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P53-Mediated Accumulation of Hypophosphorylated Prb After the G1 Restriction Point Fails to Halt Cell Cycle Progression

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P53-Mediated Accumulation of Hypophosphorylated Prb After the G1 Restriction Point Fails to Halt Cell Cycle Progression Oncogene (1997) 15, 337 ± 345 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00 p53-mediated accumulation of hypophosphorylated pRb after the G1 restriction point fails to halt cell cycle progression Steven P Linke1,2,*, Matthew P Harris3,*, Sarah E Neugebauer3, Kristie C Clarkin1, H Michael Shepard3, Daniel C Maneval3 and Georey M Wahl1 1Gene Expression Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037; 2Department of Biology, University of California - San Diego, La Jolla, California 92093; 3Canji, Inc, 3030 Science Park Road, Suite 302, San Diego, California 92121, USA This study analyses whether the inability of p53 to and inactivate p53 (Momand et al., 1992). Mdm2 was induce G1 arrest after the restriction point relates to an later shown to interact with pRb and pRb-regulated inability to modulate pRb phosphorylation. Transient E2F complexes as well (Martin et al., 1995; Xiao et al., p53 overexpression in normal human diploid ®broblasts 1995), although recent studies suggest that deletion of and p53-de®cient cancer cells led to increased levels of Mdm2 has no additional eect on cell proliferation, the cyclin-dependent kinase inhibitor p21Cip1/Waf1/Sdi1 and cell cycle control or tumorigenesis when p53 is absent an accumulation of hypophosphorylated pRb in cells (Jones et al., 1996; McMasters et al., 1996). growing asynchronously and in cells synchronized in late p53 and pRb participate in several cell cycle G1 or M. Similarly, g-irradiation of asynchronous, late- checkpoint responses that contribute to maintaining G1, or S phase ®broblasts led to an increase in genetic stability. DNA damage activates p53 (Kastan et hypophosphorylated pRb. Experiments with ®broblasts al., 1991), inducing a prolonged G0/G1 cell cycle arrest expressing the HPV16 E6 protein indicated that resembling senescence in human ®broblasts (Di accumulation of hypophosphorylated pRb required func- Leonardo et al., 1994; Linke et al., 1997). Ribonucleo- tional p53. Progression into and through S phase was not tide depletion activates a reversible p53-dependent altered by the presence of hypophosphorylated pRb in arrest in G0 or early G1, resembling quiescence (Linke late G1, consistent with the failure of p53 to mediate G1 et al., 1996). p53 has also recently been implicated in arrest in cells that are past the restriction point. These regulating centrosome replication (Fukasawa et al., data indicate that accumulation of hypophosphorylated 1996) and in preventing DNA rereplication when pRb has signi®cantly dierent eects on cell cycle spindle assembly is blocked (Cross et al., 1995; Di progression in early G1 versus late G1 or S phase. Leonardo et al., 1997). Inactivation of p53 allows for the emergence of aneuploid cells and mutants with Keywords: p53; pRb; restriction point; DNA damage; structural chromosome changes such as gene amplifica- Glarrest tion (Livingstone et al., 1992; Yin et al., 1992). Similarly, inactivation of pRb compromises the DNA damage-dependent arrest response (Almasan et al., 1995; Demers et al., 1994; Slebos et al., 1994) and the Introduction checkpoint activated by spindle inhibitors (Di Leonar- do et al., 1997) and enables cells to enter the cycle Retinoblastoma (RB) and p53 gene de®ciencies occur when certain deoxyribonucleotides become limiting in diverse human neoplasms. p53 alterations have been after treatment with antimetabolites such as metho- reported in approximately half of human malignancies trexate (Almasan et al., 1995). However, inactivation of encompassing a wide spectrum of tissues (Greenblatt et pRb in cells expressing wild-type p53 can lead to p53- al., 1994) and although RB alterations are more tissue- dependent apoptosis in vitro (Almasan et al., 1995) and speci®c (Horowitz et al., 1990), some member of the in vivo (Howes et al., 1994; Morgenbesser et al., 1994; pRb pathway (e.g., p16INK4a, cyclin D1, or pRb) is Pan and Griep, 1994). Thus, a collateral de®ciency in frequently disrupted in many tumors (Sherr, 1996). p53 pRb and p53 allows cells to enter S phase under was initially discovered as a cellular protein bound by suboptimal conditions that contribute to genetic SV40 T-antigen, and it was subsequently found that instability and accelerate tumorigenesis (Di Leonardo pRb is also inactivated by this viral oncoprotein et al., 1993; Hartwell, 1992; Hartwell and Kastan, (DeCaprio et al., 1988; Lane and Crawford, 1979; 1994). Linzer and Levine, 1979). Replication of many DNA pRb modulation of G1-S phase progression is viruses requires inactivation of both p53 and pRb, regulated, at least in part, by cell cycle-dependent which is typically achieved by expression of one or phosphorylation (Buchkovich et al., 1989; Chen et al., more such oncoproteins (Levine et al., 1991). In 1989; DeCaprio et al., 1989). pRb which is found addition, regulation of the activity of p53 and pRb is predominantly in a hypophosphorylated form in early mediated by endogenous binding proteins. For G1 phase, binds to a subset of E2F complexes, thereby example, the Mdm2 protein was ®rst reported to bind inhibiting their transcriptional activation potential (Hiebert et al., 1992). As cells reach the restriction point in mid to late G , pRb is phosphorylated by Correspondence: GM Wahl 1 *These authors contributed equally cyclin/cyclin-dependent kinase (Cdk) complexes, most Received 1 August 1996; revised 14 April 1997; accepted 16 April notably cyclin D/Cdk4 and cyclin D/Cdk6. In addition, 1997 it is further phosphorylated near the G1-S transition by p53 modulation of pRb phosphorylation state SP Linke et al 338 cyclin A/Cdk2 and, perhaps, by cyclin E/Cdk2 the inability of p53 to mediate cell cycle arrest beyond (reviewed by Bartek et al., 1996). Phosphorylation of this point relates to dierences in its ability to pRb prevents its inhibitory association with E2F modulate pRb phosphorylation. We show that p53 complexes, allowing transcriptional activation of gene can be activated after the restriction point, leading to products required for S phase entry and DNA an accumulation of hypophosphorylated pRb. replication (DeGregori et al., 1995; Farnham et al., Although these late-G1 cells contained predominantly 1993; Slansky et al., 1993). pRb then remains hypophosphorylated pRb, they progressed into S hyperphosphorylated under normal conditions until phase. The data imply that the barrier to arrest in the transition from metaphase to anaphase (Durfee et late G1 is not due to an inability of p53 to receive al., 1993; Ludlow et al., 1993). damage signals or to mediate the accumulation of The activity of the Cdks that phosphorylate pRb can hypophosphorylated pRb. Rather, cells in late G1 be modi®ed by Cdk inhibitors, such as p21Cip1/Waf1/Sdi1 appear to have activated an irreversible program to (p21), members of the INK family (reviewed by Hunter enter S phase that is refractory to the functions of both and Pines, 1994), and other kinases (reviewed by p53 and pRb. Morgan, 1995; Sherr, 1994). Since p21 is induced by p53 (el-Deiry et al., 1993; Harper et al., 1995), pRb phosphorylation can be aected by the diversity of Results stresses that activate p53 in G1, including DNA damage and ribonucleotide depletion (Demers et al., Overexpression of p53 in asynchronous cells causes cell 1994; Di Leonardo et al., 1994; Dulic et al., 1994; cycle arrest, induction of p21 and accumulation of Linke et al., 1996; Slebos et al., 1994). Thus the net hypophosphorylated pRb result of p53 activation in G1 is to maintain pRb in its hypophosphorylated form, preventing activation of We previously showed that transient overexpression of E2F-responsive gene products required for S phase p53 by adenoviral transduction in p53-de®cient cell progression (DeGregori et al., 1995; Slansky et al., lines resulted in a dramatic decrease in S phase 1993). progression as measured by [3H]thymidine uptake Normal human diploid ®broblasts (NDF) that are g- (Harris et al., 1996). The use of adenovirus for p53 irradiated in early to mid G1 undergo a dose-dependent gene transfer provides an attractive tool to assess the permanent G0/G1 arrest (Di Leonardo et al., 1994). In eects of p53 expression on cell cycle regulation, as contrast, NDF irradiated in late G1 fail to arrest. This high gene transfer eciencies are obtained in many cell suggests that a fundamental change occurs in the lines, and the episomal nature of the vector obviates transduction of DNA damage signals at this juncture. the need for selection procedures. The H358 tumor cell The mechanism by which the damage response line was chosen for initial studies because it lacks p53 pathway becomes desensitized in late G1 has not been protein and contains apparently normal pRb (Otterson elucidated. This study looks at the eects of p53 et al., 1994; Takahashi et al., 1989). NDF strain activation on pRb phosphorylation and cell cycle MRC-9 was studied in parallel to address the function progression in late G1 and S phase to determine if of overexpressed p53 in normal human cells. ac b d ppRb ppRb — — pRb — pRb — p53 — p53 — p21 — p21 Figure 1 p53 overexpression in asynchronous cells leads to cell cycle arrest accompanied by p21 induction and accumulation of hypophosphorylated pRb. Cultures of p53-de®cient H358 or NDF strain MRC-9 were infected with adenovirus containing wild- type p53 (V53) or control vector with an empty expression cassette (V), or they were left untreated (UN). Infections were at 36106 and 16107 infectious units/ml for H358 and MRC-9, respectively. After 24 h samples were pulse-labeled with BrdU for 1 h and harvested for cell cycle or Western blot analysis. Cells harvested for cell cycle analysis were stained with propidium iodide and anti- BrdU-FITC and analysed by bivariate ¯ow cytometry (a and c). Numbers represent the percentage of cells in each cell cycle phase. The lower-left region represents G0 and G1 phase cells, the upper region represents S phase cells, and the lower-right region represents G2 and M phase cells.
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