Inhibition Of Ribosomal RNA Synthesis Research Articles

Abstract 4526: Inhibition of RNA Pol I transcription and induction of nucleolar stress in cancer cells treated with the experimental alpha-adrenergic receptor antagonist JP-1302

Abstract The inhibition of ribosome biogenesis through pharmacological means has drawn attention as a potential strategy in cancer therapy. In our previous study we highlighted a novel property of the FDA-approved antimalarial drug amodiaquine, demonstrating its ability to dose-dependently inhibit ribosomal RNA (rRNA) transcription, a critical step in ribosome biogenesis (Espinoza JA, et al Cell Death Diff, 2020). Amodiaquine was found to induce the degradation of the catalytic subunit of RNA polymerase I, known as POLR1A (RPA194), and resulting in nucleolar stress and the stabilization of p53. Notably, RNA Pol I shutdown occurred independently of DNA damage. Motivated by these findings, we sought to find additional inhibitors of ribosome biogenesis. Acridine derivatives display anti-cancer properties. The activity of acridines is mainly attributed to the planarity of these structures, which can intercalate within the DNA structure, and inhibition of chromatin associated protein targets including topoisomerases. JP-1302 is an experimental drug, on a 9-aminoacridine structural scaffold, acting as a selective alpha-2 adrenergic receptor antagonist. It was recently shown that JP-1302 inhibits transcription, blocking RNA Pol II phosphorylation and inhibiting p21 expression at a concentration of 10 μM (Mitchell DC, et al Nat Biotech, 2023). Given JP-1302's structural similarity to other acridine derivatives such as aminacrine, ethacridine, and the experimental compound BMH-21, all known to inhibit RNA Pol I transcription, we hypothesized that JP-1302 might share similar properties. The purpose of our new study was to evaluate JP-1302´s effect on ribosome biogenesis, p53, and cancer cell growth. We show here that JP-1302 acts as an inhibitor of RNA Pol I transcription. Treatment with JP-1302 induced a rapid decrease in the levels of POLR1A, cessation of rRNA synthesis and nucleolar stress already within six hours and at a concentration of 0.5 μM. JP-1302 treatment stabilized wild type p53 and enhanced p21 expression in the osteosarcoma line U2OS. RNA Pol I shutdown occurred in the absence of DNA damage response, as determined by gH2A.X staining. JP-1302 impaired the growth of both p53 wild type and p53 null HCT116 cells with GI50 values of around 0.6 μM, as well as in U2OS cells at 0.8 μM, as determined after 48 hours. Paradoxically, at very low concentrations JP-1302 appeared to transiently stimulate RNA synthesis, a phenomenon that will require further investigation. In conclusion, we identified JP-1302 as a novel inhibitor of ribosomal RNA synthesis, exerting this effect at concentrations that do not induce DNA damage, and independently of p53. Given that JP-1302 passes the blood-brain barrier in rodents, and accumulating in different brain regions, it is of future interest to evaluate this drug in brain tumor models. Citation Format: Sheetanshu Saproo, Styliani Papadaki, Jaime A. Espinoza, Jiri Bartek, Mikael S. Lindström. Inhibition of RNA Pol I transcription and induction of nucleolar stress in cancer cells treated with the experimental alpha-adrenergic receptor antagonist JP-1302 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4526.

Abstract 3524: Leveraging ChatGPT for literature-based inference of drug-gene relationships in cancer

Abstract Approximately 5,000 new articles are indexed by PubMed daily, many providing critical insights into novel gene-drug relationships. However, searching and distilling the wealth of biomedical literature to infer specific pharmacogenomic relationships between a gene and a drug poses a significant burden for researchers. Recent advances in large language models, such as ChatGPT, offer a promising solution to tackle this challenge. This study aims to prototype a literature-based inference for drug-gene relationships in the context of cancer using ChatGPT, marking an exploratory effort in this emerging domain. Our approach involves developing an automated pipeline that integrates the Application Programming Interfaces (APIs) of PubMed and ChatGPT, specifically GPT-3.5. Given a gene, a drug, and a disease, the pipeline searches PubMed for relevant articles and extracts their abstracts. Leveraging prompt engineering techniques, we formulated a prompt that facilitates accurate summarization of these abstracts. The output of our pipeline includes three key components: 1) a concise, one-sentence summary elucidating the relationship between the drug-gene pair; 2) a step-by-step explanation of the inference process; and 3) the confidence level associated with the generated summary. Our approach was able to confirm well-known gene-drug relationships in cancer, such as palbociclib and CDK4. We further examined a challenging case of CX-5461, an inhibitor of ribosomal RNA synthesis currently under clinical investigation for various cancers. Until recent evidence emerged in 2021, this drug was mischaracterized as an RNA-Pol I inhibitor, whereas it primarily targets topoisomerase II beta (TOP2B). Notably, our pipeline correctly identified TOP2B as the primary target of CX-5461, despite an extensive body of literature prior to 2021 supporting the RNA-Pol I relationship. To evaluate the sensitivity of our pipeline, we conducted a systematic assessment using a mixture of relevant and irrelevant abstracts. Utilizing bevacizumab, VEGF, and hepatocellular carcinoma as a demonstrating example, we showed that ChatGPT achieved nearly 95% accuracy even when only 30% of the abstracts were relevant to the case. In summary, this pilot research serves as a foundational step towards the utilization of large language models in the field of drug discovery and development. Our ongoing efforts involve the rigorous evaluation of our approach across a diverse spectrum of drug targets and cancer types, as well as the optimization of prompts through state-of-the-art prompt engineering techniques. Citation Format: Yuna Shin, Michael Ning, Li-Ju Wang, Yufei Huang, Yu-Chiao Chiu. Leveraging ChatGPT for literature-based inference of drug-gene relationships in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3524.

Meis1 supports leukemogenesis through stimulation of ribosomal biogenesis and Myc.

The homeobox transcription factors HoxA9 and Meis1 are causally involved in the etiology of acute myeloid leukemia. While HoxA9 alone immortalizes cells, cooperation with Meis1 is necessary to induce a full leukemic phenotype. Here, we applied degron techniques to elucidate the leukemogenic contribution of Meis1. Chromatin immunoprecipitation experiments revealed that Meis1 localized mainly to H3K27 acetylated and H3K4 mono-methylated enhancers preactivated by HoxA9. Chromatin association of Meis1 required physical presence of HoxA9 and all Meis1 DNA interactions were rapidly lost after HoxA9 degradation. Meis1 controlled a gene expression pattern dominated by Myc, ribosome biogenesis and ribosomal RNA synthesis genes. While Myc accounted for the cell cycle stimulating effect of Meis1, overexpression of this oncogene alone did not accelerate leukemogenesis. Besides its effect on Myc, Meis1 induced transcription of ribosomal biogenesis genes. This was accompanied by an elevated resistance against inhibition of ribosomal RNA synthesis and translation, but without affecting steady-state protein synthesis. Finally, we demonstrate that HoxA9 and Meis1 proteins are stabilized by post-translational modification. Mutation of HoxA9/Meis1 phosphorylation sites or inhibition of casein kinase 2 lead to rapid protein degradation suggesting a potential pathway for pharmacological intervention.

The chemotherapeutic agent CX-5461 irreversibly blocks RNA polymerase I initiation and promoter release to cause nucleolar disruption, DNA damage and cell inviability.

In the search for drugs to effectively treat cancer, the last 10 years have seen a resurgence of interest in targeting ribosome biogenesis. CX-5461 is a potential inhibitor of ribosomal RNA synthesis that is now showing promise in phase I trials as a chemotherapeutic agent for a range of malignancies. Here, we show that CX-5461 irreversibly inhibits ribosomal RNA transcription by arresting RNA polymerase I (RPI/Pol1/PolR1) in a transcription initiation complex. CX-5461 does not achieve this by preventing formation of the pre-initiation complex nor does it affect the promoter recruitment of the SL1 TBP complex or the HMGB-box upstream binding factor (UBF/UBTF). CX-5461 also does not prevent the subsequent recruitment of the initiation-competent RPI–Rrn3 complex. Rather, CX-5461 blocks promoter release of RPI–Rrn3, which remains irreversibly locked in the pre-initiation complex even after extensive drug removal. Unexpectedly, this results in an unproductive mode of RPI recruitment that correlates with the onset of nucleolar stress, inhibition of DNA replication, genome-wide DNA damage and cellular senescence. Our data demonstrate that the cytotoxicity of CX-5461 is at least in part the result of an irreversible inhibition of RPI transcription initiation and hence are of direct relevance to the design of improved strategies of chemotherapy.

Treacle controls the nucleolar response to rDNA breaks via TOPBP1 recruitment and ATR activation.

Induction of DNA double-strand breaks (DSBs) in ribosomal DNA (rDNA) repeats is associated with ATM-dependent repression of ribosomal RNA synthesis and large-scale reorganization of nucleolar architecture, but the signaling events that regulate these responses are largely elusive. Here we show that the nucleolar response to rDNA breaks is dependent on both ATM and ATR activity. We further demonstrate that ATM- and NBS1-dependent recruitment of TOPBP1 in the nucleoli is required for inhibition of ribosomal RNA synthesis and nucleolar segregation in response to rDNA breaks. Mechanistically, TOPBP1 recruitment is mediated by phosphorylation-dependent interactions between three of its BRCT domains and conserved phosphorylated Ser/Thr residues at the C-terminus of the nucleolar phosphoprotein Treacle. Our data thus reveal an important cooperation between TOPBP1 and Treacle in the signaling cascade that triggers transcriptional inhibition and nucleolar segregation in response to rDNA breaks.

First-In-Class Inhibitor of Ribosomal RNA Synthesis with Antimicrobial Activity against Staphylococcus aureus

We report the discovery of the first bacterial ribosomal RNA (rRNA) synthesis inhibitor that has specific antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA). A pharmacophore model was constructed on the basis of the protein-protein interaction between essential bacterial rRNA transcription factors NusB and NusE and employed for an in silico screen to identify potential leads. One compound, (E)-2-{[(3-ethynylphenyl)imino]methyl}-4-nitrophenol (MC4), demonstrated antimicrobial activity against a panel of S. aureus strains, including MRSA, without significant toxicity to mammalian cells. MC4 resulted in a decrease in the rRNA level in bacteria, and the target specificity of MC4 was confirmed at the molecular level. Results obtained from this work validated the bacterial rRNA transcription machinery as a novel antimicrobial target. This approach may be extended to other factors in rRNA transcription, and MC4 could be applied as a chemical probe to dissect the relationship among MRSA infection, MRSA growth rate, and rRNA synthesis, in addition to its therapeutic potential.

Abstract B23: Inhibition of ribosomal RNA synthesis as a new therapeutic approach to treat advanced prostate cancer

Abstract Background: Prostate epithelium is exquisitely sensitive to the overexpression of the proto-oncogene MYC which causes neoplastic transformation. Indeed, MYC protein is almost universally overexpressed in metastatic castration-resistant prostate cancer (CRPC) making targeting MYC an attractive option for treating advanced stage disease. Unfortunately, the development of therapeutic agents directly targeting MYC has been largely unsuccessful, thus emphasizing the need to indirectly target MYC activity through inhibition of downstream cellular processes it regulates. One of the main effects of MYC in cancer cells is to accelerate proliferative growth via stimulation of high levels of ribosome biogenesis. Accordingly, the control of protein synthesis rate has emerged as the “Achilles' heel” of a wide array of tumors. MYC also regulates and cooperates with PIM kinases to increase the activity of the eIF4F translation initiation complex and MYC-driven tumors are addicted to eIF4E. Here, we investigate the efficacy of a single and dual approach targeting ribosome biogenesis and function to treat prostate cancer (PC). Experimental design: We employed numerous models of PC, including a novel CRPC patient derived xenograft system, which showed the pre-clinical efficacy of therapies that combine to target MYC directed signaling to the ribosome. The inhibition of ribosomal RNA (rRNA) synthesis with CX-5461, a potent, selective and orally bioavailable inhibitor of RNA polymerase I (Pol I) transcription has been successfully exploited therapeutically, but only in models of hematological malignancy. CX-5461 and CX-6258, a pan-PIM kinase inhibitor, were tested alone and in combination in PC cell lines, in Hi-MYC and PTEN-deficient mouse models and in patient derived xenografts (PDX) of metastatic tissue obtained from a castration-resistant PC patient. Results: CX-5461 inhibited anchorage-independent growth and induced cell cycle arrest in PC cell lines at nanomolar concentrations. Oral administration of 50 mg/kg CX-5461 induced p53 expression and activity and reduced proliferation (Ki-67) and invasion (loss of ductal actin) in Hi-MYC tumors, but not in PTEN null (low MYC) tumors. While 100 mg/kg CX-6258 showed limited effect alone, its combination with CX-5461 further suppressed proliferation and dramatically reduced large invasive lesions in both models. This rational combination strategy significantly inhibited proliferation and induced cell death in PDX of PC. Conclusion: Our results demonstrate preclinical efficacy of targeting the ribosome at multiple levels and provide a new approach for the treatment of PC. In addition, a key conclusion of our study is that the androgen receptor (AR) presence or activity has no significant impact on the therapeutic activity of our novel combination therapy. Therefore, we believe our new exciting combination therapy could be used in the clinic in combination with current anti-androgens or as salvage therapy in multi-drug resistant CRPC. Citation Format: Richard J. Rebello, Eric Kusnadi, Don P. Cameron, Helen B. Pearson, Analia Lesmana, Jennifer R. Devlin, Denis Drygin, Ashlee K. Clark, Laura Porter, John Pedersen, Shahneen Sandhu, Gail P. Risbridger, Richard B. Pearson, Ross D. Hannan, Luc Furic. Inhibition of ribosomal RNA synthesis as a new therapeutic approach to treat advanced prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr B23.

The Potential of Targeting Ribosome Biogenesis in High-Grade Serous Ovarian Cancer.

Overall survival for patients with ovarian cancer (OC) has shown little improvement for decades meaning new therapeutic options are critical. OC comprises multiple histological subtypes, of which the most common and aggressive subtype is high-grade serous ovarian cancer (HGSOC). HGSOC is characterized by genomic structural variations with relatively few recurrent somatic mutations or dominantly acting oncogenes that can be targeted for the development of novel therapies. However, deregulation of pathways controlling homologous recombination (HR) and ribosome biogenesis has been observed in a high proportion of HGSOC, raising the possibility that targeting these basic cellular processes may provide improved patient outcomes. The poly (ADP-ribose) polymerase (PARP) inhibitor olaparib has been approved to treat women with defects in HR due to germline BRCA mutations. Recent evidence demonstrated the efficacy of targeting ribosome biogenesis with the specific inhibitor of ribosomal RNA synthesis, CX-5461 in v-myc avian myelocytomatosis viral oncogene homolog (MYC)-driven haematological and prostate cancers. CX-5461 has now progressed to a phase I clinical trial in patients with haematological malignancies and phase I/II trial in breast cancer. Here we review the currently available targeted therapies for HGSOC and discuss the potential of targeting ribosome biogenesis as a novel therapeutic approach against HGSOC.

Abstract B39: Identification and targeting mediators of chemoresistance using the patient-derived xenograft model of ovarian cancer

Abstract Objectives: The objectives of this study were to use the heterogeneity of the patient-derived xenograft (PDX) model to characterize the surviving population for de novo mediators of chemotherapy resistance, and subsequently target pathways contributing to that resistance. Methods: Tumors removed during primary tumor-reductive surgery were implanted directly into SCID mice, either subcutaneously and intraperitoneally. Mice with PDX tumors were treated with combination carboplatin/paclitaxel for 4 weeks. In addition, chemoresistant models were developed by treatment until complete response was achieved, and recurrent tumors retreated until resistant. Patient tumors, developing PDX tumors, and post-chemo surviving residual tumors were subjected to qPCR arrays, RNA-Seq, Methyl-Seq, and proteomic analysis; and IHC for Ki-67 and cancer stem cell markers CD133, ALDH1, and CD44. The Pol-I inhibitor CX-5461 was used to treat five PDX models. Results: At the mRNA level, the original patient tumors were similar to PDX tumors in mice as assessed by an 84-oncogene expression panel. Subcutaneous and intraperitoneal tumors were also remarkably similar. Patient responses correlated well with responses in mice. Proteomic analysis identified 14,023 unique proteins, of which approximately 30% were murine, and 70% were human in each sample. A total of 1,202 of human proteins were found to be differently expressed (p-value < 0.01) between patient and PDX tumors. Of these, 996 (82.9%) were reduced in the PDX model, and predominantly participants in the immune system. PDX tumors collected after chemotherapy showed that the surviving population was significantly more dormant than the original tumor (Ki-67 67% versus 31%, P<0.01), and enriched in all three cancer stem cell populations (2.1-4.7 fold, p<0.05). Principle component analysis of both RNA-Seq and proteomic data show that sensitive and resistant tumors were much more similar to one another than to tumors from different patients, suggesting a limited number of genes contributing to chemoresistance. IPA analysis of RNA-Seq data from treated and untreated PDX tumor pairs demonstrated that the most commonly enriched pathways are those of Protein Kinase A, GNRH, Sphingosine-1-phosphate, and alpha-adrenergic signaling. Additionally, numerous regulators of ribosomal synthesis were significantly altered by treatment. In vivo, 5 PDX models were treated with CX-5461; two had progression, one had stable disease, one had a 60% reduction in tumor growth, and one had a complete response to single agent therapy, still without recurrence after 3 months. Conclusion: The ovarian-PDX model exhibits consistent molecular and biologic similarity to patient tumors. Post-chemo tumors reveal multiple pathways to be consistently altered. Taking this information back to the PDX model demonstrated that inhibition of ribosomal RNA synthesis was highly effective, offering a novel opportunity to preferentially target the chemoresistant population in ovarian cancer. Citation Format: Zachary Dobbin, Robert Cornelison, Ashwini K. Katre, David A. Schneider, Karen Watters, Fabian Coscia, Ernst Lengyel, Aaron Buechlein, Fang Fang, Kenneth Nephew, Charles N. Landen, Jr.. Identification and targeting mediators of chemoresistance using the patient-derived xenograft model of ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B39.

Abstract 4809: Combination therapy targeting ribosome biogenesis and mRNA translation provides a novel and potent therapeutic approach to treat MYC-driven malignancy

Abstract MYC-driven malignancies are associated with elevated rates of ribosome biogenesis and mTORC1/eIF4E-driven protein synthesis suggesting they may be vulnerable to therapeutic strategies that target the ribosome. We investigated the therapeutic efficacy of targeting multiple nodes of the network controlling the ribosome in mouse models of MYC-driven lymphoma (Eì-Myc) and prostate cancer (HI-MYC). Simultaneous inhibition of ribosomal RNA synthesis and repression of protein translation was achieved by utilizing the novel RNA polymerase I inhibitor CX-5461 and PI3K/AKT/mTORC1 and PIM 1 signaling inhibitors. Combined inhibition of ribosome biogenesis and function significantly improved therapeutic outcomes in lymphoma and prostate cancer models. CX-5461 and Everolimus (mTORC1 inhibitor) co-treatment more than doubled the survival of Eì-Myc lymphoma-bearing mice. While both classes of inhibitor suppress rDNA transcription, they treat MYC-driven malignancy through distinct molecular mechanisms facilitating their combinatorial effects. In contrast to CX-5461, PI3K/AKT/mTOR pathway inhibitors did not activate a nucleolar stress response and p53-dependent apoptosis but instead induce B-lymphoma cell death via the upregulation of the BH3-only protein BMF (Devlin et al Cancer Discovery 2015 Oct 21. pii: CD-14-0673. [Epub ahead of print]). PIM kinase has been shown to regulate eIF4Edriven protein synthesis on prostate cancer cell lines. Co-treatment of HI-MYC mice with CX-5461 and the PIM kinase inhibitor CX-6258 reverted highly invasive disease to low-grade prostate intraepithelial neoplasia. These findings demonstrate that MYC driven tumors are addicted to multiple regulatory steps associated with ribosome synthesis and function and coordinated targeting of these addictions provides an effective new therapeutic approach to treat MYC driven cancers. Citation Format: Jennifer R. Devlin, Richard J. Rebello, Katherine M. Hannan, Carleen Cullinane, Denis Drygin, Gail P. Risbridger, Luc Furic, Ross D. Hannan, Richard B. Pearson. Combination therapy targeting ribosome biogenesis and mRNA translation provides a novel and potent therapeutic approach to treat MYC-driven malignancy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4809.

Depletion of the cisplatin targeted HMGB-box factor UBF selectively induces p53-independent apoptotic death in transformed cells.

Cisplatin-DNA adducts act as strong decoys for the Upstream Binding Factor UBF (UBTF) and have been shown to inhibit transcription of the ribosomal RNA genes by RNA polymerase I. However, it is unclear if this plays a significant role in the chemotherapeutic activity of cis- or carboplatin. We find that cisplatin in fact induces a very rapid displacement of UBF from the ribosomal RNA genes and strong inhibition of ribosomal RNA synthesis, consistent with this being an important factor in its cytotoxicity. Using conditional gene deletion, we recently showed that UBF is an essential factor for transcription of the ribosomal RNA genes and for ribosome biogenesis. We now show that loss of UBF arrests cell proliferation and induces fully penetrant, rapid and synchronous apoptosis, as well as nuclear disruption and cell death, specifically in cells subjected to oncogenic stress. Apoptosis is not affected by homozygous deletion of the p53 gene and occurs equally in cells transformed by SV40 T antigens, by Myc or by a combination of Ras & Myc oncogenes. The data strongly argue that inhibition of UBF function is a major factor in the cytotoxicity of cisplatin. Hence, drug targeting of UBF may be a preferable approach to the use of the highly toxic platins in cancer therapy.

Regulation of ribosomal RNA synthesis in T cells: requirement for GTP and Ebp1

AbstractMycophenolic acid (MPA) is the active metabolite of mycophenolate mofetil, an effective immunosuppressive drug. Both MPA and mycophenolate mofetil are highly specific inhibitors of guanine nucleotide synthesis and of T-cell activation. However, the mechanism by which guanine nucleotide depletion suppresses T-cell activation is unknown. Depletion of GTP inhibits ribosomal RNA synthesis in T cells by inhibiting transcription initiation factor I (TIF-IA), a GTP-binding protein that recruits RNA polymerase I to the ribosomal DNA promoter. TIF-IA–GTP binds the ErbB3-binding protein 1, and together they enhance the transcription of proliferating cell nuclear antigen (PCNA). GTP binding by TIF-IA and ErbB3-binding protein 1 phosphorylation by protein kinase C δ are both required for optimal PCNA expression. The protein kinase C inhibitor sotrastaurin markedly potentiates the inhibition of ribosomal RNA synthesis, PCNA expression, and T-cell activation induced by MPA, suggesting that the combination of the two agents are more highly effective than either alone in inducing immunosuppression.

Pre-surgical window study of metformin in obesity-driven endometrial cancer

population was significantly more dormant than the original tumor. RNA sequencing analysis of treated and untreated PDX tumors (n = 6 pair) demonstrated that numerous factors with roles in ribosomal synthesis were significantly altered by treatment. In vitro, an inhibitor of ribosomal synthesis, CX-5461, caused significant G2/M-phase arrest, and effects were much more profound in chemoresistant cells than corresponding chemosensitive cells. In vivo, of the five PDX models that were treated with CX-5461, two had progression, one had stable disease, one had a 60% reduction in tumor growth, and one had a complete response to single-agent therapy, still without recurrence after 3 months. Conclusions: The ovarian PDX model exhibits similarity to patient tumors. Postchemotherapy tumors reveal severe alteration of ribosomal synthesis. Treatment with an inhibitor of ribosomal RNA synthesis was highly effective in vitro and in vivo, offering a novel opportunity to specifically target the chemoresistant population in ovarian cancer.

Effects of S-adenosylmethionine decarboxylase, polyamines, amino acids, and weak bases (amines and ammonia) on development and ribosomal RNA synthesis in Xenopus embryos

We have been studying control mechanisms of gene expression in early embryogenesis in a South African clawed toad Xenopus laevis, especially during the period of midblastula transition (MBT), or the transition from the phase of active cell division (cleavage stage) to the phase of extensive morphogenesis (post-blastular stages). We first found that ribosomal RNA synthesis is initiated shortly after MBT in Xenopus embryos and those weak bases, such as amines and ammonium ion, selectively inhibit the initiation and subsequent activation of rRNA synthesis. We then found that rapidly labeled heterogeneous mRNA-like RNA is synthesized in embryos at pre-MBT stage. We then performed cloning and expression studies of several genes, such as those for activin receptors, follistatin and aldolases, and then reached the studies of S-adenosylmethionine decarboxylase (SAMDC), a key enzyme in polyamine metabolism. Here, we cloned a Xenopus SAMDC cDNA and performed experiments to overexpress the in vitro-synthesized SAMDC mRNA in Xenopus early embryos, and found that the maternally preset program of apoptosis occurs in cleavage stage embryos, which is executed when embryos reach the stage of MBT. In the present article, we first summarize results on SAMDC and the maternal program of apoptosis, and then describe our studies on small-molecular-weight substances like polyamines, amino acids, and amines in Xenopus embryos. Finally, we summarize our studies on weak bases, especially on ammonium ion, as the specific inhibitor of ribosomal RNA synthesis in Xenopus embryonic cells.

Chromophore‐assisted light inactivation of pKi‐67 leads to inhibition of ribosomal RNA synthesis

Expression of the nuclear Ki-67 protein (pKi-67) is strongly associated with cell proliferation. For this reason, antibodies against this protein are widely used as prognostic tools for the assessment of cell proliferation in biopsies from cancer patients. Despite this broad application in histopathology, functional evidence for the physiological role of pKi-67 is still missing. Recently, we proposed a function of pKi-67 in the early steps of ribosomal RNA (rRNA) synthesis. Here, we have examined the involvement of pKi-67 in this process by photochemical inhibition using chromophore-assisted light inactivation (CALI). Anti-pKi-67 antibodies were labelled with the fluorochrome fluorescein 5(6)-isothiocyanate and were irradiated after binding to their target protein. Performing CALI in vitro on cell lysates led to specific cross-linking of pKi-67. Moreover, the upstream binding factor (UBF) necessary for rRNA transcription was also partly subjected to cross-link formation, indicating a close spatial proximity of UBF and pKi-67. CALI in living cells, using micro-injected antibody, caused a striking relocalization of UBF from foci within the nucleoli to spots located at the nucleolar rim or within the nucleoplasm. pKi-67-CALI resulted in dramatic inhibition of RNA polymerase I-dependent nucleolar rRNA synthesis, whereas RNA polymerase II-dependent nucleoplasmic RNA synthesis remained almost unaltered. Our data presented here argue for a crucial role of pKi-67 in RNA polymerase I-dependent nucleolar rRNA synthesis.

Modulation of RNA editing by functional nucleolar sequestration of ADAR2.

The adenosine deaminases that act on RNA (ADARs) catalyze the site-specific conversion of adenosine to inosine (A to I) in primary mRNA transcripts, thereby affecting the splicing pattern or coding potential of mature mRNAs. Although the subnuclear localization of A-to-I editing has not been precisely defined, ADARs have been shown to act before splicing, suggesting that they function near nucleoplasmic sites of transcription. Here we demonstrate that ADAR2, a member of the vertebrate ADAR family, is concentrated in the nucleolus, a subnuclear domain disparate from the sites of mRNA transcription. Selective inhibition of ribosomal RNA synthesis or the introduction of mutations in the double-stranded RNA-binding domains within ADAR2 results in translocation of the protein to the nucleoplasm, suggesting that nucleolar association of ADAR2 depends on its ability to bind to ribosomal RNA. Fluorescence recovery after photobleaching reveals that ADAR2 can shuttle rapidly between subnuclear compartments. Enhanced translocation of endogenous ADAR2 from the nucleolus to the nucleoplasm results in increased editing of endogenous ADAR2 substrates. These observations indicate that the nucleolar localization of ADAR2 represents an important mechanism by which RNA editing can be modulated by the sequestration of enzymatic activity from potential RNA substrates in the nucleoplasm.

Subcellular localization of IRS-1 in cell proliferation and differentiation.

The type 1 insulin-like growth factor receptor (IGF-IR) and its docking protein, insulin receptor substrate-1 (IRS-1), play important roles in cell transformation, cell differentiation and aging. IRS-1 and other IRS proteins can, under certain conditions, localize to the nuclei of cells, where they undergo interactions with nuclear and nucleolar proteins. In this study, we confirm and extend these observations, demonstrating that IRS-1 is preferentially nuclear in growing cells. Differentiation and inhibition of ribosomal RNA synthesis cause subcellular redistribution of IRS-1 and other nuclear proteins to the cytoplasm.

High temperature of development and selection of Bacillus thuringiensis-supernatant-resistant females in a Drosophila melanogaster Oregon R strain

The supernatant of Bacillus thuringiensis cultures contains a thermostable toxin: the beta exotoxin or thuringiensin, which in vivo acts as a preferential inhibitor of ribosomal RNA synthesis. Added to Drosophila melanogaster culture medium, it induces, during the flies' development, a lethal effect that is, in our Oregon R strain, greater for females than for males. The authors have previously shown that a diminution of the ribosomal DNA amount increases the sensitivity to the lethal effect of the supernatant. From a stock subjected for several generations to a 28 degrees C temperature and more, they have spontaneously obtained a variant population where males and females have similar resistance. It is shown here that this difference between the two populations is expressed in the X/X female genotypes, and that there is a correlation between the sensitivity to the lethal effect of the Berliner Bacillus thuringiensis supernatant and the sensitivity to the effect of the 28 degrees C developmental temperature; genotypes resistant to the lethal effect of the temperature were positively selected when the temperature of development was increased. The better resistance of these genotypes could be related to more active ribosomal units on the X chromosome.

Heavy Ion Effects on Yeast: Inhibition of Ribosomal RNA Synthesis

Diploid wild-type yeast cells were exposed to beams of heavy ions covering a wide range of linear energy transfer (LET) (43-13,700 keV/microns). Synthesis of ribosomal RNA (rRNA) was assessed as a functional measure of damage produced by particle radiation. An exponential decrease of relative rRNA synthesis with particle fluence was demonstrated in all cases. The inactivation cross sections derived were found to increase with LET over the entire range of LET studied. The corresponding values for relative biological effectiveness were slightly less than unity. Maximum cross sections measured were close to 1 micron 2, implying that some larger structure within the yeast nucleus (e.g., the nucleolus) might represent the target for an impairment of synthetic activity by very heavy ions rather than the genes coding for rRNA. Where tested, an oxygen effect for rRNA synthesis could not be demonstrated.

Level of ubiquitinated histone H2B in chromatin is coupled to ongoing transcription

The relationship between transcription and ubiquitination of the histones was investigated. Previous studies have shown that ubiquitinated (u) histone H2B and, to a lesser extent, mono- and polyubiquitinated histone H2A are enriched in transcriptionally active gene-enriched chromatin fractions. Here, we show that treatment of T-47D-5 human breast cancer cells with actinomycin D (10 micrograms/mL) or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, inhibitors of heterogeneous nuclear RNA synthesis, selectively reduced the level of uH2B, but not uH2A, uH2A.Z, or polyubiquitinated H2A, in chromatin. Treatment of the cells with low levels (0.04 micrograms/mL) of actinomycin D slightly reduced the level of uH2B, suggesting that inhibition of ribosomal RNA synthesis does not have a profound effect on the level of uH2B in chromatin. The level of the ubiquitinated histones was not affected by treating the cells with inhibitors of DNA synthesis (sodium butyrate or aphidicolin), but heat-shock treatment resulted in the loss of all the monoubiquitinated histone species. These results demonstrate that maintenance of the levels of uH2B in chromatin is dependent upon ongoing transcription, particularly the synthesis of hnRNA. Thus, histone H2B would be ubiquitinated when the nucleosome was opened during transcription. Ubiquitination of histone H2B may impede nucleosome refolding, facilitating subsequent rounds of transcription.