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First published online April 3, 2008
doi: 10.1242/10.1242/jcs.023259

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Influence of irofulven, a transcription-coupled repair-specific antitumor agent, on RNA polymerase activity, stability and dynamics in living mammalian cells

¹ Laboratory of Cancer Biology and Therapeutics, Centre de Recherche Saint-Antoine, Paris, France
² Institut National de la Santé et de la Recherche Médicale U893, Paris, France
³ Université Pierre et Marie Curie (UPMC06), Paris, France
⁴ Centre National de la Recherche Scientifique FRE 2939, Institut Gustave Roussy, Villejuif, France
⁵ Université Paris XI, Paris, France
⁶ Institut Gustave-Roussy, Villejuif 94805, France
⁷ Sir William Dunn School of Pathology, University of Oxford, OX1 3RE, Oxford, UK

View larger version (52K): [in this window] [in a new window]   Fig. 1. Influence of irofulven on RNA synthesis. (A) HeLa cells were incubated with the indicated concentrations of irofulven for 1 hour and the influence on RNA synthesis was measured by incorporation of radiolabeled uridine. Error bars represent standard errors. (B) HeLa cells were exposed for 1 hour to actinomycin D, DRB or irofulven at the indicated concentrations, permeabilized and the engaged polymerases allowed to extend their transcripts in the presence of bromo-UTP. Nascent RNAs were revealed by immunolabeling of bromo-labeled nucleotides. (C) HeLa cells were exposed for 1 hour to irofulven at the indicated concentrations. 15 minutes before fixation, bromo-uridine was added to the medium. Bromo-labeled RNAs were revealed by immunolabeling.

View larger version (40K): [in this window] [in a new window]   Fig. 2. Influence of irofulven on the dynamics of Pol II LS and induction of post-translational modifications. (A) Mammalian cells expressing GFP-Pol II were subjected to FLIP analysis under different experimental conditions: untreated cells, cells treated with 100 µM DRB, cells treated with 5 µg/ml actinomycin D, cells treated with 5 µg/ml irofulven and cells treated with 5 µg/ml irofulven and 100 µM DRB together. All curves represent an average of at least 12 independent measurements. (B) HeLa cells were incubated with 1 µg/ml irofulven for the indicated times. Total (upper panel), H14-reactive (second panel) and H5-reactive (third panel) levels of Pol II LS were assessed by immunoblotting, with actin (last panel) serving as an internal control for equal loading. Band intensities were measured, and the indicated IIO ratios were calculated as [(IIO/total Pol II LS)x100]. (C) Overexposed Pol II LS immunoblot.

View larger version (38K): [in this window] [in a new window]   Fig. 3. Irofulven treatment is accompanied by polyubiquitylation of Pol II LS followed by its proteasome-mediated proteolysis. (A) HeLa cells were incubated in the absence or presence of 1 µg/ml irofulven for 30 minutes, polyubiquitylated proteins were affinity purified and the resulting proteins were subjected to gel electrophoresis and the blots probed with the H14 monoclonal antibody. (B) HeLa cells were incubated in the absence or presence of 1 µg/ml irofulven for 30 minutes, Pol II LS was immunoprecipitated and the resulting proteins were subjected to gel electrophoresis and the blots probed with the P4D1 monoclonal antibody against ubiquitin. (C) HeLa cells were treated for the indicated times with 1 µg/ml irofulven in the absence (upper panel) or presence (lower panel) of 5 µM MG-132. (D) HeLa cells were treated for 6 hours with 1 µg/ml irofulven in the absence or presence of increasing amounts of velcade (bortezomib/PS-341) ranging from 20 to 1000 nM. (E) HeLa cells were incubated with 1 µg/ml irofulven for the indicated times in the presence () or absence () of 100 µM DRB. The total amounts of Pol II LS were assessed by immunoblotting and the relative intensities measured and standardized with respect to actin levels. Error bars represent standard errors and are indicated when they exceed symbol size.

View larger version (31K): [in this window] [in a new window]   Fig. 4. Degradation of Pol II LS is required for transcriptional restart following irofulven-exposure and influences cell viability. (A) HeLa cells were incubated for 30 minutes in the presence (, ) or absence () of 150 nM velcade before addition of 1 µg/ml irofulven (time –1). After 1 hour of irofulven exposure, cells were post-incubated for the indicated times in the presence () or absence () of 150 nM velcade. Cells continuously exposed to velcade alone were included as control (). Bromo-uridine was added for the last 15 minutes of incubation and the cells were fixed. Nascent RNA was revealed by immunolabeling, and the intensities were measured. All curves represent the average of at least three independent experiments in which the intensities of bromo-uridine labeling were assessed for more than 100 cells per time-point. Error bars represent standard errors and are indicated when they exceed symbol size. (B) The same conditions as above. The relative contents of H14 (left panel) and H5 (right panel) species were assessed by immunoblot and the relative intensities of each band measured and standardized versus actin. All curves represent an average of three independent experiments. Error bars represent standard errors and are indicated when they exceed symbol size. Filled symbols: cells treated for 1 hour with 1 µg/ml irofulven and post-incubated in drug-free medium; open symbols: cells pre-, co- and post-incubated with 150 nM velcade. (C) HeLa cells were preincubated for 30 minutes in the absence (left panel) or presence (right panel) of 10 µg/ml cycloheximide (CHX) followed by coincubation with 1 µg/ml irofulven for 1 hour. Cells were then post-incubated for the indicated times in the absence or presence of 10 µg/ml cycloheximide, and the relative content of Pol II LS forms was assessed by immunoblotting. Actin was included as a control for equal loading (lower panels). (D) HeLa cells were treated with irofulven for 1 hour, incubated in drug-free media for 5 days, and cellular viability was assessed by MTT assay. , cells treated with irofulven and post-incubated in drug-free medium; , cells pre-, co- and post-incubated with 500 nM velcade; , cells pre- co- and post-incubated with 100 µM DRB. All curves represent an average of at least three independent experiments, each done in duplicate. Error bars represent standard errors and are indicated when they exceed symbol size.

View larger version (21K): [in this window] [in a new window]   Fig. 5. Model depicting how transcription copes with irofulven lesions. Under normal conditions (upper-left panel), two main populations of Pol II coexist: the major one corresponds to a noncommitted fraction of Pol II, the second represents the fraction of engaged enzyme. Among the engaged fraction, two subtypes can be distinguished: the initiating Pol II, recognized by the H14 antibody, and the elongating subtype, recognized by the H5 antibody. The monoclonal H14 antibody specifically recognizes phosphorylated Ser5, whereas the H5 antibody specifically recognizes phosphorylated Ser2. Both serine residues are present within a heptapeptide that is repeated 52 times in the C-terminal domain of the human Pol II LS. In the presence of irofulven (upper-right panel), lesions are formed. These lesions are specifically processed by TCR after stalling of the RNA polymerase. Irofulven treatment is followed by an accumulation of initiating Pol II LS and a concomitant loss of the non-committed fraction of Pol II (lower-right panel). In contrast to the H14 species, the H5 fraction is rapidly lost. This suggests that the initiating Pol II cannot proceed efficiently to elongation. Three main hypothesis can be raised to explain this phenotype: (1) the presence of numerous lesions in the vicinity of the promoter region, (2) the inefficient recycling of other subunits that form, with Pol II LS, the holoenzyme and/or (3) the preferential association with repair sites of factors involved in both transcription and repair. Stalled polymerases become ubiquitylated and degraded through the proteasome. New protein synthesis is required to restore the initial pool of noncommitted enzyme necessary for transcription restart (lower-left panel). Proteasome inhibitors block Pol II LS recycling, inhibiting transcriptional recovery and increasing the probability of secondary DNA lesions. Cycloheximide (CHX), by blocking new protein synthesis, induces a rapid and complete loss of Pol II LS owing to the continuous degradation of newly engaged enzymes.

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