Filter elution assay to measure apoptotic DNA fragmentation

DNA filter elution is commonly used to study the effects and mechanisms of action of chemotherapeutic drugs and carcinogens (38, 39). The basic DNA elution filter methods were originally designed to assay DNA damage in intact cells or tissues from living animals (38). More recently, DNA elution filter assays were adapted to study drug mechanisms in isolated nuclei (40,41) and in a reconstituted cell-free system (13,16). Altogether, the various elution methods are currently applied in the study of the DNA effects of a variety of anticancer agents (topoisomerase inhibitors, DNA cross-linking and alkylating drugs) in cells in culture, and in the analysis of DNA fragmentation associated with programmed cell death (apoptosis).

A variety of DNA lesions can be detected and quantitated by filter elution methods. Such lesions include DNA single-strand breaks (SSB), as well as double-strand breaks (DSB). SSB and DSB are either protein-associated (PASB) or protein-free ('frank') breaks. Alkaline elutions can also measure DNA-protein cross-links (DPC), interstrand DNA cross-links (ISC), and alkali-labile sites (ALS). More recently, we have used a simple filter elution method to quantitatively measure apoptosis-associated DNA fragmentation (42). We will only describe here this filter elution method. Details of the other alkaline elution methods can be found in detailed reviews and methods chapters (e.g. 38,39,43).

The filter elution assay can detect both internucleosomal and high molecular weight DNA fragmentation in apoptotic cells. Comparison with the pulse-field electrophoresis method indicated that filter elution can detect DNA fragmentation in the absence of internucleosomal DNA fragmentation and that DNA fragments up to 50-80 kb were detectable by filter elution.

Was this article helpful?

0 0

Post a comment