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. 2008 Jan 25;283(4):2353-62.
doi: 10.1074/jbc.M705986200. Epub 2007 Nov 26.

RhoBTB2 (DBC2) is a mitotic E2F1 _target gene with a novel role in apoptosis

Scott N Freeman  1 , Yihong Ma, W Douglas Cress
Affiliations

RhoBTB2 (DBC2) is a mitotic E2F1 _target gene with a novel role in apoptosis

Scott N Freeman et al. J Biol Chem. .

Abstract

We have identified the RhoBTB2 putative tumor suppressor gene as a direct _target of the E2F1 transcription factor. Overexpression of E2F1 led to up-regulation of RhoBTB2 at the level of mRNA and protein. This also occurred during the induction of E2F1 activity in the presence of cycloheximide, thus indicating that RhoBTB2 is a direct _target. RNAi-mediated knockdown of E2F1 resulted in decreased RhoBTB2 protein expression, demonstrating that RhoBTB2 is a physiological _target of E2F1. Because E2F1 primarily serves to transcribe genes involved in cell cycle progression and apoptosis, we explored whether RhoBTB2 played roles in either of these processes. We found RhoBTB2 expression highly up-regulated during mitosis, which was partially dependent on the presence of E2F1. Furthermore, overexpression of RhoBTB2 induced a short term increase in cell cycle progression and proliferation, while long term expression had a negative effect on these processes. We similarly found RhoBTB2 up-regulated during drug-induced apoptosis, with this being primarily dependent on E2F1. Finally, we observed that knockdown of RhoBTB2 levels via siRNA delayed the onset of drug-induced apoptosis. Collectively, we describe RhoBTB2 as a novel direct _target of E2F1 with roles in cell cycle and apoptosis.

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Figures

Fig. 1
Fig. 1. E2F1 overexpression upregulates RhoBTB2
A: H1299s treated with equal amounts of Ad-GFP, Ad-E2F1 or Ad-E2F1(1–283) adenovirus with subsequent real-time PCR analysis of RhoBTB2/18S at 24- and 48-hour time points B: MCF7s or T98Gs were treated with either Ad-GFP or Ad-E2F1 with subsequent real-time PCR analysis for RhoBTB2/18S at 24- and 48 hour time points. C: Immunofluorescent microscopy (IFM) for RhoBTB2 in either non-trasfected H1299s at 40x (top), H1299s transfected with a control siRNA at 63x (middle) or transfected with siRNA specific to RhoBTB2 at 63x (bottom)—DAPI: blue; RhoBTB2: red. Quantification of RhoBTB2 signal intensity per area of whole-field images is provided on the right. D: A western blot for RhoBTB2 in either non-transfected, transfected with p3XFlag-RhoBTB2-myc or co-transfected with p3XFlag-RhoBTB2-myc and increasing amounts of a shRNA _targeted toward RhoBTB2 (shRhoBTB2) in H1299s. E. IFM at 63x of two different fields of H1299s transiently cotransfected with pcDNA3-HA-E2F1 and pBB14, a membrane GFP plasmid—DAPI: blue; GFP (transfected cells): green; RhoBTB2: red. Quantification of RhoBTB2 signal intensity per area of whole-field images is provided on the right.
Fig. 2
Fig. 2. RhoBTB2 expression is directly and physiologically regulated by E2F1
A: Real-time PCR analysis of RhoBTB2/18S in the H1299-ER-E2F1 cell line treated with CHX, 4-OHT or both at 8- and 24-hour time points. B: A western blot for E2F1 in the H1299-pBS/U6 and H1299-shE2F1 cell lines demonstrating efficient knockdown of E2F1 (top). IFM for RhoBTB2 with our rabbit polyclonal antibody conducted on the H1299-pBS/U6 and H1299-shE2F1 cell lines—DAPI: blue; RhoBTB2: red at 63x (bottom). Quantification of RhoBTB2 signal intensity per area of whole-field images is provided on the right.
Fig. 3
Fig. 3. RhoBTB2 is upregulated during mitosis, which is partially dependent on E2F1
A: IFM for RhoBTB2 at 63x of cells in interphase, prophase, metaphase, anaphase and telophase/cytokinesis on an asynchronously growing population of H1299s (top) and MCF10As (bottom). B. Quantification of RhoBTB2 signal intensity per area of images provided in 3A. C. Western blot for RhoBTB2 in H1299s released from a double thymidine block, with accompanying DNA content analysis for cell cycle status as described in experimental procedures. D. IFM as in 3A, but in the H1299-pBS/U6 and H1299-shE2F1 cell lines. E. Quantification of RhoBTB2 signal intensity per area of images is provided in 3D.
Fig. 4
Fig. 4. Overexpression of RhoBTB2 both positively and negatively influences cell cycle progression and proliferation
A: A western blot for RhoBTB2 and actin upon infection with equal amounts of Ad-GFP or Ad-RhoBTB2 (left). DNA content and cell cycle status derivations of H1299s infected in triplicate with equal amounts of either the Ad-GFP or Ad-RhoBTB2 adenovirus via propidium iodide staining and flow cytometry. B: BrdU incorporation assay of H1299s infected in triplicate with equal amounts of either the Ad-GFP or Ad-RhoBTB2 adenovirus at 48 hours post infection. C. An MTS assay of the fold increase in MTS signal intensity over a 96-hour time course of triplicate samples of H1299s infected with either Ad-GFP or Ad-RhoBTB2. D. A BrdU incorporation assay as in 4B, but over a 96-hour time course. Data displayed reflects BrdU signal intensity percentage of Ad-RhoBTB2 infected cells to Ad-GFP infected cells.
Fig. 5
Fig. 5. RhoBTB2 is upregulated during drug induced apoptosis, which is primarily dependent on E2F1
A: IFM at 63x for RhoBTB2 in H1299s after 24 hours of treatment of either no treatment control, 20 uM cisplatin, 200 nM flavopiridol, or 20 uM etoposide. Quantification of RhoBTB2 signal intensity per area of images is provided on the right. B: IFM as in 5A, but with the H1299-pBS/U6 and H1299-shE2F1 cell lines --DAPI: blue; RhoBTB2: red (left). A western blot for E2F1 expression in H1299-pBS/U6 and H1299-shE2F1 cell lines for the drug treatments utilized in the left panel (right). C. Quantification of RhoBTB2 signal intensity per area of whole-field images of the treatments presented in 5B.
Fig. 6
Fig. 6. siRNA-mediated knockdown of RhoBTB2 impairs the induction of drug-induced apoptosis
A-C: H1299s were transiently transfected with either a negative control siRNA, or siRNA against RhoBTB2, detached at 24 post-transfection, counted, and transferred to 96 well plates where an MTS assay was performed to analyze cell viability after 24, 48 and 72 hours. The cytotoxic drugs used were 20 uM cisplatin (A), 200 nM flavopiridol (B) and 20 uM etoposide (C). D-E: Western blots for cleaved PARP and actin control after 0, 16, 24, 32, 40 and 48 hours of treatment with either 20 uM cisplatinum (D), 200 nM flavopiridol (E) and 20 uM etoposide (F) in H1299s transfected with either a negative control siRNA (siControl) or siRNA against RhoBTB2 (siRhoBTB2).
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