosis and cell necrosis: difficulties in identification of dead cells in such cultures.

BACKGROUND: Apoptosis and necrosis ("accidental cell death") are distinct modes of cell death. The feature that often distinguishes apoptotic from necrotic cells is preservation of the plasma membrane integrity, reflected by ability of the former cells to exclude cationic dyes such as propidium iodide (PI) for a certain length of time. During necrosis, the plasma membrane is rapidly ruptured and necrotic cells stain intensely with PI. While studying cytostatic effects of the anti-inflammatory sesquiterpene parthenolide (PRT), we have noticed that, concurrent with apoptosis, the cells were dying by necrosis in the same cultures. Furthermore, because apoptosis was atypical, reflected by rapid loss of plasma membrane integrity, it was difficult to distinguish apoptotic from necrotic cells based on this feature. METHODS: Three methods were used to distinguish apoptosis from necrosis: (a) HL-60 cells treated with PRT were subjected to analysis of caspases activation using antibody that detects activated (cleaved) caspase-3, (b) apoptotic cells were identified by binding of fluorochrome-labeled inhibitor of caspases FAM-VAD-FMK combined with the PI exclusion assay, and (c) cellular DNA and RNA were differentially stained with acridine orange (AO). RESULTS: Apoptotic cells were characterized by (a) caspase-3 activation detected immunocytochemically and (b) binding of FAM-VAD-FMK followed by or concurrent with (c) loss of ability to exclude PI, (d) deficit in DNA content, and (e) relatively little changed RNA content. Necrotic cells showed (a) no evidence of caspase-3 activation, (b) no binding of FAM-VAD-FMK, (c) inability to exclude PI, (d) rapid loss of RNA, and (e) unchanged DNA content. CONCLUSIONS: Identification of apoptotic cells versus necrotic cells was possible either based on the evidence of caspase-3 activation, by labeling with FAM-VAD-FMK combined with PI or by differential staining of cellular DNA and RNA with AO. The data indicate that plasma membrane appears to be one of the targets of PRT, because its integrity is lost very early during cell death, which is reflected by atypical apoptosis and by primary necrosis (lysis of the membrane).

Acridine Orange↗

PubMed2002

TH1/TH2 balance in the subretinal fluid of patients with rhegmatogenous retinal detachment.

BACKGROUND: The pathogenesis of rhegmatogenous retinal detachments remains unclear, but some data suggest immune system involvement. Cytokines are substances secreted by cells in order to modulate the function of other cells. They exert primarily local effects. Because the TH1/TH2 balance is crucial in the immune response, the predominance of one cytokine profile in subretinal fluid may be of particular importance in understanding its pathogenesis. MATERIAL/METHODS: We measured the titres of IL-10 and tIL-12 in the subretinal fluid of 36 rhegmatogenous retinal detachment patients using ELISA assays. RESULTS: The mean level of IL-10 and tIL-12 in the subretinal fluid was 4.75 pg/ml and 106.5 pg/ml, respectively. CONCLUSIONS: It would appear that IL-10 and IL-12 are produced by cells in subretinal fluid, and their presence may be evidence for an existing inflammatory process. Despite the tendency to decreased cytokine concentration over time and the tendency to higher concentrations of IL-10 and tIL-12 when the detachment of the retina is more extensive, no predominance of TH1- or TH2-type response was observed.

Adolescent↗

PubMedDate not supplied

Cell cycle effects and caspase-dependent and independent death of HL-60 and Jurkat cells treated with the inhibitor of NF-kappaB parthenolide.

The sesquiterpene parthenolide (PRT) is an active component of Mexican-Indian medicinal plants and also of the common herb of European origin feverfew. PRT is considered to be a specific inhibitor of NF-kappaB. Human leukemic HL-60, Jurkat, and Jurkat IkappaBalphaM cells, the latter expressing a dominant-negative IkappaBalpha and thus having non-functional NF-kappaB, were treated with PRT and activation of caspases, plasma membrane integrity, DNA fragmentation, chromatin condensation (probed by DNA susceptibility to denaturation), and changes in cell morphology were determined. As a positive control for apoptosis cells were treated with topotecan (TPT) and H2O2. At 2-8 microM concentration PRT induced transient cell arrest in G2 and M followed by apoptosis. A narrow range of PRT concentration (2-10 microM) spanned its cytostatic effect, induction of apoptosis and induction of necrosis. In fact, necrotic cells were often seen concurrently with apoptotic cells at the same PRT concentration. Atypical apoptosis was characterized by loss of plasma membrane integrity very shortly after caspases activation. In contrast, a prolonged phase of caspase activation with preserved integrity of plasma membrane was seen during apoptosis induced by TPT or H2O2. Necrosis induced by PRT was also atypical, characterized by rapid rupture of plasma membrane and no increase in DNA susceptibility to denaturation. Using Jurkat cells with inactive NF-kappaB we demonstrate that cell cycle arrest and the mode of cell death induced by PRT were not caused by inhibition of NF-kappaB. The data suggest that regardless of caspase activation PRT targets plasma membrane causing its destruction. 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.

Anti-Inflammatory Agents, Non-Steroidal↗