Steven W. Sherwood

Cell cycle analysis of apoptosis using flow cytometry.

Publisher Summary Using flow cytometry to analyze asynchronous and synchronized cell populations, this chapter describes a number of features of drug-induced apoptosis in cultured cells: (1) apoptosis can occur in any cell cycle phase; (2) a given agent can induce apoptosis in more than one cell cycle phase; (3) there is a clear temporal relationship between cell cycle “arrest” induced by different agents and the onset of apoptosis; (4) proteolysis of specific intracellular proteins represents an early event of apoptosis; ( 5 ) aberrant mitosis precedes apoptosis under a variety of conditions, including exposure to inhibitors of DNA synthesis; (6) cells with sub-G, DNA content are not always apoptotic cells; (7) DNA content can be an unreliable index of cell cycle position; and (8) under all conditions, cell cycle “stasis” precedes drug-induced apoptosis. The chapter describes specific examples of applications of standard flow cytometric methods to the analysis of drug-induced apoptosis in HeLa S3 cells.

A novel method for measuring CTL and NK cell-mediated cytotoxicity using annexin V and two-color flow cytometry

An assay based on two-color flow cytometry has been developed to measure CTL and NK cell-mediated cytotoxicity. After effector/target cells are incubated together, CTL or NK populations are stained with an effector cell specific PE-conjugated mAb. Subsequently, annexin V-FITC binds to cells expressing phosphatidylserine (an early marker of apoptosis) on the cell surface. Target cells are gated upon as PE-negative and quantified with respect to their annexin V positivity. The shift from annexin Vneg to annexin Vhi is a discrete event such that all target cells fall within discernible populations with respect to annexin V. There is a strong correlation between cytotoxicity measured with our assay and a standard 51Cr release assay (r2 = 0.989). The PE/annexin V assay shows increased sensitivity at early timepoints after target/effector cell mixing. In addition, this method allows for analysis of target cells at the single cell level. Therefore, we have described a promising new technique to measure in vitro cell-mediated cytotoxicity. It avoids the potential difficulties of working with radioactive isotopes, and offers increased sensitivity and versatility.

Flow cytometric analysis of the molecular mechanisms of immunosuppressive action of the active metabolite of leflunomide and its malononitrilamide analogues in a novel whole blood assay

Malononitrilamides (MNAs) are a new class of immunomodulatory drug highly effective in in vivo models of allo- and xenotransplantation. Knowledge of their effects on immune cells, however, is limited and has been derived solely from investigations using isolated mononuclear cells. This use of purified cells to investigate drug activity is not ideal, so we have combined the analytical power of flow cytometry with our mitogen-driven, whole blood lymphocyte activation and proliferation assays to investigate the in vitro mechanism of action of MNAs. We first show that MNAs (A77 1726, HMR1279, and HMR1715), as well as brequinar (BQR) and cyclosporine (CsA), effectively inhibit cell activation antigen expression and lymphocyte proliferation. We next show that the inhibitory effects of MNAs and BQR, but not CsA, are reversed by the addition of uridine to the culture. These results suggest that inhibition of pyrimidine biosynthesis may be a mechanism by which MNAs suppress both lymphocyte activation and proliferation since these effects were reversed when uridine nucleotide pools were replenished. This novel finding of suppression of activation antigen expression by MNAs in whole blood expands our understanding of the effects of this new class of drug.

Induction of Apoptosis by Cell-Cycle Phase Specific Drugs

The events of apoptosis represent the outcome of processes defining a common physiological pathway of dying cells, and the inputs to this pathway are complex and highly varied1–5. It is clear that the actions of cytotoxic agents in mammalian cells frequently feed into the apoptosis pathway, and an extremely wide variety of agents/treatments have been shown to induce the events of apoptosis in cultured mammalian cells6,7. It is important to note, however, that cells vary widely in responses to specific cytotoxic agents and the induction of apoptosis by a given agent can vary between cell lines.

Flow-cytometric analysis of spermatogenesis in Teleogryllus commodus

Abstract The development of the testis of the cricket Teleogryllus commodus has been studied using flow cytometry. From a laboratory-reared population of insects, total testis tissue was prepared at approx. 48-h intervals for the entire development period extending from the fifth instar (28 days after eclosion) to the imaginal moult (about 60 days after eclosion). Cells were stained with DNA-specific fluorochromes and the temporal sequence of the development of the testis as well as the timing of specific meiotic events quantitatively determined. Testis development studied in this fashion is seen as changes in the relative frequency of cells with 1, 2, and 4C DNA content. Pre-spermiogenic processes of spermatogenesis (change in the frequency of cells with 2 and 4C DNA content) occur continuously during the fifth-eighth instars, while the appearance of 1C cells (initiation of spermiogenesis) occurs abruptly at the transition between the sixth and seventh instars.

Cyclin B Degradation as a Target of Antiproliferative Drug Action

For a number of years we have been examining the genotoxic and cytotoxic effects of antiproliferative drugs as well as cytotoxic treatments such as u.v. irradiation and hypoxia in cultured mammalian cells. The approach we have taken is to examine cellular response to treatment rather than drug-target interaction, and in particular, we have been interested in determining the relationship of treatment-induced cell cycle perturbation to cytotoxicity and genotoxicity (1–5).