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.Chemotherapeutic drugs (hereinafter drugs) are used for the treatment of neoplastic diseases for more than 50 years. The mode of action and specifically the therapeutic relevant targets of many drugs, however, are often less defined. Recent studies revealed that some drugs like 5-fluorouracil (5-FU), 4 which were first assumed to interfere with DNA metabolism actually act mainly on RNA metabolism (1-9). In fact an increasing number of analyses identifies RNA metabolism as an important target of cancer drugs.In a hallmark study, Rubbi and Milner (10) showed that accumulation of the tumor suppressor p53 in UV-or drug-damaged cells occurs only if nucleolar functions are affected. Local, severe UV irradiation of the nucleoplasm could not stabilize p53 accumulation. In contrast, UV damage in the nucleolus induced a strong p53 response suggesting that the major sensor controlling the stability and degradation of p53 is located in the nucleolus, the place of ribosome biogenesis.The stability of the p53 protein is controlled by the ubiquitin ligase Mdm2, which targets p53 to the proteasome for degradation. Strikingly, several ribosomal proteins, including L5, L11, L23, and S7 proteins can bind and inactivate . Conditional knockdown of these ribosomal protein genes prevents Mdm2 inactivation and p53 stabilization in 5-FU-treated cells (15), consistent with the assumption that destruction of nucleolar functions by 5-FU inhibits ribosome biogenesis and results in liberation of ribosomal proteins followed by Mdm2 inactivation and p53 stabilization. The inhibition of rRNA transcription by knockout of the gene for the RNA polymerase I (Pol I) transcription factor TIF-1A (16), by blockage of the transcription factor UBF after microin...

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Background: Processing of ribosomal RNA is sensitive to Cdk inhibitors. Results: Inhibition of Cdk9 activity blocks 47 S rRNA processing and stabilizes a 3Ј extended 47 S primary transcript. Defective 3Ј processing negatively feeds back on RNAPI transcription. Conclusion: Cdk9 facilitates processing of 47 S rRNA by RNAPII-dependent synthesis of U8 snoRNA. Significance: Cdk9 may be a critical regulator of rRNA processing to harmonize RNAPII transcription activity with a ribosome biogenesis rate.

ABSTRACT:Since its first description more than 30 years ago p53 has become a paradigm for a protein with versatile functions. P53 sensitizes a large variety of genetic alterations and has been entitled the guardian of the genome. Stabilization of p53 upon DNA damage is accompanied by a complex pattern of modifications, which ascertain the cellular response either in the direction of a reversible or irreversible cell cycle arrest or programmed cell death. More recently it became evident that p53 also responds to non-genotoxic cell stress, in particular if ribosome biogenesis is affected. P53 degradation requires ribosome biogenesisThe nucleolus is the place of ribosome biogenesis. Here, the ribosomal RNA precursor is transcribed and processed into mature 28S, 18S, and 5.8S rRNAs. Ribosomal RNAs assemble with ribosomal proteins in 40S and 60S ribosomal subunits and are exported via the nucleoplasm into the cytoplasm. In a hallmark study, Rubbi and Milner [1] identified the nucleolus as the key structure in the control of p53 stability in UV-irradiated cells. They found that localized UV-induced pyrimidine dimers in nucleoplasmic DNA failed to stabilize p53, while the same DNA damage in nucleolar DNA stabilized p53. How does DNA damage in the nucleolus differ from DNA damage in the nucleoplasm? The genes for rRNA are organized in clusters on mammalian chromosomes, and transcription of rRNA genes leads to the establishment of nucleolar structures. Thus, it was tempting to speculate that not DNA damage itself was critical for p53 stabilization, but rather impaired expression of the rRNA genes. To test this assumption, Rubbi and Milner applied chemical drugs, genetic knockdowns, or microinjection experiments with antibodies to interfere with rRNA transcription. From these studies a model emerged with ribosome biogenesis as an essential prerequisite for p53 degradation.The production of ribosomes in the nucleolus is comparable with an assembly line in a modern car factory. Each component is delivered just in time at the right place. Already during synthesis the nascent 47S rRNA precursor associates with ribosomal and non-ribosomal proteins and assembles in mammals in the 90S pre-ribosome. The nonribosomal proteins regulate a multitude of different steps, which involve the modification of the rRNA by methylation and pseudo-uridinylation, the removal of external and internal transcribed rRNA sequences (ETS and ITS) from the primary transcript by endo-and exonucleases, the separation of the preribosomal 90S complex into the 40S and 60S ribosomal subunits, and finally the transport of the subunits from the nucleolus into the cytoplasm. In growing cells the production of ribosomes consumes up to twothirds of the cellular energy and the assembly line can be interrupted at many different sites.Here we have interrupted ribosome biogenesis by knockdown of three assembly factors for the 60S subunit. The factors Pes1, Bop1, and WDR12 are constituents of the PeBoW-complex. Knockdown of each component ( Figure 1A) or expression ...