Emetine Regulates the Alternative Splicing of Bcl-x through a Protein Phosphatase 1-Dependent Mechanism

Boon-Unge, Kritsanapol; Yu, Qian; Zou, Tie; Zhou, An; Govitrapong, Piyarat; Zhou, Jianhua

doi:10.1016/j.chembiol.2007.11.004

Cited by 57 publications

(43 citation statements)

References 37 publications

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“…S-Adenosylmethionine as well as its metabolites is proapoptotic by stimulating the PP1-catalyzed dephosphorylation of RS proteins, resulting in the formation of the alternative variant Bcl-x S (28). Emetine, an inhibitor of protein synthesis, can also regulate the alternative splicing of Bcl-x transcript to produce proapoptotic Bcl-x S through a PP1-dependent mechanism (29). Based on our results and these previous studies, we speculate that PP1 may play a key role in modulating the pre-mRNA alternative splicing and that any molecule capable of activating PP1 might have the ability to regulate the alternative splicing of apoptotic gene transcripts.…”

Section: Discussionsupporting

confidence: 76%

Amiloride Modulates Alternative Splicing in Leukemic Cells and Resensitizes Bcr-AblT315I Mutant Cells to Imatinib

et al. 2011

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The antihypertensive drug amiloride is being considered as a tactic to improve cancer therapy including that for chronic myelogenous leukemia. In this study, we show that amiloride modulates the alternative splicing of various cancer genes, including Bcl-x, HIPK3, and BCR/ABL, and that this effect is not mainly related to pH alteration, which is a known effect of the drug. Splice modulation involved various splicing factors, with the phosphorylation state of serine-arginine-rich (SR) proteins also altered during the splicing process. Pretreatment with okadaic acid to inhibit protein phosphatase PP1 reversed partially the phosphorylation levels of SR proteins and also the amiloride-modulated yields of Bcl-x s and HIPK3 U(-) isoforms. Genome-wide detection of alternative splicing further revealed that many other apoptotic genes were regulated by amiloride, including APAF-1, CRK, and SURVIVIN. Various proteins of the Bcl-2 family and MAPK kinases were found to be involved in amiloride-induced apoptosis. Moreover, the effect of amiloride on mRNA levels of Bcl-x was demonstrated to translate to the protein levels. Cotreatment of K562 and BaF3/Bcr-AblT315I cells with amiloride and imatinib induced more loss of cell viability than either agent alone. Our findings suggest that amiloride may offer a potential treatment option for chronic myelogenous leukemia either alone or in combination with imatinib.Cancer Res; 71(2); 383-92. Ó2011 AACR.

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Section: Discussionsupporting

confidence: 76%

Amiloride Modulates Alternative Splicing in Leukemic Cells and Resensitizes Bcr-AblT315I Mutant Cells to Imatinib

et al. 2011

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“…To identify small molecules that regulate alternative splicing, 1,040 FDA-approved drugs and compounds were screened using RT-PCR in C33A cells, a cervical cancer line. As described in our previous study, Emetine regulated alternative splicing of the Bcl-x gene (15). Notably, Emetine was also found to increase the smaller caspase 9b mRNA with a concomitant increase of the larger caspase 9 mRNA (Fig.…”

Section: Resultssupporting

confidence: 79%

“…We previously described that Emetine regulated alternative splicing of Bcl-x, increasing the smaller pro-apoptotic Bcl-xS isoform and decreasing the larger anti-apoptotic Bcl-xL isoform, resulting in the possible sensitization of tumor cells to chemotherapy (15). We further demonstrated that Emetine modulated Bcl-x splicing by affecting PP1 phosphatase.…”

Section: Discussionmentioning

confidence: 82%

“…As discussed in our earlier study (15), Emetine acts as an effective chemotherapeutic agent by increasing the lifespan of tumor-bearing mice (16,17) and thus has the possibility for clinical advantage (18,19). However, although no previous study explains the mechanism responsible for the anti-tumor effect of Emetine, our results regarding the effects of Emetine on Bcl-x splicing (15) suggested that Emetine promoted the expression of pro-apoptotic proteins by regulating alternative splicing. However, based on the results in this study, we deduce that Emetine sensitizes cells to chemotherapy in certain types of tumors but does not sensitize them in others by regulating alternative splicing of caspase 9 in various ways.…”

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confidence: 97%

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Emetine regulates the alternative splicing of caspase 9 in tumor cells

et al. 2011

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Abstract. Exons 3 to 6 in the caspase 9 gene undergo alternative splicing in which the larger caspase 9 splice variant promotes apoptosis, in contrast to the dominant negative anti-apoptotic splice variant, the smaller caspase 9b. In this study, the regulation of the alternative splicing of caspase 9 pre-mRNA was examined in response to Emetine. Treatment of C33A cells, breast cancer MCF-7 cells and MCF-7/Adr cells with Emetine dihydrochloride upregulated the level of smaller caspase 9b mRNA and concomitantly decreased the mRNA level of larger caspase 9 in a dose-and time-dependent manner, indicating that Emetine desensitizes C33A, MCF-7 and MCF-7/Adr to cell death. In contrast, treatment of PC3 cells, a prostate cancer cell line, manifested an opposite effect: a greater production of the larger caspase 9 mRNA with a concomitant decrease of caspase 9b mRNA. Pretreatment with calyculin A, an inhibitor of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) blocked Emetine-induced alternative splicing in cells, in contrast to okadaic acid, a specific inhibitor of PP2A, demonstrating a PP1-mediated mechanism. These results suggest that the various splicing patterns of the caspase 9 gene that are regulated by chemotherapy reagents may contribute to the resistance or sensitization of the tumors to other cell death inducers.

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“…Six compounds were found to inhibit luciferase expression by Ͼ75% and reduce cell viability by Ͻ25% (Table 2). Cephaeline and emetine are closely related alkaloid components of ipecac (19) (a wellknown substance used to induce vomiting), and emetine has been shown to inhibit protein synthesis and promote apoptosis in mammalian cells (3,9,13). Emetine appears to be an especially potent inhibitor of translation of viral mRNA in some systems (16).…”

Section: Fig 2 Establishment Of Ebolavirus Minireplicon Assays For mentioning

confidence: 99%

Minigenome-Based Reporter System Suitable for High-Throughput Screening of Compounds Able To Inhibit Ebolavirus Replication and/or Transcription

Jasenosky

Neumann

Kawaoka

2010

Antimicrob Agents Chemother

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We describe an Ebolavirus minigenome-based system that is suitable for high-throughput screening of compounds able to impair Ebolavirus virus replication and/or transcription. The assay is robust (Z factor, >0.6) and can be carried out in low-biosafety containment. Results from a pilot screen of 960 compounds are presented.Ebolaviruses and the closely related Marburgviruses cause hemorrhagic fever in humans and nonhuman primates, with high mortality rates (18). Currently, treatment of these infections is limited to supportive care (1). The need for efficacious therapeutics is further underscored by the increasing frequency of Ebolavirus outbreaks in both humans and endangered great ape populations (2,20,21).High-throughput screening (HTS) systems should be robust with high reproducibility and should be amenable to work in low-biosafety containment. Ebolavirus is categorized as a biosafety level 4 agent, and HTS systems that rely on live Ebolavirus are thus impractical. Here, we describe a minireplicon system to screen for compounds that interfere with viral replication and/or transcription. In these systems, a virus-like RNA encoding a reporter protein is amplified by the viral proteins required for replication and transcription, i.e., the Ebolavirus polymerase (L), nucleoprotein (NP), and VP30 and VP35 proteins (10,14).A minireplicon system based on Zaire Ebolavirus developed in our laboratory was taken as a starting point (22). First, we generated a reporter construct (p3E5E-Luc) in which the open reading frame (ORF) for the luciferase gene (in antisense orientation) is flanked by the Zaire Ebolavirus leader and trailer regions (which contain the viral promoters for replication and transcription) and by T7 RNA promoter and terminator sequences (Fig. 1A). Next, we transfected human embryonic kidney (293T) cells with p3E5E-Luc, pC-T7 (for T7 polymerase expression [15]), and pCEZ-NP, pCEZ-VP35, pCEZ-VP30, and pCEZ-L (for the expression of the Zaire Ebolavirus NP, VP35, VP30, and L proteins, respectively [15]) using Trans-IT LT1 reagent (Mirus); the amounts of the respective plasmids are listed in Table 1, Setup number 1. To determine background expression levels of luciferase in the absence of a functional nucleocapsid complex (negative control), we substituted empty vector for pCEZ-L. Forty-eight hours posttransfection, an equal volume of Steady-Glo luciferase assay substrate (Promega) was mixed with each sample and luminescence was measured. Mean luciferase expression and standard deviations were obtained by measuring luminescence in eight separate wells for both the positive (with pCEZ-L) and negative (without pCEZ-L) controls. For this and all subsequent experiments, we next calculated the ZЈ factor (24) as a measure of the robustness of the assay:, where p is the standard deviation of the positive control (reporter protein expression from the minireplicon), n is the standard deviation of the negative control (reporter protein expression in the absence of the L protein), p is the mean of the positive control...

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Emetine Regulates the Alternative Splicing of Bcl-x through a Protein Phosphatase 1-Dependent Mechanism

Cited by 57 publications

References 37 publications

Amiloride Modulates Alternative Splicing in Leukemic Cells and Resensitizes Bcr-AblT315I Mutant Cells to Imatinib

Amiloride Modulates Alternative Splicing in Leukemic Cells and Resensitizes Bcr-AblT315I Mutant Cells to Imatinib

Emetine regulates the alternative splicing of caspase 9 in tumor cells

Minigenome-Based Reporter System Suitable for High-Throughput Screening of Compounds Able To Inhibit Ebolavirus Replication and/or Transcription

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