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. 1999 Mar 30;96(7):3993-8.
doi: 10.1073/pnas.96.7.3993.

V体育ios版 - Induction of ARF tumor suppressor gene expression and cell cycle arrest by transcription factor DMP1

K Inoue  1 , M F Roussel, C J Sherr
Affiliations
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Induction of ARF tumor suppressor gene expression and cell cycle arrest by transcription factor DMP1

K Inoue (V体育安卓版) et al. Proc Natl Acad Sci U S A. .
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Abstract

Expression of the DMP1 transcription factor, a cyclin D-binding Myb-like protein, induces growth arrest in mouse embryo fibroblast strains but is devoid of antiproliferative activity in primary diploid fibroblasts that lack the ARF tumor suppressor gene. DMP1 binds to a single canonical recognition site in the ARF promoter to activate gene expression, and in turn, p19(ARF) synthesis causes p53-dependent cell cycle arrest. Unlike genes such as Myc, adenovirus E1A, and E2F-1, which, when overexpressed, activate the ARF-p53 pathway and trigger apoptosis, DMP1, like ARF itself, does not induce programmed cell death. Therefore, apart from its recently recognized role in protecting cells from potentially oncogenic signals, ARF can be induced in response to antiproliferative stimuli that do not obligatorily lead to apoptosis. VSports手机版.

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Figures

Figure 1
Figure 1
Mouse ARF promoter. The nucleotide sequence of 300 bp 5′ to the transcriptional start site (+1) is shown. Putative binding sites for DMP1/ETS (boldface), Sp1, and E2F-1 (both underlined) are indicated; (+) indicates sense and (−) the antisense strand. The translational initiation codon (ATG, boldface) is at +59. The BamHI site (italics) at −225 used to construct a promoter-reporter expression plasmid is indicated by the right arrow.
Figure 2
Figure 2
DMP1 binds and transactivates the ARF promoter. (A) Electrophoretic mobility-shift assays were performed with a radiolabeled 281-bp ARF promoter fragment (bracketed by arrows in Fig. 1) using recombinant DMP1 made in insect Sf9 cells. Lane 1 shows results with uninfected Sf9 lysates and lane 2 with extracts of cells expressing DMP1. Competition was performed with an Ets-specific (M3, lane 3), DMP1-specific (BS2, lane 4) or a cognate ARF promoter consensus oligonucleotide (lane 5). Nonimmune rabbit serum (lane 6) or different antibodies to DMP1 (lanes 7–9) were added before probe. (B) Electrophoretic mobility-shift assays were performed with a recombinant Ets1 protein (lanes 2–6) or its DNA-binding domain (DBD, lane 1). Competition was performed using the ARF promoter DMP1 consensus site (lane 3). Antibodies to Ets1 (lane 5) or DMP1 (lane 6) were added before probe. (C) Transactivation of the ARF promoter-reporter in NIH 3T3 cells was performed by cotransfection with vectors encoding DMP1, a DMP1 mutant (M11) that cannot bind to DNA, or the indicated Ets family members (abscissa). Plasmid inputs (μg DNA) are indicated at the left. Activation (ordinate) is normalized to SEAP expression.
Figure 3
Figure 3
DMP1 induces p19ARF and cell cycle arrest in wild-type but not ARF-null MEFs. (A) Infection of wild-type MEF strains with a DMP1 virus (lanes 2 and 3) or a Myc virus (lanes 4 and 5) induces p19ARF protein. Amounts of protein loaded in lanes 3 and 5 were 40% of those in lanes 2 and 4. All viruses expressed the T cell coreceptor CD8; whereas 95% of Myc-infected cells were CD8 positive (lane 4), only 35% of cells infected with DMP1 virus expressed the CD8 marker (lane 2). NIH 3T3 cells (lane 6) have sustained ARF deletions, whereas 10-1 cells (lane 7) lack p53 and overexpress p19ARF through loss of feedback control. (B) Wild-type (■) or ARF-null (░⃞) MEFs infected for 36 hr with the indicated viruses (abscissa) were labeled for 14 hr with BrdUrd and scored for protein expression and BrdUrd incorporation as in Fig. 4. (C) NIH 3T3 cells were cotransfected with the ARF promoter-reporter plasmid together with vectors encoding E2F-1 or both E2F-1 and DMP1. Input plasmid DNAs (μg) are noted on the abscissa and activation was normalized to coexpressed SEAP (ordinate). (D) Cells infected as in B were deprived of serum for 24 hr and then scored for viability by trypan blue exclusion. Viability was confirmed using fluorescence-activated cell sorter/terminal deoxynucleotidyltransferase-dUTP end labeling assay and by scoring representative aliquots for subdiploid DNA content.
Figure 4
Figure 4
DMP1-induced arrest depends on ARF. Wild-type or ARF-null MEFs (Right) were infected for 36 hr with the different expression vectors (Left) and scored for vector-induced protein expression (red fluorescence, Left), BrdUrd incorporation (green fluorescence, Center), and mixed fluorescence (yellow, Right). Wild-type DMP1-overexpressing cells failed to incorporate BrdUrd (a–c), whereas ARF-null cells entered S phase (d–f). ARF arrests both MEF cell types (g–l) and Myc arrests neither (m–r).
Figure 5
Figure 5
Conditional ARF induction and growth arrest of wild-type MEFs by DMP1-ER. (A) NIH 3T3 cells treated with 4-HT for the indicated times (hr, abscissa), were scored for activation (normalized to SEAP) of a cotransfected ARF-promoter-reporter plasmid. (B) Wild-type (■, ●) or ARF-null (○, □) MEFs expressing DMP1-ER were left untreated (○, ●) or were treated with 4-HT (□, ■) for the indicated times (abscissa). Cells were pulsed with BrdUrd for 3 hr before analysis. (C) ARF and actin mRNA were quantitated by reverse transcription–PCR in lysates of wild-type cells treated with 4-HT as in B or with 4-HT plus the protein synthesis inhibitor cycloheximide (CHX). (D) ARF, p53, p21Cip1, and actin protein levels were determined by immunoblotting in lysates of wild-type (Left) and ARF-null (Right) MEFs treated with 4-HT as in B.
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