Cell-cycle dependent biosynthesis and localization of p53 protein in untransformed human cells

Biol Cell (1995) 84, 167-173

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© Elsevier, Paris

Original article

Cell-cycle dependent biosynthesis and localization of p53 protein in untransformed human cells Zetsuo Katsumoto a, Katsumi Higaki b, Kousaku Ohno b, Kazukiyo Onodera c a Laboratory of Electron Microscopy; b Department of Neurobiology, Faculty of Medicine, Tottori University, 86 Nishimachi, Yonago 683," c Division of Agriculture and Agricultural Life Science, The University of Tokyo, 1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan (Received 9 June 1995; accepted 19 June 1995)

Summary - Localization of p53 in human cultured lymphocytes and in cultured skin fibroblasts was studied by immuno-fluorescent microscopy and post-embedded immunoelectron microscopy using Lowicryl K4M. In quiescent lymphocytes, p53 was found in small amounts in both the cytoplasm and the nucleus, p53 in the nucleus was found associated with the non-chromatin structure. At 24 h or 72 h of PHA stimulation, p53 increased markedly just beneath the plasma membrane and in the nucleus, which stained diffusely with anti-p53. In resting fibroblasts, small amounts of p53 were present in both the cytoplasm and the nucleus. After 16 h of stimulation of confluent-resting fibroblasts by trypsinization and replating, a phase just prior to the initiation of DNA synthesis, p53 slightly increased in both the cytoplasm and the nucleus. Afterwards, p53 was present predominantly in the cytoplasm, closely associated with the cytoskeletal actin filaments. In mitotic cells, p53 was distributed throughout the cytoplasm. When fibroblasts were extracted with saponin, p53 was still associated with the actin filaments, as well as mitochondrial membranes and granular structures of the nuclear matrix. Our data suggest that the initial increase of p53 in cells that enter the cell cycle through G1 first bind to the actin cytoskeleton, and that some of the p53 then move into the nucleus to initiate gene activation and DNA synthesis for cell proliferation. This implies that there is some functionally significant interaction between p53 and actin in the cells. p53 / actin filaments / immunocytochemistry / electron microscopy / cell proliferation

Introduction

Inactivation of the p53 tumor suppresser is a common occurrence in the development of various types of human cancers [4]. Wild type p53 has been shown to inhibit neoplastic transformation [9]. It can block transformation by activated oncogenes in cells in vitro and can prevent tumor formation in animal models. It has been shown that p53 induces cell cycle arrest in response to DNA damage. Mutations in p53 eliminate this response and result in an enhanced frequency of genomic rearrangements [5]. Moreover, several viral proteins such as adenovirus, simian vires 40, human papilloma virus, and Epstein-Barr virus were shown to bind to p53 and resulted in inactivation and stabilization [8, 25-27]. Recently a protein of cytomegalovirus (CMV) was added to this collection [24]. Biochemical and structural analyses of p53 have been carried out and its characteristics have become clear at the molecular level [3]. The p53 protein is composed of four domains: an NH2-terminal transactivation domain, a central DNA binding domain, an oligomerization domain, and a basic COOH-terminal nuclear localization domain. It is thought, therefore, that not only DNA-protein but protein-protein interaction is important for the function of p53. However, information about the subcellular localization of p53 in cells during the cell cycle is scant. Human fibroblasts or mouse 3T3 cells made quiescent by serum starvation or human blood mononuclear cells have very low levels of p53 mRNA, but, when these cells are stimulated with fresh serum or mitogen, an increase of p53 mRNA precedes the synthesis of DNA, and the highest lev-

els occur at the transition from GI(G0) to S phase [2, 19]. When quiescent Balb/c 3T3 cells are stimulated with serum, p53 initially appears in the cytoplasm, then accumulates in the nucleus before the beginning of DNA synthesis, and thereafter p53 is no longer found in the nuclear compartment, but rather accumulates in the cytoplasm [22]. This evidence suggests that the subcellular localization, in addition to the increased synthesis, of the p53 protein, is essential for the functional role of p53 on cell proliferation. In this paper, we report the synthesis of p53 and the subcellular localization of p53 preceding and during the DNA synthesis of PHA-stimulated human peripheral mononuclear cells and confluent-resting human skin fibroblasts stimulated by trypsinization and replating in fresh serum, as model systems of GI(G0)-S transition in normal cells, by an immunofluorescence method and by an ultrastructural post-embedding immunogold staining method using Lowicryl K4M resin.

Materials and m e t h o d s

Cells and cell culture Peripheral blood mononuclear (PBM) cells were purified from the heparinized whole blood of a healthy donor and cultured at a cell density of 1 x 106 per ml in Dulbecco's modified Eagle's medium (DMEM), supplemented with 15% fetal calf serum (FCS), 0.3% phytohemagglutinin (PHA-M) (Difco Laboratories) and antibiotics (penicillin G and streptomycin sulfate ) at 37°C in a humidified CO 2 incubator. Primary culture of human skin fibroblast, obtained from the forearm skin biopsy of a 14-year-

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old patient with Lesch-Nyhan syndrome, was maintained as described previously [15] in DMEM with 10% FCS and antibiotics. To make confluent-resting culture, cells were inoculated at a density of 1 x 105 per 60 mm dish and were cultured toward confluence without medium renewals. Three days after attainment of stationary cell density, cells were trypsinized and replated in fresh DMEM with 10% FCS at a density of 1 x 105 cells in 35 mm plastic dishes with or without a glass coverslip or a plastic sheet. At the same time [3H]thymidine (1/l Ci/ml, NEN) was added to each dish. At intervals, cells were lysed in 10% SDS, and TCA insoluble macuiomolecules in each dish were trapped onto a glass filter (Whatman GF/C). Radioactivites of each filter were counted in scintillation solution (ACS II). Indirect immunofluorescence The cultured PBM cells were washed with phosphate-buffered saline (PBS) and fixed with 3.7% formaldehyde in PBS for 15 min at room temperature and washed with PBS. These cells were placed on slide-glass coated with poly-L-iysine, and then extracted with cold (-20°C) acetone for 5 min and air dried. The fibroblasts cultured on coverglass were washed briefly with PBS and then fixed and extracted in the same manner as the lymphocytes. The extracted PBM cells and skin fibroblasts were then stained at room temperature with mouse monoclonal antip53(Ab-2) (diluted 1:1 in PBS) (Oncogene Science), ((Ab-2) reacts preferentially with p53 of human cellular origin and with an epitope located near the amino end of all known forms of p53), and goat anti-mouse antibodies coupled to fluorescein (Zymed Laboratories Inc) for 1 h each. The cells were then observed by an Olympus BH-2 microscope. Immunoelectron microscopy For immunoelectron microscopy, the cultured PBM cells were washed with PBS, fixed with 2% paraformaldehyde and 0.2% glutaraldehyde in PBS for 30 rain at room temperature, and then washed again with PBS. These cells were placed on a poly-Llysine coated plastic-sheet (Wako Pure Chemical Industries Ltd). Skin fibroblasts cultured on the plastic-sheet were fixed as described above. In some of the experiments, primary cultures were extracted with saponin (50/,tg/ml) in PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA and 2 mM MgCI 2, pH 6.9) [21] for 5 min at room temperature prior to aldehyde fixation. After cells on plastic sheets were dehydrated with ethanol, the sheet was cut into small pieces and embedded in Lowicryl K4M, which was then polymerized at -30°C by UV irradiation. Immunogold staining was done as reported previously [7]. Protein A-gold, 15 nm in diameter (EY Laboratories), was used as the secondary labeling. For the control experiment, treatment with antibodies prior to protein A-gold was omitted.

Results

Indirect immunofluorescence Judging by morphological criteria, the majority of enriched PBM cells were found to be lymphocytes. Imrnunofluorescent staining of P B M cells with p53 monoclonal antibody showed a weak stain in the unstimulated resting lymphocytes. On both the next and 3rd day after stimulation with PHA, the cell size became larger and the intensity of immunofluorescence b e c a m e brighter (fig 1). In fibroblast cultures stimulated at a confluent-resting state, incorporation of [3H]-thymidine into a T C A insoluble fraction started 16 h after replating them in fresh m e d i u m , and e x p o n e n t i a l l y increased until 24 h, showing that the S-phase under these c o n d i t i o n s o c c u r s 16-24 h after s t i m u l a t i o n . In cells at G I ( G 0 ) phase, the i m m u n o f l u o r e s c e n t staining with p53 monoclonal antibody revealed in the nucleus and the cytoplasm was weak (fig 2a); in cells at early S phase (at 16 h after stimulation) the fluorescence mildly increased in both the nucleus and the cytoplasm, exhibiting a homogeneous staining pattern (fig 2b). Afterwards, the fluorescence in the c y t o p l a s m showing a thread-like staining pattern, became predominant, with the fluorescence in the nucleus becoming weak (fig 2c). At the randomly proliferating phase, cells had a similar staining pattern as in figure 2c, and mitotic cells showed intense staining in the entire cytoplasm (fig 2d). lmmunoelectron microscopy The fine structure and the antigenicity o f p53 protein in cultured cells after mild aldehyde fixation and low-temperature e m b e d d i n g in L o w i c r y l K 4 M , were g e n e r a l l y w e l l - p r e served. Ultrastructural localization o f p53 in P H A - s t i m u l a t e d l y m p h o c y t e s was s t u d i e d b y the p o s t - e m b e d d i n g irnmunogold staining method. In resting lymphocytes, the nucleus occupied nearly all o f the cell volume. Within the nucleus, chromatin formed a larger mass at the periphery, the configuration o f w h i c h has been c o n s i d e r e d as condensed chromatin (fig 3a). W h e n the resting lymphocytes were labeled with anti-p53 immunogold, the gold particles were sparsely found in the c y t o p l a s m and the nucleus. In the nucleus, the particles were found in the nuclear matrix, but not associated with the condensed chromatin (fig 3b). At 72 h after P H A stimulation, the cells became enlarged, and the chromatin in the nucleus was fully dispersed. Both

Fig 1. Indirect immunofluorescent staining of p53 protein in lymphocytes, a. As the control, treatment with primary antibody was omitted. b. Resting, unstimulated cells, e. Cells PHA-stimulated for 24 h. d. Cells PHA-stimulated for 72 h. Bar = 25/.tin.

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Fig 2. Indirect immunofluorescent staining of p53 protein in fibroblasts, a. Cells 6 h after release from resting state, b. Cells 16 h after the release, c. Cells 24 h after the release, showing a thread-like staining pattern in the cytoplasm(arrows), d. A cell in mitotic phase (arrowhead). Bar = 50 #m.

the nucleus and the cytoplasm were labeled with a number of gold particles (fig 3c). In the cytoplasm, the gold particles were predominantly on the cell periphery and on the dense intra-cytoplasmic organelles, probably the mitochondria. The particles in the nucleus were distributed diffusely among the chromatin. In fibroblasts at the randomly proliferating phase, actin filament bundles in the cytoplasm were strongly labeled with the gold particles, whereas other parts of the cytoplasm and the nucleus were poorly labeled (fig 4b). In control experiments, the levels of background labeling were very low and negligible (fig 4a). In fibroblasts, 16 h after stimulation, just prior to the S phase, the gold particles in the nucleus increased. However, the distribution and the density of the gold particles in the cytoplasm were almost the same as those in fibroblasts at the randomly proliferating phase (data not shown). In mitotic cells in randomly proliferating cultures, the cytoplasm was heavily labeled but the chromosomes themselves were rarely labeled (fig 4c). Because a close association of p53 protein with actin filaments was suggested as shown in figure 4b, we studied p53 localization in cells extracted with saponin to determine whether or not p53 is present in association with soluble proteins. In the cytoplasm of the randomly proliferating fibroblasts extracted with saponin, the gold particles were present on the actin filament bundles, and on the mitochondrial membranes (fig 5a). The nucleus of the saponin-extracted cell was shown as being composed of granular and fine fibrous matrix structures, and the gold particles were found on the granular matrix structures rather than the fibrous structures

(fig 5b). Discussion Non-dividing normal lymphocytes have undetectable levels of p53 protein [12, 13] or p53 mRNA [16, 18]. However, when stimulated by PHA to enter the division cycle, induction and accumulation of p53 mRNA and protein are observed, suggesting that the p53 protein is coded by a cell cycle-dependent gene. Our results also showed that immuno-histochemically detected p53 increased markedly after PHA stimulation. The ultrastructural study here, however, showed that freshly isolated human lymphocytes contain a considerable amount of p53 in both the cyto-

plasm and nucleus. In lymphoblastic cells after 24-72 h of PHA stimulation, the condensed chromatin in the nucleus is dispersed as well-characterized by Pompidou et al [17]. In such l y m p h o b l a s t i c cells, p53 i n c r e a s e d and was p r e s e n t d i f f u s e l y a m o n g d i s p e r s e d c h r o m a t i n in the nucleus and just beneath the p l a s m a m e m b r a n e . The results suggest that p53 increases in both the cytoplasm and the nucleus in the transition from resting to proliferating state. It has been found that the p53 molecule has a domain of nuclear localization signals at the C-terminus [22]. Shaulsky et al [23] have shown that the p53 protein accumulates in the nucleus of Balb/c 3T3 cells at a specific phase around the beginning of the S phase and, following the initial step of DNA synthesis, p53 is no longer found in the nucleus but rather accumulates in the cytoplasm. Human skin fibroblasts released from confluentresting states by trypsinization and replating showed a similar pattern (fig 2), but the specific nuclear accumulation at the beginning of the S phase was not observed. Ultrastructurally, the p53-reactive gold particles in the randomly proliferating cells were found abundantly on the actin filaments. The results also suggest that an increased amount of both the cytoplasmic and the nuclear p53 may be important for the initiation of DNA synthesis in normal human cells. p53 in the n o n - t r a n s f o r m e d f i b r o b l a s t s has been detected in the cytoplasm in a Triton X-100 soluble fraction, and associated with the cytoskeleton [20]. p53 has also been detected at the plasma membrane of both normal and transformed cells during mitosis [14] and on microtubules in the cytoplasm of transformed cells [11]. Our observation showed that p53 was p r e d o m i n a n t l y located near the plasma membrane of lymphoblastic ceils and on the actin filaments of fibroblasts. It is well-known that most animal cells possess a special dense network of actin filaments just beneath the plasma membrane [1]. It is probable that p53 found near the plasma membrane in mitotic cells [14] and in lymphoblastic cells observed here is also associated with the actin filaments. Extraction with saponin well-preserves the cytoskeleton by removing soluble intracellular proteins [6]. After extraction with saponin, p53 was found to be associated with the actin filaments and the mitochondrial membranes, suggesting that p53 is directly associated with the actin filaments and the mitochondrial m e m b r a n e s in the cyto-

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CI

b

Fig 3. Immunogold staining of p53 protein in lymphocytes, a. A resting, unstimulated cell. As the control, treatment with primary antibody was omitted, b. Resting, unstimulated cell. c. Cell 72 h after PHA-stimulation. N, nucleus; asterisks, condensed chromatin. x 20000.

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Fig 4. Immunogold staining of p53 protein in fibroblasts, a. As the control, treatment with primary antibody was omitted, b. A cell at randomly proliferating phase, e. A mitotic cell. N, nucleus, F, actin filament bundles; asterisks, chromosomes. × 18000.

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Fig 5. Immunogold staining of p53 protein in fibroblasts. The cell was extracted with saponin, a. A portion of the cytoplasm, b. A portion of the nucleus. F, actin filament bundles; m, mitochondria; N, nucleus. × 20000.

plasm. We propose that the protein-protein interaction domain in the tetramerization of p53 [3] is also involved in the interaction between actin and p53. In the resting nucleus of the lymphocytes, p53 was associated with the non-chromatin nuclear structure. In the saponin-extracted nucleus of proliferating fibroblasts, p53 was associated with granular structures in the nucleus. Recently, the retinoblastoma gene product (Rb), which is known as one of the tumor suppressor genes and has similar functions as p53, has been seen to regulate cell proliferation through interaction with the nuclear matrix [10]. It was shown that hypophosphorylated Rb associated with the nuclear matrix only during early G1, suggesting that the nuclear pool of Rb was in a dynamic, cell cycle-dependent equilibrium between the soluble (nucleoplasmic) and the insoluble (nuclear matrix) states, depending on phosphorylation. In this study, so far as chromatin and chromosomal structures were identified ultrastructurally, p53 was associated with non-chromatin structures. A more detailed study on the substructural localization in the nucleus during cell cycle progression or after UV irradiation is in progress.

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