Did you see your cells under microscope? If morphology is too bad then it might be possible that membrane is completely damaged and thats why you should do TUNEL assay for detecting apoptosis.

It's interesting. You can try and find out:

1. if the downstream events are not effected though the caspase 3/7 activity increased.

2. if the growth or viability of the cells is decreased.

Good luck

Caspase 3/7 activity is a luminescent assay that may have some limitations

1.test the cell viability (direct counting), did you see a major decrease?

2.western blot for PARP clevage and caspase 3 cleavage to confirm caspase activity

3.The gold-standard for determination apoptosis involves morphologic confirmation of characteristic features: condensation and fragmentation of nuclear contents, plasma membrane blebbing, and loss of plasma membrane integrity, ethidium bromide/acridine orange may be used.

4.to prove the involment of caspases you may use caspase inhibitors to rescued the cells from the treatment

5. As mentioned TUNEL is also a good assay

That sounds very interesting!! Caspases which classically known as apoptotic caspases (Casp3, Casp7) have nonapoptotic roles in macrophage activation and differentiation. Also, Casp6 activation leads to a protracted form of axonal degeneration in neurons, without killing the cell soma (cell body). Here are some of the key references on this topic:

Nature. 2011 Apr 21;472(7343):319-24. Epub 2011 Mar 9.

Caspase signalling controls microglia activation and neurotoxicity.

Burguillos MA, Deierborg T, Kavanagh E, Persson A, Hajji N, Garcia-Quintanilla A, Cano J, Brundin P, Englund E, Venero JL, Joseph B.

Selective and protracted apoptosis in human primary neurons microinjected with active caspase-3, -6, -7, and -8.

Zhang Y, Goodyer C, LeBlanc A.

J Neurosci. 2000 Nov 15;20(22):8384-9.

Nature. 2009 Feb 19;457(7232):981-9.

APP binds DR6 to trigger axon pruning and neuron death via distinct caspases.

Nikolaev A, McLaughlin T, O'Leary DD, Tessier-Lavigne M.

Specific involvement of caspases in the differentiation of monocytes into macrophages

Olivier Sordet, Cédric Rébé, Stéphanie Plenchette, Yaël Zermati, Olivier Hermine, William Vainchenker, Carmen Garrido, Eric Solary, and Laurence Dubrez-Daloz

Blood. 2002 Dec 15;100(13):4446-53. Epub 2002 Aug 8.

Thanks to all for reply.@Amaury Dumont

For caspase activity assay, I used caspase inhibitors which shown negligible or no activity. and positive control shown almost 8-9 fold increase indicating that caspase assay proper. Cell viability (direct counting),western PARP,caspase activity i will do.

@Aiken Huang

I have not seen cells under microscope ,but in annexin V FITC +PI experiment we observed treatment with our compound almost 50%cells in early apoptosis(EA,lower right portion).where as PI3K+our compound treatment apporox 1%in EA.95%cells in lower left portion indicating live.

Really TUNNEL assay how could it be helpful.

@Dalia Halawani

thanks for references iam using leukemia cell lines.

increase in caspase 3 activity associated with apoptosis, however level of oxidative stress determines the actual caspase activity i.e. caspase activity in inversely to oxidative stress and directly to apoptosis, other caspase are executors

Some papers reviewing non-apoptotic roles of caspases

http://www.ncbi.nlm.nih.gov/pubmed/21338340

http://www.ncbi.nlm.nih.gov/pubmed/19373560

http://www.ncbi.nlm.nih.gov/pubmed/19273205

http://www.ncbi.nlm.nih.gov/pubmed/22244258

http://www.ncbi.nlm.nih.gov/pubmed/21338340 http://www.ncbi.nlm.nih.gov/pubmed/19373560 http://www.ncbi.nlm.nih.gov/pubmed/19273205 http://www.ncbi.nlm.nih.gov/pubmed/22244258

Leukemia cell lines are hematopoietic in origin (myeloid ?), where this phenomenon is well documented. You may want to consider the possibility that your compound is altering the differentiation state of the cells in a caspase depending manner. Good luck.

Thanks @Dalia Halawani could u please give me reference.

the cell lines iam using are jurkat,CEM-CM3 lymphoid in origin.

There is a whole section on lymphocytes in this review, which was suggested by Bernard Mignotte, http://www.ncbi.nlm.nih.gov/pubmed/19273205.

Likely in normal cells, a high level of caspase-activity increases apoptosis, but cancer cells, including leukemia cells, are resistant to apoptosis. On the other hand, apoptosis inhibitors, such as hand cIAP2 and XIAP are regulated through PI3k or AKT regulatory pathways; likely the expression of these apoptosis inhibitors was lowered by the PIK3 inhibitor, thus increasing caspase 9,3 or 7activity; but not necessarily this is an proapoptotic caspase-activity.

Please check the apoptosis using some other assays because some times due to limitation of annexiv/PI apoptosis is not detected. you can just think activity cant be enhanced randomly however affinity can be inhibited.

E2F1 regulation may play a role in this you mention. Have a look to this papers:

http://www.ncbi.nlm.nih.gov/pubmed/12954980

http://www.ncbi.nlm.nih.gov/pubmed/18167336

The annexin V FITC+PI assay is an early marker for apoptosis, you should try apoptosis markers down stream to caspase-3 activity, for example, ICAD (inhibitor of Caspase-activated DNase ) or PARP cutting and tunnel assays. In addition, optimal apoptosis times occurs around 8-12h of the apoptotic stimulus, check the timing in your experiments.