[Cell Cycle 4:12, 1741-1743, December 2005]; ©2005 Landes Bioscience
Histone Deacetylase Inhibition Induces Apoptosis in Neuroblastoma Perspective
ABSTRACT
Received 09/28/05; Accepted 09/29/05
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Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=2212
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*Correspondence to: Roland P.S. Kwok; 6428 Medical Science I; 1301 E. Catherine Street; Ann Arbor, Michigan 48109 USA; Tel.: 734-615-1384; Fax: 734-936-8617; Email:
[email protected]
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Departments of, 1Obstetrics and Gynecology, 2Pediatrics, and 3Biological Chemistry; University of Michigan; Ann Arbor, Michigan USA
CBP, HAT, Ku70, Bax, mitochondria, cytochrome c, acetylation
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histone acetyl transferases CREB-binding protein histone deacetylases histone deacetylase inhibitors neuroblastoma p300/CBP-associated protein neuronal stromal
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ABBREVIATIONS
Acetylation of histones correlates with activation of gene transcription.1 Histone acetyl transferases (HATs), such as CREB-binding protein (CBP) and p300, were first identified as transcriptional coactivators, and despite the nomenclature, acetylate many nonhistone proteins as well as histones.2 Similarly, enzymes that remove the acetyl group from acetylated histone and nonhistone proteins are referred to as histone deacetylases (HDACs) and are classified into one of three families based on structure. Acetylation of nonhistone proteins affects cellular functions, ranging from mitotic spindle formation3 to receptor signal response coupling.4 Consequently, enzymes that regulate the acetylation status of histones and other proteins have attracted considerable attention to better understand the molecular regulation of cellular processes and as potential therapeutic targets. Small molecule inhibitors of class 1 and 2 HDACs generate responses in cells that are associated with anti-tumor agents, triggering apoptosis, arresting the cell cycle, and inducing further differentiation.5 The toxicity and therapeutic effects of a number of these compounds have been evaluated in Phase I and Phase II clinical trials involving various types of tumors.6 Even so, the molecular mechanisms linking histone deacetylase inhibitors (HDACIs) with their effects on cells in culture, or against tumors growing in animals or people, remain unclear. Because many known substrates of HDACs, such as histones and transcription factors, are related to gene expression and transcription functions, it is not surprising that microarray transcriptome analysis has identified genes that mediate cell proliferation, cell cycle progression and survival among those that are differentially regulated by HDACIs.7 However, it is far from clear whether the anti-cancer effects attributed to HDACIs depend on altered gene transcription. Nonchromatin linked mechanisms of HDACI effects are highly likely; and to date an important example includes HDACI-induced activation of TRAIL and Fas signaling pathways in leukemic cells.8-10 Our recent studies using neuroblastoma (NB) cells provide an additional, non-chromatin mechanism to account for the anti-tumor activity of HDACIs.11 This work identified the cytoplasmic and nuclear protein Ku70 as an acetylated protein that is not only sensitive to the action of HDACIs but mediates the apoptotic response of NB cells to these agents. Ku70 was originally discovered as an auto-antigen protein.12,13 It was subsequently shown to be essential for the repair of nonhomologous DNA double-strand breaks. In this role, Ku70 complexes with Ku80 to form the DNA binding component of DNA-dependent protein kinase.14,15 Ku70 expression in tumors is associated with unfavorable responses to conventional chemotherapy and radiation treatment. Ku70 expression in cervical carcinomas, for example, is inversely correlated with radiation sensitivity and patient survival.16 Experimentally, ectopic over-expression of Ku70 leads to resistance to agents including curcumin and gamma radiation.17 Conversely, strategies that lower Ku70 enhance chemoand radiation sensitivity of cells and tissues.18-20
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KEY WORDS
HATs CBP HDACs HDACIs NB P/CAF N S
Histone deacetylase inhibitors constitute a promising new treatment for cancer due to their novel site of action and low toxicity. Almost all histone deacetylase inhibitors currently in clinical development have anti-proliferate activities against cells in cultures, and specifically cause cell cycle arrest, differentiation and apoptosis. Interestingly, despite their rapid advance into clinical use, the cellular responses leading to these effects remain unclear. We recently reported that histone deacetylase inhibitor treatment induces apoptosis of neuroblastoma cells by increasing the acetylation of Ku70 in the cytoplasm, resulting in the release of Bax from Ku70. Subsequently, Bax releases cytochrome c from mitochondria causing apoptosis. Here we will discuss these findings and the implications of our model for the further clinical development of histone deacetylase inhibitors in the treatment of cancer.
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Chitra Subramanian2 Anthony W. Opipari Jr.1 Valerie P. Castle2 Roland P.S. Kwok1,3,*
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Histone Deacetylase Inhibition Induces Apoptosis in Neuroblastoma
The mechanistic basis by which Ku70 altered tumor cell survival was not clear until recent studies showed that Ku70 is also an acetylation-sensitive binding partner for Bax, an apoptotic protein.21 Ku70 binds to Bax and inhibits Bax’s apoptotic activity through its carboxyl terminal,22 and that the same domain interacts with HATs (CBP and p300/CBP-associated factor, P/CAF).21 Moreover, two lysine residues (K539 and K542) within this domain when acetylated by CBP and P/CAF prevent binding with Bax. Inhibition of HDAC activity, using Trichostatin A (to inhibit Class I and II HDACs) and nicotinamide (to inhibit Class III HDACs), results in increased acetylation of these residues,21 suggesting that, on balance, the acetylation status of these same residues is also determined by HDACs.23 In our studies, we focused on the effectiveness of HDACI on the neuronal-type (N) NB cells. NB tumors are heterogeneous, being principally comprised of tumor cells that are classified as either N- or stromal (S)-type cells. N-type cells express high level of the N-myc protein, and are more commonly isolated from high-risk tumor explants.24,25 Thus, N-type cells are used to model the behavior and drug responsiveness of aggressive, highly transformed NB cells that characterize high-risk tumors. The results of our study demonstrate that treatment of the N-type NB cells with HDACI induces the release of Bax from Ku70, is followed by Bax translocation to mitochondria, which leads to release of cytochrome c in the cytoplasm.11 Most importantly, however, expression of an acetylation-sensitive site mutant of Ku70 in NB cells blocks the HDACI-induced apoptotic effect. These results suggest that in these cells, the ability of Ku70 to bind and sequester Bax offers an appealing explanation for its anti-apoptotic effects. However, because acetylation specifically interferes with Bax-Ku70 binding, HDACI treatment modifies this protein’s function in a highly specific way such that it becomes a trigger to induce apoptosis from which Bax is released and activated. Consequently, this model predicts that high levels of Ku70 expression, which limit cellular sensitivity to radiation and some types of chemotherapy, may facilitate sensitivity to HDACIs. Our results raise several important questions: First, is the release of Bax from Ku70 in response to HDACI treatment unique to NB cells? Ku70 is ubiquitously expressed in many cell types,26 and whether cytoplasmic Ku70 binds to Bax in other tumor cell types has not been reported. At this point, we have tested whether cytoplasmic Ku70 binds to Bax in S-type NB cells, which in contrast to N-type cells do not synthesize neurotransmitters, are not N-myc amplified, and generally are incapable of forming tumors in mice.27,28 Both N-type and S-type cells contain similar amounts of Ku70 and Bax, and Bax is bound to Ku70. But unlike N-type cells, HDAC treatment fails to release Ku70 from Bax in S-type cells (manuscript in preparation). Moreover, S-type cells are much less sensitive to the apoptotic effect of HDACI treatment. One interpretation of these results is that because the cells response to HDACI treatment depends on the acetylation status of cytoplasmic Ku70 in cells, an apoptotic response to HDACI treatment requires sufficient acetyltransferase activity to result in increase Ku70 acetylation. If Ku70 remains unacetylated even after HDACI treatment, Bax will still be bound to Ku70, thus apoptosis will not occur. This model predicts that the level of acetylation of cytoplasmic Ku70 is higher in N-type cells than that of S-type cells after HDACI treatment. This possibility remains to be tested. Consequently, the acetyltransferase activity that acetylates cytoplasmic Ku70 appears to be an important determinant in the response to HDACI treatment. Cohen et al have demonstrated that CBP and P/CAF are capable of acetylating Ku70.21 We have recently found 1742
Figure 1. A model for apoptosis in neuroblastoma cells induced by histone deacetylase inhibition.
that the level of CBP in S-type cells is much lower than that in N-type cells, and that increasing CBP levels in S-type cells by transfection reduces cell viability and enhances the sensitivity of these cells to HDACI treatment (manuscript in preparation). This may explain the difference in responsiveness between these two cell types. Together, these data support a model in which the acetylation of cytoplasmic Ku70 following HDACI treatment determines whether cells undergo HDACI-induced apoptosis (Fig. 1). This model would seem to also require that CBP acts in the cytoplasm, even though this gene product is primary known as a coactivator of many transcription factor that functions in the nucleus. Cohen et al. have partially addressed this question by showing that after HDACI treatment of 293T cells, CBP translocates from the nucleus to the cytoplasm;21 although the mechanism for this migration remains unknown. In NB cells and other cell lines, such as HeLa and HEK 293 cells, we have demonstrated that both CBP and p300 are found in the cytoplasm as well as in the nucleus, although the nuclear levels are 5 to 10 fold higher than that in the cytoplasm (manuscript in preparation). These results suggest that CBP is positioned to acetylate Ku70 in the cytoplasm. In conclusion, we propose a model in which the acetylation of Ku70 underlies the responsiveness of NB cells to HDACI treatment. The extent to which this model is applicable to other tumor cell types and to which it operates in vivo remains to be determined. Furthermore, this model is consistent with the idea that the level of CBP in cells critically determines sensitivity to HDACI treatment. Whether CBP expression is a variable that predicts therapeutic outcome is presently unknown. Ongoing work is focused on determining whether it is indeed possible to measure CBP to optimally select NB patients for therapy with HDACI. Finally, this work provides an interesting example of a post-translational modification converting protein function from pro-survival to pro-apoptotic. In this sense, it is interesting to speculate whether there are other anti-apoptotic factors that undergo a “role-reversal” following HDACI-induced acetylation.
Cell Cycle
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Histone Deacetylase Inhibition Induces Apoptosis in Neuroblastoma
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