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In addition to caspases and Annexin V, there are several other proteins important for the study of apoptosis, including the Bcl-2 family, tumor necrosis factor receptor (TNFR) family, PARP, and other signaling molecules.

Bcl-2 family members, identified by the presence of conserved BCL2 homology (BH3) domains, are versatile key regulators of apoptosis. Bcl-2, for example, protects cells from apoptosis by associating with the mitochondrial membrane and preventing the release of cytochrome c from the mitochondria. In contrast other Bcl-2 family members such as Bax promote apoptosis. Increased levels of Bcl-2 have been reported in cancer.

The TNFR family contains many members, including CD95, that can be divided into three major groups based on structure. Signaling through the TNFR pathway leads to apoptosis.

Additional Proteins for the Study of Apoptosis

PARPs are DNA repair enzymes that are activated by DNA strand breaks. Cleavage of PARP by caspase-3 into 24- and 89-kDa fragments inactivates the PARP enzyme.

BD Biosciences carries antibodies specific for cleavage products of PARP that are useful markers of apoptosis. These antibodies are available in a variety of formats and can be combined with other markers to gain additional information about the cell.

Simultaneous Studies of Apoptosis, Cell Cycle, and DNA Damage

Apoptosis and cell proliferation assays are particularly useful for basic cancer research and drug discovery. Comparing data across different experiments can be challenging due to variability introduced by sample handling, timing, and variability within the sample.

Multicolor flow cytometry addresses these challenges and is an excellent tool to study apoptosis and cell proliferation. Relevant markers can be combined with cell phenotyping markers to look at events within subpopulations of cells. Antibodies to phosphoproteins can be used to examine phosphorylation events.

In this experiment, Jurkat cells were treated with camptothecin, a potent inhibitor of topoisomerase I and apoptosis inducer.

In this experiment, Jurkat cells were treated with camptothecin, a potent inhibitor of topoisomerase I and apoptosis inducer. Phosphorylation of H2AX, a protein important for maintaining genome integrity, has been shown to correlate with levels of DNA damage. Using multicolor flow cytometry, cell proliferation (BrdU), apoptosis (cleaved PARP), and DNA damage (histone H2AX pS140) were evaluated in the same experiment.

Immunofluorescence of cleaved PARP
Immunofluorescence of cleaved PARP

HeLa cells grown were either left untreated (A) or treated with staurosporine (1.0 mM, 4 h) to induce apoptosis (B). Cells were then fixed with 3.7% formaldehyde (15 min on ice), then permeabilized in 0.25% Triton™ X-100/3% BSA/PBS (15 min on ice). Cells were then washed twice with 3% BSA/PBS and stained with 4 μL/mL of FITC-labeled anti-PARP in 3% BSA/PBS (1 h at RT). Cells were washed twice with 3% BSA/PBS and then visualized by immunofluorescence microscopy. A' and B' represent phase correlates of A and B, respectively. The results indicate that untreated cells were primarily negative for cleaved PARP (A), whereas a significant percentage of the staurosporine-treated population is positive for cleaved PARP.