Clark W. Distelhorst

Bcl-2 suppresses Ca2+ release through inositol 1,4,5-trisphosphate receptors and inhibits Ca2+ uptake by mitochondria without affecting ER calcium store content.

Cell survival is promoted by the oncoprotein Bcl-2. Previous studies have established that one of the pro-survival actions of Bcl-2 is to reduce cellular fluxes of Ca2+ within cells. In particular, Bcl-2 has been demonstrated to inhibit the release of Ca2+ from the endoplasmic reticulum. However, the mechanism by which Bcl-2 causes reduced Ca2+ release is unclear. In the accompanying paper [C.J. Hanson, M.D. Bootman, C.W. Distelhorst, T. Maraldi, H.L. Roderick, The cellular concentration of Bcl-2 determines its pro- or anti-apoptotic effect, Cell Calcium (2008)], we described that only stable expression of Bcl-2 allowed it to work in a pro-survival manner whereas transient expression did not. In this study, we have employed HEK-293 cells that stably express Bcl-2, and which are, therefore, protected from pro-apoptotic stimuli, to examine the effect of Bcl-2 on Ca2+ homeostasis and signalling. We observed that Bcl-2 expression decreased the Ca2+ responses of cells induced by application of submaximal agonist concentrations. Whereas, decreasing endogenous Bcl-2 concentration using siRNA potentiated Ca2+ responses. Furthermore, we found that Bcl-2 expression reduced mitochondrial Ca2+ uptake by raising the threshold cytosolic Ca2+ concentration required to activate sequestration. Using a number of different assays, we did not find any evidence for reduction of endoplasmic reticulum luminal Ca2+ in our Bcl-2-expressing cells. Indeed, we observed that Bcl-2 served to preserve the content of the agonist-sensitive Ca2+ pool. Endogenous Bcl-2 was found to interact with inositol 1,4,5-trisphosphate receptors (InsP3Rs) in our cells, and to modify the profile of InsP3R expression. Our data suggest that the presence of Bcl-2 in the proteome of cells has multiple effects on agonist-mediated Ca2+ signals, and can abrogate responses to submaximal levels of stimulation through direct control of InsP3Rs.

Targeting Bcl-2 based on the interaction of its BH4 domain with the inositol 1,4,5-trisphosphate receptor

Bcl-2 is the founding member of a large family of apoptosis regulating proteins. Bcl-2 is a prime target for novel therapeutics because it is elevated in many forms of cancer and contributes to cancer progression and therapy resistance based on its ability to inhibit apoptosis. Bcl-2 interacts with proapoptotic members of the Bcl-2 family to inhibit apoptosis and small molecules that disrupt this interaction have already entered the cancer therapy arena. A separate function of Bcl-2 is to inhibit Ca2+ signals that promote apoptosis. This function is mediated through interaction of the Bcl-2 BH4 domain with the inositol 1,4,5-trisphosphate receptor (IP3R) Ca2+ channel. A novel peptide inhibitor of this interaction enhances proapoptotic Ca2+ signals. In preliminary experiments this peptide enhanced ABT-737 induced apoptosis in chronic lymphocytic leukemia cells. These findings draw attention to the BH4 domain as a potential therapeutic target. This review summarizes what is currently known about the BH4 domain of Bcl-2, its interaction with the IP3R and other proteins, and the part it plays in Bcl-2s anti-apoptotic function. In addition, we speculate on how the BH4 domain of Bcl-2 can be targeted therapeutically not only for diseases associated with apoptosis resistance, but also for diseases associated with accelerated cell death.

The cellular concentration of Bcl-2 determines its pro- or anti-apoptotic effect.

Bcl-2 is an oncoprotein that is widely known to promote cell survival by inhibiting apoptosis. We explored the consequences of different expression paradigms on the cellular action of Bcl-2. Using either transient or stable transfection combined with doxycycline-inducible expression, we titrated the cellular concentration of Bcl-2. With each expression paradigm Bcl-2 was correctly targeted to the endoplasmic reticulum and mitochondria. However, with protocols that generated the greatest cellular concentrations of Bcl-2 the structure of these organelles was dramatically altered. The endoplasmic reticulum appeared to be substantially fragmented, whilst mitochondria coalesced into dense perinuclear structures. Under these conditions of high Bcl-2 expression, cells were not protected from pro-apoptotic stimuli. Rather Bcl-2 itself caused a significant amount of spontaneous cell death, and sensitised the cells to apoptotic agents such as staurosporine or ceramide. We observed a direct correlation between Bcl-2 concentration and spontaneous apoptosis. Expression of calbindin, a calcium buffering protein, or an enzyme that inhibited inositol 1,4,5-trisphosphate-mediated calcium release, significantly reduced cell death caused by Bcl-2 expression. We further observed that high levels of Bcl-2 expression caused lipid peroxidation and that the deleterious effects of Bcl-2 could be abrogated by the reactive oxygen species (ROS) scavenger Trolox. When stably expressed at low levels, Bcl-2 did not corrupt organelle structure or trigger spontaneous apoptosis. Rather, it protected cells from pro-apoptotic stimuli. These data reveal that high cellular concentrations of Bcl-2 lead to a calcium- and ROS-dependent induction of death. Selection of the appropriate expression paradigm is therefore crucial when investigating the biological role of Bcl-2.

Glucocorticoid-Induced Apoptosis

Publisher Summary In present days, glucocorticoids are a mainstay of the therapeutic armamentarium for lymphomas and other lymphocytic malignancies. When treated with glucocorticoids, malignant lymphocytes isolated from blood or bone marrow of patients undergo programmed cell death, or apoptosis. However, the fact that malignant lymphocytes have a propensity to become resistant to glucocorticoids is significantly limiting its therapeutic efficacy. A detailed understanding of the mechanism of glucocorticoid-induced apoptosis is likely to translate into new therapeutic possibilities for lymphoid malignancies, including methods for circumventing or overcoming glucocorticoid resistance. This chapter addresses a working model of glucocorticoid-induced apoptosis. This model is founded on existing knowledge regarding the mechanism of glucocorticoid-induced cell death in lymphocytes and thymocytes, together with emerging evidence from other experimental systems. Based on the genetic pathway for programmed cell death recently unraveled in the primitive nematode Caeizorhahditis elgans, cell death in glucocorticoid-treated lymphocytes is viewed as a genetically regulated, stepwise process beginning with the decision of the healthy lymphocyte to die, the execution of the death program, engulfment by other cells, and degradation. The model reveals existing gaps in understanding, thereby identifying both long-standing unresolved questions and new questions of pressing importance.

Unreliability of the cytochrome c-enhanced green fluorescent fusion protein as a marker of cytochrome c release in cells that overexpress Bcl-2.

A cytochrome c-enhanced green fluorescent protein chimera (cyt-c·EGFP) was used to monitor the release of cytochrome c from mitochondria in Bcl-2-negative and Bcl-2-positive MDA-MB-468 breast cancer cells. A comparison was made with the intracellular distribution of endogenous cytochrome c based on Western blotting of cell fractions and immunocytochemistry. The release of endogenous cytochrome c from mitochondria into the cytoplasm was detected in Bcl-2-negative cells treated with the kinase inhibitor staurosporine or the calcium-ATPase inhibitor thapsigargin. No release of endogenous cytochrome c was evident in Bcl-2-positive cells, consistent with earlier evidence that Bcl-2 overexpression inhibits cytochrome c release from mitochondria. Cyt-c·EGFP appeared to be localized to the mitochondria in Bcl-2-negative cells and to be released into the cytoplasm following treatment with either staurosporine or thapsigargin. However, in Bcl-2-positive cells the pattern of distribution of cytochrome c-EGFP was inconsistent with that of endogenous cytochrome c, due to accumulation of both cyt-c·EGFP and free EGFP in the cytoplasm of both treated and untreated cells. In summary, cyt-c·EGFP may be useful for monitoring cytochromec release in living cells that do not express high levels of Bcl-2 but is an unreliable marker of cytochrome crelease in cells that overexpress Bcl-2.

Abstract B42: The regulation of the ER-mitochondria-Ca2+ cross-talk by Bcl-2 and Bcl-XL: A new scenario for the development of selective tools in oncology?

The process of “programmed cell suicide”, referred to as apoptosis is classically controlled by the B-cell lymphoma-2 (Bcl-2) family members, a group of intracellular proteins with a pro-death (e.g. Bax and Bak) or anti-death (e.g. Bcl-2, Bcl-Xl) role. Thus, the excessive expression of the protective Bcl-2 members is a typical trademark associated with the survival advantage of many hematological as well as solid cancers [1-2]. The recently identified interaction of the anti-apoptotic Bcl-2 protein with the inositol 1,4,5-trisphosphate receptor (IP 3 R), a ubiquitous Ca 2+ -release channel in the endoplasmic reticulum (ER), represents one of the stratagems by which Bcl-2 extends the life-span of cancer cells [3]. Bcl-2 can thereby inhibit the IP 3 R-mediated Ca 2+ firing and reduce the lethal Ca 2+ transfer from the ER to the mitochondria. The only Bcl-2 molecular component essential and sufficient for this inhibitory activity on the IP 3 R is the homology domain 4 (Bcl-2-BH4) [4]. In our study, we first revealed that although Bcl-2 shares high structural and biochemical similarity with Bcl-Xl, its closest protein relative, the latter could not curb the pro-apoptotic ER-Ca 2+ discharge. This differential action of Bcl-2 versus Bcl-Xl on the IP3R could be attributable to one critical amino acid difference in their BH4 domains [5]. Furthermore, we found that BH4-Bcl-Xl, which is also able to protect against Ca 2+ -mediated apoptosis, acted downstream of IP 3 R signaling, by physically interacting with the voltage-dependent anion channel 1 (VDAC1), an outer mitochondrial Ca 2+ gateway. Our findings opened novel avenues to selectively suppress malignant Bcl-2 or Bcl-Xl functions in cancer cells by targeting their BH4 domain biology. Accordingly, we screened for apoptosis induction a set of diffuse large-B-cell lymphoma cells (DL-BCL) treated with a cell-permeable version of a peptide that selectively antagonized Bcl-2-BH49s binding to the IP 3 R (IP 3 R-derived peptide: IDP). As a result, we observed that the most responsive subclasses of DL-BCL are also the ones that express the highest levels of the type 2 IP 3 R (IP 3 R2), which is the most sensitive isoform to IP3 signaling [6]. This is compatible with the higher metabolic needs of the DL-BCL, which in the responsive DL-BCL, was reflected in: 1) a general chronic activation of the B-cell receptor (BCR) signaling [7]; 2) an up-regulation of IP 3 R2 and 3) an addiction to Bcl-2 in order to suppress the potentially lethal IP 3 R hyperactivity. Collectively, our work provided a better understanding of the molecular and functional conversation between the Ca 2+ channels at the ER/mitochondria interface and the Bcl-2/Bcl-Xl proteins. These new findings could lead to the development of more selective and safer peptidomimetics targeting the adaptive Ca 2+ -signaling dysregulation of cancer cells. [1] (http://www.ncbi.nlm.nih.gov/pubmed/18362212) [2] (http://www.ncbi.nlm.nih.gov/pubmed/14996506) [3] (http://www.ncbi.nlm.nih.gov/pubmed/15263017) [4] (http://www.ncbi.nlm.nih.gov/pubmed/19706527) [5] (http://www.ncbi.nlm.nih.gov/pubmed/21818117) [6] (http://www.ncbi.nlm.nih.gov/pubmed/23681227) [7] (http://www.ncbi.nlm.nih.gov/pubmed/23449308) Citation Format: Haidar Akl, Giovanni Monaco, Elke Decrock, Rita La Rovere, Kirsten Welkenhuyzen, Tomas Luyten, Santeri Kiviluoto, Tim Vervliet, Jordi Molgo, Ludwig Missiaen, Katsuhiko Mikoshiba, Luc Leybaert, Jan B. Parys, Humbert De Smedt, Clark W. Distelhorst, Geert Bultynck. The regulation of the ER-mitochondria-Ca2+ cross-talk by Bcl-2 and Bcl-XL: A new scenario for the development of selective tools in oncology? [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B42.

Steroid Hormones and Hormone Receptors in Neoplastic Diseases

Publisher Summary This chapter describes the role of steroid hormones and hormone receptors in promoting the growth of neoplastic diseases. There is considerable evidence that different classes of steroid hormone receptors in tumor cells are under autoregulatory control; thus, the dominant factor regulating steroid hormone receptor levels in tumor cells appears to be the cognate hormonal ligand itself. The effects of steroid hormones on cells are mediated by receptors that interact with DNA and regulate gene transcription. Estrogens have a direct mitogenic effect on breast cancer cells and shorten the duration of their cell cycle. In vivo, growth factors, and estrogen probably act in concert with other systemic mitogens to promote tumor growth. In addition to intrinsic oncogenic potential, steroid hormone receptors may be involved in the pathogenesis of cancer through their interaction with environmental toxins. Both estrogen and progesterone receptors are detectable in endometrial adenocarcinoma cells, where the level of these receptors correlates with the degree of cellular differentiation. Immunocytochemical assays of fresh frozen sections of endometrial carcinoma tissue demonstrate considerable heterogeneity in terms of the cellular distribution of estrogen and progesterone receptors.