Biomedical subjects

Beverly Z Packard

Publications and source records attributed to Beverly Z Packard.

6 recordsLinked to original sources

PubMed2004

Assessment of lymphocyte-mediated cytotoxicity using flow cytometry.

Cytotoxic lymphocytes, including cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, kill target cells by releasing granules containing perforin and granzymes, and/or via Fas-Fas ligand interactions. Both pathways lead to prompt activation within target cells of caspase cascades responsible for apoptosis induction and cell death. We have utilized cell-permeable fluorogenic caspase substrates and multiparameter flow cytometry to detect caspase activation in target cells, and applied these tools to quantify and visualize cytotoxic lymphocyte activities. This novel assay, referred to as the flow cytometric cytotoxicity (FCC) assay, is a nonradioactive single-cell-based assay that provides a more rapid, biologically informative, and sensitive approach to measure cytotoxic lymphocyte activity when compared to other assays such as the 51chromium (51Cr) release assay. In addition, the FCC assay can be used to study CTL-mediated killing of primary target cells of different cell lineages that are frequently not amenable to study by the 51Cr release assay. Furthermore, the FCC assay enables evaluation of the phenotype and fate of both target and effector cells, and as such, provides a useful new approach to illuminate the biology of cytotoxic lymphocytes.

Apoptosis↗

PubMed2004

Multiparametric analysis of apoptosis by flow and image cytometry.

Flow cytometric assays for apoptosis are now in widespread use. The multiparametric nature of flow cytometry allows multiple assays for several apoptotic characteristics to be combined in a single sample, providing a powerful tool for elucidating the complex progression of apoptotic death in a variety of cell types. This chapter describes one such assay, allowing simultaneous analysis of caspase activation, annexin V binding to "flipped" phosphatidylserine residues and membrane permeability to DNA binding dyes. This multidimensional approach to analyzing apoptosis provides far more information than single-parameter assays that provide only an ambiguous "percent apoptotic" result, given that multiple early, intermediate, and late apoptotic stages can be visualized simultaneously. This multiparametric approach is also amenable to a variety of flow cytometric instrumentation, both old and new.

Animals↗

PubMed2003

Caspase activity is not sufficient to execute cell death.

Molecular studies of the physiological cell death process have focused attention on the role of effector caspases as critical common elements of the lethal mechanism. Diverse death signals act afferently via distinct signaling pathways to activate these resident proenzyme molecules post-translationally. Whether this molecular convergence represents the mechanistic point of irreversible commitment to cell death has not been established. That a number of caspase substrates are proteins that serve important roles in cellular homeostasis has led to the view that the acquisition of this activity must be the determinative step in cell death. Observations that caspases serve in a regulatory role to catalyze the appearance of new activities involved in orderly cellular dissolution challenge this model of death as a simple process of proteolytic destruction. We found previously that caspase-dependent nuclear cyclin dependent kinase 2 (Cdk2) activity appears to be necessary for cell death. Employing direct cytofluorimetric analyses of intracellular caspase activity and colony forming assays, we now show that transient blockade of caspase-dependent Cdk2 activity confers long-lived sparing from death on cells otherwise triggered to die and fully replete with caspase activity. These data demonstrate that caspases, while necessary for apoptosis, are not sufficient to exert lethality. Caspase activation per se does not represent an irreversible point of commitment to physiological cell death.

CDC2-CDC28 Kinases↗

PubMed2002

The effector phase of physiological cell death relies exclusively on the posttranslational activation of resident components.

Inhibitors of transcription and translation can protect cells from physiological cell deaths induced by a variety of stimuli. These observations have been taken to suggest that de novo macromolecular synthesis may be an essential component of the cell death process. Paradoxically, the same inhibitors, at higher concentrations, themselves trigger the death of cells. Previously, we have mapped a conserved and ordered sequence of events that exerts physiological cell death. Diverse signals converge to activate this lethal pathway, composed of a proteolytic cascade of caspases and subsequent cyclin-dependent kinases. Here we report that inhibitors of nuclear gene expression, when they block cell death, act upstream of this lethal process to prevent its activation. In contrast, when cell death is triggered by high doses of the inhibitors, these same essential molecules are activated, despite the essentially complete blockade of macromolecular synthesis. This inhibitor-induced death response is associated with the release of cytochrome c from mitochondria and the activation of apical caspase 9 and is blocked by overexpression of Bcl-2. These data demonstrate that all essential molecules that exert lethality already are resident within cells and are activated posttranslationally upon stimulation. De novo macromolecular synthesis pertains idiosyncratically only to upstream, modulatory elements of particular death responses.

Apoptosis↗

PubMed2002

Detection of localized caspase activity in early apoptotic cells by laser scanning cytometry.

BACKGROUND: Caspase activation is a critical early step in the onset of apoptosis. Cell-permeable fluorogenic caspase substrates have proven valuable in detecting caspase activation by flow cytometry. Nevertheless, detection of early low-level caspase activation has been difficult using conventional area or peak fluorescence analysis by flow cytometry, despite the apparent presence of these cells as observed by microscopy. We describe a method utilizing maximum fluorescence pixel analysis by laser scanning cytometry (LSC) to detect early apoptotic cells. METHODS: The PhiPhiLux-G(1)D(2) caspase 3/7 substrate was used in combination with DNA dye exclusion and annexin V binding to identify several stages of apoptosis in EL4 murine thymoma cells by both traditional flow and LSC. LSC analysis of maximum pixel brightness in individual cells demonstrated an intermediate caspase-low subpopulation not detectable by flow or LSC integral analysis. LSC analysis of caspase activity was then carried out using the larger UMR-106 rat osteosarcoma cell line to determine if this apparent early caspase activity could be correlated with localized, punctate caspase activity observed by microscopy. RESULTS: The caspase-low subpopulation found in apoptotic EL4 cells was also observable in UMR-106 cells. Relocation to cells with low fluorescence due to caspase activity and subsequent examination by microscopy demonstrated that these latter cells indeed show punctate, highly localized caspase activation foci that might represent an early stage in caspase activation. CONCLUSIONS: Cells with low-level, localized caspase expression can be detected using maximum pixel analysis by LSC. This methodology allows an early step of apoptotic activation to be resolved for further analysis.

Animals↗

PubMed2002

Visualization and quantification of T cell-mediated cytotoxicity using cell-permeable fluorogenic caspase substrates.

We have developed a non-radioactive flow-cytometry assay to monitor and quantify the target-cell killing activities mediated by cytotoxic T lymphocytes (CTLs). This flow-cytometry CTL (FCC) assay is predicated on measurement of CTL-induced caspase activation in target cells through detection of the specific cleavage of fluorogenic caspase substrates. Here we show that this assay reliably detects antigen-specific CTL killing of target cells, and demonstrate that it provides a more sensitive, more informative and safer alternative to the standard 51Cr-release assay most often used to quantify CTL responses. The FCC assay can be used to study CTL-mediated killing of primary host target cells of different cell lineages, and enables the study of antigen-specific cellular immune responses in real time at the single-cell level. As such, the FCC assay can provide a valuable tool for studies of infectious disease pathogenesis and development of new vaccines and immunotherapies.

Animals↗