Question

Treatment of cells with the BOBA drug induces apoptosis of cells and leads to DNA fragmentation.

A) (4pts) Describe the cellular mechanism that causes DNA fragmentation during apoptosis to occur. (4 sentence max)

B) (8pts) Describe an experiment, step by step, that can be used to determine how many hours after BOBA treatment DNA fragmentation occurs. (8 sentence max)

C) (4pts) One of your friends said they would use an Annexin V and PI staining technique to identify how many hours post BOBA treatment DNA fragmentation occurs. Why won’t this experimental method work to answer this experimental question? (4 sentence max)

Answer 1

A) Cellular mechanism causing DNA fragmentation during apoptosis

Apoptosis is programmed cell death. The DNA fragmentation is activated by an enzyme caspase activated DNase (CAD). A protein inhibits CAD which is called as ICAD (inhibitor CAD). During apoptosis caspase-3, cleaves ICAD - CAD complex to free the CAD enzyme. The CAD cleaves the inter-linker sites at the nucleosomes to cleave the chromosomal DNA leading to fragmentation.

B) Step to determine BOBA treatment and DNA fragmentation

BOBA is 3-α-Butyryloxy-β-boswellic acid. It is an analog of boswellic acid. It induces apoptosis and cytotoxicity thereby displaying anti-cancer activity. BOBA promotes signaling pathway to mediate apoptotic cell death. The PI3K-Akt pathway is an important pathway involve in tissue repairing and cell survival. This pathway affects several inflammatory molecules such as PARP and NF-kB leading to migration and inflammation. The BOBA inhibits PARP and NF-kB thereby preventing the repair of damaged DNA leading to apoptosis. The BOBA were treated on HeLa cells grown in titre plates for time period of 24 and 48 hrs. The cultures were incubated with MTT and mean inhibitory concentration after a particular time period is calculated. PI3Kinase activity and inhibitor assay was done to understand if BOBA targets PI3K pathway. PARP activity using western blot was analyzed in HeLa cells with increased concentration of BOBA. The BOBA produced concentration dependent cell inhibition and DNA fragmentation at 48 hours of incubation.

C) Use an Annexin V and PI staining technique on DNA fragmentation

The annexin V protein and propidium iodide (PI) allows the proper estimation of apoptotic incidence. Using a flow cytometer the apoptotic cells could be determined. It can easily distinguish viable cells (annexin -/PI -), early apoptosis cells (annexin +/PI -), late apoptotic cells (annexin+/PI+) and necrotic cells (annexin -/ PI+). This method cannot distinguish apoptotic cells because of inadequate strand breaks when internucleosomal DNA degradation does not occur properly. The fragmentation stops abruptly during the initial DNA cleavage. Hence this assay cannot be used after BOBA treatment.