No abstract

Exposure of mammalian cells to ionizing radiation (IR) induces a complex array of cellular responses including cell cycle arrest and͞or apoptosis. IR-induced G 1 arrest has been shown to depend on the presence of the tumor suppressor p53, which acts as a transcriptional activator of several genes. p53 also plays a role in the induction of apoptosis in response to DNA damage, and this pathway can be activated by both transcription-dependent and -independent mechanisms. Here we report the identification of a novel transcript whose expression is induced in response to IR in a p53-dependent manner, and that shows homology to the type 2C protein phosphatases. We have named this novel gene, wip1. In vitro, recombinant Wip1 displayed characteristics of a type 2C phosphatase, including Mg 2؉ dependence and relative insensitivity to okadaic acid. Studies performed in several cell lines revealed that wip1 accumulation following IR correlates with the presence of wild-type p53. The accumulation of wip1 mRNA following IR was rapid and transient, and the protein was localized to the nucleus. Similar to waf1, ectopic expression of wip1 in human cells suppressed colony formation. These results suggest that Wip1 might contribute to growth inhibitory pathways activated in response to DNA damage in a p53-dependent manner.

To investigate the effect that human wild-type p53 (wt-p53) expression has on cell proliferation we constructed a recombinant plasmid, pM47, in which wt-p53 cDNA is under transcriptional control of the hormone-inducible mouse mammary tumor virus promoter linked to the dominant biochemical selection marker gene Eco gpt. The pM47 plasmid was introduced into T98G cells derived from a human glioblastoma multiforme tumor, and a stable clonal cell line, GM47.23, was derived that conditionally expressed wt-p53 following exposure to dexamethasone. We show that induction of wt-p53 expression in exponentially growing cells inhibits cell cycle progression and that the inhibitory effect is reversible upon removal of the inducer or infection with simian virus 40. Moreover, when growth-arrested cells are stimulated to proliferate, induction of wt-p53 expression inhibits Go/Gj progression into S phase and the cells accumulate with a DNA content equivalent to cells arrested in the Go/Gj phase of the cell cycle. Taken together, these studies suggest that wt-p53 may play a negative role in growth regulation.

The proliferating cell nuclear antigen (PCNA or cyclin) is a nuclear protein recently identified as a cofactor of DNA polymerase delta. When exponentially growing Balb/c3T3 cells are exposed to antisense oligodeoxynucleotides to PCNA, both DNA synthesis and mitosis are completely suppressed. A corresponding sense oligodeoxynucleotide has no inhibitory effects. These experiments indicate that PCNA (cyclin) is important in cellular DNA synthesis and in cell cycle progression.

Conditional expression of wild-type (wt) p53 protein in a glioblastoma tumor cell line has been shown to be growth inhibitory. We have now more precisely localized the position in the cell cycle where growth arrest occurs. We show that growth arrest occurs prior to or near the rici on point in late G1 phase of the cell cycle. The effect of wt p53 protein on the expression of four iediate-earl genes (c-FOS, c-JUN, JUN-B, and c-MYC), one delayed-early gene (ornithine decarboxylase), and two late-G1/S-phase genes (B-MYB and DNA polymerase a) was also examined. Of this subset of growth response gene, only the expression ofB-MYB and DNA polymerase a was slgicantly repressed. The possibility that decreased expression of B-MYB may be an important component of growth arrest mediated by wt p53 protein is diss. Cell cycle controls that regulate the orderly flow of cells in and out of G1 phase are the main determinant of the rate of postembryonic cell proliferation (8, 9). Two major control points have been defined in animal cells. One control point appears to operate in early G1 phase and allows cells to exit the cell cycle and enter a nonproliferative state of arrest termed Go (8,9). The second control point, the restriction point (R-point) occurs in late G1 phase (8, 10). Time-course experiments indicate that protein synthesis is required throughout early G1 phase for cells to pass the R-point and become committed to enter S phase and initiate DNA synthesis. The R-point is thus defined as the time after which inhibition of protein synthesis fails to inhibit entry into S phase (8, 10).We have shown that induction ofwt p53 protein expression in a human glioblastoma tumor cell line can inhibit cell cycle progression (5). In this model, growth inhibition was associated with a significant decrease in the steady-state mRNA levels of the replication-dependent histone H3 gene (5), which is known to be coordinately regulated with the onset of DNA replication (11), and the replication-independent proliferating cell nuclear antigen (PCNA) gene (12). On the contrary, the steady-state mRNA levels for the genes encoding endogenous p53, (32-microglobulin, (-actin, and thymidine kinase were not found to be significantly affected (5,12).In the present communication, we have more precisely localized the position in the cell cycle where growth suppression occurs, and we have examined the effect of induction of wt-p53 protein on the expression of seven additional growth response genes. Evidence is presented that growth suppression mediated by wt p53 protein occurs prior to or near the R-point in late G1 phase ofthe cell cycle and that only a subset of late-G1/S-phase growth response genes are affected. MATERIALS AND METHODSCell Lines and Culture Conditions. The parental human glioblastoma cell line T98G and the sublines GM47.23 and Mdel 4A were cultured in Earle's minimal essential medium containing 10% fetal calf serum (GIBCO) at 370C as described (5, 12). The Mdel 4A subline was derived by transfection with the pMdel plasmid (4) ...

The p53 gene is a frequent target of mutation in a wide variety of human cancers. Previously, it was reported that conditional expression ofwild-type p53 protein in a cell line (GM47.23) derived from a human glioblastoma multiform tumor had a negative effect on cell proliferation. We have now investigated the effect that induction of wild-type p53 protein in this cell line has on the expression of the proliferating-cell nuclear antigen gene. The proliferating-cell nuclear antigen gene encodes a nuclear protein that is an auxiliary factor of DNA polymerase 8 and part of the DNA replication machinery of the cell. We show that inhibition ofcell cycle progression into S-phase after induction ofwild-type p53 protein is accompanied by selective down-regulation of proliferating-cell nuclear antigen mRNA and protein expression. (14). Thus these studies support the hypothesis that the inhibitory effect of wt-p53 on oncogene-mediated transformation may be directly linked to its growth-suppressing activity and that this function can be inactivated by mutation.Direct experimental evidence for a growth-suppressing function of wt-p53 was provided by the demonstration that conditional expression of wt-p53 protein in a cell line derived from a human glioblastoma multiform tumor blocks cell cycle progression (15). In this system, inhibition of cell cycle progression was associated with a marked decrease in histone H3 mRNA expression, an S-phase marker gene whose regulation is tightly linked to DNA replication. This finding suggested that the wt-p53 protein induced in these cells may specifically effect a function (or functions) required for progression from G1 into S phase. To gain further insight into the possible mechanism(s) of growth suppression induced by wt-p53, we studied the effect of wt-p53 protein on the expression of the gene encoding proliferating-cell nuclear antigen (PCNA Plasmids and Probe Preparation. The probes used for hybridization were from plasmids digested with the appropriate restriction enzyme and followed by gel purification. They include a human p53 cDNA Xba I fragment of p53H (21), a /3-actin BamHI fragment of pHFf3A-1 (22), a human histone H3 EcoRI fragment of pFO422 (23), a human PCNA cDNA fragment of pPCNA-G3, a human thymidine kinase (TK) cDNA fragment ofpTK11 (kind gifts ofRenato Baserga, Abbreviations: wt, wild type; PCNA, proliferating-cell nuclear antigen; Dex, dexamethasone; TK, thymidine kinase. tTo whom reprint requests should be addressed at: 3400 North Broad Street, Philadelphia, PA 19140. 1958 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Monoclonal antibody directed against the transformation-related protein p53 was microinjected manually into the nuclei of quiescent Swiss 3T3 mouse cells. The cells were subsequently stimulated with 10% fetal calf serum. Microinjection of p53 antibody at or around the time of serum stimulation clearly inhibited the subsequent entry of Swiss 3T3 cells into the S phase of the cell cycle. p53 antibody had no effect on serum-stimulated DNA synthesis when it was microinjected 4 hr or later after serum stimulation. Monoclonal antibody to an unrelated antigen, Lyt-2.2, had no effect on serum-stimulated DNA synthesis regardless of the-time it was microinjected. Under similar experimental conditions, p53 antibody had no effect on simian virus 40-or adenovirus 2-induced DNA synthesis. These experiments add strength to the suggestion that p53 is involved in the regulation of cell proliferation.A protein with a molecular weight ofapproximately 53,000 (p53) has been identified in mouse cells transformed by viruses, chemicals, orx-radiation (1-6). This protein, although in smaller amounts, has also been detected in proliferating normal cells (7). Milner and Milner (8) reported that p53 is not synthesized in nondividing Go lymphocytes but is synthesized in the same lymphocytes when they are stimulated to proliferate by the addition of concanavalin A. Because p53 has been closely associated with cellular proliferation and transformation, it has been suggested that it is involved in the regulation ofthe mammalian cell cycle.Several monoclonal antibodies have now been described that react with p53 of mouse cells by immunofluorescence and immunoprecipitation (5,6,9,10 Antibodies. Two p53 monoclonal antibodies of mouse origin were used in these studies. One was a y2a product of hybridoma 200-47, described by Dippold et aL (9), and the second was a y2b product ofhybridoma PAb 122, described by Gurney et aL (5). Lyt-2.2 monoclonal antibody (aLyt-2.2) was a y2a product of hybridoma 19/178, originally derived by Ulrich Hammerling. aLyt-2.2 was used as a microinjection control, because expression of Lyt-2.2 is restricted to cells of T-cell lineage. The reactivity ofhamster antisera to the Mr 72,000 protein of adenovirus 2 (Ad2) has been described by Rossini et al (11). Antisera having specificity for the SV40 T antigen were derived from hamsters bearing SV40 tumors (12).Immunoglobulin Preparations. Immunoglobulin fractions were prepared by ammonium sulfate precipitation of (i) sera from nu/nu mice bearing hybridoma 20047 (ap53) or hybridoma 19/178 (aLyt-2.2), (ii) supernatant fluid of cultures of hybridoma PAb 122 (ap53), or (iii) sera of hamsters bearing SV40-induced tumors (aSV40 T antigen). The precipitates obtained after the addition of 45% saturated ammonium sulfate were resuspended in phosphate-buffered saline and dialyzed extensively against the same buffer at 4°C prior to microinjection. In all cases, the concentration of microinjected protein was 2.5 mg/ml.Microinjection Procedure. The microinjection procedure was c...

The MDMX gene product is related to the MDM2 oncoprotein, both of which interact with the p53 tumor suppressor. We have identified a novel transcript of the MDMX gene that is expressed in a variety of cell lines, and in particular, in growing and transformed cells. This transcript is identical to the published sequence yet it has a short internal deletion of 68 base pairs. This deletion produces a shift in the reading frame after codon 114, resulting in the inclusion of a stop codon at amino acid residue 127 (full-length MDMX is 489 residues). This truncated MDMX protein is termed MDMX-S ("short form"), represents only the p53-binding domain, and appears to bind p53 better than full-length MDMX. The MDMX-S protein can be detected in cell extracts and when overexpressed is much more effective than MDMX at inhibiting p53-mediated transcriptional activation and induction of apoptosis. Since MDMX-S lacks the central and carboxyl-terminal regions contained within full-length MDMX, it is likely to play a key role in the regulation of cell proliferation and apoptosis in a way distinct from MDMX.The p53 tumor suppressor protein plays an important role in regulating movement through a number of cell cycle checkpoints (1-3, 38). The activity of p53 is in turn modulated through the action of the MDM2 protein which is amplified in a variety of tumors (4 -6). MDM2 can associate with p53 (7) and directly inhibit p53's ability to activate transcription of target genes, such as p21. MDM2 performs this function in a number of ways. First, the amino-terminal domain MDM2 binds tightly to the transcriptional transactivation domain of p53 (4 -6, 8, 9). Through this interaction, MDM2 blocks the ability of p53 to activate transcription of specific target genes by repressing the formation of the preinitiation complex mediated through the TFIIE and TATA-binding protein subunits of RNA polymerase II (10, 11). The second blocking activity of MDM2 is that it targets p53 for proteolytic degradation by the ubiquitin-proteasome pathway (12)(13)(14)40). Third, MDM2 also functions to shuttle p53 out of the nucleus and into the cytoplasm, via a nuclear export signal located within the MDM2 protein (33).While MDM2 was first identified as a protein that inhibits the action of p53 it has recently been shown to affect other components of the G 1 /S transition. MDM2 was found to associate with both the E2F1 transcription factor (15) and the retinoblastoma tumor suppressor (pRb) 1 (16). The interaction with pRb was found to be repressive, in that the association of MDM2 with the carboxyl-terminal domain of pRb inhibited the growth regulatory function of pRb (16). The interaction with E2F1, over the amino terminus of MDM2 and the carboxyl terminus of E2F1, was stimulatory in the sense that MDM2 enhanced the ability of E2F1 to activate target gene expression (15). Thus MDM2 appears to regulate at least three of the important players in the G 1 /S transition: p53, pRb, and E2F1. It has therefore been concluded that MDM2 functions in the fashion of a domi...