PMID: 10907640

Activity of the caspase-3/CPP32 enzyme is increased in `` early stage '' myelodysplastic syndromes with excessive apoptosis , but caspase inhibition does not enhance colony formation in vitro .

[ OBJECTIVE ]

Excessive apoptosis may have a role in the ineffective hematopoiesis and cytopenias observed in myelodysplastic syndromes .

The goals of this study were 1 ) to quantify apoptosis in patients with `` early stage '' myelodysplasia [ including patients with refractory anemia ( RA ) , RA with ringed sideroblasts ( RARS ) , RA with excess blasts and with less than 10 % blasts ( RAEB ( < 10 ) ) ] , and in patients with `` late stage '' myelodysplasia [ including RAEB with more than 10 % blasts ( RAEB ( > 10 ) ) , RAEB in transformation ( RAEB-t ) , and acute myeloid leukemia secondary to myelodysplasia ( LAM2 ) ] ; 2 ) to study the activation of the caspase-3/CPP32 enzyme , a major `` effector '' caspase in hematopoiesis , in patients with `` early stage '' myelodysplasia , and 3 ) to evaluate the effect of caspase inhibition on the apoptotic phenotype and clonogenicity of hematopoietic progenitors in vitro in these patients .

[ MATERIALS AND METHODS ]

Patients : Fifty-four patients with myelodysplastic syndromes , including 30 with `` early stage '' myelodysplasia and 24 with `` late stage '' myelodysplasia were studied .

Study of apoptosis : TUNEL assay performed on bone marrow smears and/or quantification of annexin V positive bone marrow mononuclear cells by flow cytometric analysis .

Caspacse-3/CPP32 activity : Quantitative measurement of caspase-3/CPP32 activity on total bone marrow mononuclear cells using a fluorogenic substrate .

Effect of the caspase-inhibitor Z-VAD-FMK : 1 ) on the apoptotic phenotype of total bone marrow mononuclear cells and 2 ) on the clonogenicity of hematopoietic progenitor cells .

[ RESULTS ]

The group of 30 patients with `` early stage '' myelodysplasia had statistically increased apoptosis compared to the group of 24 patients with `` late stage '' myelodysplasia ( 44.1 % +/- 4.8 vs 21.8 % +/- 3.6 ; p = 0.02 ) using the TDT-mediated dUTP nick-end labeling ( TUNEL ) assay .

In the group of patients with RAEB , those with MDS ( RAEB < 10 ) had excessive apoptosis compared to those with MDS ( RAEB > 10 ) ( 44.0 % +/- 3.5 % vs 29.5 % +/- 3.6 % ; p = 0.042 ) The median caspase-3 activity in 20 `` early stage '' myelodysplasia patients was 19,000 U ( range 3,460-41,000 ) and significantly increased compared to normal individuals ( 4,256 U , range 3,200-5,200 ; p = 0.032 ) Bone marrow mononuclear cells from 12 `` early stage '' MDS patients ( including 11 from the 20 studied for caspase-3 activity ) were incubated with or without the broad-spectrum caspase inhibitor Z-VAD-FMK .

In 4 of 9 evaluable patients ( 44.4 % ) with excessive apoptosis , the number of annexin V positive cells decreased in a dose-dependent manner in the presence of Z-VAD-FMK .

However , in none of these patients was caspase inhibition with Z-VAD-FMK able to enhance colony formation in vitro .

[ CONCLUSION ]

These results confirm that a major characteristic of patients with `` early stage '' myelodysplasia is increased apoptosis .

The results also indicate that excessive apoptosis in these patients is accompanied by increased caspase-3/CPP32 activity .

However , caspase inhibition with the broad-spectrum inhibitor Z-VAD-FMK can not improve hematopoiesis in this group of patients , even when apoptosis is attenuated .