Mitochondria Superoxide Is an Early Trigger of Apoptosis in an Ex Vivo Model of Human Beta Thalassemia.

Abstract 2131

Apoptotic cell death is a central feature of beta-thalassemia and other anemias caused by ineffective erythropoiesis. In this study, markers of apoptosis were examined using an ex vivo model of human beta-thalassemia to explore the cause(s) and timing of erythroblast cell death. Beta-globin gene and protein expression were knocked down by lentiviral transduction of beta-globin shRNA (beta-KD) in adult human CD34+ cells. Beta-globin mRNA (Control=4.0E+07 ± 1.4E+06 copies/ng, vs. beta-KD=2.5E+06 ± 1.6E+06 copies/ng, p=0.014) and protein were reduced in beta-KD cells by 90% compared to controls while alpha globin expression was maintained. The effects of imbalanced globin chain synthesis were therefore studied according to the stage of erythroblast maturation. Erythroid progenitor cell commitment and proliferation occur over the first two weeks in culture. During the third week of culture (days 14–21), the proerythroblasts undergo terminal differentiation with the characteristic loss of CD71 from the cell surface.

Prior to culture day 14, phenotypic analyses demonstrated low levels of apoptosis in beta-KD and control cultures. On culture day 14, a small but significant increase in active caspase 3 was detected in the beta-KD cells compared to controls (beta-KD=4.0±1.0%, Control=0.7±0.3%, p=0.02) suggesting that apoptosis was initiated during the early stages of terminal maturation. Increases in annexin V staining did not achieve statistical significance on day 14 beta-KD cells (beta-KD=13.1±3.9%, Control=7.6±2.2%, p=0.16). By culture day 18, when orthochromic normoblasts are the most prevalent population in control cultures, a large population of apoptotic cells was detected in the beta-KD. The beta-KD erythroblasts demonstrated further increases in active caspase 3 (beta-KD=11.4±2.2% vs. Control=1.1±0.1%, p=0.014), as well as significant increases in surface annexin V (beta-KD=75.8±3.3%, vs. Control=35.9±12.7%, p=0.024). Western analysis of culture day 18 beta-KD membranes demonstrated a marked increase in alpha-chains compared with culture day 14.

Since cleaved caspase 3 was increased in beta-KD cells near the beginning of their terminal maturation and prior to the accumulation of alpha chains in the cell membranes, other triggers of apoptosis were investigated. Mitochondrial superoxide is a reactive species that can be generated by iron or other mitochondrial toxins. Increased levels of mitochondrial superoxide cause apoptosis. The cultured erythroblasts were stained with MitoSOX, a cell-permeable dye that specifically detects mitochondrial superoxide. On culture day 11, mitochondrial superoxide was barely detectable in beta-KD and control cell populations (beta-KD=5.2±3.3% vs. Control=3.6±2.9%, p=0.051). Thereafter, the superoxide detection was increased significantly in beta-KD cells on culture day 14 (beta-KD=54.2±6.7% vs. Control=9.1±2.9%, p=0.003), and culture day 18 (beta-KD=81.1±3.2% vs. Control=34.6±3.8%, p=0.007).

Oxidation of cellular membranes by hemichromes and free alpha chains damages thalassemic erythrocytes and their precursors. These data suggest that imbalanced globin chain synthesis also triggers apoptosis during the early stages of terminal differentiation by increasing superoxide formation in the mitochondria.