Chemotherapeutic Drugs Inhibit Ribosome Biogenesis at Various Levels

Abstract

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...

5-Fluorouracil signaling through a calcium–calmodulin-dependent pathway is required for p53 activation and apoptosis in colon carcinoma cells

“…37 According to the literature, 5-FU and its metabolites have several direct or indirect target points within cells, including DNA, RNA and ribosomal biogenesis. 1,4,38 Although DNA lesion represent the most well-documented incident causing activation of p53, recent reports also involve transcriptional stress and ribosome damage in signaling events, leading to specific posttranslational modifications of the tumor suppressor. 5,34 An accumulation of double-strand breaks in response to 5-FU in HCT116 cells is evident as the number of gH2AX foci observed by immunostaining increased over time (data not shown), but our data involving ATM inhibition by KU55933 and Chk2-deficient cells excluded the DNA damage response as a starting point for Ca 2 þ -induced p53 phospho activation.…”

The PML nuclear bodies-associated protein TTRAP regulates ribosome biogenesis in nucleolar cavities upon proteasome inhibition

“…28 On the other hand, absence of factors essential for rRNA transcription or treatment with drugs that inhibit RNA polymerase I has a negative impact on the levels of both prerRNA and processing intermediates. 26 One can speculate that TTRAP might function at multiple steps, as downregulation of TTRAP leads to a decrease of pre-rRNA and a concomitant increase of processing species. Accumulation of processing intermediates might be due to impaired cleavage or to inhibition of degradation of cleaved fragments.…”

“…25 Although it is accepted that proteotoxic stress triggers substantial alterations in rRNA biogenesis, its specific effects are variable depending on drug concentrations, cell lines and kinetics of treatments. 21,26,27 Transcription and processing of pre-rRNA require a plethora of proteins and enzymatic activities to generate mature molecules. By qPCR and in vivo pulse labeling we found TTRAP regulates rRNA biogenesis exclusively under proteasome impairment.…”

The balance between rRNA and ribosomal protein synthesis up- and downregulates the tumour suppressor p53 in mammalian cells

“…For this purpose, we used IGF-1-stimulated MCF-7 cells treated with either hydroxyurea or gemcitabine. These drugs were chosen because they do not directly inhibit ribosome biogenesis (Derenzini et al, 1981;Burger et al, 2010 for hydroxyurea; Supplementary Figure S5, for gemcitabine). Western blot analysis indicated that IGF-1 stimulation caused a reduction of p53 expression in MCF-7 cells treated with both drugs in comparison with unstimulated, drug-treated cells, and that amount of p53 was even lesser in cells treated with hydroxyurea and stimulated by IGF-1 than in control cells (Figure 6b).…”