Piotr Pozarowski

Interactions of fluorochrome-labeled caspase inhibitors with apoptotic cells: A caution in data interpretation

Fluorochrome‐labeled inhibitors of caspases (FLICA, e.g., FAM‐VAD‐FMK, FITC‐VAD‐FMK) have been designed as affinity labels of the enzyme active center of caspases Their binding by apoptotic cells was interpreted as reflecting activation of caspases. We have recently observed, however, that their binding is more complex and may involve additional mechanisms. Our goal in this study was to clarify the ongoing utility of these probes.

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.

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 G1 and (b) post-mitotic arrest in G1. Post-mitotic cells were multinucleated (MN) with either 2C (near-diploid) or 4C (tetraploid) DNA content. At PTX concentrations above12 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 G1 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 G1 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 G1 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.

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

The regulatory subunit (RIα) 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 RIα and malignancy is still unclear. We have recently identified a novel interaction between RIα 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 non-conventional nuclear localization signal was identified for RIα. Nuclear transport of RFC40 was found to be dependent on RIα, 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 RIα 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.

Flow Cytometry of Apoptosis

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.

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

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.

Cell Cycle Effects and Caspase-Dependent and Independent Death of HL-60 and Jurkat Cells Treated with the Inhibitor of NF-6B 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-6B. Human leukemic HL-60, Jurkat, and Jurkat IκB·M cells, the latter expressing a dominant-negative IκB· and thus having non-functional NF-6B, 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 μM concentration PRT induced transient cell arrest in G2 and M followed by apoptosis. A narrow range of PRT concentration (2–10 μM) 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-κB we demonstrate that cell cycle arrest and the mode of cell death induced by PRT were not caused by inhibition of NF-κB. 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-κB.

Laser scanning cytometry: principles and applications-an update.

Laser scanning cytometer (LSC) is the microscope-based cytofluorometer that offers a plethora of unique analytical capabilities, not provided by flow cytometry (FCM). This review describes attributes of LSC and covers its numerous applications derived from plentitude of the parameters that can be measured. Among many LSC applications the following are emphasized: (a) assessment of chromatin condensation to identify mitotic, apoptotic cells, or senescent cells; (b) detection of nuclear or mitochondrial translocation of critical factors such as NF-κB, p53, or Bax; (c) semi-automatic scoring of micronuclei in mutagenicity assays; (d) analysis of fluorescence in situ hybridization (FISH) and use of the FISH analysis attribute to measure other punctuate fluorescence patterns such as γH2AX foci or receptor clustering; (e) enumeration and morphometry of nucleoli and other cell organelles; (f) analysis of progeny of individual cells in clonogenicity assay; (g) cell immunophenotyping; (h) imaging, visual examination, or sequential analysis using different probes of the same cells upon their relocation; (i) in situ enzyme kinetics, drug uptake, and other time-resolved processes; (j) analysis of tissue section architecture using fluorescent and chromogenic probes; (k) application for hypocellular samples (needle aspirate, spinal fluid, etc.); and (l) other clinical applications. Advantages and limitations of LSC are discussed and compared with FCM.

All that glitters is not gold: all that FLICA binds is not caspase. A caution in data interpretation--and new opportunities.

variety of cytometric methods that found application in cellnecrobiology have been developed during the past two decades(review, 1). One such methodology is based on use of thefluorochrome-labeled inhibitors of caspases (FLICA; alsoknown under names CaspaTag, CaspACE, CaspGLOW, orFLIVO). FLICA were designed as affinity ligands to enzymeactive centers to detect activation of caspases (2–4). Throughthe reactive fluoromethylketone (fmk) moiety they covalentlybind to cysteine of the active center of caspase to form a thio-methyl ketone and thereby irreversibly inactivate the targetenzyme (4,5). The specificity towards individual caspases isprovided by the sequence of amino acids in the tetra-peptidemoiety (5). Carboxyfluorescein (FAM) (1–4), fluorescein(FITC) (6), or sulforhodamine serves as a florescent tag toreport its binding and localization in the cell. FLICA ligandshave proven to be convenient and reliable reporters of caspaseactivation and induction of apoptosis (1–4,6).Using FLICA reagents we observed that their binding tocells undergoing apoptosis was not fully consistent with theexpected specificity, namely, it was not restricted to enzymaticcenters of respective activated caspases (7). We postulated,therefore, that FLICA binding sites may involve other cell con-a minor fraction of total FLICA binding to apoptotic could beattributed to its interaction with the respective caspases (7).The elegant studies of Kuzelova et al. in this issue of Cyto-metry (8) provide further evidence of the contribution of cellconstituents other than caspases in FLICA binding. Theauthors observed that extent of staining of apoptotic cells withFAM-DEVD-fmk was minimally changed after pretreatmentwith the unlabeled z-DEVD-fmk under conditions when cas-pase-3 activity was inhibited. Yet, FAM-DEVD-fmk boundirreversibly to large subunit of caspase-3 and its binding wasprevented by z-DEVD-fmk, as detected by Western blotting.FLICA, thus, binds to the enzymatic active center of their re-spective activated caspases, but this contribution is minimalwith respect to the overall cell staining. While the extent ofFLICA binding by cell constituents other than caspases mayvary between cell types and may also depend on inducer ofapoptosis, the data of Kuzelova et al. (8) and our findings (7)reveal that specificity of staining of individual caspases withFLICA is far from that of the originally anticipated or adver-tised by the vendors. These findings (7,8) call for re-examina-tion of interpretation of the data obtained with these reagents.While FLICA binding reports caspase activation and is amarker of apoptosis, the overall fluorescence intensity of apo-ptotic cell labeled with this ligand does not represent its bind-ing to enzyme active center of caspases. The same cautionshould be exercised interpreting data obtained with the use of

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

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.