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Biomedical subjects

Z Darzynkiewicz

Publications and source records attributed to Z Darzynkiewicz.

At least 73 records · Page 4Linked to original sources
PubMed1999

Laser-scanning cytometry: A new instrumentation with many applications.

The laser-scanning cytometer (LSC) is a microscope-based cytofluorometer which has attributes of both flow and image cytometry. Laser-excited fluorescence emitted from fluorochromed individual cells on a microscope slide is measured at multiple wavelengths rapidly with high sensitivity and accuracy. Though the instrument has been available commercially for only 3 years, it is already used in a variety of different applications in many laboratories. This review focuses on the following unique analytical capabilities of LSC which complement those of flow cytometry and fluorescence image analysis: (a) the cells are positioned on slides during measurement so they may be examined repeatedly over time, a feature useful for studies of enzyme kinetics and other time-resolved processes; (b) sequential analysis of the same cells can be carried out using different immuno- or cytochemical stains or genetic probes, merging information on cell immunophenotype, cell functions, expression of particular proteins, DNA ploidy and cell cycle position, and/or cytogenetic profile for each measured cell; (c) any of the cells measured can be relocated to correlate with visual examination by fluorescence or brightfield microscopy or with any other parameter; (d) topographic distribution of fluorescence measurements within the cell, in cytoplasm vs nucleus, permits analysis of the translocation of regulatory molecules such as NFkappaB, p53, etc., and is essential for FISH analysis; (e) hyperchromicity of nuclear DNA as measured by maximal pixel fluorescence intensity allows one to identify cell types differing in degree of chromatin condensation such as mitotic or apoptotic cells; (f) analysis of tissue section architecture and of the constituents in transected cells within tissue sections by ratiometric assays normalized to DNA content extends applications of LSC in clinical pathology; (g) because cell loss during sample preparation and staining is minimal, samples with a paucity of cells can be analyzed; and (h) analyzed cells can be stored indefinitely, e.g., for archival preservation or additional analysis. Potential future applications of LSC are discussed.

Animals↗
PubMed1999

High affinity binding of fluorescein isothiocyanate to eosinophils detected by laser scanning cytometry: a potential source of error in analysis of blood samples utilizing fluorescein-conjugated reagents in flow cytometry.

BACKGROUND: In samples of peripheral blood cells processed using the commercial kits for detection of apoptosis based on DNA strand break labeling, a subpopulation of cells characterized by high green fluorescence, similar in intensity to that of apoptotic cells but more uniform, was consistently observed by flow cytometry. The labeled cells had no other features of apoptosis. The labeling was observed regardless of the fixative used and was evident in control samples lacking terminal deoxynucleotidyltransferase. Common to all the kits that generated this labeling pattern was the presence of fluorescein (f) conjugated reagents, f-dUTP, f-avidin, or f-antibody. METHODS: Laser scanning cytometry was used to identify the labeled cells and study the mechanism of labeling. Because it was suspected that the traces of unconjugated f-isothiocyanate (FITC) that may contaminate the reagents were responsible for the labeling, FITC binding affinity to white blood cells was studied. Gel electrophoresis was used to detect the presence of unconjugated FITC in the reagents. RESULTS: After staining with Giemsa, the strongly fluorescent objects were identified as eosinophils with normal morphology and no evidence of apoptosis. The fluorescence was localized exclusively within the cytoplasmic granules. Labeling of eosinophils was observed at 2 nM concentration of FITC, which was over three orders of magnitude lower than that needed to label neutrophils, monocytes, or lymphocytes. Gel electrophoresis of the f-conjugated reagents revealed only minor contamination with FITC. CONCLUSIONS: (1) Trace amounts of unconjugated FITC contaminating the reagents are adequate to strongly label eosinophils thereby introducing experimental bias in analysis of apoptosis and in other studies on blood cells utilizing f-labeled antibodies, e.g., in detecting cytokines. (2) FITC at concentration 2-500 nM can be used as a marker of eosinophiles; (3) Because of high affinity to FITC, eosinophiles (or the protein from these cells) may serve as a means of removing traces of unconjugated FITC from the reagents during their manufacture or prior to use.

Apoptosis↗
PubMed1999

Identification and characterization of endothelial glycoprotein Ib using viper venom proteins modulating cell adhesion.

The expression and function of a glycoprotein Ib (GPIb) complex on human umbilical vein endothelial cells (HUVECs) is still a matter of controversy. We characterized HUVEC GPIb using viper venom proteins: alboaggregins A and B, echicetin, botrocetin, and echistatin. Echicetin is an antagonist, and alboaggregins act as agonists of the platelet GPIb complex. Botrocetin is a venom protein that alters von Willebrand factor (vWF) conformation and increases its binding affinity for the GPIb complex. Echistatin is a disintegrin that blocks alphavbeta3. Echistatin, but not echicetin, inhibited the adhesion to vWF of Chinese hamster ovary (CHO) cells transfected with alphavbeta3. We found the following: (1) Binding of monoclonal antibodies against GPIbalpha to HUVECs was moderately increased after stimulation with cytokines and phorbol ester. Echicetin demonstrated an inhibitory effect. (2) Both echicetin and echistatin, an alphavbeta3 antagonist, inhibited the adhesion of HUVECs to immobilized vWF in a dose-dependent manner. The inhibitory effect was additive when both proteins were used together. (3) Botrocetin potentiated the adhesion of HUVECs to vWF, and this effect was completely abolished by echicetin, but not by echistatin. (4) CHO cells expressing GPIbalphabeta/IX adhered to vWF (in the presence of botrocetin) and to alboaggregins; GPIbalpha was required for this reaction. Echicetin, but not echistatin, inhibited the adhesion of cells transfected with GPIbalphabeta/IX to immobilized vWF. (5) HUVECs adhered strongly to immobilized vWF and alboaggregins with extensive spreading, which was inhibited by LJ1b1, a monoclonal antibody against GPIb. The purified alphavbeta3 receptor did not interact with the alboaggregins, thereby excluding the contribution of alphavbeta3 in inducing HUVEC spreading on alboaggregins. In conclusion, our data confirm the presence of a functional GPIb complex expressed on HUVECs in low density. This complex may mediate HUVEC adhesion and spreading on immobilized vWF and alboaggregins.

Animals↗
PubMed1999

Analysis of apoptosis by laser scanning cytometry.

Flow cytometry techniques that are widely used in studies of cell death, and particularly in the identification of apoptotic cells, generally rely on the measurement of a single characteristic biochemical or molecular attribute. These methods fail to recognize cell death lacking that attribute, as in some examples of atypical apoptosis. Since apoptosis was originally defined by morphologic criteria, we suggest that for any new cell system the cytometry-defined apoptosis be confirmed by morphologic examination. This quality assurance measure is now provided by laser scanning cytometry (LSC). LSC measurements of cell fluorescence are precise and highly sensitive, comparable to flow cytometry (FCM), and can be carried out on cells on slides, permitting cell by cell correlation of fluorescence cytometry with visual microscopic morphology. In this report we describe adaptations of various flow cytometry techniques for detection of apoptosis by laser scanning cytometry. We also describe features unique to LSC that are useful in recognizing apoptosis. Hyperchromicity of DNA, reflecting chromatin condensation, is evidenced by high maximal pixel values for fluorescence of the DNA-bound fluorochrome. Mitochondrial probes that have been adapted to LSC to measure the drop in mitochondrial transmembrane potential that occurs early in apoptosis include rhodamine 123, 3,3'-dihexiloxadicarbocyanine [DiOC6(3)], and the aggregate dye 5,5',6,6'tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1). The changes in plasma membrane phospholipids and transport function, also early in apoptosis, are probed by a combination of the fluoresceinated annexin V and DNA fluorochromes such as propidium or 7-aminoactinomycin D. We also review methods of detection of apoptosis based on analysis of DNA fragmentation and their application to clinical oncology. Visual examination of the presumed apoptotic cells detected by cytometry makes it possible to discriminate those that are genuine from monocytes/macrophages that have ingested nuclear fragments via apoptotic bodies. Applications of flow cytometry and laser scanning cytometry in analysis of cell death are discussed and their respective advantages and disadvantages compared.

Annexin A5↗
PubMed1999

Histone H3 phosphorylation in human monocytes and during HL-60 cell differentiation.

Phosphorylation of the nucleosome core histone H3 (H3) on Ser-10 is thought to be a prerequisite for chromatin condensation at mitosis. Although during interphase, cell differentiation, or mitogenic activation of quiescent cells, changes in chromatin structure that involve local chromatin condensation/decondensation also occur, little is known about H3 phosphorylation during these transitions. Using the recently developed sensitive marker to monitor H3 phosphorylation, namely, the mAb that recognizes the phosphorylated epitope of H3 (anti-H3-P mAb), the status of H3 phosphorylation was assayed in individual human lymphocytes after their mitogenic stimulation (G0 to G1 transition) and in human leukemic HL-60 cells induced to differentiate by all-trans-retinoic acid (RA), 1,25-dihydroxyvitamin D3 (vit D3), dimethyl sulfoxide (DMSO), or phorbol myristate acetate (PMA). Multiparameter flow cytometry was used to correlate H3 phosphorylation with cell cycle position. The specificity of the anti-H3-P mAb was confirmed by the loss of its binding following cell treatment with alkaline phosphatase. The presence of phosphorylated H3 was detected during interphase in HL-60 cells and in normal lymphocytes at a level severalfold lower than during mitosis. No significant changes in H3 phosphorylation were observed during lymphocyte stimulation. Unexpectedly, the level of H3 phosphorylation was over fourfold higher in monocytes than in lymphocytes or granulocytes from peripheral blood. The punctate pattern of labeling with anti-H3-P mAb in monocyte nuclei suggests that H3 is phosphorylated in small clusters of adjacent nucleosomes. Differentiation of HL-60 cells was accompanied by a rise in H3 phosphorylation, which was higher after induction by RA, vit D3, and PMA (approx. threefold) than after DMSO (approximately 20%). The data indicate that in addition to being a critical event during chromatin condensation at mitosis, H3 phosphorylation plays a role during chromatin changes accompanying differentiation of HL-60 cells, in particular, along the monocytic lineage. 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.

Antibodies, Monoclonal↗
PubMed1999

Cell cycle effects and control of gene expression by resveratrol in human breast carcinoma cell lines with different metastatic potentials.

Trans-resveratrol, a polyphenol present in red wines and various human foods, is an antioxidant also with reported chemopreventive properties. However, whether resveratrol may exert different effects in malignant cells with a common anatomical origin yet displaying different invasive characteristics is not known. Since invasiveness and metastasis are considered to be the most insidious and life-threatening aspects for all cancers, we compared the ability of resveratrol to control growth and cell cycle transition in the highly invasive MDA-MB-435 with the minimally invasive MCF-7 breast carcinoma cells. The data revealed that resveratrol exerted a greater inhibitory effect on the MDA-MB-435 cells. A diminution of percentage of cells in G1 phase and a corresponding accumulation of cells in S phase of the cell cycle was observed. We also studied the effect of resveratrol on a panel of MDA-MB-435 cells transfected with nm23-H1 and nm23-H2 genes, which have been suggested to play a role in controlling metastasis in breast cancer cells. These cells are designated as Vbeta, 1beta, 1Tbeta, 2beta, and 2Tbeta, respectively. The control Vbeta consists of MDA-MB-435 cells transfected with bacterial beta-glucuronidase. Cells labeled 1beta and 1Tbeta correspond to those carrying beta-glucuronidase and overexpressed wild-type (His118) or mutant (Tyr118, catalytically inactive) nm23-H1 genes. The 2beta and 2Tbeta refer to cells transfected with wild-type and mutant nm23-H2 genes. The responses of these cells to resveratrol were assessed by measuring proliferation, cell cycle phase distribution, and changes in expression of several genes. These studies have shown that resveratrol (25 microM, 3 days) reduced growth of all cell types by 60-80%. Overexpression of both wild-type and catalytically inactive nm23-H1 (1beta, 1Tbeta) but not nm23-H2 (2beta, 2Tbeta) reduced the proportion of cells in G1 phase, compared to the Vbeta control cells. Little changes in expression of PCNA, Rb, p53, and bcl-2 were observed in the five cell types treated with resveratrol, compared to untreated cells. Noted exceptions included reduced expression of Rb protein and increased expression of p53 in 2beta and 2Tbeta cells, and increased expression of bcl-2 in 2beta cells, treated with resveratrol. In contrast, resveratrol upregulated expression of cathepsin D by 50-100% in all cell lines except 1beta. These results suggest that the intrinsic metastatic potential of cancer cells may affect their responses to chemopreventive agents such as resveratrol.

Adenocarcinoma↗
PubMed1999

Differences in induction of p53, p21WAF1 and apoptosis in relation to cell cycle phase of MCF-7 cells treated with camptothecin.

The DNA topoisomerase I (topI) inhibitor camptothecin (CPT), stabilizes so-called cleavable complexes which consist of topI covalently attached to 3' OH ends of DNA nicks. Collisions between the progressing DNA replication forks (occurring in S phase cells) or between the transcription driven RNA polymerase molecules (occurring in G1, S and G2 cells) and these complexes convert the latter into secondary DNA lesions which are unrepairable and lethal to the cell. Changes induced by CPT in the level of the tumor suppressor p53, cyclin-dependent kinase inhibitor p21WAF1 and proapoptotic protein Bax (all detected immunocytochemically), were measured separately in the nucleus and cytoplasm of individual human breast carcinoma MCF-7 cells by laser scanning cytometry (LSC) in relation to cell cycle position and induction of apoptosis. The initial transient cell arrest at the G1 checkpoint seen at 8-16 h of treatment with 0.15 microM CPT was accompanied by the rapid accumulation of p53 (preventable by cycloheximide) in the nucleus; the rise (>20-fold) in p53 was maximal for S phase cells. The magnitude of the nuclear p53 increase induced by CPT, at maximum, was 2-fold higher than that induced by the proteasome inhibitor N-acetyl-Leu-Leu-norleucinal (LLnL). While the accumulation of p53 was seen in all phases of the cycle, only G1 cells responded by induction ( approximately 60-fold increase) of p21WAF1. Inhibition of DNA replication by aphidicolin prevented the accumulation of p53 in S and G2/M but had no effect on its induction in G1 cells. Perturbation of cell progression through S phase was seen between 24-72 h of treatment, and it coincided with induction of Bax and apoptosis (both maximal in S phase cells). Thus, the changes observed in S phase cells (nuclear accumulation of p53 preventable by aphidicolin, induction of Bax, apoptosis), triggered by the collisions of DNA replication forks with the CPT-induced lesions, were distinct from the changes in G1 (nuclear p53 accumulation unaffected by aphidicolin, induction of p21WAF1) presumably triggered by collisions of RNA polymerase with the CPT-lesions. Great heterogeneity in expression of p53 and p21WAF1 of the G1 cell population in response to CPT was observed, which may reflect the intercellular variability in the rate of transcription (i.e., frequencies of collisions of RNA polymerase with the lesions). Thus, differences in the transcriptional activity of G1 cells may play a role in their sensitivity to CPT and similar topI inhibitors.

Apoptosis↗
PubMed1999

Comparison of methods based on annexin-V binding, DNA content or TUNEL for evaluating cell death in HL-60 and adherent MCF-7 cells.

HL-60 and MCF-7 cells were treated with 0.15 microM camptothecin (CPT) or with the solvent dimethylsulfoxide (DMSO) for the controls, for 2, 3 and 4 h or for 24, 48 and 72 h, respectively. The apoptotic index (AI) was then evaluated in parallel by the following flow cytometric methods: (1) double staining of unfixed cells with fluoresceinated annexin V and propidium iodide (PI), this after detachment by trypsinization in the case of MCF-7 cultures; (2) prefixation in 70% ethanol, extraction of degraded, low molecular weight DNA with 0.2 M phosphatecitrate buffer and analysis of the DNA content stained with PI; (3) TUNEL, i.e. labelling of DNA strand breaks with biotin-dUTP, followed by staining with streptavidin-fluorescein and counterstaining with PI. In HL-60 cells, the three methods gave similar results for the AI (3-4% in the controls and at 2 h of CPT treatment, and 35-43% at 3 and 4 h after CPT). This indicates that CPT-induced membrane alteration and DNA fragmentation occurred concomitantly in those cells. For MCF-7 cells, CPT-induced apoptosis developed more slowly, the AI, whether based on annexin V or on DNA content, remained unchanged at 24 h, then was increasing to 8% at 48 h and to 25% at 72 h of treatment. In these cells, the TUNEL index did not increase prior to 72 h, and the increase was minor (up to 9% vs. 2-3% in the controls) at 72 h of the treatment. This indicates that in MCF-7 cells DNA strand breaks cannot be effectively labelled, which may be due to inaccessibility of 3'-OH ends in the breaks to exogenous terminal deoxynucleotidyl transferase. The mechanism of endonucleolytic DNA fragmentation thus may be different, depending on the cell type.

Annexin A5↗
PubMed1998

Activation of nuclear factor kappa B (NF-kappaB) assayed by laser scanning cytometry (LSC).

Nuclear factor kappa B (NF-kappaB)/rel is the family of ubiquitous transcriptional activators involved in regulation of diverse immune and inflammatory responses. It also plays a role in control of cell growth and apoptosis. In its inactive form NF-kappaB remains in the cytoplasm sequestered through interaction with IkappaB protein. Rapid translocation of NF-kappaB from cytoplasm to nucleus that occurs in response to extracellular signals is considered to be a hallmark feature of its activation. The translocation of NF-kappaB in HL-60, U-937 and Jurkat leukemic cells as well as in human fibroblasts induced by tumor necrosis factor alpha (TNF-alpha) or phorbol myristate acetate (PMA) was presently measured by laser scanning cytometry (LSC). NF-kappaB was detected immunocytochemically with FITC-tagged antibody and its presence in the nucleus vis-a-vis cytoplasm was monitored by measuring the green fluorescence integrated over the nucleus, which was counterstained with propidium iodide (PI), and over the cytoplasm, respectively. Activation of NF-kappaB led to a rapid increase in NF-kappaB-associated fluorescence measured over the nucleus (FN) concomitant with a decrease in fluorescence over the cytoplasm (F(C)), which was reflected by an increase in F(N)/F(C) ratio. This rapid assay of NF-kappaB activation can be combined with morphological identification of the activated cells or with their immunophenotype. Bivariate analysis of NF-kappaB expression versus cellular DNA content makes it possible to correlate its activation with the cell cycle position. The described method has a potential to be used as a functional assay to monitor intracellular translocation of other transcriptional activators such as p53 tumor suppressor protein or signal transduction molecules.

Cell Cycle↗
PubMed1998

Phosphorylation of retinoblastoma protein assayed in individual HL-60 cells during their proliferation and differentiation.

Expression of pRb and its state of phosphorylation were immunocytochemically assayed in individual HL-60 cells during their proliferation and after induction of differentiation, using mAb which detects hypophosphorylated pRb (pRbP-) combined with mAb which reacts with pRb regardless of its phosphorylation (total pRb; pRbT). Correlated measurements of pRbP-, pRbT, a ratio of pRbP-/pRbT, and cellular DNA content by flow cytometry revealed expression of total pRb and its phosphorylation state vis-à-vis the cell cycle position. Following mitosis (during the exponential phase of cell growth) a mixture of hypo- and hyperphosphorylated pRb was present within the cell for less than 2 h, i.e., early in G1; no hypophosphorylated pRb was detected throughout remainder of the cycle. Cellular pRb content was increasing primarily during G1 and the cell entrance to S was correlated with attainment of a distinct threshold level of pRb. No correlation was seen between the content of pRb per cell and its state of phosphorylation during G1. Cell differentiation whether induced by 1,25-dihydroxyvitamin D3, retinoic acid, or phorbol myristate acetate led to cell arrest primarily in G0/1. The G0/1 cells in these cultures, compared to G1 cells from the untreated cultures, had increased level of both pRbT and pRbP-. However, because the relative increase of pRbP- was disproportionally greater than of pRbT, the pRbP-/pRbT ratio of the differentiating cells was markedly elevated. The cells that still were in S and G2/M in the differentiating cultures also showed the presence of hypophosphorylated pRb. Our data suggest that the mechanism of irreversible cell cycle arrest during terminal differentiation involves both the increase in content of pRb and dephosphorylation of pRb already present within the cell. This provides a large pool of hypophosphorylated pRb that can effectively remove all free E2F, thereby precluding activation of the genes whose transcription is needed to pass the G1 restriction point. In contrast to terminal differentiation the transient quiescence (G0 state) manifests only by dephosphorylation of pRb, without a change in its cellular level.

Cell Differentiation↗
PubMed1998

Enzyme kinetic reactions and fluorochrome uptake rates measured in individual cells by laser scanning cytometry.

This study was designed to explore the utility of a microscope-based cytofluorometer, the laser scanning cytometer (LSC), for time-resolved kinetic measurements. This instrument measures fluorescence of individual cells rapidly, with high sensitivity and accuracy. Also recorded in a list mode fashion are the spatial X-Y coordinates of the cell on the slide as well as the time of individual cell measurement. Repeated measurement of each of a group of cells within a selected area of the slide, thus, yields information on their fluorescence parameters (integrated value, maximal pixel intensity, or fluorescence area) as a function of time. Using the fluorogenic substrate di-(leucyl)-rhodamine 110, we measured the kinetic activity of L-aminopeptidase in HL-60 cells and in monocytes, granulocytes, and lymphocytes from human blood. Likewise, the rate of fluorescein diacetate (FDA) hydrolysis by esterases was measured in HL-60 cells. Also studied was the rate of uptake of the lysosomo-trophic fluorochrome acridine orange (AO) by human leukocytes. Several hundred cells per sample were measured rapidly with a time resolution of 20-60 s. The resolution was inversely proportional to the number of cells within the measured population. The kinetic curves constructed for individual cells had clearly defined slope and plateau regions. During data analysis the kinetic plots were matched with the respective cells; the latter were identified by their position on the slide and classified by their fluorescence or image after staining with Giemsa. Great intercellular variability was noted in enzyme kinetics among cells of the same type, and differences were seen in rate of AO uptake between granulocytes, monocytes, and lymphocytes. Fluorescence fading and recovery was observed especially in the case of FDA hydrolysis and AO uptake. Our results indicate that LSC can be used to rapidly measure the rate of uptake or accumulation of a particular fluorochrome or the kinetics of enzymatic reactions in individual cells of large populations to reveal intercellular variability or the presence of a cell subpopulation with different kinetic properties.

Acridine Orange↗
PubMed1998

Cell cycle effects and induction of apoptosis caused by infection of HL-60 cells with human granulocytic ehrlichiosis pathogen measured by flow and laser scanning cytometry.

Human granulocytic ehrlichiosis (HGE) is an occasionally severe and even fatal disease caused by an agent closely related to Ehrlichia equi and Ehrlichia phagocytophila, which is transmitted by ticks. Little is known about the pathogen itself, which only very recently has been isolated. The agent can be cultivated in vitro because it replicates in human promyelocytic leukemic HL-60 cells. Using multiparameter flow cytometry and laser scanning cytometry (LSC) we have investigated changes in HL-60 cells following their infection with the pathogen. Its presence within the infected HL-60 cells was detected and its intracellular level measured inmmunocytochemically using antibodies obtained from HGE-infected patients. The percentage of the infected cells measured by flow cytometry or LSC correlated well with the estimates by microscopy on the Giemsa-stained specimens. In the infected cultures, the cells had diminished levels of cyclins D3 and E as well as the cyclin dependent kinase inhibitor p21WAF1/CIP1 and were arrested predominantly in G0/1. The apoptosis-associated regulatory proteins were also affected by cell infection: expression of Bcl-2 was decreased in the infected cells whereas expression of Bax become more variable, with some cells showing higher levels of this protein. The infected cells developed numerous DNA strand breaks characteristic of apoptosis. The presence of the pathogen was also detected by LSC in cells from peripheral blood of the infected patients; after relocation and visual inspection ("CompuSort") the pathogen-positive cells were identified as leukocytes. This unique ability of LSC to detect, quantify, and visualize HGE in infected cells made this instrument particularly useful to measure the degree of infection in peripheral blood of the patients and study effects of the infectious agent on the cell cycle and apoptosis of the host cells.

Apoptosis↗
PubMed1998

Histone H3 phosphorylation and expression of cyclins A and B1 measured in individual cells during their progression through G2 and mitosis.

Phosphorylation of histone H3 (H3) on Ser-10 correlates with chromatin condensation at mitosis. A new monoclonal antibody (anti-H3-P) was developed that recognizes phosphorylated H3 (H3-P). This antibody was used in multiparameter flow cytometric analysis to relate H3 phosphorylation in individual human leukemic cells to the cells' position in the cycle as well as their expression of cyclins A and B1. Mitotic cells, from prophase to telophase, reacted with anti-H3-P; the binding of the antibody to chromatin of interphase cells was several times weaker. Cell growth in the presence of staurosporine, an inhibitor of the kinase(s) that phosphorylate H3, abolished the cells' reactivity with the antibody. The reactivity also was abolished by incubation of permeabilized mitotic cells with alkaline phosphatase. These data indicate that, within permeabilized cells, the antibody is indeed specific for H3-P and does not detect the unphosphorylated epitope. All cells reacting with anti-H3-P, with the exception of prophase and early prometaphase, were cyclin A negative; the expression of cyclin B1 in these cells was threefold higher than in G2 cells. The analysis of phosphorylation of H3 in individual cells when combined with multiparameter analysis of their cycle position and expression of other proteins offers new possibilities to study molecular mechanisms associated with the G2 to M transition and chromatin condensation. 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.

Alkaline Phosphatase↗
PubMed1998

Phosphorylation of retinoblastoma susceptibility gene protein assayed in individual lymphocytes during their mitogenic stimulation.

Phosphorylation of the protein encoded by retinoblastoma susceptibility gene (pRb) is the key event of the cell cycle committing the cell to enter S phase and also required for progression through S and G2. We describe a new methodology to monitor pRb phosphorylation in individual cells and correlate it with the cell cycle position. Specifically, pRb phosphorylation in human lymphocytes was assayed immunocytochemically using mAb which recognizes underphosphorylated pRb (pRbP-) conjugated with a fluorochrome of one color combined with mAb which reacts with pRb regardless of its phosphorylation (total pRb; pRbT) tagged with another color fluorochrome. DNA was stained with still another color fluorochrome and cell fluorescence was measured by multiparameter flow cytometry. Specificity of anti-pRbP- mAb was confirmed by preincubation of the permeabilized cells with phosphatase. Analysis of pRbP- or a ratio of pRbP-/pRbT revealed that pRb was underphosphorylated in over 98% of the nonstimulated lymphocytes. The proportion of cells with underphosphorylated pRb dropped to 20% between 3 and 8 h after addition of the mitogen phytohemagglutinin (PHA). Phosphorylation of pRb within a cell was rapid and complete since reactivity of individual lymphocytes with anti-pRbP-mAb was lost abruptly rather than step-wise during stimulation. Phosphorylation of pRb coincided with the appearance of cyclin D3, which was induced 3 h and peaked 12 h after addition of PHA. The nonspecific protein kinase inhibitor staurosporine at a concentration known to arrest lymphocytes in G1 but not to interfere with the induction of cyclin D3 (20 nM) prevented pRb phosphorylation. The present assay can be applied for screening antitumor drugs targeting CDKs and be useful for monitoring pRb phosphorylation in human tumors, the feature of a possible prognostic value in oncology.

Cell Cycle↗
PubMed1998

Measurement of apoptosis.

The cell dying by apoptosis undergoes a sequence of morphological, biochemical, and molecular changes which are characteristic, and often unique, to this mode of cell death. Specific features of apoptotic cells resulting from these changes, which serve as markers used to reveal the apoptotic mode of cell death and to quantify the extent of apoptosis in cultures or in tissue, are reviewed. Analysis of these features by flow or image cytometry is the most commonly used approach to detect, quantify, and study various aspects of apoptosis. Flow or laser scanning cytometry also offer all the advantages of rapid, accurate and multiparametric measurements to investigate the biological processes associated with cell death. Numerous methods have been developed to identify apoptotic and necrotic cells, which are widely used in various disciplines, particularly in oncology and immunology. The methods based on changes in cell morphology, plasma membrane molecular structure and transport function, function of cell organelles, DNA stability to denaturation and endonucleolytic DNA degradation are reviewed and their applicability in the research laboratory and in the clinical setting is discussed. The most common pitfalls and improper use of the methodology in analysis of cell death and in data interpretation are also discussed.

Animals↗