Cell Killing Process Research Articles

Modulation of TNF-mediated cell lysis in vitro: further analysis of intracellular signaling

We have investigated the post-receptor events governing the Tumor Necrosis Factor (TNF)-mediated cytotoxicity in vitro. As calcium has been reported to be an essential mediator in the cell killing processes, we asked whether an early increase in intracellular calcium could be involved during TNF-induced cell death. Using the ACAS methodology (adherent cell analysis and sorting), we could not detect any significant increase in intracellular calcium following TNF treatment (40 s) within the TNF-sensitive human breast carcinoma MCF7 cell line. In addition, A23187 (0.1–0.4 μM) did neither enhance TNF-mediated MCF7 cell lysis, further confirming that TNF-mediated cell lysis can occur in the absence of an early calcium increase. Given the potentiating effect of cAMP-inducing agents, such as forskolin, on TNF-mediated cytotoxicity, we have investigated the relationship between cAMP accumulation and the TNF signaling pathway during cell death. Our results indicate that the potentiating effect of forskolin (50 μM) on TNF-mediated MCF7 cell lysis did not involve a modulation in the TNF-induced activation of the nuclear factor NF-kB but was associated with an increase in the DNA fragmenting capacity of TNF as assessed by agarose gel electrophoresis of target cell DNA.

Potentiation of tnf‐mediated cell killing by VP‐16: Relationship to DNA single‐strand break formation

Interaction between tumor necrosis factor (TNF) and the DNA topoisomerase II inhibitor, etoposide VP-16, in cell killing has been studied. To accurately investigate the nature of DNA damage during the cell killing process, experiments were assessed using the highly TNF-sensitive WEHI164.13 murine fibrosarcoma clone and DNA filter elution methodology. Concomitant treatment of cells with combination of TNF/VP-16 resulted in marked enhancement of cell lysis. Using the alkaline elution technique, we show that TNF fails to induce DNA single-strand breaks as compared to those generated by VP-16. In addition, the potentiating effect of VP-16 on TNF-mediated WEHI164.13 cell killing was not associated with an increase in its intrinsic activity with respect to DNA single-strand break formation. While the 2 phospholipase A2 inhibitors, quinacrine and dexamethasone, were efficient in inhibiting TNF-mediated cell lysis, only quinacrine was efficient in selectively abrogating the TNF/VP-16 cell killing pathway. The inhibitory effect of quinacrine on VP-16/TNF-mediated cell lysis was accompanied by a marked decrease in VP-16-mediated DNA single-strand break generation. Taken together, our findings suggest that TNF and TNF/VP-16 treatments may involve different events during cell killing and support the hypothesis that 2 signals are required for optimal induction of cell lysis by the combination of VP-16/TNF: one signal provided by VP-16 resulting in topoisomerase II inhibition and subsequent DNA single-strand break generation, and a second signal involving TNF.

Cell surface effects of human immunodeficiency virus.

Cell killing by human immunodeficiency virus (HIV) is thought to contribute to many of the defects of the acquired immunodeficiency syndrome (AIDS). Two types of cytopathology are observed in HIV-infected cultured cells: cell-cell fusion and killing of single cells. Both killing processes appear to involve cell surface effects of HIV. A model is proposed for the HIV-mediated cell surface processes which could result in cell-cell fusion and single cell killing. The purpose of this model is to define the potential roles of individual viral envelope and cell surface molecules in cell killing processes and to identify alternative routes to the establishment of persistently-infected cells. Elucidation of HIV-induced cell surface effects may provide the basis for a rational approach to the design of antiviral agents which are selective for HIV-infected cells.

Interpretation of UV-survival curves of Aspergillus conidiospores

Semi-logarithmic dose-response curves for survival of UV-irradiated conidiospores of A. nidulans have an initial shoulder (at low doses) followed by a decline which becomes linear. To explain the initial shoulder and the resulting extrapolation number (log S intercept of the linear extrapolation line) a general model is presented, which includes multi-target ( n) and multi-hit ( h) effects and allows for the effect of initial repair and of a compound parameter k, which stands for inherent sensitivity of the spores and for dose received inside the spores. From experiments on (a) the modification of k (spore wall colour and shelter effects), (b) a repair-deficient strain (shoulderless) and (c) preincubation during which DNA-replication takes place, it is concluded that the shoulder is generated by initial repair rather than by a multi-hit nature of the cell-killing process. In experiments where k takes different values (sub a and c), notably the position of the point of intersection of the linear lines gives conclusive information. In general, the log S intercept of the linear extrapolation line cannot be used to estimate the target number.

The time course of DNA repair following methyl nitro-nitrosoguanidine (MNNG) treatment of P388F lymphoma cells in culture: I. Alkali dissociable DNA lesions and their restitution

The pattern of formation and rejoining of P388F cell DNA single strand breaks revealed by alkaline sedimentation centrifugation studies following treatment with methyl nitro-nitrosoguanidine (MNNG) has been studied. Three periods of single strand break formation have been recognised, an initial period during the first 4 h where breaks are rapidly formed and rejoined, a second (medial) time range of 10–15 h following treatment and a final stage where the formation of breaks appears to be related to cell killing processes. The initial phase is considered to be due to the production and repair of X-ray like DNA lesions whereas those of the medial time range are more comparable to the enzymatic processes associated with the repair of ultraviolet light-induced damage.