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Cancer Research 68, 5257, July 1, 2008. doi: 10.1158/0008-5472.CAN-07-6207
© 2008 American Association for Cancer Research
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Experimental Therapeutics, Molecular Targets, and Chemical Biology

Triptolide-Induced Transcriptional Arrest Is Associated with Changes in Nuclear Substructure

Stephanie J. Leuenroth1 and Craig M. Crews1,2,3

Departments of 1 Molecular, Cellular, and Developmental Biology, 2 Chemistry, and 3 Pharmacology, Yale University, New Haven, Connecticut

Requests for reprints: Craig M. Crews, Department of Molecular, Cellular, and Developmental Biology, Yale University, P. O. Box 208103, New Haven, CT 06520-8103. Phone: 203-432-9364; Fax: 203-432-5713; E-mail: craig.crews{at}yale.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Triptolide, an active component of the medicinal herb lei gong teng, is a potent anticancer and anti-inflammatory therapeutic. It potently inhibits nuclear factor-{kappa}B transcriptional activation after DNA binding, although a precise mechanism is as yet unknown. Here, we report that triptolide also induces distinct nuclear substructural changes in HeLa cells. These changes in the nucleolus and nuclear speckles are reversible and dependent on both time and concentration. Furthermore, nuclear changes occurred within hours of triptolide treatment and were calcium and caspase independent. Rounding of nuclear speckles, an indication of transcriptional arrest, was evident and was associated with a decrease in RNA polymerase II (RNA Pol II) COOH-terminal domain Ser2 phosphorylation. Additionally, the nucleolus disassembled and RNA Pol I activity declined after RNA Pol II inhibition. We therefore conclude that triptolide causes global transcriptional arrest as evidenced by inactivity of RNA Pol I and II and the subsequent alteration in nuclear substructure. [Cancer Res 2008;68(13):5257–66]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
The small-molecule triptolide, a natural product isolated from the "thunder god vine," has been of great interest as a therapeutic for diseases such as cancer, arthritis, and autoimmune disorders. Research into its mechanism of action has revealed that it potently inhibits transcription of numerous proinflammatory mediators (1, 2), can activate caspases and other proapoptotic cascades (3–5), has separable mechanisms of action as defined by calcium concentration (6), and can activate calcium release through the polycystin-2 cation channel (7). One biological effect of triptolide that has received the most interest has been its ability to suppress cytokine-mediated transcriptional activity. Although there are reports in the literature citing the transcriptional effects of triptolide as both specific (2, 8) and global (9), it is clear that in many different cell lines triptolide is efficient at inhibiting expression of nuclear factor-{kappa}B (NF-{kappa}B)-mediated gene targets, hence its anti-inflammatory effects. However, because triptolide has also been reported most recently to reduce total RNA levels (9), it is of interest to further characterize how triptolide might be causing global transcriptional arrest.

The nucleus is highly compartmentalized to ensure efficient cellular functions, such as ribosome biogenesis and RNA polymerase II (RNA Pol II)-driven transcription. One specialized domain formed around actively transcribed rRNA genes is the nucleolus. This nuclear substructure not only is crucial to rRNA transcription and ribosome assembly but has also been shown to be involved in cell cycle regulation, proliferation, and the cellular stress response (10). Perturbations in cellular transcription are mirrored by changes in nucleolar structure; RNA Pol II inhibitors 5,6-dichloro-β-D-ribofuranosylbenzimidazole (DRB) and {alpha}-amanitin have previously been shown to alter nucleolar integrity, resulting in the dispersal of this subnuclear structure (11). Additionally, actinomycin D can be used to inhibit RNA Pol I at low concentrations (0.04–0.05 µg/mL) leading to condensation of rDNA, whereas high concentrations (2 µg/mL) additionally inhibit RNA Pol II activity and induce nucleolar disassembly (12, 13).

The nucleolar proteome is currently estimated to have ~700 proteins, many of which are of unknown function (14, 15). However, several proteins are well described, such as nucleolin (C23), nucleophosmin (NPM; B23), and upstream binding factor (UBF). Nucleolin is a multifunctional protein described as having roles in ribosome biogenesis, transcription, cell cycle regulation, and nuclear-cytoplasmic shuttling capabilities (16). NPM is a phosphoprotein involved in numerous cellular activities, such as transcriptional regulation, centrosome duplication (17), nuclear chaperoning (18), ribosome biogenesis, and nucleic acid binding and processing (19). Additionally, NPM is frequently up-regulated or is subject to genetic mutation or translocation in leukemias and lymphomas (20). UBF is a crucial cotranscriptional activator of the RNA Pol I complex; loss of UBF from the nucleolus is highly indicative of Pol I inactivity (21, 22).

Another subnuclear domain is the nuclear speckle, a region specialized in mRNA transcript splicing. These structures are highly dynamic as splicing factors are continually shuttling to sites of active transcription (23). They are characterized by their irregular shape and size and are frequently identified by the localization of the splicing factor SC35. Nuclear speckles are thought to form on the periphery of active RNA Pol II transcriptional complexes and, in addition to splicing components, may also contain cyclin-dependent kinase (Cdk)/cyclin complexes necessary for Pol II activity (24). These Cdk/cyclin complexes phosphorylate the COOH-terminal domain (CTD) of RNA Pol II, composed of 52 heptapeptide repeats in humans, and are therefore responsible for regulation of transcriptional activity. The TFIIH complex, containing Cdk7, phosphorylates Ser5 in the CTD and promotes initiation of transcription and promoter clearance (25, 26). Next, Ser2 is phosphorylated by the positive transcription elongation factor b complex (P-TEFb) composed of Cdk9 and cyclin T1 and promotes elongation of the mRNA transcript (27, 28). On RNA Pol II inhibition, nuclear speckles enlarge, become rounded, and decrease in number due to the accumulation of the splicing machinery (29, 30). As with nucleolar disruption, this effect has been observed with actinomycin D, DRB, and {alpha}-amanitin, signifying a link between active transcription and the maintenance of a structurally ordered nucleus.

In this study, we focused on the transcriptional effect of triptolide as assessed in the cervical carcinoma cell line HeLa. Previous work with this in vitro model showed that although transcriptional inhibition of NF-{kappa}B is concentration dependent, calcium was not required for this effect (6). Here, we have taken a cell biological approach to correlate differences in nuclear structure to the known inhibitory effect of triptolide on NF-{kappa}B–mediated transcription. Therefore, we examined changes in the nucleolus and nuclear speckles, two nuclear substructures that are indicative of changes in the global transcriptional activity of the cell. Our results support a concentration-dependent model of triptolide-induced RNA Pol II inactivity leading to transcriptional arrest and cell death.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Cell culture and reagents. HeLa cells were cultured as previously described (6). Triptolide was obtained from Sinobest, Inc. and dissolved in DMSO. Actinomycin D, calcimycin, and ionomycin were obtained from EMD Biosciences. Caspase-3 activity assay was purchased from Biomol and zVAD-fmk was purchased from Genotech. Antibodies purchased were as follows: nucleolin, NPM, UBF, Cdk9, and cyclin T1 from Santa Cruz Biotechnology; SC35 from Abcam; phospho-Thr199 NPM from Cell Signaling Technology; and phospho-Ser2 (BL2894) and Ser5 (BL2896) RNA Pol II antibodies from Bethyl Laboratories. NF-{kappa}B-luciferase assay was performed as previously described (6).

Generation of Clone-2. HeLa cells were incubated first with 100 nmol/L triptolide for 96 h and then with 1 µmol/L triptolide for an additional 96 h. Remaining cells were grown with 100 nmol/L triptolide until clonal populations were evident. Triptolide-resistant clones were found to grow in triptolide concentrations up to 1 µmol/L, where 5 µmol/L induced cell death. Routine administration of 100 nmol/L triptolide is added to cells to confirm maintenance of triptolide resistance.

Western blotting. Total cell lysates [0.5% Triton X-100, 50 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 500 mmol/L EDTA, and Complete protease inhibitors (Roche)] were prepared and protein levels were normalized. For nuclear fractions, cytosolic extract was removed following hypotonic lysis [10 mmol/L HEPES (pH 7.4), 10 mmol/L KCl, 0.1 mmol/L EDTA, 1 mmol/L DTT, 0.5 mmol/L phenylmethylsulfonyl fluoride] and 0.15% NP40 was added immediately before centrifugation. The pellet was resuspended in nuclear extract buffer [20 mmol/L HEPES (pH 7.4), 1 mmol/L EDTA, 0.4 mol/L NaCl, 1 mmol/L DTT] and centrifuged and the supernatant was retained. For RNase A experiments, the cytosolic and nuclear fractions were cleared with 0.5% Triton X-100 lysis buffer, and the resulting pellet was resolubilized in nuclear lysis buffer plus 0.1 mg/mL RNase A for 30 min on ice. Antibodies for nucleolin, NPM, UBF, Cdk9, and cyclin T1 (1:1,000) and RNA Pol II Ser2 and Ser5 antibodies (1:5,000) were incubated in 5% milk/TBS-Tween 20 (TBST) before enhanced chemiluminescence detection (GE Healthcare).

Immunofluorescence. For nucleolin, NPM, and UBF [1:200, 1.5% normal goat serum (NGS)/PBS], cells were fixed in methanol and blocked in 5% NGS/PBS; for phospho-Thr199 NPM [1:100, 1% bovine serum albumin (BSA)/TBS], cells were fixed in methanol and blocked in 10% NGS/1% BSA/TBS. SC35 (1:1,000, PBS) and Ser2 and Ser5 RNA Pol II (1:400, 5% BSA/PBS) detection required 4% paraformaldehyde/0.2% Triton X-100/PBS followed by methanol fixation. Cdk9 detection (1:600) required 4% paraformaldehyde/0.5% Triton X-100/PBS fixation and blocking in 5% milk/TBST. Secondary antibodies were anti-rabbit or anti-mouse Alexa Fluor 488 (green) or Alexa Fluor 594 (red; Molecular Probes), and all samples were mounted in Vectashield (Vector Laboratories).

All parvalbumin-green fluorescent protein (PV-GFP) constructs (31) were a gift of Anton Bennett (Yale University, New Haven, CT). Cells were transiently transfected using Lipofectamine 2000 (Invitrogen) plus 1 µg of one of the following plasmids for 24 h: cytomegalovirus (CMV)-GFP, CMV-nuclear export signal (NES)-PV-GFP, or CMV-nuclear localization signal (NLS)-PV-GFP. Following confirmation of GFP expression by microscopy, 100 nmol/L triptolide was added for 6 h. Cells were fixed and stained for nucleolin as described above.

Images were acquired using an Olympus CK40 inverted microscope and an Olympus digital camera. Confocal images were acquired under 63x oil immersion objective using a Zeiss Inverted Axiovert microscope and a Bio-Rad MRC 1024 laser source. Images were captured in single color channels as .pic files using LaserSharp software and then converted with Image J (NIH). For colocalization studies, images were artificially merged using Adobe Photoshop CS where indicated.

Reverse transcription-PCR. Total RNA was extracted using RNeasy (Qiagen) and each sample was normalized. Reverse transcription-PCR (RT-PCR) was performed using SuperScript One-Step RT-PCR with Platinum Taq kit (Invitrogen) and S14 primers were previously described (32). Total RNA (350 ng) was used for S14, reverse transcription proceeded 30 min/50°C, and the PCR amplification protocol was as follows: 94°C/2 min; 35 cycles of 94°C/15 s, 58°C/30 s, 72°C/1 min; 72°C/5 min. Total RNA (2 µg) was used for 5' external transcribed spacer (ETS) 45S amplification (33), reverse transcription proceeded at 50°C/30 min, and then 5 µL of the 50 µL reverse transcription reaction were used with the following protocol: 95°C/3 min; 25 cycles of 95°C/1 min, 65°C/1 min, 72°C/1 min; 72°C/5 min.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
Triptolide-mediated disruption of nucleolar structure is time and concentration dependent. We began our experiments by examining the nucleolus as its integrity has been used as an indicator of both RNA Pol I and II transcriptional activity. Previous work in our laboratory has shown that, in HeLa cells, 50 nmol/L triptolide for 6 h is the minimum concentration required to cause NF-{kappa}B transcriptional inhibition (6). We therefore incubated HeLa cells for 6 h with triptolide concentrations up to 1 µmol/L and examined nucleolar substructure. At concentrations of ≥50 nmol/L, we observed that the integrity of the nucleolus was compromised as nucleolin staining became irregular in shape and diffuse through the nucleoplasm (Fig. 1A ). This change in localization was prominent at 100 nmol/L in the majority of cells, and at 1 µmol/L, the nucleolus had completely dispersed (Fig. 1A). Because we have previously described that 100 nmol/L triptolide is effective at causing robust transcriptional inhibition and cell death, and we could now correlate that with clear nucleolar disassembly, we chose to focus on this concentration in the majority of the remaining experiments. Over a 6-h time course with 100 nmol/L triptolide, we observed that nucleolin localization changes began after 3 h of continual culture (Fig. 1B). Furthermore, this effect was predominantly reversible if cells were incubated for no more than 4 h before triptolide removal (Fig. 1C). However, if triptolide was left in culture for 6 h before removal, the nucleolus did not recover and cells were committed to cell death (Fig. 1C). This first set of experiments led us to believe that general transcription may be affected by triptolide, so we next examined another subnuclear marker that changes on transcriptional arrest—the nuclear speckle.


Figure 1
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  		Figure 1. Triptolide induces nucleolar disassembly. HeLa cells were treated with triptolide before fixation and staining for nucleolin by indirect immunofluorescence under the following conditions: increasing concentrations of triptolide for 6 h (A) and 100 nmol/L triptolide over a 6-h time course (B). C, cells were pretreated with 100 nmol/L triptolide for the indicated times, washed, and allowed to recover for 16 h. All images were acquired under x25 magnification. Scale bar, 10 µm.

 
Nuclear speckles become enlarged and rounded following triptolide treatment. We used an antibody to the splicing factor SC35 to assess nuclear speckle morphology in response to 100 nmol/L triptolide or DMSO over a 6-h time course. Whereas DMSO control cells had numerous speckles of various size and shape, cells treated with triptolide displayed a distinct rounding and enlargement of speckles beginning after 2 h of incubation (Fig. 2A ). This morphologic change, a marker of transcriptional arrest, became more pronounced over the 6-h time course that was in marked contrast to DMSO control (Fig. 2A). We next examined the concentration-dependent effect of triptolide on speckle morphology after 6 and 16 h of continual incubation. After 6 h (the time at which NF-{kappa}B inhibition has been studied), only 50 and 100 nmol/L of triptolide caused observable speckle rounding. Following 16 h, 25 to 100 nmol/L triptolide (concentrations shown to cause cell death in HeLa cells; ref. 6) induced prominent speckle rounding and an increase in size (Fig. 2B). Therefore, our data indicate that nuclear speckle rounding begins at 50 nmol/L with acute triptolide addition (6 h), although 25 nmol/L is also sufficient for this effect, although over a longer time course.


Figure 2
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  		Figure 2. Triptolide induces nuclear speckle rounding. A, HeLa cells were incubated with 100 nmol/L triptolide (T) or DMSO (D) over a 6-h time course. Following fixation, cells were stained for the splicing factor SC35 by indirect immunofluorescence. B, cells were incubated with 0, 25, 50, or 100 nmol/L of triptolide for either 6 or 16 h of continual culture before assessing SC35 localization. All images were acquired by confocal microscopy under x63 magnification. Scale bars, 10 µm.

 
Subnuclear structures are unaltered in a triptolide-resistant clonal line. We have isolated a triptolide-resistant clonal cell line derived from HeLa, named Clone-2, which remains viable in the presence of up to 1 µmol/L triptolide. Because 100 nmol/L triptolide causes rapid death in HeLa, but is permissive for growth in Clone-2 cells, we compared nucleolar and nuclear speckle differences in these lines as an indicator of cell death and transcriptional activity. After 6 h of incubation with 100 nmol/L triptolide, HeLa cells exhibited the characteristic nucleolar disassembly as assessed by both differential interference contrast (DIC) microscopy and localization of nucleolin (Fig. 3A ). This was in contrast to Clone-2 cells that retained nucleolar integrity; however, nucleolar borders became slightly irregular in shape (Fig. 3A). To confirm that Clone-2 cells are capable of a cellular stress response and RNA Pol I inhibition, we incubated cells with 10 nmol/L actinomycin D and found that in both HeLa and Clone-2 cells nucleolar structure was affected (Fig. 3A).


Figure 3
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  		Figure 3. A HeLa-derived triptolide-resistant clonal population does not undergo nuclear structure changes. A, HeLa and Clone-2 cells were assessed for nucleolar changes by either DIC or indirect immunofluorescence of nucleolin. Cells were treated with either 100 nmol/L triptolide or 10 nmol/L actinomycin D (Act D) for 6 h. Arrow, nucleolus. DIC images were acquired by confocal microscopy using x63 magnification, and immunofluorescence images were acquired under x25 magnification. B, transcriptional activity was assessed using a NF-{kappa}B-luciferase construct. HeLa or Clone-2 cells were incubated with vehicle only (control), TNF-{alpha}, or TNF-{alpha} plus 100 nmol/L triptolide for a total of 6 h. C, HeLa and Clone-2 cells were assessed for survival following 72 h of 100 nmol/L triptolide incubation. D, cells were incubated with DMSO or 100 nmol/L triptolide for 16 h before fixation and indirect immunofluorescent detection of nucleolin or SC35. Images were acquired by confocal microscopy under x63 magnification. Scale bars, 10 µm.

 
Because our triptolide-resistant cell line showed only a minor change in the shape of the nucleolar compartment, we next determined if NF-{kappa}B–mediated transactivation was affected. Following tumor necrosis factor-{alpha} (TNF-{alpha}) incubation alone, both HeLa and Clone-2 cells responded with similar levels of NF-{kappa}B-luciferase induction (Fig. 3B). Coincubation of 100 nmol/L triptolide plus TNF-{alpha} resulted in a 4-fold reduction of activity in HeLa, whereas Clone-2 cells retained the majority of its transcriptional activation (Fig. 3B). The resistance of Clone-2 to both nucleolar disassembly and transcriptional inhibition ultimately correlated with cell survival. Whereas 72 h of incubation with 100 nmol/L triptolide resulted in cell detachment and death for HeLa cells, Clone-2 cells survived and continued to proliferate with a doubling time of ~28 h (Fig. 3C; data not shown). Because only minor effects on nucleolar integrity or transcription were observed for Clone-2 cells with an acute triptolide time course, we next assessed nucleolar changes after 16 h of continual culture with triptolide. After 16 h of 100 nmol/L triptolide, many HeLa cells detached indicating cell death and those remaining adherent were characterized by a dispersal of nucleolin throughout the nucleoplasm, and several large rounded nuclear speckles (Fig. 3D). Clone-2 cells remained adherent, the nucleolus was intact, nuclear speckles looked similar to DMSO-treated control, and cells continued to grow (Fig. 3D). Taken together, our results thus far indicate that changes in the structure of both the nucleolus and nuclear speckles correlate to transcriptional inactivation and cell death due to high triptolide concentrations.

Triptolide-induced changes in nucleolar and nuclear speckle morphology are calcium, stress, and caspase independent. To begin examining a putative mechanism for the observed subnuclear changes due to triptolide, we next focused on the role of calcium. We have previously shown that NF-{kappa}B transcriptional inhibition is calcium independent and therefore wanted to determine if changes to both the nucleolus and nuclear speckles (and their role in transcriptional regulation) were also calcium independent. Because triptolide can elicit calcium release (7), we first wanted to establish if calcium ionophores could mimic triptolide-induced nucleolar disassembly. Whereas incubation with 100 nmol/L triptolide for 6 h led to a dramatic loss of nucleolar integrity, neither calcimycin nor ionomycin could mimic the effect (Fig. 4A ). We next incubated cells in the absence of extracellular calcium and still observed triptolide-induced nucleolin dispersal and speckle rounding (Fig. 4B). As a third method to test the requirement of calcium-mediated signaling on triptolide-mediated nuclear changes, we used GFP-PV constructs (31) that were localized to either the nucleus (NLS-PV-GFP) or cytosol (NES-PV-GFP). Expression of these constructs in their specific cellular compartments acts to buffer the response of a calcium signal. Expression of the GFP constructs alone did not affect nucleolar morphology as assessed by nucleolin immunofluorescent localization (Fig. 4C). In the presence of triptolide, nucleolar structure dispersed regardless of either cytosolic or nuclear calcium buffering (Fig. 4C). We therefore conclude that the subnuclear changes we observe with high triptolide concentrations are calcium independent.


Figure 4
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  		Figure 4. Nuclear changes are independent of calcium, stress kinases, and caspase activation. A, HeLa cells were incubated for 6 h with DMSO, 100 nmol/L triptolide, 5 µmol/L calcimycin, or 3 µmol/L ionomycin before fixation and nucleolin detection by indirect immunofluorescence. Magnification, x25. Scale bar, 10 µm. B, HeLa cells were incubated in calcium-free medium for 16 h before addition of DMSO or 100 nmol/L triptolide for 6 h. Cells were stained for SC35 (red) or nucleolin (green) by indirect immunofluorescence (confocal microscopy, 40x). C, HeLa cells were transiently transfected with GFP vector, NES-PV-GFP, or NLS-PV-GFP constructs for 24 h before a 6-h incubation with DMSO or 100 nmol/L triptolide. Green, GFP localization; red, nucleolin is detected by indirect immunofluorescence. Images acquired separately under x40 magnification and artificially merged. D, HeLa cells were treated with DMSO, 5 µmol/L SB203580, 10 µmol/L SP600125, or 2 µmol/L zVAD-fmk ± 100 nmol/L triptolide for 6 h. Green, nucleolar integrity was detected by indirect immunofluorescence of nucleolin. Graph represents caspase-3 activity in HeLa cells treated with DMSO or 100 nmol/L triptolide over an 18-h time course. Cell lysates were prepared and activity was assessed by cleavage of the colorimetric substrate DEVD-pNA. Columns, mean (n = 3); bars, SE.

 
Nucleolar disassembly has also been used to detect activation of the cellular stress response through p38 kinase or c-Jun NH2-terminal kinase (JNK) signaling (34). To determine if triptolide induced a general stress response that was then reflected on nucleolar integrity, we incubated cells with the p38 kinase inhibitor SB203580 or the JNK inhibitor SP600125 ± 100 nmol/L triptolide for 6 h. Treatment with DMSO or either inhibitor alone did not alter nucleolar structure and, in addition, neither could rescue the effect of triptolide on nucleolar dispersal (Fig. 4D). Because caspase activation by triptolide has been well documented in the literature, we assessed whether the observed changes in nuclear structure could be due to proteolytic cleavage by active caspases. Incubation with the irreversible broad-spectrum caspase inhibitor zVAD-fmk failed to rescue triptolide-induced nucleolar disassembly (Fig. 4D). Furthermore, triptolide-induced caspase-3 activation was first evident after 9 h of incubation, with strong activation beginning at 14 h (Fig. 4D). Because nucleolar and speckle changes begin as early as 2 h after triptolide addition, it is unlikely that caspases are inducing these effects. We therefore continued to probe changes in proteins associated with either the nucleolus or nuclear speckles to determine a possible mechanism for triptolide-induced transcriptional inhibition.

Nucleolar disassembly is characterized by changes in nucleolin, NPM, and UBF localization. Because we had previously characterized the loss of nucleolar integrity only by nucleolin immunofluorescent localization, we wanted to determine if NPM and UBF were also connected with this observed effect. Following triptolide incubation, nucleolin, NPM, and UBF dispersed into the nucleoplasm, leaving only a small remnant of the nucleolus in some cells (Fig. 5A ). To examine if there were any protein stability or modification changes, nuclear lysates from an 8-h triptolide time course were separated by SDS-PAGE; however, all protein levels remained constant and there was no apparent cleavage or shift in mobility (Fig. 5B).


Figure 5
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  		Figure 5. Nucleolar disassembly is characterized by nucleolin, NPM, and UBF localization. A, HeLa cells were incubated with DMSO or 100 nmol/L triptolide for 6 h before fixation and immunofluorescent detection of nucleolin, NPM, UBF, or SC35. Left, acquired under x25 magnification; right, acquired by confocal microscopy at x63 magnification. B, nuclear cell lysates were prepared from cells treated with 100 nmol/L triptolide over an 8-h time course and separated by SDS-PAGE. Shown are representative Western blots of nucleolin, NPM, and UBF protein levels. Left, molecular weight markers in kDa. C, following a 6-h time course with 100 nmol/L triptolide, total nuclear lysates were first prepared (Nuclear). The remaining pellet was resolubilized with nuclear lysis buffer plus 0.1 mg/mL RNase A (+RNase A). All samples were separated by SDS-PAGE and NPM expression was detected in each fraction. D, HeLa cells were treated with DMSO vehicle or 100 nmol/L triptolide for 6 h and cells were costained with p-NPM (green) and SC35 (red). Representative images were acquired in separate channels and artificially merged. Scale bars, 10 µm.

 
Because NPM has been found to bind RNA and aid in processing, we assessed if triptolide could alter this interaction. Following nuclear protein extraction, the remaining pellet was treated with RNase A to release any bound NPM, as Triton X-100 extraction alone is insufficient to solubilize this population of NPM (35). Although total nuclear levels of NPM before RNase treatment again remained stable following triptolide incubation, it was evident that the functionally active NPM population bound to RNA decreased following 3 h (Fig. 5C). This finding suggests that RNA processing and transcription is altered in the presence of triptolide.

NPM phosphorylation at Thr199 (p-NPM) has previously been reported to target NPM to nuclear speckles and repress pre-mRNA processing in the presence of the RNA Pol II inhibitor {alpha}-amanitin (36). Because we observed a change in both nucleolar integrity and nuclear speckle rounding following triptolide addition, we next examined p-NPM expression and localization in relation to nuclear speckles. In control-treated HeLa cells, p-NPM expression was nucleolar with little or no overlap with the nuclear speckle marker SC35 (Fig. 5D). In contrast, immunofluorescent staining of triptolide-treated cells showed colocalization of punctate p-NPM regions with nuclear speckles (Fig. 5D). It remained to be elucidated whether nucleolar dispersal was due to an inhibition of RNA Pol I or was an indirect effect following RNA Pol II inhibition. To address this, we next studied key phosphorylation sites on the CTD of the large subunit of RNA Pol II and its subsequent transcriptional activity.

RNA Pol II phosphorylation and transcriptional activity decrease on triptolide addition. The CTD in mammalian cells is composed of >50 repeats of a heptapeptide sequence in which Ser5 and Ser2 are required for initiation and elongation of transcription, respectively (25). To determine if triptolide could affect general transcription by influencing either Ser2 or Ser5 phosphorylation, we examined nuclear lysates prepared from a triptolide time course. Western blot analysis indicated that Ser2 phosphorylation levels significantly decreased between 2 and 3 h of incubation, whereas Ser5 levels remained largely elevated, although some fluctuation was evident (Fig. 6A ). It is also noteworthy that a higher molecular weight band is detected in the 3- and 6-h time points and may be indicative of a sustained or increased level of Ser5 phosphorylation over the length of the CTD. As an additional method to evaluate changes in CTD phosphorylation, we examined Ser5 and Ser2 by immunofluorescence microscopy and again observed only a significant decrease in Ser2 phosphorylation that was not dependent on calcium (Fig. 6A). To examine the effect of triptolide concentration on RNA Pol II phosphorylation, we incubated HeLa cells with 10 to 100 nmol/L of triptolide for 6 h. Beginning at 50 nmol/L, a mild inhibition of Ser2 phosphorylation was evident and this decrease became prominent at the 75 to 100 nmol/L concentrations (Fig. 6A). This regulation of RNA Pol II seemed to be relevant to the proapoptotic or transcriptional inhibitory effects of triptolide as Clone-2 was not Ser2 responsive (Fig. 6B).


Figure 6
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  		Figure 6. Triptolide inhibits RNA Pol II activity. A, HeLa cells were treated with 100 nmol/L triptolide over a 6-h time course or increasing triptolide concentrations for 6 h before nuclear lysates were prepared. RNA Pol II phosphorylation at Ser2 or Ser5 of the CTD was assessed by Western blot analysis. Left, molecular weight markers in kDa. Immunofluorescence localization of RNA Pol II Ser2 or Ser5 phosphorylation in the presence or absence [(–) Ca2+] of calcium-containing medium was also completed. Results are representative of four separate experiments. Scale bar, 10 µm. B, Clone-2 cells were treated with 100 nmol/L triptolide for 6 h and assessed for differences in Ser2 and Ser5 phosphorylation by Western blot analysis and immunofluorescence localization. Results are representative of three separate experiments. C, HeLa cells were treated with 100 nmol/L triptolide over a 6-h time course and total RNA was extracted from each sample. RNA was normalized per sample and RT-PCR was performed to assess Pol I and II activity. The ribosomal protein S14 transcript is transcribed by RNA Pol II and the 5' ETS of 45S pre-rRNA is transcribed by RNA Pol I. Results shown are representative of three separate experiments. D, HeLa cells were incubated in the presence of 100 nmol/L triptolide during a 6-h time course. Total cyclin T1 and Cdk9 protein levels (left), Cdk9/cyclin T1 complex interaction by Cdk9 immunoprecipitation (IP; middle), and Cdk9 immunolocalization (right) were assessed. A/G represents immunoprecipitation with protein A/G agarose beads and DIC inset shows nucleolar integrity. Results are representative of three separate experiments.

 
All of our data collected thus far linked nucleolar disruption, nuclear speckle rounding, and a decrease in Ser2 phosphorylation of the CTD of RNA Pol II to the same time frame (6 h) and triptolide concentrations (≥50 nmol/L) that are associated with NF-{kappa}B inhibition in HeLa cells. We next wanted to determine if RNA Pol I and/or Pol II transcriptional activity was inhibited and, if so, to know if there was a chronological order to this effect. To do this, we assessed RNA Pol II activity by transcript levels of ribosomal subunit S14 and RNA Pol I by the 5' ETS region of the pre-ribosomal 45S transcript. Total RNA from an equal number of cells per time point was extracted following incubation with 100 nmol/L triptolide over 6 h. Regardless of triptolide addition, total RNA levels seemed similar in all samples (Fig. 6C). Following RT-PCR analysis, we observed a dramatic reduction in RNA Pol II transcriptional activity that was evident as early as 1 h after triptolide addition (Fig. 6C). This effect was most prominent following 3 h of incubation that corresponds with a clear rounding of nuclear speckles (Fig. 2A). Because the 5' ETS of the 45S rRNA is rapidly cleaved and processed following transcription, we used this sequence as a target for RT-PCR amplification to assess RNA Pol I activity. Levels of this transcript did not start to decline until 3 h after triptolide addition, which is concomitant with the onset of nucleolar disassembly (Fig. 6C). Because the Cdk9/cyclin T1 complex (P-TEFb) is known to phosphorylate Ser2 of the RNA Pol II CTD, we next examined the expression of these two proteins. Over a 6-h, 100 nmol/L triptolide time course, total nuclear levels of Cdk9 or cyclin T1 were constant (Fig. 6D). Complex formation was tested by coimmunoprecipitation experiments and again Cdk9/cyclin T1 interaction remained stable (Fig. 6D). Additionally, Cdk9 localization remained nuclear even as nucleolar disassembly became evident (Fig. 6D).

Taken together, our data support a model in which high proapoptotic concentrations of triptolide cause RNA Pol II transcriptional inhibition through a decrease in Ser2 phosphorylation. After RNA Pol II inhibition, RNA Pol I transcriptional activity is also attenuated, affecting RNA ribosome biogenesis as early as 3 h following triptolide addition. Therefore, we conclude that triptolide affects global transcription in a concentration-dependent manner as evidenced by the following: (a) attenuation of transcripts associated with ribosome biogenesis, (b) RNA Pol II activity and its regulatory phosphorylation decreases, (c) nuclear speckle rounding is evident indicating mRNA splicing has ceased, and (d) normal nucleolar structure is lost and RNA Pol I activity decreases.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
Triptolide, a natural product isolated from a Chinese medicinal plant, may be therapeutically relevant for a variety of proliferative disorders, such as cancer (37, 38) and autosomal dominant polycystic kidney disease (7), or autoimmune diseases and inflammation, such as systemic lupus erythematosus and arthritis (39, 40). Its cellular effects and mechanisms of action are highly complex and undoubtedly involve multiple biological pathways. Ongoing studies in several laboratories have elucidated key points in the mode of action of triptolide, such as concentration-dependent and cell type–dependent effects (37), a partial dependence on calcium (6), modulation of apoptosis-activating proteins (3–5), and inhibition of the transcription factor NF-{kappa}B at a step following DNA binding (8). Although the emphasis of triptolide-induced transcriptional inhibition has focused on NF-{kappa}B and its proinflammatory gene targets, triptolide has also been reported to suppress AP-1 (41), NFAT (8), and HSF1 (42) transactivation as well. In contrast to the hypothesis that triptolide targets transcription factors with specificity, triptolide has also been reported to suppress total RNA synthesis (9); however, a mechanism has not been described. We began a careful analysis of nuclear substructure changes indicative of global transcriptional arrest at triptolide concentrations and time points known to inhibit NF-{kappa}B–mediated transcription (6). Furthermore, we were able to characterize changes in RNA polymerase activity describing a potential mechanistic action.

Known transcriptional inhibitors that affect RNA Pol II, such as actinomycin D, DRB, and {alpha}-amanitin, have all been previously shown to cause nuclear speckle rounding and/or nucleolar disruption. It should, however, be noted that coordination of RNA Pol I, II, and III activity is tightly regulated as inhibition of one will affect the efficiency of the others (43, 44). This coordinated action of all RNA polymerases ensures sufficient protein subunits and rRNA to complete ribosome assembly. We first observed a reversible nucleolar disassembly induced by triptolide and further explored if it was an indication of global transcriptional changes in the cell as assessed by nuclear speckle morphology. Whereas speckle size, shape, and number can vary per cell, RNA Pol II transcriptional inhibition is marked by an accumulation of the splicing machinery and a concomitant increase in speckle size. Although 100 nmol/L triptolide incubation resulted in a decrease in RNA Pol II transcription after only 1 h, changes in speckle morphology and a decrease in Ser2 RNA Pol II phosphorylation were readily observed after 2 h. Additionally, residual p-NPM was found associated with nuclear speckles, an event previously correlated with transcriptional inactivity (36). Ser5 phosphorylation was relatively constant throughout our time course; however, we did observe minor fluctuations. This could be due to cell cycle–dependent effects or may reflect differences in the number of Ser5 phosphorylations along the full length of the CTD. Although we believe that Ser2 is the site of primary regulation by triptolide, we cannot rule out an additional or indirect effect on Ser5 phosphorylation as well.

If we examine the timeline of data stemming from these experiments, a model emerges where triptolide (100 nmol/L) has an effect on RNA Pol II efficiency in just 1 h as assessed by transcript levels of the S14 ribosomal subunit. After 2 h, nuclear speckles become rounded, Pol II transcriptional efficiency continues to decline, and RNA Pol II Ser2 phosphorylation decreases. Following 3 h, the nucleolus begins to unravel, and RNA Pol I activity now begins to decrease. It is of note that, at this time, these effects are still reversible and cells may fully recover provided that triptolide is removed from culture. However, after 6 h of continual culture, RNA Pol I and II activities are absent and nucleolar organization has dissolved, which may therefore be the turning point toward the commitment to cell death. We should also note that this timeline of cellular effects may be shifted by triptolide concentration. For example, our data with 25 nmol/L triptolide (previously shown to induce cell death in HeLa) do not show any effect on transcription, nucleolar disassembly, or speckle rounding at the 6-h time point. However, after 16 h in culture, 25 nmol/L causes prominent enlargement and rounding of speckles, ultimately leading to cell death.

We additionally generated a triptolide-resistant clonal population of HeLa (Clone-2) to test if these nuclear changes were specific to both transcription and cell death. Although Clone-2 cells exhibited a very minor fluctuation in nucleolar integrity and transcriptional inhibition, cells were able to escape from death and dissolution of the nucleolus during an extended incubation with 100 nmol/L triptolide. The cells from this clonal line retain the majority of their transcriptional activity and do not undergo nuclear speckle rounding or inhibition of Ser2 phosphorylation. We have yet to discover why these cells are relatively resistant to triptolide-induced nucleolar disruption or RNA Pol II inactivation, but we are currently investigating these questions as we further dissect the mechanism of action of triptolide.

Taken together, we conclude that NF-{kappa}B transcriptional inhibition in HeLa cells correlates with global transcriptional and structural changes in the cell. Because the RelA subunit of NF-{kappa}B has been shown to recruit P-TEFb to stimulate RNA Pol II transcriptional elongation in response to cytokine stimulation (45), it is possible that this explains why the NF-{kappa}B pathway is exquisitely sensitive to triptolide regulation in multiple cell lines. In fact, treatment of cells with the P-TEFb inhibitor DRB sensitizes cells to TNF-{alpha}–induced apoptosis, similar to the effect observed with triptolide. Although we found no obvious changes in Cdk9/cyclin T1 interaction and no changes in protein levels or localization in Cdk9, it is likely that other mechanisms of regulation exist, such as cofactor recruitment to the RNA Pol II transcriptional complex, acetylation events, or regulation of Cdk9 binding to HEXIM1 and 7SK small nuclear RNA (46, 47).

Triptolide is a small molecule with multiple physiologic effects, such as immune modulation and chemotherapeutic. Its biological effects on cell growth and transcription vary by concentration, duration of exposure, and cell type. Here, we have shown that triptolide induces nuclear substructural changes in both the nucleolus and nuclear speckles that are associated with a rapid decrease in RNA Pol II transcriptional efficiency. Use of these nuclear substructures as surrogate markers for transcriptional arrest and possibly cytotoxicity will be of great utility in delineating the separate biological functions of triptolide and its derivatives. As the mode of action of triptolide continues to unfold, it may eventually be possible to understand its multiple regulatory actions on the cell as well as its therapeutic potentials.


   Disclosure of Potential Conflicts of Interest
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
No potential conflicts of interest were disclosed.


   Acknowledgments
 
Grant support: NIH grant AI055914 and a Postdoctoral Fellowship from the American Cancer Society (S.J. Leuenroth).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 11/12/07. Revised 3/14/08. Accepted 4/25/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

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