Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2015 Nov 1.
Published in final edited form as: Mol Cancer Res. 2014 Jul 17;12(11):1586–1596. doi: 10.1158/1541-7786.MCR-14-0229

Selective Inhibition of rDNA Transcription by a Small-Molecule Peptide that Targets the Interface Between RNA Polymerase I and Rrn3

Katrina Rothblum 1, Qiyue Hu 2, Yvonne Penrod 1, Lawrence I Rothblum 1,*
PMCID: PMC4233170  NIHMSID: NIHMS613753  PMID: 25033839

Abstract

The interface between the polymerase I associated factor Rrn3 and the 43 kDa subunit of RNA polymerase I is essential to the recruitment of Pol I to the preinitiation complex on the rDNA promoter. In silico analysis identified an evolutionarily conserved 22 amino acid peptide within rpa43 that is both necessary and sufficient to mediate the interaction between rpa43 and Rrn3. This peptide inhibited rDNA transcription in vitro while a control peptide did not. To determine the effect of the peptide in cultured cells, the peptide was coupled to the HIV TAT peptide to facilitate transduction into cells. The wild type peptide, but not control peptides, inhibited Pol I transcription and cell division. In addition, the peptide induced cell death; consistent with other observations that “nucleolar stress” results in the death of tumor cells. The 22mer is a small molecule inhibitor of rDNA transcription that is specific for the interaction between Rrn3 and rpa43, as such it represents an original way to interfere with cell growth.

Implications

These results demonstrate a potentially novel pharmaceutical target for the therapeutic treatment of cancer cells.

Keywords: RNA polymerase I, ribosomal RNA, nucleolus, Rrn3, rpa43, cell death

Introduction

Pleiomorphic nucleoli, a marker of the malignant phenotype for over a century (1), result from the high rate of ribosome biogenesis associated with cancer. Ribosome biogenesis is a tightly regulated, energetically costly process (2–4). The rate-limiting step in ribosome biogenesis is the synthesis of 47S pre-rRNA, the precursor of the 18S, 5.8S and 28S rRNAs, by RNA polymerase I (Pol I) (5–7). Indeed, recent studies have shown that selectively targeting Pol I transcription is a promising avenue for the therapeutic treatment of hematologic malignancies (8, 9). The targeting of Pol I transcription for cancer therapy will require complementary approaches. This in turn requires detailed knowledge of the key regulatory steps in Pol I transcription to allow for the rational design of selective inhibitors.

Transcription by RNA polymerase I requires Pol I-specific transcription factors (2, 10–12). In mammals, the cooperative interaction between two transcription factors is required to efficiently commit the rDNA promoter (13–15); SL1, containing TBP and TBP-associated factors (TAFs), and UBF (15). Pol I transcription is subject to multiple levels of regulation (16–20), including regulation by both anti-oncogenes and oncogenes (21–24).

The recruitment of Pol I to the committed template is complex (25, 26) and references therein). Only those polymerase molecules that contain Rrn3, a polymerase-associated factor, are capable of promoter-specific transcription (26). Hence, Rrn3 is essential for rDNA transcription (27, 28). Rrn3 acts as a bridge between RNA polymerase I and the committed rDNA promoter (29–31). Rrn3 interacts with the 43-kDa subunit of RNA polymerase I (rpa43) and with the TAFI110 and TAFI68 subunits of mammalian SL1. Further, Rrn3 is a DNA-binding protein and DNA-binding by Rrn3 is required for transcription (32). The predominant model is that active Rrn3 functions in the correct recruitment of RNA polymerase I to the committed template (33, 34).

We found that the binding site of mouse rpa43 to Rrn3 required a conserved region of 22 amino acids in mouse rpa43. We hypothesized that if this 22 amino acid peptide could sequester Rrn3, then the addition of the peptide would block rDNA transcription in vitro. When this was tested, we observed that the addition of the wild-type peptide, but not a peptide consisting of random amino acids (randomized 22mer/Ψ), to an in vitro transcription reaction blocked rDNA transcription in a dose-dependent manner. In order to study the effect of the peptide in intact cells, we fused the 22mer to a cell transducing peptide based on the HIV TAT protein transduction domain (35). Transduction of the 22mer into cultured cells resulted in the dose-dependent inhibition of rDNA transcription. Interestingly, the peptide demonstrated differential effects on cell growth. The peptide inhibited the growth of non-transformed cells, e.g. WI38 cells. In contrast, rat, mouse and human tumor cell lines underwent cell death within 8–48hrs in response to the peptide, but not in response to control peptides. The rate at which the cells died was not proportional to the rate of cell division. Our data indicate that the introduction into cells of a peptide that can bind to Rrn3, based on the sequence of rpa43, has the ability to inhibit rDNA transcription and induce cell death and has the potential to form the basis of a novel therapeutic mechanism to selectively treat cancer cells.

Materials and Methods

Yeast two-hybrid studies of protein-protein interactions

The Hybrid Hunter System (Invitrogen) was used to study the interaction between mouse rpa43 (mRPA43) and human Rrn3 (hRrn3) or mouse Rrn3 (mRrn3). The bait was a fusion protein consisting of the a LexA DNA-Binding Domain (DBD) and mRPA43. The prey was a fusion between the B42 activation domain (AD) and human or mouse Rrn3. S. cerevisiae L40 cells were transformed with pHybLexA/zeo driving the expression of the bait, and maintained in the presence of zeocin. These cells were then transformed with pYesTrp2 harboring the prey and allowing for selection by tryptophan prototrophy (W). The interaction of bait and prey proteins results in the expression of the reporter genes, HIS3 and LacZ, which can be detected by selection on plates lacking histidine (YC-WHU+Z), or by assaying for β-galactosidase activity (36).

Pull-down Assays

FLAG tagged Rrn3 was expressed in Sf9 cells and purified as previously described (37). Pull-down assays using biotin coupled peptide were carried out using avidin-coated beads and the protocol recommended by the supplier (Invitrogen). SDS-PAGE and electroblotting were carried out as described previously (21). Antibodies to caspase 3 and PARP (Cell Signaling) and FLAG (Sigma) were used as recommended by the suppliers.

Tissue culture and the measurement of cell proliferation

In some experiments, cell numbers were obtained using either a haemocytometer or a Cellometer (Nexcelom Bioscience LLC) and counterstaining with trypan blue. In other experiments, cell numbers were measured using the cyquant assay (Molecular Probes-Invitrogen) using the protocol recommended by the supplier. Cells counted with a haemocytometer were used to calibrate the assay. N1S1 cells (CRL-1604) and WI-38 cells (CL-75) were obtained from the ATCC. Mouse lymphosarcoma P1798 cells were a gift from Dr Aubrey Thompson, Jr. The human B-lymphoma cell lines used in this study were provided by Dr. Carol Webb (OMRF) and have been characterized as follows: Daudi, EBV-positive, LMP1-negative, Burkitt lymphoma (38); K562 and Raji, Burkitt’s lymphoma; and CL-01, EBV positive.

Twenty-four hours prior to treatment, adherent cells were plated at approximately 30–40% confluency. Suspenson cells were plated at 3–6×105 cells/ml. Cells were then treated as indicated. Detection of apoptotic, DNA fragmentation was carried out as described (39).

In vitro rDNA transcription

S100 extracts from N1S1 cells were prepared essentially as described (40, 41). In vitro transcription reactions were carried as described previously using 0.1 μg template/assay (41).

Measurement of RNA synthesis in vivo

N1S1 cells, plated in 24 well dishes, were treated as indicated. 5 μCi [3H]-uridine was added to the cell cultures for two hours and the cells were collected by centrifugation. The amount of radioactive uridine incorporated into RNA was determined by TCA precipitation on GFC filters and liquid scintillation spectrophotometry. Alternatively, whole cell RNA was isolated (36) and the specific radioactivity of the isolated RNA was determined. In a third assay, after 2×106 cells were treated with vehicle, TAT-eGFP (TAT peptide linked to amino acids 221–240 of eGFP) or TAT-22mer (60 μM), [3H]-uridine was added to the cell cultures (20 μCi/ml) for 30–60 min and whole cell RNA was isolated (36). Seven μg of RNA was then fractionated on formamide-formaldehyde agarose gels, transferred to Hybond-N (GE Healthcare), uv cross-linked and subjected to autoradiography at −80C after impregnating the membrane with En3Hance (Perkin Elmer) (36).

FACS Analysis

Cells were stained with propidium iodide and annexin V using the Annexin V–PI - FACS kit from Invitrogen-Molecular Probes as recommended by the supplier. The cells were then sorted on a FACSCalibur flow cytometer (BD Biosciences).

Results

Interaction between RPA43 with Rrn3 in Yeast Two Hybrid System

In the yeast two hybrid system, human Rrn3 and mouse Rrn3 interacted with mouse rpa43 (Figure 1). Yeast L40 cells transformed with pHybLex mRPA43 and pYesTrp2 hRrn3 produced colonies on the selective plate, YC-WHU+Zeocin (histidine selection medium, Right side, top panel, Figure 1A), but the cells transformed with pHybLex mRPA43 and empty pYesTrp2 did not grow in the absence of histidine (Left side, top panel, Figure 1A). This confirms that the protein-protein interaction between mRPA43 and hRrn3 can be studied in this system. The control cells transformed with pYesTrp2 hRrn3 grew on YC-WU (Left side, bottom panel, Figure 1A) but did not grow on YC-WHU medium (Right side, bottom panel, Figure 1A). This demonstrates that pYesTrp2 hRrn3 itself does not activate the reporters in L40. Confirmation of the interaction was demonstrated by the expression of β-galactosidase (Data not shown).

Figure 1.

Figure 1

Figure 1

Figure 1

Open in a new tab

A 22 amino acid long region in mouse rpa43, amino acid 136 to amino acid157, is conserved in eukaryotes and required for the interaction with Rrn3. Mouse RPA43 and hRrn3 interact in a yeast two hybrid system. (A). Yeast L40 cells transformed with pHybLexmRPA43 and pYesTrp2 hRrn3 grew on selective plate versus different controls In the upper panel the cells must express human Rrn3 to be viable. (B) Alignment of the amino acid sequences of rpa43 from various organisms. The top sequence is the conserved sequence found in mammals. The bottom sequence is the consensus for this region. (c) Deletion of the 22 amino acids (mRPA43Δ) or randomization of the 22 amino acids (mRPA43Ψ), as described in Materials and Methods, inhibits the interaction between rpa43 and Rrn3 in a dual-hybrid assay. Yeast L40 cells were transformed with the corresponding vectors and grown on YC-WHU+Zeocin selective medium. (D). Deletion or randomization of the 22 amino acids inhibits the interaction between Rrn3 and rpa43 in coimmunoprecipitation assays. Western blot after immunoprecipitation with anti-mRPA43 following in vitro transcription and translation of mRPA43, mPRA43Δ, mRPA43Ψ and hRrn3 and mixing of hRrn3 with mRPA43 and its mutants respectively. Ippt=immunoprecipitate. (E). Co-immunoprecipitation of mouse Pol I (rpa127) and wild-type and mutant mouse rpa43 with anti-rpa43 antibody after transfection of NIH 3T3 cells with wild-type rpa43 (lane 2) or mutant rpa43Ψ (lane 3). Lane 4 is a control immunoprecipitation when NIH 3T3 cells were transfected with pCDNA3 vector.

In mapping the binding site of rpa43 with Rrn3, we compared the sequences of various forms of rpa43 including human, mouse and fungus and found a highly conserved region of 22 amino acids, NKVSSSHIGCLVHGCFNASIPK, from position 136 to 157 (Figure 1B). As the interaction between rpa43 and Rrn3 is conserved from yeast to humans, we hypothesized that this conserved region might play an important role in this binding. Accordingly, we made two mutants of rpa43. One of them is rpa43Δ in which the 22 conservative amino acids were deleted. The other mutant is mRPA43Ψ in which the sequence order of the 22 amino acids deleted in mRPA43Δ was randomized as PGICVVLICPISNSSAGCIKFG, without regard to the relative amount of each amino acid. We cloned the mutants into the bait vector and examined their interaction with human and mouse Rrn3. Neither of the mutants interacted with either human or mouse Rrn3 (Figure 1C). These results support our hypothesis that the 22 conservative amino acids play an important role in the interaction between rrn3 and rpa43. These results were confirmed in pull down assays (Figure 1D), in which cotransfected Rrn3 coimmunoprecipitated with wild-type rpa43, but not with either of the mutants.

Incorporation of rpa43, rpa43Δ and rpaΨ into Pol I

To determine if the mutagenesis of amino acids 136 to 157 affected the overall structure of rpa43, we examined the interactions of wild type, rpa43Δ and rpa43Ψ with Pol I. 3T3 cells were transfected with vectors driving the expression of FLAG-tagged versions of the constructs. Lysates from the transfected cells were immunoprecipitated with anti-rpa43 antiserum bound to protein G agarose beads and the precipitates were analyzed by western blotting. Anti-rpa127 antiserum (42) was used to report for Pol I and anti-FLAG antibodies were used to report for rpa43 (Figure 1E). Both wild type rpa43 and the randomization mutant (rpa43Ψ) were immunoprecipitated with anti-rpa43 serum and Pol I (rpa127) coimmunoprecitatecd with both (lane 2 and 3). These data demonstrated that ectopically expressed, mutant forms of rpa43 can be incorporated into Pol I, and serve as controls demonstrating that the mutants are not significantly misfolded.

Rrn3 binds to the 22mer

The above experiments demonstrated that amino acids 136 to 157 of rpa43 were required for the interaction with Rrn3. We next asked if this region was sufficient for the interaction with Rrn3. The 22mer was synthesized with an N-terminal biotin and bound to avidin coated beads. In parallel, FLAG-Rrn3 was purified from infected Sf9 cells cells (32). The FLAG-Rrn3 was then incubated with blank avidin-coated beads or with beads that had been incubated with biotin-22mer (Figure 2 A, cartoon). The proteins that bound to the beads were analyzed by western blotting. Rrn3 did not bind to blank beads, but did bind to the peptide coated beads (Figure 2A, lanes 2 and 3). These results indicated that amino acids 136 to 157 (22mer) were sufficient to bind Rrn3. Taken together with the previous results, this domain is both necessary and sufficient to bind Rrn3.

Figure 2.

Figure 2

Open in a new tab

The amino acids from N136 to K157 of rpa43 are sufficient to mediate the interaction between rpa43 and Rrn3 and inhibit transcription in vitro. (A) Rrn3 copurifies with biotinylated 22-mer on streptavidin beads (lane 3) but not on streptavidin beads alone (lane 2). An N-terminal biotinylated synthetic peptide consisting of amino acids 136–157 of mouse rpa43 was incubated with FLAG-purified FLAg-Rrn3 and then the biotinylated peptide and complexed Rrn3 were purified on streptavidin beads as described in Materials and Methods and the affinity purified Rrn3 identified by western blotting. Input=10% of FLAG-Rrn3 in the binding reaction. (B) The 22mer inhibits rDNA transcription in vitro. Increasing amounts of the 22mer (lanes 2–5; 2,4,6 and 8 μg) or the random (Ψ) peptide (lanes 6–9; 2,4,6 and 8 μg) were added to an otherwise complete in vitro transcription system consisting of an S100 from N1S1 cells and the rat rDNA promoter. Transcription was carried out and the products analyzed as described in Materials and Methods. Int. Std., a radioactive fragment of DNA added as an internal standard for the efficiency of nucleic acid recovery.

The “22mer” can inhibit transcription in vitro

The binding of Rrn3 to RNA polymerase I is essential for the recruitment of Pol I to a committed template (33, 34). As our data indicated that the 22mer itself could bind Rrn3, we considered that addition of an excess amount of the 22mer to a transcription reaction would block transcription by competing with the polymerase associated rpa43 for Rrn3. This was tested using an in vitro transcription system. In this system, if the 22mer interacted with Rrn3, it would sequester it. In turn, the sequestration of Rrn3 would result in an inhibition of rDNA transcription. As shown in Figure 2, the addition of increasing amounts of wild type peptide inhibited rDNA transcription (lanes 2–5), while the addition of the Ψ peptide did not inhibit transcription (lanes 6–9).

The “22mer” can inhibit transcription in vivo

A BLAST search (NCBI) of all non-redundant GENBANK CDS failed to detect any conserved domains in rpa43. Further, the only proteins with Total scores of ≥46.9 or Identities of 75% or greater that were identified were rpa43 subunits from various species. This then led us to examine the possibility that the 22-mer might have the ability to target rDNA transcription in vivo. To do this, we coupled the 22-mer to a cell penetrating peptide (GGGRKKRRQRRR) based on the HIV TAT protein (35). The new peptide had the sequence GGGRKKRRQRRRNKVSSSHIGCLVHGCFNASIPK. N1S1 cells were treated with either vehicle, TAT peptide or TAT-22mer for various times, [3H]-uridine was added to the cell cultures for two hours, and the amount of radioactive uridine incorporated into RNA determined by TCA precipitation. As shown in Figure 3A, the TAT peptide or vehicle had no effect on the synthesis of RNA. In contrast, the TAT-22mer inhibited RNA synthesis in a dose-dependent manner to 40% of control. As rRNA synthesis accounts for approximately 70% of the total RNA synthesis in exponentially growing cells, these data are consistent with the model that the TAT-22mer targeted rDNA transcription. In a second set of experiments, [3H]-uridine was added to N1S1 cell cultures for one-half hour, four or six hours after TAT-22mer, TAT-eGFP or vehicle was added to their medium. RNA was isolated and the specific radioactivity determined. As shown, in Figure 3B, treatment with the TAT-22mer inhibited synthesis by 60% by four hours and this was maintained for at least another two hours. This is consistent with a nearly total inhibition of rDNA transcription. Similar results were obtained when WI38 cells were treated with the 22-mer (data not shown). We have used TAT peptide (GGGRKKRRQRRR) (Figure 3A) and TAT-eGFP (Figure 3B) as controls. Neither of these control peptides had an effect on RNA synthesis Finally, we examined the effect of the TAT-22mer on the labeling of the 47/45S pre-rRNA precursor. As shown in Figure 3C, treatment with the TAT-22mer inhibited incorporation into the 47/45S precursor by 70%. This is direct evidence that we have targeted rDNA transcription.

Figure 3.

Figure 3

Figure 3

Open in a new tab

Introduction of the 22mer into a cell inhibits RNA synthesis in vivo. (A). N1S1 hepatoma cells were treated with the indicated amounts of the TAT peptide (TAT) or the TAT peptide linked to the 22mer (TAT-22mer) for eight hours. At that time [3H]-uridine was added to the culture medium and the cells processed to determine the incorporation of uridine into RNA as described in Materials and Methods. (B) N1S1 hepatoma cells were treated with vehicle (6 hr) or the TAT peptide linked to 22 amino acids of eGFP (TAT-eGFP) (6 hr), or TAT- 22mer for four or six hours. At those times radioactive uridine was added to the culture medium and the cells incubated for an additional thirty minutes, RNA was isolated and its specific radioactivity determined as described in Materials and Methods. (C) N1S1 hepatoma cells were treated with vehicle or TAT-eGFP, or TAT-22mer for four hours. At that time [3H]-uridine was added to the culture medium. After the cells were incubated for an additional thirty minutes, whole cell RNA was isolated and analyzed by formamide agarose gel electrophoresis and ethidium bromide staining to control for load and autoradiography as described (36).

The “22-mer” can reversibly inhibit cell proliferation in vivo

Several laboratories have demonstrated that the inhibition of rDNA transcription, using either shRNAs or small molecules targeting components of the rDNA transcription apparatus, prevents cell proliferation (8, 9). We tested the possibility that the 22merwould have a similar effect. WI38 cells were incubated with varying doses of the TAT-22mer or TAT peptide. As shown in Figure 4A, the cells treated with TAT-22mer ceased to proliferate after 72 hours of treatment. While we did not observe a significant increase in trypan blue uptake, it was formally possible that the apparent inhibition of cell division was due to a combination of cell death and the inhibition of cell division. FACS analysis (data not shown) did not demonstrate either a specific block in cell cycle progression or an increase in the number of apoptotic cells (cells with subG0/G1 amounts of DNA). Thus, the decrease in proliferation was most likely not due to an increase in apoptotic cell death. Subsequently, we asked if the effect was reversible; would the cells reinitiate proliferation if we removed TAT-22mer from the medium? Twenty-four hours after WI38 cells were plated, vehicle, 90 mM TAT peptide or 90mM TAT-22mer was added to the media. After 72 hours of treatment (96 hr), duplicate wells were counted, and then the media on the treated cells was replaced with fresh media. The cell numbers were determined in these “recovering” wells after 24 and 48 hr. As shown in Figure 4B, the treatment with TAT-22mer for 72 hrs significantly inhibited proliferation. When the peptide was removed from the media, the cells demonstrated a robust recovery with nearly normal doublings over the next 24–48 hrs.

Figure 4.

Figure 4

Open in a new tab

Introduction of the 22mer into WI38 cells reversibly inhibits cell proliferation. (A) WI38 cells were plated at 3 × 103 per 0.32cm2 well (96 well plate) and vehicle, TAT peptide or TAT-22mer were added to the media at the concentrations indicated. Forty-eight and seventy-two hours later the cell number was determined from triplicate wells using the Cyquant assay as described in Methods and Materials. (B) WI38 cells were plated as described in (A). Twenty-four hours later, the cells were treated with vehicle, TAT peptide or TAT-22mer at the concentrations. Cell number was determined after seventy-two hours of treatment. Seventy-two hours after TAT-22mer was added to the media, duplicate wells of cells received fresh media and cell numbers were measured 24 and forty-eight hours later.

Treatment with the TAT-22-mer causes tumor cell death

It has been reported that the inhibition of ribosome biogenesis can result in cell death; a phenomenon that is sometimes referred to as nucleolar stress (8, 43–45). Our experiments with WI-38 cells did not provide evidence for cell death after 72 hours of treatment. WI-38 cells are a diploid cell line derived from normal, embryonic lung tissue that senesces in culture (adapted from the ATCC web site). As most of the studies demonstrating cell death were based on cultured tumor cells, we investigated the responses of several different tumor cell lines to the 22mer.

N1S1 hepatoma cells divide approximately every eight hours. After N1S1 cells were treated with 20, 40 or 60 μM TAT-22mer for varying periods of time, the cells were counted after staining with trypan blue (total and viable cells). As shown in Figure 5, when the cells were treated with 40μM TAT- 22mer, the number of viable cells decreased by 50% in the first 24 hours and by 90% after another 24 hours of treatment. Cells treated with vehicle, TAT peptide or TAT-eGFP did not demonstrate a significant decrease in proliferation or viability.

Figure 5.

Figure 5

Figure 5

Figure 5

Open in a new tab

Transduction of the 22mer into tumor cells causes cell death. (A) N1S1 cells growing in suspension were treated with vehicle (⬠), 40 μM TAT peptide (□) or 40 μM TAT-eGFP (★) or 40 μM TAT-22mer (○). (B) P1798 cells were cultured at 5×105 cells per well and allowed to grow treated with vehicle (⬠), 40 μM TAT peptide (□) or with 40 μM TAT-22mer (○). (C–F) Introduction of the 22mer into cells from various human hematological malignancies causes significant cell death. (C) Raji,(D) Daudi, (E) CL-01 and (F) K562 cells growing in suspension, were treated with vehicle (⬠), TAT peptide ((□), 60 or (❖) 100 μM), TAT-eGFP ((●) 40, (■) 60 or (▲) 80 μM) or TAT-22mer ((◆) 40, (▼) 60, (✖) 80 or (✚) 100 μM). Aliquots were taken at the indicated times of exposure, and viable cells counted using Trypan blue exclusion and the Nexcelom cellometer. (G) K562 cells were treated with TAT-eGFP (80 μM) and various doses of TAT-22mer for 24 hours, after which the viable cells were counted with the Nexcelom cellometer following Trypan blue staining.

When mouse lymphoma cells (P1798 cells), were treated with varying doses of TAT-22mer for 24 hours, we found that the cell number was greatly reduced from the starting density and more than 90% of the “surviving” cells failed to exclude trypan blue (data not shown). When we carried out time course studies to determine when the P1798 cells began to die after being treated with the TAT-22mer, we found that the cell number was significantly reduced after two hours of treatment, and that after six hours of treatment the number of viable cells was reduced by more than 90% (Figure 5B).

We then examined the effect of the TAT-22mer on the growth of other lymphoblast-like cell lines, Raji cells, Cl-01 and Daudi cells, cell lines derived from subjects with Burkitt’s lymphomas, and K562 cells, a chronic myelogenous leukemia. All of these cell lines were sensitive to the peptide (Figure 5, panels C–F). However, the time to <25% trypan blue exclusion varied for each cell line. After six hours of treatment with 60 μM TAT-22mer, more than 80% of the Daudi cells took up trypan blue (5D). In contrast, the Raji cells (5C) demonstrated a 25% decrease in viability after 8 hours and an 80% decrease after 24 hours. Treatment of the K562 cells (5F) with 60 μM TAT-22mer resulted in a 60% reduction in viability at 24 hours and a 95% reduction at 48 hours. CL-01 cells demonstrated a similar response, 50% reduction in viability after 24 hours and a 75 % reduction in viability after 48 hours (5E). The dose response curve of K562 cells after 24 hours of treatment is presented in Figure 5G.

Mechanism of cell death

Donati et al. have reported that the inhibition of ribosome biogenesis causes cell death through both p53-dependent and p53-independent apoptotic pathways triggered by nucleolar stress (43, 44). The N1S1 cells are p53 wild type. In order to determine if the cells were undergoing apoptotic death, we examined assayed for several apoptosis markers, e.g. caspase 3 and PARP cleavage, the formation of DNA ladders and propidium iodide-annexin V staining. As a positive control for apoptosis, cells were treated with staurosporine, a non-specific phosphatase inhibitor (46).

After six hours, cells treated with either vehicle or TAT-eGFP did not demonstrate significantly increased amounts of cleaved caspase (Figure 6A, lanes 1 and 2). However, cells treated with staurosporine and TAT-22mer demonstrated enhanced levels of cleaved caspase 3 (lanes 3 and 4). Similarly, treatment with TAT-22mer or staurosporine resulted in PARP cleavage (Figure 6B, lanes 3 and 4) and DNA fragmentation (Figure 6C, lanes 2 and 4). When N1S1 cells were treated with staurosporine, there was a significant increase in a population of cells that stained with annexin V, but not propidium iodide (lower right quadrant) that was not observed when the cells were treated with vehicle or TAT-eGFP (Figure 6D). However, when the cells were treated with TAT-22mer two populations were observed; one that stained with annexin V and a second population that stained with both annexin V and propidium iodide. This is consistent with both apoptosis (the shift to the lower right quadrant) and necrosis (the shift to the upper right quadrant).

Figure 6.

Figure 6

Open in a new tab

Treatment with the 22mer causes N1S1 cells to undergo apoptosis. (A and B) Eight hours after N1S1 cells were treated with vehicle or 40 μM TAT-eGFP or 40 μM TAT-22mer or 2 μM staurosporine, cells lysates were analyzed for activated caspase 3 (A) or PARP (B) as described in Materials and Methods. (C) DNA ladders isolated from cells treated for 22 hr with vehicle, 60 μM peptide or 2μM staurosporine. M, Trackit 1 KB Plus DNA ladder (Invitrogen). (D) Annexin-V/propidium iodide FACS analysis of N1S1 cells treated with vehicle or 40 μM TAT-eGFP or 40 μM TAT-22mer or 2 μM staurosporine for four hours and then processed for Annexin-V/Propidium iodide staining as described in Materials and Methods.

We examined the possibility that the inhibition of rRNA synthesis by the TAT-22mer induced apoptosis in other cell lines. As shown in Figure 7, we measured the levels of activated caspase 3 in P1798 cells treated with the peptide for 3 hours, a time point in which we see a significant percent of cell death. Treatment with either the TAT peptide or TAT-22mer did not cause the activation of caspase 3 as shown in Figure 7A. In contrast, treatment with staurosporine for three hours caused the accumulation of cleaved caspase 3 (lanes 2 and 3). Similarly, we did not find evidence for PARP cleavage when P1798 cells were treated with TAT-22mer (Figure 7B, lane 6). Consistent with the caspase and PARP cleavage assays, treatment of p1798 cells with TAT-22mer did not cause DNA ladders (Figure 7C, lane 5). When p1798 cells were treated with TAT-22mer for 2 hours, stained with annexin V/propidium iodide and sorted by FACS, we observed a significant increase in the number of cells in the upper right quadrant (72%) consistent with necrosis (Figure 7D). In contrast, only 2–3% of cells treated with vehicle or TAT-eGFP were in the upper right quadrant. Treatment with 1 mM staurosporine resulted in 26% of the cells sorting to the lower right quadrant, a result consistent with apoptosis.

Figure 7.

Figure 7

Open in a new tab

Introduction of the 22mer into P1798 lymphoma cells does not produce phenotypic changes consistent with apoptosis. (A and B) Three hours after P1798 cells were treated with vehicle or 60 μM TAT-eGFP or 60 μM TAT-22mer or 0.3 or 1.0 μM staurosporine, the cells were collected by centrifugation and lysates were fractionated by SDS-PAGE and analyzed for activated caspase 3 (A) or PARP (B). (C) High molecular weight DNA was isolated from cells treated for three hours as in A and analyzed by 2% agarose gel electrophoresis. (D) FACS analysis of P1798 mouse lymphoma cells treated with vehicle or 60 μM TAT-eGFP or 60 μM TAT-22mer for two hours or N1S1 cells treated with 2 μM staurosporine for four hours as described in Materials and Methods.

We have carried out similar analyses on K562 cells treated with the TAT-22mer (supplemental Figure 1). DNA isolated from cells treated with either TAT-eGFP or TAT-22mer (lanes 2 and 3) did not demonstrate a DNA ladder. Interestingly, annexin V/PI FACS analysis of K562 cells treated with TAT-22mer for twenty-four or forty-eight hours (supplemental Figure 2) demonstrated a distribution consistent with necrosis, but not apoptosis. These experiments provide initial evidence that the cell death that results from treatment with the TAT-22mer does not necessarily occur through an apoptotic pathway.

Discussion

One of the long-term goals of our laboratory has been to develop a method for targeting transcription by RNA polymerase I. Studies on the recruitment of Pol I to the committed template, suggested that preventing the formation of a Rrn3-Pol I complex, mediated by rpa43, would inhibit rDNA transcription (30, 34, 37). Thus, we sought to characterize the Rrn3-rpa43 interaction. It has been reported that mutation of several amino acids in the region between amino acids 42 and 172 of yeast rpa43 impaired specific transcription by Pol I (30). In our analysis of the sequence of rpa43, we identified a smaller conserved region of mouse rpa43, amino acids 136 to 157. When we tested the model that these amino acids would be important for the interaction with Rrn3, we found that these 22 amino acids are necessary and sufficient for rpa43 to bind to Rrn3 in either pull-down assays or in a yeast two hybrid system. While our data demonstrate the importance of the 22 amino acids in the interaction with Rrn3, neither deletion nor randomization mutants of the 22mer inhibited the incorporation of rpa43 into endogenous RNA polymerase I, arguing that the mutant forms of rpa43 are not fully misfolded.

As the basic model for transcription by Pol I requires an interaction between Rrn3 and rpa43, we examined the possibility that an excess amount of the 22mer would inactivate transcription when added to an in vitro transcription reaction. Our rationale was that the peptide would compete with rpa43-Pol I for Rrn3 in the reaction. Once we confirmed that this had happened, we next sought to determine if this could be duplicated in vivo. To facilitate entry of the peptide into living cells, we added the cell penetrating peptide from the transactivator of transcription (TAT) of human immunodeficiency virus to the 22mer (TAT-22mer). When N1S1 hepatoma cells were treated with the peptide we observed a robust and dose-dependent inhibition of RNA synthesis. TAT peptide by itself, or other control peptides, had no effect on RNA synthesis. As rRNA synthesis is approximately 70% of the total RNA synthesis in these cells, the fact that we observed 60% inhibition of incorporation of [3H]-uridine at a dose of 80μM, is consistent with the near total inhibition of rRNA synthesis. These observations were confirmed when we examined the synthesis of the 47/45S rRNA precursors. We measured a 60–70% inhibition of rRNA synthesis; again control peptides did not have a similar effect. Interestingly, we also observed evidence that pre-rRNA processing was inhibited as well (data not shown); we failed to observe the generation of the 38S and 30S processing products. This suggests that the residual labeling of 47/45S rRNA that we have observed, might in fact be caused by the accumulation of the precursor due to the lack of processing, and that the inhibition of pre-rRNA synthesis was greater than 70%. Quantitative RT-PCR analysis of several mRNAs, e.g. GAPDH, actin or peptidylprolyl isomerase, failed to demonstrate any significant changes in the steady state-levels of those mRNAs (data not shown); consistent with the model that we had not inhibited transcription by Pol II.

In the simplest model, one might predict that the inhibition of rDNA transcription and the resultant inhibition in the accumulation of ribosomes would result in the inhibition of cell cycle progression. In fact, this is what we observed when we treated WI38 cells with the peptide for periods up to 72 hours, and we did not observe an increased percentage of cells staining with trypan blue. It has been reported that the inhibition of rDNA transcription can lead to cell death via apoptosis (43, 47). However, FACS analysis did not demonstrate an increased number of subG1 cells, and when we removed the peptide from the media, the WI38 cells resumed cell division, suggesting that there had not been a significant amount of cell death.

In contrast, we found that when transformed cell lines, including a rat hepatoma, a mouse lymphoma and various human hematological malignancies were treated with the peptide, they demonstrated cell death. In some cases, more than 90% of the cells stained with trypan blue after six hours of treatment. However, the time to 90% cell death varied. Our first model was that the time to cell death would be proportional to cell cycle time; more rapidly dividing cells would be more dependent upon a constant supply of ribosomes. However, P1798 cells divide every 24 hr, Raji cells divide approximately every 39 hr and N1S1 cells divide approximately every 8 hours. There was no correlation between time to cell death and cell cycle duration. It is possible that there are variations in the levels of effectors of cell death downstream of Pol I transcription that would explain these results.

There is a growing recognition that ribosome biogenesis can serve as a therapeutic target in the treatment of cancer. Rubi et al. (45) reported on the importance of the nucleolus and its function in ribosome biogenesis in cellular homeostasis. They demonstrated that the p53 response triggered by UV irradiation derives from the disruption of nucleolar integrity rather than from damage to the DNA itself. They suggested that the nucleolus is involved in repair process monitoring. Observations from several laboratories (9, 43, 44, 48) have confirmed and extended these observations. Several commercial drugs used for cancer treatment are both pro-apoptotic and inhibitors of ribosome biogenesis, e.g. actinomycin D and cysplatin. In addition, as pointed out by Drygin et al. (9), the disruption of ribosome biogenesis can be lethal for cancer cells. Indeed, the recent reports that CX-5461(49) and ellipticine (50) which target SL1 and transcription by RNA polymerase I and cause cell death would confirm the importance of this process to cellular homeostasis.

Our studies provide evidence for a new target in the rDNA transcription apparatus. We have taken advantage of our knowledge of the biochemistry of transcription initiation to “design” a peptide that can specifically target a critical component of the rDNA transcription machinery. The interaction between rpa43 and Rrn3 is a rate-limiting step in the formation of the preinitiation complex and is unique for Pol I (37). Hence, this interaction and possibly others that mediate initiation or elongation may provide additional, specific molecular targets through which rDNA transcription can be targeted.

As mentioned above, the inhibition of rDNA transcription leads to cell death through apoptotic pathways, both p53-dependent as well as p53-independent pathways (43, 44, 47). When we examined the effects of the TAT-22mer on N1S1 cells, we observed caspase and PARP cleavage and DNA degradation ladders consistent with apoptosis. In addition, we observed annexin V-propidium staining FACS analyses consistent with apoptosis. Western blots demonstrated that these cells contain wild-type p53. However, we have been unable to demonstrate apoptosis in p1798 lymphoma cells that also contain wild-type p53. We have not observed DNA-laddering, caspase activation or PARP cleavage in cells that are clearly dying, i.e. failing to exclude trypan blue. Further, when the cells were sorted by annexin V/propidium iodide staining, the TAT-22mer treated cells demonstrated a shift consistent with necrosis. The Raji, Cl-01, Daudi and K562 cells we have used, have all been reported to be p53 mutants (51). Hence, it is possible that the inhibition of rDNA transcription by the peptide will induce apoptosis through p53-independent pathways as described by Donati et al. (44, 47). This needs to be examined in greater detail. p53 is mutated in many human tumors and defects in p53-dependent apoptotic pathway(s) present an obstacle for cancer therapeutics. Hence, the finding that disruption of the Rrn3-rpa43 interaction leads to cell death in p53 mutant cells opens the possibility of a novel chemotherapeutic pathway.

Supplementary Material

1
2
3

Acknowledgments

Financial support: This work was supported by GM069841 and HL077814 awarded to LIR and funds from the University of Oklahoma.

Footnotes

The authors have no potential conflicts of interest to disclose.

References Cited

  • 1.Busch H, Smetana K. The nucleolus [by] Harris Busch and Karel Smetana. New York: Academic Press; 1970. [Google Scholar]
  • 2.Moss T, Langlois F, Gagnon-Kugler T, Stefanovsky V. A housekeeper with power of attorney: the rRNA genes in ribosome biogenesis. Cell Mol Life Sci. 2007;64:29–49. doi: 10.1007/s00018-006-6278-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Grummt I. Life on a planet of its own: regulation of RNA polymerase I transcription in the nucleolus. Genes Dev. 2003;17:1691–702. doi: 10.1101/gad.1098503R. [DOI] [PubMed] [Google Scholar]
  • 4.Jorgensen P, Rupes I, Sharom JR, Schneper L, Broach JR, Tyers M. A dynamic transcriptional network communicates growth potential to ribosome synthesis and critical cell size. Genes & development. 2004;18:2491–505. doi: 10.1101/gad.1228804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Bernstein KA, Bleichert F, Bean JM, Cross FR, Baserga SJ. Ribosome biogenesis is sensed at the Start cell cycle checkpoint. Molecular biology of the cell. 2007;18:953–64. doi: 10.1091/mbc.E06-06-0512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Moss T. At the crossroads of growth control; making ribosomal RNA. Curr Opin Genet Dev. 2004;14:210–7. doi: 10.1016/j.gde.2004.02.005. [DOI] [PubMed] [Google Scholar]
  • 7.Milkereit P, Kuhn H, Gas N, Tschochner H. The pre-ribosomal network. Nucleic Acids Research. 2003;31:799–804. doi: 10.1093/nar/gkg165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Bywater MJ, Poortinga G, Sanij E, Hein N, Peck A, Cullinane C, et al. Inhibition of RNA polymerase I as a therapeutic strategy to promote cancer-specific activation of p53. Cancer Cell. 2012;22:51–65. doi: 10.1016/j.ccr.2012.05.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Drygin D, Rice WG, Grummt I. The RNA polymerase I transcription machinery: an emerging target for the treatment of cancer. Annu Rev Pharmacol Toxicol. 2010;50:131–56. doi: 10.1146/annurev.pharmtox.010909.105844. [DOI] [PubMed] [Google Scholar]
  • 10.Goodfellow SJ, Zomerdijk JC. Basic mechanisms in RNA polymerase I transcription of the ribosomal RNA genes. Subcell Biochem. 2012;61:211–36. doi: 10.1007/978-94-007-4525-4_10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Steffan JS, Keys DA, Vu L, Nomura M. Interaction of TATA-binding protein with upstream activation factor is required for activated transcription of ribosomal DNA by RNA polymerase I in Saccharomyces cerevisiae in vivo. Molecular and cellular biology. 1998;18:3752–61. doi: 10.1128/mcb.18.7.3752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Keys DA, Lee BS, Dodd JA, Nguyen TT, Vu L, Fantino E, et al. Multiprotein transcription factor UAF interacts with the upstream element of the yeast RNA polymerase I promoter and forms a stable preinitiation complex. Genes & development. 1996;10:887–903. doi: 10.1101/gad.10.7.887. [DOI] [PubMed] [Google Scholar]
  • 13.Comai L. Mechanism of RNA polymerase I transcription. Adv Protein Chem. 2004;67:123–55. doi: 10.1016/S0065-3233(04)67005-7. [DOI] [PubMed] [Google Scholar]
  • 14.Bell SP, Learned RM, Jantzen HM, Tjian R. Functional cooperativity between transcription factors UBF1 and SL1 mediates human ribosomal RNA synthesis. Science. 1988;241:1192–7. doi: 10.1126/science.3413483. [DOI] [PubMed] [Google Scholar]
  • 15.Smith SD, Oriahi E, Lowe D, Yang-Yen HF, O’Mahony D, Rose K, et al. Characterization of factors that direct transcription of rat ribosomal DNA. Mol Cell Biol. 1990;10:3105–16. doi: 10.1128/mcb.10.6.3105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.O’Mahony DJ, Xie WQ, Smith SD, Singer HA, Rothblum LI. Differential phosphorylation and localization of the transcription factor UBF in vivo in response to serum deprivation. In vitro dephosphorylation of UBF reduces its transactivation properties. The Journal of biological chemistry. 1992;267:35–8. [PubMed] [Google Scholar]
  • 17.Heix J, Vente A, Voit R, Budde A, Michaelidis TM, Grummt I. Mitotic silencing of human rRNA synthesis: inactivation of the promoter selectivity factor SL1 by cdc2/cyclin B-mediated phosphorylation. The EMBO journal. 1998;17:7373–81. doi: 10.1093/emboj/17.24.7373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Stefanovsky VY, Pelletier G, Hannan R, Gagnon-Kugler T, Rothblum LI, Moss T. An immediate response of ribosomal transcription to growth factor stimulation in mammals is mediated by ERK phosphorylation of UBF. Molecular cell. 2001;8:1063–73. doi: 10.1016/s1097-2765(01)00384-7. [DOI] [PubMed] [Google Scholar]
  • 19.Voit R, Grummt I. Phosphorylation of UBF at serine 388 is required for interaction with RNA polymerase I and activation of rDNA transcription. Proceedings of the National Academy of Sciences of the United States of America. 2001;98:13631–6. doi: 10.1073/pnas.231071698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Hannan KM, Sanij E, Rothblum LI, Hannan RD, Pearson RB. Dysregulation of RNA polymerase I transcription during disease. Biochimica et biophysica acta. 2013;1829:342–60. doi: 10.1016/j.bbagrm.2012.10.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Cavanaugh AH, Hempel WM, Taylor LJ, Rogalsky V, Todorov G, Rothblum LI. Activity of RNA polymerase I transcription factor UBF blocked by Rb gene product. Nature. 1995;374:177–80. doi: 10.1038/374177a0. [DOI] [PubMed] [Google Scholar]
  • 22.Hannan KM, Hannan RD, Smith SD, Jefferson LS, Lun M, Rothblum LI. Rb and p130 regulate RNA polymerase I transcription: Rb disrupts the interaction between UBF and SL-1. Oncogene. 2000;19:4988–99. doi: 10.1038/sj.onc.1203875. [DOI] [PubMed] [Google Scholar]
  • 23.Zhai W, Comai L. Repression of RNA polymerase I transcription by the tumor suppressor p53. Molecular and cellular biology. 2000;20:5930–8. doi: 10.1128/mcb.20.16.5930-5938.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Zhai W, Comai L. A kinase activity associated with simian virus 40 large T antigen phosphorylates upstream binding factor (UBF) and promotes formation of a stable initiation complex between UBF and SL1. Molecular and cellular biology. 1999;19:2791–802. doi: 10.1128/mcb.19.4.2791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Hanada K, Song CZ, Yamamoto K, Yano K, Maeda Y, Yamaguchi K, et al. RNA polymerase I associated factor 53 binds to the nucleolar transcription factor UBF and functions in specific rDNA transcription. The EMBO journal. 1996;15:2217–26. [PMC free article] [PubMed] [Google Scholar]
  • 26.Milkereit P, Tschochner H. A specialized form of RNA polymerase I, essential for initiation and growth-dependent regulation of rRNA synthesis, is disrupted during transcription. The EMBO Journal. 1998;17:3692–703. doi: 10.1093/emboj/17.13.3692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Moorefield B, Greene EA, Reeder RH. RNA polymerase I transcription factor Rrn3 is functionally conserved between yeast and human. Proceedings of the National Academy of Sciences of the United States of America. 2000;97:4724–9. doi: 10.1073/pnas.080063997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Bodem J, Dobreva G, Hoffmann-Rohrer U, Iben S, Zentgraf H, Delius H, et al. TIF-IA, the factor mediating growth-dependent control of ribosomal RNA synthesis, is the mammalian homolog of yeast Rrn3p. EMBO reports. 2000;1:171–5. doi: 10.1093/embo-reports/kvd032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Yamamoto RT, Nogi Y, Dodd JA, Nomura M. RRN3 gene of Saccharomyces cerevisiae encodes an essential RNA polymerase I transcription factor which interacts with the polymerase independently of DNA template. The EMBO journal. 1996;15:3964–73. [PMC free article] [PubMed] [Google Scholar]
  • 30.Peyroche G, Milkereit P, Bischler N, Tschochner H, Schultz P, Sentenac A, et al. The recruitment of RNA polymerase I on rDNA is mediated by the interaction of the A43 subunit with Rrn3. The EMBO journal. 2000;19:5473–82. doi: 10.1093/emboj/19.20.5473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Miller G, Panov KI, Friedrich JK, Trinkle-Mulcahy L, Lamond AI, Zomerdijk JC. hRRN3 is essential in the SL1-mediated recruitment of RNA Polymerase I to rRNA gene promoters. The EMBO journal. 2001;20:1373–82. doi: 10.1093/emboj/20.6.1373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Stepanchick A, Zhi H, Cavanaugh AH, Rothblum K, Schneider DA, Rothblum LI. DNA binding by the ribosomal DNA transcription factor rrn3 is essential for ribosomal DNA transcription. The Journal of biological chemistry. 2013;288:9135–44. doi: 10.1074/jbc.M112.444265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Aprikian P, Moorefield B, Reeder RH. New model for the yeast RNA polymerase I transcription cycle. Molecular and cellular biology. 2001;21:4847–55. doi: 10.1128/MCB.21.15.4847-4855.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Cavanaugh AH, Evans A, Rothblum LI. Mammalian Rrn3 is required for the formation of a transcription competent preinitiation complex containing RNA polymerase I. Gene Expr. 2008;14:131–47. [PMC free article] [PubMed] [Google Scholar]
  • 35.Vives E, Brodin P, Lebleu B. A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. The Journal of biological chemistry. 1997;272:16010–7. doi: 10.1074/jbc.272.25.16010. [DOI] [PubMed] [Google Scholar]
  • 36.Green MR, Sambrook J. Molecular cloning: a laboratory manual. 4. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory Press; 2012. [Google Scholar]
  • 37.Cavanaugh AH, Hirschler-Laszkiewicz I, Hu Q, Dundr M, Smink T, Misteli T, et al. Rrn3 phosphorylation is a regulatory checkpoint for ribosome biogenesis. J Biol Chem. 2002;277:27423–32. doi: 10.1074/jbc.M201232200. [DOI] [PubMed] [Google Scholar]
  • 38.Carter KL, Cahir-McFarland E, Kieff E. Epstein-barr virus-induced changes in B-lymphocyte gene expression. J Virol. 2002;76:10427–36. doi: 10.1128/JVI.76.20.10427-10436.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Herrmann M, Lorenz HM, Voll R, Grunke M, Woith W, Kalden JR. A rapid and simple method for the isolation of apoptotic DNA fragments. Nucleic acids research. 1994;22:5506–7. doi: 10.1093/nar/22.24.5506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Haglund RE, Rothblum LI. Isolation, fractionation and reconstitution of a nuclear extract capable of transcribing ribosomal DNA. Molecular and cellular biochemistry. 1987;73:11–20. doi: 10.1007/BF00229371. [DOI] [PubMed] [Google Scholar]
  • 41.Smith SD, Oriahi E, Lowe D, Yang-Yen HF, O’Mahony D, Rose K, et al. Characterization of factors that direct transcription of rat ribosomal DNA. Molecular and cellular biology. 1990;10:3105–16. doi: 10.1128/mcb.10.6.3105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Hannan RD, Hempel WM, Cavanaugh A, Arino T, Dimitrov SI, Moss T, et al. Affinity purification of mammalian RNA polymerase I. Identification of an associated kinase. J Biol Chem. 1998;273:1257–67. doi: 10.1074/jbc.273.2.1257. [DOI] [PubMed] [Google Scholar]
  • 43.Donati G, Bertoni S, Brighenti E, Vici M, Trere D, Volarevic S, et al. The balance between rRNA and ribosomal protein synthesis up- and downregulates the tumour suppressor p53 in mammalian cells. Oncogene. 2011;30:3274–88. doi: 10.1038/onc.2011.48. [DOI] [PubMed] [Google Scholar]
  • 44.Donati G, Brighenti E, Vici M, Mazzini G, Trere D, Montanaro L, et al. Selective inhibition of rRNA transcription downregulates E2F-1: a new p53-independent mechanism linking cell growth to cell proliferation. Journal of cell science. 2011;124:3017–28. doi: 10.1242/jcs.086074. [DOI] [PubMed] [Google Scholar]
  • 45.Rubbi CP, Milner J. Disruption of the nucleolus mediates stabilization of p53 in response to DNA damage and other stresses. The EMBO journal. 2003;22:6068–77. doi: 10.1093/emboj/cdg579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Zhang H, Hoang T, Saeed B, Ng SC. Induction of apoptosis in prostatic tumor cell line DU145 by staurosporine, a potent inhibitor of protein kinases. Prostate. 1996;29:69–76. doi: 10.1002/(SICI)1097-0045(199608)29:2<69::AID-PROS1>3.0.CO;2-C. [DOI] [PubMed] [Google Scholar]
  • 47.Donati G, Montanaro L, Derenzini M. Ribosome biogenesis and control of cell proliferation: p53 is not alone. Cancer research. 2012;72:1602–7. doi: 10.1158/0008-5472.CAN-11-3992. [DOI] [PubMed] [Google Scholar]
  • 48.Montanaro L, Trere D, Derenzini M. The emerging role of RNA polymerase I transcription machinery in human malignancy: a clinical perspective. Onco Targets Ther. 2013;6:909–16. doi: 10.2147/OTT.S36627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Drygin D, Lin A, Bliesath J, Ho CB, O’Brien SE, Proffitt C, et al. Targeting RNA polymerase I with an oral small molecule CX-5461 inhibits ribosomal RNA synthesis and solid tumor growth. Cancer research. 2011;71:1418–30. doi: 10.1158/0008-5472.CAN-10-1728. [DOI] [PubMed] [Google Scholar]
  • 50.Andrews WJ, Panova T, Normand C, Gadal O, Tikhonova IG, Panov KI. Old drug, new target: ellipticines selectively inhibit RNA polymerase I transcription. The Journal of biological chemistry. 2013;288:4567–82. doi: 10.1074/jbc.M112.411611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Forbes S, Clements J, Dawson E, Bamford S, Webb T, Dogan A, et al. Cosmic 2005. British journal of cancer. 2006;94:318–22. doi: 10.1038/sj.bjc.6602928. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS613753-supplement-1.docx (10.7KB, docx)
2
NIHMS613753-supplement-2.tif (5.7MB, tif)
3
NIHMS613753-supplement-3.tif (11.7MB, tif)

RESOURCES