Resultados en el aspecto ambiental

S2 cells u ndergo apoptosis w hen treated w ith the D N A -intercalating and -dam aging agent etoposide (Fig 4.1a), w ith the protein synthesis inhibitor cycloheximide (Fig 4.1b) and following X-irradiation (Fig 4.2a). In all cases, the cells fragm ent and become perm eable to the dye try p an blue (data only show n for X-irradiation). M am m alian lam ins are know n to be cleaved by caspases d u rin g apoptosis (Lazebnik et a l, 1993; O berham m er et al., 1994; Rao et al., 1996) and lam in DmO, the D rosophila lam in B hom o lo g u e (G ruenbaum et a l, 1988), is cleaved d uring apoptosis in S2 cells (Fig 4.2b). A p o p to sis of S2 cells in d u ced by eith er cyclohexim ide or eto p o sid e treatm ent is prevented by addition of the caspase inhibitor zVAD.fmk (Slee et al., 1996) (data show n for cycloheximide in Fig 4.3a) im plying that caspase activation is required for S2 cell apoptosis. Caspase inhibitors also prevent S2 cell death induced by over expression of rpr (Pronk et al., 1996) or grim (Chen et ah, 1996b), potent triggers of Drosophila cell death. Finally, S2 cells express drlCE protein, and it has identical electrophoretic m obility to drlCE expressed in Drosophila embryos (Fig 4.3b).

60 5 0 - Ui

I

Fig 4.1a Etoposide induces apoptosis in S2 cells.

S2 cells were treated with etoposide at the concentrations shown and cell deaths monitored by time-lapse videomicroscopy.

60 4 0 - s ■D 3 0 - % 3 E 3 Ü 2 0- 10 0 5 15 20 25 25p,g/ml CHX time (hrs)

Fig 4.1b Cycloheximide induces apoptosis in S2 cells.

S2 cells were treated with cycloheximide at a final concentration of 25 |xg/m l and cell deaths m o nitored by tim e -la p s e videomicroscopy.

60 50 "O 03 CD ■o CD O) c CD 40 30 20 CD CL 96 0 24 48 ■A- 0 rads 600 rads 1200 rads 2000 rads time (hrs.)

Fig 4.2a S2 cells die following X-irradiation.

S2 cells were irradiated with the doses of X-irradiation shown and the percentage of cells that stained positive by Trypan blue staining were counted at different time points.

Ohr 24hr 48hr

DmO

DmO large fragment

Fig 4.2b Lamin DmO is cleaved during S2 cell X-irradiation- induced apoptosis.

S2 cells irradiated with 2000 rads of X-irradiation were harvested at the time points shown and lysed in Reducing Sample Buffer. The lysates were subjected to SDS-PAGE on a 12.5% gel, blotted onto an Immobilon-P nitrocellulose filter and the blots probed with a monoclonal antibody to DmO. An HRP-conjugated anti-mouse secondary antibody (Amersham) was used followed by ECL to detect the protein. The blot was exposed for 30 seconds against Kodak XAR-5 film.

+zVAD.fmk

-zVAD.fmk

+CHX

-CHX

Fig 4.3a Cycloheximide induces apoptosis in S2 cells via caspases.

S2 cells were treated with cycloheximide (CHX) at a final concentration of 25 lag/ml either in the presence or absence of zVAD.fmk at a concentration of 50 pM. Cell deaths were monitored by visual inspection.

S2 cells 2-6hr embryo

drICE

Fig 4.3b 82 cells and 2-6hr embryos express drICE.

S2 cells and embryos harvested 2-6 hours after egg laying were harvested and lysed in Reducing Sample Buffer. The samples were subjected to SDS- PAGE on a 12.5% polyacrylamide gel, blotted onto Immobilon-P and probed with a rabbit polyclonal antibody raised against amino-acids 222-238 of drICE (AbSpacer).

4.3 S um m ary and D iscussion

The Drosophila S2 cell line undergoes apoptosis follow ing treatm ent w ith either D N A -dam aging agents (etoposide or X-irradiation) or cycloheximide or u p o n over expression of know n pro-apoptotic genes rpr and grim. These deaths are caspase dependent and lam in DmO, a putative caspase substrate, is cleaved d uring apoptosis. S2 cells also express drICE. S2 cells therefore seem to be a suitable cell line for analysing the function of com ponents of the Drosophila cell death m achinery in general, and the effects of drICE over expression in particular.

Chapter 5

Analysis of drICE function in S2 cells

5.1 Introduction

O ver expression of caspases has been show n in m any contexts to induce apoptosis. This has been dem onstrated both in vivo in C. elegans, in w hich expression of ced-3 from a neuron-specific prom oter reduces the num ber of neurons in the adult w orm (Shaham and Horvitz, 1996), and in a variety of m am m alian cell lines in w hich over expression of m ost caspases has been show n to induce cell death (reviewed in (Takahashi and Earnshaw, 1996)). It is th o u g h t th at high levels of expression of inactive pro-caspase molecules allow s them som ehow to escape the norm ally stringent regulation of their activity and to autoprocess spontaneously and hence become active. While th is cell d e a th in d u ce d by caspase over ex p ressio n is clearly n o t 'p h y s io lo g ic a l' (the a c tiv ity of m o st casp ases is p rim a rily p o st- translationally regulated and not regulated at the level of expression), it is none the less a useful starting-point in determ ination of w hether a caspase could have a role in apoptosis. I therefore decided to use this approach to determ ine w hether drICE could induce apoptosis in S2 cells.