Triptolide-induced transcriptional arrest is associated with changes in nuclear substructure

Abstract

Triptolide, an active component of the medicinal herb lei gong teng, is a potent anticancer and anti-inflammatory therapeutic. It potently inhibits nuclear factor-kappaB 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 Ser(2) 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.

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: A) Increasing concentrations of triptolide for 6 hours B) 100 nM triptolide over a 6-hour time course C) Cells were pre-treated with 100 nM triptolide for the indicated times, washed, and allowed to recover for 16 hours. All images were acquired under 25X magnification and scale bar is 10 µm.

Figure 2

Triptolide Induces Nuclear Speckle Rounding. A) HeLa cells were incubated with 100 nM triptolide (T) or DMSO (D) over a 6-hour 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 nM triptolide for either 6- or 16- hours of continual culture before assessing SC35 localization. All images were acquired by confocal microscopy under 63X magnification and scale bars are 10 µm.

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 nM triptolide or 10 nM actinomycin D (Act D) for 6 hours. Arrow indicates nucleolus, DIC images were acquired by confocal microscopy using 63X magnification, and immunofluorescence images were acquired under 25X magnification. B) Transcriptional activity was assessed using an NFκB-luciferase construct. HeLa or Clone-2 cells were incubated with vehicle only (control), TNF-α, or with TNF-α plus 100 nM triptolide for a total of 6 hours. C) HeLa and Clone-2 cells were assessed for survival following 72 hours of 100 nM triptolide incubation. D) Cells were incubated with DMSO or 100 nM triptolide for 16 hours before fixation and indirect immunofluorescent detection of nucleolin or SC35. Images were acquired by confocal microscopy under 63X magnification. All scale bars represent 10 µm.

Figure 4

Nuclear Changes are Independent of Calcium, Stress Kinases, and Caspase Activation. A) HeLa cells were incubated for 6 hours with DMSO, 100 nM triptolide, 5 µM calcimycin, or 3 µM ionomycin before fixation and nucleolin detection by indirect immunofluorescence (25X magnification). Scale bar is 10 µm. B) HeLa cells were incubated in calcium free media for 16 hours before addition of DMSO or 100 nM triptolide for 6 hours. Cells were stained for SC35 (red) or nucleolin (green) by indirect immunofluorescence (confocal microscopy, 40X). C) HeLa cells were transiently transfected with GFP vector, nuclear excluded (NES) or nuclear localized (NLS) parvalbumin (PV) – GFP constructs for 24 hours before a 6-hour incubation with DMSO or 100 nM triptolide. GFP localization is in green, nucleolin is detected by indirect immunofluorescence (red). Images acquired separately under 40X magnification and artificially merged. D) HeLa cells were treated with DMSO, 5 µM SB203580, 10 µM SP600125, or 2 µM zVAD-fmk ± 100 nM triptolide for 6 hours. Nucleolar integrity was detected by indirect immunofluorescence of nucleolin (green). Graph represents caspase-3 activity in HeLa cells treated with DMSO or 100 nM triptolide over an 18-hour time course. Cell lysates were prepared and activity assessed by cleavage of the colorimetric substrate, DEVD-pNA. Data represent Mean±SE, n=3.

Figure 5

Nucleolar Disassembly is Characterized by Nucleolin, Nucleophosmin, and UBF Localization. A) HeLa cells were incubated with DMSO or 100 nM triptolide for 6 hours before fixation and immunofluorescent detection of nucleolin, nucleophosmin (NPM), upstream binding factor (UBF), or SC35. Left panel images were acquired under 25X magnification, right panel images were acquired by confocal microscopy at 63X. B) Nuclear cell lysates were prepared from cells treated with 100 nM triptolide over an 8-hour time course and separated by SDS-PAGE. Shown are representative western blots of nucleolin, NPM, and UBF protein levels. Molecular weight markers in kD are represented on the left. C) Following a 6-hour time course with 100 nM 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 nM triptolide for six hours and cells were co-stained with p-NPM (green) and SC35 (red). Representative images were acquired in separate channels and artificially merged. All scale bars represent 10 µm.

Figure 6

Triptolide Inhibits RNA Pol II Activity. A) HeLa cells were treated with 100 nM triptolide over a 6-hour time course or increasing triptolide concentrations for 6 hours before nuclear lysates were prepeared. RNA Pol II phosphorylation at Ser2 or Ser5 of the CTD was assessed by western blot analysis (molecular weight markers in kD are indicated to the left of each panel). Immunofluorescence localization of RNA Pol II Ser2 or Ser5 phosphorylation in the presence or absence (− Ca²⁺) of calcium containing media was also completed. Results are representative of four separate experiments. Scale bar is 10 µm. B) Clone-2 cells were treated with 100 nM triptolide for 6 hours 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 nM triptolide over a 6-hour 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 Pol II activity. The ribosomal protein S14 transcript is transcribed by RNA Pol II and the 5’ ETS of 45S pre-ribosomal RNA is transcribed by RNA Pol I. Results shown are representative of three separate experiments. D) HeLa cells were incubated in the presence of 100 nM triptolide during a 6-hour time course. Total Cyclin T1 and Cdk9 protein levels (left panels), Cdk9/ Cyclin T1 complex interaction by Cdk9 IP (center panels), and Cdk9 immunolocalization (right panels) were assessed. A/G represents IP with Protein A/G agarose beads and DIC inset shows nucleolar integrity. Results are representative of three separate experiments.