Downloaded from genesdev.cshlp.org on October 7, 2021 - Published by Cold Spring Harbor Laboratory Press p53-Dependent and independent expression of p21 during cell growth, differentiation, and DNA damage Kay F. Macleod, 1 Nicole Sherry, 1 Greg Hannon, 2 David Beach, 2 Takashi Tokino, 3 Kenneth Kinzler, 3 Bert Vogelstein, 3 and Tyler Jacks L4 tHoward Hughes Medical Institute, Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 USA; 2Howard Hughes Medical Institute, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724 USA; 3The Oncology Center, The John Hopkins University School of Medicine, Baltimore, Maryland 21231 USA Expression of p21 has been shown to be up-regulated by the p53 tumor suppressor gene in vitro in response to DNA-damaging agents. However, p21 expression can be regulated independently of p53, and here we show that expression of p21 in various tissues during development and in the adult mouse occurs in the absence of p53 function. However, most tissues tested did require p53 for p21 induction following exposure of the whole animal to ~/irradiation. These results show that normal tissue expression of p21 to high levels is not dependent on p53 and confirm that induction of p21 by DNA-damaging agents does require p53. p21 is expressed upon differentiation of p53-deficient murine erythroleukemia (MEL1 cells, and the kinetics of induction of p21 in this system suggest that it may be involved in the growth arrest that precedes terminal differentiation. The gene is up-regulated in mouse fibroblasts in response to serum restimulation but the kinetics and levels of induction differ between wild-type and mutant cells. Expression of p21 message following serum restimulation is superinducible by cycloheximide in wild-type but not in p53-deficient cells. The increases in p21 mRNA are reflected in changes in p21 protein levels, p21 expression also appears to be regulated at the post-transcriptional level because moderate increases in mRNA expression, during differentiation of MEL cells and upon serum restimulation of fibroblasts, are followed by large increases in protein levels. Regulation of the mouse p21 promoter by p53 depends on two critical p53-binding sites located 1.95 and 2.85 kb upstream from the transcriptional initiation site. The sequences mediating serum responsiveness of the promoter map to a region containing the proximal p53 site. p53 appears to play a critical role in p21 induction following DNA damage. Moreover, p21 can be regulated independently of p53 in several situations including during normal tissue development, following serum stimulation, and during cellular differentiation. [Key Words: DNA damage; p53; p21~ cellular differentiation) Received January 27, 1995; revised version accepted March 3, 1995. Cell-cycle progression has been shown to be modulated cyclin/cdk inhibitor or as an assembly factor may de- by a new class of regulators known as cyclin kinase in- pend on the stoichiometry of components within these hibitors. The first of these proteins to be identified and complexes (Zhang et ah 19941, such that at low concen- cloned was p21, which is the product of the WAF- 1 / CIP 1 tration p21 may promote complex assembly, whereas at gene and is variously referred to as p21/WAF-1/CIP1 (E1- higher concentrations it might be inhibitory. Deiry et al. 1993; Harper et al. 1993; Xiong et al. p21 was cloned independently by virtue of its induc- 1993a, b). p21 forms a quaternary complex with cyclins, tion by p53 (E1-Deiry et al. 1993), its association with cyclin-dependent kinases (cdks), and proliferating cell cyclin/cdk complexes (Xiong et al. 1993b; Harper et al. nuclear antigen (PCNA)(Xiong et al. 1993a} and acts to 1993) and its up-regulation in senescent cells INoda et al regulate cyclin/cdk activity and to directly affect DNA 19941. Other cyclin/cdk inhibitors have since been iden- replication through its interaction with PCNA (Waga et tified and cloned (Serrano et al. 1993; Hannon and Black al. 1994; Li et al. 1994). p21 can promote cyclin/cdk 1994; Polyak et al. 1994), including p16 (Serrano et al. complex assembly in vitro (Zhang et al. 1994), and be- 19931 and p15 (Hannon and Black 19941, which appear to cause p21 exists in normal cycling cells, it may perform specifically inhibit cyclin D/cdk4 complexes, p16 is fre- this function in vivo as well. The role of p21 as either a quently mutated in a variety of tumors (Hussussian et al. 1994; Kamb et al. 1994a,b; Nobori et al. 1994). The p27 4Conesponding author. cyclin/cdk inhibitor is also involved in mediating GENES & DEVELOPMENT 9:935-944 91995 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/95 $5.00 935 Downloaded from genesdev.cshlp.org on October 7, 2021 - Published by Cold Spring Harbor Laboratory Press Macleod et al. growth arrest and inhibits cyclin E/cdk2 and cyclin These results suggest that regulation of expression of A/cdk2 complexes, p27 is induced in T cells in response p21 mRNA in normal mouse tissues occurs by a mech- to interleukin-2 and in the HL60 cell line in response to anism not involving p53. However, previous reports retinoic acid as these cells differentiate to form granulo- have clearly shown the importance of p53 in the induc- cytes (Polyak et al. 1994). However, only p21 has been tion of p21 following exposure to ionizing radiation and shown to affect DNA replication (Waga et al. 1994; Li et other DNA-damaging agents (Di Leonardo et al. 1994; al. 1994). E1-Deiry et al. 1994). Thus, we examined the pattern of p21 is up-regulated by p53 following DNA damage p21 expression in wild-type and mutant animals follow- (Xiong et al. 1993a, b; E1-Deiry et al. 1993, 1994; Di Le- ing ~/irradiation. As shown in Figure 1B, significant in- onardo et al. 1994) and is thought to be an integral part of duction of p21 occurs in all wild-type tissues examined, the p53-mediated growth-arrest pathway (E1-Deiry et al. except for the small intestine in which p21 expression is 1994). Regulation of p21 by p53 presumably occurs at the very high prior to irradiation. No induction of p21 level of transcriptional initiation because it is known mRNA was observed in tissues of irradiated p53-defi- that the ability of p53 to induce growth arrest requires its cient animals (Fig. 1B), confirming the importance of p53 trans-activation domain (Pietenpol et al. 1994). Also, in the irradiation response. loss of p53 results in dramatic reductions in p21 message Given the results with adult tissues, we next exam- levels (Xiong et al. 1993a; E1-Deiry et al. 1993, 1994). The ined p21 mRNA levels in tissues from wild-type and putative role of p21 in growth arrest mediated by p53 p53-/- embryos. Most embryonic cell types are still makes it an important gene with respect to the under- proliferating and the role of p21 in modulating cell-cycle standing of loss of growth control in transformation pro- progression may result in a different pattern of p21 ex- cesses. Mice that are homozygous for a targeted disrup- pression in embryonic tissues compared with adult tis- tion of the p53 gene are predisposed to tumor formation sues. As shown in Figure 1C, p21 is expressed in the but develop normally (Donehower et al. 1992; Jacks et al. carcass of wild-type embryos at 13.5 days of gestation 1994). Thus, either p21 itself is not essential for normal but not in fetal liver, brain, lungs, or heart. As was ob- development or p21 expression is not fully dependent on served in adult tissues, expression in embryonic carcass p53. is not dependent on p53 because high levels of expres- To characterize more fully the normal regulation of sion are also seen in p53-/- embryos (Fig. 1C). Quite p21, we have compared its expression in a variety of cells unexpectedly, absence of p53 actually resulted in in- and tissues with or without functional p53. We conclude creased expression of p21 in embryonic brain. that p21 expression during development and in the adult mouse can be p53-independent and that the gene can be induced by serum and upon differentiation in the ab- p21 mRNA and protein are induced during MEL sence of p53 function. However, p53 appears to be nec- cell differentiation essary for p21 induction following DNA damage, a func- Murine erythroleukemia (MEL) cells are derived from tion that may be critical for its tumor suppressor prop- erythoid tumors that develop in mice infected by the erties. Friend virus complex. These leukemic cells are growth- factor independent, carry activated forms of Ets family oncogenes, and are mutant for p53 (Ben-David and Bern- Results stein 1991). The cell line is effectively blocked in ery- p21 mRNA is expressed in various tissues throid differentiation and proliferates in culture until in- independently of p53 duced to terminally differentiate by various agents such as dimethylsulfoxide (DMSO)or N, N'-hexamethylene- Given the role of p21 in regulating cell-cycle progression bis-acetamide (HMBA). Alterations in the cell cycle of and DNA replication (Harper et al. 1993; Xiong et al. MEL cells following their induction to differentiate are 1993b; Li et al. 1994; Waga et al. 1994), we were inter- well characterized. Induction of differentiation in MEL ested in determining whether p21 expression was cells results in a prolonged G~ phase, up-regulated ex- strictly dependent on the presence of functional p53. Our pression of the retinoblastoma protein (Rb) tumor sup- initial studies focused on expression of p21 in tissues pressor gene, and increased levels of hypophosphorylated from wild-type and p53-deficient mice.