On my work, flow cytometry experiments with treated tumor cells point to cell cycle arrest on G1 and apoptotic death, but there's no p53 and caspase expression. PCR array experiments were done with 6-hours and 24-hours treatments.
Actually, I am testing MCF-7! Thank you very much, Dr. Shankar! The PCR array experiments show only a very low caspase 2 up-regulation, and the Ct is higher than the default cut-off (=35).
Thanks!
Luis, Apoptosis can generally be divided into two kinds: caspase-dependent apoptosis and caspase-independent apoptosis, the former is usually performed by the activated caspase-3 at the final step, while the latter is usually performed by some factors released from mitochondria, e.g. AIF, EndoG etc. Down-regulation of Bcl-2 and up-regulation of Bax and CIDEA do not necessarily means apoptosis, especially only detected by PCR. Western blot is the best choice to detect whether caspases were activated and involved in apoptosis. In addition, sub-G1 curve appearance usually means the occurrence of cell apoptosis, p53 and caspases are not necessarily involved the process. Just for reference. wish a little help for you! Best!
Caspase expression does not equal their activation, neither, in fact does their cleavage. Endogenous factors such as XIAP may prevent full caspase activation and cell death, downstream of cytochrome c release from the mitochondria. If you're doing FACS, I suggest do perform annexin V staining of unpermeabilized/unfixed cells. This is by far the easiest and generally reliable way to determine apoptosis. MCF7 cells only undergo apoptosis very slowly because they lack caspase-3. They have caspase-7, which performs similar functions, but that's a very slow enzyme.
Thanks, Dr. Ramm.
I've already done annexin V/PI tests and the results pointed late and early apoptosis. I have also performed MTT, LDH and comet assay, and cell cycle test. I wonder if this data is enough to confirm that it is apoptosis. Is it?
The golden standard of cell apoptosis is DNA ladder,one typical biochemical characteristic of apoptosis. DNA ladder is the final convincing proof of apoptosis.
As you are seeing upregulation of Bax and decrease in Bcl2, its worth to investigate the changes in mitochondrial ROS and mitochondrial membrane potential. For early events Annexin V/PI works well in MCF7. For late events TUNEL and DNA ladder are the convincing proof.
You observed apoptosis, without p53 or caspase involvement. That can happen, given that many other factors (overtly working through redundantly processes) may be activated, in both caspase-dependent and caspase-independent pathways. Additionally, caspase-dependent pathways may belong to the intrinsic or the extrinsic pathways. Thus, I think you should measure a lot of other parameters to ascertain if the apoptotis should be attributed to one of these pathways. As you observed an "early" apoptotic response, this kind of programmed cell death is usually triggered by caspase-independent processes and you should look to AIF activity (we have published a paper on that topic).
bests,
Mariano
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