Induction of Ca2+-driven apoptosis in chronic lymphocytic leukemia cells by peptide-mediated disruption of Bcl-2–IP3 receptor interaction

Abstract

IntroductionThe Bcl-2 protein contributes to the pathophysiology of cancer and the resistance of cancer to therapeutic agents by virtue of its ability to inhibit apoptosis. 1,2 The Bcl-2-positive lymphoid malignancies follicular lymphoma and chronic lymphocytic leukemia (CLL) are prime examples. They are associated with an elevation of Bcl-2 because of the t(14;18) chromosomal translocation in follicular lymphoma 3 and the loss of miR-15a and miR16-1 in CLL. 4,5 Cure of these malignancies is infrequently achieved with current therapeutic modalities, and thus a major challenge remains to develop new treatment modalities based on an understanding of the fundamental disease mechanisms. 6 Bcl-2 blocks apoptosis in part by binding its proapoptotic relatives, thus preserving mitochondrial integrity and preventing cytochrome c release. 7,8 Therefore, considerable investment has been made in the development of novel therapeutic agents, such as , that disrupt the inhibitory interaction of Bcl-2 with its proapoptotic relatives. 1,2,9-11 Several of these agents are currently undergoing clinical testing. However, Bcl-2 also inhibits apoptosis by regulating the release of Ca 2ϩ from the endoplasmic reticulum (ER). [12][13][14] This recently characterized mechanism involves a physical interaction of Bcl-2 with the inositol 1,4,5-trisphosphate receptor (IP 3 R) Ca 2ϩ -release channel on the ER. Through this interaction, Bcl-2 prevents cytoplasmic Ca 2ϩ elevation sufficient to trigger apoptosis. However, the potential contribution of the Bcl-2-IP 3 R interaction to the survival of CLL has not been investigated, so the opportunity for targeting this interaction therapeutically has not yet been realized.The IP 3 R is an IP 3 -gated Ca 2ϩ channel that is highly conserved, represented by 3 isoforms, and present in virtually all cell types. 15,16 IP 3 -dependent release of Ca 2ϩ from the ER into the cytoplasm produces Ca 2ϩ signals, generally in the form of Ca 2ϩ oscillations, which govern diverse cellular functions including cell proliferation and survival. 17,18 Ca 2ϩ oscillations support cell survival in part by positively regulating mitochondrial metabolism, but sustained high-amplitude elevations of Ca 2ϩ induce mitochondrial Ca 2ϩ overload and apoptosis. [19][20][21] Bcl-2 inhibits high-amplitude, proapoptotic Ca 2ϩ elevation but does not interfere with physiologic Ca 2ϩ oscillations. 22 In fact, under certain circumstances Bcl-2 and its homolog Bcl-xl enhance Ca 2ϩ oscillations, [22][23][24][25][26] and through this mechanism are predicted to promote efficient mitochondrial bioenergetics. 27 Thus, Bcl-2 supports cell survival both by enhancing physiologic Ca 2ϩ signals and by blocking proapoptotic Ca 2ϩ elevation.A major focus of our work has been to understand how Bcl-2 inhibits proapoptotic elevation of Ca 2ϩ based on evidence that Bcl-2 binds to the IP 3 R and thus inhibits ER Ca 2ϩ release. [28][29][30][31][32] Although this interaction has been mainly detected in cell extracts by coimmunoprecipitation or blue native gel ...

BAX inhibitor-1 is a Ca2+ channel critically important for immune cell function and survival

“…However, this experimental approach was not possible because BAPTA-AM treatment rapidly killed primary B cells (not shown), as has previously been shown for leukemic cells. 31 Thus, the observed defects in mitochondrial Ca 2+ buffering capacity seen in cultured BI-1 KO B cells are correlated with caspase activation and cell death, but a direct cause and effect relationship was not demonstrable.…”

Feedback regulation mediated by Bcl-2 and DARPP-32 regulates inositol 1,4,5-trisphosphate receptor phosphorylation and promotes cell survival

“…T-cell lines are used in the present work, as in previous studies (8,13,16). Jurkat is a human CD4 + T-cell line that expresses readily detectable levels of Bcl-2 (Fig.…”

“…A 20-amino acid peptide (InsP 3 Rderived peptide, or IDP) corresponding to the Bcl-2 binding site on the InsP 3 R inhibits Bcl-2-InsP 3 R interaction, thus eliminating Bcl-2's control over InsP 3 R-mediated Ca 2+ elevation (6,7). This peptide induces marked Ca 2+ elevation and Ca 2+ -mediated apoptosis in primary chronic lymphocytic leukemia (CLL) cells and in B-cell lymphoma lines, indicating that Bcl-2-InsP 3 R interaction contributes to the apoptosis-resistance characteristic of these lymphoid malignancies (13,14).…”

Biphasic regulation of InsP 3 receptor gating by dual Ca 2+ release channel BH3-like domains mediates Bcl-x L control of cell viability

“…Nevertheless, under conditions of cell stress the close proximity of mitochondria to Ca 2+ release sites may result in mitochondrial Ca 2+ overload and initiate Ca 2+ -dependent forms of cell death, including necrosis and apoptosis (11)(12)(13). It has been suggested that high levels of ER Ca 2+ (14)(15)(16) and enhanced activity of the InsP 3 R (17)(18)(19) promote cell death by providing a higher quantity of released Ca 2+ to mitochondria (3,20,21).…”

“…Conversely, the Bcl-x L BH4 domain may lack this interaction (36). Inhibition of the Bcl-2 BH4 domain interaction with the channel enhanced InsP 3 R-mediated Ca 2+ signals and apoptosis sensitivity in white blood cells (18,35,37).…”