'Functional assays allowing phenotypic characterization of different cell death parameters at a single-cell level are important tools for preclinical anticancer drug screening. Currently, the selection of cytometric assays is limited by the availability of fluorescent probes with overlapping spectral characteristics. Following on our earlier reports on green and orange fluorescent ... More
Mapping mechanisms and charting the time course of premature cell senescence and apoptosis: lysosomal dysfunction and ganglioside accumulation in endothelial cells.
AuthorsPatschan S, Chen J, Gealekman O, Krupincza K, Wang M, Shu L, Shayman JA, Goligorsky MS,
JournalAm J Physiol Renal Physiol
PubMed ID17928415
'Endothelial cells subjected to glycated collagen I develop premature senescence within 3-5 days, as revealed by increased senescence-associated beta-galactosidase activity, decreased proliferation, and an increase in cell size. Here, we analyzed the time course and possible mechanisms of this process. Lysosomal integrity studies revealed a rapid collapse of pH gradient ... More
Activation of caspases measured in situ by binding of fluorochrome-labeled inhibitors of caspases (FLICA): correlation with DNA fragmentation.
AuthorsBedner E, Smolewski P, Amstad P, Darzynkiewicz Z
JournalExp Cell Res
PubMed ID10942603
'Activation of caspases is the key event of apoptosis and new methods are needed to assay this event, particularly in situ, in individual cells. To measure in situ caspases activation in the present study we employed fam-VAD-fmk and fam-VEID-fmk, the fluorochrome (fam)-labeled inhibitors of caspases (FLICA), which through the fluoromethylketone ... More
Multiparametric evaluation of apoptosis: effects of standard cytotoxic agents and the cyanoguanidine CHS 828.
AuthorsLövborg H, Nygren P, Larsson R
JournalMol Cancer Ther
PubMed ID15141009
'A multiparametric high-content screening assay for measurement of apoptosis was developed. HeLa cells and lymphoma U-937 cells were exposed to cytotoxic drugs in flat-bottomed optical microtiter plates. After incubation, the DNA-binding dye Hoechst 33342, fluorescein-tagged probes that covalently bind active caspases and chloromethyl-X-rosamine to detect mitochondrial membrane potential (MMP) were ... More
Flow cytometry-based apoptosis detection.
AuthorsWlodkowic D, Skommer J, Darzynkiewicz Z,
JournalMethods Mol Biol
PubMed ID19609746
'An apoptosing cell demonstrates multitude of characteristic morphological and biochemical features, which vary depending on the stimuli and the cell type. The gross majority of classical apoptotic hallmarks can be rapidly examined by flow and image cytometry. Cytometry thus became a technology of choice in diverse studies of cellular demise. ... More
Histone H1 dephosphorylation is not a general feature in early apoptosis.
AuthorsGréen A, Sarg B, Koutzamani E, Genheden U, Lindner HH, Rundquist I,
JournalBiochemistry
PubMed ID18558721
Histone H1 is a family of nucleosomal proteins that exist in a number of subtypes. These subtypes can be modified after translation in various ways, above all by phosphorylation. Increasing levels of H1 phosphorylation has been correlated with cell cycle progression, while both phosphorylation and dephosphorylation of histone H1 have ... More
Assay of caspase activation in situ combined with probing plasma membrane integrity to detect three distinct stages of apoptosis.
AuthorsSmolewski P, Grabarek J, Halicka HD, Darzynkiewicz Z
JournalJ Immunol Methods
PubMed ID12072182
Activation of cysteine-aspartic acid specific proteases (caspases) in situ, in live cells, can be detected using fluorochrome-labeled inhibitors of caspases (FLICA), the reagents that covalently bind to the active center of these enzymes. In the present study, this assay was combined with a probe of plasma membrane capacity to exclude ... More
Detection of caspases activation by fluorochrome-labeled inhibitors: Multiparameter analysis by laser scanning cytometry.
AuthorsSmolewski P, Bedner E, Du L, Hsieh TC, Wu JM, Phelps DJ, Darzynkiewicz Z
JournalCytometry
PubMed ID11309811
BACKGROUND: The fluorochrome-labeled inhibitors of caspases (FLICA) were recently used as markers of activation of these enzymes in live cells during apoptosis (Bedner et al.: Exp Cell Res 259:308-313, 2000). The aims of this study were to (a) explore if FLICA can be used to study intracellular localization of caspases; ... More
Interactions of fluorochrome-labeled caspase inhibitors with apoptotic cells: a caution in data interpretation.
AuthorsPozarowski P, Huang X, Halicka DH, Lee B, Johnson G, Darzynkiewicz Z
JournalCytometry A
PubMed ID12938188
BACKGROUND: 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. ... More
Enhancement of activation-induced apoptosis of lymphocytes by the cytotoxic ribonuclease onconase (Ranpirnase).
AuthorsHalicka DH, Pozarowski P, Ita M, Ardelt WJ, Mikulski SM, Shogen K, Darzynkiewicz Z
JournalInt J Oncol
PubMed ID12429974
Onconase (Onc) is an amphibian ribonuclease of the pancreatic RNase family that is cytostatic and cytotoxic to several tumor lines. It also shows anti-tumor activity in mouse tumor models and is currently in phase III clinical trials. In animal tests and clinical trials Onc shows lesser toxicity and fewer side ... More
Detection of caspase activation in situ by fluorochrome-labeled caspase inhibitors.
AuthorsAmstad PA, Yu G, Johnson GL, Lee BW, Dhawan S, Phelps DJ
JournalBiotechniques
PubMed ID11570504
Apoptosis is dependent on the activation of a group of proteolytic enzymes called caspases. Caspase activation can be detected by immunoblotting using caspase-specific antibodies or by caspase activity measurement employing pro-fluorescent substrates that become fluorescent upon cleavage by the caspase. Most of these methods require the preparation of cell extracts ... More
Tannic acid inhibits EGFR/STAT1/3 and enhances p38/STAT1 signalling axis in breast cancer cells.
AuthorsDarvin P, Joung YH, Kang DY, Sp N, Byun HJ, Hwang TS, Sasidharakurup H, Lee CH, Cho KH, Park KD, Lee HK, Yang YM
JournalJ Cell Mol Med
PubMed ID27862996
'Tannic acid (TA), a naturally occurring polyphenol, is a potent anti-oxidant with anti-proliferative effects on multiple cancers. However, its ability to modulate gene-specific expression of tumour suppressor genes and oncogenes has not been assessed. This work investigates the mechanism of TA to regulate canonical and non-canonical STAT pathways to impose ... More
5-Hydroxytryptophan (5-HTP)-induced intracellular syndrome in mouse non-neural embryonic cells is associated with inhibited proliferation and cell death.
AuthorsGordeeva O, Safandeev V
JournalNeuropharmacology
PubMed ID31778690
'Biogenic monoamines are involved in the regulation of various processes in both neural and non-neural cells during development. The present study aimed to identify the regulatory effects of serotonin (5-HT) and its precursors (l-tryptophan and 5-hydroxytryptophan, 5-HTP) on proliferation and cell death in mouse embryonic stem cells (ESCs) and embryonic ... More
Pathogen reduction with riboflavin and ultraviolet light induces a quasi-apoptotic state in blood leukocytes.
AuthorsTran JQ, Muench MO, Heitman JW, Jackman RP
JournalTransfusion
PubMed ID31599981
'Alloimmunization to platelet-rich plasma (PRP) transfusions can cause adverse reactions such as platelet refractoriness or transplant rejection. Pathogen reduction treatment with ultraviolet light and riboflavin (UV?+?R) of allogeneic PRP was shown to reduce allogeneic antibody responses and confer partial antigen-specific immune tolerance to subsequent transfusions in mice. Studies have shown ... More
Synergistic anticancer activity of dietary tea polyphenols and bleomycin hydrochloride in human cervical cancer cell: Caspase-dependent and independent apoptotic pathways.
AuthorsAlshatwi AA, Periasamy VS, Athinarayanan J, Elango R
JournalChem Biol Interact
PubMed ID26800624
'Bleomycin is a chemotherapeutic agent that is frequently used in the treatment of various cancers. Bleomycin causes serious adverse effects via antioxidant defense abnormalities against reactive oxygen species (ROS). However, the current cervical cancer monodrug therapy strategy has failed to produce the expected outcomes; hence, combinational therapies are gaining great ... More
Induction of Mitochondria Mediated Apoptosis in Human Breast Cancer Cells (T-47D) by Annona reticulata L. Leaves Methanolic Extracts.
Annona reticulata Linn. (Common name: Bullock's-heart) (Annonaceae family) is a semi-evergreen and small deciduous tree. The extracts of various parts of Annona reticulata L. have been reported as cytotoxic to many cancer cells. Annona reticulata L. leaves' methanolic extract (ARME) was prepared and used against the breast cancer cells. The ... More
Multiparametric Analysis of Apoptosis by Flow Cytometry.
AuthorsTelford WG
JournalMethods Mol Biol
PubMed ID29071681
Flow cytometry is the most widely used method for detecting and quantifying apoptosis in mammalian cells. The multiparametric nature of flow cytometry allows several apoptotic characteristics to be combined in a single sample, making it a powerful tool for analyzing the complex progression of apoptotic death. This chapter provides guidelines ... More