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3. Zebrafish as a model fo r PCD

The effect of D EV D prodrug on norm al PCD w as dose d ep en d en t w ith peak inhibition detected at 10|iM, lOOpM and 300|liM w ith a m odest p ercen tag e red u ctio n of TUNEL positive cells of 24%, 20% an d 16% respectively. At a concentration of 500pM, D EV D prodrug had a slightly toxic effect (Fig. 3.5A). D E V D prodrug w as also effective in red u cin g s ta u ro s p o rin e -in d u c e d (lOpM , 2hr e x p o su re) cell d e a th ; w ith a concentration of 300|iM the num ber of TUNEL positive cells were reduced by approxim ately 30% (Fig. 3.5B).

c) The caspase inhibitor BocAsp had a slight effect in preventing both normal and staurosporine induced PCD.

BocAsp at an optim um concentration of lOOpM reduced norm al cell d e ath TUNEL positive cells by approxim ately 4% (Fig. 3.5A). At an o p tim um concentration of 300|iM BocAsp rescued staurosporine induced cell d eath TUNEL positive cells by approxim ately 10% (Fig. 3.5B). This show s the drug has a very slight effect on reducing cell death.

d) The caspase inhibitor YVAD does not prevent normal or staurosporine induced PCD.

From the slight am ount of rescued cell death seen at lOpM w ith an 8% decrease in norm al TUNEL positive cells, increasing concentrations of YVAD proved to be toxic. A concentration of BOOpM of YVAD induced a 116% increase in TUNEL positive cell death in norm al embryos (Fig. 3.5A). T reatm en t w ith YVAD on stau ro sp o rin e treated em bryos h a d a slight effect, in cre asin g th e n o rm a l a m o u n t of cell d e a th in d u c e d by staurosporine (Fig. 3.5B).

e) The stress activated protein kinase inhibitor SB203580 had a m odest effect on preventing normal cell death but did not prevent staurosporine induced cell death.

A t an optim um concentration of lOOjiiM SB203580 reduced norm al TUNEL positive cell death by 40% (Fig. 3.5A). SB20358Ü h ad no effect on staurosporine induced PCD (Fig. 3.5B).

3. Zebrafish as a model for PCD

The control d rug MACH (a chemically im provised drug, sim ilar to SB203580 b u t w ith o u t jun kinase inhibitor properties) had no effect on n o rm al or stau ro sp o rin e in d u ced PCD, show ing it is the ju n kinase properties w hich prevents norm al developm ental PCD (data not shown). 3.2.4. Protection fro m PCD by z V A D fm k is only transient.

Zebrafish incubated w ith zVADfmk (300pM) betw een 90% epiboly an d 24hpf show ed reduced cell death at 24hpf. The caspase inhibitor was th en rem oved and em bryos then exam ined at 33hpf and 48hpf show ed a re tu rn of TUNEL positive cells, show ing p ro tectio n is only transient. Em bryos treated as above w hich w ere given another dose of zVADfmk (300|liM) at 24hpf also show ed no increase in protection from PCD at 34 h o u rs. A d etailed in v estig atio n into the p ro tectio n of R ohon Beard n eu ro n s w ith the caspase inhibitor zVADfmk show ed that Rohon Beard cell d eath is delayed b u t death is inevitable and at the end of norm al Rohon Beard developm ent, previously rescued cell have died (chapter 4, Fig. 4.3).

3.2.5. The caspase, caspase-3/7 is a ctivated d u r in g sp on ta n eo u s and staurosporine-induced apoptosis in zebrafish .

To characterise a type of caspase involved in zebrafish apoptosis, the presence of caspase-1 or caspase-3/7 activity in cytoplasm ic extracts w as in v estig ated , by m easuring the cleavage of their respective su b strates, YVAD-AMC or DEVD-AMC. A poptotic cells w ere to tally d ev o id of caspase-1 activity (data not show n). In contrast figure 3.6. show s th at spontaneous as well as staurosporine induced apoptotic cells cleaved the casp ase-3 /7 DEVD-AMC substrate (*p<0.04, *^*p<0.02). This agrees w ith d ata show n above th at zVADfmk and D EV D prodrug can reduced both sta u ro sp o rin e an d n o rm al p ro g ram m ed cell d eath , b u t YVADfmk is unable. This data implicates the importance of caspase-3/7 proteases in the zebrafish.

3. Zebrafish as a model for PCD

Fig. 3.6. DEVD-AMC assay show ing the effect of staurosporine on Caspase- 3 activity. Z ebrafish (24hpf) developed for 2 ho u rs in the presence or absence of staurosporine (lOpM). H ydrolysis of DEVD-AMC substrate was m easured at 405nm. Staurosporine causes an increase of caspase-3 activity in zebrafish embryos.

V alues rep resen t m eans ± SEM of 3 separate determ inations. ***p<.01/ ’^'^p<.02, *p<.04, according to the paired t test.