Michael Hölzel

Chemotherapeutic drugs inhibit ribosome biogenesis at various levels

Drugs for cancer therapy belong to different categories of chemical substances. The cellular targets for the therapeutic efficacy are often not unambiguously identified. Here, we describe the process of ribosome biogenesis as a target of a large variety of chemotherapeutic drugs. We determined the inhibitory concentration of 36 chemotherapeutic drugs for transcription and processing of ribosomal RNA by in vivo labeling experiments. Inhibitory drug concentrations were correlated to the loss of nucleolar integrity. The synergism of drugs inhibiting ribosomal RNA synthesis at different levels was studied. Drugs inhibited ribosomal RNA synthesis either at the level of (i) rRNA transcription (e.g. oxaliplatin, doxorubicin, mitoxantrone, methotrexate), (ii) early rRNA processing (e.g. camptothecin, flavopiridol, roscovitine), or (iii) late rRNA processing (e.g. 5-fluorouracil, MG-132, homoharringtonine). Blockage of rRNA transcription or early rRNA processing steps caused nucleolar disintegration, whereas blockage of late rRNA processing steps left the nucleolus intact. Flavopiridol and 5-fluorouracil showed a strong synergism for inhibition of rRNA processing. We conclude that inhibition of ribosome biogenesis by chemotherapeutic drugs potentially may contribute to the efficacy of therapeutic regimens.

Stringent doxycycline-dependent control of gene activities using an episomal one-vector system

Conditional expression systems are of pivotal importance for the dissection of complex biological phenomena. Here, we describe a novel EBV-derived episomally replicating plasmid (pRTS-1) that carries all the elements for conditional expression of a gene of interest via Tet regulation. The vector is characterized by (i) low background activity, (ii) high inducibility in the presence of doxycycline (Dox) and (iii) graded response to increasing concentrations of the inducer. The chicken beta actin promoter and an element of the murine immunoglobin heavy chain intron enhancer drive constitutive expression of a bicistronic expression cassette that encodes the highly Dox-sensitive reverse tetracycline controlled transactivator rtTA2S-M2 and a Tet repressor-KRAB fusion protein (tTSKRAB) (silencer) placed downstream of an internal ribosomal entry site. The gene of interest is expressed from the bidirectional promoter Ptetbi-1 that allows simultaneous expression of two genes, of which one may be used as surrogate marker for the expression of the gene of interest. Tight down regulation is achieved through binding of the silencer tTSKRAB to Ptetbi-1 in the absence of Dox. Addition of Dox releases repression and via binding of rtTA2S-M2 activates Ptetbi-1.

Mammalian WDR12 is a novel member of the Pes1-Bop1 complex and is required for ribosome biogenesis and cell proliferation.

Target genes of the protooncogene c-myc are implicated in cell cycle and growth control, yet the linkage of both is still unexplored. Here, we show that the products of the nucleolar target genes Pes1 and Bop1 form a stable complex with a novel member, WDR12 (PeBoW complex). Endogenous WDR12, a WD40 repeat protein, is crucial for processing of the 32S precursor ribosomal RNA (rRNA) and cell proliferation. Further, a conditionally expressed dominant-negative mutant of WDR12 also blocks rRNA processing and induces a reversible cell cycle arrest. Mutant WDR12 triggers accumulation of p53 in a p19ARF-independent manner in proliferating cells but not in quiescent cells. Interestingly, a potential homologous complex of Pes1–Bop1–WDR12 in yeast (Nop7p–Erb1p–Ytm1p) is involved in the control of ribosome biogenesis and S phase entry. In conclusion, the integrity of the PeBoW complex is required for ribosome biogenesis and cell proliferation in mammalian cells.

Dissection of transcriptional programmes in response to serum and c-Myc in a human B-cell line

Proliferation of higher eukaryotic cells is triggered by the proto-oncogene c-myc (myc), which is induced downstream of a large number of growth factor receptors. Myc, a basic helix–loop–helix leucine zipper transcription factor, transmits growth signals by up- and downregulation of target genes. The importance of Myc in growth control is well established. However, the number of growth control genes requiring Myc as an essential factor for regulation after mitogenic stimulation of cells is not yet clear. Here, we have studied the transcriptional programme of a human B-cell line, P493-6, in response to Myc and serum. P493-6 cells do not express the endogenous myc, nor is it induced by serum stimulation. Proliferation of the cells is dependent upon both the expression of a tetracycline-regulated myc gene and serum stimulation. Using DNA microarrays, expression profiling was performed following stimulation of cells with serum, with Myc, or with both. We observed serum regulation of >1000 genes. A number of these genes were synergistically or antagonistically regulated by Myc. Moreover, we identified >300 Myc-regulated genes that were almost unresponsive to serum. Gene ontology analysis revealed that a high proportion of Myc target genes are involved in ribosome biogenesis and tRNA metabolism. The data support our current notion that Myc is essential for the regulation of a large number of growth-related genes in B cells, and cannot be replaced by other serum-induced factors.

Interdependence of Pes1, Bop1, and WDR12 Controls Nucleolar Localization and Assembly of the PeBoW Complex Required for Maturation of the 60S Ribosomal Subunit

ABSTRACT The PeBoW complex is essential for cell proliferation and maturation of the large ribosomal subunit in mammalian cells. Here we examined the role of PeBoW-specific proteins Pes1, Bop1, and WDR12 in complex assembly and stability, nucleolar transport, and preribosome association. Recombinant expression of the three subunits is sufficient for complex formation. The stability of all three subunits strongly increases upon incorporation into the complex. Only overexpression of Bop1 inhibits cell proliferation and rRNA processing, and its negative effects could be rescued by coexpression of WDR12, but not Pes1. Elevated levels of Bop1 induce Bop1/WDR12 and Bop1/Pes1 subcomplexes. Knockdown of Bop1 abolishes the copurification of Pes1 with WDR12, demonstrating Bop1 as the integral component of the complex. Overexpressed Bop1 substitutes for endogenous Bop1 in PeBoW complex assembly, leading to the instability of endogenous Bop1. Finally, indirect immunofluorescence, cell fractionation, and sucrose gradient centrifugation experiments indicate that transport of Bop1 from the cytoplasm to the nucleolus is Pes1 dependent, while Pes1 can migrate to the nucleolus and bind to preribosomal particles independently of Bop1. We conclude that the assembly and integrity of the PeBoW complex are highly sensitive to changes in Bop1 protein levels.

c-MYC activation impairs the NF-κB and the interferon response: Implications for the pathogenesis of Burkitt's lymphoma

Deregulation of the proto‐oncogene c‐myc is a key event in the pathogenesis of many tumors. A paradigm is the activation of the c‐myc gene by chromosomal translocations in Burkitt lymphoma (BL). Despite expression of a restricted set of Epstein–Barr viral (EBV) antigens, BL cells are not recognized by antigen‐specific cytotoxic T cells (CTLs) because of their inability to process and present HLA class I‐restricted antigens. In contrast, cells of EBV‐driven posttransplant lymphoproliferative disease (PTLD) are recognized and rejected by EBV‐specific CTLs. It is not known whether the poor immunogenicity of BL cells is due to nonexpression of viral antigens, overexpression of c‐myc, or both. To understand the basis for immune recognition and escape, we have compared the mRNA expression profiles of BL and EBV‐immortalized cells (as PTLD model). Among the genes expressed at low level in BL cells, we have identified many genes involved in the NF‐κB and interferon response that play a pivotal role in antigen presentation and immune recognition. Using a cell line in which EBNA2 and c‐myc can be regulated at will, we show that c‐MYC negatively regulates STAT1, the central player linking the Type‐I and Type‐II interferon response. Switching off c‐myc expression leads to STAT1 induction through a direct and indirect mechanism involving induction of Type‐I interferons. c‐MYC thus masks an interferon‐inducing activity in these cells. Our findings imply that immune escape of tumor cells is not only a matter of in vivo selection but may be additionally promoted by activation of a cellular oncogene.

Defects in 18 S or 28 S rRNA processing activate the p53 pathway.

The p53 tumor suppressor pathway is activated by defective ribosome synthesis. Ribosomal proteins are released from the nucleolus and block human double minute-2 (Hdm2) that targets p53 for degradation. However, it remained elusive how abrogation of individual rRNA processing pathways contributes to p53 stabilization. Here, we show that selective inhibition of 18 S rRNA processing provokes accumulation of p53 as efficiently as abrogated 28 S rRNA maturation. We describe hUTP18 as a novel mammalian rRNA processing factor that is specifically involved in 18 S rRNA production. hUTP18 was essential for the cleavage of the 5′-external transcribed spacer leader sequence from the primary polymerase I transcript, but was dispensable for rRNA transcription. Because maturation of the 28 S rRNA was unaffected in hUTP18-depleted cells, our results suggest that the integrity of both the 18 S and 28 S rRNA synthesis pathways can be monitored independently by the p53 pathway. Interestingly, accumulation of p53 after hUTP18 knock down required the ribosomal protein L11. Therefore, cells survey the maturation of the small and large ribosomal subunits by separate molecular routes, which may merge in an L11-dependent signaling pathway for p53 stabilization.

Dominant-negative Pes1 mutants inhibit ribosomal RNA processing and cell proliferation via incorporation into the PeBoW-complex

The nucleolar PeBoW-complex, consisting of Pes1, Bop1 and WDR12, is essential for cell proliferation and processing of ribosomal RNA in mammalian cells. Here we have analysed the physical and functional interactions of Pes1 deletion mutants with the PeBoW-complex. Pes1 mutants M1 and M5, with N- and C-terminal truncations, respectively, displayed a dominant-negative phenotype. Both mutants showed nucleolar localization, blocked processing of the 36S/32S precursors to mature 28S rRNA, inhibited cell proliferation, and induced high p53 levels in proliferating, but not in resting cells. Mutant M1 and M5 proteins associated with large pre-ribosomal complexes and co-immunoprecipitated Bop1 and WDR12 proteins indicating their proper incorporation into the PeBoW-complex. We conclude that the dominant-negative effect of the M1 and M5 mutants is mediated by the impaired function of the PeBoW-complex.

Myc/Max/Mad regulate the frequency but not the duration of productive cell cycles

Upregulation of the proto‐oncoprotein Myc, a basic, helix–loop–helix, leucin zipper domain transcription factor has profound consequences on cell proliferation, cell growth and apoptosis. Cell cultures of somatic c‐myc−/− rat fibroblasts show extremely prolonged doubling times of 52 h. Using time‐lapse microscopy, we show here that individual c‐myc−/− cells proceeded within ∼24 h through the cell cycle as fast as c‐myc+/+ cells. However, c‐myc−/− cells were highly sensitive to contact inhibition and readily arrested in the cell cycle already at low density. Activation of conditional MycER overcame cell cycle arrest in c‐myc−/− cells and led to continuous proliferation at the expense of increased apoptosis at high cell density. Conditional expression of Mad1, a Myc antagonist, represses proliferation of different cell types including U2OS cells. In analogy to the effect of Myc, this occurs mainly by reducing the probability of cells remaining in the cycle. Our data demonstrate that the Myc/Max/Mad network does not regulate the duration of the cell cycle, but the decision of cells to enter or exit the cell cycle.

c-Myc and Rel/NF-kappaB are the two master transcriptional systems activated in the latency III program of Epstein-Barr virus-immortalized B cells.

ABSTRACT The Epstein-Barr virus (EBV) latency III program imposed by EBNA2 and LMP1 is directly responsible for immortalization of B cells in vitro and is thought to mediate most immunodeficiency-related posttransplant lymphoproliferative diseases in vivo. To answer the question whether and how this proliferation program is related to c-Myc, we have established the transcriptome of both c-Myc and EBV latency III proliferation programs using a Lymphochip specialized microarray. In addition to EBV-positive latency I Burkitt lymphoma lines and lymphoblastoid cell lines (LCLs), we used an LCL expressing an estrogen-regulatable EBNA2 fusion protein (EREB2-5) and derivative B-cell lines expressing a constitutively active or tetracycline-regulatable c-myc gene. A total of 897 genes were found to be fourfold or more up- or downregulated in either one or both proliferation programs compared to the expression profile of resting EREB2-5 cells. A total of 661 (74%) of these were regulated similarly in both programs. Numerous repressed genes were known targets of STAT1, and most induced genes were known to be upregulated by c-Myc and to be involved in cell proliferation. In keeping with the gene expression patterns, inactivation of c-Myc by a chemical inhibitor or by conditional expression of dominant-negative c-Myc and Max mutants led to proliferation arrest of LCLs. Most genes differently regulated in both proliferation programs corresponded to genes induced by NF-κB in LCLs, and many of them coded for immunoregulatory and/or antiapoptotic molecules. Thus, c-Myc and NF-κB are the two main transcription factors responsible for the phenotype, growth pattern, and biological properties of cells driven into proliferation by EBV.