Klin Padiatr 2004; 216 - 15
DOI: 10.1055/s-2004-828560

Quantification of drug induced mitochondria- and caspase-mediated apoptosis signaling in primary leukemia cells by flow cytometry

SM Eckhoff 1, LH Meyer 1, KM Debatin 1, K Stahnke 1
  • 1Universität Ulm, Klinik und Poliklinik für Kinder- und Jugendmedizin, Ulm, Germany

Analysis of apoptosis pathways in primary leukemia cells for assessment of drug sensitivity or resistance has been hampered by the lack of convenient methods for measurement of activated apoptosis signaling. Caspase activation and mitochondrial alterations are key components of apoptosis signal transduction. We developed and characterized a flow cytometric method for combined measurement of caspase activation by cleavage of (Asp)2-rhodamine 110 (D2R) and mitochondrial membrane potential in living cells.

Upon apoptosis induction, in Jurkat T cell leukemia cells, the D2R cleavage signal precedes phosphatidyl-serine externalization. No such signal is obtained in post-apoptotic cells, indicating that the rhodamine substrate measures an early and specific step of apoptosis signaling. The derivatives of D2R, (Z-DEVD)2R and (Z-IETD)2R, also applicable in live cells, proofed to specifically measure activity of recombinant caspase-3 and caspase-8, respectively. Simultaneous measurement of caspase activity and mitochondrial membrane potential in the same cell identified a differential induction of mitochondrial dependent and independent caspase activation by different cytostatic drugs.

In primary leukemia cells, flow cytometric analysis with the derivative (Z-DEVD)2R proved to specifically detect proficient and deficient caspase activation in T ALL and B precursor ALL cells. Despite similar induction of cell death, a differential activation of caspases by Cytarabin and Cyclophosphamide could be quantified, indicating that in addition to deficient caspase activation, drug specific differences in activation of apoptosis signaling can be quantified.

The combination of rhodamine 110 coupled peptide substrates and potential sensitive mitochondrial dyes provide a sensitive tool to study caspase and mitochondria dependent apoptosis pathways induced by anti-leukemic drugs in primary leukemia cells.