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G1 Cell Cycle Arrest and Apoptosis Induction by Nuclear Smad4

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G1 Cell Cycle Arrest and Apoptosis Induction by Nuclear Smad4 Proc. Natl. Acad. Sci. USA Vol. 96, pp. 1427–1432, February 1999 Cell Biology G1 cell cycle arrest and apoptosis induction by nuclear Smad4yDpc4: Phenotypes reversed by a tumorigenic mutation (pancreatic cancerytype b transforming growth factorysignaling pathwayyDNA-binding proteinynuclear translocation) JIA LE DAI,RAVI K. BANSAL, AND SCOTT E. KERN† Departments of Oncology and Pathology, The Johns Hopkins Medical Institutions, Baltimore, MD 21205 Communicated by Bert Vogelstein, Johns Hopkins Oncology Center, Baltimore, MD, December 17, 1998 (received for review December 3, 1998) ABSTRACT The tumor suppressor Smad4yDpc4 is a Tumor-derived SMAD4 mutations consistently abrogate transcription activator that binds specific DNA sequences and Smad4-inducible gene activation by at least three mechanisms: whose nuclear localization is induced after exposure to type b missense mutations in the MH1 domain abolish binding to the transforming growth factor-like cytokines. We explored an SBE, missense mutations in the MH2 domain prevent Smad4 inducible system in which Smad4 protein is activated by nuclear translocation, and truncation mutations in the MH2 translocation to the nucleus when cell lines that stably express domain ablate the transactivation ability (22). The universally wild-type or mutant Smad4 proteins fused to a murine null function of mutant Smad4 in SBE-mediated transcription estrogen receptor domain are treated with 4-hydroxytamox- activation strongly indicates that Smad4-inducible gene acti- ifen. This induced Smad4-mediated transcriptional activation vation is the underlying mechanism for its tumor-suppressor and a decrease in growth rate, attributable to a cell cycle functions, although direct genetic targets for these Smad4 arrest at the G1 phase and an induction of apoptosis. A functions remain unknown. The phenotypic changes induced tumor-derived mutation (Arg-100 3 Thr) affecting a residue by Smad4 are also largely undefined. Although some of these critical for DNA-binding demonstrated an ‘‘oncogenic’’ phe- questions have been addressed by murine knock-out studies, notype, having decreases in both the G1 fraction and apoptosis the SMAD4-null mouse is early embryonic lethal (23, 24). The and, consequently, an augmentation of population growth. replacement of the SMAD4 gene into SMAD4-null cells rep- This model should be useful in the exploration and control of resents another attractive approach. Most studies of this type components that lie further downstream in the Smad4 tumor- have used transient protein overexpression that precludes a suppressor pathway. systematic examination of the phenotypic changes mediated by Smad4. In the few studies of stable integration of the wild-type SMAD4yDPC4yMADH4 belongs to a family of human SMAD SMAD4 gene, constitutive effects appear to be generated, genes (reviewed in ref. 1). The structural homologies are perhaps initiated by an autocrine or paracrine loop (25, 26). As clustered at the N-terminal Mad homology 1 (MH1) domain a result, only few clones survive, and they maintain a much and the C-terminal MH2 protein domain. A significant struc- slower growth rate compared with control cells having inte- tural feature that distinguishes Smad4 from other Smads is the grated a mutant SMAD4 gene (J.L.D. and S.E.K., unpublished lack in Smad4 of the SSXS motif at the tail of the MH2 domain data). terminal that can be phosphorylated by the cognate receptor It appeared that some of these technical limitations might be kinases (2–6). Based on biochemical studies, Smad4 is thought overcome with a system that sequesters Smad4 protein in the to act as a common Smad protein that couples the pathway- cytoplasm. Therefore, we applied an established modified restricted Smads, those activated by TGF-b-like ligands, with mouse estrogen receptor (MER) model (27) that has been downstream transcriptional events (2, 7). Phosphorylation of successfully used in the functional studies of nuclear factors the pathway-restricted Smads is accompanied by their hete- such as c-Myc (28), c-Raf-1 (29), and p53 (30) proteins. We rooligomerization with Smad4, which accompanies their trans- previously used this system to control ligand-initiated signaling port into the nucleus (4, 7–11). This nuclear translocation of as detected by reporter gene activation and have proposed the Smad4 is correlated with its activation of gene transcription. In direct control of Smad4 nuclear localization as a potential the nucleus, Smad4 binds specific short sequences of DNA therapeutic strategy (22). Here we present our evaluation of [Smad-binding elements (SBE)] and functions as a transcrip- some general biological effects with this model, our finding tion activator (9, 12–14). that Smad4 regulates both cell cycle progression and apoptosis, Although the biochemical properties of Smad proteins have and related properties of a Smad4 mutant. been intensely studied, the tumor-suppressor pathway of the SMAD4 gene remains unclear. The SMAD4 gene is inactivated MATERIALS AND METHODS frequently in pancreatic (15, 16), biliary (15, 17), and colorec- tal (15, 18, 19) tumors and less frequently in other tumor types Cell Culture and Generation of Stably Transfected Clones. (16). The TGF-b pathways are in part mediated by Smad4 (7, Constructs used in this study have been reported (22). Wild- 20), but some human colorectal and pancreatic cancers suffer type and mutant SMAD4-coding sequences fused 39 to MER inactivation of both the SMAD4 and TGF-b receptor genes ligand-binding domain (27) were constructed in pcDNA3.1 (ref. 21; L. Meyeroff, W. Grandy, S. Thiagalingam, B. Vo- (Smad4-MER or 100T-Smad4-MER, respectively; pcDNA3.1 gelstein, and S. Markowitz, personal communication). These from Invitrogen). The SMAD4-null breast cancer cell line findings specifically raise interest in the evaluation of suppres- MDA-MB-468 (ATCC) was transfected with the above plas- sive roles for Smad4 that may be independent of TGF-b signaling. Abbreviations: 4-OHT, 4-hydroxytamoxifen; TGF-b, type b trans- forming growth factor; SBE, Smad-binding element; MH1 and MH2, The publication costs of this article were defrayed in part by page charge Mad-homology domains 1 and 2; TUNEL, terminal deoxynucleoti- dyltransferase-mediated dUTP nick end labeling. payment. This article must therefore be hereby marked ‘‘advertisement’’ in †To whom reprint requests should be addressed at: 632 Ross Building, accordance with 18 U.S.C. §1734 solely to indicate this fact. The Johns Hopkins University School of Medicine, Baltimore, MD PNAS is available online at www.pnas.org. 21205-2196. e-mail: [email protected]. 1427 Downloaded by guest on September 26, 2021 1428 Cell Biology: Dai et al. Proc. Natl. Acad. Sci. USA 96 (1999) mids and FuGENE6 reagent (Boehringer Mannheim) and to the untreated group (fold changes). A trapezoidal window m y y selected with G418 (350 g ml, active concentrations, from was used to reflect the higher apoptotic labeling in G2 M Life Technologies, Gaithersburg, MD). G418-resistent clones phase cells having a higher DNA content. were screened for protein expression by immunoblot analysis Gene Expression Analysis. Clones stably expressing Smad4– (see below). MER fusion proteins were screened by immunoblot. Total Reporter Assays. Stable cell lines were plated on six-well cellular protein was harvested in Laemmli buffer (without cluster dishes and transfected with 0.5 mg of 6SBE-Luc (22), b-mercaptoethanol), and protein concentrations were deter- which contains Smad4 consensus palindromic binding sites, mined with bicinchoninic acid reagents (Pierce). Total protein 6MBE-Luc, which contains the mutant form of SBE, or a (20 mg) were separated by SDSyPAGE (10% gel) and detected TGF-b-responsive reporter, 3TP-lux (31). In all experiments, by polyclonal antiserum against the estrogen receptor ligand- 0.5 mgofb-galactosidase expression vector pCMVb (CLON- binding domain (working dilution 1:200, Santa Cruz Biotech- TECH) was cotransfected for normalization of transfection nology). efficiency. Transfected cells were then treated with 100 nM Immunodetection of hyper- or hypophosphorylated Rb pro- 4-hydroxytamoxifen (4-OHT, Sigma), 1 ngyml TGF-b1 (R&D tein was performed with the monoclonal antibody recognizing Systems), or vehicle. Reporter assays were performed as both forms of human Rb (clone G3–245, from PharMingen). described (22). Briefly, 20 h after ligand treatment, cells were Cells were trypsinized and disrupted in lysis buffer [50 mM harvested for b-galactosidase and luciferase reporter assays, TriszHCl, pH 7.5y250 mM NaCly1% Nonidet P-40y1mM according to the manufacturer’s instructions (Promega). Lu- EDTAy1 mM phenylmethanesulfonyl fluoridey13 protease ciferase activities were normalized with b-galactosidase. b-Ga- inhibitor mixture (Boehringer Mannheim)]. After 10 min of lactosidase activities remained stable among all transfection incubation, the lysates were centrifuged and the supernatants experiments. collected. Protein extracts (25 mg total) were separated by Cell Counting and Cell Cycle Analysis. Cells stably express- SDSyPAGE (7% gel) and detected by the Rb antibody (2 ing MER fusion proteins were plated in six-well cluster dishes mgyml). Blots were incubated with a horseradish peroxidase- at a density of 2 3 105 cells per plate. After the cells had conjugated secondary antibody (Pierce). Detection was af- incubated overnight, media were replaced with 10% fetal forded by SuperSignal substrates (Pierce). bovine serum in the presence of 100 nM 4-OHT or vehicle.
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